Nonstructural Protein 5B Research Articles

In silico targeting of non-structural 4B protein from dengue virus 4 with spiropyrazolopyridone: study of molecular dynamics simulation, ADMET and virtual screening.

Dengue fever is one of the most prevalent disease in tropical and sub-tropical regions of the world. According to the World Health Organisation (WHO), approximately 3.5billion people have been affected with dengue fever. Four serotypes of dengue virus (DENV) i.e. DENV1, DENV2, DENV3 and DENV4 have up to 65% genetic variations among themselves. dengue virus 4 (DENV4) was first reported from Amazonas, Brazil and is spreading perilously due to lack of awareness of preventive measures, as it is the least targeted serotype. In this study, non-structural protein 4B of dengue virus 4 (DENV4-NS4B) is computationally characterised and simulations are performed including solvation, energy minimizations and neutralisation for the refinement of predicted model of the protein. The spiropyrazolopyridone is considered as an effective drug against NS4B of DENV2, therefore, a total of 91 different analogues of spiropyrazolopyridone are used to analyse their inhibitory action against DENV4-NS4B. These compounds are docked at the binding site with various binding affinities, representing their efficacy to block the binding pocket of the protein. Pharmacological and pharmacokinetic assessment performed on these inhibitors shows that these are suitable candidates to be used as a drug against the dengue fever. Among all these 91 compounds, Analogue-I and Analogue-II are analysed to be the most effective inhibitor having potential to be used as drugs against dengue virus.

Proteome analysis of differential protein expression in porcine alveolar macrophages regulated by porcine reproductive and respiratory syndrome virus nsp1β protein.

Porcine reproductive and respiratory syndrome virus (PRRSV), an acute infectious disease agent in swine, causes enormous economic losses to the global swine industry. PRRSV nonstructural protein 1β (nsp1β) plays a critical role in viral subgenomic mRNA synthesis and host immune regulation. However, the global changes of cellular gene expression in natural target cells regulated by the nsp1β have not yet been identified. Here, isobaric tags for relative and absolute quantification (iTRAQ) labeling coupled with liquid chromatography-tandem mass spectrometry were used to quantitatively identify cellular proteins in porcine alveolar macrophage (PAM) 3D4/21 cells transduced with recombinant lentivirus expressing PRRSV nsp1β that are differentially expressed compared with PAM 3D4/21 cells transduced with recombinant lentivirus expressing GFP. Of the 425 cellular proteins detected as differentially expressed, 186 were upregulated and 239 were downregulated. Based on the identities of the differentially expressed cellular proteins and the essential role of nsp1β in interferon (IFN) activation and inflammatory factor antagonism during PRRSV infection, we propose a potential mechanism in which nsp1β inhibits IFN induction and nuclear factor κB (NF-κB) signaling pathways. Our results suggest that mitochondrial antiviral signaling (MAVS) protein and translocases of outer membrane complex 70 (TOM70), involved in type I IFN induction, were downregulated, while protein phosphatase 1A (PPM1A), related to the inhibition of NF-κB pathway activation, was upregulated in nsp1β-overexpressed PAM 3D4/21 cells. These data provide valuable information for better understanding the potential biological function of nsp1β during PRRSV infection and the mechanism of virus escape from host immune surveillance of viral replication.

