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.

In the era of rapid mRNA-based vaccines: Why is there no effective hepatitis C virus vaccine yet?

Hepatitis C virus (HCV) is responsible for no less than 71 million people chronically infected and is one of the most frequent indications for liver transplantation worldwide. Despite direct-acting antiviral therapies fuel optimism in controlling HCV infections, there are several obstacles regarding treatment accessibility and reinfection continues to remain a possibility. Indeed, the majority of new HCV infections in developed countries occur in people who inject drugs and are more plausible to get reinfected. To achieve global epidemic control of this virus the development of an effective prophylactic or therapeutic vaccine becomes a must. The coronavirus disease 19 (COVID-19) pandemic led to auspicious vaccine development against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) virus, which has renewed interest on fighting HCV epidemic with vaccination. The aim of this review is to highlight the current situation of HCV vaccine candidates designed to prevent and/or to reduce HCV infectious cases and their complications. We will emphasize on some of the crossroads encountered during vaccine development against this insidious virus, together with some key aspects of HCV immunology which have, so far, hampered the progress in this area. The main focus will be on nucleic acid-based as well as recombinant viral vector-based vaccine candidates as the most novel vaccine approaches, some of which have been recently and successfully employed for SARS-CoV-2 vaccines. Finally, some ideas will be presented on which methods to explore for the design of live-attenuated vaccines against HCV.

Resistance-associated substitutions and response to treatment in a chronic hepatitis C virus infected-patient: an unusual virological response case report.

BACKGROUND: Direct-Acting agents (DAAs) target and inhibit essential viral replication proteins. They have revolutionized the treatment of Hepatitis C virus (HCV) infection reaching high levels of sustained virologic response. However, the detection of basal resistance-associated substitutions (RASs) to DAAs in naïve patients could be important in predicting the treatment outcome in some patients exhibiting failures to DAA-based therapies. Therefore, the aim of this work was to evaluate the presence of RASs as minority variants within intra-host viral populations, and assess their relationship to response to therapy on a multiple times relapser patient infected chronically with HCV. CASE PRESENTATION: A male HCV infected-patient with a genotype 1a strain was evaluated. He had previously not responded to dual therapy (pegylated interferon-α plus ribavirin) and was going to start a direct-acting agent-based therapy (DAAs). He showed no significant liver fibrosis (F0). Viral RNA was extracted from serum samples taken prior and after therapy with DAAs (sofosbubir/ledipasvir/ribavirin). NS5A and NS5B genomic regions were PCR-amplified and the amplicons were sequenced using Sanger and next-generation sequencing (NGS) approaches. RASs were searched in in-silico translated sequences for all DAAs available and their frequencies were determined for those detected by NGS technology. Sanger sequencing did not reveal the presence of RASs in the consensus sequence neither before nor after the DAA treatment. However, several RASs were found at low frequencies, both before as well as after DAA treatment. RASs found as minority variants (particularly substitutions in position 93 within NS5A region) seem to have increased their frequency after DAA pressure. Nevertheless, these RASs did not become dominant and the patient still relapsed, despite perfect adherence to treatment and having no other complications beyond the infection (no significant fibrosis, no drug abuse). CONCLUSIONS: This report shows that some patients might relapse after a DAA-based therapy even when RASs (pre- and post-treatment) are detected in very low frequencies (< 1%) within intra-host viral populations. Increased awareness of this association may improve detection and guide towards a personalized HCV treatment, directly improving the outcome in hard-to-treat patients.

Evaluation of SYBR Green real time PCR for detecting SARS-CoV-2 from clinical samples.

The pandemic caused by SARS-CoV-2 has triggered an extraordinary collapse of healthcare systems and hundred thousand of deaths worldwide. Following the declaration of the outbreak as a Public Health Emergency of International Concern by the World Health Organization (WHO) on January 30th, 2020, it has become imperative to develop diagnostic tools to reliably detect the virus in infected patients. Several methods based on real time reverse transcription polymerase chain reaction (RT-qPCR) for the detection of SARS-CoV-2 genomic RNA have been developed. In addition, these methods have been recommended by the WHO for laboratory diagnosis. Since most of these protocols are based on the use of fluorogenic probes and one-step reagents (cDNA synthesis followed by PCR amplification in the same tube), these techniques can be difficult to perform given the limited supply of reagents in low- and middle-income countries. In order to develop an inexpensive SARS-CoV-2 detection protocol using available resources we evaluated the SYBR Green based detection of SARS-CoV-2 to establish a suitable assay. To do so, we adapted one of the WHO recommended TaqMan-based one-step real time PCR protocols (from the University of Hong Kong) to SYBR Green. Our results indicate that SYBR-Green detection of ORF1b-nsp14 target represents a reliable cost-effective alternative to increase the testing capacity.

Pretreatment Hepatitis C Virus NS5A/NS5B Resistance-Associated Substitutions in Genotype 1 Uruguayan Infected Patients.

