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OBJECTIVE: In Guangdong Province, hepatitis C virus (HCV) had been found to confer resistance to direct-acting antivirals (DAAs). There were few studies of HCV subtypes and resistance-associated substitutions (RASs) of HCV in different high-risk populations. In this study, we aimed to determine the subtype distribution and the RASs in high-risk population groups, including drug users (DU), men who have sex with men (MSM), female sex workers (FSW), and male patients with sexually transmitted diseases (STD) in Guangdong Province (a highly developed province with a large population). METHODS: Using a city-based sampling strategy,1356 samples were obtained from different population groups. Phylogenetic analyses determined subtypes based on Core, NS5B, or NS5A sequences. HCV subtype distribution and RASs in various risk groups and regions were analyzed. RESULTS: Ten subtypes, of which 6 h and 6 k were novel in Guangdong, were identified. The primary subtype among all risk groups was 6a. RASs in 1b and 3a were different from those observed in other studies. Subtype 3b in western Guangdong was higher than the other three regions. No RASs were found in 6a or any other genotype 6. CONCLUSIONS: The HCV subtypes are expanding in high-risk populations in Guangdong. Drug use by other risk groups and commercial sex by DU may bridge the dissemination of 6a from DU to other populations. The RAS profiles of 1b and 3a differed from those reported in studies conducted in southwestern China. Further research is required to determine the reason for this discrepancy. Moreover, the combination of RASs was high in subtype 3b. To guide HCV treatment of subtype 3b, pretreatment subtyping of HCV genotype 3 should be considered in western cities in the near future.
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Direct acting antivirals (DAAs) have been developed for hepatitis C virus (HCV) therapy, and they are usually effective, however resistance to DAA regimens has also been reported to have a significant impact. Resistance associated substitutions (RASs) in the NS5A region are known to be correlated with failure of DAA therapy. HCV genotypes 3a and 1 are the most prevalent genotypes in Thailand. This study analyzed the type and frequency of RASs associated with DAA failure, focusing on the NS5A region. Serum samples of HCV genotype 3a, 1a, and 1b infection from Thai blood donors were selected. The NS5A region was amplified using reverse transcription-polymerase chain reaction (RT-PCR). A phylogenetic tree was constructed to identify the genotypes of HCV. Nucleotide sequencing and amino acid sequencing were conducted to determine the prevalence of RASs. Construction of the phylogenetic tree indicated that 29 samples were genotype 3a, 11 samples were genotype 1a, and 9 were genotype 1b. Both HCV genotypes 1a and 3a can be categorized into two subclades. Results showed that the NS5A substitutions A30V/K, A62T/V/I/M/P/S/L, and S98G were present in HCV genotype 3a. In HCV genotype 1a, only NS5A RASs H54Y was detected. NS5A amino acid substitutions Q54H and P58L were found in HCV genotype 1b. In conclusion, NS5A RASs at amino acid positions 30, 62, 54, 58, and 98 are present within HCV genotypes 3a and 1. While keeping in mind that additional information was not available on the anonymous blood donors tested in this study, these findings can contribute to understand the NS5A mutation. Further study with known patients under drug treatment is recommended.
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Hepatitis C Crónica , Hepatitis C , Humanos , Hepacivirus/genética , Antivirales/farmacología , Antivirales/uso terapéutico , Tailandia/epidemiología , Donantes de Sangre , Filogenia , Hepatitis C/tratamiento farmacológico , Hepatitis C/epidemiologíaRESUMEN
Hepatitis C virus (HCV) is the most common infection worldwide. The correlation between HCV and renal cell carcinoma (RCC) is still mysterious. Therefore, the relationship between HCV and RCC was investigated. The study included 100 patients with RCC; 32 with HCV infection, and 68 without HCV infection. Expressions of viral proteins (NS3 and NS5A) were tested using an immune electron-microscope (IEM) and immunohistochemistry (IHC). IHC and quantitative real time-PCR investigated the presentation of human proteins TP53 and p21 genes. Transmission electron (TEM) detected viral-like particles in infected RCC tissues. The gene and protein expression of P53 was higher in HCV positive versus HCV negative patients and p21 was lower in HCV positive versus HCV negative in both tumor and normal tissue samples. Viral like particles were observed by TEM in the infected tumor and normal portion of the RCC tissues and the plasma samples. The IEM showed the depositions of NS3 and NS5A in infected renal tissues, while in noninfected samples, were not observed. The study hypothesizes that a correlation between HCV and RCC could exist through successfully detecting HCV-like particles, HCV proteins, and (p53 and p21) in RCC-infected patients.
