Drug-related mutational patterns in hepatitis B virus (HBV) reverse transcriptase proteins from Iranian treatment-Naïve chronic HBV patients

Background: Immunomodulators and Nucleotide analogues have been used globally for the dealing of chronic hepatitis B virus (HBV) infection. However, the development of drug resistance is a major limitation to their long-term effectiveness. Objectives: The aim of this study was to characterize the hepatitis B virus reverse transcriptase (RT) protein variations among Iranian chronic HBV carriers who did not receive any antiviral treatments. Materials and Methods: Hepatitis B virus partial RT genes from 325 chronic in active carrier patients were amplified and directly sequenced. Nucleotide/amino acid substitutions were identified compared to the sequences obtained from the database. Results: All strains belonging to genotype D.365 amino-acid substitutions were found. Mutations related to lamivudine, adefovir, telbivudine, and entecavir occurred in (YMDD) 4% (n = 13), (SVQ) 17.23% (n = 56), (M204I/V + L180M) 2.45% (n = 8) and (M204I) 2.76% (n = 9) of patients, respectively. Conclusions: RT mutants do occur naturally and could be found in HBV carriers who have never received antiviral therapy. However, mutations related to drug resistance in Iranian treatment-naïve chronic HBV patients were found to be higher than other studies published formerly. Chronic HBV patients should be monitored closely prior the commencement of therapy to achieve the best regimen option. © 2013, KOWSAR Corp

Mutations in pre-core and basal-core promoter regions of hepatitis B virus in chronic HBV patients from Golestan, Iran

Objective(s): It has been reported that the mutation of the pre-core (PC) and basal-core promoter (BCP) may play an important role in the development of HBV-related hepatocellular carcinoma (HCC). In this study the PC and BCP mutations were investigated in chronic HBV patients. Materials and Methods: In this study, 120 chronic HBV patients from Golestan, Northeast of Iran who were not vaccinated against HBV, were recruited from the year 2008 to 2012. HBV-DNA extraction from plasma and PCR were performed and positive PCR products were subjected to automated sequencing. Results: One hundred out of 120 (83.3%) patients were HBeAg negative. Comparison of our nucleotide sequences with reference sequence showed high rate mutation in BCP and PC region (96.66%). Frame shift mutation was found in 78 (65%) of patients in BCP region, among them 8 (6.6%) patients showed mutation in PC region. Conclusion: Our results demonstrated high rate of mutations in BCP and PC regions among HBV chronic patients in Northeast of Iran

The landscape of viral associations in human cancers

Here, as part of the Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium, for which whole-genome and—for a subset—whole-transcriptome sequencing data from 2,658 cancers across 38 tumor types was aggregated, we systematically investigated potential viral pathogens using a consensus approach that integrated three independent pipelines. Viruses were detected in 382 genome and 68 transcriptome datasets. We found a high prevalence of known tumor-associated viruses such as Epstein–Barr virus (EBV), hepatitis B virus (HBV) and human papilloma virus (HPV; for example, HPV16 or HPV18). The study revealed significant exclusivity of HPV and driver mutations in head-and-neck cancer and the association of HPV with APOBEC mutational signatures, which suggests that impaired antiviral defense is a driving force in cervical, bladder and head-and-neck carcinoma. For HBV, HPV16, HPV18 and adeno-associated virus-2 (AAV2), viral integration was associated with local variations in genomic copy numbers. Integrations at the TERT promoter were associated with high telomerase expression evidently activating this tumor-driving process. High levels of endogenous retrovirus (ERV1) expression were linked to a worse survival outcome in patients with kidney cancer

Correlation between hepatitis B G1896A precore mutations and HBeAg in chronic HBV patients

