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In this paper, experimental studies on blend gel polymer electrolyte membranes comprising of poly(vinylidene fluoride) (PVDF) and poly(methyl methacrylate) (PMMA) saturated with 0.1 M liquid electrolyte of sodium tetrafluoroborate (NaBF4) in the mixture of ethylene carbonate (EC) and diethyl carbonate (DEC) are presented. Membranes are prepared by phase inversion technique. The effect of blending on the ionic conductivity, electrochemical stability window, ionic transference number, and cation transport number has been investigated using complex impedance spectroscopy, linear sweep voltammetry and DC polarization technique. Ion dynamics in the electrolyte membranes has also been investigated using dielectric studies. The optimized electrolyte membrane with composition PVDF:PMMA (95:5) + 0.1 M NaBF4 + EC + DEC shows highest ionic conductivity of 0.6 mS cm−1 which follows Vogel–Tamman–Fulcher (VTF) behavior with temperature. The membrane shows an electrochemical stability window of 3.5 V and sodium ion transport number as ∼0.33.

While somatic variants of TRAF7 (Tumor necrosis factor receptor-associated factor 7) underlie anterior skull-base meningiomas, here we report the inherited mutations of TRAF7 that cause congenital heart defects. We show that TRAF7 mutants operate in a dominant manner, inhibiting protein function via heterodimerization with wild-type protein. Further, the shared genetics of the two disparate pathologies can be traced to the common origin of forebrain meninges and cardiac outflow tract from the TRAF7- expressing neural crest. Somatic and inherited mutations disrupt TRAF7–IFT57 interactions leading to cilia degradation. TRAF7 -mutant meningioma primary cultures lack cilia, and TRAF7 knockdown causes cardiac, craniofacial, and ciliary defects in Xenopus and zebrafish, suggesting a mechanistic convergence for TRAF7 -driven meningiomas and developmental heart defects.

Path to another drug against COVID-19 The rapid development of vaccines has been crucial in battling the ongoing COVID-19 pandemic. However, access challenges remain, breakthrough infections occur, and emerging variants present increased risk. Developing antiviral therapeutics is therefore a high priority for the treatment of COVID-19. Some drug candidates in clinical trials act against the viral RNA-dependent RNA polymerase, but there are other viral enzymes that have been considered good targets for inhibition by drugs. Owen et al . report the discovery and characterization of a drug against the main protease involved in the cleavage of polyproteins involved in viral replication. The drug, PF-07321332, can be administered orally, has good selectivity and safety profiles, and protects against infection in a mouse model. In a phase 1 clinical trial, the drug reached concentrations expected to inhibit the virus based on in vitro studies. It also inhibited other coronaviruses, including severe acute respiratory syndrome coronavirus 1 and Middle East respiratory syndrome coronavirus, and could be in the armory against future viral threats. —VV

New variants of SARS-CoV-2 with potential for enhanced transmission, replication, and immune evasion capabilities continue to emerge causing reduced vaccine efficacy and/or treatment failure. As of January 2021, the WHO has defined five ‘variants of concern’ (VOC): B.1.1.7 (Alpha, α), B.1.351 (Beta, β), P.1 (Gamma, γ), B.1.617.2 (Delta, δ), and B.1.1.529 (Omicron, o). To provide a therapeutic option for the treatment of COVID-19 and variants, Nirmatrelvir, the antiviral component of PAXLOVID™, an oral outpatient treatment recently authorized for conditional or emergency use treatment of COVID-19, was developed to inhibit SARS-CoV-2 replication. Nirmatrelvir (PF-07321332) is a specific inhibitor of coronavirus main protease (Mpro, also referred to as 3CLpro), with potent antiviral activity against several human coronaviruses, including SARS-CoV-2, SARS-CoV, and MERS (Owen et al, Science 2021. doi: 10.1126/science.abl4784). Here, we evaluated PF-07321332 against the five SARS-CoV-2 VOC (α, β, γ, δ,, o) and two Variants of Interest or VOI, C.37 (λ) and B.1.621 (μ), using qRT-PCR in VeroE6 cells lacking the P-glycoprotein (Pgp) multidrug transporter gene (VeroE6 P-gp knockout cells). Nirmatrelvir potently inhibited USA-WA1/2020 strain, and α, β, γ, λ, δ, μ, and o variants in VeroE6 P-gp knockout cells with mean EC50 values 38.0 nM, 41.0 nM, 127.2 nM, 24.9 nM, 21.2 nM, 15.9 nM, 25.7 nM and 16.2 nM, respectively. Sequence analysis of the Mpro encoded by the variants showed ~100% identity of active site amino acid sequences, reflecting the essential role of Mpro during viral replication leading to ability of Nirmatrelvir to exhibit potent activity across all the variants.

Intracranial aneurysm (IA) rupture leads to subarachnoid hemorrhage, a sudden-onset disease that often causes death or severe disability. Although genome-wide association studies have identified common genetic variants that increase IA risk moderately, the contribution of variants with large effect remains poorly defined. Using whole-exome sequencing, we identified significant enrichment of rare, deleterious mutations in PPIL4, encoding peptidyl-prolyl cis-trans isomerase-like 4, in both familial and index IA cases. Ppil4 depletion in vertebrate models causes intracerebral hemorrhage, defects in cerebrovascular morphology and impaired Wnt signaling. Wild-type, but not IA-mutant, PPIL4 potentiates Wnt signaling by binding JMJD6, a known angiogenesis regulator and Wnt activator. These findings identify a novel PPIL4-dependent Wnt signaling mechanism involved in brain-specific angiogenesis and maintenance of cerebrovascular integrity and implicate PPIL4 gene mutations in the pathogenesis of IA.