COMBINATORIAL PHARMACOPHORE MODELING AND ATOM BASED 3D QSAR STUDIES OF BENZOTHIADIAZINES AS HCV-NS5B INHIBITORS

Objective: The objective of the current study was to elucidate the 3D pharmacophoric features of benzothiadiazine derivatives that are crucial for inhibiting Hepatitis C virus (HCV) Non-structural protein 5B (NS5B) and quantifying the features by building an atom based 3D quantitative structure-activity relationship (3D QSAR) model.Methods: Generation of QSAR model was carried out using PHASE 3.3.Results: A five-point pharmacophore model with two hydrogen bond acceptors, one negative ionization potential and two aromatic rings (AANRR) was found to be common among a maximum number of benzothiadiazine based NS5B inhibitors. A statistically significant 3D QSAR model was obtained from AANRR.6 which had correlation-coefficient (R2) value of 0.924, cross-validated correlation-coefficient (Q2) of 0.774, high Fisher ratio of 138 and low root mean square standard error (RMSE=0.29). There is another parameter, Pearson’s R, its value emphasizes correlation between predicted and observed activities of the test set. For the current model, Pearson’s R-value is 0.90, hence underlining the good quality of the model. The present study suggests that nitrogen atom of benzothiadiazine sulfamide ring, oxyacetamide group attached to C7 carbon of benzothiadiazine and sulfonamide oxygens are crucial for NS5B inhibitory activity. Prediction of activities of hit drugs generated in earlier research suggests that Aprepitant (Phase predicted activity: 6.9) could be a potential NS5B inhibitor.Conclusion: This 3D QSAR model developed was statistically good and can be used to predict the activities of newly designed NS5B inhibitors and virtual screening as well. Predict the activities of newly designed NS5B inhibitors and virtual screening as well.

A Fluorescent Cell-Based System for Imaging Zika Virus Infection in Real-Time.

Zika virus (ZIKV) is a re-emerging flavivirus that is transmitted to humans through the bite of an infected mosquito or through sexual contact with an infected partner. ZIKV infection during pregnancy has been associated with numerous fetal abnormalities, including prenatal lethality and microcephaly. However, until recent outbreaks in the Americas, ZIKV has been relatively understudied, and therefore the biology and pathogenesis of ZIKV infection remain incompletely understood. Better methods to study ZIKV infection in live cells could enhance our understanding of the biology of ZIKV and the mechanisms by which ZIKV contributes to fetal abnormalities. To this end, we developed a fluorescent cell-based reporter system allowing for live imaging of ZIKV-infected cells. This system utilizes the protease activity of the ZIKV non-structural proteins 2B and 3 (NS2B-NS3) to specifically mark virus-infected cells. Here, we demonstrate the utility of this fluorescent reporter for identifying cells infected by ZIKV strains of two lineages. Further, we use this system to determine that apoptosis is induced in cells directly infected with ZIKV in a cell-autonomous manner. Ultimately, approaches that can directly track ZIKV-infected cells at the single cell-level have the potential to yield new insights into the host-pathogen interactions that regulate ZIKV infection and pathogenesis.

Quantum molecular modeling of hepatitis C virus inhibition through non-structural protein 5B polymerase receptor binding of C5-arylidene rhodanines

We have carried out high-level quantum chemical computations followed by molecular docking studies on a set of 17C5-arylidene rhodanine isomers to provide insights into the binding modes with different reported binding pockets of the nonstructural protein 5B (NS5B) polymerase that contribute to the hepatitis C virus (HCV) inhibition. We optimized the multi-target profile of the selected rhodanine analogs to investigate potential non-nucleotide inhibitors (NNIs) by quantum chemical optimization of the 18 isomers followed by docking with quantum chemically optimized structures of each isomer with NS5B polymerase at multiple binding pockets. The binding affinities of the PP-I, PP-II and TP-II pockets of NS5B polymerase were analyzed for all the 17 isomers of 2-[(5Z)-5-(2,4-dichlorobenzylidene)-4-oxo-2-thioxo-1,3-thiazolidin-3-yl]-3-phenylpropanoic acid. On the basis of binding propensity at the different pockets and inhibitor constants, we ranked these isomers as potential candidates for the HCV inhibition. We have identified four isomers as promising NNIs of NS5B polymerase with comparable binding and inhibition to the standard (1,3) dichloro substituted isomer that exhibits in vitro activity and several other isomers as candidates in a “multi-targeted drug” approach.

The global origins of resistance-associated variants in the non-structural proteins 5A and 5B of the hepatitis C virus.

