title: Development and validation of a concentration method for the detection of influenza a viruses from large volumes of surface water.
abstract: 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.
authors.affiliation: Institut Pasteur de Lille, Unité de Sécurité Microbiologique, 1 Rue du Professeur Calmette, 59019 Lille, France. nathalie.deboosere@pasteur-lille.fr
authors.forename: Nathalie
authors.initials: N
authors.lastname: Deboosere
authors.forename: Srey Viseth
authors.initials: SV
authors.lastname: Horm
authors.forename: Anthony
authors.initials: A
authors.lastname: Pinon
authors.forename: Jessica
authors.initials: J
authors.lastname: Gachet
authors.forename: Chloé
authors.initials: C
authors.lastname: Coldefy
authors.forename: Philippe
authors.initials: P
authors.lastname: Buchy
authors.forename: Michèle
authors.initials: M
authors.lastname: Vialette
mesh_terms: D005591:Chemical Fractionation; D005374:Filtration / Q000379:methods; D005602:France; D053124:Influenza A Virus, H5N1 Subtype / Q000302:isolation & purification*; D011092:Polyethylene Glycols / Q000737:chemistry; D014773:Virology / Q000379:methods*; D014871:Water Microbiology
chemical_list: D011092:Polyethylene Glycols