Abstract: 【Objective】 The objective of this study was to investigate the activity and mechanism of ethyl acetate extract from Alternanthera philoxeroides (AETAC) against H9N2 subtype Avian influenza (AIV-H9N2) virus in vitro and in vivo.【Method】 The inhibitory effect of AETAC at different concentrations on chicken embryo fibroblasts (DF-1) at different time was detected by CCK-8 method.The toxicity of AETAC to DF-1 cells was detected by preventing infection, influence replication, preventing adsorption and directly kill, and the best anti-AIV-H9N2 mode was selected in vitro.The maximum safe concentration of AETAC was determined by inoculation of different concentrations in allantoic cavity.AETAC was administered to AIV-H9N2 infected chicken embryo by three different administration methods, and the best administration method was selected in vivo.AETAC and AIV-H9N2 were simultaneously inoculated through allantoic cavity, the survival number of chicken embryos in each group was counted, the hemagglutination titer was determined, and the development of embryo and pathological changes of the bursa of Fabricius of chicken embryos were observed.【Result】 Within a certain range, as the concentration of AETAC increases or the duration of action prolongs, the inhibitory rate of AETAC on DF-1 cells increased.Compared with blank control group, the cell survival rate of virus control and drug groups was significantly decreased under four anti-AIV-H9N2 modes (P＜0.05).Compared with virus control group, the cell survival rate of the four anti-AIV-H9N2 modes was significantly increased when AETAC concentration was 5-30 μg/mL (P＜0.05).The survival rates of DF-1 cells were 8.15%-64.96% (prevent infection), 8.56%-83.83% (influence replication), 1.82%-5.90% (prevent adsorption) and 6.90%-40.12% (directly kill), respectively.The maximum safe concentration of AETAC for chicken embryos was 16.41 mg/mL, and the best administration method for infected chicken embryos was mixed AETAC and virus inoculation.After the optimal in vivo administration, the survival rate of embryos in medium and low AETAC concentration groups was 40%-60% and those in high concentration group were all alive. In high concentration group, the beak development of embryos was obvious, the lymphoid follicles of the bursa of Fabricius of chicken embryos were intact, the size and number of follicles were normal, the amount of congestion was less, and the histopathological status was improved.【Conclusion】 AETAC had significant inhibitory effect on AIV-H9N2 both in vivo and in vitro, and was mainly related to affecting the mode of viral replication.

Key words: ethyl acetate extract from Alternanthera philoxeroides (AETAC); H9N2 subtype Avian influenza virus (AIV-H9N2); DF-1 cells; chicken embryo