1. Single-reaction genomic amplification accelerates sequencing and vaccine production for classical and Swine origin human influenza a viruses.
- Author
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Zhou B, Donnelly ME, Scholes DT, St George K, Hatta M, Kawaoka Y, and Wentworth DE
- Subjects
- Animals, Base Sequence, Genomics, Models, Genetic, Molecular Sequence Data, Mutation, Plasmids metabolism, RNA, Viral genetics, Reverse Transcriptase Polymerase Chain Reaction, Sequence Analysis, DNA, Swine, Influenza A Virus, H1N1 Subtype genetics, Influenza A Virus, H3N2 Subtype genetics, Influenza A virus genetics, Influenza Vaccines genetics
- Abstract
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).
- Published
- 2009
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