Nucleotide sequence of XhoI O fragment of ectromelia virus DNA reveals significant differences from vaccinia virus.

The nucleotide sequence of the 3913 base pair XhoI O fragment located in an evolutionary variable region adjacent to the right end of the genome of ectromelia virus (EMV) was determined. The sequence contains two long open reading frames coding for putative proteins of 559 amino acid residues (p65) and 344 amino acid residues (p39). Amino acid database searches showed that p39 is closely related to vaccinia virus (VV), strain WR, B22R gene product (C12L gene product of strain Copenhagen), which belongs to the family of serine protease inhibitors (serpins). Despite the overall high conservation, differences were observed in the sequences of p39, B22R, and C12L in the site known to interact with proteases in other serpins, suggesting that the serpins of EMV and two strains of VV may all inhibit proteases with different specificities. The gene coding for the ortholog of p65 is lacking in the Copenhagen strain of vaccinia virus; the WR strain contains a truncated variant of this gene (B21R) potentially coding for a small protein (p16) corresponding to the C-terminal region of p65. p65 is a new member of the family of poxvirus proteins including vaccinia virus proteins A55R, C2L and F3L, and a group of related proteins of leporipoxviruses, Shope fibroma and myxoma viruses (T6, T8, T9, M9). These proteins are homologous to the Drosophila protein Kelch involved in egg development. Both Kelch protein and the related poxvirus proteins contain two distinct domains. The N-terminal domain is related to the similarly located domains of transcription factors Ttk, Br-C (Drosophila), and KUP (human), and GCL protein involved in early development in Drosophila. The C-terminal domain consists of an array of four to five imperfect repeats and is related to human placental protein MIPP. Phylogenetic analysis of the family of poxvirus proteins showed that their genes have undergone a complex succession of duplications, and complete or partial deletions.

[Insertion mutants of the vaccinia virus. The effect of inactivating E7R and D8L genes on the biological properties of the virus]. / Insertsionnye mutanty virusa ospovaktsiny. Vliianie inaktivatsii genov E7R i D8L na biologicheskie svoistva virusa.

The integrative plasmids with the expressive marker gene for beta-galactosidase were constructed for insertional inactivation of nonessential genes E7R and D8L of vaccinia virus located in the central region of the viral genome. Inactivation of the D8L gene in the strains WR and LIVP results in smaller viral plaques in the culture of chicken embryo cells, decreases the viral ability to propagate in mouse brain and has no effect on the size and character of damage in intracutaneous infection of rabbits. Inactivation of E7R gene did not affect the known biological properties of the virus. The existence of the nonessential genes in the central region of the viral genome, inactivation of which does not result in viral attenuation, can be used for increase of antigenic activity of the live attenuated vaccines.

Gene A32 product of vaccinia virus may be an ATPase involved in viral DNA packaging as indicated by sequence comparisons with other putative viral ATPases.

Statistically significant sequence similarity was revealed between the gene A32 product of vaccinia virus (VV), gene I products (gpI) of filamentous single-stranded DNA bacteriophages, and IVa2 gene products of adenoviruses. Four conserved sequence motifs were delineated, the two N-proximal of which correspond to the A and B motifs of the purine NTP-binding pattern. Based on the role of gpI and IVa2 proteins in virion morphogenesis, and on the conservation of the NTP-binding pattern in these proteins, we hypothesize that the A32 gene product might be involved in an ATP-consuming function in VV virion formation, e.g., packaging of the DNA in the virus particle.

[Search for unique segments of the ectromelia virus genome by cross blot hybridization with DNA from other orthopoxviruses]. / Poisk unikal'nykh uchastkov genoma virusa éktromelii metodom perekrestnoi blot-gibridizatsii s DNK drugikh ortopoksvirusov]

In order to identify ectromelia virus (EMV) genome regions which may contain genes responsible for the specific pathogenicity of this virus, blot cross-hybridization of EMV DNA with those of other orthopoxviruses was performed. Two hybridization schemes were employed: one of them included hybridization of labelled cloned fragments of EMV with digests of other viral DNAs, the other, reciprocal, consisted in hybridization of labelled total DNAs of various orthopoxviruses with digests of the region of EMV DNA adjacent to the right-terminal inverted repeat. It was demonstrated that the counterpart to an approximately 8-kilobase pair portion of EMV genome flanking the inverted repeat could be detected only in the cowpox virus genome but not in the genomes of vaccinia and rabbitpox viruses. XhoI-O and XhoI-K fragments of EMV DNA contained, along with genes found in other poxviruses, certain genes which appeared to be unique for EMV. It is postulated that some of these genes may determine the specific biological properties of EMV, including its pathogenicity for mice.

