A TMPRSS2/TMPRSS13-Expressing Porcine Testis Cell Platform for Trypsin-Independent Porcine Epidemic Diarrhea Virus Culture and Isolation
Description
Porcine epidemic diarrhea virus (PEDV) is a major causative agent of acute, severe enteric disease in swine. Despite considerable research, in vitro propagation of PEDV continues to rely on exogenous trypsin, a practice associated with cytotoxicity, variable viral stability, and inconsistent batch performance. Host-encoded type II transmembrane serine proteases—in particular TMPRSS2 and TMPRSS13—are known to promote coronavirus cell entry via proteolytic activation of the spike (S) protein. However, whether the combined expression of these proteases in a porcine cell line can functionally replace trypsin in PEDV culture has remained unexplored. Here, we generated a stable swine testis (ST) cell line co-expressing TMPRSS2 and TMPRSS13 (designated ST/TMPRSS2-TMPRSS13) using a lentiviral delivery system. The resulting cells exhibited normal proliferation, unaltered cell cycle progression, and minimal apoptosis. In the absence of trypsin, the engineered line supported productive PEDV infection and multicycle replication, producing cytopathic effects, viral protein levels, and viral titers that were similar to or even exceeded those obtained in parental ST cells supplemented with trypsin. The utility of this line was further validated by the successful isolation and sustained propagation of two clinical PEDV isolates (HN202505 and HN202510) from fecal samples without trypsin. Phylogenetic analysis placed both isolates within the GIb subtype, with marked genetic relatedness to the classical DR13 strain. Collectively, these results establish that TMPRSS2 and TMPRSS13 co-expression enables trypsin-independent PEDV replication in ST cells. The ST/TMPRSS2-TMPRSS13 cell line offers a simplified, reliable tool for PEDV isolation and propagation, and should facilitate basic investigations and vaccine development efforts for this virus.