APPLICATION OF BIOLOGICAL AND MOLECULAR TECHNIQUES TO THE DIAGNOSIS AND CONTROL OF AVIAN INFLUENZA AND OTHER EMERGING POULTRY PATHOGENS
Location: Exotic and Emerging Avian Viral Diseases Research Unit
Title: Development and bench validation of real time RT-PCR protocols for rapid detection of the subtypes H6, H9 and H11 of avian influenza viruses in experimental samples
Submitted to: Journal of Veterinary Diagnostic Investigation
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: June 25, 2007
Publication Date: November 13, 2007
Citation: Das, A., Suarez, D.L. 2007. Development and bench validation of real-time reverse transcription polymerase chain reaction protocols for rapid detection of the subtypes H6, H9, and H11 of avian influenza viruses in experimental samples. Journal of Veterinary Diagnostic Investigation. 19:625-634.
Interpretive Summary: Avian influenza virus can cause important disease outbreaks in poultry, including chickens and turkeys. The rapid and accurate diagnosis of an outbreak can greatly aid the control effort and prevent the spread of the virus to other poultry flocks. Avian influenza can be separated into 16 groups based on differences in the surface protein hemagglutinin. The determination of the hemagglutinin subtype is a valuable way to initially characterize a virus. Currently we have rapid PCR tests to identify the H5 and H7 subtypes of avian influenza. This paper describes the development and testing of rapid PCR tests for the H6, H9, and H11 subtypes of avian influenza. The test was evaluated by testing experimentally infected birds and comparing it to another rapid PCR test that can identify any avian influenza virus. The results show good correlation with the different tests and the ability to rapidly identify the H6, H9, and H11 subtypes.
Real time RT-PCR (RRT-PCR) is commonly used for the rapid detection of avian influenza viruses (AIV) from clinical samples. Samples are typically screened for type A influenza by targeting the matrix gene, and then positive samples are further tested for hemagglutinin (HA) and neuraminidase (NA) subtypes. There are 16 known serologically distinct HA subtypes of AIV described. Currently only RRT-PCR tests to the H5 and H7 subtypes have been widely tested, which greatly restricts the use of RRT-PCR for subtyping. In this study, we developed and tested RRT-PCR assays for rapid detection of the AIV subtypes H6, H9 and H11. The primers and probes for RRT-PCR were designed from nucleotide sequences of the HA-genes which were either downloaded from GenBank (for H6 and H9) or sequenced for this study. The specificity and sensitivity of the RRT-PCR assays were tested based on the detection of the virus from a proficiency panel consisting of 15 different HA subtypes of AIVs and from serial dilutions of target viral RNA. The subtype specific RRT-PCR assays were used to detect the virus in cloacal and oro-pharyngeal swabs of experimental chickens inoculated with H6, H9 and H11 AIVs and the test results were compared with validated RRT-PCR assays based on the amplification of AI matrix gene. The percent agreements of test results between the two protocols for the detection of H6, H9 and H11 in experimental samples were 93%, 75% and 100%, respectively, for cloacal swabs, and 91%, 97%, and 97%, respectively, for oro-pharyngeal swabs, indicating high accuracy in the detection of H6, H9 and H11 by RRT-PCR assays developed in this study.