Two vaccinia virus recombinants expressing HBsAg with different concentration of A- and pre-S2 antigenic determinants.

Comparative studies of two vaccinia virus (VV) recombinants expressing the hepatitis B virus (HBV) surface antigen (HBsAg) including the pre-S2 region (M-protein) showed that the L-pre-S2/15 recombinant expressed 5-fold more HBsAg as determined by the content of a-determinant than the recombinant v137. However, both recombinants expressed comparable amounts of the pre-S2 antigenic determinant as assessed by enzyme immunoassay with monoclonal antibodies. According to our calculations, one HBsAg unit expressed by the recombinant v137 contained 7-9 times more pre-S2 antigen than did one HBsAg unit expressed by the L-pre-S2/15 recombinant. Binding of pre-S2 region to polymerized human serum albumin was shown not to be an efficient assay at low pre-S2 concentration. HBsAg expressed by the v137 recombinant was less extensively secreted from cells as compared to that expressed by L-pre-S2/15 recombinant. Both recombinants induced the production of antibodies to the pre-S2 antigenic determinant in rabbits. L-pre-S2/15 induced anti-HBsAg a-determinant antibody as well.

[Synthesis of the E-antigen of the hepatitis B virus (HBeAg) in eukaryotic cells by a recombinant strain of the vaccinia virus]. / Sintez E-antigena virusa gepatita B (HBeAg) v éukarioticheskikh kletkakh rekombinantnym shtammom virusa ospovaktsiny.

The recombinant plasmids containing the gene for hepatitis B viral core-antigen with the pre-core-sequence controlled by the early-late promoter of the 7.5' K protein gene were constructed. The recombinant strains of vaccinia virus were obtained on their basis (vHBe42-1 and vHBe42-3) selectively expressing HBeAg of hepatitis B virus. The kinetics of HBeAg synthesis was studied in infected cells as well as secretion of the protein into culturing medium. Three proteins were found by blotting technique in the cells infected by vHBe42-3 that react with the specific antiserum to HBeAg and have the mol. masses 25, 22 and 17 kD. The completely processed HBeAg 17 kD was found in the culturing medium. The rabbit serums from the animals immunized by recombinant vHBe42-3 contained antibodies to HBeAg but not to HBcAg. This makes it possible to study the structural and functional organization, immunological properties and role of this antigen in pathogenesis of hepatitis virus B and to construct the specific test systems for screening HBeAg and corresponding antibodies.

[Verification of the safety, inoculability, reactogenicity and antigenic properties of a live recombinant smallpox-hepatitis B vaccine in an experiment in volunteers]. / Proverka bezopasnosti, privivaemosti, reaktogennosti i antigennykh svoistv zhivoi rekombinantnoi ospenno-gepatitnoi B-vaktsiny v opyte ne dobrovol'tsakh.

Trials of the first Soviet live recombinant smallpox-hepatitis B vaccine (SHBV) in volunteers (20 men aged 18-20 years) showed its safety, good "take"-rate, and lower reactogenicity as compared with the standard smallpox vaccine (LIVP strain). Smallpox virus-neutralizing antibodies in response to SHBV were produced as well as in response to the smallpox vaccine. Revaccination of human subjects with smallpox vaccine and SHBV 45 days after the previous vaccination resulted in antibody booster to vaccinia virus. After two inoculations of SHBV at an interval of 45 days no anti-HBsAg antibodies were found for 3 months after the last vaccination. However, even a single vaccination with SHBV induced priming to HBsAg. This could be demonstrated after inoculation of the subjects vaccinated with SHBV with one dose of plasma hepatitis vaccine. In the subjects vaccinated with SHBV antibody in response to the plasma vaccine formed more frequently and in higher titres than in those prevaccinated with smallpox vaccine or placebo.

[Vaccinia and ectromelia recombinant viruses, causing an infection, characteristic for ectromelia, in mice]. / Rekombinanty virusov ospovaktsiny i éktromelii, vyzyvaiushchie kharakternye dlia virusa éktromelii porazheniia u myshei.

Ten recombinants between the viruses of vaccinia and ectromelia were isolated that cause the ectromelia virus specific lesions in mice. The structure of recombinant viral genomes, the efficiency of viral propagation in mice, the nature of lesions induced by viruses have been studied. Eight of obtained recombinants have a DNA insertion originating from the right end of ectromelia viral genome, nine recombinants have an insertion originating from the left end, seven recombinants possess both insertions. The latter recombinants have more pronounced pathogenicity for mice. Both revealed regions are supposed to define the specific pathogenicity of ectromelia virus for mice.

