Hepatitis C Virus Genetic Diversity Research Articles

Mixed Infections Unravel Novel HCV Inter-Genotypic Recombinant Forms within the Conserved IRES Region.

Hepatitis C virus (HCV) remains a significant global health challenge, affecting millions of people worldwide, with chronic infection a persistent threat. Despite the advent of direct-acting antivirals (DAAs), challenges in diagnosis and treatment remain, compounded by the lack of an effective vaccine. The HCV genome, characterized by high genetic variability, consists of eight distinct genotypes and over ninety subtypes, underscoring the complex dynamics of the virus within infected individuals. This study delves into the intriguing realm of HCV genetic diversity, specifically exploring the phenomenon of mixed infections and the subsequent detection of recombinant forms within the conserved internal ribosome entry site (IRES) region. Previous studies have identified recombination as a rare event in HCV. However, our findings challenge this notion by providing the first evidence of 1a/3a (and vice versa) inter-genotypic recombination within the conserved IRES region. Utilizing advanced sequencing methods, such as deep sequencing and molecular cloning, our study reveals mixed infections involving genotypes 1a and 3a. This comprehensive approach not only confirmed the presence of mixed infections, but also identified the existence of recombinant forms not previously seen in the IRES region. The recombinant sequences, although present as low-frequency variants, open new avenues for understanding HCV evolution and adaptation.

Polyvalent immunization elicits a synergistic broadly neutralizing immune response to hypervariable region 1 variants of hepatitis C virus

A hepatitis C virus (HCV) vaccine is urgently needed. Vaccine development has been hindered by HCV's genetic diversity, particularly within the immunodominant hypervariable region 1 (HVR1). Here, we developed a strategy to elicit broadly neutralizing antibodies to HVR1, which had previously been considered infeasible. We first applied a unique information theory-based measure of genetic distance to evaluate phenotypic relatedness between HVR1 variants. These distances were used to model the structure of HVR1's sequence space, which was found to have five major clusters. Variants from each cluster were used to immunize mice individually, and as a pentavalent mixture. Sera obtained following immunization neutralized every variant in a diverse HCVpp panel (n = 10), including those resistant to monovalent immunization, and at higher mean titers (1/ID50 = 435) than a glycoprotein E2 (1/ID50 =205) vaccine. This synergistic immune response offers a unique approach to overcoming antigenic variability and may be applicable to other highly mutable viruses.

Sub-genotypes of hepatitis C virus in the Middle East and North Africa: Patterns of distribution and temporal changes.

Infection with the hepatitis C virus (HCV) remains a considerable public health concern in the Middle East and North Africa (MENA). The objectives of this study were to analyze the HCV genotype (GT) and sub-genotype (SGT) distribution in the MENA region and to assess the temporal change in the number of sequences within the MENA region. All HCV molecular sequences collected in the MENA region had been retrieved from GenBank as of 1 August 2022. The number of HCV sequences retrieved was 6740 representing sequences from a total of 17 MENA countries with a majority from Iran (n=1969, 29.2%), Egypt (n=1591, 23.6%), Tunisia (n=1305, 19.4%) and Saudi Arabia (n=1085, 16.1%). The determination of GT/SGT was based on the NCBI genotyping and Blast tool. Genotype 1 (GT1) dominated infections in the MENA (n=2777, 41.2%), followed by GT4 (n=2566, 39.0%). Additionally, SGT4a (1515/6393, 23.7%) was the most common SGT in the MENA, and SGT4a was dominant in Egypt and Saudi Arabia, followed by SGT1b (n=1308, 20.5%), which was dominant in Morocco and Tunisia, while SGT1a (n=1275, 19.9%) was common in Iran, Iraq and Palestine. Furthermore, significant temporal increase in the number of HCV MENA sequences was observed. On the SGT level, specific patterns of HCV genetic diversity were seen in the MENA region, with the most common SGT being 4a, in addition to increasing the availability of HCV sequences in the MENA region.

Mechanisms and Consequences of Genetic Variation in Hepatitis C Virus (HCV).

