Hepatitis C Virus Genome Research Articles

N6-methyladenosine modification of HCV RNA genome regulates cap-independent IRES-mediated translation via YTHDC2 recognition

Hepatitis C virus (HCV) infections are associated with the risk of progression to fibrosis, cirrhosis, and hepatocellular carcinoma. The HCV RNA genome is translated by an internal ribosome entry site (IRES)-dependent mechanism. The structure and function of the HCV IRES have been investigated by both biological and biophysical criteria. Recently, the role of N6-methyladenosine (m6A) in cellular RNA and viral transcripts has been intensely investigated. The HCV RNA genome is m6A-methylated, and this modification regulates the viral life cycle. In this study, we investigated the role of m6A modification of the HCV genome in the IRES-dependent translation function by mutating m6A consensus motifs (DRACH) within the IRES element in stem-loop III and IV regions and studied their effect on translation initiation. There are several DRACH motifs within the IRES element. Of these, the DRACH motif at nucleotide (nt) 329-333, located about 7 nt upstream of initiator AUG (iAUG) codon, regulates IRES-mediated translation initiation. Mutational analysis showed that m6A methylation of the adenosine at nt 331 is essential for the IRES-dependent translation. m6A reader protein YTHDC2, containing the RNA helicase domain, recognizes m6A-methylated adenosine at nt 331 and, in concert with the cellular La antigen, supports HCV IRES-dependent translation. The RNA helicase dead YTHDC2 (E332Q) mutant failed to stimulate HCV translation initiation. This report highlights the functional roles of m6A modification and YTHDC2 in the HCV IRES-dependent translation initiation, thus offering alternative therapeutic avenues to interfere with the infectious process.

Comparative Analysis of Hepatitis C Virus NS5A Dynamics and Localization in Assembly-Deficient Mutants.

The hepatitis C virus (HCV) life cycle is a tightly regulated process, during which structural and non-structural proteins cooperate. However, the interplay between HCV proteins during genomic RNA replication and progeny virion assembly is not completely understood. Here, we studied the dynamics and intracellular localization of non-structural 5A protein (NS5A), which is a protein involved both in genome replication and encapsidation. An NS5A-eGFP (enhanced green fluorescent protein) tagged version of the strain JFH-1-derived wild-type HCV was compared to the corresponding assembly-deficient viruses Δcore, NS5A basic cluster 352–533 mutant (BCM), and serine cluster 451 + 454 + 457 mutant (SC). These analyses highlighted an increase of NS5A motility when the viral protein core was lacking. Although to a lesser extent, NS5A motility was also increased in the BCM virus, which is characterized by a lack of interaction of NS5A with the viral RNA, impairing HCV genome encapsidation. This observation suggests that the more static NS5A population is mainly involved in viral assembly rather than in RNA replication. Finally, NS4B exhibited a reduced co-localization with NS5A and lipid droplets for both Δcore and SC mutants, which is characterized by the absence of interaction of NS5A with core. This observation strongly suggests that NS5A is involved in targeting NS4B to lipid droplets (LDs). In summary, this work contributes to a better understanding of the interplay between HCV proteins during the viral life cycle.

MicroRNA-122 and Poly-C Binding Protein 2 Regulate Hepatitis C Replication by Competing for Overlapping Binding Sites

Hepatitis C virus (HCV) is a human pathogen that causes a persistent infection in the liver that requires precise control over viral replication to be maintained. Successful HCV replication is promoted by many host factors; of importance to this study is microRNA-122 (miR-122) and Poly-C Binding Protein-2 (PCBP2). The HCV 5’ UTR contains two miR-122 binding sites near the first stem-loop (SLI) and a PCBP2 binding region that overlaps the second miR-122 binding site. PCBP2 also binds to the 3’ UTR and has been shown to circularize the HCV genome.

