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Updated: May 26, 2026

Reverse Genetics to Engineer Positive-Sense RNA Virus Variants

Reverse Genetics to Engineer Positive-Sense RNA Virus Variants

Published on: June 9, 2022

Reverse genetics approach for arteriviruses using circular polymerase extension reaction.

Rabab T Hassanien^1,2, Wellesley Dittmar¹, Udeni B R Balasuriya¹

¹Department of Pathobiological Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA, USA.

Access Microbiology

|May 25, 2026

Summary

This summary is machine-generated.

The circular polymerase extension reaction (CPER) efficiently generates infectious arterivirus clones, enabling rapid creation of reporter viruses for studying viral functions. This method proves versatile for equine arteritis virus and porcine reproductive and respiratory syndrome virus research.

Keywords:

cDNA clones circular polymerase extension reaction (CPER)equine arteritis virus (EAV)porcine reproductive and respiratory syndrome virus (PRRSV)reporter viruses reverse genetics

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Area of Science:

Virology
Molecular Biology
Genetics

Background:

Positive-sense, single-stranded RNA viruses require efficient methods for generating infectious cDNA clones.
Arteriviridae family viruses, including equine arteritis virus (EAV) and porcine reproductive and respiratory syndrome virus (PRRSV), pose significant challenges for clone generation.
Existing methods may lack the speed and fidelity required for rapid manipulation of viral genomes.

Purpose of the Study:

To implement and evaluate the circular polymerase extension reaction (CPER) for generating infectious cDNA clones of EAV and PRRSV.
To create reporter viruses expressing fluorescent proteins (mCherry and GFP) to facilitate viral studies.
To assess the viability and characteristics of CPER-generated viruses and reporter constructs.

Main Methods:

Overlapping cDNA fragments covering the full viral genomes of EAV KY84 and PRRSV VR2332 were synthesized.
Fragments were assembled using CPER with a linker containing essential expression elements, creating circularized cDNA genomes.
Infectious viruses were recovered post-transfection, and reporter genes (mCherry, GFP) were inserted into specific viral locations.

Main Results:

Infectious EAV KY84 and PRRSV VR2332 strains were successfully recovered using the CPER method.
CPER-generated viruses exhibited high sequence identity to parental strains, confirmed by next-generation sequencing.
Reporter viruses showed varying stability and expression; rPRRSV-GFP had reduced titers, while rEAV-mCherry lost the reporter gene over passages.

Conclusions:

The CPER-based strategy is a rapid and effective tool for generating infectious arterivirus cDNA clones.
This method facilitates the creation of reporter viruses and mutants for investigating viral gene functions and host interactions.
While CPER is robust, reporter gene stability requires further optimization for long-term studies.