Insights into the role of the cobalt(III)-thiosemicarbazone complex as a potential inhibitor of the Chikungunya virus nsP4.

Chikungunya virus (CHIKV) is the causative agent of chikungunya fever, a disease that can result in disability. Until now, there is no antiviral treatment against CHIKV, demonstrating that there is a need for development of new drugs. Studies have shown that thiosemicarbazones and their metal complexes possess biological activities, and their synthesis is simple, clean, versatile, and results in high yields. Here, we evaluated the mechanism of action (MOA) of a cobalt(III) thiosemicarbazone complex named [CoIII(L1)2]Cl based on its in vitro potent antiviral activity against CHIKV previously evaluated (80% of inhibition on replication). Furthermore, the complex has no toxicity in healthy cells, as confirmed by infecting BHK-21 cells with CHIKV-nanoluciferase in the presence of the compound, showing that [CoIII(L1)2]Cl inhibited CHIKV infection with the selective index of 3.26. [CoIII(L1)2]Cl presented a post-entry effect on viral replication, emphasized by the strong interaction of [CoIII(L1)2]Cl with CHIKV non-structural protein 4 (nsP4) in the microscale thermophoresis assay, suggesting a potential mode of action of this compound against CHIKV. Moreover, in silico analyses by molecular docking demonstrated potential interaction of [CoIII(L1)2]Cl with nsP4 through hydrogen bonds, hydrophobic and electrostatic interactions. The evaluation of ADME-Tox properties showed that [CoIII(L1)2]Cl presents appropriate lipophilicity, good human intestinal absorption, and has no toxicological effect as irritant, mutagenic, reproductive, and tumorigenic side effects.

Characterization of primary direct-acting antiviral (DAA) drugs resistance mutations in NS5A/NS5B regions of hepatitis C virus with genotype 1a and 1b from patients with chronic hepatitis.

The Hepatitis C virus (HCV) infection is a public health problem. The high level of HCV replication and its lack of post-transcriptional correction mechanisms results in the emergence of viral variants and the difficulty in determining polymorphisms and variants that contain the substitutions associated with resistance towards new antivirals. The main focus of this study was to map the NS5A and NS5B polymorphisms and resistance mutations to new antiviral drugs in HCV strains genotype 1 from patients with chronic hepatitis C infection. Serum samples were collected from patients who underwent routine viral load tests at the Instituto Adolfo Lutz, Sao Paulo city, Brazil. A total of 698 and 853 samples were used for the characterization of NS5A and NS5B regions, respectively, which comprise the HCV genotypes 1a and 1b. The prevalence of resistance mutations found in the NS5A region was 6.4%, with Y93H, L31M, Q30R, and Y93N as the main resistance-associated substitutions (RAS). No NS5B-associated RAS was observed for any of the analyzed drugs. These findings support that the RAS test should be offered to individuals with poor response to double combination regimens prior to treatment initiation, thereby assisting strain vigilance and selection of effective treatment or retreatment options using DAA regimens.

Identification of NS5B resistance against SOFOSBUVIR in hepatitis C virus genotype 3a, naive and treated patients.

