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.

First National Prevalence in Italian Horse Population and Phylogenesis Highlight a Fourth Sub-Type Candidate of Equine Hepacivirus.

Equine hepacivirus (EqHV, Flaviviridae, hepacivirus) is a small, enveloped RNA virus generally causing sub-clinical hepatitis with occasional fatalities. EqHV is reported in equids worldwide, but for Italy data are limited. To address this, a survey study was set up to estimate prevalence at a national level and among different production categories (equestrian; competition; work and meat; reproduction) and national macro-regions (North, Central, South, and Islands). Data obtained testing 1801 horse serum samples by Real-Time RT PCR were compared within the categories and regions. The NS3 fragment of the PCR-positive samples was sequenced by Sanger protocol for phylogenetic and mutational analysis. The tertiary structure of the NS3 protein was also assessed. The estimated national prevalence was 4.27% [1.97-6.59, 95% CI] and no statistical differences were detected among production categories and macro-regions. The phylogenesis confirmed the distribution in Italy of the three known EqHV subtypes, also suggesting a possible fourth sub-type that, however, requires further confirmation. Mutational profiles that could also affect the NS3 binding affinity to the viral RNA were detected. The present paper demonstrates that EqHV should be included in diagnostic protocols when investigating causes of hepatitis, and in quality control protocols for blood derived products due to its parental transmission.

Hepatitis C virus subtype diversity and transmission clusters characteristics among drug users in Zhuhai, South China.

BACKGROUND: Hepatitis C virus (HCV) infection poses a major public health challenge globally, especially among injecting drug users. China has the world's largest burden of HCV infections. However, little is known about the characteristics of transmission networks among drug user populations. This study aims to investigate the molecular epidemiology and transmission characteristics of HCV infections among drug users in Zhuhai, a bustling port city connecting Mainland China and its Special Administrative Regions. METHODS: Participants enrolled in this study were drug users incarcerated at Zhuhai's drug rehabilitation center in 2015. Their sociodemographic and behavioral information, including gender, promiscuity, drug use method, and so forth, was collected using a standardized questionnaire. Plasmas separated from venous blood were analyzed for HCV infection through ELISA and RT-PCR methods to detect anti-HCV antibodies and HCV RNA. The 5'UTR fragment of the HCV genome was amplified and further sequenced for subtype identifications and phylogenetic analysis. The phylogenetic tree was inferred using the Maximum Likelihood method based on the Tamura-Nei model, and the transmission cluster network was constructed using Cytoscape3.8.0 software with a threshold of 0.015. Binary logistic regression models were employed to assess the factors associated with HCV infection. RESULTS: The overall prevalence of HCV infection among drug users was 44.37%, with approximately 19.69% appearing to clear the HCV virus successfully. Binary logistic regression analysis revealed that those aged over 40, engaging in injecting drug use, and being native residents were at heightened risk for HCV infection among drug user cohorts. The predominant HCV subtypes circulating among those drug users were 6a (60.26%), followed by 3b (16.7%), 3a (12.8%), 1b (6.41%) and 1a (3.85%), respectively. Molecular transmission network analysis unveiled the presence of six transmission clusters, with the largest propagation cluster consisting of 41 individuals infected with HCV subtype 6a. Furthermore, distinct transmission clusters involved eight individuals infected with subtype 3b and seven with subtype 3a were also observed. CONCLUSION: The genetic transmission networks revealed a complex transmission pattern among drug users in Zhuhai, emphasizing the imperative for a targeted and effective intervention strategy to mitigate HCV dissemination. These insights are pivotal for shaping future national policies on HCV screening, treatment, and prevention in port cities.

Influence of Hepatitis C virus genotype and other factors on the viral load.

