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Murine gammaherpesvirus 68 (MHV-68) is used as a model for the study of gammaherpesvirus infection and pathogenesis. In the left region of the genome MHV-68 encodes four unique genes, eight viral tRNA-like molecules (vtRNAs) and nine miRNAs. The vtRNAs have a predicted cloverleaf-like secondary structure like cellular tRNAs and are processed into mature tRNAs with the addition of 3’ CCA termini, but are not aminoacylated. Their function is unknown; however they have been found to be expressed at high levels during both lytic and latent infection and are packaged in the virion. The miRNAs are expressed from the vtRNA primary transcripts during latent infection. All herpesviruses examined to date have been found to express miRNAs. These are thought to aid the viruses in avoiding the host immune response and to establish and maintain latency. The aim of this project was to investigate the functions of the vtRNAs and miRNAs of MHV-68. MHV-76 is a natural deletant mutant lacking the unique genes, vtRNAs and miRNAs. This virus was previously used in our lab to construct two insertion viruses encoding vtRNAs1-5 and miRNAs1-6. The only difference between MHV-76 and the insertion viruses is therefore the vtRNAs and miRNAs. The B-cell line NS0 was latently infected with the various viruses and the infected cells characterised. In situ hybridisation and antibody staining showed that all viruses infect the same proportion of cells. The insertion viruses were confirmed to express the vtRNAs during latency by RT-PCR. In addition, using Northern blot analysis the insertion viruses were shown to express miRNA1 during lytic infection of fibroblast cells; however, not during latent infection of NS0 cells. The lack of miRNA1 expression during latency was confirmed using qRT-PCR and miRNAs3-6 were found to be expressed at a lower level than in MHV-68 infected cells. Replication and reactivation kinetics of latently infected NS0 cells showed that introduction of vtRNAs and miRNAs into MHV-76 causes a reduction in reactivation and production of lytic virus. To determine if the reduction in reactivation was caused by the miRNAs, they were introduced into infected cells by transfection. Transfection of miRNAs1-6 into MHV-76 infected cells or miRNA1 into insertion virus infected cells did not lead to an increase or decrease in reactivation. It was confirmed by qRT-PCR that the transfection did result in miRNA levels higher than in insertion virus infected cells. Further, down-regulation of miRNAs using a siRNA against DICER did not lead to a reduction in reactivation. This supports the hypothesis that the vtRNAs rather than the miRNAs are responsible for the reduction of reactivation seen in insertion virus latently infected cells. To determine the effect of the non-coding RNAs on protein expression, NS0 cells latently infected with MHV-76 and insertion virus were analysed using cleavable ICAT and 1-D PAGE cleavable ICAT. In an ICAT analysis the proteins are labelled and the levels of individual proteins in two samples can be compared using mass spectrometry. These techniques were optimised and several proteins with differences in expression between the viruses were identified. It was, however, difficult to determine any specific functions of the non-coding RNAs from the data.

Purpose: To investigate the immune responses in cornea, ocular draining lymph nodes and spleen as well as the priming site of HSV-specific lymphocytes and their possible role in HSK development. To explore the possible treatment of HSK by transplanting corneal allograft and collagen artificial cornea. Methods: BALB/c mice corneas were infected with RE strain HSV-1. Immunohistochemistry of eye sections was performed and flow cytometry was carried out for cell suspension of cornea, TG, submandibular ocular draining lymph node (SMDLN),a non-ocular related lymph node and spleen at various times post HSV-1 eye inoculation. Results: There were strong immune responses in ocular draining lymph nodes post HSV-1 ocular infection, with a significant increasing number of innate cells as well as B cells and T cells. These changes were not observed in non-ocular related lymph nodes or spleen. An antigen specific response to HSV-1 antigen stimulation was observed <i>in vitro</i> for crude cells from ocular draining lymph node and to a lesser extent for spleen, but no changes were observed for the cells from non-ocular related lymph node. Interestingly, removal of ocular draining lymph nodes or spleen prior to HSV inoculation did not prevent HSK, but adversely impaired the control of viral replication, which was indicated by severe blepharitis and encephalitis. Both corneal allograft and collagen artificial cornea failed to survive in HSK eye, and retro-corneal membrane and degradation were the main obstacles for artificial cornea. Conclusions: HSV-specific lymphocytes are primed mainly in ocular draining lymph nodes, however, these cells might not be necessarily required for HSK development. Prevention of retro-corneal membrane seems to be important for survival of both corneal allograft and artificial cornea in HSK eyes.