Vero cells are widely used for antiviral tests and virology research for SARS-CoV-2 as well as viruses from various other families. However, Vero cells generally express high levels of multi-drug resistance 1 (MDR1) or Pgp protein, the efflux transporter of foreign substances including many antiviral compounds, affecting the antiviral activity as well as interpretation of data. To address this, a Pgp gene knockout VeroE6 cell line (VeroE6-Pgp-KO) was generated using CRISPR-CAS9 technology. These cells no longer expressed the Pgp protein as indicated by flow cytometry analysis following staining with a Pgp-specific monoclonal antibody. They also showed significantly reduced efflux transporter activity in the calcein acetoxymethyl ester (calcein AM) assay. The VeroE6-Pgp-KO cells and the parental VeroE6 cells were each infected with SARS-CoV-2 to test antiviral activities of remdesivir and nirmatrelvir, two known Pgp substrates, in the presence or absence of a Pgp inhibitor. The compounds showed antiviral activities in VeroE6-Pgp-KO cells similar to that observed in the presence of the Pgp inhibitor. Thus, the newly established VeroE6-Pgp-KO cell line adds a new in vitro virus infection system for SARS-CoV-2 and possibly other viruses to test antiviral therapies without a need to control the Pgp activity. Removal of the Pgp inhibitor for antiviral assays will lead to less data variation and prevent failed assays.

The worldwide outbreak of COVID-19 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a global pandemic. Alongside vaccines, antiviral therapeutics are an important part of the healthcare response to countering the ongoing threat presented by COVID-19. Here, we report the discovery and characterization of PF-07321332, an orally bioavailable SARS-CoV-2 main protease inhibitor with in vitro pan-human coronavirus antiviral activity and excellent off-target selectivity and in vivo safety profiles. PF-07321332 has demonstrated oral activity in a mouse-adapted SARS-CoV-2 model and has achieved oral plasma concentrations exceeding the in vitro antiviral cell potency in a phase 1 clinical trial in healthy human participants.

The worldwide outbreak of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become an established global pandemic. Alongside vaccines, antiviral therapeutics are an important part of the healthcare response to counter the ongoing threat presented by COVID-19. Here, we report the discovery and characterization of PF-07321332, an orally bioavailable SARS-CoV-2 main protease inhibitor with in vitro pan-human coronavirus antiviral activity, and excellent off-target selectivity and in vivo safety profiles. PF-07321332 has demonstrated oral activity in a mouse- adapted SARS-CoV-2 model and has achieved oral plasma concentrations exceeding the in vitro antiviral cell potency, in a phase I clinical trial in healthy human participants. ClinicalTrials.gov Identifier: NCT04756531 One-Sentence Summary PF-07321332 is disclosed as a novel, orally active, investigational small-molecule inhibitor of the SARS-CoV-2 main protease, which is being evaluated in clinical trials for the treatment of COVID-19.

Many RNA viruses encode a proof-reading deficient, low-fidelity RNA-dependent polymerase (RdRp), which generates genetically diverse populations that can adapt to changing environments and thwart antiviral therapies. 3D(pol), the RdRp of the foot-and-mouth disease virus (FMDV), is responsible for replication of viral genomes. The 3D(pol) N terminus encodes a nuclear localization signal (NLS) sequence,MRKTKLAPT, important for import of the protein to host nucleus. Previous studies showed that substitutions at residues 18 and 20 of the NLS are defective in proper incorporation of nucleotides and RNA binding. Here, we use a systematic alanine scanning mutagenesis approach to understand the role of individual residues of the NLS in nuclear localization and nucleotide incorporation activities of 3D(pol). We identify two residues of 3D(pol) NLS, T19 and L21, that are important for the maintenance of enzyme fidelity. The 3D(pol) NLS alanine substitutions of T19 and L21 results in aberrant incorporation of nucleoside analogs, conferring a low fidelity phenotype of the enzyme. A molecular dynamics simulation of RNA- and mutagen (RTP)-bound 3D(pol) revealed that the T19 residue participates in a hydrogen bond network, including D165 in motif F and R416 at the C terminus of the FMDV 3D(pol) and RNA template-primer. Based on these findings and previous studies, we conclude that at least the first six residues of theMRKTKLAPT sequence motif play a vital role in the maintenance of faithful RNA synthesis activity (fidelity) of FMDV 3D(pol), suggesting that the role of the NLS motif in similar viral polymerases needs to be revisited. IMPORTANCE In this study, we employed genetic and molecular dynamics approaches to analyze the role of individual amino acids of the FMDV 3D(pol) nuclear localization signal (NLS). The NLS residues were mutated to alanine using a type A full-genome cDNA clone, and the virus progeny was analyzed for defects in growth and in competition with the parental virus. We identified two mutants in 3D(pol), T19A and L21A, that exhibited high rate of mutation, were sensitive to nucleotide analogs, and displayed reduced replicative fitness compared to the parental virus. Using molecular dynamics simulation, we demonstrated that residues T19 and L21 played a role in the structural configuration of the interaction network at the 3D(pol) palm subdomain. Cumulatively, our data suggest that the T19 and L21 3D(pol) amino acids are important for maintaining the fidelity of the FMDV polymerase and ensuring faithful replication of the FMDV genome.

The highly contagious foot-and-mouth disease virus (FMDV) afflicts cloven-hoofed animals, resulting in significant costs because of loss of trade and recovery from disease. We developed a sensitive, specific, and rapid competitive ELISA (cELISA) to detect serum antibodies to FMDV. The cELISA utilized a monoclonal blocking antibody specific for a highly conserved FMDV nonstructural 3B epitope, a recombinant mutant FMDV 3ABC coating protein, and optimized format variables including serum incubation for 90 min at 20–25°C. Samples from 16 animals experimentally infected with one FMDV serotype (A, O, Asia, or SAT-1) demonstrated early detection capacity beginning 7 d post-inoculation. All samples from 55 vesicular stomatitis virus antibody-positive cattle and 44 samples from cloven-hoofed animals affected by non-FMD vesicular diseases were negative in the cELISA, demonstrating 100% analytical specificity. The diagnostic sensitivity was 100% against sera from 128 cattle infected with isolates of all FMDV serotypes, emphasizing serotype-agnostic results. Diagnostic specificities of U.S. cattle ( n = 1135) and swine ( n = 207) sera were 99.4% and 100%, respectively. High repeatability and reproducibility were demonstrated with 3.1% coefficient of variation in percent inhibition data and 100% agreement using 2 kit lots and 400 negative control serum samples, with no difference between bench and biosafety cabinet operation. Negative results from vaccinated, uninfected cattle, pig, and sheep sera confirmed the DIVA (differentiate infected from vaccinated animals) capability. This rapid (