Background: Hepatitis B virus (HBV) infection is an important health concern worldwide, with critical outcomes. Hepatitis B e antigen (HBeAg) negative chronic hepatitis B is frequently caused by a mutation (G1896A) in the hepatitis B virus (HBV) precore (PC) reading frame, which creates a stop codon, causing premature termination of the HBe protein. Objectives: This study aimed to investigate the G1896A PC mutation and its effect on HBeAg detection in chronic HBV patients. Patients and Methods: In this study, 120 chronic HBV patients neither vaccinated or who had benefited from immunoglobulin therapy, were recruited. The HBV-DNA was extracted from plasma and polymerase chain reaction (PCR) was performed. Positive PCR products were subjected to automated sequencing. The HBV serological markers hepatitis B s antigen (HBsAg), HBeAg were tested. Results: One hundred out of 120 (83.3%) patients were HBeAg negative and 100% were HBsAg positive. The comparison of nucleotide sequences with the reference sequence (Accession number: AB033559) in HBeAg negative patients showed that there was a high rate of mutations in G1896A (93.18%). Conclusions: This study indicates that the rate of G1896A mutation at the PC region among HBeAg negative patients, in the Golestan province of Iran, was similar to the average rate encountered in other parts of Iran. The PC stop codon mutation was detected in 93.18% of HBeAg negative patients. Further studies with larger sample sizes are required to elucidate the exact role of these mutations in the clinical course of chronic HBV infection. © 2015, Ahvaz Jundishapur University of Medical Sciences

Re-evaluation of the carcinogenic significance of hepatitis B virus integration in hepatocarcinogenesis

To examine the role of hepatitis B virus (HBV) integration in hepatocarcinogenesis, a systematic comparative study of both tumor and their corresponding non-tumor derived tissue has been conducted in a cohort of 60 HBV associated hepatocellular carcinoma (HCC) patients. By using Alu-polymerase chain reaction (PCR) and ligation-mediated PCR, 233 viral-host junctions mapped across all human chromosomes at random, no difference between tumor and non-tumor tissue was observed, with the exception of fragile sites (P = 0.0070). HBV insertions in close proximity to cancer related genes such as hTERT were found in this study, however overall they were rare events. No direct correlation between chromosome aberrations and the number of HBV integration events was found using a sensitive array-based comparative genomic hybridization (aCGH) assay. However, a positive correlation was observed between the status of several tumor suppressor genes (TP53, RB1, CDNK2A and TP73) and the number of chromosome aberrations (r = 0.6625, P = 0.0003). Examination of the viral genome revealed that 43% of inserts were in the preC/C region and 57% were in the HBV X gene. Strikingly, approximately 24% of the integrations examined had a breakpoint in a short 15 nt viral genome region (1820-1834 nt). As a consequence, all of the confirmed X gene insertions were C-terminal truncated, losing their growth-suppressive domain. However, the same pattern of X gene C-terminal truncation was found in both tumor and non-tumor derived samples. Furthermore, the integrated viral sequences in both groups had a similar low frequency of C1653T, T1753V and A1762T/G1764A mutations. The frequency and patterns of HBV insertions were similar between tumor and their adjacent non-tumor samples indicating that the majority of HBV DNA integration events are not associated with hepatocarcinogenesis

HDV can constrain HBV genetic evolution in hbsag: Implications for the identification of innovative pharmacological targets

Chronic HBV + HDV infection is associated with greater risk of liver fibrosis, earlier hepatic decompensation, and liver cirrhosis hepatocellular carcinoma compared to HBV mono-infection. However, to-date no direct anti-HDV drugs are available in clinical practice. Here, we identified conserved and variable regions in HBsAg and HDAg domains in HBV + HDV infection, a critical finding for the design of innovative therapeutic agents. The extent of amino-acid variability was measured by Shannon-Entropy (Sn) in HBsAg genotype-D sequences from 31 HBV + HDV infected and 62 HBV mono-infected patients (comparable for demographics and virological-parameters), and in 47 HDAg genotype-1 sequences. Positions with Sn = 0 were defined as conserved. The percentage of conserved HBsAg-positions was significantly higher in HBV + HDV infection than HBV mono-infection (p = 0.001). Results were confirmed after stratification for HBeAg-status and patients’ age. A Sn = 0 at specific positions in the C-terminus HBsAg were correlated with higher HDV-RNA, suggesting that conservation of these positions can preserve HDV-fitness. Conversely, HDAg was characterized by a lower percentage of conserved-residues than HBsAg (p < 0.001), indicating higher functional plasticity. Furthermore, specific HDAg-mutations were significantly correlated with higher HDV-RNA, suggesting a role in conferring HDV replicative-advantage. Among HDAg-domains, only the virus-assembly signal exhibited a high genetic conservation (75% of conserved-residues). In conclusion, HDV can constrain HBsAg genetic evolution to preserve its fitness. The identification of conserved regions in HDAg poses the basis for designing innovative targets against HDV-infection

