A Novel Complex A/C/G Intergenotypic Recombinant of Hepatitis B Virus Isolated in Southern China

Hepatitis B virus (HBV) genotypes and subgenotypes may vary in geographical distribution and virological features. Previous investigations, including ours, showed that HBV genotypes B and C were respectively predominant in South and North China, while genotypes A and D were infrequently detected and genotype G was not found. In this study, a novel A/C/G intergenotype was identified in patients with chronic HBV infection in Guilin, a city in southern China. Initial phylogenetic analysis based on the S gene suggested the HBV recombinant to be genotype G. However, extended genotyping based on the entire HBV genome indicated it to be an A/C/G intergenotype with a closer relation to genotype C. Breakpoint analysis using the SIMPLOT program revealed that the recombinant had a recombination with a arrangement of genotypes A, G, A and C fragments. Compared with the HBV recombinants harboring one or two genotype G fragments found in Asian countries, this Guilin recombinant was highly similar to the Vietnam (98–99%) and Long An recombinants (96–99%), but had a relatively low similarity to the Thailand one (89%). Unlike those with the typical genotype G of HBV, the patients with the Guilin recombinant were seropositive for HBeAg. Moreover, a relatively high HBV DNA viral load (>2×10⁶ IU/ml) was detected in the patients, and the analysis of viral replication capacity showed that the Guilin recombinant strains had a competent replication capacity similar to genotypes B and C strains. These findings can aid in not only the clarification of the phylogenetic origin of the HBV recombinants with the genotype G fragment found in Asian countries, but also the understanding of the virological properties of these complicated HBV recombinants.     

A type-specific nested PCR assay established and applied for investigation of HBV genotype and subgenotype in Chinese patients with chronic HBV infection

ABSTRACT: BACKGROUND: Many studies have suggested that hepatitis B virus (HBV) genotypes show not only geographical distribution and race specificity, but also are associated with disease progression and response to interferon treatment. The objective of this study was to develop a nested polymerase chain reaction (nPCR) assay for genotypes A-D and subgenotypes B1, B2, C1 and C2 of hepatitis B virus (HBV) and to investigate the distribution characteristics of HBV genotypes/subgenotype in China. METHODS: After redesigning the primers and optimizing the reaction conditions using common Taq polymerase, the sensitivity, specificity and reproducibility of the method were evaluated using plasmids and serum samples. In total, 642 serum samples from patients with chronic HBV infection were applied to investigate the distribution of HBV genotype and subgenotype in China. RESULTS: The genotype and subgenotype could be identified when the HBV DNA load of a sample was ≥10(2.3) IU/mL. For the 639 successfully genotyped samples, the sequencing results of 130 randomly selected samples (20.3%, 130/639) were consistent with those of the nPCR method. The present study showed that HBV genotype B (11.2%, 72/642), C (68.2%, 438/642) and D (7.2%, 46/642) were circulating in China, while genotype C was the dominant strain except for western region where genotype D was the prevalent strain. The main subgenotypes of genotypes B and C were B2 (87.5%, 63/72) and C2 (92.9%, 407/438), respectively. CONCLUSIONS: The low-cost nPCR method would be a useful tool for clinical and epidemiological investigation in the regions where genotypes A-D are predominant.     

A Rare HBV Subgenotype D4 with Unique Genomic Signatures Identified in North-Eastern India –An Emerging Clinical Challenge?

Background/Aims

HBV has been classified into ten genotypes (A–J) and multiple subgenotypes, some of which strongly influence disease outcome and their distribution also correlate with human migration. HBV infection is highly prevalent in India and its diverse population provides an excellent opportunity to study the distinctiveness of HBV, its evolution and disease biology in variegated ethnic groups. The North-East India, having international frontiers on three sides, is one of the most ethnically and linguistically diverse region of the country. Given the paucity of information on molecular epidemiology of HBV in this region, the study aimed to carry out an in-depth genetic characterization of HBV prevailing in North-East state of Tripura.

Methods

From sera of chronically HBV infected patients biochemical/serological tests, HBV DNA quantification, PCR-amplification, sequencing of PreS/S or full-length HBV genomes were done. HBV genotype/subgenotype determination and sequence variability were assessed by MEGA5-software. The evolutionary divergence times of different HBV subgenotypes were estimated by DNAMLK/PHYLIP program while jpHMM method was used to detect any recombination event in HBV genomes.

