In silico, in ovo and in vitro antiviral efficacy of phosphorylated derivatives of abacavir: an experimental approach.

Outstanding increase of oral absorption, bioavailability, and antiviral efficacy of phosphorylated nucleosides and basic antiviral influence of abacavir is the central idea for the development of new series of phosphorylated abacavir (ABC) derivatives. The designed compounds were primarily screened for antiviral nature against HN protein of NDV and VP7 protein of BTV using the molecular environment approach. Out of all the designed compounds, the compounds which are having higher binding energies against these two viral strains were prompted for the synthesis of the target compounds (5A-K). Among the synthesized title compounds (5A-K), the compounds which have exhibited higher dock scores akin to the rest of the compounds were then selected and screened for the antiviral activity against NDV and BTV infected embryonated eggs and BHK 21 cell lines through the in ovo and in vitro approaches. The results revealed that all the designed compounds have formed higher binding energies against both the targets. Among all, the compounds which are selected based on their dock scores such as 5A, 5F, 5G, 5H, 5I, and 5K against NDV and 5J, 5E, 5I, 5C, 5A, and 5K against BTV have shown significant antiviral activity against HN protein of NDV, VP7 protein of Bluetongue virus in both NDV- and BTV-treated embryonated eggs and BHK 21 cell lines. Hence, it is concluded that, the best lead compounds will stand as the potential antiviral agents and prompted them as virtuous therapeutics against NDV and BTV in future.

Inhibition of Orbivirus Replication by Aurintricarboxylic Acid.

Bluetongue virus (BTV) and African horse sickness virus (AHSV) are vector-borne viruses belonging to the Orbivirus genus, which are transmitted between hosts primarily by biting midges of the genus Culicoides. With recent BTV and AHSV outbreaks causing epidemics and important economy losses, there is a pressing need for efficacious drugs to treat and control the spread of these infections. The polyanionic aromatic compound aurintricarboxylic acid (ATA) has been shown to have a broad-spectrum antiviral activity. Here, we evaluated ATA as a potential antiviral compound against Orbivirus infections in both mammalian and insect cells. Notably, ATA was able to prevent the replication of BTV and AHSV in both cell types in a time- and concentration-dependent manner. In addition, we evaluated the effect of ATA in vivo using a mouse model of infection. ATA did not protect mice against a lethal challenge with BTV or AHSV, most probably due to the in vivo effect of ATA on immune system regulation. Overall, these results demonstrate that ATA has inhibitory activity against Orbivirus replication in vitro, but further in vivo analysis will be required before considering it as a potential therapy for future clinical evaluation.

Replication-Deficient Particles: New Insights into the Next Generation of Bluetongue Virus Vaccines.

Bluetongue virus (BTV) is endemic in many parts of the world, often causing severe hemorrhagic disease in livestock. To date, at least 27 different serotypes have been recognized. Vaccination against all serotypes is necessary to protect susceptible animals and to prevent onward spread of the virus by insect vectors. In our previous studies, we generated replication-deficient (disabled infectious single-cycle [DISC]) virus strains for a number of serotypes and reported preliminary data on their protective efficacy in animals. In this report, to advance the DISC vaccines to the marketplace, we investigated different parameters of these DISC vaccines. First, we demonstrated the genetic stabilities of these vaccine strains and also the complementing cell line. Subsequently, the optimal storage conditions of vaccines, including additives, temperature, and desiccation, were determined and their protective efficacies in animals confirmed. Furthermore, to test if mixtures of different vaccine strains could be tolerated, we tested cocktails of DISC vaccines in combinations of three or six different serotypes in sheep and cattle, the two natural hosts of BTV. Groups of sheep vaccinated with a cocktail of six different vaccines were completely protected from challenge with individual virulent serotypes, both in early challenge and after 5 months of challenge without any clinical disease. There was no interference in protection between the different vaccines. Protection was also achieved in cattle with a mixture of three vaccine strains, albeit at a lesser level than in sheep. Our data support and validate the suitability of these virus strains as the next-generation vaccines for BTV. IMPORTANCE: Bluetongue (BT) is a debilitating and in many cases lethal disease that affects ruminants of economic importance. Classical vaccines that afford protection against bluetongue virus, the etiological agent, are not free from secondary and undesirable effects. A surge in new approaches to produce highly attenuated, safer vaccines was evident after the development of the BTV reverse-genetics system that allows the introduction of targeted mutations in the virus genome. We targeted an essential gene to develop disabled virus strains as vaccine candidates. The results presented in this report further substantiate our previous evidence and support the suitability of these virus strains as the next-generation BTV vaccines.

