A dual role for adeno-associated virus in human health.

Adeno-associated virus (AAV) differs from most other viruses, as it requires the simultaneous presence of a helper virus for an active infection. Up to 80% of the human population is seropositive for AAV antibodies. AAV has been known to be a non-pathogenic virus and an inhibitor of carcinogenesis caused by coinfecting viruses. However, the recent reports associating AAV infection with hepatocellular carcinoma development and the mysterious cases of acute severe hepatitis in children have challenged the idea that AAV is a harmless virus. Herein, we explore the usefulness of AAV in gene therapy and the importance of AAV as a protector or perpetrator in human carcinogenesis, ultimately reflecting on the dual role of AAV in human health.

Hepatitis B Virus Genotype D: An Overview of Molecular Epidemiology, Evolutionary History, and Clinical Characteristics.

The hepatitis B virus (HBV) genotype D (HBV/D) is the most extensively distributed genotype worldwide with distinct molecular and epidemiological features. This report provides an up-to-date review on the history of HBV/D subgenotyping and misclassifications, along with large-scale analysis of over 1000 HBV/D complete genome sequences, with the aim of gaining a thorough understanding of the global prevalence and geographic distribution of HBV/D subgenotypes. We have additionally explored recent paleogenomic findings, which facilitated the detection of HBV/D genomes dating back to the late Iron Age and provided new perspectives on the origins of modern HBV/D strains. Finally, reports on distinct disease outcomes and responses to antiviral therapy among HBV/D subgenotypes are discussed, further highlighting the complexity of this genotype and the importance of HBV subgenotyping in the management and treatment of hepatitis B.

Adeno-associated virus infection and its impact in human health: an overview.

Discovered as a contaminant of adenovirus stocks in the 1960s, adeno-associated virus (AAV) is a mono-stranded DNA virus that depends on helper factors to replicate. Even though AAV is endemic in the human population (35-80%), it is remarkable that many issues concerning the natural infection by this virus remain unanswered. In this study, we reflect on the main basic aspects of AAV biology and provide an overview of the studies exploring the impact of AAV infection on human health, focusing on three major research areas including, (i) cervical and (ii) liver cancer, and (iii) reproductive system disorders. Conflicting results have been obtained into the association of AAV infection with the occurrence of adverse reproductive outcomes, such as placental complications, spontaneous abortion, and fertility disorders, or with a protective role in HPV-related cervical carcinogenesis. Noteworthy, recent reports have identified AAV insertional mutagenesis as a novel risk factor for the development of hepatocellular carcinoma. This latest finding raises concern regarding the widespread usage of AAV vectors in liver-targeted gene therapy.

Hepatitis B Virus Genotype G: The Odd Cousin of the Family.

With a widespread distribution but low prevalence worldwide, the hepatitis B virus (HBV) genotype G (HBV/G) is a recently described genotype for which the origin and biology are poorly understood. Some unique features make HBV/G the most peculiar of all genotypes. In this review, we reflect on the major milestones in HBV/G research, highlighting the main aspects of its discovery, molecular epidemiology, and virological and clinical characteristics. We also illustrate common pitfalls in the routine detection, which may lead to underestimated rates of HBV/G infection. Large-scale analysis of data from dozens of articles was further performed, with the aim of gaining comprehensive insights into the epidemiological aspects of HBV/G. Finally, we point out recent findings on HBV/G origins and discuss new perspectives regarding the evolutionary history of HBV/G and the plausibility of an African geographic re-emergence of this genotype.

Association of Polymorphisms in the Glutathione S-Transferase Theta-1 Gene with Cirrhosis and Hepatocellular Carcinoma in Brazilian Patients with Chronic Hepatitis C.

Oxidative stress contributes to hepatitis C virus (HCV)-induced liver damage. Host genetic factors may be involved in progression of HCV infection. The present study was conducted to determine the influence of glutathione S-transferase (GST)-M1 and T1 gene polymorphisms during different stages of HCV infection, including chronic hepatitis, cirrhosis, and hepatocellular carcinoma (HCC). The study population comprised 190 patients (47 with chronic hepatitis, 83 with cirrhosis (without HCC), and 60 with HCC). GSTM1 and GSTT1 gene polymorphisms were analyzed via multiplex polymerase chain reaction. The GSTT1-null genotype was more commonly detected in patients with cirrhosis (n = 17; 20.5%) and HCC (n = 13; 21.7%) than those with chronic hepatitis (n = 3; 6.4%). The differences in GSTT1-null genotype frequencies were significant for cirrhosis vs. chronic hepatitis (odds ratio, OR, 3.778 (95% confidence interval, CI, 1.045-13.659); p = 0.043) and HCC vs. chronic hepatitis (OR, 4.057 (95% CI, 1.083-15.201); p = 0.038) groups. However, the incidence of individual GSTM1-null or combined GSTM1/GSTT1 double-null genotypes did not vary significantly between the groups. Our collective findings support the utility of the GSTT1-null genotype as a useful biomarker for liver disease progression in Brazilian patients with chronic hepatitis C.

