RESUMO
The role of environmental reservoirs in avian influenza virus (AIV) transmission has been investigated during AIV-associated outbreaks. To date, no method has been defined for detection of AIV from mud samples. A procedure using elution and polyethylene glycol (PEG) concentration steps was designed to detect AIV by RT-PCR from 42g of raw mud, corresponding to 30g of the solid fraction of mud. RNA was recovered with MagMAX AI/ND Viral RNA Isolation kit (Ambion, Austin, TX). Three elution buffers were studied and viral recoveries higher than 29% were yielded by elution with a 10% beef extract solution (pH 7). The overall method showed that, under some conditions, virus was not detectable in PEG samples, whereas viruses were detected in the elution fractions. PCR curves were improved significantly by running the amplification reaction with a mixture containing a PCR additive for inhibitor removal, such as T4 gene 32 protein (Gp32), although PCR inhibitors from mud were removed partially from PEG samples. A theoretical detection threshold of 5×10(5) RNA copies of H5N1 virus per 30g of solid mud could be obtained by elution. The overall method has proved successful for detecting H5N1 virus contamination of mud specimens collected during outbreak investigations of avian influenza in Cambodia.
Assuntos
Microbiologia Ambiental , Virus da Influenza A Subtipo H5N1/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Esgotos/virologia , Camboja , Virus da Influenza A Subtipo H5N1/genética , RNA Viral/genética , RNA Viral/isolamento & purificação , Sensibilidade e Especificidade , Manejo de Espécimes/métodosRESUMO
Contamination of lakes and ponds plays an essential role as a reservoir of avian influenza A virus (AIV) in the environment. A method to concentrate waterborne AIV is a prerequisite for the detection of virus present at low levels in water. The aim of this study was to develop and validate a method for the concentration and detection of infectious AIV from large volumes of surface water samples. Two filtration systems, glass wool and electropositive NanoCeram filter, were studied. The individual effects of filtration-elution and polyethylene glycol (PEG) concentration parameters on the recovery efficiency of the H1N1 strain from 10-liter surface water samples were assessed. An ultimate 1% recovery rate of infectious viruses was achieved with the optimal protocol, corresponding to filtration through glass wool, followed by a viral elution step and then a PEG concentration. This method was validated for the detection of highly pathogenic H5N1 strains from artificially contaminated larger water volumes, from 10 to up to 50 liters, from different sources. The viral recovery efficiencies ranged from 0.01% to 7.89% and from 3.63% to 13.79% with lake water and rainwater, respectively. A theoretical detection threshold of 2.25 × 10(2) TCID(50) (50% tissue culture infectious dose) in the filtered volume was obtained for seeded lake waters by M gene reverse transcriptase PCR (RT-PCR). Moreover, the method was used successfully in field studies for the detection of naturally occurring influenza A viruses in lake water in France.