Results

HBV genotypes D (89.5%), C (6.6%) and A (3.9%) were detected among chronic carriers. While all HBV/A and HBV/C isolates belonged to subgenotype-A1 and C1 respectively, five subgenotypes of HBV/D (D1–D5) were identified including the first detection of rare D4. These non-recombinant Indian D4 (IndD4) formed a distinct phylogenetic clade, had 2.7% nucleotide divergence and recent evolutionary radiation than other global D4. Ten unique amino acids and 9 novel nucleotide substitutions were identified as IndD4 signatures. All IndD4 carried T¹²⁰ and R¹²⁹ in ORF-S that may cause immune/vaccine/diagnostic escape and N¹²⁸ in ORF-P, implicated as compensatory Lamivudine resistance mutation.

Conclusions

IndD4 has potential to undermine vaccination programs or anti-viral therapy and its introduction to North-East India is believed to be linked with the settlement of ancient Tibeto-Burman migrants from East-Asia.     

Subgenotyping of Genotype C Hepatitis B Virus: Correcting Misclassifications and Identifying a Novel Subgenotype

Background

More than ten subgenotypes of genotype C Hepatitis B virus (HBV) have been reported, including C1 to C16 and two C/D recombinant subgenotypes (CD1 and CD2), however, inconsistent designations of these subgenotypes still exist.

Methodology/Principal Findings

We performed a phylogenetic analysis of all full-length genotype C HBV genome sequences to correct the misclassifications of HBV subgenotypes and to study the influence of recombination on HBV subgenotyping. Our results showed that although inclusion of the recombinant sequences changed the topology of the phylogenetic tree, it did not affect the subgenotyping of the non-recombinant sequences, except subgenotype C2. In addition, most of the subgenotypes have been properly designated. However, several misclassifications of HBV subgenotypes have been identified and corrected. For example, C11 proposed by Utsumi and colleagues in 2011 was found to be grouped with C12 proposed by Mulyanto and colleagues. Two sequences, {"type":"entrez-nucleotide","attrs":{"text":"GQ358157","term_id":"282929781","term_text":"GQ358157"}}GQ358157 and {"type":"entrez-nucleotide","attrs":{"text":"GU721029","term_id":"289721059","term_text":"GU721029"}}GU721029, previously designated as C6 have been re-designated as C12 and C7, respectively. Moreover, a quasi-subgenotype C2 was proposed, which included the old C2, several previously unclassified sequences and previously designated C14. In particular, we identified a novel subgenotype, tentative C14, which was well supported by phylogenetic analysis and sequence divergence of >4%.

Conclusions/Significance

A number of misclassifications in the subgenotyping of genotype C HBV have been identified in this study. After correcting the misclassifications, we proposed a better classification for the subgenotyping of genotype C HBV, in which a novel quasi-subgenotype C2 and a novel subgenotype, tentative C14, were described. Based on this large-scale analysis, we propose that a novel subgenotype should only be reported after a complete comparison of all relevant sequences rather than a few representative sequences only.     

Research on Hepatitis B virus Genotypes and Subgenotypes among Bai Nationality in Dali, Yunnan Province

To investigate the distribution of hepatitis B virus （HBV） genotypes and subgenotypes among the Bai nationality in Dali, a total of 100 serum samples from patients with chronic HBV-infection were collected for the detection of HBV genotypes and subgenotypes by genotype-specific primers and restriction fragment length polymorphism （RLFP）, respectively. Among the 100 samples, the proportions of genotype B, C and mixed genotype （B＋C） were 41%, 25% and 34%, respectively. All the genotype B strains belonged to subgenotype Ba In genotype C, 84% were Subgenotype Cs and 12% were subgenotype Ce. The distribution of genotypes B, C and B＋C showed no significant difference between male and female patients （P=0.182） and among the age groups of patients （P=0.812）. The rates of HBeAg/HBeAg positivity were no significantly different among genotypes B, genotype C and mixed genotype （B＋C） （P=0.077/P=0.663）. In Dali, genotypes B, B＋C and C existed among Bai nationality with chronic HBV-infection, and genotype B was the major genotype. Subgenotypes Ba and Cs were the predominant strains in patients with HBV genotype B/C infection. The most prominent characteristic was the higher prevalent rate of mixed genotype （B＋C） in patients.     

