Viral Pathogenesis

Research Interests

Herpesviruses are a major cause of disease worldwide and are among the most accomplished human pathogens, with specific viruses infecting more than 90% of the world population. Their main biological feature is their ability to persist and reactivate in a primed immunocompetent host. The control of herpesvirus infections thus represents an important clinical goal. To achieve this we must understand the basic mechanisms of viral pathogenesis. The overall aim of the viral pathogenesis unit is to gain an understanding of basic molecular mechanisms of the modulation of cellular function and immune evasion afforded by herpesviruses. To this end, we use a laboratory animal model of infection. Our model consists of infection of laboratory mice with murine gamma-herpesvirus-68 (MHV-68) that establishes latent infection in B-lymphocytes. The ability to genetically manipulate both the virus and the host offers the potential to dissect the molecular mechanisms, involved in the virus/host interplay.

Group Members

Research Project

Molecular interactions in Murine Herpesvirus 68 Latent Infection of B-Lymphocytes

Studies into the molecular basis of gammaherpesvirus latency have been hindered by the lack of amenable animal model systems and the lack of fully permissive cell lines, which are required for the genetic manipulation of these viruses. Research on the utilisation of a gammaherpesvirus, designated murine herpesvirus 68 (MHV-68), whose pathogenesis can be readily investigated in the laboratory mouse (for recent reviews see Simas & Efstathiou, 1998; Doherty and Christensen, 2000). MHV-68 is genetically related to Epstein-Barr virus and Kaposi¹s sarcoma associated herpesvirus, which are important Human pathogens. Experimental infection of inbred strains of mice with MHV-68 results in acute productive infection of the lung followed by latent infection of B-lymphocytes. Comparison of the genomic organisation of MHV-68 with other gammaherpesviruses shows the presence of virus specific ORFs and a number of cellular homologues, which are predicted to determine the particular biological properties of these viruses, e.g. host range, immune evasion, latency and disease. MHV-68 has 14 unique such genes, designated M1 to M14, and several cellular homologues, including a complement control protein, a D-type cyclin and an IL8 receptor. In addition to these cellular homologues two of the ŒM¹ genes, M1 and M11, show low level similarity to serpins and bcl2 cellular protein, respectively. Our research interests are focused in trying to understand how co-ordinated expression of these cellular homologues and unique ORFs, in a developing B-lymphocyte, result in immune evasion and latent infection. To this end, we have adopted the following strategies; (i) to determine the tropism and virus transcription pattern, during the establishment and maintenance of latent infection in B-cells and, (ii) to construct recombinant viruses with specific genes deleted and study their phenotype upon infection of mice and, (iii) to identify cellular molecular targets for virus latency-associated genes.

Funding

SAPIENS/34240/99
Molecular interactions in Murine Herpesvirus 68 Latent Infection of B-Lymphocytes

Collaborators

University of Cambridge, UK
Stacey Efstathiou and Philip Stevenson

Research Project

Antiviral peptides blocking herpes simplex virus type 1 entry into cells

Human herpes simplex virus 1 (HSV-1) affects over 50% of the European Union adult population and is highly transmittable: No vaccines, despite repeated attempts, are yet available for prevention, but classical antivirals such as nucleosides are the choice chemotherapies. Nevertheless, HSV resistant strains and the failure to completely relieve symptoms (especially in immunocompromised individuals) make it imperative that antivirals that utilise new mechanisms of action are developed and marketed. Symptoms of HSV-1 infections include erythromatous vesicles, at mucocutaneous sites, which can ulcerate and be quite painful. The mouth, lips and genitalia are the dominant sites of these lesions. HSV-1 however causes herpes keratitis, a recurrent infection of the eye as well as the much feared manifestation of HSV-1 infection, herpes encephalitis, which has a 1/500,000 incidence and high mortality if not treated. Systemic infection of the newborn and the chronic lesions of immunocompromised patients (especially HIV and cancer patients) and patients undergoing organ transplantation (which necessitates use of immunosuppressive drugs) can often be complicated by HSV-1 infections. All of the above, including the pain, loss of work and productivity, are potent reasons for possessing effective new and possibly multiple therapies against HSV-1 infections. New therapies and in particular those which act by novel antiviral mechanisms should be developed. Many arguments against the use of peptide therapeutic agents, due principally to their rapid proteolysis and short half-lives are now dissipated. An excellent example is the anti-HIV aspartyl protease which enters cells and inhibit viral assembly. A second development, the existence of virions technologies to stabilize and deliver therapeutic peptides, has permitted administration of peptide chemotherapeutics by oral, I.V., nasal and other routes. The objective of this research program is to develop of a new class of anti-HSV-1 drugs – a peptide that acts at the cell surface blocking virus entry – when used alone or in combination with, for example, nucleoside antivirals should increase the quality of life of many in the EU who suffer pain, discomfort and sometimes morbidity. It is hopped that this work will form a basis for developing peptides moieties as anti-HSV (not part of this proposal) and provide an example of this class of antiviral that block virus entry into cells. When sold in the EU and globally it should prove economically beneficial to the EU and provide R&D training to young scientists. Because it operates via an extracellular novel mechanism these anti-HSV peptides will prove complementary not competitive with current antiviral chemotherapies; predictably combined therapy, as with anti-HIV therapies, may prove the most common use of antiviral peptides that block virus entry.

Funding

EU-QLK2-CT-2002-00810
Antiviral peptides blocking herpes simplex virus type 1 entry into cells.

