Heterologous gene expression from transmissible gastroenteritis virus replicon particles

Area of Science:

• Virology
• Molecular Biology
• Vaccinology

Background:

• Transmissible Gastroenteritis Virus (TGEV) is a significant swine pathogen.
• Previous research established infectious TGEV constructs for genetic manipulation.
• Understanding TGEV replication and assembly is crucial for disease control and vaccine development.

Purpose of the Study:

• To construct and characterize infectious TGEV replicon particles (TGEV VRPs).
• To develop a packaging system for TGEV VRPs using a Venezuelan equine encephalitis (VEE) replicon vector.
• To evaluate the potential of TGEV VRPs for studying viral replication and as a basis for novel swine vaccines.

Main Methods:

• Generation of a recombinant TGEV expressing green fluorescent protein (GFP) by replacing ORF 3A.
• Construction of TGEV replicon RNAs lacking essential viral budding genes (ORF 3B, E, and M).
• Development of a VEE-based packaging system to produce TGEV VRPs encoding the TGEV E protein.

Main Results:

• Recombinant TGEV-GFP2 replicated efficiently and expressed GFP in baby hamster kidney (BHK) cells.
• TGEV replicon RNAs replicated but did not produce infectious virus, demonstrating single-hit dependence.
• Packaging TGEV replicon RNAs with a VEE-TGEV(E) helper system produced infectious TGEV VRPs capable of gene expression and replication in swine testicular (ST) cells without generating infectious virus.

Conclusions:

• The developed TGEV VRP system is replication-competent and capable of expressing heterologous genes.
• This system provides a valuable tool for investigating TGEV replication and assembly mechanisms.
• TGEV VRPs represent a promising platform for the development of novel swine vaccines against TGEV.