Antiviral Properties of Streptomyces tuirus DBZ39 Mediated Gold Nanoparticles Against Bluetongue virus.

<b>Background and Objective:</b> The proposed study involves the approach from the point of anti-viral activity of gold nanoparticles against the <i>Bluetongue virus</i>. Among viral diseases, Bluetongue is regarded as an economically scouring disease. Neither a vaccine nor an antiviral drug is available for the prevention or treatment of this disease. The antiviral activity of gold nanoparticles synthesized by a novel isolate of <i>Streptomyces tuirus</i> DBZ39 is the breakthrough of the study. <i>Streptomyces tuirus </i>DBZ39, a novel isolate obtained from alkaline soil was proved to be efficient actinomycetes, for the extracellular synthesis of gold nanoparticles. <b>Materials and Methods:</b> An upstream bioprocess was optimized and developed for the synthesis of controlled size gold nanoparticles with solitary mono dispersal pattern in aurum chloride solution. The characterization and confirmation of gold nanoparticles were illustrated by Scanning Electron Microscopy (SEM), Transmission Electron Microscopy (TEM), Energy Dispersive X-ray Analysis (EDAX) and Fourier Transmission Infrared Radiation Analysis (FTIR). <b>Results:</b> Biomass size of 3 g, substrate concentration of 1 mM, pH of 8.5 and temperature of 45°C were observed as optimum conditions for the synthesis of 15-24 nm size gold nanoparticles. The <i>Bluetongue virus</i> (BTV) which belongs to the genus Orbivirus in the family Reoviridae with 26 serotypes is an etiological agent of infectious and non-contagious Bluetongue disease of main sheep and several other domestic animals. <b>Conclusion:</b> Gold nanoparticles for the 1st time, at a higher concentration of 1:64 dilutions revealed a very promising and novel antiviral property against the <i>Bluetongue virus</i>.

Inhibition of Orbivirus Replication by Fluvastatin and Identification of the Key Elements of the Mevalonate Pathway Involved.

Statin derivatives can inhibit the replication of a range of viruses, including hepatitis C virus (HCV, Hepacivirus), dengue virus (Flavivirus), African swine fever virus (Asfarviridae) and poliovirus (Picornaviridae). We assess the antiviral effect of fluvastatin in cells infected with orbiviruses (bluetongue virus (BTV) and Great Island virus (GIV)). The synthesis of orbivirus outer-capsid protein VP2 (detected by confocal immunofluorescence imaging) was used to assess levels of virus replication, showing a reduction in fluvastatin-treated cells. A reduction in virus titres of ~1.7 log (98%) in fluvastatin-treated cells was detected by a plaque assay. We have previously identified a fourth non-structural protein (NS4) of BTV and GIV, showing that it interacts with lipid droplets in infected cells. Fluvastatin, which inhibits 3-hydroxy 3-methyl glutaryl CoA reductase in the mevalonic acid pathway, disrupts these NS4 interactions. These findings highlight the role of the lipid pathways in orbivirus replication and suggest a greater role for the membrane-enveloped orbivirus particles than previously recognised. Chemical intermediates of the mevalonic acid pathway were used to assess their potential to rescue orbivirus replication. Pre-treatment of IFNAR(-/-) mice with fluvastatin promoted their survival upon challenge with live BTV, although only limited protection was observed.

Beta-propiolactone inactivated bivalent bluetongue virus vaccine containing Montanide ISA-71VG adjuvant induces long-term immune response in sheep against serotypes 4 and 16 even after 3 years of controlled vaccine storage.

In this study, we developed and evaluated the beta-propiolactone inactivated bivalent bluetongue virus (BTV) serotypes 4 and 16 vaccine delivered with Montanide™ ISA-71VG adjuvant. The safety, stability and immunological profile of the fresh and after three years of long-term storage of the vaccine formulation was analyzed. We observed after long-term storage that the vaccine emulsion was stable as indicated by unchanged pH and viscosity. The stored vaccine formulation induced virus neutralizing antibodies (VNA) in sheep against both the bluetongue virus serotypes at 7-10 day post-vaccination (dpv). VNA titers reached the peak by 60 dpv and detectable during the entire study period. Antibodies against bluetongue virus structural protein VP7 were detected by ELISA in all BTV vaccinated experimental animal groups. Partial clinical protection was observed in vaccinates against challenge virulent BTV-4 and BTV-16 serotypes by 10 dpv, while complete protection was observed at 14 dpv. The levels of viremia was decreased in challenged sheep by 10 dpv while the viremia was undetectable by 14 dpv. In summary, our newly formulated bivalent BTV (BTV-4 and BTV-16) vaccine delivered with Montanide™ ISA-71VG adjuvant was found safe and stable for over three years and induced protective response in sheep.

