RESUMO
Respiratory syncytial virus (RSV) causes bronchiolitis in young children and common colds in adults. There is no licensed vaccine, and prophylactic treatment with palivizumab is very expensive and limited to high-risk infants. Ribavirin is used as an antiviral treatment in infants and immunosuppressed patients, and its use is limited due to side-effects, toxicity to the recipient and staff, and evidence of marginal clinical efficacy. Therefore, we studied the in vivo kinetics, and the antiviral and protective properties of a novel candidate for RSV disease treatment. The drug is a small molecule (TMC353121) discovered by screening for fusion inhibitory properties against RSV in a cellular infection model. The pharmacokinetics of TMC353121 was studied in BALB/c mice and antiviral effects determined by testing viral loads in lung tissue by quantitative RT-PCR and plaque assay after intranasal RSV infection. At doses of 0.25-10 mg · kg(-1), TMC353121 significantly reduced viral load, bronchoalveolar lavage cell accumulation and the severity of lung histopathological change after infection. Treatment remained effective if started within 48 h of infection, but was ineffective thereafter. Therefore, TMC353121 is a novel potent antiviral drug, in vivo reducing RSV replication and inhibiting consequential lung inflammation, with a great potential for further clinical development.
Assuntos
Antivirais/uso terapêutico , Benzimidazóis/uso terapêutico , Pulmão/efeitos dos fármacos , Piridinas/uso terapêutico , Vírus Sinciciais Respiratórios/efeitos dos fármacos , Internalização do Vírus/efeitos dos fármacos , Animais , Líquido da Lavagem Broncoalveolar/virologia , Feminino , Pulmão/virologia , Pneumopatias/tratamento farmacológico , Pneumopatias/virologia , Camundongos , Camundongos Endogâmicos BALB C , Infecções por Vírus Respiratório Sincicial/tratamento farmacológico , Resultado do Tratamento , Carga Viral/efeitos dos fármacos , Replicação Viral/efeitos dos fármacosRESUMO
Tn5 lacZ mutants were generated with Azospirillum brasilense 7030 by mating that strain with Escherichia coli strains carrying suicide plasmid pCIB100 or pCIB110. Kanamycin-resistant Azospirillum colonies were obtained with a maximum frequency of 10(-6) per recipient cell. The potential of Tn5 lacZ for random transposon mutagenesis coupled to transcription analysis in A. brasilense 7030 was demonstrated. Sixty percent of all Kmr A. brasilense 7030 mutants expressed beta-galactosidase activity. Mutants affected in motility (Fla-) and general chemotaxis (Che-) were identified. Chromosomal insertions of Tn5 lacZ are involved, except for two Che- mutants. The latter che loci reside on a 90-megadalton plasmid. Expression of an acidic protein (Mr, 110,000) was abolished in these mutants.
Assuntos
Azospirillum brasilense/genética , Azospirillum brasilense/fisiologia , Movimento Celular/genética , Quimiotaxia/genética , Elementos de DNA Transponíveis , DNA Bacteriano/genética , Escherichia coli/genética , Óperon Lac , Peso Molecular , Mutação , PlasmídeosRESUMO
Tn5-induced insertion mutants were generated in Azospirillum brasilense Sp7 and A. lipoferum SpBr17 by mating with Escherichia coli strains carrying suicide plasmid vectors. The sources of Tn5 were the suicide plasmids pGS9 and pSUP2021. Kanamycin-resistant Azospirillum colonies appeared from crosses with E. coli at maximum frequencies of 10 per recipient cell. Transposon Tn5 also conferred streptomycin resistance on Azospirillum colonies as was observed earlier for Rhizobium sp. Eight Tn5-induced Km SmA. brasilense Sp7 mutants with reduced nitrogen-fixing capacity were isolated. The potential use of Tn5-Mob for labeling and mobilization of Azospirillum-indigenous plasmids was demonstrated by isolating Tn5-Mob insertions in the megaplasmids of A. brasilense Sp7.