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

Abstract

Transfer of influenza A viruses from animal hosts to man may lead to the emergence of new human pandemic strains. The early detection and identification of such events are therefore paramount in the surveillance of influenza viruses. To detect and partially characterize influenza A viruses from different animal species, a combined reverse transcription (RT)-PCR heteroduplex mobility assay (HMA) was designed. This M gene RT-PCR was shown to be sensitive and specific for the detection of human, avian, and swine influenza A viruses. PCR amplicons from human, avian, and swine viruses of 15 different subtypes, with between 1.9 and 21.4% nucleotide divergence, were differentiated by HMA. Sequencing of the amplicons showed that the heteroduplex mobility patterns correlated with the sequence divergence between test and reference DNA. The application of the RT-PCR HMA method for rapid screening of samples was assessed with a reference panel of viruses of human, avian, and swine origin. The avian H9N2 virus A/HongKong/1073/99, which crossed the species barrier to humans, was screened against the reference panel. It was 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 to offer a rapid and sensitive means for screening for novel or unusual influenza viruses.

FIG. 1

Detection of influenza A viruses from different animal species, by M gene RT-PCR. Influenza A viruses with M genes of human, swine, and avian origin (Table 1) were assayed by RT-PCR with primers specific for the M gene of all influenza A viruses. Amplicons (413 bp) were analyzed by electrophoresis on a 2% agarose gel stained with ethidium bromide. Lanes: 1, Wuh/359/95 (human); 2, Wuh/371/95 (human); 3, Sw/Tw/70 (human); 4, Sw/OMS/82 (swine); 5, Sw/OMS/84 (swine); 6, Dk/Ger/73 (avian); 7, Dk/Ukr/63 (avian); 8, Shearw/Aus/72 (avian). M, DNA molecular weight markers.

FIG. 2

Heteroduplex formation between influenza A viruses from different host species with a human H1N1 reference strain, Bay/95. Amplicons from viruses assayed by the M gene RT-PCR were mixed with the reference virus amplicon, heat denatured, cooled, and then analyzed by electrophoresis on 8% TBE polyacrylamide gels. Homoduplexes and heteroduplexes were visualized by staining with SYBR Green II. Lanes show Bay/95 mixed with H₂O (lane 1), Wuh/371/95 (lane 2), Sw/OMS/82 (lane 3), Dk/Ger/73 (lane 4), Wuh/359/95 (lane 5), Syd/97 (lane 6), Dk/Ukr/63 (lane 7), Dk/Sing/97 (lane 8), Shearw/Aus/72 (lane 9), AfrStarl/Eng/79 (lane 10), Ty/Ont/68 (lane 11), Ty/Wis/66 (lane 12), Ck/Ger/49 (lane 13), Ty/Wey/79 (lane 14), Dk/Alb/76 (lane 15), Gull/MD/77 (lane 16), and Bay/95 (lane 17). M, DNA molecular weight markers.

FIG. 3

Characterization of the virus A/HK/1073/99 by HMA. (A) A panel of nine reference viruses from different host species and the test virus, A/HK/1073/99, were assayed by the M gene RT-PCR. Amplicons of the expected size (413 bp) were visualized on 2% agarose gels stained with ethidium bromide. Lanes: 1, Syd/97; 2, Bay/95; 3, HK/1774/99; 4, Sw/Scot/94; 5, Ty/Wis/66; 6, Dk/Alb/76; 7, Qa/HK/G1/97; 8, Dk/Sing/97; 9, Sw/HK/93; 10, HK/1073/99. (B) Lanes 1 to 9: amplicons of the reference viruses as in panel A and A/HK/1073/99 were mixed and subjected to HMA. Homoduplexes and heteroduplexes were analyzed by polyacrylamide gel electrophoresis on an 8% TBE gel. M, DNA molecular weight markers.