A set of universal insertion plasmids was designed that makes possible the insertion of foreign genetic information into intergene region of viral DNA without alteration of functionally important virus genome sequences. The plasmids include the highly conserved region of orthopoxviruses carrying the thymidine kinase (TK) gene and adjacent sequences of viral DNA. This region is localized between open reading frames (ORF) K2R (TK gene) and K3R of vaccinia virus (strain Lister) genome. The recipient strains of vaccinia and ectromelia viruses providing for highly efficient construction and selection of recombinants on the basis of two selective systems (TK-/TK+ and LacZ(+)/LacZ(-)) were obtained. These recipient strains were used to design recombinants carrying in their intergene region a synthetic sequence encoding beta-endorphin gene. Insertions into the intergene region downstream of the TK gene do not alter the recombinant virulence. The local effect of beta-endorphin expressed by ectromelia recombinants was studied on the chorioallantoic membrane of chicken embryos and in mongrel mice. In this system the recombinant virus reproduction results in local enhancement of chorion epithelial cell proliferation and increased infiltration with leukocytes in the zone of virus reproduction, as compared with the wild-type parental virus.

Genomes of vaccinia strain L-IVP and its cloned variants were investigated by restriction and Southern blotting analysis. The strain L-IVP was shown to be heterogeneous and to consist of variants which differed in the genome structure and properties. Clone 1 derived from L-IVP was less reactogenic than the original strain for rabbits inoculated intradermally and had mutations in the right terminus of the genome. All the clones were stable in passages in the chorioallantoic membranes of developing chick embryos and roller BHK-21 cell cultures.