Phylogenetic Analysis of NS5B Region of Hepatitis C Genotype 3a Virus among Different Risk Groups in Iran

Background: Hepatitis C virus (HCV) is a major cause of liver diseases. Transmission of HCV, as a blood-borne virus, is a major concern for the safety of blood products. In Iran, intravenous drug abuse is a major risk factor for HCV transmission. Recently a change in the distribution of HCV genotype among Iranian blood donors has been reported, and genotype 3a is reported as the most frequent genotype. Also, genotype 3a is the dominant genotype among IDUs. Objective: To investigate the association between HCV genotype 3a circulating in the community, sequences of HCV genotype 3a were analyzed among different risk groups in Iran. Methods: In this cross-sectional study, a partial sequence of the NS5B region of the HCV genome was isolated from 58 blood donors, 48 intravenous drug users (IDUs), and 31 patients with inherited bleeding disorders (IBDs) infected with HCV genotype 3a were included. The published sequence data were obtained from the Gen Bank database and were compared to construct the phylogenetic trees. Results: Of 58 blood donors infected with subtype 3a, 31 (53.5%) reported the risk of intravenous drug abuse. In the phylogenetic tree, no separate cluster was formed to differentiate between HCV sequences in blood donors and IDUs. A cluster at the middle level was found in the phylogenetic tree formed by sequences from blood donors, IDUs, and patients with inherited bleeding disorders. Conclusions: The phylogenetic tree showed the phylogenetic mixing of genotype 3a sequences in BDs, IBD patients, and IDUs. The results confirmed the extensive transmission of IDU-related genotype 3a towards the general population.

The Impact of Hepatitis C Virus Genotypes on Oxidative Stress Markers and Catalase Activity.

Hepatitis C virus (HCV) is a major cause of liver disease worldwide. Chronic HCV infections are usually associated with increased oxidative stress in the liver tissue. The intensity of oxidative stress may be a detrimental factor in liver injury and may determine the severity of the disease. The aim of the present case-control study was to determine the level of lipid peroxidation (TBARS), protein oxidative modification (AOPP), and catalase activity in sera of patients infected with HCV, in relation to different HCV genotypes and viral load. Considering the HCV patients with chronic hepatitis C (52) and control subject (50) recruitment, the study was designed as a case–control-type. The HCV RNA isolation, viral load, and HCV genotyping were performed according to the standard protocols. A significant difference compared to control healthy subjects was reported for TBAR (p < 0.001), AOPP (p = 0.001), and catalase activity (p = 0.007). In a gender-based comparison, a significantly higher level of AOPP for females was reported (p < 0.001). As stratified by HCV genotype, the most common was HCV-1 (HCV-1a and HCV 1b), with the overall participation of more than 60%, followed by genotype 3, while the least represented was genotype 2. No significant difference was documented among genotypes in regard to oxidative stress markers, although somewhat higher TBARS level, but not significant, was registered in patients infected with genotype 1b. A statistically significant positive correlation was found between the concentration of HCV genome copies and AOPP (r = 0.344; p = 0.012). A high level of HCV viral load was more likely to have a higher TBARS, but still without statistical significance (p = 0.072). In conclusion, the results obtained confirmed an imbalance between the ROS production and antioxidative defense system in HCV-infected patients. Since oxidative stress may have a profound influence on disease progression, fibrosis, and carcinogenesis, our results may meet the aspirations of mandatory introduction of antioxidants as early HCV therapy to counteract ROS consequences.

Acute Hepatitis C Virus Infections in Spouses: The Utility of a Genetic Analysis of the Hepatitis C Virus Hypervariable Region Sequence for Identifying the Infectious Source

A married couple who developed hepatitis was referred to our hospital. The husband was diagnosed with acute hepatitis C virus (HCV) infection based on HCV RNA positivity and seroconversion to HCV antibody. The wife was also diagnosed with HCV-related hepatitis; however, she could not be confirmed to have acute hepatitis due to the lack of information on her HCV negativity just before this event. The HCV strains recovered from the couple were genotype 2b and shared 100% identities within the 5’-untranslated region-core region sequence (655 nucleotides/nt) and non-structural (NS)5B region sequence (502 nt). The amplified hypervariable region 1 (HVR-1) sequence indicated that all 10 clones from the wife shared 100% identity and were identical to 3 of 10 heterogeneous clones (separable into 4 groups) from the husband. The husband had a history of intravenous drug use. These results suggested that one of four quasispecies 2b HCV strains was transmitted from the husband to the wife, with the husband being the infectious source for acute HCV infection in the wife, most likely via sexual intercourse. A sequence analysis of the HCV genomes and the further comparison of the HVR-1 amino acid sequence variability may be useful for defining the infectious source of HCV, especially in couples or cluster cases.