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Carcinoma de Células Renales , Genotipo , Hepacivirus , Neoplasias Renales , Proteína p53 Supresora de Tumor , Proteínas no Estructurales Virales , Humanos , Carcinoma de Células Renales/virología , Carcinoma de Células Renales/genética , Carcinoma de Células Renales/patología , Hepacivirus/genética , Proteínas no Estructurales Virales/genética , Neoplasias Renales/virología , Neoplasias Renales/patología , Neoplasias Renales/genética , Masculino , Proteína p53 Supresora de Tumor/genética , Femenino , Persona de Mediana Edad , Hepatitis C/virología , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Anciano , Adulto , Inmunohistoquímica , Proteasas Virales , ARN Polimerasa Dependiente del ARN , ARN Helicasas DEAD-box , Nucleósido-Trifosfatasa , Serina EndopeptidasasRESUMEN
Classical swine fever (CSF) has been eradicated from Western and Central Europe but remains endemic in parts of Central and South America, Asia, and the Caribbean. CSF virus (CSFV) has been endemic in Cuba since 1993, most likely following an escape of the highly virulent Margarita/1958 strain. In recent years, chronic and persistent infections with low-virulent CSFV have been observed. Amino acid substitutions located in immunodominant epitopes of the envelope glycoprotein E2 of the attenuated isolates were attributed to positive selection due to suboptimal vaccination and control. To obtain a complete picture of the mutations involved in attenuation, we applied forward and reverse genetics using the evolutionary-related low-virulent CSFV/Pinar del Rio (CSF1058)/2010 (PdR) and highly virulent Margarita/1958 isolates. Sequence comparison of the two viruses recovered from experimental infections in pigs revealed 40 amino acid differences. Interestingly, the amino acid substitutions clustered in E2 and the NS5A and NS5B proteins. A long poly-uridine sequence was identified previously in the 3' untranslated region (UTR) of PdR. We constructed functional cDNA clones of the PdR and Margarita strains and generated eight recombinant viruses by introducing single or multiple gene fragments from Margarita into the PdR backbone. All chimeric viruses had comparable replication characteristics in porcine monocyte-derived macrophages. Recombinant PdR viruses carrying either E2 or NS5A/NS5B of Margarita, with 36 or 5 uridines in the 3'UTR, remained low virulent in 3-month-old pigs. The combination of these elements recovered the high-virulent Margarita phenotype. These results show that CSFV evolution towards attenuated variants in the field involved mutations in both structural and non-structural proteins and the UTRs, which act synergistically to determine virulence.
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Virus de la Fiebre Porcina Clásica , Peste Porcina Clásica , Animales , Porcinos , Virulencia/genética , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/química , MutaciónRESUMEN
Hepatitis C virus (HCV) infection is recognized as a major causative agent of chronic hepatitis, cirrhosis, and hepatocellular carcinoma. HCV non-structural protein 5A (NS5A) is a dimeric phosphoprotein with a hyperphosphorylated form to act as a switch that regulates HCV replication and assembly. NS5A inhibitors have been utilized as the scaffold for combination therapy of direct-acting antiviral agents (DAA). However, the mode of action of NS5A inhibitors is still unclear due to the lack of mechanistic detail regarding NS5A phosphorylation and dimerization in the HCV life cycle. It has been demonstrated that phosphorylation of NS5A at Ser235 is essential for RNA replication of the JFH1 strain. In this report, we found that NS5A phosphomimetic Ser235 substitution (Ser-to-Asp mutation) formed a dimer that was resistant to disruption by NS5A inhibitors as was the NS5A resistance-associated substitution Y93H. Phosphorylation of NS5A at Ser235 residue was required for the interaction of two NS5A-WT molecules in JFH1-based cell culture system but not absolutely required for dimerization of the NS5A-Y93H mutant. Interestingly, HCV nonstructural proteins from the subgenomic replicon NS3-5A was required for NS5A-WT dimerization but not required for NS5A-Y93H dimerization. Our data suggest that spontaneous Ser235 phosphorylation of NS5A and ensuing dimerization account for resistance of the JFH1/NS5A-Y93H mutant to NS5A inhibitors.