Chronic infection with hepatitis C virus (HCV) is an important contributor to the global incidence of liver diseases, including liver cirrhosis and hepatocellular carcinoma. Although common for single-stranded RNA viruses, HCV displays a remarkable high level of genetic diversity, produced primarily by the error-prone viral polymerase and host immune pressure. The high genetic heterogeneity of HCV has led to the evolution of several distinct genotypes and subtypes, with important consequences for pathogenesis, and clinical outcomes. Genetic variability constitutes an evasion mechanism against immune suppression, allowing the virus to evolve epitope escape mutants that avoid immune recognition. Thus, heterogeneity and variability of the HCV genome represent a great hindrance for the development of vaccines against HCV. In addition, the high genetic plasticity of HCV allows the virus to rapidly develop antiviral resistance mutations, leading to treatment failure and potentially representing a major hindrance for the cure of chronic HCV patients. In this chapter, we will present the central role that genetic diversity has in the viral life cycle and epidemiology of HCV. Incorporation errors and recombination, both the result of HCV polymerase activity, represent the main mechanisms of HCV evolution. The molecular details of both mechanisms have been only partially clarified and will be presented in the following sections. Finally, we will discuss the major consequences of HCV genetic diversity, namely its capacity to rapidly evolve antiviral and immunological escape variants that represent an important limitation for clearance of acute HCV, for treatment of chronic hepatitis C and for broadly protective vaccines.

From Bench to Bedside: Clinical and Biomedical Investigations on Hepatitis C Virus (HCV) Genotypes and Risk Factors for Albuminuria.

An extrahepatic manifestation of nephropathies can be a feature of the chronic hepatitis C virus (HCV) infection. Albuminuria is a major risk factor for nephropathies and chronic kidney disease (CKD). The correlation between HCV genotypes and albuminuria is still unclear. In this study, investigations have been done for the biomedical tools and methodologies used in the National Health and Nutrition Examination Survey (NHANES) public database. We searched the 2007–2016 NHANES public database to retrieve data regarding the different HCV genotypes and clinical scenarios. This study attempted to investigate the impacts of HCV genetic diversity, associated comorbidities, and racial differences on albuminuria. The urine albumin/creatinine ratio (ACR) was the primary endpoint. Among 40,856 participants, 336 participants with positive and 237 with negative HCV RNA tests were analyzed, excluding 14,454 participants with negative HCV antibodies and 25,828 which were missed. After controlling for sex, race, education level, smoking, diabetes mellitus, hepatitis B, alcohol use, and body mass index (BMI) with a generalized linear equation, HCV genotype 2 was more likely than any other genotype to cause albuminuria based on the urine ACR (p < 0.001). The generalized linear equation also demonstrated a significantly higher urine ACR, including hepatitis B (p < 0.001), diabetes mellitus (p < 0.001), and smoking (p = 0.026). In summary, the patients with HCV genotype 2 presented with increased albuminuria in comparison with other HCV genotypes in this 10-year retrospective analysis. HCV infection could be a risk factor of CKD; early diagnosis and appropriate treatment may improve clinical outcomes.

Hepatitis C Vaccination: Where We Are and Where We Need to Be

The hepatitis C virus (HCV) is a common cause of chronic liver disease and liver cancer worldwide. Despite advances in curative therapies for HCV, the incidence of new infections is not decreasing at the expected rate to hit the World Health Organization (WHO) target for the elimination of HCV by 2030. In fact, there are still more new cases of infection in the United States and worldwide than are being cured. The reasons for the rise in new cases include poor access to care and the opioid epidemic. The clinical burden of HCV requires a multimodal approach to eradicating the infection. Vaccination would be an excellent tool to prevent incidence of new infections; however, the genetic diversity of HCV and its ability to generate quasispecies within an infected host make creating a broadly reactive vaccine difficult. Multiple vaccine candidates have been identified, but to date, there has not been a target that has led to a broadly reactive vaccine, though several of the candidates are promising. Additionally, the virus is very difficult to culture and testing candidates in humans or chimpanzees is ethically challenging. Despite the multiple barriers to creating a vaccine, vaccination still represents an important tool in the fight against HCV.

