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Quantitative analysis of the splice variants expressed by the major hepatitis B virus genotypes.

ABSTRACT: Hepatitis B virus (HBV) is a major human pathogen that causes liver diseases. The main HBV RNAs are unspliced transcripts that encode the key viral proteins. Recent studies have shown that some of the HBV spliced transcript isoforms are predictive of liver cancer, yet the roles of these spliced transcripts remain elusive. Furthermore, there are nine major HBV genotypes common in different regions of the world, these genotypes may express different spliced transcript isoforms. To systematically study the HBV splice variants, we transfected human hepatoma cells, Huh7, with four HBV genotypes (A2, B2, C2 and D3), followed by deep RNA-sequencing. We found that 13-28 % of HBV RNAs were splice variants, which were reproducibly detected across independent biological replicates. These comprised 6 novel and 10 previously identified splice variants. In particular, a novel, singly spliced transcript was detected in genotypes A2 and D3 at high levels. The biological relevance of these splice variants was supported by their identification in HBV-positive liver biopsy and serum samples, and in HBV-infected primary human hepatocytes. Interestingly the levels of HBV splice variants varied across the genotypes, but the spliced pregenomic RNA SP1 and SP9 were the two most abundant splice variants. Counterintuitively, these singly spliced SP1 and SP9 variants had a suboptimal 5' splice site, supporting the idea that splicing of HBV RNAs is tightly controlled by the viral post-transcriptional regulatory RNA element.

SUBMITTER: Lim CS

PROVIDER: S-EPMC8115900 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:We previously sequenced the 5' noncoding region of 44 isolates of hepatitis C virus (HCV), as well as the envelope 1 (E1) gene of 51 HCV isolates, and provided evidence for the existence of at least 6 major genetic groups consisting of at least 12 minor genotypes of HCV (i.e., genotypes I/1a, II/1b, III/2a, IV/2b, 2c, V/3a, 4a-4d, 5a, and 6a). We now report the complete nucleotide sequence of the putative core (C) gene of 52 HCV isolates that represent all of these 12 genotypes as well as two additional genotypes provisionally designated 4e and 4f that we identified in this study. The phylogenetic analysis of the C gene sequences was in agreement with that of the E1 gene sequences. A major division in the genetic distance was observed between HCV isolates of genotype 2 and those of the other genotypes in analysis of both the E1 and C genes. The C gene sequences of 9 genotypes have not been reported previously (i.e., genotypes 2c, 4a-4f, 5a, and 6a). Our analysis indicates that the C gene-based methods currently used to determine the HCV genotype, such as PCR with genotype-specific primers, should be revised in light of these data. We found that the predicted C gene was exactly 573 nt long in all 52 HCV isolates, with an N-terminal start codon and no in-frame stop codons. The nucleotide and predicted amino acid identities of the C gene sequences were in the range of 79.4-99.0% and 85.3-100%, respectively. Furthermore, we mapped universally conserved, as well as genotype-specific, nucleotide and deduced amino acid sequences of the C gene. The predicted C proteins of the different HCV genotypes shared the following features: (i) high content of proline residues, (ii) high content of arginine and lysine residues located primarily in three domains with 10 such residues invariant at positions 39-62, (iii) a cluster of 5 conserved tryptophan residues, (iv) two nuclear localization signals and a DNA-binding motif, (v) a potential phosphorylation site with a serine-proline motif, and (vi) three conserved hydrophilic domains that have been shown by others to contain immunogenic epitopes. Thus, we have extended analysis of the predicted C protein of HCV to all of the recognized genotypes, confirmed the existence of highly conserved regions of this important structural protein, and demonstrated that the genetic relatedness of HCV isolates is equivalent when analyzing the most conserved (i.e., C) and the most variable (i.e., E1) genes of the HCV genome.

Project description:Background and objectivesThe variable response of hepatitis C virus (HCV) genotypes towards anti-viral treatment requires prior information on the genotype status before planning a therapeutic strategy. Although assays for typing or subtyping of HCV are available, however, a fast and reliable assay system is still needed. The present study was planned to develop a single-step multiplex quantitative real time polymerase chain reaction (qPCR) assay to determine HCV genotypes in patients' sera.MethodsThe conserved sequences from 5' UTR, core and NS5b regions of HCV genome were used to design primers and hydrolysis probes labeled with fluorophores. Starting with the standardization of singleplex (qPCR) for each individual HCV-genotype, the experimental conditions were finally optimized for the development of multiplex assay. The sensitivity and specificity were assessed both for singleplex and multiplex assays. Using the template concentration of 102 copies per microliter, the value of quantification cycle (Cq) and the limit of detection (LOD) were also compared for both singleplex and multiplex assays. Similarly, the merit of multiplex assay was also compared with sequence analysis and restriction fragment length polymorphism (RFLP) techniques used for HCV genotyping. In order to find the application of multiplex qPCR assay, it was used for genotyping in a panel of 98 sera positive for HCV RNA after screening a total number of 239 patients with various liver diseases.ResultsThe results demonstrated the presence of genotype 1 in 26 of 98 (26.53%) sera, genotype 3 in 65 (66.32%) and genotype 4 in 2 (2.04%) sera samples, respectively. One sample showed mixed infection of genotype 1 and 3. Five samples could not show the presence of any genotype. Genotypes 2, 5 and 6 could not be detected in these sera samples. The analysis of sera by singleplex and RFLP indicated the results of multiplex to be comparable with singleplex and with clear merit of multiplex over RFLP. In addition, the results of multiplex assay were also found to be comparable with those from sequence analysis. The sensitivity, specificity, Cq values and LOD values were compared and found to be closely associated both for singleplex and multiplex assays.ConclusionThe multiplex qPCR assay was found to be a fast, specific and sensitive method that can be used as a technique of choice for HCV genotyping in all routine laboratories.

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Quantitative analysis of the splice variants expressed by the major hepatitis B virus genotypes.

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