[Mapping of "nonessential" regions in the genome of vaccinia virus]. / Kartirovanie "nesushchestvennykh" oblastei v genome virusa ospovaktsiny.

The special molecular probe for mapping the "nonessential" regions in the genome of vaccinia virus has been obtained by the genetic engineering methods. The probe included the gene for beta-galactosidase of E. coli under the control of vaccinia virus 7.5 K protein promoter as well as the gene for kanamycin resistance. In its final version the probe is obtainable from the plasmid pUCZ beta using the restriction endonucleases SalI, BamHI, EcoRI. The probe included by the BamHI fragment of DNA was inserted into the HindIII-E-fragment of the vaccinia virus (cloned into a plasmid) in 8 of the existing 9 BglII cleavage sites. The latter plasmids were introduced into the chicken embryo cells infected by the vaccinia virus. The plasmid having the probe inserted into the 5th BglII site (from the left end) of the HindIII-E fragment permitted to obtain the live vaccinia strain expressing the beta-galactosidase. Thus, the "nonessential" region of vaccinia virus, that was not described previously, is mapped.

Antigenic properties of vaccinia virus and of the virus recombinant strains expressing heterologous genes.

Immunologic properties of vaccinia virus (VV), strain LIPV, and VV recombinant strains containing the gene of hepatitis B virus surface antigen and the TK gene of herpes simplex virus (HSV) have been studied. Production of antibodies against the majority of VV structural proteins, including nucleocapsid internal proteins was demonstrated in rabbits. Insertion of heterologous genes into the VV genome was without effect on the spectrum of antibodies produced against VV virion proteins. The data obtained in volunteers indicate that not only virus-neutralizing antibodies but also antibodies against most VV structural proteins are preserved in humans over many years. Reimmunization of volunteers with VV recombinant stimulates synthesis of antibodies against virion proteins whereas the spectrum of antibodies remains unchanged. Humans and rabbits did not differ in the spectrum of antibodies to VV virion proteins.

[Analysis of antibody formation to the vaccinia virus in human subjects and rabbits in response to the administration of a recombinant vaccinia-hepatitis B vaccine]. / Analiz obrazovaniia antitel k virusu ospovaktsiny u liudei i krolikov v otvet na vvedenie rekombinantnoi ospenno-gepatitnoi (B) vaktsiny.

Immune blotting analysis was used to study antibody production to virion proteins of vaccinia virus (VV) in response to inoculation of human volunteers and rabbits with VV (LIVP strain) and its genetic engineering recombinant variants carrying a gene of hepatitis B virus surface antigen (LIOGEN-HB/C2-TK-) and, plus to that, the TK gene of herpes simplex virus (LIOGEN-HB/C2-TK+). In the blood of human subjects vaccinated 10-15 years earlier antibodies to many virion proteins were demonstrated: not only to surface 42K, 35K, 11K, but also to nucleoid proteins 135K, 88K, 62K, 60K, 26K. Vaccination with genetic engineering viruses results in stimulation of synthesis of antibodies to VV virion proteins. No differences in the antigenic properties of recombinant viruses were found. The results obtained in model experiments in rabbits verified those obtained in human volunteers.

[Expression of beta-galactosidase in recombinant nonintegrated plasmids in evaluating the functional activity of vaccinia virus promoters]. / Ispol'zovanie ékspressii beta-galaktozidazy rekombinantnymi neintegrirovannymi plazmidami dlia otsenki funktsional'noi aktivnosti promotorov virusa ospovaktsiny.

The recombinant plasmids pVL1 and pVL2 were constructed for insertion and expression of alien genetic information in HindIII-F fragment of vaccinia virus DNA under the control of the strong early-late promoter of the protein 7.5. The late promoter of the main late protein 11K of vaccinia virus was cloned. These as well as other vector plasmids have been used to express the procaryotic beta-galactosidase gene. Functional activity of the genetic engineering constructions was estimated by transitory expression of beta-galactosidase after plasmid DNA transfection into the chicken fibroblasts embryo culture infected with vaccinia virus. The promoters of the genes for 7.5K and 11K proteins permitted the high level of beta-galactosidase expression. Using of the early promoter of the central part of HindIII-F fragment DNA from vaccinia virus was less efficient for expression of the enzyme.

Biologic properties and genome structure of the recombinants between ectromelia and rabbitpox viruses.

