The study of orthopoxvirus genes for Kelch-like proteins. II. Construction of cowpox virus variants with targeted gene deletions

Integrative plasmids p delta C, p delta D, and p delta G were designed to contain a selective marker beyond the region of homology to virus DNA and to allow construction of recombinant cowpox viruses (CPV) that lack C18L, D11L, or G3L coding for kelch-like proteins. CPV mutants lacking one (C18L, D11L, or G3L), two (D11L/G3L or C18L/D11L), or three (D11L/G3L/C18L, that is, all) kelch-like protein genes of the left variable region of the virus genome were obtained. Impaired reproduction was observed for the triple mutant. Pocks produced by the triple mutant and the original virus differed in size and morphology. In addition, the two CPV variants differed in destructive changes caused in the chorioallantoic membrane of chicken embryos.     

Neutralizing Monoclonal Antibodies Cross-React with Fusion Proteins Encoded by <Emphasis Type="Italic">129L</Emphasis> of the Ectromelia Virus and <Emphasis Type="Italic">A30L</Emphasis> of the Variola Virus

PCR fragments containing the fusion protein genes 129L of the ectromelia virus (EV) and A30L of the variola virus (VARV) were cloned in pQE32. The expression products, recombinant prA30L and pr129L, were isolated from Escherichia coli cell lysates by metal-chelate affinity chromatography. The recombinant proteins retained the capability of oligomerization, characteristic of their natural analogs. ELISA and immunoblotting were used to test 22 monoclonal antibodies (mAbs) to orthopoxviruses (19 mAbs to EV, 2 mAbs to the vaccinia virus (VACV), and 1 mAb to the cowpox virus (CPXV)) for interaction with prA30L, pr129L, and orthopoxviruses. Twelve species-specific epitopes were found in the EV fusion protein 129L and its recombinant analog. Ten cross-reacting epitopes were found in the EV, CPXV, and VACV fusion proteins. Of these, nine epitopes were present both in prA30L and in the VARV fusion protein. Five mAbs interacting with cross-reacting epitopes were capable of efficient neutralization of VACV; two of these mAbs neutralized VARV. It was demonstrated that there are species-specific epitopes in EV 129L and cross-reacting epitopes in the EV, VARV, CPXV, and VACV fusion proteins, including epitopes that induced synthesis of virus-neutralizing antibodies against VACV and VARV.     

Maintenance stability of the pBR322 and pBR327 vector plasmids in Escherichia coli cells during multiple passages

Stability of pBR322 and pBR327 plasmids was studied. Plasmid-containing Escherichia coli strains were grown in liquid growth medium without selection pressure. Plasmid pBR327 was shown to be more stable in E. coli CSH54 cells than pBR322. Essential heterogenity of individual plasmid-containing clones was recognized by the maintenance stability of plasmid DNA. The indicated clones with high stability failed to be cured from pBR327 plasmid by means of acridine orange. High stability of plasmid maintenance and the failure to cure cells containing this plasmid are suggested to correlate with and to be essentially determined by the cell functions.     

Expression of genes for orthopoxviral TNF-binding proteins in insect cells and investigation of the recombinant TNF-binding proteins

Genes for TNF-binding proteins (CrmBs) of the variola virus (VARV), monkeypox virus (MPXV) or cowpox virus (CPXV) were isolated by PCR from viral genomes and expressed in a baculovirus system in the Sf21 insect cell line. Properties of the purified recombinant proteins were studied by various physicochemical and immunological methods. Using solid-phase enzyme-linked immunosorbent assay, it was shown that viral proteins inhibited hTNF binding with polyclonal anti-hTNF antibodies, with the efficiency of inhibition decreasing in the series VARV-CrmB > CPXV-CrmB > MPXV-CrmB. Biological activity of the recombinant protein preparations was assessed by their ability to neutralize TNF cytotoxicity on the L929 murine fibroblast cells line. CrmBs were shown to neutralize cytotoxicity of human, mouse, and rabbit TNF in a species-specific manner. It was also shown that the efficiency of VARV-CrmB in inhibiting hTNF cytotoxicity exceeded that of polyclonal anti-hTNF antibodies. Orthopoxviral CrmB proteins can provide a basis for development of new anti-TNF drugs.__________Translated from Molekulyarnaya Biologiya, Vol. 39, No. 2, 2005, pp. 245–254.Original Russian Text Copyright © 2005 by Gileva, Ryazankin, Nepomnyashchikh, Totmenin, Maxutov, Lebedev, Afinogenova, Pustoshilova, Shchelkunov.     

Comparative analysis of variable regions in the genomes of variola virus

Nucleotide sequences of two extended segments of the terminal variable regions in variola virus genome were determined. The size of the left segment was 13.5 kbp and of the right, 10.5 kbp. Totally, over 540 kbp were sequenced for 22 variola virus strains. The conducted phylogenetic analysis and the data published earlier allowed us to find the interrelations between 70 variola virus isolates, the character of their clustering, and the degree of intergroup and intragroup variations of the clusters of variola virus strains. The most polymorphic loci of the genome segments studied were determined. It was demonstrated that that these loci are localized to either noncoding genome regions or to the regions of destroyed open reading frames, characteristic of the ancestor virus. These loci are promising for development of the strategy for genotyping variola virus strains. Analysis of recombination using various methods demonstrated that, with the only exception, no statistically significant recombinational events in the genomes of variola virus strains studied were detectable.