New, costly, fast acting, therapies targeting the non-structural proteins 5A and 5B (NS5A and NS5B) regions of the hepatitis C virus (HCV) genome are curative in the majority of cases. Variants with certain mutations in the NS5A and NS5B regions of HCV have been shown to reduce susceptibility to direct-acting NS5A and NS5B therapy and are found in treatment naïve patients. Despite this, the ease with which these variants evolve is poorly known, as are their evolutionary and geographic origins. To address this crucial gap we inferred the evolutionary and geographic origins of resistance-associated variants (RAVs) in the HCV NS5A and NS5B regions of subtypes 1a, 1b, and 3a sequences available from global databases. We found that RAVs in the NS5A region of HCV, when prevalent, were widely dispersed throughout the phylogenetic tree of HCV with multiple independent origins and that these variants are globally distributed. In contrast, most of the NS5B C316N variants came from one of two clades in the phylogenetic tree of HCV subtype 1b. The presence of serine (S) at codon 218 of HCV NS5B appears to facilitate the evolution of the C316N RAV. Other NS5B RAVs did not arise very frequently in our data set, except for S556G in subtype 1b and with respect to geography NS5B RAVs were also globally distributed. The inferred distribution of RAVs in the NS5A region and frequency of their origin suggest a low fitness barrier without the need for co-evolution of compensatory mutations. A low fitness barrier may allow rapid selection of de novo resistance to NS5A inhibitors during therapy.

Bioactivation of cyclopropyl rings by P450: an observation encountered during the optimisation of a series of hepatitis C virus NS5B inhibitors

1. Due to its unique C–C and C–H bonding properties, conformational preferences and relative hydrophilicity, the cyclopropyl ring has been used as a synthetic building block in drug discovery to modulate potency and drug-like properties. During an effort to discover inhibitors of the hepatitis C virus non-structural protein 5B with improved potency and genotype-coverage profiles, the use of a pyrimidinylcyclopropylbenzamide moiety linked to a C6-substituted benzofuran or azabenzofuran core scaffold was explored in an effort to balance antiviral potency and metabolic stability.2. In vitro metabolism studies of two compounds from this C6-substituted series revealed an NADPH-dependent bioactivation pathway leading to the formation of multiple glutathione (GSH) conjugates. Analysis of these conjugates by LC-MS and NMR demonstrated that the cyclopropyl group was the site of bioactivation. Based on the putative structures and molecular weights of the cyclopropyl-GSH conjugates, a multi-step mechanism was proposed to explain the formation of these metabolites by P450. This mechanism involves hydrogen atom abstraction to form a cyclopropyl radical, followed by a ring opening rearrangement and reaction with GSH.3. These findings provided important information to the medicinal chemistry team which responded by replacing the cyclopropyl ring with a gem-dimethyl group. Subsequent compounds bearing this feature were shown to avert the bioactivation pathways in question.

Resistance analysis in patients with genotype 1–6 HCV infection treated with sofosbuvir/velpatasvir in the phase III studies

