Influenza A Virus Molecular Virology Techniques
Molecular biological techniques for genomic analysis and for creation of recombinant viruses are critical tools in our efforts to understand and combat influenza A viruses. These molecular virology approaches are used in diagnostics, basic research, molecular epidemiology, bioinformatics, and vaccine development. The majority of the techniques used to study this segmented negative-sense RNA virus begin by purifying RNA from the virus, or infected cells, and converting it to cDNA, then to dsDNA, and amplifying that dsDNA using reverse transcription in combination with the polymerase chain reaction (RTPCR). The RTPCR amplicons can be probed, sequenced, or cloned into a variety of vectors for further analysis and to create recombinant influenza A viruses by plasmid-based reverse genetics. To accelerate the amplification and cloning process, we developed multi-segment-RTPCR (M-RTPCR) techniques that efficiently amplify the eight genomic viral RNA segments (vRNAs) of influenza A virus in a single reaction, irrespective of the virus strain. The M-RTPCR amplicons are ideal for nucleotide sequence analysis and cloning full-length vRNAs into plasmids or other vectors designed for protein expression or reverse genetics. Therefore, we also developed modified reverse-genetics plasmids that are designed to rapidly clone M-RTPCR products, or other full-length vRNA amplicons, using recombination-based techniques. The combination of M-RTPCR and recombination-based cloning confers sensitivity, speed, fidelity, and flexibility to the analysis and rescue of any strain/subtype of influenza A virus, without the need for in vitro propagation. The specific topics described in this chapter include purification of high-quality viral RNA, genomic amplification using two different M-RTPCR schemes, sequencing vRNA amplicons, and cloning vRNA amplicons into our modified reverse-genetics plasmids, or commercially available plasmids.
Key wordsInfluenza A virus RTPCR amplification M-RTPCR Cloning Recombination Ligase independent Reverse genetics Sequencing
The authors would like to thank Derek T. Scholes and Mathew E. Donnelly for their assistance in developing the techniques discussed above. Work in our lab is/was supported by NIH/NIAID 2 U54 AI057178-06, NIH/NIAID P01AI059576-05, and NIH/NIAID R21AI 057941-02 (supported the development of these techniques).
- 1.Palese, P. and M. L. Shaw. 2007. Orthomyxoviridae: The Viruses and Their Replication, p. 1647–1690. In: D. M. Knipe, P. M. Howley, D. E. Griffin, R. A. Lamb, S. E. Straus, M. A. Martin, and B. Roizman (eds.), Fields Virology. 5 ed., vol. 2. Lippincott Williams and Wilkins, Philadelpha, PA.Google Scholar
- 4.Wang, S., Q. Liu, J. Pu, Y. Li, L. Keleta, Y. W. Hu, J. Liu, and E. G. Brown. 2008. Simplified recombinational approach for influenza A virus reverse genetics. J.Virol.Methods.Google Scholar
- 7.Zhou, B., M. E. Donnelly, D. T. Scholes, K. St.George, M. Hatta, Y. Kawaoka, and D. E. Wentworth. 2009. Single-Reaction Genomic Amplification Accelerates Sequencing and Vaccine Production for Classical and Swine Origin Human Influenza A Viruses. J.Virol. 83:10309–10313.Google Scholar