Development and characterization of ORF68 negative equine herpes virus type-1, Ab4p strain.

The study focuses on the development and analysis of a mutant strain of equine herpes virus type-1 (EHV-1) known as Ab4p, which lacks the open reading frame-68 (ORF68) gene. The aim of this research was to understand the functional role of this gene during the virus’s infection cycle at a cellular level, which could potentially help in the improved development of vaccines against this disease.

Development of Mutant Strains

• The researchers used a bacterium called Escherichia coli DH10β containing the Ab4p bacterial artificial chromosome (pAb4pBAC) and a Red/ET expression vector to create different versions of ORF68 mutant strains of the EHV-1 Ab4p virus.
• Growth properties of these mutant viruses were then evaluated through multi-step growth kinetic experiments.

Analysis of Growth Properties

• The results demonstrated that the growth of the Ab4pΔORF68 mutant yielded the lowest virus titer, as compared to other Ab4p strains carrying altered forms of ORF68, however, the difference was not statistically significant. This suggests that the deletion of ORF68 gene may not have a major impact on virus replication.
• Additionally, an almost similar growth rate of these mutant viruses was observed across various cell types, but the growth seemed more efficient in fetal horse kidney (FHK) cells, indicating that the FHK cell line could be more susceptible to EHV-1 Ab4p infection.

Role of ORF68 in Virus Transmission

• The study identified that the mutant virus lacking the ORF68 gene produced significantly smaller plaques than the viruses with this gene, suggesting that ORF68 may play an important role in cell-to-cell transmission of EHV-1.
• The subcellular localization technique revealed that the gene product of ORF68 showed a delayed expression inside the nucleus of cells infected with the virus, emphasizing that it may play a part in virus penetration and transmission within the cell.

Implications and Future Directions

• The findings imply that while ORF68 might not be crucial for the growth of EHV-1 Ab4p virus, it plays a pivotal role in the virus’s penetration and transmission at the cellular level.
• These insights suggest that the generated EHV-1 ORF68 negative mutant strain could be a potential candidate for the development of a marker vaccine, aimed at controlling the spread of the disease.