Combined PCR-heteroduplex mobility assay for detection and differentiationof influenza A viruses from different animal species

Transfer of influenza A viruses from animal hosts to man may lead to the em ergence of new human pandemic strains. The early detection and identificati on of such events are therefore paramount in the surveillance of influenza viruses. To detect and partially characterize influenza A viruses from diff erent animal species, a combined reverse transcription (RT)-PCR heteroduple x mobility assay (HMA) was designed. This M gene RT-PCR was shown to be sen sitive and specific for the detection of human, avian, and swine influenza A viruses. PCR amplicons from human, avian, and swine viruses of 15 differe nt subtypes, with between 1.9 and 21.4% nucleotide divergence, were differe ntiated by HMA. Sequencing of the amplicons showed that the heteroduplex mo bility patterns correlated with the sequence divergence between test and re ference DNA. The application of the RT-PCR HMA method for rapid screening o f samples was assessed with a reference panel of viruses of human, avian, a nd swine origin. The avian H9N2 virus A/HongKong/1073/99, which crossed the species barrier to humans, was screened against the reference panel. It wa s found to be most closely related to the avian A/Quail/HongKong/G1/97 H9N2 reference PCR product. Sequence analysis showed a nucleotide divergence of 1.1% between the A/Quail/HongKong/G1/97 and A/HongKong/1073/99 amplicons. From the results of our work, we consider the RT-PCR HMA method described t o offer a rapid and sensitive means for screening for novel or unusual infl uenza viruses.