Francisco C. A. Mello

Hepatitis B virus genotypes circulating in Brazil: molecular characterization of genotype F isolates

BackgroundHepatitis B virus (HBV) isolates have been classified in eight genotypes, A to H, which exhibit distinct geographical distributions. Genotypes A, D and F are predominant in Brazil, a country formed by a miscegenated population, where the proportion of individuals from Caucasian, Amerindian and African origins varies by region. Genotype F, which is the most divergent, is considered indigenous to the Americas. A systematic molecular characterization of HBV isolates from different parts of the world would be invaluable in establishing HBV evolutionary origins and dispersion patterns. A large-scale study is needed to map the region-by-region distribution of the HBV genotypes in Brazil.ResultsGenotyping by PCR-RFLP of 303 HBV isolates from HBsAg-positive blood donors showed that at least two of the three genotypes, A, D, and F, co-circulate in each of the five geographic regions of Brazil. No other genotypes were identified. Overall, genotype A was most prevalent (48.5%), and most of these isolates were classified as subgenotype A1 (138/153; 90.2%). Genotype D was the most common genotype in the South (84.2%) and Central (47.6%) regions. The prevalence of genotype F was low (13%) countrywide. Nucleotide sequencing of the S gene and a phylogenetic analysis of 32 HBV genotype F isolates showed that a great majority (28/32; 87.5%) belonged to subgenotype F2, cluster II. The deduced serotype of 31 of 32 F isolates was adw4. The remaining isolate showed a leucine-to-isoleucine substitution at position 127.ConclusionThe presence of genotypes A, D and F, and the absence of other genotypes in a large cohort of HBV infected individuals may reflect the ethnic origins of the Brazilian population. The high prevalence of isolates from subgenotype A1 (of African origin) indicates that the African influx during the colonial slavery period had a major impact on the circulation of HBV genotype A currently found in Brazil. Although most genotype F isolates belonged to cluster II, the presence of some isolates belonging to clusters I (subgroup Ib) and IV suggests the existence of two or more founder viral populations of genotype F in Brazil.

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.

Occult hepatitis B virus infection and lamivudine‐resistant mutations in isolates from renal patients undergoing hemodialysis

Background and Aims:  Patients undergoing hemodialysis are at risk of infection with both hepatitis B virus (HBV) and hepatitis C virus (HCV). Occult HBV infection is usually associated with low levels of HBV and is frequently detected in HCV‐infected patients. The aims of the present study were to compare the prevalence of occult HBV infection among anti‐HCV‐positive and anti‐HCV‐negative patients undergoing hemodialysis, and characterize the molecular patterns of HBV isolates from patients with occult infection.

Patterns of hepatitis B virus infection in Brazilian human immunodeficiency virus infected patients: high prevalence of occult infection and low frequency of lamivudine resistant mutations

Hepatitis B virus (HBV) molecular profiles were determined for 44 patients who were infected with human immunodeficiency virus (HIV) type 1 and had antibodies to the hepatitis B core antigen (anti-HBc), with and without other HBV serological markers. In this population, 70% of the patients were under lamivudine treatment as a component of antiretroviral therapy. HBV DNA was detected in 14 (32%) patients. Eight out of 12 (67%) HBsAg positive samples, 3/10 (30%) anti-HBc only samples, and 3/22 (14%) anti-HBs positive samples were HBV DNA positive. HBV DNA loads, measured by real time polymerase chain reaction, were much higher in the HBsAg positive patients (mean, 2.5 x 10(9) copies/ml) than in the negative ones (HBV occult infection; mean, 2.7 x 10(5) copies/ml). Nine out of the 14 HBV DNA positive patients were under lamivudine treatment. Lamivudine resistant mutations in the polymerase gene were detected in only three patients, all of them belonging to the subgroup of five HBsAg positive, HBV DNA positive patients. A low mean HBV load (2.7 x 10(5) copies/ml) and an absence of lamivudine resistant mutations were observed among the cases of HBV occult infection.

Epidemiological study of hepatitis A, B and C in the largest Afro-Brazilian isolated community

This study was conducted to estimate the prevalence and molecular epidemiological features of viral hepatitis A, B and C in the Kalunga population, which represents the largest Afro-Brazilian isolated community. Among 878 individuals studied, the overall prevalence of anti-hepatitis A virus antibodies was 80.9%, with a significant rise from 44.8% to near 100% between the first and fourth decade of life. Rates for hepatitis B surface antigen (HBsAg) and antibody to hepatitis B core antigen (anti-HBc) of 1.8% and 35.4%, respectively, were found. Increasing age, male gender, illiteracy and history of multiple sexual partners were associated with hepatitis B virus (HBV) infection. An occult HBV infection rate of 1.7% (5/295) was found among anti-HBc-positive individuals. HBV genotype A (subtype Aa) was dominant in this community. Only 5/878 individuals (0.6%) were positive for anti-hepatitis C virus (HCV). HCV RNA was detected in three of them, who were infected with genotype 1 (subtype 1a). These findings point out high, intermediate and low endemicity for hepatitis A, B and C, respectively, in the Kalunga community in Brazil. Circulation of HBV genotype A (subtype Aa) in this Afro-Brazilian isolated community indicates the introduction of this virus during the slave trade from Africa to Brazil.

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.

