Background: Influenza surveillance and drug resistance testing have always been central to clinical efforts. Therefore, researching the virus characteristics and antiviral drugs is essential.

Method: The HA and NA activities were assessed in influenza strains, and mutations were identified through gene sequencing. The effects of oseltamivir, molnupiravir, and baloxavir treatments were evaluated in vitro. The effectiveness of molnupiravir monotherapy and its combination with baloxavir was also evaluated in a mouse model. Changes in body weight and lung tissue were examined, including pathological changes, virus replication, and inflammation levels.

Results: Forty-one seasonal influenza H1N1 strains from 2023 were used. The EC50 of oseltamivir was significantly increased compared to the 2009 reference strain. Correlation analysis showed that the increase in EC50 was related to the HA and NA activities. The antiviral effects of molnupiravir and baloxavir significantly inhibited virus replication; the combination treatment of molnupiravir/baloxavir showed more potent and synergistic inhibitory effects in vitro. In the mouse model, molnupiravir treatment effectively inhibited virus replication and lung inflammation, but the treatment did not improve weight loss or reduce mortality. With the molnupiravir/baloxavir treatment, viral replication was significantly inhibited and proved to be more effective than either monotherapy. The combination therapy also showed the lowest inflammatory response along with a higher survival rate.

Conclusions: The increase in HA and NA activities of seasonal influenza reduced the efficacy of oseltamivir treatment, but the effectiveness of molnupiravir and baloxavir was retained. Combination therapy showed a significant antiviral effect, which provides a reference for the clinical treatment.