The fixed-dose combination of sofosbuvir/velpatasvir was highly efficacious in patients infected with genotype (GT)1-6 hepatitis C virus (HCV) in the ASTRAL studies. This analysis evaluated the impact of baseline resistance-associated substitutions (RASs) on treatment outcome and emergence of RASs in patients infected with HCV GT1-6 who were treated with sofosbuvir/velpatasvir. Non-structural protein 5A and 5B (NS5A and NS5B) deep sequencing was performed at baseline and at the time of relapse for all patients treated with sofosbuvir/velpatasvir for 12 weeks (n = 1,778) in the ASTRAL-1-3, ASTRAL-5 and POLARIS-2-3 studies. Patients with 37 known and 19 novel HCV subtypes were included in these analyses. Overall, 28% (range 9% to 61% depending on genotype) had detectable NS5A class RASs at baseline, using a 15% sequencing assay cut-off. There was no significant effect of baseline NS5A class RASs on sustained virologic response at week 12 (SVR12) with sofosbuvir/velpatasvir; the SVR12 rate in the presence of NS5A class RASs was 100% and 97%, in patients with GT1a and GT1b infection, respectively, and 100% in patients with GT2 and GT4-6 infections. In GT3 infection, the SVR rate was 93% and 98% in patients with and without baseline NS5A class RASs, respectively. The overall virologic failure rate was low (20/1,778 = 1.1%) in patients treated with sofosbuvir/velpatasvir. Single NS5A class resistance was observed at virologic failure in 17 of the 20 patients. Sofosbuvir/velpatasvir taken for 12 weeks once daily resulted in high SVR rates in patients infected with GT1-6 HCV, irrespective of baseline NS5A RASs. NS5A inhibitor resistance, but not sofosbuvir resistance, was detected in the few patients with virologic failure. These data highlight the high barrier to resistance of this regimen for the treatment of chronic HCV across all genotypes in the vast majority of patients. Sofosbuvir/velpatasvir taken once daily for 12 weeks resulted in high sustained virologic response rates in patients infected with HCV, irrespective of the presence of NS5A resistance-associated variants prior to treatment. Single class NS5A inhibitor resistance, but not sofosbuvir resistance, was detected in the few patients with virologic failure. These data highlight the high barrier to resistance of this regimen for the treatment of chronic HCV across all genotypes in the vast majority of patients.

Glycine Zipper Motifs in Hepatitis C Virus Nonstructural Protein 4B Are Required for the Establishment of Viral Replication Organelles.

Hepatitis C virus (HCV) RNA replication occurs in tight association with remodeled host cell membranes, presenting as cytoplasmic accumulations of single-, double-, and multimembrane vesicles in infected cells. Formation of these so-called replication organelles is mediated by a complex interplay of host cell factors and viral replicase proteins. Of these, nonstructural protein 4B (NS4B), an integral transmembrane protein, appears to play a key role, but little is known about the molecular mechanisms of how this protein contributes to organelle biogenesis. Using forward and reverse genetics, we identified glycine zipper motifs within transmembrane helices 2 and 3 of NS4B that are critically involved in viral RNA replication. Foerster resonance energy transfer analysis revealed the importance of the glycine zippers in NS4B homo- and heterotypic self-interactions. Additionally, ultrastructural analysis using electron microscopy unraveled a prominent role of glycine zipper residues for the subcellular distribution and the morphology of HCV-induced double-membrane vesicles. Notably, loss-of-function NS4B glycine zipper mutants prominently induced single-membrane vesicles with secondary invaginations that might represent an arrested intermediate state in double-membrane vesicle formation. These findings highlight a so-far-unknown role of glycine residues within the membrane integral core domain for NS4B self-interaction and functional as well as structural integrity of HCV replication organelles.IMPORTANCE Remodeling of the cellular endomembrane system leading to the establishment of replication organelles is a hallmark of positive-strand RNA viruses. In the case of HCV, expression of the nonstructural proteins induces the accumulation of double-membrane vesicles that likely arise from a concerted action of viral and coopted cellular factors. However, the underlying molecular mechanisms are incompletely understood. Here, we identify glycine zipper motifs within HCV NS4B transmembrane segments 2 and 3 that are crucial for the protein's self-interaction. Moreover, glycine residues within NS4B transmembrane helices critically contribute to the biogenesis of functional replication organelles and, thus, efficient viral RNA replication. These results reveal how glycine zipper motifs in NS4B contribute to structural and functional integrity of the HCV replication organelles and, thus, viral RNA replication.

Prevalence of NS5B resistance-associated variants in treatment-naïve Asian patients with chronic hepatitis C

