RESUMO
The Orthopoxvirus (OPV) genus of the Poxviridae family contains several human pathogens, including Vaccinia virus (VACV), which have been implicating in outbreaks of a zoonotic disease called Bovine Vaccinia in Brazil. So far, no approved treatment exists for OPV infections, but ST-246 and Cidofovir (CDV) are now in clinical development. Therefore, the objective of this work was to evaluate the susceptibility of five strains of Brazilian VACV (Br-VACV) to ST-246 and Cidofovir. The susceptibility of these strains to both drugs was evaluated by plaque reduction assay, extracellular virus's quantification in the presence of ST-246 and one-step growth curve in cells treated with CDV. Besides that, the ORFs F13L and E9L were sequenced for searching of polymorphisms associated with drug resistance. The effective concentration of 50% (EC50) from both drugs varies significantly for different strains (from 0.0054 to 0.051⯵M for ST-246 and from 27.14 to 61.23⯵M for CDV). ST-246 strongly inhibits the production of extracellular virus for all isolates in concentrations as low as 0.1⯵M and it was observed a relevant decrease of progeny production for all Br-VACV after CDV treatment. Sequencing of the F13L and E9L ORFs showed that Br-VACV do not present the polymorphism(s) associated with resistance to ST-246 and CDV. Taken together, our results showed that ST-246 and CDV are effective against diverse, wild VACV strains and that the susceptibility of Br-VACV to these drugs mirrored the phylogenetic split of these isolates into two groups. Thus, both ST-246 and CDV are of great interest as compounds to treat individuals during Bovine Vaccinia outbreaks in Brazil.
Assuntos
Antivirais/farmacologia , Benzamidas/farmacologia , Cidofovir/farmacologia , Isoindóis/farmacologia , Vaccinia virus/classificação , Vaccinia virus/efeitos dos fármacos , Vacínia/virologia , Brasil , Humanos , Filogenia , Vacínia/tratamento farmacológico , Vaccinia virus/genética , Vaccinia virus/fisiologiaRESUMO
In the present work, the antiviral activity of brequinar (BQR) against the replication of Cantagalo virus was evaluated. BQR is a potent inhibitor of cellular dihydroorotate dehydrogenase, an enzyme of the de novo pyrimidine biosynthetic pathway. Infection in the presence of 0.5µM BQR reduced virus progeny production by >90%, revealing an EC(50) (drug concentration required to inhibit 50% of virus replication) of 0.017µM. Replication of other orthopoxviruses was also inhibited by BQR at similar levels. In the presence of the drug, virus early proteins accumulated to control levels, whereas late gene expression was severely impaired. This result was confirmed by indirect immunofluorescence assays and analysis of time-regulated expression of a reporter gene under the control of a virus promoter. Both assays revealed nearly 90% inhibition of late gene expression. BQR also blocked virus DNA replication, which accounted for the subsequent inhibition of virus late gene expression. The ablation of virus DNA replication, late gene expression and infectious progeny production was restored to control levels when infected cells were co-treated with uridine (URD) and BQR. These data demonstrated that BQR targeted virus DNA synthesis by depleting the cellular pyrimidine pool, which was bypassed by the salvage pathway when URD was added to the cell cultures.
