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The cloning and expression of herpes simplex virus type 1 glycoprotein c in vaccinia virus.

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posted on 19.11.2015, 09:10 by Caroline Mary. Griffiths
The Herpesviridae family contains numerous virus types, several of which can infect man. The virus which is most widely spread in terms of infection is herpes simplex virus (HSV), of which there are two serotypes (HSV-1 and HSV-2). Following a primary infection, the virus can establish a latent infection within neurons of sensory ganglia, where it remains throughout the lifetime of the host. HSV is able to reactivate from the latent state, and may produce clinical infection at the site of the initial virus invasion (recrudescent lesions). Although most HSV-1 infections are mild or subclinical, infection can lead to life-threatening illness. The search for an effective HSV vaccine has met with limited success to date. In recent years, vaccine research has turned toward the use of viral vectors, for the expression of individual virus proteins with a view to stimulating host immunity. A great deal of attention has been focussed on the vaccinia virus, which is able to accept large amounts of foreign DNA into it's genome with no loss of viability. Several HSV proteins have been expressed from recombinant vaccinia viruses, and this project outlines the cloning of DNA sequences encoding the HSV-1 glycoprotein C, and expression of that protein from a recombinant vaccinia virus. The HSV DNA sequences encoding the glycoprotein were cloned from an existing library, into plasmid vector pSC11. This plasmid allows insertion of gC sequences into the vaccinia virus genome by way of homologous recombination. The plasmid also provides a vaccinia virus promoter for the control of gC transcription during infection of cells with the resulting recombinant viruses. Recombinant vaccinia viruses produced on cloning of a 1.75kb NheI/SphI fragment containing the HSV-1 gC gene did not express the glycoprotein during infections. Vaccinia virus DNA polymerase is known to be sensitive to secondary structures within DNA, and the 5' non-coding region of the HSV gC gene is rich in GC basepairs, which could readily form such structures. Site-directed mutagenesis removed the 5' non-coding region (34 nucleotides) in an attempt to remove any such block to gC transcription. Vaccinia viruses containing the mutated sequences were able to express gC within infected cells, as determined by immunofluorescence. Mice inoculated with a gC-expressing recombinant vaccinia virus were able to induce HSV-neutralising antibodies and displayed 50% protection against a lethal HSV-1 challenge. The implications of gC-expressing vaccinia recombinant viruses, with respect to the search for an HSV vaccine and to the understanding of the role of gC during HSV infections is discussed.


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University of Leicester

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