A cosmid-based system for inserting mutations and foreign genes into the simian varicella virus genome.
Simian varicella is a natural varicella-like disease of nonhuman primates. The etiologic agent, simian varicella virus (SVV), is genetically related to varicella-zoster virus (VZV) and SVV infection of nonhuman primates is a useful model to investigate VZV pathogenesis and latency. In this study, we report development of a cosmid-based genetic system to generate SVV mutant viruses. SVV subgenomic DNA fragments (32-38kb) that span the viral genome were cloned into cosmid vectors. Co-transfection of Vero cells with four overlapping cosmid clones representing the entire SVV genome resulted in recombination and generation of infectious virus. SVV mutants were produced by manipulation of one cosmid and substitution into the genetic system. This genetic approach was used to insert a site-specific mutation within the SVV open reading frame 14 which encodes the nonessential glycoprotein C gene. In a subsequent experiment, the green fluorescent protein (GFP) gene was inserted into the SVV genome within ORF 14. These SVV mutants replicate as efficiently as wild-type SVV in cell culture. This cosmid-based genetic system will be useful to investigate the effect of viral mutations on SVV pathogenesis and latency and also to develop and evaluate recombinant varicella vaccines that express foreign antigens.
Gray
Wayne L
WL
Department of Microbiology and Immunology, Slot 511, University of Arkansas for Medical Sciences, 4301 W. Markham St., Little Rock, AR 72205, USA. :graywaynel@uams.edu
Mahalingam
Ravi
R
D000818:Animals; D002522:Chlorocebus aethiops; D003360:Cosmids; D016679:Genome, Viral / Q000235:genetics*; D016254:Mutagenesis, Insertional; D016865:Reassortant Viruses; D018140:Varicellovirus / Q000235:genetics*; D014709:Vero Cells; D014759:Viral Envelope Proteins / Q000096:biosynthesis / Q000235:genetics
D014759:Viral Envelope Proteins; C038535:glycoprotein gC, herpes simplex virus type 1