Here, we engineered two FMD viruses with histidine residues inserted into or fused to the FMDV capsid. Both 6xHis viruses exhibited growth kinetics, plaque morphologies and antigenic characteristics similar to wild-type virus. The 6xHis tag allowed one-step purification of the mutant virions by Co(2+) affinity columns. Electron microscopy and biochemical assays showed that the 6xHis FMDVs readily assembled into antigen: adjuvant complexes in solution, by conjugating with Ni(2+)-chelated nanolipoprotein and monophosphoryl lipid A adjuvant (MPLA:NiNLP). Animals Immunized with the inactivated 6xHis-FMDV:MPLA:NiNLP vaccine acquired enhanced protective immunity against FMDV challenge compared to virions alone. Induction of anti-6xHis and anti-FMDV neutralizing antibodies in the immunized animals could be exploited in the differentiation of vaccinated from infected animals needed for the improvement of FMD control measures. The novel marker vaccine/nanolipid technology described here has broad applications for the development of distinctive and effective immune responses to other pathogens of importance.

Foot-and-mouth disease virus (FMDV) utilizes four integrins (αvβ1, αvβ3, αvβ6, and αvβ8) as its primary cell receptor. During cell culture propagation, FMDV frequently adapts to use heparan sulfate (HS), and rarely utilizes an unidentified third receptor. Capsid mutations acquired by a soluble integrin resistant FMDV cause (i) adaptation to CHO-677 cells (ii) increased affinity to membrane-bound Jumonji C-domain containing protein 6 (JMJD6) (iii) induced JMJD6 re-localization from the cell surface and cytoplasm to the nucleus. Interestingly, pre-treatment of cells with N- and C-terminal JMJD6 antibodies or by simultaneous incubation of mutant virus with soluble JMJD6 (but not by treatment with HS or αvβ6) impaired virus infectivity in cultured cells. JMJD6 and mutant virus co-purified by reciprocal co-immunoprecipitation. Molecular docking predictions suggested JMJD6 C-terminus interacts with mutated VP1 capsid protein. We conclude when specific VP1 mutations are displayed, JMJD6 contributes to FMDV infectivity and may be a previously unidentified FMDV receptor.

Bovine rhinitis viruses (BRVs) cause mild respiratory disease of cattle. In this study, a near full-length genome sequence of a virus named RS3X (formerly classified as bovine rhinovirus type 1), isolated from infected cattle from the UK in the 1960s, was obtained and analyzed. Compared to other closely related Aphthoviruses, major differences were detected in the leader protease (Lpro), P1, 2B, and 3A proteins. Phylogenetic analysis revealed that RS3X was a member of the species bovine rhinitis A virus (BRAV). Using different codon-based and branch-site selection models for Aphthoviruses, including BRAV RS3X and foot-and-mouth disease virus, we observed no clear evidence for genomic regions undergoing positive selection. However, within each of the BRV species, multiple sites under positive selection were detected. The results also suggest that the probability (determined by Recombination Detection Program) for recombination events between BRVs and other Aphthoviruses, including foot-and-mouth disease virus was not significant. In contrast, within BRVs, the probability of recombination increases. The data reported here provide genetic information to assist in the identification of diagnostic signatures and research tools for BRAV.

Foot-and-mouth disease (FMD) is a highly contagious viral disease that severely impacts global food security and is one of the greatest constraints on international trade of animal products. Extensive viral population diversity and rapid, continuous mutation of circulating FMD viruses (FMDVs) pose significant obstacles to the control and ultimate eradication of this important transboundary pathogen. The current study investigated mechanisms contributing to within-host evolution of FMDV in a natural host species (cattle). Specifically, vaccinated and non-vaccinated cattle were infected with FMDV under controlled, experimental conditions and subsequently sampled for up to 35 days to monitor viral genomic changes as related to phases of disease and experimental cohorts. Consensus-level genomic changes across the entire FMDV coding region were characterized through three previously defined stages of infection: early, transitional, and persistent. The overall conclusion was that viral evolution occurred via a combination of two mechanisms: emergence of full-genomic minority haplotypes from within the inoculum super-swarm, and concurrent continuous point mutations. Phylogenetic analysis indicated that individuals were infected with multiple distinct haplogroups that were pre-existent within the ancestral inoculum used to infect all animals. Multiple shifts of dominant viral haplotype took place during the early and transitional phases of infection, whereas few shifts occurred during persistent infection. Overall, this work suggests that the establishment of the carrier state is not associated with specific viral genomic characteristics. These insights into FMDV population dynamics have important implications for virus sampling methodology and molecular epidemiology.

In this study we describe the adaptive changes fixed on the capsid of several foot-and-mouth disease virus serotype A strains during propagation in cell monolayers. Viruses passaged extensively in three cell lines (BHK-21, LFBK and IB-RS-2) consistently gained positively charged amino acids in the putative heparin-sulfate-binding pocket (VP2 βE-βF loop, VP1 C-terminus and VP3 β-B knob) surrounding the fivefold symmetry axis (VP1 βF-βG loop) and at other discrete sites on the capsid (VP3 βG-βH loop, VP1 C-terminus, VP2 βC strand and VP1 βG-βH loop). A lysine insertion in the VP1 βF-βG loop of two of the BHK-21-adapted viruses supports the biological advantage of positively charged residues acquired in cell culture. The charge transitions occurred irrespective of cell line, suggesting their possible role in ionic interaction with ubiquitous negatively charged cell-surface molecules such as glycosaminoglycans (GAG). This was supported by the ability of the cell-culture-adapted variants to replicate in the integrin-deficient, GAG-positive CHO-K1 cells and their superior fitness in competition assays compared with the lower passage viruses with WT genotypes. Substitutions fixed in the VP1 βG-βH loop (-3, -2 and +2 'RGD' positions) or in the structural element known to be juxtaposed against that loop (VP1 βB-βC loop) suggest their possible role in modulating the efficiency and specificity of interaction of the 'RGD' motif with αv-integrin receptors. The nature and location of the substitutions described in this study could be applied in the rapid cell culture adaptation of viral strains for vaccine production.

