Optimization and application of a DNA-launched infectious clone of equine arteritis virus.

This research explores the development and application of a new system to control and manipulate Equine Arteritis Virus (EAV), a prototype member of the Equartevirus. Using a modified cDNA plasmid and reverse genetic techniques, a genetically engineered version of EAV was created which could be potentially useful for scientific investigation into the virus’s pathogenic mechanisms.

Development of a New Reverse Genetics System

• The researchers created a cDNA plasmid, a piece of DNA that can replicate in the host cell, by flanking the EAV’s viral cDNA sequence with hammerhead ribozyme (HamRz) and hepatitis delta virus ribozyme (HdvRz) sequences. These special sequences were placed at both ends of the viral genome.
• They then optimized the promoter and terminator regions, which control the start and end of transcription, to enable the replication of the viral genome by the cell’s RNA polymerase II enzyme.

Characteristics of the Rescued Virus

• The rescued virus, ic-EAV, exhibited similar growth characteristics and morphology as the wild-type (WT) virus.
• Additionally, a unique BspEI restriction site was engineered into the nsp3 gene of the ic-EAV, which allowed differentiation of the rescued virus from the wild type.

Mutation and its Impact

• Through the established method, a G-to-C silent mutation was introduced at site 12642, which triggered a significant change in the plaque size of the rescued virus. Although the mutation does not alter the protein produced, it seems to considerably impact the virus’s growth behavior.

Creation of a Labeled EAV

• An eGFP-labeled EAV was built by introducing the gene for Green Fluorescent Protein (eGFP) into the EAV’s infectious clone.
• This genetic modification allowed the observation of EAV’s infection process in the target cells, highlighting the potential of this reverse genetics method in facilitating further research into the virus’s pathogenesis.

In conclusion, the reverse genetics method presented in this study provides a manipulable tool to enable detailed investigations into the mechanics of EAV.