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Journal of Virology, October 2009, p. 10309-10313, Vol. 83, No. 19
0022-538X/09/$08.00+0     doi:10.1128/JVI.01109-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Single-Reaction Genomic Amplification Accelerates Sequencing and Vaccine Production for Classical and Swine Origin Human Influenza A Viruses {triangledown}

Bin Zhou,1,2 Matthew E. Donnelly,1 Derek T. Scholes,1 Kirsten St. George,1 Masato Hatta,3 Yoshihiro Kawaoka,3,4 and David E. Wentworth1,2*

Wadsworth Center, New York State Department of Health,1 Department of Biomedical Sciences, School of Public Health, State University of New York, Albany, New York 12201,2 Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin—Madison, Madison, Wisconsin 53706,3 Division of Virology, Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan4

Received 31 May 2009/ Accepted 8 July 2009

Pandemic influenza A viruses that emerge from animal reservoirs are inevitable. Therefore, rapid genomic analysis and creation of vaccines are vital. We developed a multisegment reverse transcription-PCR (M-RTPCR) approach that simultaneously amplifies eight genomic RNA segments, irrespective of virus subtype. M-RTPCR amplicons can be used for high-throughput sequencing and/or cloned into modified reverse-genetics plasmids via regions of sequence identity. We used these procedures to rescue a contemporary H3N2 virus and a swine origin H1N1 virus directly from human swab specimens. Together, M-RTPCR and the modified reverse-genetics plasmids that we designed streamline the creation of vaccine seed stocks (9 to 12 days).

* Corresponding author. Mailing address: Wadsworth Center, NYSDOH, Griffin Laboratory, 5668 State Farm Road, Slingerlands, NY 12159. Phone: (518) 862-4311. Fax: (518) 869-6487. E-mail: dwentwor{at}wadsworth.org

{triangledown} Published ahead of print on 15 July 2009.

Journal of Virology, October 2009, p. 10309-10313, Vol. 83, No. 19
0022-538X/09/$08.00+0     doi:10.1128/JVI.01109-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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