Assuntos
Antivirais/farmacologia , Compostos de Bifenilo/farmacologia , Inibidores Enzimáticos/farmacologia , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/antagonistas & inibidores , Vaccinia virus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Animais , Linhagem Celular , Replicação do DNA/efeitos dos fármacos , Di-Hidro-Orotato Desidrogenase , Regulação da Expressão Gênica , Rim/citologia , Rim/virologia , Pirimidinas/biossíntese , Vaccinia virus/classificação , Vaccinia virus/enzimologia , Vaccinia virus/fisiologia , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
Vaccinia virus (VACV) is the cause of bovine vaccinia (BV), an emerging zoonotic disease that affects dairy cows and milkers. Some chemical disinfectants have been used on farms affected by BV to disinfect cow teats and milkers' hands. To date, there is no information about the efficacy of disinfectants against VACV. Therefore, this study aimed to assess the virucidal activity of some active disinfectants commonly used in the field. Sodium hypochlorite, quaternary ammonium combined with chlorhexidine, and quaternary ammonium combined with glutaraldehyde were effective in inactivating the virus at all concentrations tested. Iodine and quaternary ammonium as the only active component were partially effective. The presence of bovine feces as organic matter and light decreased the effectiveness of sodium hypochlorite. These results show that an appropriated disinfection and asepsis of teats and hands may be helpful in the control and prevention of BV and other infections with VACV.
Assuntos
Desinfetantes/farmacologia , Vaccinia virus/efeitos dos fármacos , Animais , Chlorocebus aethiops , Testes de Sensibilidade Microbiana , Células VeroRESUMO
AIMS: To evaluate the antiviral activity of Bignoniaceae species occurring in the state of Minas Gerais, Brazil. METHODS AND RESULTS: Ethanol extracts of different anatomical parts of bignoniaceous plant species have been evaluated in vitro against human herpesvirus type 1 (HSV-1), vaccinia virus (VACV) and murine encephalomyocarditis virus (EMCV) by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. A total of 34 extracts from 18 plant species selected according to ethnopharmacological and taxonomic criteria were screened. Fifteen of the 34 extracts (44.1%) have disclosed antiviral activity against one or more of the viruses assayed with EC(50) values in the range of 23.2 ± 2.5-422.7 ± 10.9 µg ml(-1). CONCLUSIONS: Twelve of the 34 extracts (35.3%) might be considered promising sources of antiviral natural products, as they have shown EC50 ≤ 100 µg ml(-1). The present screening discloses the high potential of the Bignoniaceae family as source of antiviral agents. SIGNIFICANCE AND IMPACT OF THE STUDY: Active extracts were identified and deserve bioguided studies for the isolation of antiviral compounds and studies on mechanism of action.
Assuntos
Antivirais/farmacologia , Bignoniaceae/química , Vírus da Encefalomiocardite/efeitos dos fármacos , Herpesvirus Humano 1/efeitos dos fármacos , Extratos Vegetais/farmacologia , Vaccinia virus/efeitos dos fármacos , Animais , Bignoniaceae/classificação , Brasil , Chlorocebus aethiops , Humanos , Células L , Camundongos , Testes de Sensibilidade Microbiana/métodos , Extratos Vegetais/química , Células VeroRESUMO
Cidofovir (CDV) is one of the most effective antiorthopoxvirus drugs, and it is widely accepted that viral DNA replication is the main target of its activity. In the present study, we report a detailed analysis of CDV effects on the replicative cycles of distinct vaccinia virus (VACV) strains: Cantagalo virus, VACV-IOC, and VACV-WR. We show that despite the approximately 90% inhibition of production of virus progeny, virus DNA accumulation was reduced only 30%, and late gene expression and genome resolution were unaltered. The level of proteolytic cleavage of the major core proteins was diminished in CDV-treated cells. Electron microscopic analysis of virus-infected cells in the presence of CDV revealed reductions as great as 3.5-fold in the number of mature forms of virus particles, along with a 3.2-fold increase in the number of spherical immature particles. A detailed analysis of purified virions recovered from CDV-treated cells demonstrated the accumulation of unprocessed p4a and p4b and nearly 67% inhibition of DNA encapsidation. However, these effects of CDV on virus morphogenesis resulted from a primary effect on virus DNA synthesis, which led to later defects in genome encapsidation and virus assembly. Analysis of virus DNA by atomic force microscopy revealed that viral cytoplasmic DNA synthesized in the presence of CDV had an altered structure, forming aggregates with increased strand overlapping not observed in the absence of the drug. These aberrant DNA aggregations were not encapsidated into virus particles.