Effects of avermectins on olfactory responses of Culicoides imicola (Diptera: Ceratopogonidae).

The aim of this study was to examine the role of the olfactory system of the midge Culicoides imicola Kieffer as the major system mediating repellency to antihelminthic avermectins. Incidental observations indicate that treatment with Dectomax or Ivomec (commercial formula of the avermectins doramectin and ivermectin, respectively) protects sheep from infection by bluetongue (BT) viruses. Our electrophysiological data from midge antennae showed that the stimulating effectiveness of L- (+)-lactic acid, butanone, and sheep fleece odor decreased after addition of avermectins. The results show that these antihelminthics affect the olfactory sensitivity of the insect toward the animal host by reducing the response to those compounds that attract the insect, consequently reducing the possibility of biting the sheep and thereby transferring the BT virus.

Atomic model of a nonenveloped virus reveals pH sensors for a coordinated process of cell entry.

Viruses sense environmental cues such as pH to engage in membrane interactions for cell entry during infection, but how nonenveloped viruses sense pH is largely undefined. Here, we report both high- and low-pH structures of bluetongue virus (BTV), which enters cells via a two-stage endosomal process. The receptor-binding protein VP2 possesses a zinc finger that may function to maintain VP2 in a metastable state and a conserved His866, which senses early-endosomal pH. The membrane-penetration protein VP5 has three domains: dagger, unfurling and anchoring. Notably, the ß-meander motif of the anchoring domain contains a histidine cluster that can sense late-endosomal pH and also possesses four putative membrane-interaction elements. Exposing BTV to low pH detaches VP2 and dramatically refolds the dagger and unfurling domains of VP5. Our biochemical and structure-guided-mutagenesis studies support these coordinated pH-sensing mechanisms.

Application of bluetongue Disabled Infectious Single Animal (DISA) vaccine for different serotypes by VP2 exchange or incorporation of chimeric VP2.

Bluetongue is a disease of ruminants caused by the bluetongue virus (BTV). Bluetongue outbreaks can be controlled by vaccination, however, currently available vaccines have several drawbacks. Further, there are at least 26 BTV serotypes, with low cross protection. A next-generation vaccine based on live-attenuated BTV without expression of non-structural proteins NS3/NS3a, named Disabled Infectious Single Animal (DISA) vaccine, was recently developed for serotype 8 by exchange of the serotype determining outer capsid protein VP2. DISA vaccines are replicating vaccines but do not cause detectable viremia, and induce serotype specific protection. Here, we exchanged VP2 of laboratory strain BTV1 for VP2 of European serotypes 2, 4, 8 and 9 using reverse genetics, without observing large effects on virus growth. Exchange of VP2 from serotype 16 and 25 was however not possible. Therefore, chimeric VP2 proteins of BTV1 containing possible immunogenic regions of these serotypes were studied. BTV1, expressing 1/16 chimeric VP2 proteins was functional in virus replication in vitro and contained neutralizing epitopes of both serotype 1 and 16. For serotype 25 this approach failed. We combined VP2 exchange with the NS3/NS3a negative phenotype in BTV1 as previously described for serotype 8 DISA vaccine. DISA vaccine with 1/16 chimeric VP2 containing amino acid region 249-398 of serotype 16 raised antibodies in sheep neutralizing both BTV1 and BTV16. This suggests that DISA vaccine could be protective for both parental serotypes present in chimeric VP2. We here demonstrate the application of the BT DISA vaccine platform for several serotypes and further extend the application for serotypes that are unsuccessful in single VP2 exchange.

Production of murine cytotoxic T lymphocytes by bluetongue virus following various immunisation procedures.