There is little information on the association between baseline non-structural protein (NS) 5b resistance-associated variants (RAVs) and treatment failure in hepatitis C patients. This study examined the frequencies of natural hepatitis C virus (HCV) NS5B resistance-associated variants (RAVs) in an Asian cohort. Samples from Asian HCV patients enrolled between October 2009 and September 2014 were analyzed for NS5B RAVs within the region from amino acid 230 to 371. Serum samples were tested by PCR genotyping, with sequence alignment performed using the neighbor-joining method. NS5B was detected by Sanger sequencing followed by Geno2pheno analysis. NS5B RAVs were detected in 80.52% (1199/1489) of patients; 68.4% (1019/1489) and 79.7% (1186/1489) were associated with resistance to sofosbuvir (SOF) and dasabuvir (DSV), respectively. These RAVs were present in 95% (1004/1058) of genotype 1b patients. When genotypes 1b and 2a were compared, SOF-associated RAVs were detected at a higher frequency in genotype 1b (94.8% [1004/1058] vs. 2.9% [9/309]; χ2 = 1054.433, P < 0.001), C316H/N was more common in genotype 1b (94.7% [1002/1058] vs. 0% [0/309]; χ2 = 1096.014, P < 0.001), M289F/L/I/W/V had a higher frequency in genotype 2a (0.7% [7/309] vs. 2.3% [7/1058]; χ2 = 4.589, P = 0.032), DSV-associated RAVs were most often found in genotype 1b (95.0% [1005/1058] vs. 40.1% 124/309]; χ2 = 500.577, P < 0.001), and frequency of C316Y/H/N/W was higher in genotype 1b (94.7% [1002/1058] vs. 0% [0/309]; χ2 = 1096.014, P < 0.001). In conclusion, baseline SOF and DSV RAVs are common in Asian HCV patients and predominantly occur in genotype 1b.

Identification and Analysis of Novel Inhibitors against NS3 Helicase and NS5B RNA-Dependent RNA Polymerase from Hepatitis C Virus 1b (Con1).

Hepatitis C virus (HCV) leads to severe liver diseases, including liver fibrosis, cirrhosis and hepatocellular carcinoma. Non-structural protein 3 helicase (NS3h) and non-structural protein 5B RNA-dependent RNA polymerase (NS5B) are involved in the replication of HCV RNA genome, and have been proved to be excellent targets for discovery of direct-acting antivirals. In this study, two high-throughput screening systems, fluorescence polarization (FP)-based ssDNA binding assay and fluorescence intensity (FI)-based dsRNA formation assay, were constructed to identify candidate NS3h and NS5B inhibitors, respectively. A library of approximately 800 small molecules and crude extracts, derived from marine microorganisms or purchased from the National Compound Resource Center, China, were screened, with three hits selected for further study. Natural compound No.3A5, isolated from marine fungi, inhibited NS3h activity with an IC50 value of 2.8 μM. We further demonstrated that compound No.3A5 inhibited the abilities of NS3h to bind ssDNA in electrophoretic mobility shift assay and to hydrolyze ATP. The NS3h-inhibitory activity of compound No.3A5 was reversible in our dilution assay, which indicated there was no stable NS3h-No.3A5 complex formed. Additionally, compound No.3A5 exhibited no binding selectivity on NS3h or single strand binding protein of Escherichia coli. In NS5B assays, commercial compounds No.39 and No.94 previously reported as kinase inhibitors were found to disrupt dsRNA formation, and their IC50 values were 62.9 and 18.8 μM, respectively. These results highlight how identifying new uses for existing drugs is an effective method for discovering novel HCV inhibitors. To our knowledge, all inhibitors reported in this study were originally discovered with HCV anti-non-structural protein activities in vitro.

NMR reveals the intrinsically disordered domain 2 of NS5A protein as an allosteric regulator of the hepatitis C virus RNA polymerase NS5B

