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Title: Analytical validation of a real-time RT-PCR test for Pan-American lineage H7 subtype avian influenza viruses

item Spackman, Erica
item Suarez, David

Submitted to: Journal of Veterinary Diagnostic Investigation
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/13/2008
Publication Date: 9/29/2008
Citation: Spackman, E., Ip, H.S., Suarez, D.L., Slemons, R., Stallknecht, D. 2008. Analytical validation of a real-time reverse transcription polymerase chain reaction test for Pan-American lineage H7 subtype avian influenza viruses. Journal of Veterinary Diagnostic Investigation. 20:612-616.

Interpretive Summary: Avian influenza viruses can belong to any of 16 "H" subtypes. Two of these subtypes, H5 and H7, are considered especially important because sometimes they can cause severe disease in chickens and turkeys. Therefore rapid identification of these subtypes in type A influenza positive clinical diagnostic specimens and specimens from wild birds is important for disease control. Tests that detect the virus genetic material are rapid and sensitive and a genetic test for the H7 subtype was first reported in 2002, however it was eventually noted that the original test did not detect all H7 viruses from North and South America due to the high genetic variation of avian influenza virus. Since 2002, a great deal more sequence information has become available for the H7 subtype so a new test to identify the H7 subtype of avian influenza viruses from North and South America was developed and evaluated for sensitivity and specificity. The sensitivity was similar to that of the previous test and the observed specificity was expected; only North and South American H7 viruses were detected and all the American H7 viruses that were tested were detected.

Technical Abstract: Rapid detection of avian influenza virus and identification of the H5 and H7 hemagglutinin subtypes some of which are associated with high pathogenicity in poultry is critical for clinical diagnosis and wild bird monitoring programs. A real-time RT-PCR test for identification of the H7 subtype in North American AI viruses was first reported in 2002, however with the recent surveillance in wild birds and outbreaks in poultry in North and South America it was discovered that the 2002 test did not detect all H7 viruses from North and South America. Therefore, a new test was developed using recently available nucleotide sequences. The specificity of the new assay was characterized with an RNA panel comprised of 19 H7 viruses from around the world and RNA from all HA subtypes except H16. Specificity for the American lineage H7 viruses was observed. The limits of detection were determined to be between 103 to 104 gene copies with in vitro transcribed RNA and 100.0 to 100.8 50% egg infectious doses per reaction. The new 2008 Pan-American H7 test also was shown to perform similarly to the 2002 test with specimens from chickens experimentally exposed to A/Chicken/BritishColumbia/314514-2/04 H7N3 highly pathogenic avian influenza virus. Furthermore, the 2008 test was able to detect 27 H7 viruses from North American wild birds which were not detected by the 2002 H7 test and had 100% agreement with hemagglutination inhibition assay with a total of 468 avian influenza virus isolates from wild bird surveillance including the 27 positives.