Construction of mutant herpes simplex virus type 2 for the development of preventive and therapeutic vaccines.

LOSINNO A.D; DAMIANI M.T; DAMIANI A.M

Mendoza

Simposio; II INTERNATIONAL SYMPOSIUM ON TRANSLATIONAL MEDICINE XIII ANNUAL MEETING OF INVESTIGATION AT THE FACULTY OF MEDICAL SCIENCES.; 2019

Facultad de Ciencias Médicas, Universidad Nacional de Cuyo

Herpes simplex virus type 2 (HSV-2) causes a sexually transmitted disease characterized by genital ulcers. The pathology associated with HSV-2 is recurrent, which causes persistent infections that, in the face of stress, hormonal changes and immunosupression, reactivate the latent virus.Antivirals (i.e. acyclovir) can partially control the signs and symptoms of this pathology. However, these drugs do not eradicate the virus from the body. Due to the biological similarities and antigenic cross-reactivity between HSV-1 and HSV-2, the development of a vaccine for the later could provide an additional protection against HSV-1, which icreases its potential value. The aim of this work is to clone the HSV-2 DNA as bacterial artificial chromosomes (BAC) to allow an easy manipulation of its genome and the construction of mutant viruses for vaccine purpose.Metodology. Preparation of a viral stock. Construction of transfer plasmid (pUC19-VHS-2_miniF). Transfection of VERO-infected cells with the transfer plasmid DNA. Infection of VERO cells with the recombinant virus. Extraction of circular viral DNA. Construction of VHS-2 BAC. Results. We generated a viral stock of reference strain MS by infecting VERO cells. After viral DNA purification, we amplified and cloned segments from specific regions (UL40 and UL41 genes) that are conserved in HSV-2 strains. We introduced the miniF between the two amplified segments and subsequently HSV-2-infected VERO cells were transfected with the transfer plasmid. Viruses expressing the GFP were detected under the fluorescence microscope and isolated by 3 rounds of plaque purification. Extrachromosomal DNA was extracted from infected cells and used to electroporate competent bacteria. Conclusion. We succesfully constructed a HSV-2 virus expressing GFP that will allow studies about the viral infection process. Currently, we have assays for the construction of HSV-2 BAC that are in progress.