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AIM: Demonstrate the possibility of using nested PCR method for determination of Varicella Zoster virus (VZV) in clinical samples of peripheral blood of patients. MATERIALS AND METHODS: Material from 35 patients with clinical manifestations of herpes zoster and control group of 20 healthy donors was used in the study. Monocyte fraction of venous blood cells, pretreated with heparin, was isolated by centrifugation in ficoll-verografin density gradient, total DNA was then isolated from cells by phenol-chloroform extraction with subsequent precipitation with alcohol. Polymerase chain reaction was carried out in thermocyclers Tercyc and TProfessional Gradient (Biometra), amplified DNA was analyzed by electrophoresis on 1.6% agarose gel in the presence of ethidium bromide. RESULTS: Data on detection of viral DNA in blood monocytes in 17 (49%) of ill patients, as well as in 1 (out of 20 in control group) practically healthy donor were obtained. A possibility of a subclinical reactivation of the virus is discussed in the latter case. CONCLUSION: A possibility of viral DNA determination in monocytes of patient blood without using expensive equipment is shown, that could find application in clinical practice, especially for diagnostics of patients with non-characteristic clinical manifestations, as well as patients with subclinical forms of the disease.

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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Labelled vaccinia virus DNA was used in saturation-hybridization experiments with RNA extracted from virus-infected cells. An excess of "late" cytoplasmic RNA converted 45% of DNA into DNA-RNA hybrids, whereas 17% of DNA could be converted into hybrids by RNA extracted from purified nuclei. RNA-RNA hybrids obtained from cytoplasmic RNA by self-annealing and ribonuclease treatment, were melted and used in hybridization with DNA: 36% of DNA was hybridized at the maximal concentration used.

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Comparative studies of tissue culture and dermal smallpox vaccines were carried out in a strictly controlled coded trial by revaccination of adults by scarification. Two lots of tissue culture and one lot of dermal vaccines with a similar infectious titer (8.0 lg PFU/ml) were used. All the lots tested "took" in 100%. In the group revaccinated with tissue culture vaccine seroconversion was observed in 94.2 +/- 2.8%--95.0 +/- 2.8%, with a 4.6 +/- 5.3-fold rise in geometric mean antibody titer; the dermal smallpox vaccine produced seroconversion in 85.7 +/- 4.4% with 4.3-fold rise in geometric mean antibody titer. Febrile reactions in revaccines with the tissue culture vaccine were observed in 23.1%--26.1% which did not exceed this parameter in the group revaccinated with the dermal vaccine (25.2%); in all the groups febrile ractions were of mild degree (37.1 degrees--38 degrees C). Local reactions of the type of confluent erythema were observed 1.8--5.3-fold more frequently among those revaccinated with the dermal vaccine than among those revaccinated with the tissue culture vaccine. Thus, the tissue culture vaccine used epicutaneously was as good as the dermal one by the take rate and antigenic activity, but had less remarked reactogenic properties which allows to recommend it for public health practice.

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Restriction endonucleases EcoRI, BamHI, Hind III and KpnI were used for analysis of acccinia virus DNA. The number and size of restriction endonuclease fragments were determined by gel electrophoresis and the analysis of 3H-labeled vaccinia virus DNA. It was shown that many EcoRI and BamHI fragments had the same and similar sizes. The exact number of EcoRI and BamHI fragments were estimated only after analysis of [3H]-labeled vaccinia DNA. The vaccinia genome sizes calculated from HindIII, KpnI and EcoRI are very close to the actual genome weight. But the sum of BamHI fragments is much lower than those determined by electron microscope method.

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Denaturated 3H-thymidine-labeled vaccinia virus DNA was hybridized with an excess of "late" virus-specific RNA isolated from virus-infected chick embryo cell cultures 8 hours postinfection. The percentage of DNA converted into DNA-RNA hybrid under these conditions never exceeded 50%. If the RNA preparation had been self-annealed prior to hybridization, the percentage was decreased slightly. On the other hand, if the self-annealed RNA had been treated with RN-ase and the double-stranded DNA-RNA hybrids had been denaturated, they became capable of converting into hybrids at least 68% of the labeled DNA. These data indicate that transcription of both DNA strands occurs in a large portion (over 68%) of vaccinia virus genome.

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The tissue culture vaccine against smallpox has some important advantages over the dermal preparation: it is free from bacterial contamination, contains no serum proteins, and suitable for intradermal inoculation with jet injections. The virus for the tissue culture smallpox vaccine is grown in Japanese quail embryo cultures controlled for the absence of contaminating viruses. In trials of the tissue culture smallpox vaccine in 800 revaccinated volunteers no untoward reactions or complications were observed. The antigenic activity of the tissue culture smallpox vaccine was superior to that of dermal vaccine used in the same dose: the geometric mean neutralizing antibody titre after vaccination with the tissue and dermal preparations was 1 : 256 and 1 : 158, respectively, and the antibody rise was 4.5- and 2.5-fold.

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The antigenic and immunogenic properties of five ts mutants of neurovaccinia virus with markedly reduced pathogenicity for laboratory animals as compared with "wild" type virus were studied. The antigenic and immunogenic activities of the mutants correlated with their capacity to reproduce in the skin of the inoculated animals. When similar doses of UV-inactivated mutants were used for immunization of rabbits, significant differences (more than 100-fold) in their antigenic activity were found. When rabbits were immunized with active virus, neutralizing antibody in titer of 1:800 protected all the immunized animals against intracerebral challenge with log 4.0 LD50 of neurovaccinia virus. On the other hand, the neutralizing antibody formed in response to inoculation of UV-inactivated virus did not protect the immunized animals in titers exceeding 1:2560. A correlation between the antibody titer for "extracellular" virus and immunity in vaccinated rabbits has been demonstrated.

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