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The protein encoded by the vaccinia virus D4R gene has base excision repair uracil–DNA N-glycosylase (vvUNG) activity and also acts as a processivity factor in the viral replication complex. The use of a protein unlike PolN/PCNA sliding clamps is a unique feature of orthopoxviral replication, providing an attractive target for drug design. However, the intrinsic processivity of vvUNG has never been estimated, leaving open the question whether it is sufficient to impart processivity to the viral polymerase. Here, we use the correlated cleavage assay to characterize the translocation of vvUNG along DNA between two uracil residues. The salt dependence of the correlated cleavage, together with the similar affinity of vvUNG for damaged and undamaged DNA, support the one-dimensional diffusion mechanism of lesion search. Unlike short gaps, covalent adducts partly block vvUNG translocation. Kinetic experiments show that once a lesion is found it is excised with a probability ~0.76. Varying the distance between two uracils, we use a random walk model to estimate the mean number of steps per association with DNA at ~4200, which is consistent with vvUNG playing a role as a processivity factor. Finally, we show that inhibitors carrying a tetrahydro-2,4,6-trioxopyrimidinylidene moiety can suppress the processivity of vvUNG. [ABSTRACT FROM AUTHOR]

Considering that vaccination against smallpox with live vaccinia virus led to serious adverse effects in some cases, the WHO, after declaration of the global eradication of smallpox in 1980, strongly recommended to discontinue the vaccination in all countries. This led to the loss of immunity against not only smallpox but also other zoonotic orthopoxvirus infections in humans over the past years. An increasing number of human infections with zoonotic orthopoxviruses and, first of all, monkeypox, force us to reconsider a possible re-emergence of smallpox or a similar disease as a result of natural evolution of these viruses. The review contains a brief analysis of the results of studies on genomic organization and evolution of human pathogenic orthopoxviruses, development of modern methods for diagnosis, vaccination, and chemotherapy of smallpox, monkeypox, and other zoonotic human orthopoxvirus infections. [ABSTRACT FROM AUTHOR]

The conventional live smallpox vaccine based on the vaccinia virus (VACV) cannot be widely used today because it is highly reactogenic. Therefore, there is a demand for designing VACV variants possessing enhanced immunogenicity, making it possible to reduce the vaccine dose and, therefore, significantly eliminate the pathogenic effect of the VACV on the body. In this study, we analyzed the development of the humoral and T cell-mediated immune responses elicited by immunizing mice with low-dose VACV variants carrying the mutant A34R gene (which increases production of extracellular virions) or the deleted A35R gene (whose protein product inhibits antigen presentation by the major histocompatibility complex class II). The VACV LIVP strain, which is used as a smallpox vaccine in Russia, and its recombinant variants LIVP-A34R*, LIVP-dA35R, and LIVP-A34R*-dA35R, were compared upon intradermal immunization of BALB/c mice at a dose of 104 pfu/animal. The strongest T cell-mediated immunity was detected in mice infected with the LIVP-A34R*-dA35R virus. The parental LIVP strain induced a significantly lower antibody level compared to the strains carrying the modified A34R and A35R genes. Simultaneous modification of the A34R gene and deletion of the A35R gene in VACV LIVP synergistically enhanced the immunogenic properties of the LIVP-A34R*-dA35R virus. [ABSTRACT FROM AUTHOR]

On May 8, 1980, the World Health Assembly at its 33rd session solemnly declared that the world and all its peoples had won freedom from smallpox and recommended ceasing the vaccination of the population against smallpox. Currently, a larger part of the world population has no immunity not only against smallpox but also against other zoonotic orthopoxvirus infections. Recently, recorded outbreaks of orthopoxvirus diseases not only of domestic animals but also of humans have become more frequent. All this indicates a new situation in the ecology and evolution of zoonotic orthopoxviruses. Analysis of state-of-the-art data on the phylogenetic relationships, ecology, and host range of orthopoxviruses—etiological agents of smallpox (variola virus, VARV), monkeypox (MPXV), cowpox (CPXV), vaccinia (VACV), and camelpox (CMLV)—as well as the patterns of their evolution suggests that a VARV-like virus could emerge in the course of natural evolution of modern zoonotic orthopoxviruses. Thus, there is an insistent need for organization of the international control over the outbreaks of zoonotic orthopoxvirus infections in various countries to provide a rapid response and prevent them from developing into epidemics. [ABSTRACT FROM AUTHOR]