The S fragment of the FMDV 5’ UTR is predicted to fold into a long stem-loop structure and it has been implicated in virus-host protein interactions. In this study, we report the minimal S fragment sequence required for virus viability and show a direct correlation between the extent of the S fragment deletion mutations and attenuated phenotypes. Furthermore, we provide novel insight into the role of the S fragment in modulating the host innate immune response. Importantly, in an FMDV mouse model system, all animals survive the inoculation with the live A 24 FMDV-S 4 mutant, containing a 164 nucleotide deletion in the upper S fragment loop, at a dose 1000 higher than the one causing lethality by parental A 24 FMDV, indicating that the A 24 FMDV-S 4 virus is highly attenuated in vivo . Additionally, mice exposed to high doses of live A 24 FMDV-S 4 virus are fully protected when challenged with parental A 24 FMDV virus.

Bovine rhinitis B virus (BRBV) shares many motifs and sequence similarities with foot-and-mouth disease virus (FMDV). This study examined if the BRBV leader proteinase (L(pro) ) could functionally replace that of FMDV. A mutant A24LBRV3DYR FMDV engineered with the BRBV L(pro) and an antigenic marker in the 3D polymerase exhibited growth properties and eIF4G cleavage similar to parental A24WT virus. The A24LBRV3DYR type I interferon activity in infected bovine cells resembled that of A24LL virus that lacks L(pro), but this effect was less pronounced for A24LBRV3DYR infected porcine cells. In vivo studies showed that the A24LBRV3DYR virus was attenuated in cattle, and exhibited low virulence in pigs exposed by direct contact. The mutant virus induced protective immunity in cattle against challenge with parental A24WT. These results provide evidence that L(pro) of different Aphthoviruses are not fully functionally interchangeable and have roles that may depend on the nature of the infected host.

Foot-and-mouth disease (FMD) is a highly contagious disease of livestock caused by a highly variable RNA virus (FMDV) that has seven serotypes and more than sixty subtypes. Both prophylactic and post-infection means of controlling the disease outbreak, including universally applicable vaccines and emergency response measures such as therapeutic treatments, are on high demand. In this study, we analyzed the long-term exposure outcome to a previously identified inhibitor of 3D polymerase (FMDV 3Dpol) for controlling FMDV infection and for the selection of resistance mutants. The results showed that no escape mutant viruses were isolated from FMDV A24 Cruzeiro infections in cell culture treated with gradually increasing concentrations of the antiviral compound 5D9 (4-chloro-N'-thieno, [2,3-d]pyrimidin-4-ylbenzenesulfonohydrazide) over ten passages. Biochemical and plaque assays revealed that when 5D9 was used at concentrations within a non-toxic range in cells, it drove the virus to undetectable levels at passage eight to ten. This is in contrast with observations made on parallel control (untreated) passages exhibiting fully viable and stable virus progenies. Collectively, the results demonstrated that under the experimental conditions, treatment with 5D9 does not confer a resistant phenotype and the virus is unable to evade the antiviral effect of the inhibitor. Further efforts using quantitative structure-property relationship (QSPR) based modifications of the 5D9 compound may result in the successful development of an effective in vivo antiviral drug targeting FMDV.

Article history: Objective: To evaluate the antioxidant activity of aqueous extract of Moringa oleifera (M. oleifera) young leaves by in vivo as well as in vitro assays. Methods: In vitro study included estimation of total phenolic, total flavonol, total flavonoid and total antioxidant power (FRAP assay). In addition, in vivo study was done with the identified most effective dose of 200 mg/kg of its lyophilized powder on normal and diabetic rats. Its effect on different oxidative free radical scavenging enzymes, viz, superoxide dismutase (SOD), catalase (CAT), glutathione-S-transferase (GST), lipid peroxide (LPO) contents were measured. Results: Significant increase in activities of SOD, CAT, GST while, a decrease in LPO content was observed. Whereas, total phenolic, flavonoid and flavonol contents in the extract were found to be 120 mg/g of GAE, 40.5 mg/g of QE and 12.12 mg/g of QE, respectively. On the other hand, FRAP assay results of M. oleifera leaves was (85.00依5.00) 毺M of Fe ++ /g of extract powder. Conclusions: The significant antioxidant activities of M. oleifera leaves from both in vivo as well as in vitro studies suggests that the regular intake of its leaves through diet can protect normal as well as diabetic patients against oxidative damage.

Foot-and-mouth disease virus (FMDV) RNA-dependent RNA polymerase (RdRp) (3D pol ) catalyzes viral RNA synthesis. Its characteristic low fidelity and absence of proofreading activity allow FMDV to rapidly mutate and adapt to dynamic environments. In this study, we used the structure of FMDV 3D pol in combination with previously reported results from similar picornaviral polymerases to design point mutations that would alter replication fidelity. In particular, we targeted Trp237 within conserved polymerase motif A because of the low reversion potential inherent in the single UGG codon. Using biochemical and genetic tools, we show that the replacement of tryptophan 237 with phenylalanine imparts higher fidelity, but replacements with isoleucine and leucine resulted in lower-fidelity phenotypes. Viruses containing these W237 substitutions show in vitro growth kinetics and plaque morphologies similar to those of the wild-type (WT) A 24 Cruzeiro strain in BHK cells, and both high- and low-fidelity variants retained fitness during coinfection with the wild-type virus. The higher-fidelity W237F (W237F HF ) mutant virus was more resistant to the mutagenic nucleoside analogs ribavirin and 5-fluorouracil than the WT virus, whereas the lower-fidelity W237I (W237I LF ) and W237L LF mutant viruses exhibited lower ribavirin resistance. Interestingly, the variant viruses showed heterogeneous and slightly delayed growth kinetics in primary porcine kidney cells, and they were significantly attenuated in mouse infection experiments. These data demonstrate, for a single virus, that either increased or decreased RdRp fidelity attenuates virus growth in animals, which is a desirable feature for the development of safer and genetically more stable vaccine candidates. IMPORTANCE Foot-and-mouth disease (FMD) is the most devastating disease affecting livestock worldwide. Here, using structural and biochemical analyses, we have identified FMDV 3D pol mutations that affect polymerase fidelity. Recombinant FMDVs containing substitutions at 3D pol tryptophan residue 237 were genetically stable and displayed plaque phenotypes and growth kinetics similar to those of the wild-type virus in cell culture. We further demonstrate that viruses harboring either a W237F HF substitution or W237I LF and W237L LF mutations were highly attenuated in animals. Our study shows that obtaining 3D pol fidelity variants by protein engineering based on polymerase structure and function could be exploited for the development of attenuated FMDV vaccine candidates that are safer and more stable than strains obtained by selective pressure via mutagenic nucleotides or adaptation approaches.