Xenosurveillance reflects traditional sampling techniques for the identification of human pathogens: A comparative study in West Africa

BACKGROUND: Novel surveillance strategies are needed to detect the rapid and continuous emergence of infectious disease agents. Ideally, new sampling strategies should be simple to implement, technologically uncomplicated, and applicable to areas where emergence events are known to occur. To this end, xenosurveillance is a technique that makes use of blood collected by hematophagous arthropods to monitor and identify vertebrate pathogens. Mosquitoes are largely ubiquitous animals that often exist in sizable populations. As well, many domestic or peridomestic species of mosquitoes will preferentially take blood-meals from humans, making them a unique and largely untapped reservoir to collect human blood. METHODOLOGY/PRINCIPAL FINDINGS: We sought to take advantage of this phenomenon by systematically collecting blood-fed mosquitoes during a field trail in Northern Liberia to determine whether pathogen sequences from blood engorged mosquitoes accurately mirror those obtained directly from humans. Specifically, blood was collected from humans via finger-stick and by aspirating bloodfed mosquitoes from the inside of houses. Shotgun metagenomic sequencing of RNA and DNA derived from these specimens was performed to detect pathogen sequences. Samples obtained from xenosurveillance and from finger-stick blood collection produced a similar number and quality of reads aligning to two human viruses, GB virus C and hepatitis B virus. CONCLUSIONS/SIGNIFICANCE: This study represents the first systematic comparison between xenosurveillance and more traditional sampling methodologies, while also demonstrating the viability of xenosurveillance as a tool to sample human blood for circulating pathogens

ViFi: accurate detection of viral integration and mRNA fusion reveals indiscriminate and unregulated transcription in proximal genomic regions in cervical cancer.

The integration of viral sequences into the host genome is an important driver of tumorigenesis in many viral mediated cancers, notably cervical cancer and hepatocellular carcinoma. We present ViFi, a computational method that combines phylogenetic methods with reference-based read mapping to detect viral integrations. In contrast with read-based reference mapping approaches, ViFi is faster, and shows high precision and sensitivity on both simulated and biological data, even when the integrated virus is a novel strain or highly mutated. We applied ViFi to matched genomic and mRNA data from 68 cervical cancer samples from TCGA and found high concordance between the two. Surprisingly, viral integration resulted in a dramatic transcriptional upregulation in all proximal elements, including LINEs and LTRs that are not normally transcribed. This upregulation is highly correlated with the presence of a viral gene fused with a downstream human element. Moreover, genomic rearrangements suggest the formation of apparent circular extrachromosomal (ecDNA) human-viral structures. Our results suggest the presence of apparent small circular fusion viral/human ecDNA, which correlates with indiscriminate and unregulated expression of proximal genomic elements, potentially contributing to the pathogenesis of HPV-associated cervical cancers. ViFi is available at https://github.com/namphuon/ViFi