Non-structural protein 5B (NS5B) is the RNA-dependent RNA polymerase that catalyzes replication of the hepatitis C virus (HCV) RNA genome and therefore is central for its life cycle. NS5B interacts with the intrinsically disordered domain 2 of NS5A (NS5A-D2), another essential multifunctional HCV protein that is required for RNA replication. As a result, these two proteins represent important targets for anti-HCV chemotherapies. Despite this importance and the existence of NS5B crystal structures, our understanding of the conformational and dynamic behavior of NS5B in solution and its relationship with NS5A-D2 remains incomplete. To address these points, we report the first detailed NMR spectroscopic study of HCV NS5B lacking its membrane anchor (NS5BΔ21). Analysis of constructs with selective isotope labeling of the δ1 methyl groups of isoleucine side chains demonstrates that, in solution, NS5BΔ21 is highly dynamic but predominantly adopts a closed conformation. The addition of NS5A-D2 leads to spectral changes indicative of binding to both allosteric thumb sites I and II of NS5BΔ21 and induces long-range perturbations that affect the RNA-binding properties of the polymerase. We compared these modifications with the short- and long-range effects triggered in NS5BΔ21 upon binding of filibuvir, an allosteric inhibitor. We demonstrate that filibuvir-bound NS5BΔ21 is strongly impaired in the binding of both NS5A-D2 and RNA. NS5A-D2 induces conformational and functional perturbations in NS5B similar to those triggered by filibuvir. Thus, our work highlights NS5A-D2 as an allosteric regulator of the HCV polymerase and provides new insight into the dynamics of NS5B in solution.

Characterization of a dengue NS4B inhibitor originating from an HCV small molecule library

Dengue is the most important mosquito-transmitted viral disease and a major global health concern. Over the last decade, dengue virus (DENV) drug discovery and development has intensified, however, this has not resulted in approved DENV-specific antiviral treatments yet. DENV and hepatitis C virus (HCV) belong to the same Flaviviridae family and, in contrast to DENV, antiviral treatments for HCV have been licensed. Therefore, applying the knowledge gained on anti-HCV drugs may foster the discovery and development of dengue antiviral drugs. Here, we screened a library of compounds with established anti-HCV activity in a DENV-2 sub-genomic replicon inhibition assay and selected compounds with single-digit micromolar activity. These compounds were advanced into a hit-to-lead medicinal chemistry program resulting in lead compound JNJ-1A, which inhibited the DENV-2 sub-genomic replicon at 0.7 μM, in the absence of cytotoxicity. In addition, JNJ-1A showed equipotent antiviral activity against DENV serotypes 1, 2, and 4. In vitro resistance selection experiments with JNJ-1A induced mutation T108I in non-structural protein 4B (NS4B), pointing towards a mechanism of action linked to this protein. Collectively, we described the discovery and characterization of a novel DENV inhibitor potentially targeting NS4B.

Limited overlap between phylogenetic HIV and hepatitis C virus clusters illustrates the dynamic sexual network structure of Dutch HIV-infected MSM.

MSM are at increased risk for infection with HIV-1 and hepatitis C virus (HCV). Is HIV/HCV coinfection confined to specific HIV transmission networks? A HIV phylogenetic tree was constructed for 5038 HIV-1 subtype B polymerase (pol) sequences obtained from MSM in the AIDS therapy evaluation in the Netherlands cohort. We investigated the existence of HIV clusters with increased HCV prevalence, the HIV phylogenetic density (i.e. the number of potential HIV transmission partners) of HIV/HCV-coinfected MSM compared with HIV-infected MSM without HCV, and the overlap in HIV and HCV phylogenies using HCV nonstructural protein 5B sequences from 183 HIV-infected MSM with acute HCV infection. Five hundred and sixty-three of 5038 (11.2%) HIV-infected MSM tested HCV positive. Phylogenetic analysis revealed 93 large HIV clusters (≥10 MSM), 370 small HIV clusters (2-9 MSM), and 867 singletons with a median HCV prevalence of 11.5, 11.6, and 9.3%, respectively. We identified six large HIV clusters with elevated HCV prevalence (range 23.5-46.2%). Median HIV phylogenetic densities for MSM with HCV (3, interquartile range 1-7) and without HCV (3, interquartile range 1-8) were similar. HCV phylogeny showed 12 MSM-specific HCV clusters (clustersize: 2-39 HCV sequences); 12.7% of HCV infections were part of the same HIV and HCV cluster. We observed few HIV clusters with elevated HCV prevalence, no increase in the HIV phylogenetic density of HIV/HCV-coinfected MSM compared to HIV-infected MSM without HCV, and limited overlap between HIV and HCV phylogenies among HIV/HCV-coinfected MSM. Our data do not support the existence of MSM-specific sexual networks that fuel both the HIV and HCV epidemic.

