2.1. Animals, Reagents, and Viruses

Adult BALB/c mice (6- to 8-week-old, female) were purchased from Jackson Laboratory (Bar Harbor, ME, USA) and maintained in the animal facility at Georgia State University (GSU). All mouse studies were approved by GSU Institutional Animal Care and Use Committee (IACUC, {"type":"entrez-protein","attrs":{"text":"A21004","term_id":"81959","term_text":"pir||A21004"}}A21004) and carried out in compliance with the Guide for the Care and Use of Laboratory Animals of the NIH.

Groups 1 (G1) and 2 (G2) inactivated influenza viruses were used for immunizations and as coating antigens for enzyme-linked immunosorbent assay (ELISA). The inactivated viruses were prepared as previously described [18]. Briefly, G1 and G2 influenza viruses were inactivated with 1% formalin and concentrated by ultracentrifugation (SW32 Ti rotor, 123,760× g, 1 h). The inactivated virus pellet was resuspended in phosphate-buffered saline (PBS) and the total protein concentration of the inactivated viruses was determined using the DC protein assay kit (Bio-Rad Laboratories, Hercules, CA, USA). The G1 inactivated influenza A viruses were as follows: A/Puerto Rico/8/1934 H1N1 (iPR8/H1N1), A/California/04/2009 H1N1 (iCal/H1N1), A/Indonesia/5/2005 H5N1 (iIndo/H5N1), and A/Hong Kong/1073/1999 H9N2 (iHK/H9N2). The G2 inactivated viruses were as follows: A/Hong Kong/1/1968 H3N2 (iHK/H3N2) and reassortant A/Shanghai/11/2013 H7N9 with A/Puerto Rico/8/1934 (A/PR8) H1N1 backbone (iSH/H7N9) [19,20]. The Challenge viruses included G2 (A/Philippine/2/1982 H3N2 (Phil/H3N2)) and G1 (A/Hong Kong/1073/1999 H9N2 (HK/H9N2)) viruses and reverse genetics (rg) reassortant H5N1 (rgH5N1) virus containing HA and NA derived from A/Vietnam/1203/2004 and six internal genes from A/PR8 H1N1, as previously described [21]. Phil/H3N2, HK/H9N2, and rgH5N1 viruses were propagated using embryonated chicken eggs.