Comprehensive Analysis of Clinically Significant Hepatitis B Virus Mutations in Relation to Genotype, Subgenotype and Geographic Region.

Hepatitis B virus (HBV) is a highly variable DNA virus due to its unique life cycle, which involves an error-prone reverse transcriptase. The high substitution rate drives the evolution of HBV by generating genetic variants upon which selection operates. HBV mutants with clinical implications have been documented worldwide, indicating the potential for spreading and developing their own epidemiology. However, the prevalence of such mutants among the different HBV genotypes and subgenotypes has not been systematically analyzed. In the current study, we performed large-scale analysis of 6,479 full-length HBV genome sequences from genotypes A-H, with the aim of gaining comprehensive insights into the relationships of relevant mutations associated with immune escape, antiviral resistance and hepatocellular carcinoma (HCC) development with HBV (sub)genotypes and geographic regions. Immune escape mutations were detected in 10.7% of the sequences, the most common being I/T126S (1.8%), G145R (1.2%), M133T (1.2%), and Q129R (1.0%). HBV genotype B showed the highest rate of escape mutations (14.7%) while genotype H had no mutations (P < 0.001). HCC-associated mutations were detected in 33.7% of the sequences, with significantly higher frequency of C1653T, T1753V and A1762T/G1764A in genotype G than C (P < 0.001). The overall frequencies of lamivudine-, telbivudine-, adefovir-, and entecavir-resistant mutants were 7.3, 7.2, 0.5, and 0.2%, respectively, while only 0.05% showed reduced susceptibility to tenofovir. In particular, the highest frequency of lamivudine-resistant mutations was observed in genotype G and the lowest frequency in genotype E (32.5 and 0.3%; P < 0.001). The prevalence of HBV mutants was also biased by geographic location, with North America identified as one of the regions with the highest rates of immune escape, antiviral resistance, and HCC-associated mutants. The collective findings were discussed in light of natural selection and the known characteristics of HBV (sub)genotypes. Our data provide relevant information on the prevalence of clinically relevant HBV mutations, which may contribute to further improvement of diagnostic procedures, immunization programs, therapeutic protocols, and disease prognosis.

Author Correction: Caspase-8 mediates inflammation and disease in rodent malaria.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Caspase-8 mediates inflammation and disease in rodent malaria.

Earlier studies indicate that either the canonical or non-canonical pathways of inflammasome activation have a limited role on malaria pathogenesis. Here, we report that caspase-8 is a central mediator of systemic inflammation, septic shock in the Plasmodium chabaudi-infected mice and the P. berghei-induced experimental cerebral malaria (ECM). Importantly, our results indicate that the combined deficiencies of caspases-8/1/11 or caspase-8/gasdermin-D (GSDM-D) renders mice impaired to produce both TNFα and IL-1ß and highly resistant to lethality in these models, disclosing a complementary, but independent role of caspase-8 and caspases-1/11/GSDM-D in the pathogenesis of malaria. Further, we find that monocytes from malaria patients express active caspases-1, -4 and -8 suggesting that these inflammatory caspases may also play a role in the pathogenesis of human disease.

The First Complete Genome Sequences of Hepatitis C Virus Subtype 2b from Latin America: Molecular Characterization and Phylogeographic Analysis.

The hepatitis C virus (HCV) has remarkable genetic diversity and exists as eight genotypes (1 to 8) with distinct geographic distributions. No complete genome sequence of HCV subtype 2b (HCV-2b) is available from Latin American countries, and the factors underlying its emergence and spread within the continent remain unknown. The present study was conducted to determine the first full-length genomic sequences of HCV-2b isolates from Latin America and reconstruct the spatial and temporal diversification of this subtype in Brazil. Nearly complete HCV-2b genomes isolated from two Brazilian patients were obtained by direct sequencing of long PCR fragments and analyzed together with reference sequences using the Bayesian coalescent and phylogeographic framework approaches. The two HCV-2b genomes were 9318 nucleotides (nt) in length (nt 37-9354). Interestingly, the long RT-PCR technique was able to detect co-circulation of viral variants that contained an in-frame deletion of 2022 nt encompassing E1, E2, and p7 proteins. Spatiotemporal reconstruction analyses suggest that HCV-2b had a single introduction in Brazil during the early 1980s, displaying an epidemic history characterized by a low and virtually constant population size until the present time. These results coincide with epidemiological data in Brazil and may explain the low national prevalence of this subtype.