Hepatitis C Virus (HCV) infection treatment has dramatically changed with the advent of direct-acting antiviral agents (DAAs). However, the efficacy of DAAs can be attenuated by the presence of resistance-associated substitutions (RASs) before and after treatment. Indeed, RASs detected in DAA treatment-naïve HCV-infected patients could be useful for clinical management and outcome prediction. Although the frequency of naturally occurring HCV NS5A and NS5B RASs has been addressed in many countries, there are only a few reports on their prevalence in the South American region. The aim of this study was to investigate the presence of RASs to NS5A and NS5B inhibitors in a DAA treatment naïve cohort of Uruguayan patients infected with chronic hepatitis C and compare them with reports from other South American countries. Here, we found that naturally occurring substitutions conferring resistance to NS5A and NS5B inhibitors were present in 8% and 19.2%, respectively, of treatment-naïve HCV genotype 1 infected patients. Importantly, the baseline substitutions in NS5A and NS5B herein identified differ from the studies previously reported in Brazil. Furthermore, Uruguayan strains subtype 1a clustered within all major world clades, showing that HCV variants currently circulating in this country are characterized by a remarkable genetic diversity.

Correction: miRNA independent hepacivirus variants suggest a strong evolutionary pressure to maintain miR-122 dependence.

[This corrects the article DOI: 10.1371/journal.ppat.1006694.].

IL28B gene polymorphism rs12979860, but not rs8099917, contributes to the occurrence of chronic HCV infection in Uruguayan patients.

BACKGROUND: Host single-nucleotide polymorphisms (SNPs) near the interleukin 28B (IL28B) locus are associated with sustained virological response to antiviral therapy and with spontaneous Hepatitis C Virus (HCV) clearance. Prevalence of these SNPs varies depending on ethnicity. The impact of IL28B SNPs in HCV-infected patients is currently unknown in Uruguay. Therefore, the aim of this study was to evaluate and compare the distribution of polymorphisms in the IL28B gene (rs12979860 and rs8099917) among HCV-infected patients and healthy individuals in Uruguay and thus assess their possible association with the establishment of HCV infection. METHODS: DNA was recovered from 92 non-infected individuals and 78 HCV-infected patients and SNPs were determined by RFLP and allelic discrimination by real-time PCR. RESULTS: The distribution of rs12979860 genotypes for the infected population was 29.5%-CC, 47.4%-CT and 23.1%-TT and for the control group 45.7%, 42.4% and 11.9%, respectively. Prevalence in both infected and uninfected individuals is similar to that reported in other countries with admixed populations. The distribution of rs8099917 genotypes for the infected population was 57.7%-TT, 27.2%-TG and 14.1%-GG and for the control group 60.9%, 33.7% and 5.4%, respectively. The comparison of rs12979860 genotype distribution between the two populations evidenced a higher prevalence of the favourable genotype (CC) in the uninfected control group (p < 0.05). Additionally, results generated using logistic regression analysis show that individuals carrying rs12979860-TT or CT genotypes have a higher likelihood of developing chronic hepatitis upon infection with HCV, when compared to CC carriers, considering rs8099917 genotype as constant. CONCLUSION: Patients with HCV infection have a statistically significant lower prevalence of the favourable rs12979860 genotype when compared to uninfected individuals; therefore we can establish that only IL28B rs12979860-CT and TT genotypes seem to contribute to the occurrence of chronic HCV infection in the cohort of Uruguayan population studied. Considering that a trend towards a higher frequency of "good" response genotypes was observed in responder patients, we believe that IL28B rs12979860 genotyping could be a useful tool for predicting different therapies outcome, including in the DAA era.

miRNA independent hepacivirus variants suggest a strong evolutionary pressure to maintain miR-122 dependence.

Hepatitis C virus (HCV) requires the liver specific micro-RNA (miRNA), miR-122, to replicate. This was considered unique among RNA viruses until recent discoveries of HCV-related hepaciviruses prompting the question of a more general miR-122 dependence. Among hepaciviruses, the closest known HCV relative is the equine non-primate hepacivirus (NPHV). Here, we used Argonaute cross-linking immunoprecipitation (AGO-CLIP) to confirm AGO binding to the single predicted miR-122 site in the NPHV 5'UTR in vivo. To study miR-122 requirements in the absence of NPHV-permissive cell culture systems, we generated infectious NPHV/HCV chimeric viruses with the 5' end of NPHV replacing orthologous HCV sequences. These chimeras were viable even in cells lacking miR-122, although miR-122 presence enhanced virus production. No other miRNAs bound this region. By random mutagenesis, we isolated HCV variants partially dependent on miR-122 as well as robustly replicating NPHV/HCV variants completely independent of any miRNAs. These miRNA independent variants even replicate and produce infectious particles in non-hepatic cells after exogenous delivery of apolipoprotein E (ApoE). Our findings suggest that miR-122 independent HCV and NPHV variants have arisen and been sampled during evolution, yet miR-122 dependence has prevailed. We propose that hepaciviruses may use this mechanism to guarantee liver tropism and exploit the tolerogenic liver environment to avoid clearance and promote chronicity.