AIMS: Pakistan has the second highest prevalence of HCV with genotype 3a (GT-3a) being the most frequently circulating genotype. Currently, resistance-associated substitutions (RASs) are a major challenge in HCV treatment with direct-acting antivirals (DAAs). Sofosbuvir (SOF) is an FDA-approved NS5B nucleotide inhibitor. The aim of this study was to identify these RASs in the NS5B gene in naive and treated Pakistani HCV 3a isolates against SOF. METHODS AND RESULTS: Blood samples were collected from anti-HCV-positive patients, followed by HCV RNA isolation and real-time PCR quantification. HCV-positive patients were processed for HCV RNA genotyping, patients with genotype 3a were processed for NS5B gene amplification and sequencing. GT-3a was the most prevalent genotype (62.2%). S282T was identified in 2 (8.7%) patients, C316Y/G/R in 3 (13%), V321A and L320P in 1 (4.3%) each in SOF/RBV-resistant patients. Variants of S282 were detected in 3 (13%) of SOF/RBV-treated patients. While INF/RBV-associated mutations were also analysed, D244N, A333R and A334E were identified in 2 (9.5%), 3 (14.2%) and 7 (33.3%) in treatment-naive and 15 (65.2%), 7 (30.4%) and 5 (21.7%) treated patients, respectively. Q309R was observed only in one treatment-experienced patients. Some substitutions were present at higher frequency in both groups like N307G, K304R, A272D and R345H, considered that they do not have any role in sofosbuvir resistance. CONCLUSION: It was concluded that sofosbuvir RASs are present in Pakistani HCV GT-3a isolates, and they should be monitored carefully, especially in treatment-experienced patients, for further selection of treatment regimens. SIGNIFICANCE AND IMPACT OF STUDY: HCV RASs have been studied very well across the world but there is scarcity of data regarding this topic in Pakistani population, this study provides data regarding the prevalence of these RASs in Pakistani HCV isolates emphasizing the fact that these RASs must be carefully monitored before starting HCV treatment, especially in treatment failure patients.

First report on molecular docking analysis and drug resistance substitutions to approved HCV NS5A and NS5B inhibitors amongst Iranian patients.

BACKGROUND: NS5A and NS5B proteins of hepatitis C virus (HCV) are the main targets of compounds that directly inhibit HCV infections. However, the emergence of resistance-associated substitutions (RASs) may cause substantial reductions in susceptibility to inhibitors. METHODS: Viral load and genotyping were determined in eighty-seven naïve HCV-infected patients, and the amplified NS5A and NS5B regions were sequenced by Sanger sequencing. In addition, physicochemical properties, structural features, immune epitopes, and inhibitors-protein interactions of sequences were analyzed using several bioinformatics tools. RESULTS: Several amino acid residue changes were found in NS5A and NS5B proteins; however, we did not find any mutations related to resistance to the treatment in NS5B. Different phosphorylation and few glycosylation sites were assessed. Disulfide bonds were identified in both proteins that had a significant effect on the function and structure of HCV proteins. Applying reliable software to predict B-cell epitopes, 3 and 5 regions were found for NS5A and NS5B, respectively, representing a considerable potential to induce the humoral immune system. Docking analysis determined amino acids involved in the interaction of inhibitors and mentioned proteins may not decrease the drug efficiency. CONCLUSIONS: Strong interactions between inhibitors, NS5A and NS5B proteins and the lack of efficient drug resistance mutations in the analyzed sequences may confirm the remarkable ability of NS5A and NS5B inhibitors to control HCV infection amongst Iranian patients. The results of bioinformatics analysis could unveil all features of both proteins, which can be beneficial for further investigations on HCV drug resistance and designing novel vaccines.

Low level of baseline resistance in recently HCV-infected men who have sex with men with high-risk behaviours.