Research on hepatitis B virus genotypes and subgenotypes among Bai nationality in Dali, Yunnan Province

To investigate the distribution of hepatitis B virus (HBV) genotypes and subgenotypes among the Bai nationality in Dali, a total of 100 serum samples from patients with chronic HBV-infection were collected for the detection of HBV genotypes and subgenotypes by genotype-specific primers and restriction fragment length polymorphism (RLFP), respectively. Among the 100 samples, the proportions of genotype B, C and mixed genotype (B C) were 41%, 25% and 34%, respectively. All the genotype B strains belonged to subgenotype Ba. In genotype C, 84% were Subgenotype Cs and 12% were subgenotype Ce. The distribution of genotypes B, C and B C showed no significant difference between male and female patients (P=0.182) and among the age groups of patients (P=0.812). The rates of HBeAg/HBeAg positivity were no significantly different among genotypes B, genotype C and mixed genotype (B C) (P=0.077/P=0.663). In Dali, genotypes B, B C and C existed among Bai nationality with chronic HBV-infection, and genotype B was the major genotype. Subgenotypes Ba and Cs were the predominant strains in patients with HBV genotype B/C infection. The most prominent characteristic was the higher prevalent rate of mixed genotype (B C) in patients.     

Investigating the origin and spread of hepatitis C virus genotype 5a

Epidemiological and phylogenetic studies of hepatitis C virus (HCV) have identified six major HCV genotypes and have attempted to characterize their origin and spread worldwide. Putative regions of endemic infection have been identified for all HCV genotypes except HCV genotype 5a. Although HCV genotype 5a was previously thought to be largely restricted to the northern part of South Africa, this study reports an unexpected cluster of the genotype in West Flanders Province in Belgium. To investigate the molecular epidemiology of this cluster and of HCV genotype 5a in general, a rigorous phylogenetic analysis of Belgian and South African HCV genotype 5a samples was performed. Remarkably, the Belgian and South African strains form two distinct clusters of similar diversity. We used a Bayesian coalescent method to estimate the rate of virus spread through time for HCV genotype 5a in both regions. Our results indicate that HCV genotype 5a strains have been spreading independently in Belgium and South Africa for more than 100 years, with a rate of spread characteristic of an epidemic genotype. These findings have major implications for tracing the origin of HCV genotype 5a. Here, we speculate about the possible origins of these clusters.     

Replacement of previously circulating respiratory syncytial virus subtype B strains with the BA genotype in South Africa

Respiratory syncytial virus (RSV) is a major cause of bronchiolitis and pneumonia in infants, the immunocompromised, and the elderly in both developed and developing countries. Reinfections are common, and G protein variability is one mechanism to overcome herd immunity. This is illustrated by the appearance of the BA genotype with a 60-nucleotide duplication dominating the subtype B genotypes in epidemics worldwide. To investigate the evolution of subtype B in South Africa since 2002, the genetic variability of the G protein was analyzed in all recent strains isolated over 4 years (2006 to 2009) in South African hospitals. Bayesian analysis revealed a replacement of all subtype B genotypes previously identified in South Africa with the BA genotype since 2006, while subtype A genotypes identified in previous years are still circulating. Compared to BA strains from other countries, the evolutionary rate of the South African BA genotype was shown to be 2.305 × 10(-3) nucleotide substitutions/site/year and drift was evident. The most recent common ancestor (MRCA) of the South African BA viruses was determined to date back to 1996. All South African BA isolates clustered with the BA-IV subgenotype, and the appearance of new subgenotypes within this branch may occur if drift continues. Sequencing of the complete G protein of selected South African strains revealed an additional 6-nucleotide deletion. Acquisition of the 60-nucleotide duplication appeared to have improved the fitness of this virus, and more recent subtype B strains may need to be included in experimental vaccines to evaluate their efficacy in the current setting of evolved circulating strains.     