Anti-HCV reactive subjects were selected and relevant data was collected. Viral load and genotype were determined for all patients and were divided into low (<800,000 IU/mL) and high viral load (>800,000 IU/mL). Correlation of viral load with parameters like age, gender, risk factors and genotype etc. was determined by binomial regression. Higher viral load was noted with genotype 4, males and high risk groups like People Who Inject Drugs (PWIDs), blood transfusion before routine testing or frequent transfusion, Intravenous drug therapy and MTP by unregistered medical practitioners (P ≤ 0.5). Prevention and treatment strategies for HCV should be tailored around these areas.

Community-Based Screening for Hepatitis B and C Infectivity Using Two Quantitative Antigens to Identify Endemic Townships.

Screening and linkage to care are essential to achieve viral hepatitis elimination before 2030. The accurate identification of endemic areas is important for controlling diseases with geographic aggregation. Viral activity drives prognosis of chronic hepatitis B and hepatitis C virus infection. This screening was conducted in Chiayi County from 2018-2019. All residents aged 30 years or older were invited to participate in quantitative HBsAg (qHBsAg) and HCV Ag screening. Among the 4010 participants (male:female = 1630:2380), the prevalence of qHBsAg and HCV Ag was 9.9% (396/4010) and 4.1% (163/4010), respectively. High-prevalence townships were identified, three for qHBsAg > 15% and two for HCV Ag > 10%. The age-specific prevalence of qHBsAg was distributed in an inverse U-shape with a peak (16.0%, 68/424) for subjects in their 40 s; for HCV, prevalence increased with age. Concentrations of qHBsAg < 200 IU/mL were found in 54% (214/396) of carriers. The rate of oral antiviral treatment for HCV was 75.5% (114/151), with subjects younger than 75 years tending to undergo treatment (85.6% vs. 57.4%, p < 0.001). QHBsAg and HCV Ag core antigens can reflect the concentration of the viral load, which serves as a feasible screening tool. Using quantitative antigen screening for hepatitis B and C in community-based screening, two hyperendemic townships were identified from an endemic county.

A Highly Divergent Hepacivirus Identified in Domestic Ducks Further Reveals the Genetic Diversity of Hepaciviruses.

Hepaciviruses represent a group of viruses that pose a significant threat to the health of humans and animals. During the last decade, new members of the genus Hepacivirus have been identified in various host species worldwide, indicating the widespread distribution of genetically diversified hepaciviruses among animals. By applying unbiased high-throughput sequencing, a novel hepacivirus, provisionally designated Hepacivirus Q, was discovered in duck liver samples collected in Guangdong province of China. Genetic analysis revealed that the complete polyprotein of Hepacivirus Q shares 23.9-46.6% amino acid identity with other representatives of the genus Hepacivirus. Considering the species demarcation criteria for hepaciviruses, Hepacivirus Q should be regarded as a novel hepacivirus species of the genus Hepacivirus within the family Flaviviridae. Phylogenetic analyses also indicate the large genetic distance between Hepacivirus Q and other known hepaciviruses. Molecular detection of this novel hepacivirus showed an overall prevalence of 15.9% in duck populations in partial areas of Guangdong province. These results expand knowledge about the genetic diversity and evolution of hepaciviruses and indicate that genetically divergent hepaciviruses are circulating in duck populations in China.

A Novel Subtype of Bovine Hepacivirus Identified in Ticks Reveals the Genetic Diversity and Evolution of Bovine Hepacivirus.

Hepaciviruses represent a group of viruses that pose a significant threat to the health of humans and animals. New members of the genus Hepacivirus in the family Flaviviridae have recently been identified in a wide variety of host species worldwide. Similar to the Hepatitis C virus (HCV), bovine hepacivirus (BovHepV) is hepatotropic and causes acute or persistent infections in cattle. BovHepVs are distributed worldwide and classified into two genotypes with seven subtypes in genotype 1. In this study, three BovHepV strains were identified in the samples of ticks sucking blood on cattle in the Guangdong province of China, through unbiased high-throughput sequencing. Genetic analysis revealed the polyprotein-coding gene of these viral sequences herein shared 67.7-84.8% nt identity and 76.1-95.6% aa identity with other BovHepVs identified worldwide. As per the demarcation criteria adopted for the genotyping and subtyping of HCV, these three BovHepV strains belonged to a novel subtype within the genotype 1. Additionally, purifying selection was the dominant evolutionary pressure acting on the genomes of BovHepV, and genetic recombination was not common among BovHepVs. These results expand the knowledge about the genetic diversity and evolution of BovHepV distributed globally, and also indicate genetically divergent BovHepV strains were co-circulating in cattle populations in China.