Cowpox virus (CPXV) strain GRI-90 contains six genes encoding kelch-like proteins. All six proteins contain both, the N-terminal BTB domain and the C-terminal kelch domain. We constructed mutant variants of a CPXV strain with targeted deletions of one to four genes of the kelch family, namely D11L, C18L, G3L, and A57R. As kelch genes are located in terminal variable regions of the CPXV genome, we studied the relationship of these genes with integral biological characteristics such as virulence, host range, reproduction in vitro and in ovo (in chicken embryos). It was demonstrated that the following effects occurred in a gene dose dependent manner with an increase of the number of genes deleted: (1) range of sensitive cells altered – deletion mutants lacking three genes displayed a considerably decreased ability to reproduce in MDCK cells; mutants lacking four genes lost this ability completely; (2) analysis of pocks formed by mutants with deletion of three and four kelch-like genes on chorioallantoic membranes of chicken embryos demonstrated that pock size and virus yield were significantly decreased; (3) light microscopic analysis of the pocks revealed impaired proliferation and reduced vascularisation in the pock region. More alterations were detected by electron microscopic analysis: the reproduction of mutants results in a reduction of the number of mature virions formed, and in many cells this process was arrested at the stage of assembly of immature virions; and (4) the evaluation of LD50 and body weight loss in BALB/c mice infected intranasally with CPXVs revealed a reduction of the virulence of the deletion mutants, which became statistically significant when four kelch-like genes were excised. [ABSTRACT FROM AUTHOR]

Uracil–DNA glycosylases are enzymes that excise uracil bases appearing in DNA as a result of cytosine deamination or accidental dUMP incorporation from the dUTP pool. The activity of Family 1 uracil–DNA glycosylase (UNG) activity limits the efficiency of antimetabolite drugs and is essential for virulence in some bacterial and viral infections. Thus, UNG is regarded as a promising target for antitumor, antiviral, antibacterial, and antiprotozoal drugs. Most UNG inhibitors presently developed are based on the uracil base linked to various substituents, yet new pharmacophores are wanted to target a wide range of UNGs. We have conducted virtual screening of a 1,027,767-ligand library and biochemically screened the best hits for the inhibitory activity against human and vaccinia virus UNG enzymes. Although even the best inhibitors had IC50 ≥ 100 μM, they were highly enriched in a common fragment, tetrahydro-2,4,6-trioxopyrimidinylidene (PyO3). In silico, PyO3 preferably docked into the enzyme's active site, and in kinetic experiments, the inhibition was better consistent with the competitive mechanism. The toxicity of two best inhibitors for human cells was independent of the presence of methotrexate, which is consistent with the hypothesis that dUMP in genomic DNA is less toxic for the cell than strand breaks arising from the massive removal of uracil. We conclude that PyO3 may be a novel pharmacophore with the potential for development into UNG-targeting agents. [ABSTRACT FROM AUTHOR]

Mass vaccination has played a critical role in the global eradication of smallpox. Various vaccinia virus (VACV) strains, whose origin has not been clearly documented in most cases, have been used as live vaccines in different countries. These VACV strains differed in pathogenicity towards various laboratory animals and in reactogenicity exhibited upon vaccination of humans. In this work, we studied the development of humoral and cellular immune responses in BALB/c mice inoculated intranasally (i.n.) or intradermally (i.d.) with the VACV LIVP strain at a dose of 105 PFU/mouse, which was used in Russia as the first generation smallpox vaccine. Active synthesis of VACV-specific IgM in the mice occurred on day 7 after inoculation, reached a maximum on day 14, and decreased by day 29. Synthesis of virus-specific IgG was detected only from day 14, and the level increased significantly by day 29 after infection of the mice. Immunization (i.n.) resulted in significantly higher production of VACV-specific antibodies compared to that upon i.d. inoculation of LIVP. There were no significant differences in the levels of the T cell response in mice after i.n. or i.d. VACV administration at any time point. The maximum level of VACV-specific T-cells was detected on day 14. By day 29 of the experiment, the level of VACV-specific T-lymphocytes in the spleen of mice significantly decreased for both immunization procedures. On day 30 after immunization with LIVP, mice were infected with the cowpox virus at a dose of 46 LD50. The i.n. immunized mice were resistant to this infection, while 33% of i.d. immunized mice died. Our findings indicate that the level of the humoral immune response to vaccination may play a decisive role in protection of animals from orthopoxvirus reinfection. [ABSTRACT FROM AUTHOR]

