Genetic manipulation of equine arteritis virus using full-length cDNA clones: separation of overlapping genes and expression of a foreign epitope.

This research studied the equine arteritis virus (EAV), with a focus on understanding how its overlapping genes function. Through genetic manipulation, they discovered that it was possible to separate these genes and still maintain the viability of the virus. The researchers also successfully introduced a foreign epitope, a part of the antigen recognized by the immune system, into the virus.

Equine Arteritis Virus and its Genetic Structure

• The Equine arteritis virus (EAV) is a type of RNA virus from the Arteriviridae family.
• Its genome is unsegmented and infectious, measuring 12,704 nucleotides in length without the poly(A) tail.
• The genome contains eight overlapping genes, which creates a ‘nested set’ of mRNAs.

Genetic Manipulation of the Virus

• The researchers constructed a series of mutant full-length cDNA clones in order to study the importance of gene overlapping in the viral lifecycle.
• The open reading frames (ORFs) 4 and 5, ORFs 5 and 6, or ORFs 4, 5, and 6 were separated using the enzyme AflII.
• The RNA transcribed from these modified plasmids was infectious, proving that overlapping gene organization is not necessary for EAV’s viability.
• Moreover, the mutated viruses were able to replicate almost as efficiently as the wildtype virus and maintain the introduced mutations.

Introduction of a Foreign Epitope

• The AflII site between ORFs 5 and 6 was used to genetically engineer a virus with an extended ectodomain of the ORF 6-encoded M protein.
• This extension consisted of nine amino acids from a similar protein in the mouse hepatitis virus (MHV).
• The added nonapeptide contained an O-glycosylation signal and an epitope recognized by an MHV-specific monoclonal antibody.
• The recombinant virus successfully expressed both the glycosylation signal and the epitope.
• While the engineered virus showed a growth disadvantage compared to the original, it was able to retain the foreign genetic material even after three rounds of passage, or replication.