EEF1G interaction with foot-and-mouth disease virus nonstructural protein 2B: Identification by yeast two-hybrid system

Foot-and-mouth disease virus (FMDV) is a picornavirus that causes an economically significant disease in cattle and swine. Replication of FMDV is dependent on both viral proteins and cellular factors. Nonstructural protein 2B of FMDV plays multiple roles during viral infection and replication. We investigated the roles of 2B in virus–host interactions by constructing a cDNA library obtained from FMDV-infected swine tissues, and used a split-ubiquitin-based yeast two-hybrid system to identify host proteins that interacted with 2B. We found that 2B interacted with amino acids 208–437 in the C-terminal region of the eEF1G subunit of eukaryotic elongation factor 1, which is essential for protein synthesis. The 2B–eEF1G interaction was confirmed by co-immunoprecipitation of 2B and eEF1G in HEK293T cells. Collectively, our results suggest that eEF1G interacts with the 2B protein of FMDV. The identified 2B interaction partner may help to elucidate the mechanisms of FMDV infection and replication.

Molecular vaccine prepared by fusion of XCL1 to the multi-epitope protein of foot-and-mouth disease virus enhances the specific humoural immune response in cattle.

Targeting antigen to dendritic cells (DCs) is a promising way to manipulate the immune response and to design prophylactic molecular vaccines. In this study, the cattle XCL1, ligand of XCR1, was fused to the type O foot-and-mouth disease virus (FMDV) multi-epitope protein (XCL-OB7) to create a molecular vaccine antigen, and an △XCL-OB7 protein with a mutation in XCL1 was used as the control. XCL-OB7 protein specifically bound to the XCR1 receptor, as detected by flow cytometry. Cattle vaccinated with XCL-OB7 showed a significantly higher antibody response than that to the △XCL-OB7 control (P<0.05). In contrast, when XCL-OB7 was incorporated with poly (I:C) to prepare the vaccine, the antibody response of the immunized cattle was significantly decreased in this group and was lower than that in the △XCL-OB7 plus poly (I:C) group. The FMDV challenge indicated that cattle immunized with the XCL-OB7 alone or the △XCL-OB7 plus poly (I:C) obtained an 80% (4/5) clinical protective rate. However, cattle vaccinated with △XCL-OB7 plus poly (I:C) showed more effective inhibition of virus replication than that in the XCL-OB7 group after viral challenge, according to the presence of antibodies against FMDV non-structural protein 3B. This is the first test of DC-targeted vaccines in veterinary medicine to use XCL1 fused to FMDV antigens. This primary result showed that an XCL1-based molecular vaccine enhanced the antibody response in cattle. This knowledge should be valuable for the development of antibody-dependent vaccines for some infectious diseases in cattle.

Characterization of Naturally Occurring NS5A and NS5B Polymorphisms in Patients Infected with HCV Genotype 3a Treated with Direct-Acting Antiviral Agents.

Hepatitis C virus (HCV) genotype (GT)3 is associated with increased risk of steatosis, development of cirrhosis and hepatocellular carcinoma. Limited data are available regarding genetic variability and use of direct-acting antiviral agents in these patients. non-structural protein 5A (NS5A) and non-structural protein 5B (NS5B) sequencing was performed on 45 HCV GT3-infected Italian patients subsequently treated with sofosbuvir ± daclatasvir (SOF ± DCV). Novel GT3a polymorphisms were observed by Sanger sequencing in three NS5A (T79S, T107K, and T107S) and three NS5B (G166R, Q180K, and C274W) baseline sequences in patients who achieved sustained virological response (SVR). Baseline NS5A resistance-associated substitutions (RASs) A30K and Y93H were detected in 9.5% of patients; one patient with A30K did not achieve SVR. Phylogenetic analyses of sequences showed no distinct clustering. Genetic heterogeneity of NS5A and NS5B was evaluated using ultra-deep pyrosequencing (UDPS) in samples longitudinally collected in patients not achieving SVR. Some novel NS5A and NS5B polymorphisms detected at baseline may not impact treatment outcome, as they were not enriched in post-failure samples. In contrast, the clinically novel GT3 NS5A-L31F RAS emerged in one treatment failure, and I184T, G188D and N310S, located on the same NS5B haplotype, became predominant after failure. These findings suggest a potential impact of these novel substitutions on the treatment outcome; however, their significance requires further investigation.