Reconstruction of the spatial and temporal dynamics of hepatitis B virus genotype D in the Americas.

Hepatitis B virus (HBV) genotype D (HBV/D) is globally widespread, and ten subgenotypes (D1 to D10) showing distinct geographic distributions have been described to date. The evolutionary history of HBV/D and its subgenotypes, for which few complete genome sequences are available, in the Americas is not well understood. The main objective of the current study was to determine the full-length genomic sequences of HBV/D isolates from Brazil and frequency, origin and spread of HBV/D subgenotypes in the Americas. Complete HBV/D genomes isolated from 39 Brazilian patients infected with subgenotypes D1 (n = 1), D2 (n = 10), D3 (n = 27), and D4 (n = 1) were sequenced and analyzed together with reference sequences using the Bayesian coalescent and phylogeographic framework. A search for HBV/D sequences available in GenBank revealed 209 complete and 926 partial genomes from American countries (Argentina, Brazil, Canada, Chile, Colombia, Cuba, Haiti, Martinique, Mexico, USA and Venezuela), with the major circulating subgenotypes identified as D1 (26%), D2 (17%), D3 (36%), D4 (21%), and D7 (1%) within the continent. The detailed evolutionary history of HBV/D in the Americas was investigated by using different evolutionary time scales. Spatiotemporal reconstruction analyses using short-term substitution rates suggested times of the most recent common ancestor for the American HBV/D subgenotypes coincident with mass migratory movements to Americas during the 19th and 20th centuries. In particular, significant linkages between Argentina and Syria (D1), Brazil and Central/Eastern Europe (D2), USA and India (D2), and Brazil and Southern Europe (D3) were estimated, consistent with historical and epidemiological data.

Mutation and recombination in pathogen evolution: Relevance, methods and controversies.

Mutation and recombination drive the evolution of most pathogens by generating the genetic variants upon which selection operates. Those variants can, for example, confer resistance to host immune systems and drug therapies or lead to epidemic outbreaks. Given their importance, diverse evolutionary studies have investigated the abundance and consequences of mutation and recombination in pathogen populations. However, some controversies persist regarding the contribution of each evolutionary force to the development of particular phenotypic observations (e.g., drug resistance). In this study, we revise the importance of mutation and recombination in the evolution of pathogens at both intra-host and inter-host levels. We also describe state-of-the-art analytical methodologies to detect and quantify these two evolutionary forces, including biases that are often ignored in evolutionary studies. Finally, we present some of our former studies involving pathogenic taxa where mutation and recombination played crucial roles in the recovery of pathogenic fitness, the generation of interspecific genetic diversity, or the design of centralized vaccines. This review also illustrates several common controversies and pitfalls in the analysis and in the evaluation and interpretation of mutation and recombination outcomes.

Prevalence of occult hepatitis B virus infection and Torque teno virus infection and their association with hepatocellular carcinoma in chronic hepatitis C patients.

BACKGROUND: The role of occult hepatitis B virus (HBV) infection and Torque teno virus (TTV) infection in the development of hepatocellular carcinoma (HCC) in chronic hepatitis C patients is still uncertain. AIM: The aim of the present study was to investigate the prevalence and significance of OBI and TTV infection, and to examine the genetic diversity of these viruses, in chronic hepatitis C patients with and without HCC. METHODS: Sera from 151 hepatitis C virus (HCV)-infected patients (49 patients with HCC and 102 without HCC) negative for HBV surface antigen (HBsAg) were tested for the presence of OBI and TTV infection by semi-nested and group-specific multiplex PCR assays, respectively. Nucleotide sequencing of HBV S region was further performed. RESULTS: OBI and TTV infection were detected in 5 (3.3%) and 68 (45%) patients, respectively. HBV isolates were classified into genotypes A (4/5, 80%) and D (1/5, 20%), and no HBsAg escape mutation was observed. TTV phylogenetic group 3 was the most prevalent among both HCC and non-HCC patients. OBI and TTV infection were significantly more frequent in patients with HCC than patients without HCC (p=0.003, and p=0.009, respectively). Moreover, TTV infection was associated with HCC (OR=2.23, 95%CI=1.04-4.80, p=0.040), independently of liver cirrhosis. CONCLUSIONS: A low prevalence of OBI was observed in patients with HCV-related chronic liver disease, and TTV infection was an independent factor associated with the occurrence of HCC. Whether TTV influences the progression of liver disease in chronic hepatitis C patients remains to be elucidated.