The induction of bluetongue virus specific cytotoxic T lymphocytes (CTLs) in C3H mice by various live and inactivated bluetongue virus preparations was studied. Live virus preparations were shown to induce good levels of CTLs; however, inactivation of virus preparations either by beta propriolactone or glutaraldehyde induced only a low level response. The use of Freund's adjuvants and double immunisation procedures failed to improve the response of the inactivated preparations. These findings are discussed in relationship to protection from bluetongue disease with various bluetongue virus vaccines.

Enzyme-linked immunosorbent assay for detection of bluetongue virus antibodies.

The immune response to bluetongue virus in sheep and cattle was studied by applying a newly developed indirect enzyme-linked immunosorbent assay (ELISA). Purified virus obtained by sucrose gradient centrifugation was used at a concentration of 0.01 optical density units (formula: see text) to coat individual wells (200 microliter) of a microtitration plate. Dilution of antigen was performed in 0.05 M carbonate buffer, pH 9.6, and adsorption lasted for at least 16 hours at 4 C. Coated plates retained their activity for 10 weeks when stored at 4 C. Sera recovered from experimentally infected sheep and cattle were tested together with known negative sera. A good correlation between results was obtained with the modified complement-fixation test and the ELISA; however, the ELISA proved to be more sensitive. The group specificity of the ELISA was proven by testing various type-specific sheep and cattle immune sera. The ELISA has potential for the detection of group-specific antibodies to bluetongue virus infection.

Immunologic response of sheep to inactivated and virulent bluetongue virus.

Humoral and cellular immune responses of sheep to inactivated and virulent bluetongue virus (BTV) were studied. All sheep inoculated with inactivated BTV developed BTV group-specific nonneutralizing antibodies, as determined by agar-gel immunodiffusion. The development of group-specific, nonneutralizing, complement-fixing antibodies was variable and appeared to be dependent on immunizing BTV serotype, sheep breed, and individual variation. Virus-neutralizing antibodies were never detected after inoculation with the inactivated BTV. In vitro lymphocyte stimulation to BTV soluble antigen was observed with cells from all inoculated Warhill sheep and with cells from 1 of 3 inoculated Suffolk cross sheep. Complement-fixation titers did not appear to correlate with the degree of protection observed, ie, duration of postchallenge-exposure viremia. The development of postchallenge-exposure neutralizing antibody titer was inversely correlated to protective immunity. The development of a response to BTV antigen in the lymphocyte-stimulation test associated most closely with protection. Warhill sheep were afforded better protection, by inoculation with inactivated BTV, to live virus challenge exposure than were the Suffolk cross sheep. Approximately 30% of the inoculated Suffolk cross sheep responded to challenge exposure with intensified clinical signs of blue-tongue, compared with the challenge-exposed control sheep of the same breed.

[Inactivation of viruses of different taxonomic groups by cuprous sulphate]. / Inaktivatsiia virusov razlichnykh taksonomicheskikh grupp sernokisloi med'iu.

Study results of inactivated effects exerted by cuprous sulphate on viruses of different taxonomy groups are summarized in the paper. Cuprous sulphate is a simple and reliable agent in inactivation of viruses of classical porcine fever, Aujeszky's disease and bovine infectious rhinotracheitis. Its inactivation action is based on the ability to reduce the viral genome to low-molecular fragment. Apart from inactivation of the virus material, a decreased level of protective antibody determinants is observed when cuprous sulphate is used in case of sheep catarrhal fever.

Assays for the identification of novel antivirals against bluetongue virus.

To identify potential antivirals against BTV, we have developed, optimized and validated three assays presented here. The CPE-based assay was the first assay developed to evaluate whether a compound showed any antiviral efficacy and have been used to screen large compound library. Meanwhile, cytotoxicity of antivirals could also be evaluated using the CPE-based assay. The dose-response assay was designed to determine the range of efficacy for the selected antiviral, i.e. 50% inhibitory concentration (IC50) or effective concentration (EC50), as well as its range of cytotoxicity (CC50). The ToA assay was employed for the initial MoA study to determine the underlying mechanism of the novel antivirals during BTV viral lifecycle or the possible effect on host cellular machinery. These assays are vital for the evaluation of antiviral efficacy in cell culture system, and have been used for our recent researches leading to the identification of a number of novel antivirals against BTV.

Saliva proteins of vector Culicoides modify structure and infectivity of bluetongue virus particles.