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Hepatitis C Crónica , Hepatitis C , Neoplasias Hepáticas , Humanos , Hepacivirus/metabolismo , Fosforilación , Antivirales/uso terapéutico , Dimerización , Hepatitis C Crónica/tratamiento farmacológico , Hepatitis C/tratamiento farmacológico , Neoplasias Hepáticas/tratamiento farmacológico , Resistencia a Medicamentos , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismoRESUMEN
HCV NS5A is a dimeric phosphoprotein involved in HCV replication. NS5A inhibitors are among direct-acting antivirals (DAA) for HCV therapy. The Y93H mutant of NS5A is resistant to NS5A inhibitors, but the precise mechanism remains unclear. In this report, we proposed a Ser38-His93-Asn91 triad to dissect the mechanism. Using pymol 1.3 software, the homology structure of JFH1 NS5A was determined based on the dimer structure of genotype 1b extracted from the database Protein DataBank (www.ebi.ac.uk/pdbsum) with codes 1ZH1 and 3FQM/3FQQ. FLAG-NS5A-WT failed to form dimer in the absence of nonstructural proteins from subgenomic replicon (NS3-5A); however, FLAG-NS5A-Y93H was able to form dimer without the aid of NS3-5A. The Ser38-His93-Asn91 triad in the dimer of the Y93H variant predicts a structural crash of the cleft receiving the NS5A inhibitor daclatasvir. The dimerization assay revealed that the existence of JFH1-NS5A-1ZH1 and -3FQM homology dimers depended on each other for existence and that both NS5A-WT 1ZH1 and 3FQM dimers cooperated to facilitate RNA replication. However, NS5A-Y93H 1ZH1 alone could form dimer and conduct RNA replication in the absence of the 3FQM structure. In conclusion, this study provides novel insight into the functional significance of the Ser38-His93-Asn91 triad in resistance of the Y93H variant to NS5A inhibitors.
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Antivirales , Hepatitis C Crónica , Humanos , Antivirales/farmacología , Hepatitis C Crónica/tratamiento farmacológico , Genotipo , Hepacivirus/genética , Proteínas no Estructurales Virales/genética , Farmacorresistencia Viral/genéticaRESUMEN
IMPORTANCE: Autophagy is a conserved degradation process that maintains cellular homeostasis and regulates native and adaptive immunity. Viruses have evolved diverse strategies to inhibit or activate autophagy for their benefit. The paper reveals that CSFV NS5A mediates the dissociation of PP2A from Beclin 1 and the association of PP2A with DAPK3 by interaction with PPP2R1A and DAPK3, PP2A dephosphorylates DAPK3 to activate its protein kinase activity, and activated DAPK3 phosphorylates Beclin 1 to trigger autophagy, indicating that NS5A activates autophagy via the PP2A-DAPK3-Beclin 1 axis. These data highlight a novel mechanism by which CSFV activates autophagy to favor its replication, thereby contributing to the development of antiviral strategies.