Administration of rabbitpox virus (RPV) DNA, cleaved into 2 fragments by SmaI restrictase, into ectromelia virus (EMV)-infected chick fibroblast cells yielded recombinants whose properties were characteristic of both parents. Some recombinants capable of producing RPV-type lesions upon intracutaneous (i.c.) infection of rabbits could also produce EMV-specific lesions upon footpad inoculation of mice. The analysis of some recombinants as well as vaccinia virus strains has shown that the ability of the virus to reproduce when injected into the mouse footpad is a necessary, but not a sufficient condition for production of EMV-type lesions. According to restrictase analysis of recombinant DNA, the genome of recombinants mainly consists of RPV DNA sequences with insertions of small EMV DNA fragments.

[Physical mapping of DNA-temperature-sensitive mutations of vaccinia virus]. / Fizicheskoe kartirovanie DNK-temperaturochuvstvitel'nykh mutatsii virusa ospovaktsiny.

Four DNA-temperature-sensitive (ts) mutations were mapped in the genome of vaccinia virus (VV). Physical mapping of these mutations was performed by restriction analysis of the genomes of recombinants between VV DNA- ts mutants and ectromelia virus as well as by the marker rescue with cloned restriction fragments of VV DNA. One of the mutations was mapped on the HindIII-E-fragment. Biochemical studies of this mutant indicate that the mutation is not in the DNA polymerase gene which is located on the same fragment. The other three mutations were mapped in a 10 kilobase region in the middle of the HindIII-D-fragment. As shown previously, these mutations inactivate different genes, and the products of these genes participate directly in the DNA synthesis. Thus, at least three proteins involved in the VV DNA synthesis are encoded by neighboring genes in the central part of the viral genome.

[Comparative restriction analysis of HindIII-F-fragments of DNA from 4 strains of vaccinia virus]. / Sravnitel'nyi restriktsionnyi analiz HindIII-F-fragmentov DNK 4 shtammov virusa ospovaktsiny.

The localization of KpnI, SacI, XhoI, AvaI, PstI, BglI, BamHI, EcoRI, PmiI, SalI, BglII, restriction endonuclease cleavage sites in HindIII-F-fragments of DNA from vaccinia strains WR, Copenhagen, LIVP and neurovaccine has been detected. The fragments have been shown to differ in the number of AvaI, EcoRI and BamHI sites. The fragments also differ from the analogue of Tian Tan vaccinia strain in the pattern of restriction by AvaI, XhoI, PstI, EcoRI and BamHI endonucleases.

Recombinants between vaccinia and ectromelia viruses bearing the specific pathogenicity markers of both parents.

Nineteen recombinants between vaccinia virus (VV) DNA-temperature-sensitive mutants and ectromelia virus (EMV) were characterized with respect to their biological properties and genome structure. Four of these recombinants acquired the pathogenicity for mice characteristic of EMV, while the pathogenicity for rabbits characteristic of VV was not only preserved, but even enhanced. Most unexpectedly, these recombinants with 'double pathogenicity', as well as four other recombinants, acquired a stable genetic trait which was not typical of the parental viruses, i.e., the ability to form haemorrhagic lesions on the chorioallantoic membrane of chick embryos. Approximate mapping of the genomes of these recombinants with restriction endonucleases showed that their DNA contained mostly VV sequences with a single detected insert of EMV DNA. In the 'double pathogenicity' recombinants, this insert was located in the central part of the genome, and its minimal size was about 16 Mdal.

Biochemical and genetic characterization of vaccinia virus temperature-sensitive mutants with DNA- and DNAf-phenotypes.

Eighty temperature-sensitive (ts) mutants of vaccinia virus were examined for defects in synthesis of DNA. Nine ts mutants were incapable of synthesizing DNA at the restrictive temperature of 39.5 degrees C (DNA- mutants). Biochemical and genetic data indicate that all 9 DNA- mutants carry mutations in different genes. Temperature shift-up experiments have shown that 6 ts mutants with the DNA- phenotype have mutations in the genes coding for the proteins directly associated with vaccinia DNA synthesis. Temperature shift-down experiments in the presence of cytosine arabinoside revealed 5 ts mutants capable of synthesizing DNA at the elevated temperature, but this DNA failed to form infectious virions. These ts mutants were designated as DNAf- mutants. Pulse-chase experiments for the DNAf- mutant 1877 revealed that viral DNA produced at 39.5 degrees C was incapable of entering into mature virions or any subviral particles. Based on the data for recombination among ts mutants with the DNA- and DNAf- phenotype a tentative genetic map was constructed.