Reconstructing the Complex Evolutionary History of Hepatitis B Virus

A detailed analysis of the evolutionary history of hepatitis B virus (HBV) was undertaken using 39 mammalian hepadnaviruses for which complete genome sequences were available, including representatives of all six human genotypes, as well as a large sample of small S gene sequences. Phylogenetic trees of these data were ambiguous, supporting no single place of origin for HBV, and depended heavily on the underlying model of DNA substitution. In some instances genotype F, predominant in the Americas, was the first to diverge, suggesting that the virus arose in the New World. In other trees, however, sequences from genotype B, prevalent in East Asia, were the most divergent. An attempt was also made to determine the rate of nucleotide substitution in the C open reading frame and then to date the origin of HBV. However, no relationship between time and number of substitutions was found in two independent data sets, indicating that a reliable molecular clock does not exist for these data. Both the pattern and the rate of nucleotide substitution are therefore complex phenomena in HBV and hinder any attempt to reconstruct the past spread of this virus. Received: 5 December 1998 / Accepted: 23 February 1999     

High Diversity of Hepatitis B Virus Genotypes in Panamanian Blood Donors: A Molecular Analysis of New Variants

Hepatitis B Virus (HBV) is an infectious agent that causes more than half of the cases of liver disease and cancer in the world. Globally there are around 250 million people chronically infected with this virus. Despite 16% of the cases of liver disease in Central America are caused by HBV, the information regarding its genetic diversity, genotypes and circulation is scarce. The purpose of this study was to evaluate the genetic variability of the HBV genotypes from HBV-DNA positive samples obtained from screening blood donors at the Social Security System of Panama and to estimate its possible origin. From 59,696 blood donors tested for HBV infection during 2010–2012, there were 74 HBV-DNA positive subjects. Analysis of the partial PreS2-S region of 27 sequences shows that 21% of the infections were caused by genotype A, 3% by genotype D and 76% by genotype F. In addition, we were able to confirm circulation of six sub-genotypes A1, A2, A3, D4, F3, F1 and a proposed new sub-genotype denominated F5pan. We found a confinement of sub-genotypes F1 and F5pan to the western area of Panama. The tMRCA analysis suggests a simultaneous circulation of previously described sub-genotypes rather than recent introductions of the Panamanian sub-genotypes in the country. Moreover, these results highlight the need of more intensive research of the HBV strains circulating in the region at the molecular level. In conclusion, Panama has a high HBV genotype diversity that includes a new proposed sub-genotype, an elevated number of PreCore-Core mutations, and confinement of these variants in a specific geographical location.     

Estimating mutation parameters,population history and genealogy simultaneously from temporally spaced sequence data

Molecular sequences obtained at different sampling times from populations of rapidly evolving pathogens and from ancient subfossil and fossil sources are increasingly available with modern sequencing technology. Here, we present a Bayesian statistical inference approach to the joint estimation of mutation rate and population size that incorporates the uncertainty in the genealogy of such temporally spaced sequences by using Markov chain Monte Carlo (MCMC) integration. The Kingman coalescent model is used to describe the time structure of the ancestral tree. We recover information about the unknown true ancestral coalescent tree, population size, and the overall mutation rate from temporally spaced data, that is, from nucleotide sequences gathered at different times, from different individuals, in an evolving haploid population. We briefly discuss the methodological implications and show what can be inferred, in various practically relevant states of prior knowledge. We develop extensions for exponentially growing population size and joint estimation of substitution model parameters. We illustrate some of the important features of this approach on a genealogy of HIV-1 envelope (env) partial sequences.     