Collaborators

ARISTOS Scientific Ltd, UK
William Gibbons

PRIMM, Italy
Paolo Sarmientos

University of Cambridge, UK
Helena Browne

University of Napoli, Italy
Massimiliano Galdiero

Research Project

Transcriptome Analysis of Germinal Centre B cells During Gammaherpesvirus Latent Infection

Analysis of genomes from gammaherpesviruses reveals the presence of large blocks of co-linearly arranged conserved genes interspersed with virus specific ORFs and cellular homologues. Hence, there are two classes of putative viral host control proteins, namely those encoded by genes with and without sequence similarity to cellular genes. The existence of viral homologues to cellular genes suggests that during co-evolution viruses have ‘hijacked’ host genes that were subsequently modified for the benefit of the virus. Virus specific ORFs may represent novel structures with functional activities homologous to cellular proteins or could simply be an example of proteins for which the host homologues have not yet been identified. Our objectives are focused in trying to reveal the molecular function that these ORFs and cellular homologues have in a context of infection, that result in evasion of the host immune response and life-long latency. To this end we use a gammaherpesvirus designated murine herpesvirus 68, as its pathogenesis can investigated in the laboratory mice. This project investigates the effect that MHV68 latent infection has upon GC B cell physiology by analyzing their transcription profile. We propose to use a strategy involving transgenic mice with a floxed EGFP allele that only becomes functional upon Cre mediated excision. In this model, Cre will be provided by a recombinant MHV68 resulting in the fluorescent tagging of latently infected cells. This makes possible the purification of pure populations of latently infected GC B cells, a pre-requisite for DNA microarray analysis. It is hoped that this strategy will identify key cellular genes and biochemical pathways that are involved in cellular functions important for the control of gammaherpesvirus infection. Knowledge gained from this type of approach may not only help determining the molecular basis for gammaherpesvirus infection but also provide clues on what gene products (either cellular or viral) may have therapeutic uses themselves or may be targets for therapeutic intervention.

Funding

POCTI/ESP/46378/02
Transcriptome analysis of germinal centre B cells during gammaherpesvirus latent infection

Collaborators

University of Cambridge, UK
Stacey Efstathiou, Philip Stevenson, and Paul Lyons

Publications

Jensen, K.K., Chen, S.C., Hipkin, R.W., Wiekowski, M.T., Schwarz, M.A., Chou, C.C., Simas, J., Alcami, A., Lira, S.A. (2003). Disruption of CCL21-induced chemotaxis in vitro and in vivo by M3, a chemokine-binding protein encoded by murine gammaherpesvirus 68. Journal of Virology 77 :624-30

Marques, S., Efstathiou, S., Smith, K.G.C., Haury, M.and Simas, J.P. (2003). Selective Gene Expression of Latent Murine Gammaherpesvirus 68 in B Lymphocytes. Journal of Virology 77 :7308-7318

Orge, L., Galo, A., Sepúlveda, N., Simas, J.P.*, Pires, M. (2003). Scrapie genetic susceptibility in Portuguese sheep breeds. Veterinary Record 153 :508

Fowler, P., Marques, S., Simas, J.P. and Efstathiou, S. (2003). ORF73 of murine herpesvirus-68 is critical for the establishment and maintenance of latency. Journal of General Virology 84 :3405-3416

Stevenson, P.G., May, J.S., Smith, X.G., Marques, S., Adler, H., Koszinowski, U.H., Simas, J.P. and Efstathiou, S. (2002). CD8+ T cell evasion by K3 plays a critical role in the amplification of a latent g-herpesvirus Nature Immunology 3 :733-40

Bridgeman, A., Stevenson, P., Simas, J.P., Efstathiou, S. (2001). A secreted chemokine binding protein encoded by murine gammaherpesvirus 68 is necessary for the establishment of a normal latent load. Journal of Experimental Medicine 194 :1-13

Parry, C.N., Simas, J.P., Smith, V.P., Stewart, C.A., Minson, A.C., Efstathiou, S., Alcami, A. (2000). A broad spectrum secreted chemokine binding protein encoded by a herpesvirus. Journal of Experimental Medicine 191 :573-578

Orge, L., Simas, J.P., Fernandes, A.C., Ramos, M., Galo, A. (2000). The lesion profile of BSE in Portuguese cattle is similar to that described in British cattle. Veterinary Record 147 :486-488

Simas, J.P., Swann, D., Bowden, R., Efstathiou, S. (1999). Analysis of murine gammaherpesvirus-68 transcription during lytic and latent infection. Journal of General Virology 80 :75-82

Donnelly, C.A., Santos, R., Ramos, M., Galo, A., Simas, J.P. (1999). BSE in Portugal: Anticipating the decline of an Epidemic. Journal of Epidemiology and Biostatistics 4 :277-283

Simas, J.P., Bowden, R.J., Paige, V., Efstathiou, S. (1998). Four tRNA-like sequences and a serpin homologue encoded by murine gammaherpesvirus 68 are dispensable for lytic replication in vitro and latency in vivo Journal of General Virology 79 :149-53

Simas, J.P., Efstathiou, S. (1998). Murine gammaherpesvirus 68: a model for the study of gammaherpesvirus pathogenesis Trends in Microbiology 6 :276-82

Bowden, R.J., Simas, J.P., Davis, A.J., Efstathiou, S. (1997). Murine gammaherpesvirus 68 encodes tRNA-like sequences which are expressed during latency Journal of General Virology 78 :1675-87

Simas, J.P., Fazakerley, J.K. (1996). The course of disease and persistence of virus in the central nervous system varies between individual CBA mice infected with the BeAn strain of Theiler's murine encephalomyelitis virus Journal of General Virology 77 :2701-11

Simas, J.P., Dyson, H. and Fazakerley, J.K. (1995). The neurovirulent GDVII strain of Theiler¹s virus can replicate in glial cells Journal of Virology 69 :5599-5606