Detection and Quantification of Infectious Avian Influenza A (H5N1) Virus in Environmental Water by Using Real-Time Reverse Transcription-PCR

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 develo...

Full description

Saved in:

Bibliographic Details
Published in:	Applied and Environmental Microbiology Vol. 76; no. 7; pp. 2165 - 2174
Main Authors:	Dovas, C.I, Papanastassopoulou, M, Georgiadis, M.P, Chatzinasiou, E, Maliogka, V.I, Georgiades, G.K
Format:	Journal Article
Language:	English
Published:	Washington, DC American Society for Microbiology 01-04-2010 American Society for Microbiology (ASM)
Subjects:	Adsorption Avian flu Avian influenza virus Biological and medical sciences Deoxyribonucleic acid DNA Erythrocytes Fundamental and applied biological sciences. Psychology Influenza A Virus, H5N1 Subtype - isolation & purification Microbiology Poultry Public Health Microbiology Real time Reference Standards Reproducibility of Results Reservoirs Reverse Transcriptase Polymerase Chain Reaction - methods Reverse Transcriptase Polymerase Chain Reaction - standards Ribonucleic acid RNA RNA, Viral - genetics Sensitivity and Specificity Tobacco Mosaic Virus - genetics Viral infections Water Microbiology Water quality Water Reverse transcription Orthomyxoviridae Real time Virus Avian influenza Infection Polymerase chain reaction Influenzavirus A(H5N1) Influenzavirus A Influenza A virus Viral disease Detection Quantitative analysis
Online Access:	Get full text
Tags:	Add Tag No Tags, Be the first to tag this record!

Description
Summary:	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.
Bibliography:	ObjectType-Article-2 SourceType-Scholarly Journals-1 ObjectType-Feature-1 content type line 23
ISSN:	0099-2240 1098-5336 1098-6596
DOI:	10.1128/aem.01929-09