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Autofagia , Virus de la Fiebre Porcina Clásica , Peste Porcina Clásica , Proteínas no Estructurales Virales , Animales , Beclina-1/metabolismo , Peste Porcina Clásica/inmunología , Peste Porcina Clásica/virología , Virus de la Fiebre Porcina Clásica/fisiología , Porcinos , Replicación Viral , Proteínas no Estructurales Virales/metabolismoRESUMEN
Hepatitis C virus (HCV) is a hepatotropic virus that affects millions of human lives worldwide. Direct-acting antiviral (DAA) regimens are the most effective HCV treatment option. However, amino acid substitution-dependent resistance to DAAs has been a major challenge. This study aimed to determine the increasing risk of DAA resistance due to substitutions in DAA target non-structural proteins (NS3/4A, NS5A, and NS5B). Using a Sequence Retrieval System (SRS) at the virus pathogen resource (ViPR/BV-BRC), n = 32763 target protein sequences were retrieved and analyzed for resistance-associated amino acid substitutions (RAASs) by the Sequence Feature Variant Type (SFVT) antiviral-resistance assessment modeling tool. Reference target protein sequences with 100% identity were retried from UniProt following NCBI BLAST. The types and locations of RAASs were identified and visualized by AlphaFold and PyMol. Linux-r-base/R-studio was used for the data presentation. Multi-drug-resistant variants of NS3/4A in genotype 1 (n = 9) and genotype 5 (n = 5) along with DAA-specific NS3/4A, NS5A, and NS5B variants were identified pan-genotypically. A total of 27 variants (RAASs) of all the targets were identified. Fourteen genotype 1-specific substitutions: V1196A, V1158I, D1194A/T/G, R1181K, T1080S, Q1106R, V1062A, S1148G, A1182V, Y2065N, M2000T, and L2003V were identified. The most frequent substitutions were V1062L and L2003M, followed by Q2002H. L2003V, Q2002H, M2000T, Y2065N, and NL2003M of NS5A and L2003M of NS5B conferred resistance to daclatasvir. S2702T NS5B was the sofosbuvir-resistant variant. D1194A NS3/4A was triple DAA (simeprevir, faldaprevir, and asunaprevir) resistant. The double-drug resistant variants R1181K (faldaprevir and asunaprevir), A1182V and Q1106K/R (faldaprevir and simeprevir), T1080S (faldaprevir and telaprevir), and single drug-resistant variants V1062L (telaprevir), D1194E/T (simeprevir), D1194G (asunaprevir), S1148A/G (simeprevir), and Q1106L (Boceprevir) of NS3/4A were determined. The molecular phenomenon of DAA resistance is paramount in the development of HCV drug candidates. RAASs in NS3, NS5A, and NS5B reduce the susceptibility to DAAs; therefore, continuous RAAS-dependent resistance profiling in HCV is recommended to minimize the probability of DAA therapeutic failure.
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Hepatitis C virus (HCV) infects millions of people worldwide and is a leading cause of liver disease. Despite recent advances in antiviral therapies, viral resistance can limit drug efficacy and understanding the mechanisms that confer viral escape is important. We employ an unbiased interactome analysis to discover host binding partners of the HCV non-structural protein 5A (NS5A), a key player in viral replication and assembly. We identify ASPP2, apoptosis-stimulating protein of p53, as a new host co-factor that binds NS5A via its SH3 domain. Importantly, silencing ASPP2 reduces viral replication and spread. Our study uncovers a previously unknown role for ASPP2 to potentiate HCV RNA replication.
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Hepacivirus , Hepatitis C , Humanos , Hepacivirus/genética , Dominios Homologos src , Replicación Viral , Proteínas no Estructurales Virales/metabolismo , Dominios ProteicosRESUMEN
The hepatitis C virus (HCV) NS5A protein is comprised of three domains (D1-3). Previously, we observed that two alanine substitutions in D1 (V67A, P145A) abrogated replication of a genotype 2a isolate (JFH-1) sub-genomic replicon (SGR) in Huh7 cells, but this phenotype was partially restored in Huh7.5 cells. Here we demonstrate that five additional residues, surface-exposed and proximal to V67 or P145, exhibited the same phenotype. In contrast, the analogous mutants in a genotype 3a isolate (DBN3a) SGR exhibited different phenotypes in each cell line, consistent with fundamental differences in the functions of genotypes 2 and 3 NS5A. The difference between Huh7 and Huh7.5 cells was reminiscent of the observation that cyclophilin inhibitors are more potent against HCV replication in the former and suggested a role for D1 in cyclophilin dependence. Consistent with this, all JFH-1 and DBN3a mutants exhibited increased sensitivity to cyclosporin A treatment compared to wild-type. Silencing of cyclophilin A (CypA) in Huh7 cells inhibited replication of both JFH-1 and DBN3a. However, in Huh7.5 cells CypA silencing did not inhibit JFH-1 wild-type, but abrogated replication of all the JFH-1 mutants, and both DBN3a wild-type and all mutants. CypB silencing in Huh7 cells had no effect on DBN3a, but abrogated replication of JFH-1. CypB silencing in Huh7.5 cells had no effect on either SGR. Lastly, we confirmed that JFH-1 NS5A D1 interacted with CypA in vitro. These data demonstrate both a direct involvement of NS5A D1 in cyclophilin-dependent genome replication and functional differences between genotype 2 and 3 NS5A.
