Detection and Quantification of Infectious Avian Influenza A (H5N1) Virus in Environmental Water by Using Real-Time Reverse Transcription-PCR ▿
| Autor: | M. P. Georgiadis, G. K. Georgiades, E. Chatzinasiou, Varvara I. Maliogka, M. Papanastassopoulou, Chrysostomos I. Dovas |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2010 |
| Předmět: |
animal diseases
Orthomyxoviridae Hemagglutinin (influenza) Public Health Microbiology medicine.disease_cause Applied Microbiology and Biotechnology Sensitivity and Specificity Virus Microbiology medicine Tobacco mosaic virus Influenza A virus Ecology biology Influenza A Virus H5N1 Subtype Reverse Transcriptase Polymerase Chain Reaction Embryonated virus diseases Reproducibility of Results Reference Standards biology.organism_classification Virology Influenza A virus subtype H5N1 Tobacco Mosaic Virus Real-time polymerase chain reaction biology.protein RNA Viral Water Microbiology Food Science Biotechnology |
| Popis: | Routes of avian influenza virus (AIV) dispersal among aquatic birds involve direct (bird-to-bird) and indirect (waterborne) transmission. The environmental persistence of H5N1 virus in natural water reservoirs can be assessed by isolation of virus in embryonated chicken eggs. Here we describe development and evaluation of a real-time quantitative reverse transcription (RT)-PCR (qRT-PCR) method for detection of H5N1 AIV in environmental water. This method is based on adsorption of virus particles to formalin-fixed erythrocytes, followed by qRT-PCR detection. The numbers of hemagglutinin RNA copies from H5N1 highly pathogenic AIV particles adsorbed to erythrocytes detected correlated highly with the infectious doses of the virus that were determined for three different types of artificially inoculated environmental water over a 17-day incubation period. The advantages of this method include detection and quantification of infectious H5N1 AIVs with a high level of sensitivity, a wide dynamic range, and reproducibility, as well as increased biosecurity. The lowest concentration of H5N1 virus that could be reproducibly detected was 0.91 50% egg infective dose per ml. In addition, a virus with high virion stability ( Tobacco mosaic virus ) was used as an internal control to accurately monitor the efficiency of RNA purification, cDNA synthesis, and PCR amplification for each individual sample. This detection system could be useful for rapid high-throughput monitoring for the presence of H5N1 AIVs in environmental water and in studies designed to explore the viability and epidemiology of these viruses in different waterfowl ecosystems. The proposed method may also be adapted for detection of other AIVs and for assessment of their prevalence and distribution in environmental reservoirs. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|