Georgia Veterinary Scholars Program at the UGA College of Veterinary Medicine

The University of Georgia College of Veterinary Medicine

2005 Georgia Veterinary Scholars

Georgia Veterinary Scholar	Faculty Mentor

Jody Willis University of Georgia Class of 2007	Dr. David Suarez

Development and Optimization of a Real Time RT-PCR test for the H8 Hemagglutinin Subtype of Avian Influenza

Jody Willis 1,2, Erica Spackman 2, and David L. Suarez 2

1) College of Veterinary Medicine , University of Georgia , Athens , GA

2) Southeast Poultry Research Laboratory, USDA, ARS, Athens , GA

The traditional method for the detection of avian influenza is virus isolation in embryonating chicken eggs followed by serologic tests to confirm the subtype of the virus. This method is time consuming and may require up to two weeks to obtain results. In an outbreak situation, this delay in obtaining results can hamper control efforts and allow the outbreak to spread. Alternative rapid detection technologies have become widely available in the last five years, including a real-time RT-PCR (RRT-PCR) test for avian influenza. The AI RRT-PCR test could identify positive samples in as little as three hours with a sensitivity similar to virus isolation. This test was shown to be a valuable control tool during the Virginia H7N2 AI outbreak in 2002. The design of the test included a type A screening test, designed to pick up all AI isolates, and a hemagglutinin (HA) subtype confirmation test. Sixteen HA subtypes have been described, but RRT-PCR tests have been developed for only a few of these subtypes. Our goal is to create a RRT-PCR test that will distinguish H8 from the other 15 hemagglutinin subtypes of avian influenza. The determination of the subtype is necessary to characterize an influenza virus. Also, a rapid RRT-PCR test provides confirmation of the type A screening test and provides epidemiologic information useful in controlling an outbreak. To develop a H8 specific RRT-PCR test, a comparison of available sequence was made to identify conserved regions of the genome. Two sets of primers and probes were selected and synthesized by a commercial company. After an initial comparison of both tests, a single primer and probe set was selected for further work. The primers/probe set was optimized for sensitivity and specificity using a one-step RT-PCR kit comparing primer, probe, and MgCl concentration, optimal annealing temperatures, and other parameters. To test the specificity of the primers/probe set we ran RRT-PCR against a panel of all 15 known HA subtypes. The results indicated that the primers/probe set was specific for the H8 subtype of AI, and the test could detect up to a dilution of 10 -7 of a reference virus. After bench validation, experimental animal studies were performed for further comparison of the primers/probe set. We inoculated 30 Leghorn chickens with two different viruses and collected oral and cloacal swabs for testing of the primers/probe set. At the end of the animal experiment, the chickens were bled to determine the immune response and specifically the level of antibody titers that were achieved against the H8 subtype.