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

TLDR

Pandemic influenza A viruses that emerge from animal reservoirs are inevitable. The study aims to enable rapid genomic analysis and vaccine creation by developing a multisegment reverse transcription‑PCR method that amplifies all eight viral RNA segments. The M‑RTPCR approach simultaneously amplifies eight genomic RNA segments, allowing high‑throughput sequencing and cloning into reverse‑genetics plasmids, which were used to rescue contemporary H3N2 and swine‑origin H1N1 viruses directly from human swabs. This integrated method reduces vaccine seed stock production to 9–12 days.

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).