Rapid detection of reassortment of pandemic H1N1/2009 influenza virus

Poon, LLM; Mak, PWY; Li, OTW; Chan, KH; Cheung, CL; Ma, ES; Yen, HL; Vijaykrishna, D; Guan, Y; Peiris, JSM

BACKGROUND: Influenza viruses can generate novel reassortants in co-infected cells. The global circulation and occasional introductions of pandemic H1N1/2009 virus in humans and in pigs, respectively, might provide opportunities for this virus to reassort with other influenza viruses. These possible reassortment events might alter virulence and/or transmissibility of the new reassortants. Investigations that can detect such possible reassortants should therefore be included as a part of pandemic influenza surveillance plans. METHODS: We established a real-time RT-PCR-based strategy for the detection of reassortment of pandemic H1N1/2009 virus. Singleplex SYBR green-based RT-PCR assays specific for each gene segment of pandemic H1N1/2009 were developed. These assays were evaluated by influenza viruses with different genetic backgrounds. RESULTS: All human pandemic H1N1 (N=27) and all seasonal human (N=58) isolates were positive and negative, respectively, for all 8 segments. Of 48 swine influenza viruses isolated from our on-going surveillance program of influenza viruses in swine, 10 were positive in all reactions. All 8 viral segments of these 10 samples were confirmed to be of pandemic H1N1 origin, indicating that these were caused by zoonotic transmissions from human to pigs. The 38 swine viruses that are non-pandemic H1N1/2009 had 1 to 6 gene segments positive in the tests. Further characterization of these non-pandemic H1N1/2009 swine viruses indicated that these PCR positive genes are the precursor genes of pandemic H1N1/2009 virus. CONCLUSIONS: Our results demonstrated that these assays can detect re-introductions of pandemic H1N1/2009 virus in pigs. These assays might be useful screening tools for identifying viral reassortants derived from pandemic H1N1/2009 or its precursors.