A TMPRSS2/TMPRSS13-Expressing Porcine Testis Cell Platform for Trypsin-Independent Porcine Epidemic Diarrhea Virus Culture and Isolation

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.猪流行性腹泻病毒（Porcine epidemic diarrhea virus, PEDV）是引发猪群急性重症肠道疾病的主要病原体。尽管已有大量研究积累，但猪流行性腹泻病毒的体外培养仍依赖外源性胰蛋白酶，该操作易引发细胞毒性、病毒稳定性波动以及批次间性能不一致等问题。宿主编码的II型跨膜丝氨酸蛋白酶——尤其是TMPRSS2与TMPRSS13——已被证实可通过蛋白酶水解激活刺突（S）蛋白，进而促进冠状病毒侵染宿主细胞。然而，在猪源细胞系中联合表达这两种蛋白酶，能否在猪流行性腹泻病毒培养中功能性替代胰蛋白酶，此前尚未得到探索。本研究通过慢病毒递送系统，构建了稳定共表达TMPRSS2与TMPRSS13的猪睾丸（ST）细胞系，命名为ST/TMPRSS2-TMPRSS13。该工程化细胞系表现出正常的增殖能力、未受干扰的细胞周期进程以及极低的细胞凋亡水平。在不添加胰蛋白酶的条件下，该细胞系可支持猪流行性腹泻病毒的有效侵染与多周期复制，其引发的细胞病变效应、病毒蛋白表达水平以及病毒滴度，与添加胰蛋白酶的亲本ST细胞所获结果相当，甚至更优。本研究进一步验证了该细胞系的实用性：无需添加胰蛋白酶，即可从粪便样本中成功分离两株临床猪流行性腹泻病毒分离株（HN202505与HN202510）并实现持续传代培养。系统发育分析显示，这两株分离株均属于GIb亚型，与经典DR13毒株具有显著的遗传同源性。综上，本研究结果证实，共表达TMPRSS2与TMPRSS13可使ST细胞在无需胰蛋白酶的条件下支持猪流行性腹泻病毒的复制。该ST/TMPRSS2-TMPRSS13细胞系为猪流行性腹泻病毒的分离与培养提供了简便可靠的工具，将有助于推动该病毒的基础研究与疫苗开发工作。