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Ciclofilinas , Hepatitis C , Humanos , Genotipo , Hepacivirus , Replicación ViralRESUMEN
The NS5A non-structural protein of classical swine fever virus (CSFV) is a multifunctional protein involved in viral genomic replication, protein translation, assembly of infectious virus particles, and regulation of cellular signaling pathways. Previous report showed that NS5A inhibited nuclear factor kappa B (NF-κB) signaling induced by poly(I:C); however, the mechanism involved has not been elucidated. Here, we reported that NS5A directly interacted with NF-κB essential modulator (NEMO), a regulatory subunit of the IκB kinase (IKK) complex, to inhibit the NF-κB signaling pathway. Further investigations showed that the zinc finger domain of NEMO and the aa 126-250 segment of NS5A are essential for the interaction between NEMO and NS5A. Mechanistic analysis revealed that NS5A mediated the proteasomal degradation of NEMO. Ubiquitination assay showed that NS5A induced the K27-linked but not the K48-linked polyubiquitination of NEMO for proteasomal degradation. In addition, NS5A blocked the K63-linked polyubiquitination of NEMO, thus inhibiting IKK phosphorylation, IκBα degradation, and NF-κB activation. These findings revealed a novel mechanism by which CSFV inhibits host innate immunity, which might guide the drug design against CSFV in the future.
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Virus de la Fiebre Porcina Clásica , FN-kappa B , Animales , Porcinos , FN-kappa B/metabolismo , Virus de la Fiebre Porcina Clásica/metabolismo , Transducción de Señal , Quinasa I-kappa B/genética , Quinasa I-kappa B/metabolismo , Inmunidad InnataRESUMEN
BACKGROUND/AIMS: Despite the availability of direct-acting antivirals (DAAs) for chronic hepatitis C virus (HCV) infection in Korea, need remains for pangenotypic regimens that can be used in the presence of hepatic impairment, comorbidities, or prior treatment failure. We investigated the efficacy and safety of sofosbuvir-velpatasvir and sofosbuvir-velpatasvir-voxilaprevir for 12 weeks in HCV-infected Korean adults. METHODS: This Phase 3b, multicenter, open-label study included 2 cohorts. In Cohort 1, participants with HCV genotype 1 or 2 and who were treatment-naive or treatment-experienced with interferon-based treatments, received sofosbuvir-velpatasvir 400/100 mg/day. In Cohort 2, HCV genotype 1 infected individuals who previously received an NS5A inhibitor-containing regimen ≥ 4 weeks received sofosbuvir-velpatasvir-voxilaprevir 400/100/100 mg/day. Decompensated cirrhosis was an exclusion criterion. The primary endpoint was SVR12, defined as HCV RNA < 15 IU/mL 12 weeks following treatment. RESULTS: Of 53 participants receiving sofosbuvir-velpatasvir, 52 (98.1%) achieved SVR12. The single participant who did not achieve SVR12 experienced an asymptomatic Grade 3 ASL/ALT elevation on day 15 and discontinued treatment. The event resolved without intervention. All 33 participants (100%) treated with sofosbuvir-velpatasvir-voxilaprevir achieved SVR 12. Overall, sofosbuvir-velpatasvir and sofosbuvir-velpatasvir-voxilaprevir were safe and well tolerated. Three participants (5.6%) in Cohort 1 and 1 participant (3.0%) in Cohort 2 had serious adverse events, but none were considered treatment-related. No deaths or grade 4 laboratory abnormalities were reported. CONCLUSION: Treatment with sofosbuvir-velpatasvir or sofosbuvir-velpatasvir-voxilaprevir was safe and resulted in high SVR12 rates in Korean HCV patients.