African swine fever virus (ASFV) is the etiological agent of a contagious and often lethal disease of domestic pigs that has significant economic consequences for the swine industry. The viral genome encodes for more than 150 genes, and only a select few of these genes have been studied in some detail. Here we report the characterization of open reading frame Ep152R that has a predicted complement control module/SCR domain. This domain is found in Vaccinia virus proteins that are involved in blocking the immune response during viral infection. A recombinant ASFV harboring a HA tagged version of the Ep152R protein was developed (ASFV-G-Ep152R-HA) and used to demonstrate that Ep152R is an early virus protein. Attempts to construct recombinant viruses having a deleted Ep152R gene were consistently unsuccessful indicating that Ep152R is an essential gene. Interestingly, analysis of host-protein interactions for Ep152R using a yeast two-hybrid screen, identified BAG6, a protein previously identified as being required for ASFV replication. Furthermore, fluorescent microscopy analysis confirms that Ep152R-BAG6 interaction actually occurs in cells infected with ASFV. © 2016

Bovine Rhinitis B Virus (BRBV) is a picornavirus responsible for mild respiratory infection of cattle. It is probably the least characterized among the aphthoviruses. BRBV is the closest relative known to Foot and Mouth Disease virus (FMDV) with a ~43% identical polyprotein sequence and as much as 67% identical sequence for the RNA dependent RNA polymerase (RdRp), which is also known as 3D polymerase (3Dpol). In the present study we carried out phylogenetic analysis, structure based sequence alignment and prediction of three-dimensional structure of BRBV 3Dpol using a combination of different computational tools. Model structures of BRBV 3Dpol were verified for their stereochemical quality and accuracy. The BRBV 3Dpol structure predicted by SWISS-MODEL exhibited highest scores in terms of stereochemical quality and accuracy, which were in the range of 2Å resolution crystal structures. The active site, nucleic acid binding site and overall structure were observed to be in agreement with the crystal structure of unliganded as well as template/primer (T/P), nucleotide tri-phosphate (NTP) and pyrophosphate (PPi) bound FMDV 3Dpol (PDB, 1U09 and 2E9Z). The closest proximity of BRBV and FMDV 3Dpol as compared to human rhinovirus type 16 (HRV-16) and rabbit hemorrhagic disease virus (RHDV) 3Dpols is also substantiated by phylogeny analysis and root-mean square deviation (RMSD) between C-α traces of the polymerase structures. The absence of positively charged α-helix at C terminal, significant differences in non-covalent interactions especially salt bridges and CH-pi interactions around T/P channel of BRBV 3Dpol compared to FMDV 3Dpol, indicate that despite a very high homology to FMDV 3Dpol, BRBV 3Dpol may adopt a different mechanism for handling its substrates and adapting to physiological requirements. Our findings will be valuable in the design of structure-function interventions and identification of molecular targets for drug design applicable to Aphthovirus RdRps.

We report key mechanistic differences between the reverse transcriptases (RT) of human immunodeficiency virus type-1 (HIV-1) and of xenotropic murine leukemia virus-related virus (XMRV), a gammaretrovirus that can infect human cells. Steady and pre-steady state kinetics demonstrated that XMRV RT is significantly less efficient in DNA synthesis and in unblocking chain-terminated primers. Surface plasmon resonance experiments showed that the gammaretroviral enzyme has a remarkably higher dissociation rate (k(off)) from DNA, which also results in lower processivity than HIV-1 RT. Transient kinetics of mismatch incorporation revealed that XMRV RT has higher fidelity than HIV-1 RT. We identified RNA aptamers that potently inhibit XMRV, but not HIV-1 RT. XMRV RT is highly susceptible to some nucleoside RT inhibitors, including Translocation Deficient RT inhibitors, but not to non-nucleoside RT inhibitors. We demonstrated that XMRV RT mutants K103R and Q190M, which are equivalent to HIV-1 mutants that are resistant to tenofovir (K65R) and AZT (Q151M), are also resistant to the respective drugs, suggesting that XMRV can acquire resistance to these compounds through the decreased incorporation mechanism reported in HIV-1.

This study deals with the effects of freeze dried rhizome powder of Curcuma longa (C. longa) dissolved in milk on normal as well as diabetic models. Diabetes of type II and type I was within 3 days of a single administration of doses of 45 and 65 mg kg(-1) of streptozotocin respectively. Various parameters such as blood glucose levels, triglycerides, total cholesterol, high density lipoprotein, very low density lipoprotein, low density lipoprotein, serum glutamic oxaloacetic transaminase, serum glutamic pyruvate transaminase, alkaline phosphatase, creatinine, hemoglobin, urine protein and urine sugar in addition to body weight were taken in to consideration and were analyzed after administration of variable doses of rhizome powder. The dose of 200 mg kg(-1) was identified as the most effective dose as it increased HDL, Hb and bw (P0.05) with significant decrease in the levels of blood glucose, lipid profile and hepatoprotective enzymes (P0.001).