OBJECTIVES: Presence of baseline hepatitis C virus (HCV) resistance-associated substitutions (RASs) can impair treatment outcome of direct-acting antivirals. We investigated the prevalence of pre-treatment HCV resistance among recently HCV-infected men who have sex with men (MSM) with high risk behaviours, either human immunodeficiency virus (HIV) co-infected or at high risk of HIV acquisition and under pre-exposure prophylaxis (PrEP). METHODS: NS5A and NS3 fragments were deep sequenced on pre-treatment samples of 72 subjects using Illumina MiSeq paired-end sequencing technology. Ultra-deep sequencing data were analysed by SmartGene® platform. RASs mentioned in the literature were analysed and interpreted depending on genotype (GT) at 10% cut-off. RESULTS: HCV genotyping showed 36 (50.0%) GT1a, 31 (43.1%) GT4d and 5 (6.9%) GT3a infections. Fifty-five patients (76.4%) were co-infected with HIV and 15 (20.8%) received PrEP. In GT1a viruses, NS3 RASs were found in 4/30 viruses (13.3%; S122 G/N, R155 K and I170 V) and Q80 K polymorphism was present in 14/30 viruses (46.7%). No NS3 RASs were detected in GT4d and GT3a viruses. NS5A RASs were detected in 3/36 GT1a viruses (8.3%; Q30E/R, L31 M and H58 L). NS5A subtype-specific polymorphisms L30R and T58 P were found at high frequencies in 31/31 (100%) and 16/31 (51.6%) GT4d viruses, respectively. One RAS M31 L was also observed along with the polymorphisms L30R and T58 P. No NS5A RASs were detected in GT3a viruses. CONCLUSION: A low level of RASs to NS3 and NS5A inhibitors in pre-treatment samples was detected in the study population. Our findings reassure the clinical management of HCV infection in this high-risk population.

[Treatment of chronic viral hepatitis C with direct acting antiviral agent: review].

HCV infection treatment regimens are viewed from positions of HCV life cycle and replication, effects of NS3/4A protease inhibitors and NS5A/NS5B inhibitors on HCV strain replication. Evolution of HCV treatment regimens from its discovery to modern DAA agents had led to substantial progress although drug resistance poses a new issue to be addressed.

Dengue virus non-structural protein 1: a pathogenic factor, therapeutic target, and vaccine candidate.

Dengue virus (DENV) infection is the most common mosquito-transmitted viral infection. DENV infection can cause mild dengue fever or severe dengue hemorrhagic fever (DHF)/dengue shock syndrome (DSS). Hemorrhage and vascular leakage are two characteristic symptoms of DHF/DSS. However, due to the limited understanding of dengue pathogenesis, no satisfactory therapies to treat nor vaccine to prevent dengue infection are available, and the mortality of DHF/DSS is still high. DENV nonstructural protein 1 (NS1), which can be secreted in patients' sera, has been used as an early diagnostic marker for dengue infection for many years. However, the roles of NS1 in dengue-induced vascular leakage were described only recently. In this article, the pathogenic roles of DENV NS1 in hemorrhage and vascular leakage are reviewed, and the possibility of using NS1 as a therapeutic target and vaccine candidate is discussed.

Asunaprevir: A Review of Preclinical and Clinical Pharmacokinetics and Drug-Drug Interactions.

Asunaprevir is a tripeptidic acylsulfonamide inhibitor of the hepatitis C virus (HCV) NS3/4A protease. Asunaprevir undergoes rapid absorption, with a time to reach maximum plasma concentration (T max) of 2-4 h and an elimination half-life (t ½) of ≈15-20 h observed in single-ascending dose studies. Steady state was achieved by day 7 in multiple-ascending dose studies. The large food effect observed with earlier formulations was mitigated by the soft-gel capsule. Asunaprevir demonstrates high apparent oral clearance and minimal renal elimination, and is eliminated primarily via cytochrome P450 (CYP) 3A4-mediated hepatic oxidative metabolism and faecal excretion. Highly preferential distribution of asunaprevir to the liver occurs via organic anion-transporting polypeptide (OATP)-mediated transport and results in low plasma concentrations. The condition of the liver affects disposition, as higher asunaprevir plasma exposures are observed in patients infected with HCV and/or hepatic impairment. Japanese patients also have higher exposure relative to North American/European patients, but comparable safety at the registrational dose. Asunaprevir has a low potential to perpetrate drug-drug interactions via CYP3A4, P-glycoprotein and OATP, but is a moderate CYP2D6 inhibitor; concomitant drugs that are substrates of CYP2D6 or P-glycoprotein and have a narrow therapeutic index should be used with care. Asunaprevir plasma exposure is strongly affected by inhibitors of OATP transport. No clinically significant interactions were observed between asunaprevir and daclatasvir or daclatasvir/beclabuvir. Asunaprevir has a complex pharmacokinetic profile and forms part of potent and well-tolerated all-oral regimens for the treatment of chronic HCV infection.