A protracted war of human beings against virus: Interpreting for the Nobel Prize in Physiology or Medicine in 2020

On 5th October of 2020, the Nobel committee announced in the Karolinska Institute that the 2020 Nobel Prize in Physiology or Medicine was awarded to Harvey J. Alter, Michael Houghton, and Charles M. Rice to commend their contribution in the aspect of discovering hepatitis C virus (HCV). Alter and his team acquired C100-3 protein by utilizing recombinant yeast to express viral sequence segments and found the HCV antibody by performing radioimmunoassay. Houghton and his colleagues successfully created the cDNA library from the serum of non-A and non-B hepatitis patients in bacteriophage. Subsequently, the large-scale blind screening method was performed in the serum of patients, acquired clone 81 from the library screening as the clone 5-1-1 involving short sequences to be the probe, and confirmed HCV ultimately. In 1995, Rice and his collaborators completed the full-length HCV genome sequencing from the 3′ terminal sequence using a quantitatively competitive reverse transcription PCR method and verified the full-length sequence has infectivity. This is the first causative agent entirely discovered by molecular biological techniques in human being’s history. Furthermore, Wakita with his team, acquired a full-length HCV genome from acute HCV patients and transformed viral RNA to Hun-7 cell to generate HCV virion in 2005. On this basis, Rice and his team successfully established the cell-cultured system that can effectively generate HCV with infectivity. According to the WHO latest data, there were approximately 71 million chronic HCV cases and nearly 400000 deaths globally in 2016. About 14% of the total HCV infected individuals worldwide lived in China. As a common infectious pathogen globally, HCV infection may cause severe threats and damages to human health, such as chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. The high genetic variation is major characteristics of HCV, which has phylogenetically generated more than 7 genotypes and hundreds of subtypes. The various genotypes and subtypes have distinct geographic distribution and different responses on anti-virus treatment. The resistant mutation for the clinical using drugs including Epclusa (Sofosbuvir and Velpatasvir), is occurring. Moreover, the highly chronic progression render HCV as a silent slayer for human health, and plenty of HCV cases were difficult to be timely diagnosed due to its high proportion of asymptomatic. All these indicated that HCV prevention and control are still facing severe challenges.

Consensus small interfering RNA targeted to stem-loops II and III of IRES structure of 5′ UTR effectively inhibits virus replication and translation of HCV sub-genotype 4a isolates from Saudi Arabia

Being the most conserved region of all hepatitis C virus (HCV) genotypes and sub-genotypes, the 5′ untranslated region (5′ UTR) of HCV genome signifies it’s importance as a potential target for anti-mRNA based treatment strategies like RNA interference. The advent and approval of first small interference RNA (siRNA) -based treatment of hereditary transthyretin-mediated amyloidosis for clinical use has raised the hopes to test this approach against highly susceptible viruses like HCV. We investigated the antiviral potential of consensus siRNAs targeted to stem-loops (SLs) II and III nucleotide motifs of internal ribosome entry site (IRES) structure within 5′ UTR of HCV sub-genotype 4a isolates from the Saudi population. siRNA inhibitory effects on viral replication and translation of full-length HCV genome were determined in a competent, persistent, and reproducible Huh-7 cell culture system maintained for one month. Maximal inhibition of RNA transcript levels of HCV-IRES clones and silencing of viral replication and translation of full-length virus genome was demonstrated by siRNAs targeted to SL-III nucleotide motifs of IRES in Huh-7 cells. siRNA Usi-169 decreased 5′ UTR RNA transcript levels of HCV-IRES clones up to 75% (P < 0.001) at 24 h post-transfection and 80% (P < 0.001) at 48 h treatment in Huh-7 cells. 5′ UTR-tagged GFP protein expression was significantly decreased from 70 to 80% in Huh-7 cells co-transfected with constructed vectors (i.e. pCR3.1/GFP/5′ UTR) and siRNA Usi-169 at 24 h and 48 h time-span. Viral replication was inhibited by more than 90% (P < 0.001) and HCV core (C) and hypervariable envelope glycoproteins (E1 and E2) expression was also significantly degraded by intracytoplasmic siRNA Usi-169 activity in persistent Huh-7 cell culture system. The findings unveil that siRNAs targeted to 5′ UTR-IRES of HCV sub-genotype 4a Saudi isolates show potent silencing of HCV replication and blocking of viral translation in a persistent in-vitro Huh-7 tissue culture system. Furthermore, we also elucidated that siRNA silencing of viral mRNA not only inhibits viral replication but also blocks viral translation. The results suggest that siRNA potent antiviral activity should be considered as an effective anti-mRNA based treatment strategies for further in-vivo investigations against less studied and harder-to-treat HCV sub-genotype 4a isolates in Saudi Arabia.

