Phloretin inhibits transmissible gastroenteritis virus proliferation via multiple mechanisms.

Transmissible gastroenteritis virus (TGEV), an enteropathogenic coronavirus, has caused huge economic losses to the pig industry, with 100% mortality in piglets aged 2 weeks and intestinal injury in pigs of other ages. However, there is still a shortage of safe and effective anti-TGEV drugs in clinics. In this study, phloretin, a naturally occurring dihydrochalcone glycoside, was identified as a potent antagonist of TGEV. Specifically, we found phloretin effectively inhibited TGEV proliferation in PK-15 cells, dose-dependently reducing the expression of TGEV N protein, mRNA, and virus titer. The anti-TGEV activity of phloretin was furthermore refined to target the internalization and replication stages. Moreover, we also found that phloretin could decrease the expression levels of proinflammatory cytokines induced by TGEV infection. In addition, we expanded the potential key targets associated with the anti-TGEV effect of phloretin to AR, CDK2, INS, ESR1, ESR2, EGFR, PGR, PPARG, PRKACA, and MAPK14 with the help of network pharmacology and molecular docking techniques. Furthermore, resistant viruses have been selected by culturing TGEV with increasing concentrations of phloretin. Resistance mutations were reproducibly mapped to the residue (S242) of main protease (M^pro). Molecular docking analysis showed that the mutation (S242F) significantly disrupted phloretin binding to M^pro, suggesting M^pro might be a potent target of phloretin. In summary, our findings indicate that phloretin is a promising drug candidate for combating TGEV, which may be helpful for developing pharmacotherapies for TGEV and other coronavirus infections.