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Hepatitis C Crónica , Hepatitis C , Adulto , Humanos , Sofosbuvir/efectos adversos , Antivirales/efectos adversos , Hepatitis C Crónica/diagnóstico , Hepatitis C Crónica/tratamiento farmacológico , Hepatitis C/tratamiento farmacológico , Hepacivirus/genética , Quimioterapia Combinada , República de Corea , Genotipo , Resultado del TratamientoRESUMEN
Ribonucleotide reductases (RRs or RNRs) catalyze the reduction of the OH group on the 2nd carbon of ribose, reducing four ribonucleotides (NTPs) to the corresponding deoxyribonucleotides (dNTPs) to promote DNA synthesis. Large DNA viruses, such as herpesviruses and poxviruses, could benefit their replication through increasing dNTPs via expression of viral RRs. Little is known regarding the relationship between cellular RRs and RNA viruses. Mammalian RRs contain two subunits of ribonucleotide reductase M1 polypeptide (RRM1) and two subunits of ribonucleotide reductase M2 polypeptide (RRM2). In this study, expression of cellular RRMs, including RRM1 and RRM2, is found to be down-regulated in hepatitis C virus (HCV)-infected Huh7.5 cells and Huh7 cells with HCV subgenomic RNAs (HCVr). As expected, the NTP/dNTP ratio is elevated in HCVr cells. Compared with that of the control Huh7 cells with sh-scramble, the NTP/dNTP ratio of the RRM-knockdown cells is elevated. Knockdown of RRM1 or RRM2 increases HCV replication in HCV replicon cells. Moreover, inhibitors to RRMs, including Didox, Trimidox and hydroxyurea, enhance HCV replication. Among various HCV viral proteins, the NS5A and/or NS3/4A proteins suppress the expression of RRMs. When these are taken together, the results suggest that HCV down-regulates the expression of RRMs in cultured cells to promote its replication.
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Drug discovery is a difficult task, and is even more challenging when the target evolves during therapy. Antiviral drug therapy is an excellent example, exemplified by the evolution of therapeutic approaches for treatment of hepatitis C and HIV-1. Nick Meanwell and his colleagues made important contributions leading to molecules for treatment of hepatitis C and HIV-1, each with distinct mechanisms of action. This review summarizes the discovery and impact of these drugs, and will highlight, where applicable, the broader contributions of these discoveries to medicinal chemistry and drug discovery.
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Hepatitis C virus (HCV) is associated with liver damage and an increased progression rate to cirrhosis and hepatocellular carcinoma. In Portugal, it is prevalent in vulnerable populations such as injection drug users (IDU). HCV is characterized by a high intra-host variability, and the selecting driving forces could select variants containing resistance-associated substitutions (RAS) that reduce treatment effectiveness. The main goal of this study was to analyze the sequence variation of NS5A in treatment-naïve IDU. The epidemiological and clinical status of hepatitis C were analyzed, and samples were sequenced by Sanger and Next-Generation sequencing (NGS) to assess RAS and confirm HCV subtypes. Phylogenetic classification was concordant: 1a, 52.4%; 1b, 10.7%; 3a, 20.2%; 4a, 8.3%; 4d, 7.1%; and one 2k/1b recombinant. A 1a/3a mixed infection was detected by NGS. RAS were found in 34.5% (29/84) of samples using Sanger sequencing, while in 42.9% (36/84) using NGS. In sequences from subtypes 1a and 1b, RAS K24R, M28V, Q30H/R, H58D/P/Q/R, and RAS L31M and P58S were detected, respectively. In subtype 3a, RAS A30S/T, Y93H and polymorphisms in position 62 were identified. Additionally, RAS P58L was detected in genotype 4. The strategy used for the molecular survey of baseline HCV resistance is of particular importance to achieve treatment effectiveness and contribute to the elimination of hepatitis C.
