Influenza Virus Propagation and Quantification

Branched PEI was
ordered from Sigma-Aldrich. CpG ODN1826 was a product of InvivoGen,
USA. Spodoptera frugiperda (Sf9, ATCC, CRL-1711)
insect cells were cultured in protein-free ESF 921 (Expression Systems,
USA). Aichi HA (H3) and hrHA3 were expressed and purified as described
previously.^{34 (link)} Purified proteins were assayed
using a BCA assay kit (Thermo Fisher Scientific, USA).
Madin-Darby
canine kidney (MDCK, ATCC CCL-34) cells were grown in Eagle’s
Minimum Essential Medium (EMEM, ATCC 30–2003) supplemented
with 10% heat-inactivated fetal calf serum (FCS, ATCC 30–2020)
and 1% penicillin/streptomycin in a CO₂ (5%) incubator
at 37 °C. HEK 293T (ATCC CRL-3216) cells were grown in Dulbecco’s
Modified Eagle’s Medium (DMEM) (ATCC 30–2002) containing
10% FCS and 2 mM l-glutamine (ATCC 30–2214).
Influenza A/Aichi/2/1968 (Aic, H3N2) and A/Philippines/2/1982 (Phi,
H3N2) were passaged in embryonated chicken eggs. Mouse-adapted Aic
and Phi were expanded in intranasally (i.n.) infected mouse lungs.
The standard Reed and Muench method was used to measure the virus
median lethal dose (LD₅₀).