Genetic diversity of hepatitis C virus in Nanaian region, Khabarovsk territory

Examining hepatitis C virus (HCV) genetic diversity is of great practical value in molecular-epidemiological research, development of specific prevention tools and outlining therapeutic strategy.Aim of study is to conduct analysis assessing HCV genetic diversity circulating in the Nanaisky Region population of the Khabarovsk Krai.Materials and methods. Molecular and genetic analysis of 124 blood plasma samples collected from patients with chronic hepatitis C and residing in the Nanaisky Region was conducted.Results. HCV RNA was detected in 84 (67.7±4.2%) plasma samples. HCV genotyping was performed by using AmpliSens — 1/2/3 kit (Central Research Institute of Epidemiology, Moscow, Russian Federation) showing that genotype 3 dominated reaching up to 47.6±5.4% (n = 40). Genotype 1 was detected in 30 patients (35.7±5.2%). In thirteen cases (15.5±3.9%) genotype 2 was identified, whereas in one case (1.2±1.2%) virus genotype was unidentified. Phylogenetic analysis of the nucleotide sequences in HCV NS5B region was performed for 60 HCV RNA-positive samples showing subtype ratio as follows: 1a — 2 (3.3±2.3%), 1b — 23 (38.3±6.3%), 2а — 6 (10.0±3.9%), 2с — 2 (3.3±2.3%), 3а — 27 (45.0±6.4%). Three samples of RF2k/1b recombinant virus were found. A full NS2 gene nucleotide sequence was cloned in order to confirm the recombination event. The results of the study evidence about a need to conduct multi-layered examination of patients with chronic hepatitis C by using current molecular and biologic methods for assigning proper therapy coupled to characteristics of the isolated strains. The data regarding hepatitis C virus molecular and genetic parameters circulating in the Far Eastern Federal District, Russia, are rather limited. Hence, our study would contribute to current understanding of HCV genovariants circulating in territories of the Russian Federation.

Therapy Implications of Hepatitis C Virus Genetic Diversity.

Hepatitis C virus (HCV) is an important human pathogen with a high chronicity rate. An estimated 71 million people worldwide are living with chronic hepatitis C (CHC) infection, which carries the risk of progression to hepatic fibrosis, cirrhosis, and hepatocellular carcinoma (HCC). Similar to other RNA viruses, HCV has a high rate of genetic variability generated by its high mutation rate and the actions of evolutionary forces over time. There are two levels of HCV genetic variability: intra-host variability, characterized by the distribution of HCV mutant genomes present in an infected individual, and inter-host variability, represented by the globally circulating viruses that give rise to different HCV genotypes and subtypes. HCV genetic diversity has important implications for virus persistence, pathogenesis, immune responses, transmission, and the development of successful vaccines and antiviral strategies. Here we will discuss how HCV genetic heterogeneity impacts viral spread and therapeutic control.

Hepatitis C virus genotype/subtype distribution and evolution among Chineseblood donors: Revealing recent viral expansion.

Hepatitis C virus (HCV) genotype (GT) distribution in China shows significant geographical and demographic difference. As a routinely tested virus in Chinese blood bank systems, rare molecular epidemiology research in blood donors is reported. Our purpose is to investigate the HCV GT/subtypes distribution, phylogenetic analysis and population genetics in Chinese blood donors. Anti-HCV screen positive samples and donor demographics were collected. HCV Core and E1 gene fragments were amplified by RT-PCR, followed by sequencing and phylogenetic analysis to determine HCV GTs/subtypes using MEGA 7.0. The population genetics were performed using Arlequin v3.0 and Beast v1.10.4. SPSS Statistics 17.0 software was used to analyze the correlation between HCV GTs/subtypes distribution and demographic characteristics. 419 and 293 samples based on Core and E1 gene respectively were successfully amplified. HCV la, lb, 2a, 3a, 3b, 6a, 6e and 6n were found, and the corresponding proportions were 0.66% (3/455), 58.68% (267/455), 17.80% (81/455) and 5.05% (23/455), 3.52% (16/455), 12.31% (56/455), 0.88% (4/455) and 0.66% (3/455). Samples from Guangxi showed the most abundant genetic diversity with 8 subtypes were found. The number of haplotypes in HCV-1b is higher than 2a and 6a. The negative Tajima's D and Fu's Fs values of HCV-1b, 2a and 6a suggested the population expansion of those HCV subtypes. The distribution of HCV GT showed significant statistical difference by age and ethnicity. Conclusion: An abundance of HCV genetic diversity was found in Chinese blood donors with mainly 1b and then 2a subtype. There were significant geographical and demographic differences in HCV GTs/subtypes among Chinese blood donors. HCV subtype 1b has stronger viability and HCV subtype 6a has experienced significant expansion.

Substitution of the CD81 Binding Site and β-Sandwich Area in E2 of HCV in Cambodia.