Factors associated with phylogenetic clustering of hepatitis C among people who inject drugs in Baltimore

BackgroundThe availability of effective, oral direct acting antivirals (DAAs) for hepatitis C virus (HCV) treatment has put elimination of HCV as a public health challenge within reach. However, little is known about the characteristics of transmission networks of people who inject drugs (PWID).MethodsSequencing of a segment of the HCV genome was performed on samples collected from a community-based cohort of PWID between August 2005 and December 2016. Phylogenetic trees were inferred, and clusters were identified (70% bootstrap threshold; 0.04 maximum genetic distance threshold). We describe sex, race, age difference, and HIV infection status of potential transmission partners. Logistic regression was used to assess factors associated with being in an HCV cluster.ResultsOf 508 HCV genotype 1 viremic PWID, 8% (n = 41) were grouped into 20 clusters, consisting of 19 pairs and 1 triad. In adjusted analyses, female sex (odds ratio [OR] 2.3 [95% confidence interval (CI) 1.2–4.5]) and HIV infection (OR 5.7 [CI 2.7–11.9]) remained independently associated with being in an HCV infection cluster.ConclusionsMolecular epidemiological analysis reveals that, in this cohort of PWID in Baltimore, HIV infection and female sex were associated with HCV clustering. Combination HCV prevention interventions targeting HIV infected PWID and addressing HCV infection prevention needs of women have potential to advance HCV elimination efforts.

A machine learning-based treatment prediction model using whole genome variants of hepatitis C virus.

In recent years, the development of diagnostics using artificial intelligence (AI) has been remarkable. AI algorithms can go beyond human reasoning and build diagnostic models from a number of complex combinations. Using next-generation sequencing technology, we identified hepatitis C virus (HCV) variants resistant to directing-acting antivirals (DAA) by whole genome sequencing of full-length HCV genomes, and applied these variants to various machine-learning algorithms to evaluate a preliminary predictive model. HCV genomic RNA was extracted from serum from 173 patients (109 with subsequent sustained virological response [SVR] and 64 without) before DAA treatment. HCV genomes from the 109 SVR and 64 non-SVR patients were randomly divided into a training data set (57 SVR and 29 non-SVR) and a validation-data set (52 SVR and 35 non-SVR). The training data set was subject to nine machine-learning algorithms selected to identify the optimized combination of functional variants in relation to SVR status following DAA therapy. Subsequently, the prediction model was tested by the validation-data set. The most accurate learning method was the support vector machine (SVM) algorithm (validation accuracy, 0.95; kappa statistic, 0.90; F-value, 0.94). The second-most accurate learning algorithm was Multi-layer perceptron. Unfortunately, Decision Tree, and Naive Bayes algorithms could not be fitted with our data set due to low accuracy (< 0.8). Conclusively, with an accuracy rate of 95.4% in the generalization performance evaluation, SVM was identified as the best algorithm. Analytical methods based on genomic analysis and the construction of a predictive model by machine-learning may be applicable to the selection of the optimal treatment for other viral infections and cancer.

Mathematical modeling of hepatitis C RNA replication, exosome secretion and virus release.

Hepatitis C virus (HCV) causes acute hepatitis C and can lead to life-threatening complications if it becomes chronic. The HCV genome is a single plus strand of RNA. Its intracellular replication is a spatiotemporally coordinated process of RNA translation upon cell infection, RNA synthesis within a replication compartment, and virus particle production. While HCV is mainly transmitted via mature infectious virus particles, it has also been suggested that HCV-infected cells can secrete HCV RNA carrying exosomes that can infect cells in a receptor independent manner. In order to gain insight into these two routes of transmission, we developed a series of intracellular HCV replication models that include HCV RNA secretion and/or virus assembly and release. Fitting our models to in vitro data, in which cells were infected with HCV, suggests that initially most secreted HCV RNA derives from intracellular cytosolic plus-strand RNA, but subsequently secreted HCV RNA derives equally from the cytoplasm and the replication compartments. Furthermore, our model fits to the data suggest that the rate of virus assembly and release is limited by host cell resources. Including the effects of direct acting antivirals in our models, we found that in spite of decreasing intracellular HCV RNA and extracellular virus concentration, low level HCV RNA secretion may continue as long as intracellular RNA is available. This may possibly explain the presence of detectable levels of plasma HCV RNA at the end of treatment even in patients that ultimately attain a sustained virologic response.

