The University of Georgia College of Veterinary Medicine

2005 Georgia Veterinary Scholars

Georgia Veterinary Scholar	Faculty Mentor

Anusha Natarajan University of Georgia Class of 2007	Dr. David Suarez

RRT-PCR Diagnostic Test for Avian Influenza subtype H10

Anusha Natarajan 1,2, Erica Spackman 2, David L. Suarez 2

College of Veterinary Medicine , University of Georgia , Athens , GA
Southeast Poultry Research Laboratory, USDA,ARS Athens , GA

Avian influenza is an important disease in the poultry industry. It is a negative sense, segmented RNA virus in the orthomyxovirus family. Avian influenza (AI) has been at the crux of international veterinary medicine in the last three years with the numerous outbreaks in southeast Asia and around the world. There are sixteen known subtypes of the hemagglutinin protein, which is the protein responsible for attachement of the virus to the host cell. The ability to rapidly and accurately diagnose an infected flock that has avian influenza is critical for the successful control of an outbreak. A real-time RT-PCR (RRT-PCR) test was previously developed to detect any type A influenza virus from clinical samples from poultry. The type A test was further confirmed by a subtyping RRT-PCR test. However, of the sixteen AI subtypes, RRT-PCR tests are available for only a fraction of them. The goal of the project was to develop and bench validate a test for the H10 subtype.

We designed two sets of primers and probe to the H10 subtype based on the available sequence information. After an initial comparison of the two primer/probe sets, a single set was selected for optimization which included comparing primer, probe and MgCl concentration, and cycling parameters including annealing temperature. Specificity tests were conducted on a panel of all the AI subtypes. After bench validation, a comparison of RRT-PCR with the type A test was used to evaluate its effectiveness from orophayngeal and cloacal samples from experimentally infected chickens with two different H10 isolates. .Initial results are encouraging though the test is far from being finalized.