Study of Quasispecies Complexity and Liver Damage Progression after Liver Transplantation in Hepatitis C Virus Infected Patients.

Cirrhosis derived from chronic hepatitis C virus (HCV) infection is still a common indication for liver transplantation (LT). Reinfection of the engrafted liver is universal in patients with detectable viral RNA at the time of transplant and causes fast progression of cirrhosis (within 5 years) in around one-third of these patients. To prevent damage to the liver graft, effective direct-acting antiviral (DAA) therapy is required as soon as possible. However, because of post-LT clinical instability, it is difficult to determine the optimal time to start DAAs with a low risk of complications. Evaluate changes in quasispecies complexity following LT and seek a predictive index of fast liver damage progression to determine the timing of DAA initiation. HCV genomes isolated from pre-LT and 15-day post-LT serum samples of ten patients, who underwent orthotopic LT, were quantified and sequenced using a next-generation sequencing platform. Sequence alignments, phylogenetic trees, quasispecies complexity measures, biostatistics analyses, adjusted R2 values, and analysis of variance (ANOVA) were carried out. Three different patterns of reinfection were observed (viral bottlenecking, conserved pre-LT population, and mixed populations), suggesting that bottlenecking or homogenization of the viral population is not a generalized effect after liver graft reinfection. None of the quasispecies complexity measures predicted the future degree of liver damage. Higher and more uniform viral load (VL) values were observed in all pre-LT samples, but values were more dispersed in post-LT samples. However, VL increased significantly from the pre-LT to 15-day post-LT samples in patients with advanced fibrosis at 1-year post-LT, suggesting that a VL increase on day 15 may be a predictor of fast liver fibrosis progression. HCV kinetics after LT differ between patients and are not fibrosis-dependent. Higher VL at day 15 post-LT versus pre-LT samples may predict fast liver fibrosis progression.

Validation of a Genotype-Independent Hepatitis C Virus Near-Whole Genome Sequencing Assay.

Despite the effectiveness of direct-acting antiviral agents in treating hepatitis C virus (HCV), cases of treatment failure have been associated with the emergence of resistance-associated substitutions. To better guide clinical decision-making, we developed and validated a near-whole-genome HCV genotype-independent next-generation sequencing strategy. HCV genotype 1-6 samples from direct-acting antiviral agent treatment-naïve and -treated HCV-infected individuals were included. Viral RNA was extracted using a NucliSens easyMAG and amplified using nested reverse transcription-polymerase chain reaction. Libraries were prepared using Nextera XT and sequenced on the Illumina MiSeq sequencing platform. Data were processed by an in-house pipeline (MiCall). Nucleotide consensus sequences were aligned to reference strain sequences for resistance-associated substitution identification and compared to NS3, NS5a, and NS5b sequence data obtained from a validated in-house assay optimized for HCV genotype 1. Sequencing success rates (defined as achieving >100-fold read coverage) approaching 90% were observed for most genotypes in samples with a viral load >5 log10 IU/mL. This genotype-independent sequencing method resulted in >99.8% nucleotide concordance with the genotype 1-optimized method, and 100% agreement in genotype assignment with paired line probe assay-based genotypes. The assay demonstrated high intra-run repeatability and inter-run reproducibility at detecting substitutions above 2% prevalence. This study highlights the performance of a freely available laboratory and bioinformatic approach for reliable HCV genotyping and resistance-associated substitution detection regardless of genotype.

