Validated H5 Eurasian Real-Time Reverse Transcriptase-Polymerase Chain Reaction and Its Application in H5N1 Outbreaks in 2005-2006

• Published in: Avian diseases Vol. 51; no. 1 Suppl; pp. 373 - 377
• Main Authors: Slomka, M.J, Pavlidis, T, Banks, J, Shell, W, McNally, A, Essen, S, Brown, I.H
• Format: Journal Article
• Language: English
• Published: United States 01.03.2007
• Subjects: Animals; automation; avian influenza; Birds - virology; calibration; diagnostic techniques; disease detection; disease diagnosis; disease outbreaks; Disease Outbreaks - veterinary; disease surveillance; food animals; highly pathogenic avian influenza; Influenza A virus; Influenza A Virus, H5N1 Subtype - isolation & purification; Influenza in Birds - epidemiology; Influenza in Birds - virology; new methods; optimization; poultry; Reproducibility of Results; reverse transcriptase polymerase chain reaction; Reverse Transcriptase Polymerase Chain Reaction - methods; Reverse Transcriptase Polymerase Chain Reaction - veterinary; RNA; Sensitivity and Specificity; strain differences; strains; Time Factors; wild birds
• ISSN: 0005-2086; 1938-4351
• DOI: 10.1637/7664-060906R1.1

Real time reverse transcriptase (RRT)-polymerase chain reaction (PCR) for the detection of Eurasian H5 avian influenza virus (AIV) isolates was adapted from an existing protocol, optimized, and validated using a number of genetically diverse H5 isolates (n = 51). These included 34 “Asian lineage” H5N1 highly pathogenic avian influenza (HPAI) viruses (2004-2006), plus 12 other H5 isolates from poultry outbreaks and wild birds in the Eastern Hemisphere (1996-2005). All 51 were positive by H5 Eurasian RRT-PCR. Specificity was assessed by testing representative isolates from all other AI virus subtypes (n = 52), non-AI avian pathogens (n = 8), plus a negative population of clinical specimens derived from AI-uninfected wild birds and poultry (n = 604); all were negative by H5 Eurasian RRT-PCR. RNA was directly extracted from suspect HPAI H5N1 clinical specimens (Africa, Asia, and Europe; 2005-2006; n = 58) from dead poultry and wild birds, and 55 recorded as positive by H5 Eurasian RRT-PCR: Fifty-one of these 55 were in agreement with positive AIV isolation in embryonated chickens' eggs. H5 Eurasian RRT-PCR was invaluable in H5 outbreak diagnosis and management by virtue of its rapidity and high degree of sensitivity and specificity. This method provides a platform for automation that can be applied for large-scale intensive investigations, including surveillance.