The high genetic variability of hepatitis C virus (HCV) is the main obstacle to developing a vaccine. E2 has attracted attention for vaccine development because targeting this protein could potentially overcome issues related to the genetic diversity of HCV. In this study, we analyzed HCV genes in the general population of Cambodia and investigated the E2 locus as a candidate for vaccine development. HCV sero-epidemiological surveys were conducted between the period 2010 and 2014, with an HCV RNA–positive rate of 1.3% (11/868). Follow-up blood samples were collected from four anti-HCV– and HCV RNA– positive patients (genotype 1b: 2 cases, 6e: 1 case, 6r: 1 case) after 4.12 years. Analysis of HCV full-length nucleotide sequences in paired specimens revealed that the mutation rates of HCV genotypes 1b and 6e/6r were 1.61–2.03 × 10−3 and 2.52–2.74 × 10−3 substitutions/site/year, respectively. Non-synonymous substitutions were detected in HVR1, the front layer of the CD81 binding site, and the β-sandwich, but not in the N-terminal region or adjacent to the CD81 binding site. Therefore, we conclude that the CD81 binding site is a promising locus for HCV vaccine development.

Full-genome analysis of hepatitis C virus in HIV-coinfected hemophiliac Japanese patients.

More than 1400 Japanese hemophiliacs acquired HIV infection around 1983 through contaminated blood products imported from the USA, most of whom also acquired hepatitis C virus (HCV) infection. To delineate the HCV genetic relations in HIV-coinfected hemophiliacs, we analyzed stocked plasma samples of the patients seen at the largest referral center for HIV care in Japan. Hepatitis C virus full-genome sequences were amplified and determined using next-generation sequencing, and genotyping and phylogenetic analyses of these sequences were carried out. The results of these hemophiliacs were compared with those of previously studied HIV-coinfected Japanese non-hemophiliacs who had undergone similar analysis of HCV full-genome sequences. From 1997 to the end of 2017, 72 HIV-infected Japanese hemophiliacs regularly visited our outpatient clinic. Of these, 51 patients had detectable plasma HCV-RNA. The HCV full genome was successfully amplified and sequenced in 50 patients. Not only HCV genotypes 1b (28%) and 2a (6%), which are common in Japan, but also HCV genotypes 1a (32%) and 3a (22%) were identified at high frequency. A single case of intergenotypic recombinant form (2b/1a) and a single case of mixed infection (1a and 3a) were also identified. Each sequence derived from hemophiliacs was more than 0.05 genetic distance away from the other sequences in phylogenetic analysis. Various HCV genotypes were identified in Japanese hemophiliacs, a finding that reflects the HCV genotypic distribution in the USA. The genetic distance among them are the results of viral evolution in each patient plus HCV genetic diversity in the USA.

An update on hepatitis C virus genotype distribution in Jordan: a 12-year retrospective study from a tertiary care teaching hospital in Amman

BackgroundNucleic acid hybridization (NAH) of hepatitis C virus (HCV) is a practical and reliable tool for virus genotyping. Genotype assignment is an important factor in the prediction of treatment success in chronic hepatitis C patients. The aim of this study was to determine the genotype distribution among HCV clinical isolates in Jordan between 2007 and 2018.MethodsElectronic and paper-based clinical data registry records from 2007 to 2018 at the Jordan University Hospital (JUH) were retrospectively examined for individuals with HCV genotype, HCV viral load, and alanine aminotransferase (ALT) testing results. Genotype determination was based on NAH technique using the HCV 5′ untranslated region (5′ UTR) with 386 requests available from 342 unique individuals.ResultsA total of 263 out of 342 unique individuals (76.9%) had genotyping results available for final analysis with 259 individuals each having a single genotyping result. The most common HCV genotypes in the study were: genotype 4 (n = 142, 54.0%), genotype 1 (n = 87, 33.1%), genotype 3 (n = 16, 6.1%), genotype 2 (n = 9, 3.4%), other undetermined genotypes (n = 5, 1.9%) and mixed infections (n = 4, 1.5%). Sub-genotyping results were available for 46 individuals as follows: sub-genotype 4c/d (n = 13, 28.3%), sub-genotype 1a (n = 11, 23.9%), sub-genotype 1b (n = 10, 21.7%), sub-genotype 4a (n = 8, 17.4%), sub-genotype 3a (n = 2, 4.3%), sub-genotypes 2a/c and 4 h (n = 1, 2.2% for both). Individuals infected with genotype 1 showed higher viral load when compared to those infected with genotype 4 (p = 0.048, t-test). Younger HCV-infected individuals (< 52 years) had higher ALT levels compared to older individuals (p = 0.036, t-test). Self-reported risk factors for HCV acquisition included: history of previous surgery, invasive dental procedures, and blood transfusion, delivery at home, circumcision at home and wet cupping therapy (hijama).ConclusionsHigh genetic diversity of HCV was found in Jordan, with genotypes 4 and 1 as the most prevalent genotypes co-circulating in the country. Potential impact of virus genotype on disease markers (viral load, ALT) was detected and needs further assessment. The study can be helpful to plan for future prevention and management of HCV infection in Jordan.

