Analysis of ORFs 2b, 3, 4, and partial ORF5 of sequential isolates of equine arteritis virus shows genetic variation following experimental infection of horses.

The study targeted novel genetic variations in the equine arteritis virus (EAV), specifically in areas identified as ORFs 2b, 3, 4, and a part of ORF5, which occurred after experimental infection in horses.

Study Design and Sample Collection

• The horses in the study were intentionally infected with a strain of equine arteritis virus identified as “large plaque variant (LP3A+).”
• A total of 182 nasal swab samples were collected over a range of days from 1 to 14 following initial infection. In addition, 21 virus isolates were obtained from the white blood cells of those animals that had extended viraemia (presence of virus in the bloodstream) between days 30 to 72 following initial infection.

Analysis and Findings

• The researchers generated partial sequences of open reading frame 5 (ORF5) which codes for glycoprotein 5 (GP5) to find out the genetic stability of the virus and to characterise the genetic variants during extended viraemia.
• Viruses with specific changes in amino acid in GP5 were subjected to further amplification and sequencing of a fragment that included ORFs 2b, 3, and 4.
• Researchers observed that the ORF5 nucleotide sequences of the virus found in 65 out of 66 nasal swabs were identical to the original virus, indicating that the ORF5 genes remained remarkably stable during the first two weeks after infection.

Observations on Mutation

• Conversely, mutations were noticed in ORF5 as of day 30, as opposed to the high stability during the first 2 weeks p.i. These changes primarily grouped in the antigenic neutralization site C within variable region 1 of the GP5 ectodomain.
• Researchers also noticed varying sequences in ORFs 2b, 3 and 4. Among these ORFs, ORF 4 had the highest ratio of non-synonymous changes, implying that ORF 4 undergoes more genetic alterations during the infection cycle.

Significance of Findings

• The study findings provide vital insights into the genetic dynamics of equine arteritis virus during the course of infection, especially the surprising stability of the ORF5 gene in the early infection stages and significant mutations thereafter.
• These genetic changes and their locations provide critical knowledge for future studies, vaccine development, and better management of equine arteritis. The high mutation rate in ORF 4 suggests it could be a crucial area of focus in developing antiviral strategies.