Hepatitis B Virus Subgenotype A1: Evolutionary Relationships between Brazilian, African and Asian Isolates

Brazil is a country of low hepatitis B virus (HBV) endemicity in which the genotype A of HBV (HBV/A) is the most prevalent. The complete nucleotide sequences of 26 HBV/A isolates, originating from eight Brazilian states, were determined. All were adw2. Twenty-three belonged to subgenotype A1 and three to A2. By phylogenetic analysis, it was shown that all the 23 HBV/A1 isolates clustered together with isolates from Bangladesh, India, Japan, Nepal, the Philippines and United Arab Emirates, but not with those of Congo, Kenya, Malawi, Rwanda, South Africa, Tanzania, Uganda and Zimbabwe. Four amino acid residues in the polymerase (His138 in the terminal protein domain, Pro18 and His90 in the spacer, and Ser109 in the reverse transcriptase), and one (Phe17) in the precore region, predominated in Latin American and Asian HBV/A1 isolates, but were rarely encountered in African isolates, with the exception of those from Somalia. Specific variations of two adjacent amino acids in the C-terminal domain of the HBx protein, namely Ala146 and Pro147, were found in all the Brazilian, but rarely in the other HBV/A1 isolates. By Bayesian analysis, the existence of an ‘Asian-American’ clade within subgenotype A1 was supported by a posterior probability value of 0.996. The close relatedness of the Brazilian, Asian and Somalian isolates suggests that the HBV/A1 strains predominant in Brazil did not originate from the five million slaves who were imported from Central and Western Africa from 1551 to 1840, but rather from the 300–400,000 captives forcibly removed from southeast Africa at the middle of the 19th century.

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.

Detection of mixed populations of wild-type and YMDD hepatitis B variants by pyrosequencing in acutely and chronically infected patients

BackgroundLamivudine (LAM) is associated with the highest known rate of resistance mutations among nucleotide analogs used to treat chronic hepatitis B virus (HBV) infection. Despite this, LAM continues in widespread use, especially in combination therapies. The primary LAM resistance mutation (rtM204V/I) occurs in the YMDD motif of HBV polymerase. The aim of this study was to characterize Brazilian HBV isolates from acute and chronic cases by direct sequencing, and to identify HBV quasispecies in the YMDD motif using a pyrosequencing method capable of detecting single-nucleotide polymorphisms. HBV DNA from serum samples of 20 individuals with acute HBV infection and 44 with chronic infection undergoing antiviral therapies containing LAM were analyzed by direct sequencing and pyrosequencing methods.ResultsPhylogenic analyses of direct-sequenced isolates showed the expected genotypes (A, D and F) for the Brazilian population in both acute and chronic infections. However, within genotype A isolates, subgenotype A2 was more frequently detected in acute cases than in chronic cases (P = 0.012). As expected, none of the individuals with acute hepatitis B had LAM-resistant isolates as a dominant virus population, whether detected by direct sequencing or pyrosequencing. However, pyrosequencing analyses showed that 45% of isolates (9/20) had minor subpopulations (4-17%) of LAM-resistant isolates. Among chronic patients undergoing LAM treatment, YMDD mutants were frequently found as a dominant virus population. In cases where wild-type virus was the dominant population, subpopulations of YMDD variants were usually found, demonstrating the complexity of HBV quasispecies.ConclusionsYMDD variants were frequently detected as a minor population in acute HBV infection. The occurrence of pre-existing variants may lead to a high frequency of resistant mutants during antiviral therapy in the chronic phase. In chronic infection, detection of YMDD variants before virological or biochemical breakthrough might contribute to making better therapy choices and thus improving treatment outcome.

Prevalence of hepatitis B virus infection among tuberculosis patients with or without HIV in Goiânia City, Brazil

BACKGROUND Hepatitis B virus (HBV) and tuberculosis (TB) represent major public health problems. There is currently little data on HBV infection among TB patients with and without human immunodeficiency virus (HIV). OBJECTIVES To assess HBV prevalence among TB patients with and without HIV. STUDY DESIGN From April 2008 to March 2010, a cross-sectional study was conduct among TB patients attended at a reference hospital in Goiânia City, Brazil. The participants were tested for serological markers of HBV infection and HIV antibodies. HBV DNA was detected in HBsAg-positive samples, and also in HBsAg-negative/anti-HBc-positive samples to look for HBV occult infection. RESULTS Of 425 patients, 402 (94.6%) agreed to participate in the study. The overall prevalence of HBV (HBsAg and/or anti-HBc positive) and HIV infections were 25.6% (103/402) and 27.6% (111/402), respectively. A higher HBV infection rate was found among HIV-infected patients (36.9%; 41/111) compared to patients infected with TB only (20.0%; 57/285). A multivariate analysis of risk factors showed that age ≥ 50 years (p=0.03), non-injecting (p<0.01) and injecting (p<0.01) drugs use were associated with HBV infection. Among the HBsAg-positive samples (n=13), HBV DNA was detected in 10 (76.9%) samples. Of the 90 anti-HBc-positive samples, 13 were HBV DNA positive (with very low levels) resulting in an occult HBV infection rate of 14.4%. PCR-RFLP was successfully performed in 20 HBV DNA-positive samples: 15 were genotype A and 5 were genotype D. CONCLUSIONS HBV infection was common, particularly among this with HIV infection.