The use of a hybridization assay for the study of host defences against herpes simplex virus.
A rapid and simple hybridization assay was developed as an alternative for virus titration for the investigation of host resistance against HSV-1 infections in vitro. The probe which was constructed for this assay was shown to be HSV-1-specific. When a monolayer of fibroblasts was infected for 24 h before hybridization, 15 PFU were detected reliably. A plateau in hybridization levels was found when the multiplicity of infection reached 1. In order to demonstrate the applicability of the probe for the study of host defences against HSV in vitro, fibroblasts were infected with HSV in the presence of different numbers of adherent cells and different concentrations of serum containing high titres of anti-HSV antibodies and complement. After 20 h of incubation, samples were lysed, spotted on Zetaprobe filter paper and hybridized with a 32P-labelled RNA probe. Spots were counted for radioactivity. The radioactivity was taken as a measure of the success of infection. Results showed that at high (10%) concentrations of serum containing high titres of anti-HSV antibodies and complement neutralization plays an important role. At low (1%) concentrations of serum containing high titres of anti-HSV antibodies and complement the phagocytic role of adherent cells becomes the dominant factor in preventing infection of the fibroblasts. However, when the number of infectious particles is increased, the protection provided by adherent cells is overwhelmed.
Fluit
A C
AC
Eijkman Winkler Laboratory for Medical Microbiology University Hospital Utrecht, The Netherlands.
van Strijp
J A
JA
Miltenburg
L A
LA
van Kessel
C P
CP
Snippe
H
H
Verhoef
J
J
D000914:Antibodies, Viral / Q000276:immunology*; D001769:Blood; D002478:Cells, Cultured; D003001:Cloning, Molecular; D003165:Complement System Proteins / Q000276:immunology*; D015342:DNA Probes; D004279:DNA, Viral / Q000032:analysis*; D005347:Fibroblasts; D005814:Genes, Viral; D006801:Humans; D009500:Neutralization Tests; D009693:Nucleic Acid Hybridization; D010587:Phagocytosis; D018139:Simplexvirus / Q000235:genetics / Q000276:immunology* / Q000502:physiology
D000914:Antibodies, Viral; D015342:DNA Probes; D004279:DNA, Viral; D003165:Complement System Proteins