Molecular epidemiology of hepatitis C virus in Cambodia during 2016\u20132017

In Cambodia, little epidemiological data of hepatitis C virus (HCV) is available. All previous studies were limited to only small or specific populations. In the present study, we performed a characterization of HCV genetic diversity based on demography, clinical data, and phylogenetic analysis of HCV non-structural 5B (NS5B) sequences belonging to a large cohort of patients (n = 3,133) coming from majority part of Cambodia between September 2016 and December 2017. The phylogenetic analysis revealed that HCV genotype 1 and 6 were the most predominant and sharing equal proportions (46%). The remaining genotypes were genotype 2 (4.3%) and unclassified variants (3.6%). Among genotype 1, subtype 1b was the most prevalent subtype accounting for 94%. Within genotype 6, we observed a high degree of diversity and the most common viral subtypes were 6e (44%) and 6r (23%). This characteristic points to the longstanding history of HCV in Cambodia. Geographic specificity of viral genotype was not observed. Risks of HCV infection were mainly associated with experience of an invasive medical procedure (64.7%), having partner with HCV (19.5%), and blood transfusion (9.9%). In addition, all of these factors were comparable among different HCV genotypes. All these features define the specificity of HCV epidemiology in Cambodia.

High-Resolution Evolutionary Analysis of Within-Host Hepatitis C Virus Infection.

BackgroundDespite recent breakthroughs in treatment of hepatitis C virus (HCV) infection, we have limited understanding of how virus diversity generated within individuals impacts the evolution and spread of HCV variants at the population scale. Addressing this gap is important for identifying the main sources of disease transmission and evaluating the risk of drug-resistance mutations emerging and disseminating in a population.MethodsWe have undertaken a high-resolution analysis of HCV within-host evolution from 4 individuals coinfected with human immunodeficiency virus 1 (HIV-1). We used long-read, deep-sequenced data of full-length HCV envelope glycoprotein, longitudinally sampled from acute to chronic HCV infection to investigate the underlying viral population and evolutionary dynamics.ResultsWe found statistical support for population structure maintaining the within-host HCV genetic diversity in 3 out of 4 individuals. We also report the first population genetic estimate of the within-host recombination rate for HCV (0.28 × 10−7 recombination/site/year), which is considerably lower than that estimated for HIV-1 and the overall nucleotide substitution rate estimated during HCV infection.ConclusionsOur findings indicate that population structure and strong genetic linkage shapes within-host HCV evolutionary dynamics. These results will guide the future investigation of potential HCV drug resistance adaptation during infection, and at the population scale.

Hepatitis C virus surveillance and identification of human pegivirus 2 in a large Cameroonian cohort

SummaryThe prevalence of chronic hepatitis C virus (HCV) and the presence of human pegivirus 2 (HPgV‐2) have not been examined in Cameroon, although HCV has been associated with HPgV‐2 infections previously. Herein we aimed to characterize the burden and genetic diversity of HCV and the presence of HPgV‐2 in Cameroon. Retrospective plasma specimens collected from N = 12 369 consenting subjects in South Cameroon from 2013 to 2016 were included in the study. The majority (97.1%) of participants were patients seeking health care. All specimens were screened for HCV using the Abbott RealTime HCV viral load assay and positive specimens with remaining volume were also screened for HPgV‐2 antibodies on the Abbott ARCHITECT instrument, followed by molecular characterization. Overall, HCV RNA was detected in 305 (2.47%; 95% CI: 2.21%‐2.75%) specimens. Notably, the prevalence of HCV RNA was 9.09% amongst participants over age 40 and 3.81% amongst males. Phylogenetic classification of N = 103 HCV sequences identified genotypes 1 (19.4%), 2 (15.5%) and 4 (65.1%) within the study cohort. Amongst HCV RNA‐positive specimens, N = 28 (10.6%; 95% CI: 7.44%‐14.90%) specimens also had detectable HPgV‐2 antibodies. Of these, N = 2 viremic HPgV‐2 infections were confirmed by sequencing and shared 93‐94 median % identity with strains found on other continents. This is the first study to determine the prevalence of chronic HCV in Cameroon, and the discovery of HPgV‐2 in this study cohort expands the geography of HPgV‐2 to the African continent, indicating a widespread distribution exists.