Erythrocytes are prone to oxidative stress due to the presence of hemoglobin and polyunsaturated fatty acids. Oxidative stress (OS) is associated with increased osmotic fragility (OF) of erythrocytes. Organophosphate and organocarbamate pesticides are known to cause OS in erythrocytes. We have investigated the effect of a single sub-acute dose of carbofuran (CF), an organocarbamate pesticide and ameliorating role of vitamin C on OF and OS in erythrocytes of Wistar rats. OF and OS were assessed by determining membrane stability in terms of erythrocyte OF and the activities of free radicals scavenging enzymes such as superoxide dismutase (SOD), catalase (CAT) and glutathione-S-transferase (GST). We observed a significant alteration in the mean erythrocyte fragility (MEF) at relatively higher NaCl concentration (0.67%) as compared to MEF at 0.55%, 0.58% and 0.56% of NaCl in control, vitamin C- and vitamin C + CF-treated groups, respectively. The activities of CAT and SOD were observed to be elevated by 74.35% and 85.56%, respectively, with significance level of p < or = 0.001, whereas GST activity got significantly (p < or = 0.001) diminished by 46.30% in the erythrocytes of CF-treated rats. Vitamin C treatment exhibited marked (p < or = 0.05) prevention of carbofuran-induced oxidative stress as well as erythrocyte osmotic fragility in the Wistar rats. These results suggest that CF treatment induces OF and OS in the erythrocytes of rats, and pretreatment with vitamin C can mitigate these toxic effects.

In the present study, the in vivo effects of λ-cyhalothrin and cypermethrin on the activity of acetylcholinesterase (AChE, EC 3.1.1.7) were evaluated for 96 h in brain, muscle and gills of Channa punctatus. Both compounds exhibited tissue specific as well as dose dependent decrease in the activity of AChE. The treated fish showed a significant decrease in the activity of AChE in brain and a lesser inhibition in muscle and gills in response to the increasing concentrations of λ-cyhalothrin as well as cypermethrin. Our results indicated that the brain was the main target organ for both insecticides, followed by muscle and gills, as determined by AChE inhibition study. However, these organs showed variations in the degree of AChE inhibition for separate treatments of both insecticides. The λ-cyhalothrin was a more potent AChE inhibitor as compared to cypermethrin. These findings indicated that apart from the established mechanism of delayed closure of sodium ion channels, these pyrethroids inhibit the activity of AChE in C. punctatus which could further aggravate their neurotoxic effects.

The river Ganges has been one of the major recipients of industrial effluents in India. The present paper deals with the study related to occurrence and bioaccumulation of heavy metals (Cu, Cr, Cd, Pb, Zn) in the riverine water, sediment, and the muscles of two cat fish species, Channa punctatus (C. punctatus) and Aorichthys aor (A. aor) procured from the river Ganges at Allahabad. The data obtained after water analysis reflected the order of occurrence of heavy metals to be Zn > Pb > Cu > Cr > Cd, respectively. The analysis of heavy metals in sediment indicated that among the five heavy metals tested; Zn was maximally accumulated followed by Pb, Cr, Cu and Cd. The trend of heavy metals accumulation in fish muscles was found to be similar to that observed in sediment and water such as Zn > Pb > Cu > Cr > Cd. Data indicated that Zn accumulated maximally in the sediment as well as muscles of both of the fish species in comparison to other metals.

Background The nuclear protein Src-associated protein of 68 kDa in mitosis (Sam68) is known to bind RNA and be involved in cellular processes triggered in response to environmental stresses, including virus infection. Interestingly, Sam68 is a multi-functional protein implicated in the life cycle of retroviruses and picornaviruses and is also considered a marker of virus-induced stress granules (SGs). Recently, we demonstrated the partial redistribution of Sam68 to the cytoplasm in FMDV infected cells, its interaction with viral protease 3Cpro, and found a significant reduction in viral titers as consequence of Sam68-specific siRNA knockdowns. Despite of that, details of how it benefits FMDV remains to be elucidated. Methods Sam68 cytoplasmic localization was examined by immunofluorescent microscopy, counterstaining with antibodies against Sam68, a viral capsid protein and markers of SGs. The relevance of RAAA motifs in the IRES was investigated using electromobility shift assays with Sam68 protein and parental and mutant FMDV RNAs. In addition, full genome WT and mutant or G-luc replicon RNAs were tested following transfection in mammalian cells. The impact of Sam68 depletion to virus protein and RNA synthesis was investigated in a cell-free system. Lastly, through co-immunoprecipitation, structural modeling, and subcellular fractionation, viral protein interactions with Sam68 were explored. Results FMDV-induced cytoplasmic redistribution of Sam68 resulted in it temporarily co-localizing with SG marker: TIA-1. Mutations that disrupted FMDV IRES RAAA motifs, with putative affinity to Sam68 in domain 3 and 4 cause a reduction on the formation of ribonucleoprotein complexes with this protein and resulted in non-viable progeny viruses and replication-impaired replicons. Furthermore, depletion of Sam68 in cell-free extracts greatly diminished FMDV RNA replication, which was restored by addition of recombinant Sam68. The results here demonstrated that Sam68 specifically co-precipitates with both FMDV 3Dpol and 3Cpro consistent with early observations of FMDV 3Cpro-induced cleavage of Sam68. Conclusion We have found that Sam68 is a specific binding partner for FMDV non-structural proteins 3Cpro and 3Dpol and showed that mutations at RAAA motifs in IRES domains 3 and 4 cause a decrease in Sam68 affinity to these RNA elements and rendered the mutant RNA non-viable. Interestingly, in FMDV infected cells re-localized Sam68 was transiently detected along with SG markers in the cytoplasm. These results support the importance of Sam68 as a host factor co-opted by FMDV during infection and demonstrate that Sam68 interact with both, FMDV RNA motifs in the IRES and viral non-structural proteins 3Cpro and 3Dpol. Electronic supplementary material The online version of this article (doi:10.1186/s12985-015-0452-8) contains supplementary material, which is available to authorized users.

A companion study reported Jumonji-C domain containing protein 6 (JMJD6) is involved in an integrin- and HS-independent pathway of FMDV infection in CHO cells. JMJD6 localization was investigated in animal tissues from cattle infected with either wild type A24-FMDV (A24-WT) or mutant FMDV (JMJD6-FMDV) carrying E95K/S96L and RGD to KGE mutations in VP1. Additionally, pathogenesis of mutant JMJD6-FMDV was investigated in cattle through aerosol and intraepithelial lingual (IEL) inoculation. Interestingly, JMJD6-FMDV pathogenesis was equivalent to A24-WT administered by IEL route. In contrast, JMJD6-FMDV aerosol-infected cattle did not manifest signs of FMD and animals showed no detectable viremia. Immunofluorescent microscopy of post-mortem tissue revealed JMJD6-FMDV exclusively co-localized with JMJD6(+) cells while A24-WT was occasionally found in JMJD6(+) cells. In vitro, chemical uptake inhibitors demonstrated JMJD6-FMDV entered cells via clathrin-coated pit endocytosis. In vivo, JMJD6-FMDV exhibited preference for JMJD6(+) cells, but availability of this alternative receptor likely depends on route of inoculation.