Spatial and temporal dynamics of hepatitis B virus D genotype in Europe and the Mediterranean Basin

Hepatitis B virus genotype D can be found in many parts of the world and is the most prevalent strain in south-eastern Europe, the Mediterranean Basin, the Middle East, and the Indian sub-continent. The epidemiological history of the D genotype and its subgenotypes is still obscure because of the scarcity of appropriate studies. We retrieved from public databases a total of 312 gene P sequences of HBV genotype D isolated in various countries throughout the world, and reconstructed the spatio-temporal evolutionary dynamics of the HBV-D epidemic using a bayesian framework.The phylogeographical analysis showed that India had the highest posterior probability of being the location of the tree root, whereas central Asia was the most probable location of the common ancestor of subgenotypes D1-D3. HBV-D5 (identified in native Indian populations) diverged from the tree root earlier than D1-D3. The time of the most recent common ancestor (tMRCA) of the tree root was 128 years ago, which suggests that the common ancestor of the currently circulating subgenotypes existed in the second half of the XIX century. The mean tMRCA of subgenotypes D1-D3 was between the 1940s and the 1950-60s. On the basis of our phylogeographic reconstruction, it seems that HBV-D reached the Mediterranean area in the middle of the XX century by means of at least two routes: the first pathway (mainly due to the spread of subgenotype D1) crossing the Middle East and reaching north Africa and the eastern Mediterranean, and the second pathway (closely associated with D2) that crossed the former Soviet Union and reached eastern Europe and the Mediterranean through Albania. We hypothesise that the main route of dispersion of genotype D was the unsafe use of injections and drug addiction.     

Inferring epidemiological parameters on the basis of allele frequencies

In this article, I develop a methodology for inferring the transmission rate and reproductive value of an epidemic on the basis of genotype data from a sample of infected hosts. The epidemic is modeled by a birth-death process describing the transmission dynamics in combination with an infinite-allele model describing the evolution of alleles. I provide a recursive formulation for the probability of the allele frequencies in a sample of hosts and a Bayesian framework for estimating transmission rates and reproductive values on the basis of observed allele frequencies. Using the Bayesian method, I reanalyze tuberculosis data from the United States. I estimate a net transmission rate of 0.19/year [0.13, 0.24] and a reproductive value of 1.02 [1.01, 1.04]. I demonstrate that the allele frequency probability under the birth-death model does not follow the well-known Ewens' sampling formula that holds under Kingman's coalescent.     

Validation of Cross-Genotype Neutralization by Hepatitis B Virus-Specific Monoclonal Antibodies by In Vitro and In Vivo Infection

Vaccines based on hepatitis B virus (HBV) genotype A have been used worldwide for immunoprophylaxis and are thought to prevent infections by non-A HBV strains effectively, whereas, vaccines generated from genotype C have been used in several Asian countries, including Japan and Korea, where HBV genotype C is prevalent. However, acute hepatitis B caused by HBV genotype A infection has been increasing in Japan and little is known about the efficacy of immunization with genotype C-based vaccines against non-C infection. We have isolated human monoclonal antibodies (mAbs) from individuals who were immunized with the genotype C-based vaccine. In this study, the efficacies of these two mAbs, HB0116 and HB0478, were analyzed using in vivo and in vitro models of HBV infection. Intravenous inoculation of HBV genotype C into chimeric mice with human hepatocytes resulted in the establishment of HBV infection after five weeks, whereas preincubation of the inocula with HB0116 or HB0478 protected chimeric mice from genotype C infection completely. Interestingly, both HB0116 and HB0478 were found to block completely genotype A infection. Moreover, infection by a genotype C strain with an immune escape substitution of amino acid 145 in the hepatitis B surface protein was also completely inhibited by incubation with HB0478. Finally, in vitro analysis of dose dependency revealed that the amounts of HB0478 required for complete protection against genotype C and genotype A infection were 5.5 mIU and 55 mIU, respectively. These results suggested that genotype C-based vaccines have ability to induce cross-genotype immunity against HBV infection.     

New methods employing multilocus genotypes to select or exclude populations as origins of individuals

A new method for assigning individuals of unknown origin to populations, based on the genetic distance between individuals and populations, was compared to two existing methods based on the likelihood of multilocus genotypes. The distribution of the assignment criterion (genetic distance or genotype likelihood) for individuals of a given population was used to define the probability that an individual belongs to the population. Using this definition, it becomes possible to exclude a population as the origin of an individual, a useful extension of the currently available assignment methods. Using simulated data based on the coalescent process, the different methods were evaluated, varying the time of divergence of populations, the mutation model, the sample size, and the number of loci. Likelihood-based methods (especially the Bayesian method) always performed better than distance methods. Other things being equal, genetic markers were always more efficient when evolving under the infinite allele model than under the stepwise mutation model, even for equal values of the differentiation parameter F(st). Using the Bayesian method, a 100% correct assignment rate can be achieved by scoring ca. 10 microsatellite loci (H approximately 0.6) on 30-50 individuals from each of 10 populations when the F(st) is near 0.1.     

