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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., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Contaminated mud and soil may play roles as reservoirs and sources of transmission for avian influenza A virus. However, the persistence of highly pathogenic avian influenza (HPAI) H5N1 virus in soil or mud has not been well documented, and specific methods of H5N1 virus detection in mud and soil specimens have not been described. The aim of this work was to evaluate the capacities of five different commercial kits and one elution-concentration technique to extract nucleic acids from H5N1 virus and to detect infectious viral particles in experimentally infected mud specimens. The viral RNA detection thresholds for the QIAamp kit, Trizol LS and the MagNA Pure LC kit were 5 × 10(2)RNA copies per gram of mud. Trizol reagent and the RNA PowerSoil™ kit were unsuccessful in recovering any viral RNA from mud. When the elution-concentration technique was performed prior to nucleic acid extraction, the performance of the MagNA Pure kit increased to a level that allowed the detection of H5N1 nucleic acids in naturally contaminated environmental samples that had previously tested negative after direct extraction using commercial kits. The levels of detection of infectious virus after inoculation into embryonated eggs were higher in concentrates than in eluates., (Copyright © 2011 Elsevier B.V. All rights reserved.)