Comprehensive mapping of rhadinovirus dissemination and persistence

Rhadinoviruses infect both man and economically important animals. Infection predisposes to several cancers, and even though these are individually uncommon the high prevalence of infection leads to a large total disease burden, particularly in the developing world. For example Kaposi's Sarcoma is the commonest cancer in untreated HIV infection and is caused by a rhadinovirus. Once rhadinoviruses infect they persist lifelong and spread to close contacts. However, surprisingly little is known about how and where in the body they persist. White blood cells are one site but there also appear to be other sites that are important for disease.

The lack of complete information makes it very difficult to develop infection control measures such as vaccination. Large animal and human rhadinoviruses are difficult to study. However, all mammals carry their own related viruses that behave in broadly similar ways. Mice provide the standard experimental model of mammalian biology, and so by studying a murine rhadinovirus - MuHV-4 - we can go a long way to understanding how these viruses as a whole work. Here we will use MuHV-4 to establish where in the body persistent infection is established, how it gets there from the point of virus entry, and how it then gets back to the sites from which virus is released to infect new hosts.

Rhadinoviruses can exist in either lytic or latent forms. When lytic they produce new virus, but are vulnerable to attack; when latent they are relatively inactive and so difficult to attack. Persistent infection is predominantly latent. However, lytic replication is required for virus to spread between different cell types, and this seems to be essential for normal infection. By defining precisely where lytic replication is essential we can identify targets for therapeutic intervention. Thus we can start to develop new means of reducing human and animal disease.

Studies of Murid Herpesvirus-4 (MuHV-4) have transformed our understanding of how rhadinoviruses work in vivo. Gamma-herpesviruses generally establish long-term latency in lymphocytes and replicate intermittently in epithelial cells. However several rhadinoviruses, including MuHV-4, also infect myeloid cells. Using cre-lox recombination to mark genetically viral genomes in specific cell types, we have identified an important role for dendritic cells in MuHV-4 transfer from its neuroepithelial entry point to B cells, and a surprising role for macrophages in subsequent infection spread to secondary sites such as the spleen. Thus host colonization is complex, with serial lytic replication shuttling infection between different cell types.
A related, unresolved question is what acute lymphoproliferation contributes to host colonization, as long-term virus loads in B cells correlate with epitope presentation to CD8+ T cells rather than with the extent of acute lymphoproliferation. Q-PCR data suggest that lymphoproliferation might help to establish systemic, non-lymphoid sites of persistence in epithelial / endothelial cells. Thus B cells would have an important role in virus transport as well as in persistence per se, with myeloid cells providing a link between lymphoid and stromal reservoirs.
Using luciferase+ and eGFP+ MuHV-4 reporter viruses and an extensive repertoire of virus-specific monoclonal antibodies, we will map comprehensively rhadinovirus dissemination and persistence. We will then used floxed marker viruses in cre transgenic mice to establish functional links between the infections of different cell types. Finally we will establish what roles lytic replication and acute lymphoproliferation play in virus dissemination and persistence. Thus we can establish the potential of targeted, perhaps type-specific, interventions to combat the rhadinovirus infections of humans and economically important ungulates.

Gamma-herpesviruses have a significant negative impact on human and animal health. KSHV infection is relatively rare in the UK, but still infects some 3 million people. Its prevalence around the Mediterranean and in developing countries is much higher - seropositivity exceeds 50% in sub-Saharan Africa, and KS is the commonest cancer in untreated HIV infection. Malignant Catarrhal Fever is similarly uncommon in the UK, but a substantial and intractable problem in the developing world. Apart from adjunctive HIV therapies, efforts to control rhadinovirus infections have so far met with little success. Therefore academic, commercial and philanthropic groups with an interest in controlling rhadinoviruses - either through vaccination or through anti-viral drugs - would benefit greatly from having more information about what these infections entail.

The staff involved in this project will learn vital skills in pathogenesis research. This requires experience - there are no pathogenesis "kits" - and such experience is likely to be of great benefit in post-genomic analysis. As knockout mice increasingly come on line, identifying gene functions will depend on sophisticated and realistic phenotypic screens such as those provided by in vivo infection models. Establishing and maintaining expertise in pathogenesis research will therefore be crucial for deciphering a large part of the human genome.