Heterogeneous recombination among Hepatitis B virus genotypes.

The rapid evolution of Hepatitis B virus (HBV) through both evolutionary forces, mutation and recombination, allows this virus to generate a large variety of adapted variants at both intra and inter-host levels. It can, for instance, generate drug resistance or the diverse viral genotypes that currently exist in the HBV epidemics. Concerning the latter, it is known that recombination played a major role in the emergence and genetic diversification of novel genotypes. In this regard, the quantification of viral recombination in each genotype can provide relevant information to devise expectations about the evolutionary trends of the epidemic. Here we measured the amount of this evolutionary force by estimating global and local recombination rates in >4700 HBV complete genome sequences corresponding to nine (A to I) HBV genotypes. Counterintuitively, we found that genotype E presents extremely high levels of recombination, followed by genotypes B and C. On the other hand, genotype G presents the lowest level, where recombination is almost negligible. We discuss these findings in the light of known characteristics of these genotypes. Additionally, we present a phylogenetic network to depict the evolutionary history of the studied HBV genotypes. This network clearly classified all genotypes into specific groups and indicated that diverse pairs of genotypes are derived from a common ancestor (i.e., C-I, D-E and, F-H) although still the origin of this virus presented large uncertainty. Altogether we conclude that the amount of observed recombination is heterogeneous among HBV genotypes and that this heterogeneity can influence on the future expansion of the epidemic.

Complete genome sequence of a hepatitis B virus isolate of genotype D2, subtype adrq+, from Brazil.

Hepatitis B virus (HBV) has been classified into 10 distinct serological subtypes of the surface antigen (HBsAg) that can be predicted by sequencing of the corresponding S gene. HBV genotype D usually displays determinants of subtypes ayw2 or ayw3. On the other hand, subtype adrq+ has been found exclusively in association with genotype C. Here, we describe the first HBV genome (isolate BR32) belonging to genotype D with the serological subtype adrq+. This isolate had a genome length of 3,062 nucleotides (nt), and no recombination events were observed in the BR32 genome that could explain the occurrence of the subtype adr in a genotype D isolate. Analysis of the quasispecies population revealed that 28 out of 30 clones (93%) were of subtype adrq+, while the subtypes of the two remaining could not be determined, since they contained an S residue (instead of K or R) at position 122 of HBsAg. These results will contribute to further epidemiological and evolutionary studies of HBV.

Splenic differentiation and emergence of CCR5+CXCL9+CXCL10+ monocyte-derived dendritic cells in the brain during cerebral malaria.

Dendritic cells have an important role in immune surveillance. After being exposed to microbial components, they migrate to secondary lymphoid organs and activate T lymphocytes. Here we show that during mouse malaria, splenic inflammatory monocytes differentiate into monocyte-derived dendritic cells (MO-DCs), which are CD11b+F4/80+CD11c+MHCIIhighDC-SIGNhighLy6c+ and express high levels of CCR5, CXCL9 and CXCL10 (CCR5+CXCL9/10+ MO-DCs). We propose that malaria-induced splenic MO-DCs take a reverse migratory route. After differentiation in the spleen, CCR5+CXCL9/10+ MO-DCs traffic to the brain in a CCR2-independent, CCR5-dependent manner, where they amplify the influx of CD8+ T lymphocytes, leading to a lethal neuropathological syndrome.

Prevalence, Risk Behaviors, and Virological Characteristics of Hepatitis B Virus Infection in a Group of Men Who Have Sex with Men in Brazil: Results from a Respondent-Driven Sampling Survey.

