Important Factors in Reliable Determination of Hepatitis C Virus Genotype by Use of the 5′ Untranslated Region

Abstract

Accurate genotyping of hepatitis C virus (HCV) is important for determining the optimal regimen, dose, and duration of antiviral therapy for chronic HCV infection, as well as for estimating the response rate. The 5= untranslated region (UTR) of HCV RNA is used in commercial genotyping, but the probes and the lengths of the amplicons are proprietary and vary among the assays. In this study, factors involved in the reliable determination of HCV genotypes utilizing the 5= UTR were evaluated. Serum samples from four subjects with chronic HCV infection and disparate results on commercial genotyping and four controls were analyzed. HCV RNA was extracted from serum samples, and the 5= UTR and NS5B region were sequenced. Ten clones from each region were compared to prototype sequences and analyzed for genotype assignment using five programs. The results were compared to those from commercial assays. 5= UTR sequences were sequentially shortened from either the 5= end, the 3= end, or both ends, with genotyping of the resultant fragments. Sequences were obtained for the 5= UTR in all eight subjects and for the NS5B region in five subjects. The genotype assignments were identical between the two regions in the five subjects with complete sequencing. Genotyping by sequencing gave different results than those from the commercial assays in the four experimental samples but agreed in the four controls. Shortening of the sequences affected the results, and the results for sequences of <200 bases were inaccurate. Neither the Hamming distance nor the quasispecies affected the results. Sequencing of the HCV 5= UTR provided reliable genotyping results and resolved discrepancies identified in commercial assays, but genotyping by sequencing was highly dependent upon sequence length. H epatitis C virus (HCV), a member of the Flaviviridae family, possesses a single-stranded positive-sense RNA genome approximately 9.6 kb in length. The HCV genome is characterized by 5= and 3= untranslated regions (UTRs) that abut a single large open reading frame. The single polyprotein translated from the HCV genome includes an initial structural region comprising envelope (E1 and E2) and core regions and a nonstructural region that is posttranslationally processed into at least 4 polypeptides (NS2 to NS5). In a manner similar to that of other RNA viruses, HCV circulates in infected individuals as a population of closely related but diverse viral sequences, referred to as quasispecies. Heterogeneity of the HCV genome is a consequence of mutations during viral replication, which is mainly due to the error-prone viral RNA polymerase and the lack of an associated repair mechanism. Different HCV isolates display significant nucleotide sequence variability depending on the genomic region. The E1 and E2 regions are highly variable, whereas the 5= UTR is the most conserved (1, 2).The accumulation of mutations in the viral genome over time has led to the emergence of multiple HCV genotypes. Phylogenetic analysis of full-length or partial sequences of HCV strain...

A method for rapid detection and genotype identification of hepatitis C virus 1–6 by one-step reverse transcription loop-mediated isothermal amplification

“…Additionally, the assay detected and distinguished 298 the HCV genotypes without requiring post-amplification 299 genotyping procedures such as restriction enzyme analysis, 300 reverse hybridization, or nested RT-PCR. 25,[29][30][31][32][33] Moreover, this 301 assay successfully amplified HCV-RNA utilizing heated specimens 302 without conventional RNA extraction (Supplementary Material 303 Figure S3). This method of substrate preparation enhances the 304 rapidity of detection and may provide a cost-saving approach for 305 laboratories in resource-limited settings.…”

“…This 290 assay demonstrates advantages over labor-intensive methods that 291 require heavy equipment and multiple diagnostic steps for HCV 292 detection and genotype identification. [29][30][31][32][33] Conducted as a one-293 step procedure, this assay also obviates the need for extra cDNA 294 synthesis and restriction digest steps for genotype identification. 295…”

A Pan-HIV Strategy for Complete Genome Sequencing

“…In addition to the core or NS5B, genotyping of hepatitis C (HCV) typically involves interrogating the 5= untranscribed region (UTR), a region with extensive secondary structure that this method reliably sequences through (46,47). Treatment indications for new direct-acting antiviral (DAA) therapies vary by HCV viral genotype, so it may be important to consider the potential clinical impact of recombination and identify the specific genotype found at the drug target: i.e., NS3, NS5A, and NS5B versus the generic genotype identified by the 5= UTR or core (48).…”

Hepatitis C Genotype Prevalence in Monastir Region, Tunisia: Correlation between 5′ Untranslated Region (5′UTR), Non-structural 5B (NS5B), and Core Sequences in HCV Subtyping

“…In this context, genotyping assays that use sequence information from both the 5 0 UTR and the core region have allowed for more improved and accurate distinction between genotypes and subtypes [41]. Genotyping by sequence analysis of the NS5B region has been shown to correlate well with genotyping using the 5 0 UTR, with the added advantage of superior subtyping [18,27]. In our analysis, there was complete agreement in genotype assignments and overall subtype assignments with the 5 0 UTR, core, and NS5B regions.…”

“…A possible explanation for this is a high viral load in the patients' serum tested especially that none of the patients selected for the study had started antiviral therapy. Baclig and collaborators [10] and Hara and collaborators [18] reported that the 5 0 UTR is a highly conserved region which is often targeted to detect major genotypes. It is worth noticing that the high sequence conservation of the 5 0 UTR makes it difficult to distinguish between all genotypes and subtypes.…”