Genetic Subtypes and Natural Resistance Mutations in HCV Genotype 4 Infected Saudi Arabian Patients.

This study aimed to characterize the HCV genetic subtypes variability and the presence of natural occurring resistance-associated substitutions (RASs) in Saudi Arabia patients. A total of 17 GT patients were analyzed. Sequence analysis of NS3, NS5A, and NS5B regions was performed by direct sequencing, and phylogenetic analyses were used to determine genetic subtypes, RAS, and polymorphisms. Nine patients were infected by GT 4a, two with GT 4o and three with GT 4d. Two patients were infected with apparent recombinant virus (4a/4o/4a in NS3/NS5A/NS5B), and one patient was infected with a previously unknown, unclassifiable, virus of GT 4. Natural RASs were found in six patients (35%), including three infected by GT 4a, two by GT 4a/GT 4o/GT 4a, and one patient infected by an unknown, unclassifiable, virus of GT 4. In particular, NS3-RAS V170I was demonstrated in three patients, while NS5A-RASs (L28M, L30R, L28M + M31L) were detected in the remaining three patients. All patients were treated with sofosbuvir plus daclatasvir; three patients were lost to follow-up, whereas 14 patients completed the treatment. A sustained virological response (SVR) was obtained in all but one patient carrying NS3-RAS V170I who later relapsed. GT 4a is the most common subtype in this small cohort of Saudi Arabia patients infected with hepatitis C infection. Natural RASs were observed in about one-third of patients, but only one of them showed a treatment failure.

Characterization of a Novel Hepatitis C Subtype, 6xj, and Its Consequences for Direct-Acting Antiviral Treatment in Yunnan, China.

Hepatitis C virus (HCV) has a high rate of genetic variability, with eight genotypes and 91 subtypes. The genetic diversity of HCV genotype 6 (HCV-6) is the highest with 31 subtypes, and this genotype is prevalent in Southeast Asia. In this study, we investigated 160 individuals with chronic hepatitis C in Yunnan Province, China. Using reverse transcription (RT)-PCR and Sanger sequencing, 147 cases were successfully amplified and genotyped as 3b (4.9%), 3a (19.73%), 6n (12.24%), 1b (7.48%), 2a (6.12%), 6a (2.04%), 1a (0.68%), 6v (0.68%), and 6xa (0.68%), with eight sequences remaining unclassified. Subsequently, the eight nearly full-length genomes were successfully amplified and analyzed. The eight complete coding sequences formed a phylogenetic group that was distinct from the previously assigned HCV-6 subtypes and clustered with two previously unnamed HCV-6 sequences. Furthermore, Simplot analysis showed no recombination and the p-distance was more than 15% in comparison to the 6a to 6xi subtypes. Taken together, we identified a new HCV-6 subtype, 6xj, which originated approximately in 1775 according to Bayesian analyses. Moreover, all eight individuals received follow-up assessments at 44 weeks from the beginning of their 12-week treatments of sofosbuvir/velpatasvir (after-treatment week 32). One case relapsed at after-treatment week 32. Next-generation sequencing (NGS) was conducted and showed that the treatment failure case had two suspected antiviral resistance mutations, NS5A V28M (a change of V to M at position 28) and NS5B A442V, compared with the baseline. Overall, this newly identified 6xj subtype further confirmed the high diversity of the HCV-6 genotype. The newly identified resistance-associated amino acid substitutions may help inform future clinical treatments. IMPORTANCE This study investigated the genetic diversity of hepatitis C virus (HCV), particularly in relation to genotype 6, which is prevalent in Yunnan, China, and is often difficult to treat successfully. We identified a new HCV-6 subtype, 6xj, which is an ancient strain. Moreover, all eight individuals with the novel subtype received follow-up assessments at 44 weeks from the beginning of their treatments. One case relapsed after 8 months of withdrawal. NGS was conducted and showed that the isolate from the treatment failure case had two suspected antiviral resistance mutations, NS5A V28M and NS5B A442V, compared with the baseline. Overall, this newly identified 6xj subtype further confirmed the high diversity of the HCV-6 genotype. The newly identified resistance-associated amino acid substitutions may help inform future clinical treatments. We believe that our study makes a significant contribution to the literature based on the results described above.