Assuntos
Antivirais/farmacologia , Citosina/análogos & derivados , Organofosfonatos/farmacologia , Vaccinia virus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Animais , Linhagem Celular , Cidofovir , Citosina/farmacologia , DNA Viral/biossíntese , Morfogênese/efeitos dos fármacos , Vaccinia virus/fisiologia , Proteínas Virais/biossíntese , Vírion/fisiologia , Montagem de Vírus/efeitos dos fármacosRESUMO
This paper describes the antiviral evaluation of new N-amino-1,2,3-triazole derivatives, 1-(substituted-phenylamino)-5-methyl-1H-[1,2,3]-triazole-4-carboxylic acid ethyl esters, 3 and 1-(4-substituted-phenylamino)-5-methyl-1H-[1,2,3]-triazole-4-carboxylic acid hydrazides, 4, on Cantagalo virus replication. 1-(4-Fluoro-phenylamino)-5-methyl-1H-[1,2,3]-triazole-4-carboxylic acid hydrazide, 4e, exhibited a significant antiviral effect. Characterization of all compounds was confirmed by IR, (1)H and (13)C spectroscopies and elemental analysis. In addition, molecular structure of 4e was also reported.
Assuntos
Amitrol (Herbicida)/análogos & derivados , Amitrol (Herbicida)/farmacologia , Antivirais/química , Antivirais/farmacologia , Vaccinia virus/efeitos dos fármacos , Amitrol (Herbicida)/toxicidade , Animais , Antivirais/toxicidade , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Chlorocebus aethiops , Testes de Sensibilidade Microbiana , Estrutura Molecular , Relação Estrutura-Atividade , Proteínas Virais/análise , Proteínas Virais/metabolismoRESUMO
Viral manipulation of the transduction pathways associated with key cellular functions such as actin remodeling, microtubule stabilization, and survival may favor a productive viral infection. Here we show that consistent with the vaccinia virus (VACV) and cowpox virus (CPXV) requirement for cytoskeleton alterations early during the infection cycle, PBK/Akt was phosphorylated at S473 [Akt(S473-P)], a modification associated with the mammalian target of rapamycin complex 2 (mTORC2), which was paralleled by phosphorylation at T308 [Akt(T308-P)] by PI3K/PDK1, which is required for host survival. Notably, while VACV stimulated Akt(S473-P/T308-P) at early (1 h postinfection [p.i.]) and late (24 h p.i.) times during the infective cycle, CPXV stimulated Akt at early times only. Pharmacological and genetic inhibition of PI3K (LY294002) or Akt (Akt-X and a dominant-negative form of Akt-K179M) resulted in a significant decline in virus yield (from 80% to >/=90%). This decline was secondary to the inhibition of late viral gene expression, which in turn led to an arrest of virion morphogenesis at the immature-virion stage of the viral growth cycle. Furthermore, the cleavage of both caspase-3 and poly(ADP-ribose) polymerase and terminal deoxynucleotidyl transferase-mediated deoxyuridine nick end labeling assays confirmed that permissive, spontaneously immortalized cells such as A31 cells and mouse embryonic fibroblasts (MEFs) underwent apoptosis upon orthopoxvirus infection plus LY294002 treatment. Thus, in A31 cells and MEFs, early viral receptor-mediated signals transmitted via the PI3K/Akt pathway are required and precede the expression of viral antiapoptotic genes. Additionally, the inhibition of these signals resulted in the apoptosis of the infected cells and a significant decline in viral titers.