Following the WHO announcement of smallpox eradication, discontinuation of smallpox vaccination with vaccinia virus (VACV) was recommended. However, interest in VACV was soon renewed due to the opportunity of genetic engineering of the viral genome by directed insertion of foreign genes or introduction of mutations or deletions into selected viral genes. This genomic technology enabled production of stable attenuated VACV strains producing antigens of various infectious agents. Due to an increasing threat of human orthopoxvirus re-emergence, the development of safe highly immunogenic live orthopoxvirus vaccines using genetic engineering methods has been the challenge in recent years. In this study, we investigated an attenuated VACV LIVP-GFP (TK-) strain having an insertion of the green fluorescent protein gene into the viral thymidine kinase gene, which was generated on the basis of the LIVP (Lister-Institute for Viral Preparations) strain used in Russia as the first generation smallpox vaccine. We studied the effect of A34R gene modification and A35R gene deletion on the immunogenic and protective properties of the LIVP-GFP strain. The obtained data demonstrate that intradermal inoculation of the studied viruses induces higher production of VACV-specific antibodies compared to their levels after intranasal administration. Introduction of two point mutations into the A34R gene, which increase the yield of extracellular enveloped virions, and deletion of the A35R gene, the protein product of which inhibits presentation of antigens by MHC II, enhances protective potency of the created LIVP-TK--A34R*-dA35R virus against secondary lethal orthopoxvirus infection of BALB/c mice even at an intradermal dose as low as 103 plaque forming units (PFU)/mouse. This virus may be considered not only as a candidate attenuated live vaccine against smallpox and other human orthopoxvirus infections but also as a vector platform for development of safe multivalent live vaccines against other infectious diseases using genetic engineering methods. [ABSTRACT FROM AUTHOR]

The mass smallpox vaccination campaign has played a crucial role in smallpox eradication. Various strains of the vaccinia virus (VACV) were used as a live smallpox vaccine in different countries, their origin being unknown in most cases. The VACV strains differ in terms of pathogenicity exhibited upon inoculation of laboratory animals and reactogenicity exhibited upon vaccination of humans. Therefore, each generated strain or clonal variant of VACV needs to be thoroughly studied in in vivo systems. The clonal variant 14 of LIVP strain (LIVP-14) was the study object in this work. A comparative analysis of the virulence and immunogenicity of LIVP-14 inoculated intranasally (i.n.), intradermally (i.d.), or subcutaneously (s.c.) to BALB/c mice at doses of 108, 107, and 106 pfu was carried out. Adult mice exhibited the highest sensitivity to the i.n. administered LIVP-14 strain, although the infection was not lethal. The i.n. inoculated LIVP-14 replicated efficiently in the lungs. Furthermore, this virus was accumulated in the brain at relatively high concentrations. Significantly lower levels of LIVP-14 were detected in the liver, kidneys, and spleen of experimental animals. No clinical manifestations of the disease were observed after i.d. or s.c. injection of LIVP-14 to mice. After s.c. inoculation, the virus was detected only at the injection site, while it could disseminate to the liver and lungs when delivered via i.d. administration. A comparative analysis of the production of virus-specific antibodies by ELISA and PRNT revealed that the highest level of antibodies was induced in i.n. inoculated mice; a lower level of antibodies was observed after i.d. administration of the virus and the lowest level after s.c. injection. Even at the lowest studied dose (106 pfu), i.n. or i.d. administered LIVP-14 completely protected mice against infection with the cowpox virus at the lethal dose. Our findings imply that, according to the ratio between such characteristics as pathogenicity/immunogenicity/protectivity, i.d. injection is the optimal method of inoculation with the VACV LIVP-14 strain to ensure the safe formation of immune defense after vaccination against orthopoxviral infections. [ABSTRACT FROM AUTHOR]