Ribosome Pausing at Inefficient Codons at the End of the Replicase Coding Region Is Important for Hepatitis C Virus Genome Replication.

Hepatitis C virus (HCV) infects liver cells and often causes chronic infection, also leading to liver cirrhosis and cancer. In the cytoplasm, the viral structural and non-structural (NS) proteins are directly translated from the plus strand HCV RNA genome. The viral proteins NS3 to NS5B proteins constitute the replication complex that is required for RNA genome replication via a minus strand antigenome. The most C-terminal protein in the genome is the NS5B replicase, which needs to initiate antigenome RNA synthesis at the very 3′-end of the plus strand. Using ribosome profiling of cells replicating full-length infectious HCV genomes, we uncovered that ribosomes accumulate at the HCV stop codon and about 30 nucleotides upstream of it. This pausing is due to the presence of conserved rare, inefficient Wobble codons upstream of the termination site. Synonymous substitution of these inefficient codons to efficient codons has negative consequences for viral RNA replication but not for viral protein synthesis. This pausing may allow the enzymatically active replicase core to find its genuine RNA template in cis, while the protein is still held in place by being stuck with its C-terminus in the exit tunnel of the paused ribosome.

Fitness-associated substitutions following failure of direct-acting antivirals assessed by deep sequencing of full-length hepatitis C virus genomes.

In hepatitis C virus (HCV) infection, treatment failure is generally associated with the selection of resistance-associated substitutions (RAS) conferring reduced susceptibility to direct-acting antiviral (DAA) drugs. Resistant variants continue to replicate after the end of treatment with potential for transmission. This may result from the selection of "fitness-associated substitutions". To characterise potential "fitness-associated substitutions" in patients infected with genotype 3a failing DAA drugs METHODS: By means of shotgun metagenomics, we sequenced full-length HCV genomes at treatment initiation and at virological relapse in eight patients infected with genotype 3a with cirrhosis failing sofosbuvir and an NS5A inhibitor. The impact of amino acid changes occurring outside of DAA target regions selected in at least two patients were assessed on the in vitro susceptibility to an NS5A inhibitor and replication capacity. At treatment failure, besides selection of known NS5A RASs, especially Y93H, a large number of amino acid changes was observed outside of DAA target regions. We identified four amino acid positions at which observed changes substantially improved in vitro replication capacity without affecting NS5A inhibitor susceptibility. This is the first in vivo observation combined with in vitro confirmation of selection of phenotypically characterised "fitness-associated substitutions" together with RASs at the time of sofosbuvir-NS5A inhibitor treatment failure in patients infected with genotype 3a with cirrhosis. Our findings may explain the persistence of resistant HCV variants after treatment in patients who did not achieve sustained virological remission.

Location specific annealing of miR-122 and other small RNAs defines an Hepatitis C Virus 5' UTR regulatory element with distinct impacts on virus translation and genome stability.

Hepatitis C virus (HCV) replication requires annealing of a liver specific small-RNA, miR-122 to 2 sites on 5′ untranslated region (UTR). Annealing has been reported to (a) stabilize the genome, (b) stimulate translation and (c) promote the formation of translationally active Internal Ribosome Entry Site (IRES) RNA structure. In this report, we map the RNA element to which small RNA annealing promotes HCV to nucleotides 1–44 and identify the relative impact of small RNA annealing on virus translation promotion and genome stabilization. We mapped the optimal region on the HCV genome to which small RNA annealing promotes virus replication to nucleotides 19–37 and found the efficiency of viral RNA accumulation decreased as annealing moved away from this region. Then, by using a panel of small RNAs that promote replication with varying efficiencies we link the efficiency of lifecycle promotion with translation stimulation. By contrast small RNA annealing stabilized the viral genome even if they did not promote virus replication. Thus, we propose that miR-122 annealing promotes HCV replication by annealing to an RNA element that activates the HCV IRES and stimulates translation, and that miR-122 induced HCV genome stabilization is insufficient alone but enhances virus replication.