Characterization and evaluation of apoptotic potential of double gene construct pVIVO.VP3.NS1.

Viral gene oncotherapy, targeted killing of cancer cells by viral genes, is an emerging non-infectious therapeutic cancer treatment modality. Chemo and radiotherapy in cancer treatment is limited due to their genotoxic side effects on healthy cells and need of functional p53, which is mutated in most of the cancers. VP3 (apoptin) of chicken infectious anaemia (CIA) and NS1 (Non structural protein 1) of Canine Parvovirus-2 (CPV-2) have been proven to have oncolytic potential in our laboratory. To evaluate oncolytic potential of VP3 and NS1 together these genes needed to be cloned in a bicistronic vector. In this study, both these genes were cloned and characterized for expression of their gene products and its apoptotic potential. The expression of VP3 and NS1 was studied by confocal microscopy and flowcytometry. Expression of VP3 and NS1 in pVIVO.VP3.NS1 transfected HeLa cells in comparison to mock transfected cells indicated that the double gene construct expresses both the products. This was further confirmed by flowcytometry where there was increase in cells expressing VP3 and NS1 in pVIVO.VP3.NS1 transfected group in comparison with the mock control group. The apoptotic inducing potential of this characterized pVIVO.VP3.NS1 was evaluated in human cervical cancer cell line (HeLa) by DNA fragmentation assay, TUNEL assay and Hoechst staning. This double construct was observed to induce apoptosis in HeLa cells.

Alphavirus-based vaccines encoding nonstructural proteins of hepatitis C virus induce robust and protective T-cell responses.

An absolute prerequisite for a therapeutic vaccine against hepatitis C virus (HCV) infection is the potency to induce HCV-specific vigorous and broad-spectrum T-cell responses. Here, we generated three HCV vaccines based on a recombinant Semliki Forest virus (rSFV) vector expressing all- or a part of the conserved nonstructural proteins (nsPs) of HCV. We demonstrated that an rSFV vector was able to encode a transgene as large as 6.1 kb without affecting its vaccine immunogenicity. Prime-boost immunizations of mice with rSFV expressing all nsPs induced strong and long-lasting NS3-specific CD8(+) T-cell responses. The strength and functional heterogeneity of the T-cell response was similar to that induced with rSFV expressing only NS3/4A. Furthermore this leads to a significant growth delay and negative selection of HCV-expressing EL4 tumors in an in vivo mouse model. In general, as broad-spectrum T-cell responses are only seen in patients with resolved HCV infection, this rSFV-based vector, which expresses all nsPs, inducing robust T-cell activity has a potential for the treatment of HCV infections.

Hepatitis C therapy: highlights from the 2012 annual meeting of the European Association for the Study of the Liver.

The results from clinical trials testing new direct-acting antivirals (DAAs) for chronic hepatitis C were the major focus of interest at the 2012 annual meeting of the European Association for the Study of the Liver. Besides triple combinations, in which any one of the new DAAs is given along with peginterferon-α/ribavirin, clinical trials exploring interferon-free oral regimens combining several DAAs attracted major attention. The good tolerance, broad hepatitis C virus (HCV) genotype activity, and high resistance barrier of sofosbuvir make this nucleotide analogue one of the most promising DAAs. Among HCV protease inhibitors, the safety, potency, and convenient dosing of simeprevir, asunaprevir, faldaprevir, and ABT-450/r were particularly highlighted. Among NS5A inhibitors, the good performance of daclatasvir encourages further clinical development. Finally, intriguing results were released about the role of interleukin 28B (IL-28B) polymorphisms using interferon-free regimens, indirectly supporting the role of innate immunity for clearing HCV definitively.