Clinical, epidemiological and virological features of acute hepatitis B in Italy

Purpose To evaluate the association of hepatitis B virus (HBV) genotypes, basal core promoter (BCP)/precore (PC) and S gene mutations with the clinical-epidemiological characteristics of acute hepatitis B (AHB) in Italy. Methods During July 2005–January 2007, 103 symptomatic AHB patients were enrolled and prospectively followed up at 15 national hospitals. HBV genotypes, BCP/ PC and S gene variants were determined by nested-PCR and direct sequence analysis. Results Genotype D, A and F were detected in 49, 45 and 6 % of patients, respectively. BCP, PC, and BCP plus PC variants were found in 3.1, 11.3 and 7.2 % of patients, respectively. At enrollment, 68.3 % of patients were hepatitis B e antigen (HBeAg)-positive and 31.7 % HBeAg-negative. BCP/PC mutations were more common in HBeAg-negative than in HBeAg-positive patients (p < 0.0001). Compared to genotype D patients, those harboring non-D genotypes were more frequently males (p = 0.023), HBeAg-positive (p < 0.001), had higher bilirubin (p = 0.014) and viremia (p = 0.034) levels and less frequently carried BCP/PC mutations (p < 0.001). Non-D genotype patients more often were from Central Italy (p = 0.001) and reported risky sexual exposure (p = 0.021). Two patients had received vaccination before AHB: one harbored genotype F; the other showed a S gene mutation. Four patients developed fulminant AHB; mutations were found in 2 of 3 patients who underwent BCP/ PC sequencing. After a 6-month follow-up, only 2 (2.8 %) patients developed persistent infection. Conclusion AHB by non-D genotypes is increasing in Italy and is associated with risky sexual exposure. The ability of some genotypes to cause persistent and/or severe infection in Italy warrants larger studies for clarificatio

The use of chloroplast genome sequences to solve phylogenetic incongruences in Polystachya Hook (Orchidaceae Juss)

Background: Current evidence suggests that for more robust estimates of species tree and divergence times, several unlinked genes are required. However, most phylogenetic trees for non-model organisms are based on single sequences or just a few regions, using traditional sequencing methods. Techniques for massive parallel sequencing or next generation sequencing (NGS) are an alternative to traditional methods that allow access to hundreds of DNA regions. Here we use this approach to resolve the phylogenetic incongruence found in Polystachya Hook. (Orchidaceae), a genus that stands out due to several interesting aspects, including cytological (polyploid and diploid species), evolutionary (reticulate evolution) and biogeographical (species widely distributed in the tropics and high endemism in Brazil). The genus has a notoriously complicated taxonomy, with several sections that are widely used but probably not monophyletic. Methods: We generated the complete plastid genome of 40 individuals from one clade within the genus. The method consisted in construction of genomic libraries, hybridization to RNA probes designed from available sequences of a related species, and subsequent sequencing of the product. We also tested how well a smaller sample of the plastid genome would perform in phylogenetic inference in two ways: by duplicating a fast region and analyzing multiple copies of this dataset, and by sampling without replacement from all non-coding regions in our alignment. We further examined the phylogenetic implications of non-coding sequences that appear to have undergone hairpin inversions (reverse complemented sequences associated with small loops). Results: We retrieved 131,214 bp, including coding and non-coding regions of the plastid genome. The phylogeny was able to fully resolve the relationships among all species in the targeted clade with high support values. The first divergent species are represented by African accessions and the most recent ones are among Neotropical species. Discussion: Our results indicate that using the entire plastid genome is a better option than screening highly variable markers, especially when the expected tree is likely to contain many short branches. The phylogeny inferred is consistent with the proposed origin of the genus, showing a probable origin in Africa, with later dispersal into the Neotropics, as evidenced by a clade containing all Neotropical individuals. The multiple positions of Polystachya concreta (Jacq.) Garay & Sweet in the phylogeny are explained by allotetraploidy. Polystachya estrellensis Rchb.f. can be considered a genetically distinct species from P. concreta and P. foliosa (Lindl.) Rchb.f., but the delimitation of P. concreta remains uncertain. Our study shows that NGS provides a powerful tool for inferring relationships at low taxonomic levels, even in taxonomically challenging groups with short branches and intricate morphology.Swedish Research Council [B0569601]; European Research Council under the European Union's Seventh Framework Programme (ERC) [331024]; Swedish Foundation for Strategic Research; Knut and Alice Wallenberg Foundation; Biodiversity and Ecosystems in a Changing Climate programme; Wenner-Gren Foundations; David Rockefeller Center for Latin American Studies at Harvard University; Faculty of Science at the University of Gothenbur