FIG. 2.
Genomic amplification of human influenza A viruses directly from clinical swab specimens. (A). M-RTPCR of influenza A virus rescued directly from human swab specimens. RNA was isolated directly from nas and oro swab specimens collected in New York during the 2005/2006 influenza season (lanes 1 to 5), the 2001/2002 season (lane 6), and the 2008/2009 season that had previously tested positive for influenza A virus. Work with blinded patient swab specimens was done in accordance with Institutional Review Board protocols 07-022 and 09-025. The vRNA was isolated from 100 μl of swab suspension, and one-sixth of the total RNA was amplified by M-RTPCR and subjected to agarose gel electrophoresis. The amplicons shown are from the (i) H3N2 (nas), (ii) H3N2 (oro), (iii) H3N2 (nas or oro), (iv) H3N2 (nas), (v) H3N2 (oro), (vi) H1N1 (nas), and (vii) swine origin H1N1 (nas) subtypes. Where known, the real-time RT-PCR CT corresponding to a particular swab is listed below the lanes. ND, not determined; N, no-template M-RTPCR control; and L, DNA ladder (1 kb plus; Invitrogen). Amplicons were electrophoresed through a 1.5% agarose gel, which was then stained with ethidium bromide. (B) Modification of reverse-genetics plasmid for in-fusion (Clontech) cloning. Shown is a schematic diagram for modified reverse-genetics vector pG26A12 and cloning of M-RTPCR amplicons for generation of vRNA expression plasmids. Twenty-five nucleotides, comprising the 13 nucleotides of the 5′ termini (Uni13) and the 12 nucleotides of the 3′ termini (Uni12) of all influenza A vRNAs, were inserted between the RNA polymerase 1 promoter and terminator. pG26A12 is linearized by StuI digestion, exposing nucleotides identical to those at the termini of M-RTPCR amplicons. The M-RTPCR amplicons have the same termini (Uni13/Uni12 plus some sequence of the Pol I promoter/terminator) as the linearized plasmid for facilitating in-fusion cloning. The clones generated produce authentic vRNA upon transfection into appropriate cells.