Hepatitis C Virus Subtypes Novel 6g-Related Subtype and 6w Could Be Indigenous in Southern Taiwan with Characteristic Geographic Distribution.

Hepatitis C virus (HCV) genotype (GT) 6 is the most genetically diverse GT and mainly distributed in Southeast Asia and south China but not Taiwan. Earlier studies showed the major HCV GTs in Taiwan were GT 1b and 2 with very rare GT 6 except in injection drug users (IDUs), and subtype 6a is the main GT 6 subtype among IDUs. Recently, we reported a much higher prevalence (18.3%) of GT 6 in Tainan City, southern Taiwan. This study was designed to clarify the subtypes of GT 6 in this endemic area. A total of 3022 (1343 men and 1679 women) HCV viremic patients were enrolled. Subtypes of GT 6 were determined by sequencing of core/E1 and nonstructural protein 5B in 322 of 518 GT 6 patients. The overall GT 6 prevalence rate was 17.1% (518/3022), with higher prevalence districts (>25%) located in northern Tainan. A novel 6g-related subtype is the most prevalent subtype (81.0%), followed by 6w (10.8%), 6a (7.5%), and 6n (0.7%). The high GT 6 prevalence in Tainan was mainly due to a novel 6g-related subtype and 6w. These two subtypes could be indigenous in Tainan with characteristic geographic distribution.

Impact of HCV Eradication on Lipid Metabolism in HIV/HCV Coinfected Patients: Data from ICONA and HepaICONA Foundation Cohort Study.

OBJECTIVES: HCV shows complex interactions with lipid metabolism. Our aim was to examine total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) changes in HIV/HCV coinfected patients, after achieving sustained virological response (SVR), according to different HCV genotypes and specific antiretroviral use. METHODS: HIV/HCV coinfected patients, enrolled in the ICONA and HepaICONA cohorts, who achieved DAA-driven SVR were included. Paired t-tests were used to examine whether the pre- and post-SVR laboratory value variations were significantly different from zero. ANCOVA regression models were employed to estimate the causal effect of SVR and of PI/r use on lipid changes. The interaction between the effect of eradication and HCV genotype was formally tested. RESULTS: six hundred and ninety-nine HIV/HCV coinfected patients were enrolled. After HCV eradication, a significant improvement in liver function occurred, with a significant decrease in AST, ALT, GGT, and total plasmatic bilirubin. TC and LDL-C significantly increased by 21.4 mg/dL and 22.4 mg/dL, respectively (p < 0.001), after SVR, whereas there was no evidence for a change in HDL-C (p = 0.45) and triglycerides (p = 0.49). Notably, the TC and LDL-C increase was higher for participants who were receiving darunavir/ritonavir, and the TC showed a more pronounced increase among HCV genotype 3 patients (interaction-p value = 0.002). CONCLUSIONS: complex and rapid changes in TC and LDL-C levels, modulated by HCV genotype and PI/r-based ART combinations, occurred in HIV/HCV coinfected patients after SVR. Further studies are needed to evaluate the clinical impact of these changes on the long-term risk of cardiovascular disease.

SHARED: An International Collaboration to Unravel Hepatitis C Resistance.

The advent of direct-acting antivirals (DAAs) has transformed the treatment landscape of hepatitis C [...].

Expression of HCV genotype-4 core antigen in prokaryotic E. coli system for diagnosis of HCV infection in Egypt.

