Identification and subtyping of avian influenza viruses by reverse transcription-PCR

Avian influenza viruses have 15 different hemagglutinin (HA) subtypes (H1-H 15). We report a procedure for the identification and HA-subtyping of avian influenza virus by reverse transcription-PCR (RT-PCR). The avian influenza virus is identified by RT-PCR using a set of primers specific to the nucle oprotein (NP) gene of avian influenza virus. The HA-subtypes of avian influ enza virus were determined by running simultaneously 15 RT-PCR reactions, e ach using a set of primers specific to one HA-subtype. For a single virus s train or isolate. only one of the 15 RT-PCR reactions will give a product o f expected size, and thus the HA-subtype of the virus is determined. The re sult of HA-subtyping was then confirmed by sequence analysis of the PCR pro duct. A total of 80 strains or isolates of avian influenza viruses were sub typed by this RT-PCR procedure. and the result of RT-PCR gave an excellent (100%) correlation with the result of the conventional serological method. The RT-PCR procedure we developed is rapid and sensitive. and could be used for the identification and HA-subtyping of avian influenza virus in organ homogenates. (C) 2001 Elsevier Science B.V. All rights reserved.