Open AccessSingle-Reaction Genomic Amplification Accelerates Sequencing and Vaccine Production for Classical and Swine Origin Human Influenza A Viruses
Author(s) -
Bin Zhou,
Matthew E. Donnelly,
Derek T. Scholes,
Kirsten St. George,
Masato Hatta,
Yoshihiro Kawaoka,
David E. Wentworth
Publication year - 2009
Publication title -
journal of virology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.617
H-Index - 292
eISSN - 1070-6321
pISSN - 0022-538X
DOI - 10.1128/jvi.01109-09
Subject(s) - biology , reverse genetics , virology , amplicon , plasmid , virus , reverse transcriptase , reverse transcription polymerase chain reaction , influenza a virus , genetics , genome , polymerase chain reaction , gene , gene expression
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).