[Oncolytic parvoviruses. A new approaches for cancer therapy].

Parvoviruses such as parvovirus H-1 (H-1PV) may selectively infect and lysis cancer cells. The parvoviruses also induce an immune system to eliminate the tumor cells through the formation of anti-cancer immunity. One of the possible mechanisms of antitumor activity is associated with the direct induction of apoptosis by parvoviral proteins NS1 and 11 kDa. Parvovirus-based vectors are promising for gene therapy of oncological diseases and genetic disorders in humans. Parvoviruses were successfully used for the experimental treatment on animal models of human glioma, neuroblastomas, lymphomas, pancreatic carcinoma, carcinomas and breast tumors. ParvOryx is the first oncolytic preparation constructed on the base of H-1PV; its phase I/IIa clinical trials in patients with glioblastoma multiforme are in process.

In vitro analysis of virus particle subpopulations in candidate live-attenuated influenza vaccines distinguishes effective from ineffective vaccines.

Two effective (vac+) and two ineffective (vac-) candidate live-attenuated influenza vaccines (LAIVs) derived from naturally selected genetically stable variants of A/TK/OR/71-delNS1[1-124] (H7N3) that differed only in the length and kind of amino acid residues at the C terminus of the nonstructural NS1 protein were analyzed for their content of particle subpopulations. These subpopulations included total physical particles (measured as hemagglutinating particles [HAPs]) with their subsumed biologically active particles of infectious virus (plaque-forming particles [PFPs]) and different classes of noninfectious virus, namely, interferon-inducing particles (IFPs), noninfectious cell-killing particles (niCKPs), and defective interfering particles (DIPs). The vac+ variants were distinguished from the vac- variants on the basis of their content of viral subpopulations by (i) the capacity to induce higher quantum yields of interferon (IFN), (ii) the generation of an unusual type of IFN-induction dose-response curve, (iii) the presence of IFPs that induce IFN more efficiently, (iv) reduced sensitivity to IFN action, and (v) elevated rates of PFP replication that resulted in larger plaques and higher PFP and HAP titers. These in vitro analyses provide a benchmark for the screening of candidate LAIVs and their potential as effective vaccines. Vaccine design may be improved by enhancement of attributes that are dominant in the effective (vac+) vaccines.

Immunization against hepatitis C virus with a fusion protein containing the extra domain A from fibronectin and the hepatitis C virus NS3 protein.

BACKGROUND/AIMS: Vaccination strategies able to induce strong T-cell responses might contribute to eradicate hepatitis C virus (HCV) infection. We previously demonstrated that fusion of an antigen to the extra domain A from fibronectin (EDA) targets the antigen to TLR4-expressing dendritic cells (DC) and improves its immunogenicity. Here, we studied if fusion of EDA with the non-structural HCV protein NS3 might constitute an effective immunogen against HCV. METHODS: Recombinant NS3 and the fusion protein EDA-NS3 were produced and purified from E. coli, and tested in vitro for their capacity to activate maturation of DC and to favour antigen presentation. HHD transgenic mice expressing the human HLA-A2 molecule were immunized with recombinant proteins in the absence or presence of poly(I:C) and anti-CD40 agonistic antibodies and responses elicited by vaccination were tested in vitro, and in vivo, by their capacity to downregulate intrahepatic expression of HCV-NS3 RNA. RESULTS: EDA-NS3, but not NS3 alone, upregulated the expression of maturation markers, as well as Delta-like 1 and Delta-like 4 Notch ligands in DC and induced the production of IL-12. Mice immunized with EDA-NS3 had strong and long lasting NS3-specific CD4+ and CD8+ T-cell responses and, in combination with poly(I:C) and anti-CD40, downregulated intrahepatic expression of HCV-NS3 RNA. CONCLUSIONS: Recombinant EDA-NS3 may be considered for the development of vaccines against HCV infection.