Bluetongue virus (BTV) and epizootic haemorrhagic disease virus (EHDV) are related orbiviruses, transmitted between their ruminant hosts primarily by certain haematophagous midge vectors (Culicoides spp.). The larger of the BTV outer-capsid proteins, 'VP2', can be cleaved by proteases (including trypsin or chymotrypsin), forming infectious subviral particles (ISVP) which have enhanced infectivity for adult Culicoides, or KC cells (a cell-line derived from C. sonorensis). We demonstrate that VP2 present on purified virus particles from 3 different BTV strains can also be cleaved by treatment with saliva from adult Culicoides. The saliva proteins from C. sonorensis (a competent BTV vector), cleaved BTV-VP2 more efficiently than those from C. nubeculosus (a less competent/non-vector species). Electrophoresis and mass spectrometry identified a trypsin-like protease in C. sonorensis saliva, which was significantly reduced or absent from C. nubeculosus saliva. Incubating purified BTV-1 with C. sonorensis saliva proteins also increased their infectivity for KC cells ∼10 fold, while infectivity for BHK cells was reduced by 2-6 fold. Treatment of an 'eastern' strain of EHDV-2 with saliva proteins of either C. sonorensis or C. nubeculosus cleaved VP2, but a 'western' strain of EHDV-2 remained unmodified. These results indicate that temperature, strain of virus and protein composition of Culicoides saliva (particularly its protease content which is dependent upon vector species), can all play a significant role in the efficiency of VP2 cleavage, influencing virus infectivity. Saliva of several other arthropod species has previously been shown to increase transmission, infectivity and virulence of certain arboviruses, by modulating and/or suppressing the mammalian immune response. The findings presented here, however, demonstrate a novel mechanism by which proteases in Culicoides saliva can also directly modify the orbivirus particle structure, leading to increased infectivity specifically for Culicoides cells and, in turn, efficiency of transmission to the insect vector.

Assay development and high-throughput antiviral drug screening against Bluetongue virus.

Bluetongue virus (BTV) infection is one of the most important diseases of domestic livestock. There are no antivirals available against BTV disease. In this paper, we present the development, optimization and validation of an in vitro cell-based high-throughput screening (HTS) assay using the luminescent-based CellTiter-Glo reagent to identify novel antivirals against BTV. Conditions of the cytopathic effect (CPE)-based assay were optimized at cell density of 5000 cells/well in medium containing 1% FBS and a multiplicity of infection at 0.01 in 384-well plate, with Z'-values > or = 0.70, Coefficient of Variations > or = 5.68 and signal-to-background ratio > or = 7.10. This assay was further validated using a 9532 compound library. The fully validated assay was then used to screen the 194,950 compound collection, which identified 693 compounds with >30% CPE inhibition. The 10-concentration dose response assay identified 185 structures with IC(50) < or =100 microM, out of which 42 compounds were grouped into six analog series corresponding to six scaffolds enriched within the active set compared to their distribution in the library. The CPE-based assay development demonstrated its robustness and reliability, and its application in the HTS campaign will make significant contribution to the antiviral drug discovery against BTV disease.

Inhibition of bluetongue and Colorado tick fever orbiviruses by selected antiviral substances.

The effects of four ribonucleic acid virus inhibitors were evaluated in cell cultures and in mice to determine inhibitory effects against bluetongue virus and Colorado tick fever virus (CTFV). Test compounds included 1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide (ribavirin), 3-deazaguanine, 3-deazauridine, and 9-(S)-(2,3-dihydroxypropyl)adenine. Ribavirin-2',3',5'-triacetate (ribavirin triacetate) was evaluated in vivo against CTFV. Inhibition of cytopathic effect and plaque reduction were used to evaluate antiviral activity. In cytopathic effect inhibition studies, bluetongue virus was markedly inhibited by 3-deazaguanine and 3-deazauridine in Vero cells with moderate inhibition by the other agents. Ribavirin and 3-deazaguanine markedly inhibited CTFV in MA-104 cells, 3-deazauridine was slightly less active, and 9-(S)-(2,3-dihydroxypropyl)adenine was negative. Ribavirin was less effective in Vero cells against CTFV. When mice were inoculated intracerebrally with CTFV and treated by a single intracerebral injection with drug, ribavirin triacetate increased the number of survivors, 3-deazaguanine increased mean survival time, and ribavirin was negative. Intraperitoneal treatment of infected mice with ribavirin triacetate for 1 week significantly increased the number of survivors and mean survival time, providing strong evidence that the agent is active across the blood-brain barrier.