Genetic Diversity of the Hepatitis B Virus Strains in Cuba: Absence of West-African Genotypes despite the Transatlantic Slave Trade

Cuba is an HBsAg low-prevalence country with a high coverage of anti-hepatitis B vaccine. Its population is essentially the result of the population mix of Spanish descendants and former African slaves. Information about genetic characteristics of hepatitis B virus (HBV) strains circulating in the country is scarce. The HBV genotypes/subgenotypes, serotypes, mixed infections, and S gene mutations of 172 Cuban HBsAg and HBV-DNA positive patients were determined by direct sequencing and phylogenetic analysis. Phylogenetic analysis of HBV S gene sequences showed a predominance of genotype A (92.4%), subgenotype A2 (84.9%) and A1 (7.6%). Genotype D (7.0%) and subgenotype C1 (0.6%) were also detected but typical (sub)genotypes of contemporary West-Africa (E, A3) were conspicuously absent. All genotype A, D, and C strains exhibited sequence characteristics of the adw2, ayw2, and adrq serotypes, respectively. Thirty-three (19.1%) patients showed single, double, or multiple point mutations inside the Major Hydrophilic domain associated with vaccine escape; eighteen (10.5%) patients had mutations in the T-cell epitope (amino acids 28-51), and there were another 111 point mutations downstream of the S gene. One patient had an HBV A1/A2 mixed infection. This first genetic study of Cuban HBV viruses revealed only strains that were interspersed with strains from particularly Europe, America, and Asia. The absence of genotype E supports previous hypotheses about an only recent introduction of this genotype into the general population in Africa. The presence of well-known vaccine escape (3.5%) and viral resistance mutants (2.9%) warrants strain surveillance to guide vaccination and treatment strategies.     

Hepatitis B Virus Genotype D Isolates Circulating in Chapecó, Southern Brazil,Originate from Italy

Hepatitis B virus genotype A1 (HBV/A1), of African origin, is the most prevalent genotype in Brazil, while HBV/F predominates in the other South American countries. However, HBV/D is the most common in the three states of southern Brazil, where ‘islands’ of elevated prevalence, as Chapecó and other cities, have been described. In this study, 202 HBV chronic carriers attending in 2013 the viral hepatitis ambulatory of Chapecó, were investigated. In comparison with previous studies performed in the same ambulatory, a rapid aging of the HBV infected population was observed (mean age of the newly diagnosed patients increasing from 29.9 ± 10.3 years in 1996 to 44.4 ± 13.3 years in 2013), probably due to a singular vaccination schedule at Chapecó that included not only children but also adolescents. Phylogenetic and BLAST analyses (S region) classified 91 HBV isolates into genotypes A (n = 3) and D (n = 88). The majority of HBV/D isolates were closely related to D3 sequences. To understand the reasons for the absence or near absence of genotypes A and F, and how HBV/D was introduced in the south of Brazil, HBV/D infected patients were inquired about their genealogical and geographical origins. Forty-three (52%) patients have their four grandparents of Italian origin, vs. seven (8%) who have their four grandparents of Brazilian origin. At all, 65 out of 83 (78%) patients had at least one grandparent originating from Italy. Taking into consideration the fact that Italy is one of the few countries where subgenotype D3 is predominant, the results strongly suggested that HBV/D was introduced in Brazil through Italian immigration which culminated between 1870 and 1920.     