BACKGROUND: Egypt has a high prevalence of hepatitis C virus (HCV) infection with 92.5% of genotype-4. AIM: This study aimed to clone and express the core gene of HCV genotype-4 for using it to develop a highly sensitive, specific, and cost-effective diagnostic assay for detecting HCV infection. METHODS: Using synthetic HCV genotype-4 core gene, pET15b as E. coli expression vector, and 1 mM lactose as inducer, the HCV core protein (MW 17 kDa) was expressed in the form of inclusion bodies (IBs) that was purified and solubilized using 8 M guanidinium HCl. The recombinant core protein was in vitro refolded by a rapid dilution method for further purification using weak cation exchange liquid chromatography. The immunogenicity of the purified protein was tested by ELISA using 129 serum samples. RESULTS: The recombinant core protein was successfully expressed and purified. The results also showed that the in-house anti-HCV core assay is accurate, specific (~96.6%), and highly sensitive (~100%) in accordance with the commercial ELISA kit. CONCLUSION: The sensitivity, specificity, and reproducibility of the developed assay were high and promising to be used as a screening assay for detecting HCV infection.

Geographical Distribution and Genetic Diversity of Bank Vole Hepaciviruses in Europe.

The development of new diagnostic methods resulted in the discovery of novel hepaciviruses in wild populations of the bank vole (Myodes glareolus, syn. Clethrionomys glareolus). The naturally infected voles demonstrate signs of hepatitis similar to those induced by hepatitis C virus (HCV) in humans. The aim of the present research was to investigate the geographical distribution of bank vole-associated hepaciviruses (BvHVs) and their genetic diversity in Europe. Real-time reverse transcription polymerase chain reaction (RT-qPCR) screening revealed BvHV RNA in 442 out of 1838 (24.0%) bank voles from nine European countries and in one of seven northern red-backed voles (Myodes rutilus, syn. Clethrionomys rutilus). BvHV RNA was not found in any other small mammal species (n = 23) tested here. Phylogenetic and isolation-by-distance analyses confirmed the occurrence of both BvHV species (Hepacivirus F and Hepacivirus J) and their sympatric occurrence at several trapping sites in two countries. The broad geographical distribution of BvHVs across Europe was associated with their presence in bank voles of different evolutionary lineages. The extensive geographical distribution and high levels of genetic diversity of BvHVs, as well as the high population fluctuations of bank voles and occasional commensalism in some parts of Europe warrant future studies on the zoonotic potential of BvHVs.

Management and Treatment of Hepatitis C: Are There Still Unsolved Problems and Unique Populations?

Direct-acting antivirals (DAA) have revolutionized the treatment of patients with chronic hepatitis C virus (HCV) infection, possibly leading to HCV elimination by 2030 as endorsed by the World Health Organization (WHO). However, some patients belonging to the so-called unique or special populations are referred to as difficult-to-treat due to unreached sustained virological response, potential drug side effects or interactions or co-morbidities. Several years after the DAA introduction and on the basis of excellent findings in terms of efficacy and safety, some doubts arise around the exact meaning of the special population designation and whether this group of patients actually exists. The aim of this review is to discuss and analyze current evidence on the management and treatment of the so-called "unique populations". We placed particular emphasis on patients with decompensated cirrhosis, chronic kidney disease (CKD), coinfections, rare genotypes, and previous treatment failure, in order to provide physicians with an updated overview of the actual problems and needs in the current scenario.

Late Relapse and Reinfection in HCV Patients Treated with Direct-Acting Antiviral (DAA) Drugs.

The risk of hepatitis C virus (HCV) recurrence after direct-acting antiviral (DAA) treatment is <0.5%. However, the distinction between HCV RNA late relapse and reinfection still represents a challenge in virological diagnostics. The aim of this study was to employ next-generation sequencing (NGS) to investigate HCV RNA recurrence in patients achieving a sustained virologic response (SVR) at least six months post-treatment. NGS was performed on plasma samples from six HCV-positive patients (Pt1-6) treated with DAA. NGS of HCV NS5B was analyzed before treatment (T0), after HCV RNA rebound (T1), and, for Pt3, after a second rebound (T2). Reinfection was confirmed for Pt5, and for the first rebound observed in Pt3. Conversely, viral relapse was observed when comparing T0 and T1 for Pt6 and T1 and T2 for Pt3. Z-scores were calculated and used to predict whether HCV-positive patient samples at different time points belonged to the same quasispecies population. A low Z-score of <2.58 confirmed that viral quasispecies detected at T0 and T1 were closely related for both Pt1 and Pt2, while the Z-score for Pt4 was suggestive of possible reinfection. NGS data analyses indicate that the Z-score may be a useful parameter for distinguishing late relapse from reinfection.