Genotypic Profile of Hepatitis C Virus (HCV) in Patients Attending the Medical Outpatient Department in Bingham University Teaching Hospital Jos Nigeria for Chronic HCV Infection

INTRODUCTION: HCV is recognized as the major cause of transfusion associated and sexually transmitted sporadic chronic non-A non-B hepatitis. There is a high degree of genetic variability from HCV genomes isolated from infected patients. Currently, HCV isolates are classified into at least six major genotypes and numerous sub- genotypes. The genetic diversity of HCV and the clinical and virological relevance of HCV genetic classification schemes remain important areas of active research. OBJECTIVES: The objective of this study was to determine the genotypic profile of hepatitis C virus (HCV) in patients who are infected with chronic HCV with or without liver cirrhosis and hepatocellular carcinoma, receiving treatment at the medical outpatient department (MOPD) of the Bingham University Teaching Hospital Jos Nigeria with a view to seeing which of these genotypes is more prevalent. METHODS: This prospective cohort study enrolled patients who were clinically diagnosed to have chronic HCV infection and subsequently confirmed by polymerase chain reaction (PCR) using the light cycler. Patients attending the clinic from December 2012 to December 2014 who had all the relevant investigations done were enrolled in the study. This was regardless of whether they were profiled for commencement of treatment or not due to disease stage. RESULTS: A total of 48 patients were enrolled for the study during the period. Out of this figure, genotype 1 patients accounted for 34 (70.8 %) to emerge the leading genotype in the centre. This was followed by genotype 4 with a very low value of 7(14.6 %) as the second while genotype 2 had 6 (12.5 %) and genotype 3 had only 1 (2.1 %) patient. CONCLUSION: The study has revealed that HCV genotype 1 is the most prevalent among the population studied followed by genotype 4 while other genotypes 2 and 3 were also encountered but with lesser prevalence.

Genetic diversity of hepatitis C virus in Ethiopia.

Hepatitis C virus (HCV) is genetically highly divergent and classified in seven major genotypes and approximately hundred subtypes. These genotypes/subtypes have different geographic distribution and response to antiviral therapy. In Ethiopia, however, little is known about their molecular epidemiology and genetic diversity. The aim of this study was to investigate the distribution and genetic diversity of HCV genotypes/subtypes in Ethiopia, using 49 HCV RNA positive samples. HCV genotypes and subtypes were determined based on the sequences of the core and the nonstructural protein 5B (NS5B) genomic regions. Phylogenetic analysis revealed that the predominant was genotype 4 (77.6%) followed by 2 (12.2%), 1 (8.2%), and 5 (2.0%). Seven subtypes were identified (1b, 1c, 2c, 4d, 4l, 4r and 4v), with 4d (34.7%), 4r (34.7%) and 2c (12.2%) as the most frequent subtypes. Consistent with the presence of these subtypes was the identification of a potential recombinant virus. One strain was typed as genotype 2c in the NS5B region sequence and genotype 4d in the core region. In conclusion, genotype 4 HCV viruses, subtypes 4d and 4r, are most prevalent in Ethiopia. This genotype is considered to be difficult to treat, thus, our finding has an important impact on the development of treatment strategies and patient management in Ethiopia.

