Members of the Herpesviridae are large enveloped, double-stranded DNA viruses, whose virions are comprised of a viral genome-containing icosahedral capsid, a layer of tegument and a glycoprotein-embedded envelope. The tegument contains numerous viral proteins and cellular proteins. Most of the tegument proteins are poorly understood and require further investigation. This study focuses on one of the tegument proteins, Us2, and utilizes two model alphaherpesviruses: pseudorabies virus (PRV) and herpes simplex virus (HSV). Us2 is conserved among all alphaherpesvirus with the exception of varicella-zoster virus (VZV). The amino acid sequence of all Us2 orthologs share three N-terminal conserved regions whereas the C-terminal sequences are highly variable. PRV Us2 contains a C-terminal prenylation motif that targets Us2 to the plasma membrane. Although it is indispensable for virus growth in cell culture, deletion of Us2 gene in PRV caused an accumulation of virions in the cytoplasm of infected primary cells. Furthermore, PRV Us2 spatially regulates MAPK ERK activity by sequestering it to the plasma membrane. Inhibition of ERK kinase activity caused a delay in the release of extracellular viruses and the defect was more profound in PRV Us2-null virus infected cells. Altogether, these data suggest a requirement for ERK activity and significance of PRV Us2-ERK interaction in virus egress. To understand the mechanism of Us2-ERK interaction, PRV Us2 determinants for ERK interaction were mapped. Our data revealed that the N-terminal 214 residues are the minimal sequence of Us2 required for interaction with ERK. In addition, PRV Us2 oligomerizes and forms complexes with ERK via the ERK common docking (CD) domain that facilitates the interaction of ERK with many of its substrates. Unlike PRV Us2, HSV-2 Us2 does not have any putative membrane targeting signals. However, our data revealed that HSV-2 Us2 localizes to the plasma membrane and is lipid raft associated. In addition, HSV-2 Us2 interacts directly with ubiquitin. As ubiquitination is responsible for proteasomal degradation and is involved in endocytosis and lysosomal degradation, these findings suggest that Us2 may be involved in proteasomal degradation pathways that counteract host defenses, or participate in final envelopment in endocytic compartments by facilitating the endocytosis of viral envelope proteins. / Thesis (Ph.D, Microbiology & Immunology) -- Queen's University, 2013-01-21 01:25:54.103

Ruminant alphaherpesviruses related to bovine herpesvirus 1 (BoHV-1) are a cluster of viruses antigenically and genetically closely related. The prototype of this cluster, BoHV-1, is a major pathogen of cattle associated with various clinical manifestations including infectious bovine rhinotracheitis (IBR) and infectious pustular vulvovaginitis (IPV). IBR is a disease of major economic concern in many parts of the world and especially in Europe, both in countries where this infection has been eradicated and in those where the control of IBR is currently or will be undertaken. The massive use of vaccination allowed a significant reduction of the number of IBR clinical cases. However, the existence of closely related viruses to BoHV-1 is a threat for IBR eradication programmes. Consequently, the main objective of the present work is dedicated to afford a better knowledge of the interaction between alphaherpesviruses and their ruminant hosts in order to contribute to improve the control of IBR. To meet the objective, two approaches have been developed: the study of the viral diversity aiming to extend both epidemiological and virological data about ruminant alphaherpesviruses related to BoHV-1 and the study of the heterologous protection aiming to protect minor ruminant species by the concept of the cascade vaccination. Illustrating the problematic of the cluster of ruminant alphaherpesviruses related to BoHV-1, an original situation has been described recently in Belgium. During 2001 and 2002 hunting seasons, 28.9% of red deer were detected seropositive to BoHV-1. Due to an apparent lack of contact between cattle and red deer, it was suggested that a BoHV-1 related virus was spreading in the Belgian red deer population. Thus, the first isolation of cervid herpesvirus 1 (CvHV-1) in wild fauna is reported, which brings the opportunity to deeper analyse the antigenic, genomic and genetic relationship between BoHV-1 and its related ruminant alphaherpesviruses. This isolation demonstrates that a ruminant can be strongly identified as BoHV-1 positive while in actual fact it is infected with a related but distinct alphaherpesvirus and this ruminant will be declared as false positive. The problem is even greater when these viruses become latent allowing their possible reactivation and persistence for a very long time in their ecological niches. It is necessary to have tests which can differentiate related alphaherpesviruses that infect different ruminant species. The control of IBR relies on the use of BoHV-1 gB and gE blocking enzyme linked immunosorbent assays (ELISA) in order to differentiate infected and gE-negative vaccinated animals. Knowing that CpHV-1 is the most distant virus from BoHV-1, it can be hypothesised that a BoHV-1 gB blocking ELISA detects CpHV-1 but that CpHV-1 infection could be discriminated by a BoHV-1 gE blocking ELISA. CpHV-1 being mainly distributed in the Mediterranean part of Europe as Greece, Spain and Italy, the analysis was performed with field serums collected in France with the aim to update the epidemiological situation of the infection in Europe. Besides BoHV-1, CpHV-1 is the most relevant infection in Europe but is sadly neglected. The first reason is that economic losses are restricted to a herd level in contrast with IBR that brings an economical impact at a country level. The second reason is that goat is considered as a minor species. In this context, the problem is still not big enough for commercial interest towards vaccine development. The European Union has recently pointed out the problem of minor uses and minor species and allowed off label use of veterinary medicinal products or the use of a product licensed for a major species when an authorised veterinary medicinal product is not available (cascade principle). Goat being a minor species and CpHV-1 sharing close antigenic and genetic properties with BoHV-1, a live attenuated gE-negative BoHV-1 vaccine has been assessed in goats to protect against either a nasal or a genital CpHV-1 infection.