Naturally occurring NS3 resistance-associated variants in hepatitis C virus genotype 1: Their relevance for developing countries.

Hepatitis C virus (HCV) is a major cause of global morbidity and mortality, with an estimated 130-150 million infected individuals worldwide. HCV is a leading cause of chronic liver diseases including cirrhosis and hepatocellular carcinoma. Current treatment options in developing countries involve pegylated interferon-α and ribavirin as dual therapy or in combination with one or more direct-acting antiviral agents (DAA). The emergence of resistance-associated variants (RAVs) after treatment reveals the great variability of this virus leading to a great difficulty in developing effective antiviral strategies. Baseline RAVs detected in DAA treatment-naïve HCV-infected patients could be of great importance for clinical management and outcome prediction. Although the frequency of naturally occurring HCV NS3 protease inhibitor mutations has been addressed in many countries, there are only a few reports on their prevalence in South America. In this study, we investigated the presence of RAVs in the HCV NS3 serine protease region by analysing a cohort of Uruguayan patients with chronic hepatitis C who had not been treated with any DAAs and compare them with the results found for other South American countries. The results of these studies revealed that naturally occurring mutations conferring resistance to NS3 inhibitors exist in a substantial proportion of Uruguayan treatment-naïve patients infected with HCV genotype 1 enrolled in these studies. The identification of these baseline RAVs could be of great importance for patients' management and outcome prediction in developing countries.

Hepatitis C virus genetic variability and evolution.

Hepatitis C virus (HCV) has infected over 170 million people worldwide and creates a huge disease burden due to chronic, progressive liver disease. HCV is a single-stranded, positive sense, RNA virus, member of the Flaviviridae family. The high error rate of RNA-dependent RNA polymerase and the pressure exerted by the host immune system, has driven the evolution of HCV into 7 different genotypes and more than 67 subtypes. HCV evolves by means of different mechanisms of genetic variation. On the one hand, its high mutation rates generate the production of a large number of different but closely related viral variants during infection, usually referred to as a quasispecies. The great quasispecies variability of HCV has also therapeutic implications since the continuous generation and selection of resistant or fitter variants within the quasispecies spectrum might allow viruses to escape control by antiviral drugs. On the other hand HCV exploits recombination to ensure its survival. This enormous viral diversity together with some host factors has made it difficult to control viral dispersal. Current treatment options involve pegylated interferon-α and ribavirin as dual therapy or in combination with a direct-acting antiviral drug, depending on the country. Despite all the efforts put into antiviral therapy studies, eradication of the virus or the development of a preventive vaccine has been unsuccessful so far. This review focuses on current available data reported to date on the genetic mechanisms driving the molecular evolution of HCV populations and its relation with the antiviral therapies designed to control HCV infection.

An evolutionary insight into Newcastle disease viruses isolated in Antarctica.

The disease caused by Newcastle disease virus (NDV) is a severe threat to the poultry industry worldwide. Recently, NDV has been isolated in the Antarctic region. Detailed studies on the mode of evolution of NDV strains isolated worldwide are relevant for our understanding of the evolutionary history of NDV. For this reason, we have performed Bayesian coalescent analysis of NDV strains isolated in Antarctica to study evolutionary rates, population dynamics, and patterns of evolution. Analysis of F protein cleavage-site sequences of NDV isolates from Antarctica suggested that these strains are lentogenic. Strains isolated in Antarctica and genotype I reference strain Ulster/67 diverged from ancestors that existed around 1958. The time of the most recent common ancestor (MRCA) was established to be around 1883 for all class II viruses. A mean rate of evolution of 1.78 × 10(-3) substitutions per site per year (s/s/y) was obtained for the F gene sequences of NDV strains examined in this study. A Bayesian skyline plot indicated a decline in NDV population size in the last 25 years. The results are discussed in terms of the possible role of Antarctica in emerging or re-emerging viruses and the evolution of NDV populations worldwide.

Human endogenous retrovirus np9 gene is over expressed in chronic lymphocytic leukemia patients.

The human genome contains a large number of endogenous retroviruses (HERVs). Their reactivation has frequently been observed in patients with cancer. Considering their role in the carcinogenesis process, we aimed to study the possible relationship between HERVs gene expression and Chronic Lymphocytic Leukemia (CLL). We focused on two viral genes gag and np9, the latter presumably an oncogene. We found that the transcriptional activity of HERV-K np9 gene was greater in CLL patients than in healthy donors. However, gag expression was not significantly increased. These findings suggest a noteworthy relationship between CLL disease and HERV-K np9 expression.