BACKGROUND: Men who have sex with men (MSM) are at increased risk of exposure to hepatitis B virus (HBV) compared with the general population. This study aims to assess the epidemiological and virological characteristics of HBV infection in a sample of MSM in Brazil, where data are scarce. METHODS: A cross-sectional study was conducted among MSM in the City of Goiânia, Central Brazil, from March to November 2014, using Respondent-Driven Sampling (RDS). After signing the consent form, participants were interviewed and a blood sample collected. All samples were tested for HBV serological markers and HBV DNA. HBV nucleotide sequence analysis was also performed. RESULTS: A total of 522 MSM were recruited in the study. The prevalence of HBV infection (current or past [presence of anti-HBc marker]) was 15.4% (95% CI: 8.7-25.8) and the rate of HBsAg carriers was 0.6% (95% CI: 0.2-1.6). About 40% (95% CI: 32.3-48.8) of the participants had serological evidence of previous HBV vaccination (reactive for isolated anti-HBs). In addition, 44.3% (95% CI: 36.1-52.9) were seronegative for all HBV markers. Age over 25 years old, receptive anal intercourse, previous sex with women, and history of sexually transmitted infections (STIs) were factors associated with HBV infection. HBV DNA was detected only in HBsAg-positive individuals. HBV isolates were classified into genotype A (subgenotypes A1 and A2), and some mutations were identified throughout the genome. Therefore, occult HBV infection was not observed in the study population. CONCLUSIONS: Public health strategies should be improved for the MSM population in order to prevent HBV and other STIs, as well as to provide appropriate management of patients with active infections.

RASSF1A and DOK1 Promoter Methylation Levels in Hepatocellular Carcinoma, Cirrhotic and Non-Cirrhotic Liver, and Correlation with Liver Cancer in Brazilian Patients.

Hepatocellular carcinoma (HCC) is the second most common cause of cancer mortality worldwide. Most cases of HCC are associated with cirrhosis related to chronic hepatitis B virus or hepatitis C virus infections. Hypermethylation of promoter regions is the main epigenetic mechanism of gene silencing and has been involved in HCC development. The aim of this study was to determine whether aberrant methylation of RASSF1A and DOK1 gene promoters is associated with the progression of liver disease in Brazilian patients. Methylation levels were measured by pyrosequencing in 41 (20 HCC, 9 cirrhotic, and 12 non-cirrhotic) liver tissue samples. Mean rates of methylation in RASSF1A and DOK1 were 16.2% and 12.0% in non-cirrhotic, 26.1% and 19.6% in cirrhotic, and 59.1% and 56.0% in HCC tissues, respectively, showing a gradual increase according to the progression of the disease, with significantly higher levels in tumor tissues. In addition, hypermethylation of RASSF1A and DOK1 was found in the vast majority (88%) of the HCC cases. Interestingly, DOK1 methylation levels in HCC samples were significantly higher in the group of younger (<40 years) patients, and higher in moderately differentiated than in poorly differentiated tumors (p < 0.05). Our results reinforce the hypothesis that hypermethylation of RASSF1A and DOK1 contributes to hepatocarcinogenesis and is associated to clinicopathological characteristics. RASSF1A and DOK1 promoter hypermethylation may be a valuable biomarker for early diagnosis of HCC and a potential molecular target for epigenetic-based therapy.

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.

Genetic variability of hepatitis B and C viruses in Brazilian patients with and without hepatocellular carcinoma.

Most cases of hepatocellular carcinoma (HCC) are due to chronic hepatitis B virus (HBV) or hepatitis C virus (HCV) infection worldwide. The aim of this study was to determine the viral genotypes and frequency of 17 mutations (15 for HBV and 2 for HCV), described previously as able to influence the course of chronic liver disease, in patients with and without HCC. This transversal study included 157 Brazilian patients with chronic hepatitis B (n = 51) and C (n = 106). Of these, 12 and 40 patients had HBV- and HCV-related HCC, respectively. Nucleotide sequencing of core promoter, pre-core, and pre-S/S regions of HBV and core region of HCV strains was performed to determine their genotypes and the frequency of the respective mutations. Among the HBV isolates, subgenotype A1 was the most prevalent in both patients with (90%) and without (61%) HCC. Fourteen out of the 15 mutations under study, as well as five different pre-S deletions, were identified. Core promoter T1753V, A1762T, and G1764A mutations were more frequent in patients with HCC than in those without, although with no statistical difference. However, a significant correlation was observed between T1753V mutation and elevation of transaminases levels (P < 0.05). As for HCV, mutation at residue 70 in the core protein of genotype 1b strains was significantly more frequent in patients with cirrhosis (56.3%) than in those without (9.1%) (P = 0.018). The detection of some key mutations in the genomes of HBV and HCV might be helpful to predict the clinical outcome of patients with chronic liver disease.

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

BACKGROUND: Hepatitis 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. METHODS: Complete 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. RESULTS: Phylogenetic 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. CONCLUSIONS: This 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.