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Hepatitis C virus (HCV) is one of the basic culprits behind chronic liver disease, which may result in cirrhosis and hepatocarcinoma. In spite of the extensive research conducted, a vaccine against HCV has not been yet created. We have obtained human mesenchymal stem cells (hMSCs) and used them for expressing the HCV NS5A protein as a model vaccination platform. Sixteen hMSC lines of a different origin were transfected with the pcNS5A-GFP plasmid to obtain genetically modified MSCs (mMSCs). The highest efficiency was obtained by the transfection of dental pulp MSCs. C57BL/6 mice were immunized intravenously with mMSCs, and the immune response was compared with the response to the pcNS5A-GFP plasmid, which was injected intramuscularly. It was shown that the antigen-specific lymphocyte proliferation and the number of IFN-γ-synthesizing cells were two to three times higher after the mMSC immunization compared to the DNA immunization. In addition, mMSCs induced more CD4+ memory T cells and an increase in the CD4+/CD8+ ratio. The results suggest that the immunostimulatory effect of mMSCs is associated with the switch of MSCs to the pro-inflammatory phenotype and a decrease in the proportion of myeloid derived suppressor cells. Thus, the possibility of using human mMSCs for the creation of a vaccine against HCV has been shown for the first time.
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Vimentin (VIM), an indispensable protein, is responsible for the formation of intermediate filament structures within cells and plays a crucial role in viral infections. However, the precise role of VIM in classical swine fever virus (CSFV) infection remains unclear. Herein, we systematically investigated the function of VIM in CSFV replication. We demonstrated that both knockdown and overexpression of VIM affected CSFV replication. Furthermore, we observed by confocal microscopy the rearrangement of cellular VIM into a cage-like structure during CSFV infection. Three-dimensional (3D) imaging indicated that the cage-like structures were localized in the endoplasmic reticulum (ER) and ringed around the double-stranded RNA (dsRNA), thereby suggesting that VIM was associated with the formation of the viral replication complex (VRC). Mechanistically, phosphorylation of VIM at serine 72 (Ser72), regulated by the RhoA/ROCK signaling pathway, induced VIM rearrangement upon CSFV infection. Confocal microscopy and coimmunoprecipitation assays revealed that VIM colocalized and interacted with CSFV NS5A. Structurally, it was determined that amino acids 96 to 407 of VIM and amino acids 251 to 416 of NS5A were the respective important domains for this interaction. Importantly, both VIM knockdown and disruption of VIM rearrangement inhibited the localization of NS5A in the ER, implying that VIM rearrangement recruited NS5A to the ER for VRC formation. Collectively, our results suggest that VIM recruits NS5A to form a stable VRC that is protected by the cage-like structure formed by VIM rearrangement, ultimately leading to enhanced virus replication. These findings highlight the critical role of VIM in the formation and stabilization of VRC, which provides alternative strategies for the development of antiviral drugs. IMPORTANCE Classical swine fever (CSF), caused by classical swine fever virus (CSFV), is a highly infectious disease that poses a significant threat to the global pig industry. Therefore, gaining insights into the virus and its interaction with host cells is crucial for developing effective antiviral measures and controlling the spread of CSF. Previous studies have shown that CSFV infection induces rearrangement of the endoplasmic reticulum, leading to the formation of small vesicular organelles containing nonstructural protein and double-stranded RNA of CSFV, as well as some host factors. These organelles then assemble into viral replication complexes (VRCs). In this study, we have discovered that VIM recruited CSFV NS5A to form a stable VRC that was protected by a cage-like structure formed by rearranged VIM. This enhanced viral replication. Our findings not only shed light on the molecular mechanism of CSFV replication but also offer new insights into the development of antiviral strategies for controlling CSFV.
