We selected ten H1N1 and three H3N2 influenza viruses, which included examples of each genotype and evaluated their replication and virulence in BALB/c mice. Groups of nine 6-week-old BABL/c mice were inoculated i.n. with 5 × 104 TCID50 of virus in a volume of 50 µL. The average body weight loss of mice caused by two H1N1 viruses (Sw/GX/CZ7/2014 (genotype C) and Sw/GX/NNXD2023/2013 (genotype D)) reached to 15.5% and 15.1%, respectively (Fig. 3a), especially Sw/GX/CZ7/2014 caused 33% of mice death (Fig. 3b). The rest did not show obvious weight lost or death during the 2-week observation. All 13 viruses replicated in lungs of mice with titers ranging from 3.1 to 5.0 log10 TCID50/mL and 4.5 to 7.3 log10 TCID50/ml at days 3 and 5 p.i., respectively (Fig. 3c), with higher titers observed by the lethal Sw/GX/CZ7/2014 virus. There was no detection in spleens, kidneys, intestines, or brains of any mice. These results indicated that novel triple-reassortant viruses could replicate well in mice lung without prior adaptation and two H1N1 IAVs-S causing sign of disease and lethality. Interestingly, although it is known that H3N2 human viruses replicate to low levels in mice without adaptation42, this was not the case for the IAV-S H3N2 viruses isolated in our surveillance.Fig. 3Weight variation (a), survival rates (b), and replication (c) of novel reassortant IAVs-S in mice. Mice in each group were infected intranasally with 5 × 104 TCID50 of virus in a volume of 50 µL. The body weight and survival rates of mice were measured over 14 days. Virus titers of lung on 3 and 5 days post infection (d.p.i.) were shown as the mean titers of three mice