Prevalence of occult hepatitis C virus infection in beta-thalassemia major patients in Ahvaz, Iran.

Occult hepatitis C virus infection (OCI) is defined by the presence of HCV RNA in peripheral blood mononuclear cells (PBMCs) and liver tissue cells despite the absence of HCV RNA in plasma. Currently, OCI is classified into two types: seropositive OCI (anti-HCV positive and serum HCV RNA negative) and seronegative OCI (anti-HCV and serum HCV RNA negative). Beta-thalassemia is described as a blood disorder that decreases the synthesis of hemoglobin. Repeated blood transfusion is the standard treatment for patients with beta-thalassemia major (BTM), and this increases the risk of exposure to infectious agents. The aim of this study was to investigate the prevalence of OCI among BTM patients. Plasma and PBMCs were collected from 90 BTM patients who were referred to Shafa Hospital in the city of Ahvaz and were screened for HCV antibody using a commercial ELISA kit as the first step. Next, nested RT-PCR was performed on extracts of plasma and PBMCs. HCV RNA from positive PBMCs was sequenced, the sequences were aligned, and a phylogenetic tree was constructed to determine their relationship to reference sequences retrieved from the GenBank database. Seventy-nine out of 90 patients (87.8%) were negative for HCV Ab (seronegative), while 11 patients (12.2%) were seropositive. HCV RNA was found in PBMCs of four patients (66.7%) who were negative for HCV Ab (seronegative) and two patients (33.3%) who were positive for HCV Ab (seropositive). HCV RNA was not detected in plasma samples from these six patients. Six out of 90 BTM patients (6.7%) had OCI. HCV genotyping revealed that all six patients were infected with HCV subtype 3a. We found a high frequency of OCI in BTM patients, which warrants more attention, considering the importance of this infection. Further studies are needed to determine the actual prevalence of OCI in BTM patients in Iran.

Non-primate hepacivirus transmission and prevalence: Novel findings of virus circulation in horses and dogs in Morocco.

Non-primate hepacivirus (NPHV) is a homolog of hepatitis C virus and has been isolated from dogs and horses. Data on NPHV prevalence and distribution are not complete, and there is a particular lack of reports from the African continent. The present study represents the first investigation of NPHV prevalence in horses and dogs in North Africa. Blood was collected from 172 horses and 36 dogs at different locations in Morocco, and screened for NPHV RNA using nested PCR targeting 5'UTR and NS3 regions and analyzed for anti-NPHV NS3 antibody using a Gaussia luciferase immunoprecipitation system-to determine seroprevalence. Eight sequences of the NS3 region isolated from positive serum samples were targeted for phylogenetic analysis. Horses and dogs showed respective NPHV RNA positivity rates of 10.5% and 5.5%, and seroprevalences of 65.7% and 8.33%. Juvenile horses appeared more susceptible to infection, with a 23.5% NHPV RNA positivity rate. Seropositivity was more extensive in mares than stallions (77.14% vs. 46.27%, p < 0.0001). Phylogenetically, that NPHV NS3 genes isolated from horses and dog are clustered together. The NPHV strains we detected showed no correlation with geographic location within Morocco. In conclusion, Moroccan horses showed much evidence of previous and/or current NPHV infection, with young age and female sex as noted potential risk factors. Interestingly, NPHV is circulating in dogs as well as horses, suggesting that it has crossed species barriers and that horses and dogs are potential vectors by which an ancestor to hepatitis C virus was transmitted into human populations.