Development and characterization of ORF68 negative equine herpes virus type-1, Ab4p strain.
Abstract: Equine herpesvirus-1 (EHV-1) is an important pathogen, which infects horses worldwide with high morbidity but low mortality rates. The respiratory disorders and abortions are the most common indicators. Ab4p (an abortigenic and paralytic virus) is one of the most important and virulent strains. The development and functional characterization of the open reading frame-68 (ORF68) negative EHV-1 Ab4p mutants and an assessment of their roles in the infection at the cellular level were the main targets of the current study. Escherichia coli DH10β containing the Ab4p bacterial artificial chromosome (pAb4pBAC) and Red/ET expression vector were used to develop different ORF68 mutants. Multi-step growth kinetic experiments were conducted in order to evaluate the growth properties of the constructed mutant viruses. Growth of the Ab4pΔORF68 showed the lowest titer, compared to the Ab4pΔORF68R, Ab4pΔORF68R non-sense, and the parent Ab4p viruses without any significant difference (P > 0.05). The growth of the mutant viruses was almost similar across the cell types, but viruses growth was more efficient in FHK cells as judged by the number of the obtained virus particles. The plaque size of Ab4pΔORF68 was significantly (40%) smaller than those of Ab4p (P < 0.01), Ab4pΔORF68R, and Ab4pΔORF68R non-sense viruses which confirmed the importance of ORF68 protein in the cell-to-cell transmission of EHV-1. Subcellular localization of the green fluorescent protein (GFP) ORF68 gene fusion product showed late expression with intranuclear localization of the transfected cells while immunofluorescent antibody technique (IFAT) localized it at the nucleus and nuclear membranes of the infected cells. Hence, it could be concluded that ORF68 protein may not be essential for EHV-1 Ab4p growth but plays a crucial role in virus penetration and transmission at the cellular level. Therefore, the generated EHV-1 ORF68 negative mutant could be a prospective candidate for the development of a vaccine marker.
Copyright © 2018 Elsevier B.V. All rights reserved.
Publication Date: 2018-08-27 PubMed ID: 30165189DOI: 10.1016/j.jviromet.2018.08.018Google Scholar: Lookup
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- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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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.
Cite This Article
APA
Ata EB, Zaghawa A, Ghazy AA, Elsify A, Abdelrahman K, Kasem S, Nayel M.
(2018).
Development and characterization of ORF68 negative equine herpes virus type-1, Ab4p strain.
J Virol Methods, 261, 121-131.
https://doi.org/10.1016/j.jviromet.2018.08.018 Publication
Researcher Affiliations
- Department of Parasitology and Animal Diseases, Veterinary Research Division, National Research Centre (NRC), Egypt; Laboratory of Veterinary Microbiology, Faculty of Applied Biological Sciences, Gifu University, 1-1 Yanagido, Gifu, 501-1193, Japan. Electronic address: emadvet2003@yahoo.com.
- Department of Internal Medicine and Infectious Diseases, Faculty of Veterinary Medicine, Sadat City University, Egypt.
- Department of Parasitology and Animal Diseases, Veterinary Research Division, National Research Centre (NRC), Egypt.
- Department of Internal Medicine and Infectious Diseases, Faculty of Veterinary Medicine, Sadat City University, Egypt.
- Department of Parasitology and Animal Diseases, Veterinary Research Division, National Research Centre (NRC), Egypt.
- Department of Virology, Faculty of Veterinary Medicine, Kafrelsheikh University, 33516, El-Geish street, Kafrelsheikh, Egypt; Laboratory of Veterinary Microbiology, Faculty of Applied Biological Sciences, Gifu University, 1-1 Yanagido, Gifu, 501-1193, Japan.
- Department of Internal Medicine and Infectious Diseases, Faculty of Veterinary Medicine, Sadat City University, Egypt; Laboratory of Veterinary Microbiology, Faculty of Applied Biological Sciences, Gifu University, 1-1 Yanagido, Gifu, 501-1193, Japan.
MeSH Terms
- Animals
- Cell Line
- Cell Nucleus / virology
- Chromosomes, Artificial, Bacterial
- Escherichia coli / genetics
- Genes, Reporter
- Green Fluorescent Proteins / analysis
- Green Fluorescent Proteins / genetics
- Herpesvirus 1, Equid / genetics
- Herpesvirus 1, Equid / growth & development
- Horses
- Microscopy, Fluorescence
- Nuclear Envelope / virology
- Recombinant Fusion Proteins / analysis
- Recombinant Fusion Proteins / genetics
- Sequence Deletion
- Viral Load
- Viral Plaque Assay
- Viral Proteins / analysis
- Viral Proteins / genetics
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