HA and NA genes for the Perth/2009 viral strain were cloned from recombinant virus obtained from BEI Resources (NR-41803) into the pHW2000 (79 (link)) influenza reverse-genetics plasmids to create pHW-Perth09-HA and pHW-Perth09-NA.
We initially created a virus with the HA and NA from Perth/2009 and internal genes from WSN/1933 and passaged it in cell culture to test its genetic stability. To generate this virus, we transfected a coculture of 293T and MDCK–SIAT1–TMPRSS2 in D10 media (DMEM, supplemented with 10% heat-inactivated FBS, or fetal bovine serum, 2 mM L-glutamine, 100 U of penicillin per milliliter, and 100 g of streptomycin per milliliter) with equal amounts of pHW-Perth09-HA, pHW-Perth09-NA, the pHW18 series of plasmids (79 (link)) for all non-HA/NA viral genes, and pHAGE2–EF1aInt–TMPRSS2–IRES-mCherry-W. The next day, we changed the media to influenza growth media (IGM, consisting of Opti-MEM supplemented with 0.01% heat-inactivated FBS, 0.3% BSA, 100 U of penicillin per milliliter, 100 g of streptomycin per milliliter, and 100 g of calcium chloride per milliliter; no trypsin was added since there was TMPRSS2) and then collected the viral supernatant at 72 h posttransfection. This viral supernatant was blind passaged in MDCK–SIAT1–TMPRSS2 a total of six additional times. We isolated viral RNA from these passaged viruses and sequenced the HA gene. The passaged HA had two mutations, G78D and T212I, which enhanced viral growth as shown inSI Appendix, Fig. S1 . The HA with these two mutations was cloned into pHW2000 (79 (link)) and pICR2 (80 (link)) to create pHW-Perth09-HA-G78D-T212I and pICR2-Perth09-HA-G78D-T212I. For all subsequent experiments, we used viruses with the HA containing these two mutations to improve titers and viral genetic stability, and this is the HA that we refer to as Perth/2009. We used all non-HA genes (including NA) from WSN/1933 to help increase titers and reduce biosafety concerns.
The codon-mutant libraries were generated using the approach in ref. 81 (link) with the modifications in ref. 82 (link). SeeSI Appendix, Supplementary Text for full details.
We initially created a virus with the HA and NA from Perth/2009 and internal genes from WSN/1933 and passaged it in cell culture to test its genetic stability. To generate this virus, we transfected a coculture of 293T and MDCK–SIAT1–TMPRSS2 in D10 media (DMEM, supplemented with 10% heat-inactivated FBS, or fetal bovine serum, 2 mM L-glutamine, 100 U of penicillin per milliliter, and 100 g of streptomycin per milliliter) with equal amounts of pHW-Perth09-HA, pHW-Perth09-NA, the pHW18 series of plasmids (79 (link)) for all non-HA/NA viral genes, and pHAGE2–EF1aInt–TMPRSS2–IRES-mCherry-W. The next day, we changed the media to influenza growth media (IGM, consisting of Opti-MEM supplemented with 0.01% heat-inactivated FBS, 0.3% BSA, 100 U of penicillin per milliliter, 100 g of streptomycin per milliliter, and 100 g of calcium chloride per milliliter; no trypsin was added since there was TMPRSS2) and then collected the viral supernatant at 72 h posttransfection. This viral supernatant was blind passaged in MDCK–SIAT1–TMPRSS2 a total of six additional times. We isolated viral RNA from these passaged viruses and sequenced the HA gene. The passaged HA had two mutations, G78D and T212I, which enhanced viral growth as shown in
The codon-mutant libraries were generated using the approach in ref. 81 (link) with the modifications in ref. 82 (link). See