Optimized Hepatitis C Virus (HCV) E2 Glycoproteins and their Immunogenicity in Combination with MVA-HCV.

Hepatitis C virus (HCV) represents a major global health challenge and an efficient vaccine is urgently needed. Many HCV vaccination strategies employ recombinant versions of the viral E2 glycoprotein. However, recombinant E2 readily forms disulfide-bonded aggregates that might not be optimally suited for vaccines. Therefore, we have designed an E2 protein in which we strategically changed eight cysteines to alanines (E2.C8A). E2.C8A formed predominantly monomers and virtually no aggregates. Furthermore, E2.C8A also interacted more efficiently with broadly neutralizing antibodies than conventional E2. We used mice to evaluate different prime/boost immunization strategies involving a modified vaccinia virus Ankara (MVA) expressing the nearly full-length genome of HCV (MVA-HCV) in combination with either the E2 aggregates or the E2.C8A monomers. The combined MVA-HCV/E2 aggregates prime/boost strategy markedly enhanced HCV-specific effector memory CD4+ T cell responses and antibody levels compared to MVA-HCV/MVA-HCV. Moreover, the aggregated form of E2 induced higher levels of anti-E2 antibodies in vaccinated mice than E2.C8A monomers. These antibodies were cross-reactive and mainly of the IgG1 isotype. Our findings revealed how two E2 viral proteins that differ in their capacity to form aggregates are able to enhance to different extent the HCV-specific cellular and humoral immune responses, either alone or in combination with MVA-HCV. These combined protocols of MVA-HCV/E2 could serve as a basis for the development of a more effective HCV vaccine.

Major mutations in the NS3 gene region of hepatitis C virus related to the resistance to direct acting antiviral drugs: a systematic review.

Hepatitis C virus (HCV) remains a global public health problem with high prevalence rates and chronicity of infection. Present work aimed to describe the main mutations in the NS3 region of the HCV genome related to the resistance of patients to the currently available direct-acting antivirals (DAAs). To guide the study description, the preferred items in the PRISMA protocol for systematic review were used. The data collected were HCV genotypes and subtypes and mutations in HCV NS3, general and stratified by continent. The 10 papers selected for this systematic review reported studies in seven countries, on three continents, and generated data of 2937 patients. The most frequent HCV subtype was 1a. Prevalence of genotypes suggested that there were few demographic regions reached by the studies, since there were regional variations in the type of genotypes reported in the available bibliographies. Of the total study population, 35.3% (n = 1037) had mutations in the NS3 gene region of HCV, suggesting a high rate of resistance to DAAs and a low sustained virologic response among those who used some therapeutic option. Ten major mutations were identified: Q80K, V170I, S122G, V36L, T54S, D168Q, A156S, Q80G, S122R, and V55A. The Q80K mutation was the highlight of the study, appearing not only with greater representativity (61.6%) but also as the only one described in the three continents analyzed. This systematic review reinforces the need to carry out more studies of detection of these mutations to fill in all information gaps that might help in optimization of treatment.

Should a viral genome stay in the host cell or leave? A quantitative dynamics study of how hepatitis C virus deals with this dilemma.

Virus proliferation involves gene replication inside infected cells and transmission to new target cells. Once positive-strand RNA virus has infected a cell, the viral genome serves as a template for copying (“stay-strategy”) or is packaged into a progeny virion that will be released extracellularly (“leave-strategy”). The balance between genome replication and virion release determines virus production and transmission efficacy. The ensuing trade-off has not yet been well characterized. In this study, we use hepatitis C virus (HCV) as a model system to study the balance of the two strategies. Combining viral infection cell culture assays with mathematical modeling, we characterize the dynamics of two different HCV strains (JFH-1, a clinical isolate, and Jc1-n, a laboratory strain), which have different viral release characteristics. We found that 0.63% and 1.70% of JFH-1 and Jc1-n intracellular viral RNAs, respectively, are used for producing and releasing progeny virions. Analysis of the Malthusian parameter of the HCV genome (i.e., initial proliferation rate) and the number of de novo infections (i.e., initial transmissibility) suggests that the leave-strategy provides a higher level of initial transmission for Jc1-n, whereas, in contrast, the stay-strategy provides a higher initial proliferation rate for JFH-1. Thus, theoretical-experimental analysis of viral dynamics enables us to better understand the proliferation strategies of viruses, which contributes to the efficient control of virus transmission. Ours is the first study to analyze the stay-leave trade-off during the viral life cycle and the significance of the replication-release switching mechanism for viral proliferation.

