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Equine herpesvirus type 1 (EHV-1) is an important pathogen that threatens horse populations worldwide and causes spacious economic losses through recurrent outbreaks of respiratory, abortion and neurological disease. Primary infections with EHV-1 lead to the establishment of latent infection within the first weeks of life. The two main strategies for controlling EHV-1 infection and disease are strict hygiene and management practices as well as vaccination. Several vaccines exist to prevent the most severe outcomes of EHV-1 infection, but they are insufficient for long-term protection. Therefore, considerable efforts have been developed to understand the replication and growth properties of EHV-1 and the function of its gene products in order to generate information that may be useful in the design of an effective vaccine. Since the functions of the EHV-1 ORF34 and ORF59 protein (pORF34 and pORF59) products are unknown, the overall targets of this thesis are to (i) identify and characterize the EHV-1 pORF34 and pORF59 products and (ii) examine whether they have a role in pathogenesis of EHV-1.
In Chapter 2 of this thesis, we focused on the identification of ORF34 product that is expressed with early kinetics and its product is localized within the cytosol of EHV-1- infected cells.
Immunoblot analysis of EHV-1 pORF34 detected a protein with the apparent molecular mass of 28-kDa, which is larger than that predicted based on the sequence of the protein (18-kDa). Interestingly, the kinetics of pORF34 expression had shown protein stability until 10 h p.i., however, additional bands with apparent molecular masses ranging between 17- and 28-kDa appeared at later times after infection. Our findings suggested that stability of pORF34 is dependent on the time after infection and that pORF34 may only be required at early times after infection or that its function at later times after infection requires the protein be modified by ubiquitin/proteasome pathway. Furthermore, examination of the replication properties of an EHV-1 lacking ORF34 (rAb4Δ34) revealed that pORF34 is required for efficient virus growth in vitro at early stages of infection.
In Chapter 3 of this thesis, we addressed more details about the identification of EHV-1 pORF59 and its role in the EHV-1 pathogenesis. We found that pORF59 is expressed at the early stage of infection and its product is localized in the cytosol of the infected cells. Immunoblot analysis of EHV-1 pORF59, including the inserted epitope, detected a protein with a molecular mass of approximately 21-kDa, which is similar to the predicted sequence of the protein. Moreover, EHV-1 lacking ORF59 (rAb4Δ59) exhibited a small-plaque phenotype and could not be propagated even after serial passages in cell culture. Our findings suggest that the ORF59 protein plays a major role in EHV-1 growth in cultured cells.
Generally, a concise list below can show the highlights points of this thesis:
- The EHV-1 ORF34 protein is expressed at early stage of infection and its product is localized in cytosol of EHV-1 infected cells.
- The ORF34 protein is modified by ubiquitination and degraded in the proteasome at late times of infection
- The ORF34 protein is required for optimal replication of EHV-1 in cultured cells and target cells taken directly ex vivo at early times of infection.
- The EHV-1 ORF59 protein is a 21-kDa protein expressed with early kinetics.
- The product of the EHV-1 ORF59 protein localizes to cytosol of EHV-1-infected cells.
- The EHV-1 ORF59 protein is essential for EHV-1 growth in cultured cells.