New method for the assessment of molluscum contagiosum virus infectivity.
Molluscum contagiosum virus (MCV), a poxvirus pathogenic for humans, replicates well in human skin in vivo, but not in vitro in standard monolayer cell cultures. In order to determine the nature of the replication deficiency in vitro, the MCV infection process in standard culture has to be studied step by step. The method described in this chapter uses luciferase and GFP reporter constructs to measure poxviral mRNA transcription activity in cells in standard culture infected with known quantities of MCV or vaccinia virus. Briefly, MCV isolated from human tissue specimen is quantitated by PCR and used to infect human HEK293 cells, selected for ease of transfection. The cells are subsequently transfected with a reporter plasmid encoding firefly luciferase gene under the control of a synthetic early/late poxviral promoter and a control plasmid encoding a renilla luciferase reporter under the control of a eukaryotic promoter. After 16 h, cells are harvested and tested for expression of luciferase. MCV genome units are quantitated by PCR targeting a genome area conserved between MCV and vaccinia virus. Using a GFP reporter plasmid, this method can be further used to infect a series of epithelial and fibroblast-type cell lines of human and animal origin to microscopically visualize MCV-infected cells, to assess late promoter activation, and, using these parameters, to optimize MCV infectivity and gene expression in more complex eukaryotic cell culture models.
Department of Microbiology and Infectious Diseases, Cardiff Institute of Infection and Immunity, Cardiff, UK.
Subuhi
S
Sherwani
Niamh
N
Blythe
Laura
L
Farleigh
Joachim J
JJ
Bugert
D004279:DNA, Viral / Q000235:genetics / Q000302:isolation & purification; D015870:Gene Expression; D015967:Gene Expression Regulation, Viral; D017930:Genes, Reporter; D005814:Genes, Viral; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D049409:Luciferases, Firefly / Q000096:biosynthesis / Q000235:genetics; D049410:Luciferases, Renilla / Q000096:biosynthesis / Q000235:genetics; D008977:Molluscum contagiosum virus / Q000235:genetics / Q000302:isolation & purification / Q000472:pathogenicity* / Q000502:physiology; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic; D060888:Real-Time Polymerase Chain Reaction; D014616:Vaccinia virus / Q000235:genetics / Q000254:growth & development / Q000472:pathogenicity; D014776:Virus Cultivation; D014779:Virus Replication
D004279:DNA, Viral; D049452:Green Fluorescent Proteins; D049410:Luciferases, Renilla; D049409:Luciferases, Firefly