Fine mechanisms of ectromelia virus thymidine kinase-negative mutants avirulence.
Three independently selected spontaneous thymidine kinase-negative mutants (TK-phenotype) and a recombinant with Escherichia coli beta-galactosidase gene (LacZ+ phenotype) inserted in the viral thymidine kinase gene (tk) were derived from a plaque-cloned isolate of K-1 ectromelia virus strain (TK+ phenotype). Dramatically decreased virulence of TK- variants was observed for all routes of mouse inoculation. The kinetics of TK+ and TK- variants in various target organs indicated a significant decrease of production and dissemination of TK- mutants and recombinant in the organs of mice. In the spleen and liver of intranasally or intracerebrally infected mice TK- virus was not detected during the entire period of observation. Analysis of organs homogenates of mice intranasally infected by a mixture of recombinant with TK-LacZ+ phenotype and parental isolate with TK+LacZ- phenotype on the monolayers of TK- cells indicated that only white plaques (LacZ-) with the TK+ phenotype appeared from liver and spleen homogenates. Thus, the mouse acts as a live filter much more efficiently than any other selective systems. Ultrastructural studies showed that viral damage in animals infected by TK- variants was far less than that observed in mice, infected with wild type of ectromelia virus and pathological lessions were slight and reversible. Replication of ectromelia virus TK- variants was blocked at the viroplasma stage in cells with a high level of differentiation in contrast to TK+ variants. Most likely, such restriction of target cells assortment is the general reason of reduced virulence in the case of tk-gene inactivation.
Institute of Molecular Biology, NPO Vector, Koltsovo, Novosibirsk Region, Russia.
G V
GV
Kochneva
I H
IH
Urmanov
E I
EI
Ryabchikova
V V
VV
Streltsov
O I
OI
Serpinsky
D000818:Animals; D001921:Brain / Q000821:virology; D002460:Cell Line; D002522:Chlorocebus aethiops; D004481:Ectromelia virus / Q000201:enzymology / Q000235:genetics / Q000472:pathogenicity* / Q000648:ultrastructure; D004926:Escherichia coli / Q000235:genetics; D008099:Liver / Q000821:virology; D008168:Lung / Q000821:virology; D008297:Male; D051379:Mice; D009928:Organ Specificity; D010641:Phenotype; D011993:Recombinant Fusion Proteins / Q000235:genetics; D013154:Spleen / Q000821:virology; D013937:Thymidine Kinase / Q000235:genetics* / Q000502:physiology; D014764:Viral Proteins / Q000235:genetics* / Q000502:physiology; D014774:Virulence / Q000235:genetics; D014776:Virus Cultivation; D014779:Virus Replication
D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D013937:Thymidine Kinase