Diminished viral replication and compartmentalization of hepatitis C virus in hepatocellular carcinoma tissue

Analysis of hepatitis C virus (HCV) replication and quasispecies distribution within the tumor of patients with HCV-associated hepatocellular carcinoma (HCC) can provide insight into the role of HCV in hepatocarcinogenesis and, conversely, the effect of HCC on the HCV lifecycle. In a comprehensive study of serum and multiple liver specimens from patients with HCC who underwent liver transplantation, we found a sharp and significant decrease in HCV RNA in the tumor compared with surrounding nontumorous tissues, but found no differences in multiple areas of control non-HCC cirrhotic livers. Diminished HCV replication was not associated with changes in miR-122 expression. HCV genetic diversity was significantly higher in livers containing HCC compared with control non-HCC cirrhotic livers. Tracking of individual variants demonstrated changes in the viral population between tumorous and nontumorous areas, the extent of which correlated with the decline in HCV RNA, suggesting HCV compartmentalization within the tumor. In contrast, compartmentalization was not observed between nontumorous areas and serum, or in controls between different areas of the cirrhotic liver or between liver and serum. Our findings indicate that HCV replication within the tumor is restricted and compartmentalized, suggesting segregation of specific viral variants in malignant hepatocytes.

Genetic Diversity and Selective Pressure in Hepatitis C Virus Genotypes 1–6: Significance for Direct-Acting Antiviral Treatment and Drug Resistance

Treatment with pan-genotypic direct-acting antivirals, targeting different viral proteins, is the best option for clearing hepatitis C virus (HCV) infection in chronically infected patients. However, the diversity of the HCV genome is a major obstacle for the development of antiviral drugs, vaccines, and genotyping assays. In this large-scale analysis, genome-wide diversity and selective pressure was mapped, focusing on positions important for treatment, drug resistance, and resistance testing. A dataset of 1415 full-genome sequences, including genotypes 1–6 from the Los Alamos database, was analyzed. In 44% of all full-genome positions, the consensus amino acid was different for at least one genotype. Focusing on positions sharing the same consensus amino acid in all genotypes revealed that only 15% was defined as pan-genotypic highly conserved (≥99% amino acid identity) and an additional 24% as pan-genotypic conserved (≥95%). Despite its large genetic diversity, across all genotypes, codon positions were rarely identified to be positively selected (0.23%–0.46%) and predominantly found to be under negative selective pressure, suggesting mainly neutral evolution. For NS3, NS5A, and NS5B, respectively, 40% (6/15), 33% (3/9), and 14% (2/14) of the resistance-related positions harbored as consensus the amino acid variant related to resistance, potentially impeding treatment. For example, the NS3 variant 80K, conferring resistance to simeprevir used for treatment of HCV1 infected patients, was present in 39.3% of the HCV1a strains and 0.25% of HCV1b strains. Both NS5A variants 28M and 30S, known to be associated with resistance to the pan-genotypic drug daclatasvir, were found in a significant proportion of HCV4 strains (10.7%). NS5B variant 556G, known to confer resistance to non-nucleoside inhibitor dasabuvir, was observed in 8.4% of the HCV1b strains. Given the large HCV genetic diversity, sequencing efforts for resistance testing purposes may need to be genotype-specific or geographically tailored.

Diversity of 1,213 hepatitis C virus NS3 protease sequences from a clinical virology laboratory database in Marseille university hospitals, southeastern France.

Infection with hepatitis C virus (HCV) represents a major public health concern worldwide. Recent therapeutic advances have been considerable, HCV genotype continuing to guide therapeutic management. Since 2008, HCV genotyping in our clinical microbiology laboratory at university hospitals of Marseille, Southeastern France, has been based on NS3 protease gene population sequencing, to allow concurrent HCV genotype and protease inhibitor (PI) genotypic resistance determinations. We aimed, first, to analyze the genetic diversity of HCV NS3 protease obtained from blood samples collected between 2003 and 2013 from patients monitored at university hospitals of Marseille and detect possible atypical sequences; and, second, to identify NS3 protease amino acid patterns associated with decreased susceptibility to HCV PIs. A total of 1,213 HCV NS3 protease sequences were available in our laboratory sequence database. We implemented a strategy based on bioinformatic tools to determine whether HCV sequences are representative of our local HCV genetic diversity, or divergent. In our 2003-2012 HCV NS3 protease sequence database, we delineated 32 clusters representative of the majority HCV genetic diversity, and 61 divergent sequences. Five of these divergent sequences showed less than 85% nucleotide identity with their top GenBank hit. In addition, among the 294 sequences obtained in 2013, three were divergent relative to these 32 previously delineated clusters. Finally, we detected both natural and on-treatment genotypic resistance to HCV NS3 PIs, including a substantial prevalence of Q80K substitutions associated with decreased susceptibility to simeprevir, a second generation PI.