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Virus de la Fiebre Porcina Clásica , Peste Porcina Clásica , Porcinos , Animales , Virus de la Fiebre Porcina Clásica/fisiología , Vimentina/metabolismo , ARN Bicatenario , Filamentos Intermedios/metabolismo , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismo , Replicación Viral , Antivirales , Aminoácidos/genéticaRESUMEN
Several direct-acting antivirals (DAAs) are available, providing interferon-free strategies for a hepatitis C cure. In contrast to DAAs, host-targeting agents (HTAs) interfere with host cellular factors that are essential in the viral replication cycle; as host genes, they are less likely to rapidly mutate under drug pressure, thus potentially exhibiting a high barrier to resistance, in addition to distinct mechanisms of action. We compared the effects of cyclosporin A (CsA), a HTA that targets cyclophilin A (CypA), to DAAs, including inhibitors of nonstructural protein 5A (NS5A), NS3/4A, and NS5B, in Huh7.5.1 cells. Our data show that CsA suppressed HCV infection as rapidly as the fastest-acting DAAs. CsA and inhibitors of NS5A and NS3/4A, but not of NS5B, suppressed the production and release of infectious HCV particles. Intriguingly, while CsA rapidly suppressed infectious extracellular virus levels, it had no significant effect on the intracellular infectious virus, suggesting that, unlike the DAAs tested here, it may block a post-assembly step in the viral replication cycle. Hence, our findings shed light on the biological processes involved in HCV replication and the role of CypA.
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Hepatitis C Crónica , Hepatitis C , Humanos , Hepacivirus/genética , Antivirales/uso terapéutico , Ciclosporina/farmacología , Ciclosporina/uso terapéutico , Hepatitis C Crónica/tratamiento farmacológico , Proteínas no Estructurales Virales/genética , Hepatitis C/tratamiento farmacológicoRESUMEN
Hepatitis C virus (HCV) infection contributes significantly to liver cirrhosis and hepatocellular carcinoma (HCC), often requiring liver transplantation. Introducing direct-acting antiviral agents (DAAs) has radically changed HCV treatment. DAAs achieve high rates of sustained virological response (>98%). Even then, resistant-associated substitution and HCC during or after treatment have become prominent clinical concerns. Further, several clinically significant issues remain unresolved after successful HCV eradication by DAAs, including treating patients with chronic kidney disease or decompensated liver cirrhosis. Extensive and large-scale screening and treatment implementation programs are needed to make DAA therapies effective at the population level.
Asunto(s)
Carcinoma Hepatocelular , Hepatitis C Crónica , Hepatitis C , Neoplasias Hepáticas , Humanos , Hepatitis C Crónica/tratamiento farmacológico , Hepatitis C Crónica/epidemiología , Carcinoma Hepatocelular/tratamiento farmacológico , Carcinoma Hepatocelular/epidemiología , Carcinoma Hepatocelular/patología , Antivirales/uso terapéutico , Neoplasias Hepáticas/epidemiología , Neoplasias Hepáticas/etiología , Neoplasias Hepáticas/tratamiento farmacológico , Cirrosis Hepática , Hepatitis C/tratamiento farmacológico , Hepacivirus/genéticaRESUMEN
INTRODUCTION AND OBJECTIVES: In clinical trials, patients with hepatitis C virus (HCV) genotype (GT)1a infection and baseline resistance-associated substitutions (RASs) at amino acid positions 28, 30, 31, or 93 receiving elbasvir/grazoprevir for 12 weeks achieved lower rates of sustained virologic response (SVR) than those without baseline RASs. SVR rates in patients with RASs were improved when elbasvir/grazoprevir treatment duration was extended from 12 to 16 weeks and administered concomitantly with ribavirin. MATERIALS AND METHODS: This was a retrospective, observational analysis using electronic health record abstraction. Patients with HCV GT1a infection and RASs at positions 28, 30, 31, or 93 who were prescribed 16 weeks of elbasvir/grazoprevir and ≥ 1 prescription for ribavirin were included. SVR was defined as HCV RNA below the lower limit of quantification ≥ 70 days after end of treatment. RESULTS: The primary analysis included patients with baseline RASs at positions 30, 31, or 93 (n = 76); a secondary analysis included patients with RASs at positions 28, 30, 31, or 93 (n = 93). SVR was achieved by 77.6% (59/76) of patients in the primary analysis and 80.6% (75/93) of those in the secondary analysis. Of the 18 (19.4%) patients in the secondary cohort who failed to achieve SVR, 8 relapsed (4 with treatment-emergent NS5A substitutions) and 10 did not have viral sequencing to distinguish relapse from reinfection. CONCLUSIONS: This analysis highlights the opportunities in leveraging real-world data to further understand treatment outcomes in smaller, discrete subgroups of patients with HCV infection who cannot be thoroughly evaluated in clinical trials.