For centuries now, antioxidants have been known to provide better health by neutralizing the free radicals which are continuously produced in the human body. In normal circumstances, self-antioxidant defense system of the human body is capable of quantitatively managing the free radicals. However, in certain cases, which are at the threshold of developing diseases like diabetes and Alzheimer's, the human body calls for an external source of antioxidants. Since orally delivered antioxidants are easily destroyed by acids and enzymes present in the human system, only a small portion of what is consumed actually gets absorbed. Hence, there is a recognized and urgent need to develop effective methods for efficiently delivering antioxidants to the required sites. This chapter provides an in-depth overview and analysis of two such methods and processes-nano-encapsulation and nano-dendrimers. Among the various nanoscale delivery mechanisms, nano-encapsulation has emerged as a key and efficient delivery process. Designed as a spongelike polymer, nano-encapsulated antioxidants provide a protective vehicle which keeps antioxidants from being destroyed in the human gut and ensures their better absorption in the digestive tract. In fact, the nano-capsules bind themselves to the intestinal walls and pour antioxidants directly into the intestinal cells, which allow them to be absorbed directly into the blood stream. Another distinguished and popular mode for delivering antioxidants is that of nano-polymers known as dendrimers. Dendrimers involve multiple branches and sub-branches of atoms radiating out from a central core. Dendrimers afford a high level of control over their architectural design, including their size, shape, branching length or density, and surface functionality. Such flexibility makes these nanostructures ideal carriers in biomedical applications such as drug delivery, gene transfection, and imaging. Antioxidant dendrimers, made out of numerous units of antioxidants connected with each other in a branched fashion, provide numerous possible sites to couple with an active species and have enhanced free radicals scavenging potency. These dendrimer chains are biocompatible, biodegradable with nontoxic degradation products, and well suited for targeted drug delivery and other biomedical applications. Recent successes in simplifying and optimizing the synthesis of dendrimers, such as the "lego" and "click" approaches, provide a large variety of structures while at the same time reducing the cost of their production. The use of these highly branched, nanometer-sized, polymeric materials as nano-antioxidants for prevention and treatment of human diseases, associated with oxidative stress, is of immense public health relevance globally.

Background Foot-and-Mouth Disease Virus (FMDV) is a picornavirus that infects cloven-hoofed animals and leads to severe losses in livestock production. In the case of an FMD outbreak, emergency vaccination requires at least 7 days to trigger an effective immune response. There are currently no approved inhibitors for the treatment or prevention of FMDV infections. Methodology/Principal Findings Using a luciferase-based assay we screened a library of compounds and identified seven novel inhibitors of 3Dpol, the RNA-dependent RNA polymerase of FMDV. The compounds inhibited specifically 3Dpol (IC50s from 2-17 µM) and not other viral or bacterial polymerases. Enzyme kinetic studies on the inhibition mechanism by compounds 5D9 and 7F8 showed that they are non-competitive inhibitors with respect to NTP and nucleic acid substrates. Molecular modeling and docking studies into the 3Dpol structure revealed an inhibitor binding pocket proximal to, but distinct from the 3Dpol catalytic site. Residues surrounding this pocket are conserved among all 60 FMDV subtypes. Site directed mutagenesis of two residues located at either side of the pocket caused distinct resistance to the compounds, demonstrating that they indeed bind at this site. Several compounds inhibited viral replication with 5D9 suppressing virus production in FMDV-infected cells with EC50 = 12 µM and EC90 = 20 µM). Significance We identified several non-competitive inhibitors of FMDV 3Dpol that target a novel binding pocket, which can be used for future structure-based drug design studies. Such studies can lead to the discovery of even more potent antivirals that could provide alternative or supplementary options to contain future outbreaks of FMD.

The present study deals with the scientific evaluation of antioxidant potential of aqueous extract of Trichosanthes dioica fruits on diabetes-induced oxidative stress of diabetic rats. The most effective dose of mg/kg bw of fruit aqueous extract was given orally to diabetic rats for 30 days. Different oxidative stress parameters were analyzed in various tissues of control and treated diabetic rats. The observed elevated level of lipid peroxidation (LPO) comes down significantly (p0.05) and decreased activities of antioxidant enzymes such as catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), and glutathione-S-transferase (GST) got increased (p0.05) significantly of diabetic rats on extract treatment. Laser-Induced Breakdown Spectroscopy (LIBS) has been used as an analytical tool to detect major and minor elements like Mg, Fe, Na, K, Zn, Ca, H, O, C, and N present in the extract. The higher concentration of Ca(2+), Mg(2+), and Fe(2+), as reflected by their intensities are responsible for antioxidant potential of T. dioica.

Erythrocytes are readily available cells and a good model system to study the health status of individuals with pathologic complications. It can also serve as a meaningful target to study toxicant/xenobiotic-induced damages. We have prepared different concentrations of a carbamate pesticide (carbofuran) and carried out experiments to determine its toxicity on erythrocytes in terms of mean erythrocyte fragility (MEF). We observed a significant alteration in the osmotic fragility upon treatment with carbofuran. In our earlier studies we have observed a good correlation between OF and OS in diabetic subjects. Study reveals OF as a potential biomarker of oxidative membrane damage in pathologic conditions as well as toxicant/xenobiotic/pesticide-induced oxidative membrane damage to erythrocytes.

