Single-reaction genomic amplification accelerates sequencing and vaccine production for classical and Swine origin human influenza a viruses.
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).
Zhou
Bin
B
Wadsworth Center, New York State Department of Health, Albany, New York 12201, USA.
Donnelly
Matthew E
ME
Scholes
Derek T
DT
St George
Kirsten
K
Hatta
Masato
M
Kawaoka
Yoshihiro
Y
Wentworth
David E
DE
D000818:Animals; D001483:Base Sequence; D023281:Genomics; D053118:Influenza A Virus, H1N1 Subtype / Q000235:genetics*; D053122:Influenza A Virus, H3N2 Subtype / Q000235:genetics*; D009980:Influenza A virus / Q000235:genetics*; D007252:Influenza Vaccines / Q000235:genetics*; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D012367:RNA, Viral / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D013552:Swine
D007252:Influenza Vaccines; D012367:RNA, Viral