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Updated: Jul 2, 2026
Production of Pseudotyped Particles to Study Highly Pathogenic Coronaviruses in a Biosafety Level 2 Setting
Published on: March 1, 2019
[Characterization of murine leukemia virus recombinants bearing PRRSV GP5 glycoproteins]
Zhanguo Dang1, Ping'an Xia, Bin Zhou
1College of Animal Husbandry and Veterinary, Henan Agricultural University, Zhengzhou 450002, China.
Abstract:
The highly virulent PRRSV isolate strain HN-1/06 was cultivated on Marc-145. To study the viral entry mechanisms, the GP5 gene of PRRSV isolate was amplified by RT-PCR and cloned into pcDNA3.0 to generate the expressing plasmid pcDNA-GP5. pcDNA-GP5 was transfected into 293T by the calcium phosphate precipitation method. Analysis of flow cytometry confirmed that the GP5 proteins were expressed in surface of the 293T cells. Then 293T cells were transfected with pcDNA-GP5, pHIT60 and pHIT111 plasmids to generate pseudotyping virus. The pseudotyping virus supernatant was harvested 48 hours post-transfection and was detected by Western blotting and infection assay. Western blotting indicated that the GP5 glycoproteins were incorporated into the retroviral pseudotyped virus. Infection assay showed that the pseudotyped virus infected 293T and Mark-145 cell. The pseudotyped virus could be used to further study infectious mechanism of PRRSV.
Insights
This study developed a pseudotyped virus system to investigate Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) entry. This novel system successfully incorporated PRRSV GP5 proteins, enabling infection studies for understanding PRRSV mechanisms.
Area of Science:
- Virology
- Molecular Biology
Context:
- Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) is a significant pathogen in swine.
- Understanding PRRSV viral entry mechanisms is crucial for developing effective control strategies.
Purpose:
- To develop a pseudotyped virus system for studying PRRSV entry mechanisms.
- To investigate the incorporation and function of PRRSV GP5 protein in a retroviral pseudotype.
Summary:
- The GP5 gene of a virulent PRRSV strain (HN-1/06) was cloned and expressed in 293T cells.
- Retroviral pseudotyped viruses were generated displaying PRRSV GP5 on their surface.
- Western blotting confirmed GP5 incorporation, and infection assays demonstrated the pseudotyped virus's ability to infect 293T and Marc-145 cells.
Impact:
- This pseudotyped virus system provides a valuable tool for further research into PRRSV infectious mechanisms.
- Facilitates studies on viral entry and host-cell interactions without requiring infectious PRRSV.
- Potential to accelerate the development of antiviral therapies and vaccines.
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