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1.
Mem. Inst. Oswaldo Cruz ; 108(1): 36-40, Feb. 2013. graf, tab
Artigo em Inglês | LILACS | ID: lil-666041

RESUMO

ELISA in situ can be used to titrate hepatitis A virus (HAV) particles and real-time polymerase chain reaction (RT-PCR) has been shown to be a fast method to quantify the HAV genome. Precise quantification of viral concentration is necessary to distinguish between infectious and non-infectious particles. The purpose of this study was to compare cell culture and RT-PCR quantification results and determine whether HAV genome quantification can be correlated with infectivity. For this purpose, three stocks of undiluted, five-fold diluted and 10-fold diluted HAV were prepared to inoculate cells in a 96-well plate. Monolayers were then incubated for seven, 10 and 14 days and the correlation between the ELISA in situ and RT-PCR results was evaluated. At 10 days post-incubation, the highest viral load was observed in all stocks of HAV via RT-PCR (10(5) copies/mL) (p = 0.0002), while ELISA revealed the highest quantity of particles after 14 days (optical density = 0.24, p < 0.001). At seven days post-infection, there was a significant statistical correlation between the results of the two methods, indicating equivalents titres of particles and HAV genome during this period of infection. The results reported here indicate that the duration of growth of HAV in cell culture must be taken into account to correlate genome quantification with infectivity.


Assuntos
Animais , Vírus Defeituosos/fisiologia , Ensaio de Imunoadsorção Enzimática/métodos , Vírus da Hepatite A/fisiologia , Reação em Cadeia da Polimerase em Tempo Real/métodos , Replicação Viral/fisiologia , Linhagem Celular , Macaca mulatta , Fatores de Tempo , Carga Viral , Ensaio de Placa Viral
2.
Braz. j. med. biol. res ; 43(2): 217-224, Feb. 2010. ilus, graf
Artigo em Inglês | LILACS | ID: lil-538233

RESUMO

Bovine herpesvirus type 5 (BoHV-5) is an important pathogen of cattle in South America. We describe here the construction and characterization of deletion mutants defective in the glycoprotein E (gE) or thymidine kinase (TK) gene or both (gE/TK) from a highly neurovirulent and well-characterized Brazilian BoHV-5 strain (SV507/99). A gE-deleted recombinant virus (BoHV-5 gE∆) was first generated in which the entire gE open reading frame was replaced with a chimeric green fluorescent protein gene. A TK-deleted recombinant virus (BoHV-5 TK∆) was then generated in which most of the TK open reading frame sequences were deleted and replaced with a chimeric â-galactosidase gene. Subsequently, using the BoHV-5 gE∆ virus as backbone, a double gene-deleted (TK plus gE) BoHV-5 recombinant (BoHV-5 gE/TK∆) was generated. The deletion of the gE and TK genes was confirmed by immunoblotting and PCR, respectively. In Madin Darby bovine kidney (MDBK) cells, the mutants lacking gE (BoHV-5 gE∆) and TK + gE (BoHV-5 gE/TK∆) produced small plaques while the TK-deleted BoHV-5 produced wild-type-sized plaques. The growth kinetics and virus yields in MDBK cells for all three recombinants (BoHV-5 gE∆, BoHV-5 TK∆ and BoHV-5 gE/TK∆) were similar to those of the parental virus. It is our belief that the dual gene-deleted recombinant (BoHV-5 gE/TK∆) produced on the background of a highly neurovirulent Brazilian BoHV-5 strain may have potential application in a vaccine against BoHV-5.


Assuntos
Animais , Bovinos , Deleção de Genes , /genética , Timidina Quinase/genética , Proteínas do Envelope Viral/genética , Vírus Defeituosos/genética , Eletroforese em Gel de Poliacrilamida , Proteínas de Fluorescência Verde/genética , /imunologia , /patogenicidade , Immunoblotting , Reação em Cadeia da Polimerase , Recombinação Genética/genética , Timidina Quinase/imunologia , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Proteínas do Envelope Viral/imunologia , Vacinas Virais/genética , Vacinas Virais/imunologia , Virulência/genética
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