Hepatitis C virus NS5A anchor peptide disrupts human immunodeficiency virus.

In the absence of an effective vaccine, there is an urgent need for safe and effective antiviral agents to prevent transmission of HIV. Here, we report that an amphipathic alpha-helical peptide derived from the hepatitis C virus NS5A anchor domain (designated C5A in this article) that has been shown to be virocidal for the hepatitis C virus (HCV) also has potent antiviral activity against HIV. C5A exhibits a broad range of antiviral activity against HIV isolates, and it prevents infection of the three in vivo targets of HIV: CD4(+) T lymphocytes, macrophages, and dendritic cells by disrupting the integrity of the viral membrane and capsid core while preserving the integrity of host membranes. C5A can interrupt an ongoing T cell infection, and it can prevent transmigration of HIV through primary genital epithelial cells, infection of mucosal target cells and transfer from dendritic cells to T cells ex vivo, justifying future experiments to determine whether C5A can prevent HIV transmission in vivo.

[Synthetic fragments of the NS1 protein of the tick-borne encephalitis virus exhibiting a protective effect].

Potentially immunoactive regions of the NS1 nonstructural protein of the tick-borne encephalitis virus that can stimulate the antibody formation in vivo and protect animals from this disease were chosen on the basis of theoretical calculations. Eleven 16- to 27-aa peptides containing the chosen regions were synthesized. The ability of the free peptides (without any high-molecular-mass carrier) to stimulate the production of antipeptide antibodies in mice of three lines and ensure the formation of protective immunity was studied. Most of these peptides were shown to exhibit the immunogenic activity in a free state. Five fragments that can protect mice from the infection by a lethal dose of tick-borne encephalitis virus were found.

Evaluation of serum antibody responses against the rotavirus nonstructural protein NSP4 in children after rhesus rotavirus tetravalent vaccination or natural infection.

The immune response elicited by the rotavirus nonstructural protein NSP4 and its potential role in protection against rotavirus disease are not well understood. We investigated the serological response to NSP4 and its correlation with disease protection in sera from 110 children suffering acute diarrhea, associated or not with rotavirus, and from 26 children who were recipients of the rhesus rotavirus tetravalent (RRV-TV) vaccine. We used, as antigens in an enzyme-linked immunosorbent assay (ELISA), affinity-purified recombinant NSP4 (residues 85 to 175) from strains SA11, Wa, and RRV (genotypes A, B, and C, respectively) fused to glutathione S-transferase. Seroconversion to NSP4 was observed in 54% (42/78) of the children who suffered from natural rotavirus infection and in 8% (2/26) of the RRV-TV vaccine recipients. Our findings indicate that NSP4 evokes significantly (P < 0.05) higher seroconversion rates after natural infection than after RRV-TV vaccination. The serum antibody levels to NSP4 were modest (titers of < or = 200) in most of the infected and vaccinated children. A heterotypic NSP4 response was detected in 48% of the naturally rotavirus-infected children with a detectable response to NSP4. Following natural infection or RRV-TV vaccination, NSP4 was significantly less immunogenic than the VP6 protein when these responses were independently measured by ELISA. A significant (P < 0.05) proportion of children who did not develop diarrhea associated with rotavirus had antibodies to NSP4 in acute-phase serum, suggesting that serum antibodies against NSP4 might correlate with protection from rotavirus diarrhea. In addition, previous exposures to rotavirus did not affect the NSP4 seroconversion rate.

Immunization with nonstructural proteins E1 and E2 of cottontail rabbit papillomavirus stimulates regression of virus-induced papillomas.

Cottontail rabbit papillomavirus is the major animal model for cancer-associated papillomaviruses. Here we show that vaccination with the nonstructural proteins E1 and E2 induces the regression of virus-induced papillomas and that vaccination is equally effective when proteins are given with and without adjuvant. There was no correlation between antibody levels and regression, suggesting that tumor regression may be due to a cell-mediated response.