The aim of the study was to determine the effect of the elements of the extract of seed from Emblica officinalis on antioxidant enzymes and osmotic fragility of erythrocytes membrane in normal as well as streptozotocin-induced severely diabetic albino Wister rats. The results revealed that the untreated diabetic rats exhibited increase in oxidative stress as indicated by significantly diminished activities of free radical scavenging enzymes such as catalase (CAT) and superoxide dismutase (SOD) by 37.5% (p < 0.001) and 18.6% (p < 0.01), respectively. However, the E. officinalis seed extract treatment showed marked improvements in CAT and SOD activities by 47.09% (p < 0.001) and 21.61% (p < 0.001), respectively. The enhanced lipid peroxidation by 30.87% (p < 0.001) in erythrocytes of untreated diabetic rats was significantly accentuated in the extract treated animals by 23.72% (p < 0.001). The erythrocytes showed increased osmotic fragility due to diabetes in terms of hemolysis. It attained the normal level in diabetic treated group. The findings thus suggest that E. officinalis seed extract has the potential to be exploited as an agent to boost the antioxidant system in the diabetic animal model. Laser-induced breakdown spectroscopy has been used as an analytical tool to detect major and minor elements like Mg, Fe, Na, K, Zn, Ca, H, O, C, and N present in the extract. The higher concentration of Ca (II), Mg (II) and Fe (II) as reflected by their intensities are responsible for the antioxidant potential of E. officinalis.

Erythrocytes are readily available cells and a good model system to study the health status of individuals with pathologic complications. It can also serve as a meaningful target to study toxicant/xenobiotic-induced damages. We have prepared different concentrations of a carbamate pesticide (carbofuran) and carried out experiments to determine its toxicity on erythrocytes in terms of mean erythrocyte fragility (MEF). We observed a significant alteration in the osmotic fragility upon treatment with carbofuran. In our earlier studies we have observed a good correlation between OF and OS in diabetic subjects. Study reveals OF as a potential biomarker of oxidative membrane damage in pathologic conditions as well as toxicant/xenobiotic/pesticide-induced oxidative membrane damage to erythrocytes.

The present study deals with the scientific evaluation of antioxidant potential of aqueous extract of Trichosanthes dioica fruits on diabetes-induced oxidative stress of diabetic rats. The most effective dose of mg/kg bw of fruit aqueous extract was given orally to diabetic rats for 30 days. Different oxidative stress parameters were analyzed in various tissues of control and treated diabetic rats. The observed elevated level of lipid peroxidation (LPO) comes down significantly (p < 0.05) and decreased activities of antioxidant enzymes such as catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), and glutathione-S-transferase (GST) got increased (p < 0.05) significantly of diabetic rats on extract treatment. Laser-Induced Breakdown Spectroscopy (LIBS) has been used as an analytical tool to detect major and minor elements like Mg, Fe, Na, K, Zn, Ca, H, O, C, and N present in the extract. The higher concentration of Ca(2+), Mg(2+), and Fe(2+), as reflected by their intensities are responsible for antioxidant potential of T. dioica.

Monitoring of acetylcholinesterase (EC: 3.1.1.7, AChE) activity has been widely used in aquatic and terrestrial systems as an indicator of pollutant exposure. The reports regarding impact of fertilizer industry effluent on the level of AChE activity are very scanty. In this paper, an attempt has been made to investigate the in vitro impact of fertilizer industry effluent upon the levels of AChE activity and protein content in different tissues of non-target aquatic fish, Channa striatus (Bloch). The fish when exposed to three sublethal concentrations (3.5, 4.7, and 7.0%; v/v) of fertilizer industry effluent for short (96 h) and long (15 days) durations registered sharp reduction in the levels of AChE activity (15–75%) and protein (10–71%) in different fish organs. The highest effluent concentration treatment for short or long duration, the fish brain and gills registered significant (P < 0.001) inhibition (64–75%) in the activity of AChE whereas other organs such as muscles, liver, and heart exhibited slightly lower inhibition (40–59%) in enzyme activity. However, kidney of C. striatus was the only organ where very less effect (14–18%) of the effluent was observed on the activity of AChE when the fish were exposed to all the three concentrations of the effluent for both treatment durations. This effluent also induced alterations in the level of protein in different fish organs; in kidney the effect was pronounced only at higher concentrations at both treatment durations. The most affected organs were muscle and gills where in 60–71% reduction in the protein content was recorded due to highest effluent concentration treatment at short or long durations. The results of present study indicated that the fertilizer industry effluents might significantly influence the neurotransmission system and protein turnover in the non-target organisms after exposure even at very low concentrations. Further, the data suggested that the fish AChE could be used as a potential biochemical marker for fertilizer industry effluent pollution in aquatic systems.

The industrial activities pose threat to the life of aquatic organisms in many ways. This research communication presents an account of the impact of fertilizer industry effluent upon the levels of protein and the activity of lactate dehydrogenase (EC 1.1.1.28, LDH), a terminal key enzyme in glycolytic pathway, in different organs of a fresh water teleost fish, Channa striatus (Bloch). The fish exposed to different sublethal concentrations of fertilizer industry effluent (3.5, 4.7 and 7.0% v/v) equivalent to 1/20th, 1/15th and 1/10th of LC50 value (70% v/v) for varying treatment periods (96 h and 15 days) exhibited decrease in the level of protein (8–76%) in different organs of the effluent treated fish. At highest effluent concentration (7% v/v) treatment for short (96 h) or long (15 days) duration, the liver of the fish registered significant (p

Chronic exposure to carbofuran, a carbamate pesticide, via oral administration has been reported to generate reactive oxygen species (ROS) in rat brain. However, information regarding the effect of short-term intraperitoneal (i.p.) carbofuran intoxication on oxidative stress is lacking. In the present study, the effect of carbofuran on oxidative indices in brain of Wistar rats has been determined by exposing the animals to three subacute concentrations (0.2, 0.4 and 0.8 mg/kg body weight) equivalent to 10, 20, and 40%, respectively, of its LD50 (i.p.) for 24 h. Rat liver has been used as a positive control. The results demonstrated that carbofuran treatment at the 3 concentrations tested caused significant increase in lipid peroxidation (LPO) by 12.50, 34.38, and 59.38%, respectively. The increased oxidative stress at same pesticide concentrations significantly induced activities of antioxidant enzymes such as superoxide dismutase (SOD) and catalase in rat brain; the impact on catalase being more marked only at high-pesticide doses (0.4 and 0.8 mg/kg body weight). Carbofuran also caused reduction in protein content of rat tissues tested. Rat brain was more severely affected by carbofuran than liver. The results clearly demonstrated that i.p. administration of carbofuran accelerated oxidative stress in rat brain in a dose-dependent manner.