Establishment of stable Vero cell lines expressing TMPRSS2 and MSPL: A useful tool for propagating porcine epidemic diarrhea virus in the absence of exogenous trypsin

Abstract

Porcine epidemic diarrhea virus (PEDV) is the causative agent of porcine epidemic diarrhea, causing substantial economic losses to the swine industry worldwide. However, the development of PEDV vaccine is hampered by its low propagation titer in vitro, due to difficulty in adapting to the cells and complex culture conditions, even in the presence of trypsin. Furthermore, the frequent variation, recombination, and evolution of PEDV resulted in reemergence and vaccination failure. In this study, we established the Vero/TMPRSS2 and Vero/MSPL cell lines, constitutively expressing type II transmembrane serine protease TMPRSS2 and MSPL, in order to increase the stability and titer of PEDV culture and isolation in vitro. Our study revealed that the Vero/ TMPRSS2, especially Vero/MSPL cell lines, can effectively facilitate the titer and multicycle replication of cell-adapted PEDV in the absence of exogenous trypsin, by cleaving and activating PEDV S protein. Furthermore, our results also highlighted that Vero/TMPRSS2 and Vero/MSPL cells can significantly enhance the isolation of PEDV from the clinical tissue samples as well as promote viral infection and replication by cell-cell fusion. The successful construction of the Vero/TMPRSS2 and Vero/MSPL cell lines provides a useful approach for the isolation and propagation of PEDV, simplification of virus culture, and large-scale production of industrial vaccine, and the cell lines are also an important system to research PEDV S protein cleaved by host protease.

Immunogenicity and protective efficacy of recombinant adenovirus expressing a novel genotype G2b PEDV spike protein in protecting newborn piglets against PEDV

“…PED is a considerable threat to the swine industry, and vaccination is the most efficient way to prevent the disease ( 23 , 25 ); however, the emergence of novel G2b PEDV variants has led to vaccine failure, hindered the prevention and control of PED, and caused substantial economic losses in China ( 3 , 26 – 28 ). The replication of novel PEDV variants in vitro is another substantial challenge, resulting in difficulty in isolating novel strains ( 29 – 31 ). The S protein is the structural protein of the virus, containing a receptor binding domain that mediates viral invasion of host cells and an antigenic epitope that mediates the organism’s production of NAb ( 32 , 33 ).…”

Molecular Mechanism of Porcine Epidemic Diarrhea Virus Cell Tropism

“…Receptor interaction plays a key role in coronaviruses' cell, tissue, and host tropism. Unfortunately, even though porcine aminopeptidase N (pAPN), transmembrane protein 41B (TMEM41B), transmembrane serine protease (TMPRSS2), a trypsin-like serine protease (MSPL), and transferrin receptor I can effectively facilitate PEDV invasion of cells ( 22 , 44 , 45 ) and sialic acid-binding activity of the PEDV S1 subunit facilitates cell entry ( 46 ), the functional receptors of PEDV are still unclear. Isolated exchange of S1 and S2 of adaptive strains did not make PEDV adapt to cells ( Fig.…”

Comparative Review of the State of the Art in Research on the Porcine Epidemic Diarrhea Virus and SARS-CoV-2, Scope of Knowledge between Coronaviruses

“…In relation to PEDV, the successful generation of the Vero/TMPRSS2 and Vero/MSPL cell lines, which constitutively express TMPRSS2 and MSPL (trypsin-like serine protease), increased the viral titer and isolation of PEDV in vitro [ 85 , 86 ].…”