Occult HBV infection and suppression of HCV replication in the early phase of combination therapy for chronic hepatitis C

BACKGROUND: Occult HBV infection in subjects with chronic hepatitis C is related to more severe disease outcome. It has been suggested that it might reduce sensitivity to antiviral treatment. AIMS: To assess in HBsAg negative subjects with chronic hepatitis C any effect of the presence of HBV genomes in the liver on the early kinetics of HCV-RNA under PEG-IFN plus ribavirin. PATIENTS AND METHODS: Twenty-two anti-HCV and HCV-RNA positive subjects, with biopsy-proven chronic hepatitis C (M/F 15/7; 50 +/- 8.6 years, 16 genotype 1b) were given PEG-IFN alpha 2b 1.0 microg qw plus ribavirin (800 to 1,200 mg daily according to body weight) for an intended 52 week period. Early virological response was assessed over the first 4 weeks of therapy by quantifying HCV-RNA. Occult HBV infection was assessed by testing for HBV-DNA in the liver before therapy. RESULTS: HBV genomes were found in the liver of 7 of 22 (31.4%) patients, unrelated to anti-HBc status. Kinetics of HCV-RNA during the first 4 weeks of antiviral treatment was unaffected by occult HBV infection, both in terms of absolute reduction of viral load and of number of cases with a reduction of > or = 2 log10 on treatment. CONCLUSIONS: Occult HBV infection does not affect the early phase of response to combination therapy. Further follow-up of patients into the maintenance phase of antiviral treatment and after stopping it will clarify if and when occult HBV has a role in reducing sustained virological response.     

A revised evolutionary history of hepatitis B virus (HBV)

Previous studies of the evolutionary history of hepatitis B virus (HBV) have been compromised by intergenotype recombination and complex patterns of nucleotide substitution, perhaps caused by differential selection pressures. We examined the phylogenetic distribution of recombination events among human HBV genotypes and found that genotypes A plus D, and genotypes B plus C, had distinct patterns of recombination suggesting differing epidemiological relationships among them. By analyzing the nonoverlapping regions of the viral genome we found strong bootstrap support for some intergenotypic groupings, with evidence of a division between human genotypes A–E from the viruses sampled from apes and human genotype F. However, the earliest events in the divergence of HBV remain uncertain. These uncertainties could not be explained by differential selection pressures, as the ratio of nonsynonymous-to-synonymous substitutions (d _N/d _S) did not vary extensively among lineages and there is no strong evidence for positive selection across the whole tree. Finally, we provide a new estimate of the mean substitution rate in HBV, 4.2 × 10⁻⁵, which suggests that divergence of HBV in humans and apes has occurred only in the last 6000 years.     

Plant growth promotion and Penicillium citrinum

Background

Lamivudine is an oral nucleoside analogue widely used for the treatment of chronic hepatitis B. The main limitation of lamivudine use is the selection of resistant mutations that increases with time of utilization. Hepatitis B virus (HBV) isolates have been classified into eight genotypes (A to H) with distinct geographical distributions. HBV genotypes may also influence pathogenic properties and therapeutic features. Here, we analyzed the HBV genotype distribution and the nature and frequency of lamivudine resistant mutations among 36 patients submitted to lamivudine treatment for 12 to 84 months.

Results

Half of the patients were homosexual men. Only 4/36 (11%) patients were HBV DNA negative. As expected for a Brazilian group, genotypes A (24/32 positive individuals, 75%), D (3/32, 9.3%) and F (1/32, 3%) were present. One sample was from genotype C, which is a genotype rarely found in Brazil. Three samples were from genotype G, which had not been previously detected in Brazil. Lamivudine resistance mutations were identified in 20/32 (62%) HBV DNA positive samples. Mean HBV loads of patients with and without lamivudine resistance mutations were not very different (2.7 × 10⁷ and 6.9 × 10⁷ copies/mL, respectively). Fifteen patients showed the L180M/M204V lamivudine resistant double mutation. The triple mutant rt173V/180M/204V, which acts as a vaccine escape mutant, was found in two individuals. The three isolates of genotype G were entirely sequenced. All three showed the double mutation L180M/M204V and displayed a large genetic divergence when compared with other full-length genotype G isolates.

Conclusion

A high (55%) proportion of patients submitted to long term lamivudine therapy displayed resistant mutations, with elevated viral load. The potential of transmission of such HBV mutants should be monitored. The identification of genotypes C and G, rarely detected in South America, seems to indicate a genotype distribution different to that observed in non treated patients. Disparities in routes of transmission (genotype G seems to be linked to homosexual behavior) and in pathogenic properties (genotype C is very aggressive) among HBV genotypes may explain the presence of rare genotypes in the present work.