A simple restriction fragment length polymorphism-based strategy that can distinguish the internal genes of human H1N1, H3N2, and H5N1 influenza A viruses

A simple molecular technique for rapid genotyping was developed to monitor the internal gene composition of currently circulating influenza A viruses. Sequence information from recent H1N1, H3N2, and H5N1 human virus isolates was used to identify conserved regions within each internal gene, and gene -specific PCR primers capable of amplifying all three virus subtypes were d esigned, Subtyping was based on subtype-specific restriction fragment lengt h polymorphism (RFLP) patterns within the amplified regions. The strategy w as tested in a blinded fashion using 10 control viruses of each subtype (to tal, 30) and was found to be very effective. Once standardized, the genotyp ing method was used to identify the origin of the internal genes of 51 infl uenza A viruses isolated from humans in Hong Kong during and immediately fo llowing the 1997-1998 H5N1 outbreak No avian-human or H1-H3 reassortants we re detected. Less than 2% (6 of 486) of the RFLP analyses were inconclusive ; all were due to point mutations within a restriction site. The technique was also used to characterize the internal genes of two avian H9N2 viruses isolated from children in Hong Kong during 1999.