Large transmission cluster of acute hepatitis C identified among HIV-positive men who have sex with men in Bangkok, Thailand.

A rapidly emerging and highly concentrated hepatitis C virus (HCV) outbreak has recently been observed among both acute and chronic HIV-positive men who have sex with men (MSM) in Bangkok, Thailand. NS5B regions of the HCV genome were amplified using nested PCR and sequenced. Phylogenetic inference was constructed by Maximum Likelihood methods and clusters were identified with support and genetic distance thresholds of 85% and of 4.5%. Forty-eight (25 acute HIV and 23 chronic HIV) MSM with incident HCV infection were included in the analysis. HCV genotype (GT) was 85% GT 1a and 15% GT 3a or 3b. Median age at HCV diagnosis was 34 (interquartile range, 28-41) years. 83.3% (40/48) had history of syphilis infection and 36% (16/44) reported crystal methamphetamine use. Only 2 (4%) reported ever injecting drugs, both crystal methamphetamine. In the phylogenetic clustering analysis, 83% belonged to one of two clusters: one large (75%) and one small (8%) cluster. All clusters were GT 1a. MSM with acute HIV infection were more likely to be in a cluster (92%) than those with chronic infection (74%). HCV screening should be regularly performed for MSM in ART clinics, and offering direct-acting antiviral agents to all MSM with HCV infection might contain the HCV epidemic from expanding further.

Assessing routes of hepatitis C transmission in HIV-infected men who have sex with men using single genome sequencing.

The epidemic of hepatitis C virus (HCV) infection among HIV-infected men who have sex with men (MSM) is in its second decade, but the routes of transmission remain poorly understood. We hypothesized that by pairing single genome sequencing (SGS), to enumerate infecting HCV genomes (viruses), with detailed sexual and drug histories, we could gain insight into the routes of transmission among MSM. We used SGS to analyze blood specimens from eight HIV-infected MSM who had 10 episodes of acute (seronegative) or early HCV infections. Seven of eight men reported condomless receptive anal intercourse (CRAI), six with rectal exposure to semen, and all eight denied rectal trauma or bleeding. Of the 10 HCV infections, eight resulted from transmission of a single virus; one infection resulted from transmission of either one or a few (three or four) closely-related viruses; and one infection resulted from transmission of >10 distinct viruses. The participant infected by >10 viruses reported sharing injection equipment for methamphetamine during sex. Two other participants also injected methamphetamine during sex but they did not share injection equipment and were infected by a single virus. Conclusions: Most HCV infections of HIV-infected MSM without a history of either rectal trauma or bleeding or shared injection equipment were caused by a single virus. Intra-rectal exposure to semen during CRAI is therefore likely sufficient for HCV transmission among MSM. Conversely, rectal trauma or bleeding or shared injection equipment are not necessary for HCV transmission among MSM. These results help clarify routes of HCV transmission among MSM and can therefore help guide the design of much-needed behavioral and other interventions to prevent HCV transmission among MSM.

Exploring HCV genome to construct multi-epitope based subunit vaccine to battle HCV infection: Immunoinformatics based approach.

Hepatitis C Virus (HCV) infection is a major cause of chronic liver disease, hepatocellular carcinoma, and the single most common indication for liver transplantation. HCV vaccines eliciting specific T-cell responses, have been considered as potent method to prevent HCV infection. Despite several reports on progress of vaccine, these vaccine failed in mediating clinical relevance activity against HCV in humans. In this study we integrated both immunoinformatic and molecular docking approach to present a multiepitope vaccine against HCV by designating 17 conserved epitopes from eight viral proteins such as Core protein, E1, E2, NS2, NS34A, NS4B, NS5A, and NS5B. The epitopes were prioritized based on conservation among epitopes of T cell, B cell and IFN-γ that were then scanned for non-homologous to host and antigenicity. The prioritized epitopes were then linked together by AAY linker and adjuvant (β-defensin) were attached at N-terminal to enhance immunogenic potential. The construct thus formed were subjected to structural modeling and physiochemical characteristics. The modeled structure were successfully docked to antigenic receptor TLR-3 and In-silico cloning confers the authenticity of its expression efficiency. However, the proposed construct need to be validate experimentally to ensure its safety and immunogenic profile.