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Journal of Clinical Microbiology, July 2000, p. 2579-2583, Vol. 38, No. 7
0095-1137/00/$04.00+0

A Simple Restriction Fragment Length Polymorphism-Based Strategy That Can Distinguish the Internal Genes of Human H1N1, H3N2, and H5N1 Influenza A Viruses

Lynn A. Cooper* and Kanta Subbarao

Influenza Branch, Division of Viral and Rickettsial Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia 30333

Received 19 January 2000/Returned for modification 23 March 2000/Accepted 1 May 2000

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 designed. Subtyping was based on subtype-specific restriction fragment length polymorphism (RFLP) patterns within the amplified regions. The strategy was tested in a blinded fashion using 10 control viruses of each subtype (total, 30) and was found to be very effective. Once standardized, the genotyping method was used to identify the origin of the internal genes of 51 influenza A viruses isolated from humans in Hong Kong during and immediately following the 1997-1998 H5N1 outbreak. No avian-human or H1-H3 reassortants were 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.

* Corresponding author. Mailing address: Influenza Branch, Division of Viral and Rickettsial Diseases, Centers for Disease Control and Prevention, 1600 Clifton Road, N.E., MS G-16, Atlanta, GA 30333. Phone: (404) 639-3591. Fax: (404) 639-2334. E-mail: lcooperbiol{at}earthlink.net.

Journal of Clinical Microbiology, July 2000, p. 2579-2583, Vol. 38, No. 7
0095-1137/00/$04.00+0


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