GSK2878175, a pan-genotypic non-nucleoside NS5B polymerase inhibitor, in healthy and treatment-naïve chronic hepatitis C subjects.

GSK2878175 is a potent, pan-genotypic, non-nucleoside, nonstructural protein 5B palm polymerase inhibitor being developed for the treatment of chronic hepatitis C (CHC). A first-in-human, randomized, placebo-controlled, dose escalation study, evaluated the safety and pharmacokinetics of GSK2878175 administered as single and repeat oral doses (once daily for 14days) to healthy volunteers. A separate proof-of-concept, placebo-controlled, repeat dose (once daily for 2days) study evaluated the safety, pharmacokinetics and antiviral activity of GSK2878175 monotherapy in treatment-naïve, noncirrhotic, subjects with hepatitis C virus (HCV) genotype 1 [1a and 1b], 2, or 3. No deaths or SAEs were reported in either study, and treatment was well-tolerated. Across all the HCV genotypes, GSK2878175 monotherapy at doses of 10, 30 or 60mg once daily for 2days produced a statistically significant multilog reduction (P<.001) in plasma HCV RNA log10 IU/mL from Baseline to 24, 48 and 72hours after the first dose of GSK2878175 compared to placebo. The reduction in HCV RNA was sustained for a prolonged period across all of the active treatment groups, consistent with the long apparent half-life of GSK2878175 that was observed (mean t1/2 range: 60-63hours in the CHC subjects). In summary, GSK2878175, when administered to healthy subjects and subjects with CHC, did not reveal any safety concerns that would limit or preclude further clinical development. GSK2878175 monotherapy across a wide dose range produced substantial reduction in HCV RNA, irrespective of HCV genotype. The results from these studies support further evaluation of GSK2878175-based regimens.

Detection and Genomic Characterization of Senecavirus A, Ohio, USA, 2015.

Detection and Genomic Characterization of Senecavirus A, Ohio, USA, 2015.

Biophysical Mode-of-Action and Selectivity Analysis of Allosteric Inhibitors of Hepatitis C Virus (HCV) Polymerase.

Allosteric inhibitors of hepatitis C virus (HCV) non-structural protein 5B (NS5B) polymerase are effective for treatment of genotype 1, although their mode of action and potential to inhibit other isolates and genotypes are not well established. We have used biophysical techniques and a novel biosensor-based real-time polymerase assay to investigate the mode-of-action and selectivity of four inhibitors against enzyme from genotypes 1b (BK and Con1) and 3a. Two thumb inhibitors (lomibuvir and filibuvir) interacted with all three NS5B variants, although the affinities for the 3a enzyme were low. Of the two tested palm inhibitors (dasabuvir and nesbuvir), only dasabuvir interacted with the 1b variant, and nesbuvir interacted with NS5B 3a. Lomibuvir, filibuvir and dasabuvir stabilized the structure of the two 1b variants, but not the 3a enzyme. The thumb compounds interfered with the interaction between the enzyme and RNA and blocked the transition from initiation to elongation. The two allosteric inhibitor types have different inhibition mechanisms. Sequence and structure analysis revealed differences in the binding sites for 1b and 3a variants, explaining the poor effect against genotype 3a NS5B. The indirect mode-of-action needs to be considered when designing allosteric compounds. The current approach provides an efficient strategy for identifying and optimizing allosteric inhibitors targeting HCV genotype 3a.