RT.PCR As A Diagnostic Tool for The Detection of Avian Influenza Virus

Avian influenza viruses (AIVs) are important zoonotic pathogens which pose a public health threat to humans, poultry, and swine. Free living waterfowl, particularly migratory dabbling ducks (Anas spp.), are the main natural reservoirs of AIV. Several countries including the United States have implemented AIV surveillance programs in the natural waterfowl reservoir. Real-time RT-PCR has been extensively used for the detection of AIV from waterfowl because of its low cost and high throughput. However, due to frequent mutations in influenza viruses, the sequence of primer sets used in PCR-based detection must be continuously updated for the detection of currently circulating strains. Also, it is epidemiologically important to isolate the actual circulating subtypes of AIV rather than to determine only if a sample is positive or negative for viral nucleic acid by RT-PCR.We conducted four different experiments in this study. In the first experiment, a large number of qRT-PCR–negative cloacal samples (n = 1,369) was examined for the presence of AIV by virus isolation in non-SPF ECEs from commercial sources instead of using SPF embryos. Reports on the isolation of AIV from PCR-negative samples are scattered throughout the veterinary literature but no comprehensive study is available on this subject. The present study is the first systematic study on this subject. The results of the present study reinforce the observation that some PCR-negative samples can yield AIV by virus isolation. Of the 1,369 AIV qRT-PCR negative samples examined, 147<b yielded HA-positive allantoic fluids of which 82 were confirmed to be AIV by qRTPCR.Although molecular methods are considered more sensitive than virus isolation,the failure of qRT-PCR to detect all AIV in the present study can partially be explained.