Assuntos
Vírus da Varíola Bovina/fisiologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Vaccinia virus/fisiologia , Replicação Viral , Animais , Apoptose , Caspase 3/metabolismo , Linhagem Celular , Cromonas/farmacologia , Varíola Bovina/metabolismo , Vírus da Varíola Bovina/efeitos dos fármacos , Vírus da Varíola Bovina/genética , Regulação Viral da Expressão Gênica , Camundongos , Morfolinas/farmacologia , Fosforilação , Poli(ADP-Ribose) Polimerases/metabolismo , Transdução de Sinais , Vacínia/metabolismo , Vaccinia virus/efeitos dos fármacos , Vaccinia virus/genéticaRESUMO
We report a 1-step assay to screen antiviral substances combining the simultaneous application of cells, virus sample, and drug to 96-well plates. The results were obtained within 26 h when vaccinia virus plaques were counted or virus-induced cytopathic effect was measured. This fast cost-effective procedure may be used for other cytopathic viruses and is suitable to 1st screening tests.
Assuntos
Antivirais/isolamento & purificação , Avaliação Pré-Clínica de Medicamentos/economia , Avaliação Pré-Clínica de Medicamentos/métodos , Vaccinia virus/efeitos dos fármacos , Animais , Linhagem Celular , Análise Custo-Benefício , Efeito Citopatogênico Viral , Haplorrinos , Humanos , Ensaio de Placa ViralRESUMO
Solanum species are traditionally employed as antiherpes and anticancer agents in different countries. S. paniculatum has widespread ethnomedical uses in Brazil, including the treatment of viral infections. This paper reports on the isolation of neotigogenin (1) and the new compound delta25(27)-tigogenin-3-O-beta-D-glucopyranoside (2), obtained as a mixture of R and S diastereoisomers at C22 from an ethanol extract of S. paniculatum leaves, along with the determination of their cytotoxicity against Vero cells and antiviral effect against human herpes virus type 1 (HHV-1), murine encephalomyocarditis virus (EMCv), and vaccinia virus strain Western Reserve (VACV-WR). The extract of S. paniculatum inhibited HHV-1 replication [EC50 = (298.0 +/- 11.2) microg/ml] and showed no effect on EMCv and VACV-WR. On its turn, 1 was inactive against the assayed strains but presented high cytotoxicity [CC50 = (2.03 +/- 0.03) microg/ml], whereas 2 exhibited significant antiherpes [EC50 = (170.8 +/- 1.7) microg/ml] and antivaccinia virus effects [EC50 = (177.0 +/- 3.3) microg/ml], with low cytotoxicity (CC50 > 400 microg/ml). The results corroborate Solanum paniculatum as a source of cytotoxic and antiviral compounds.
Assuntos
Antivirais/farmacologia , Extratos Vegetais/farmacologia , Solanum/química , Aciclovir/farmacologia , Animais , Antivirais/química , Linhagem Celular Tumoral , Chlorocebus aethiops , Herpesvirus Humano 1/efeitos dos fármacos , Humanos , Saponinas/química , Saponinas/isolamento & purificação , Saponinas/farmacologia , Espirostanos , Estereoisomerismo , Esteroides/química , Esteroides/isolamento & purificação , Esteroides/farmacologia , Vaccinia virus/efeitos dos fármacos , Células VeroRESUMO
The antiviral effect of cidofovir was evaluated against two strains of vaccinia virus: the field strain Cantagalo virus (CTGV) and the smallpox vaccine IOC. The drug severely inhibited virus replication, revealing an EC(50) (drug concentration required to inhibit 50% of virus replication) of 7.68 microM and 9.66 microM, respectively, for CTGV and vaccine strain IOC. Similarly, other field isolates of Cantagalo-like viruses recently collected in distinct outbreaks were equally sensitive to the drug. Pre-treatment of cells prior to infection effectively established an antiviral state, inhibiting virus replication by >90% after 24h in the absence of cidofovir. CTGV infections represent an emerging zoonosis, and outbreaks have been frequently reported in several states of Brazil. Also, the possibility of resuming the manufacture of smallpox vaccine supports the need to evaluate the effect of antiviral drugs on the Brazilian vaccine strain IOC. As there is no currently approved antipoxvirus therapy, our data are extremely encouraging.