[Genotyping of varicella zoster virus strains isolated in Mongolia].

This study analyzed 50 varicella zoster virus (VZV) samples collected during 2004 to 2007 from patients with VZV infection, who were treated at the National Center of Communicable Diseases, Ulan-Bator, Mongolia. The method based on amplification of specific DNA fragments of the ORF21, ORF22, and ORF50 genes was used, followed by the sequencing and detection of the status of characteristic point mutations in these fragments. The results indicated that the collected samples belonged to genotypes J (62%), M1 (18%), E1 (12%), E2 (4%), and M2 (4%). Moreover, restriction endonuclease polymorphism in ORF 62 for the cleavage site Smal and Mspl, in ORF 38 and ORF 54 for the cleavage site Pstl and Bgll were analyzed. All the samples were Sma- Msp-. All samples with genotype E were Pst+ Bgl-; all samples with genotype M1 and M2 were Pst+ Bgl+. Out of 31 samples with genotype J, 29 and 2 were Pst+ Bgl+ and Pst+ Bgl+, respectively. The study could identify the genotypes of VZV circulating in Mongolia and confirmed the absence of mutations characteristic for the vaccine strain.

Genomic analysis of varicella-zoster virus: primers for individual open reading frames.

The genome of varicella-zoster virus (VZV) contains nearly 125,000 bp. Preliminary genomic analysis has revealed that VZV may be less immutable than once thought. Through the investigation of the VZV genome using specifically designed oligonucleotides, it has been learned that sequence variation within VZV open reading frame 62 can distinguish between vaccine and wild-type virus. Additionally, the presence of single nucleotide polymorphisms within the VZV genome has identified distinct VZV populations originating from circumscribed geographic locations. In order for future studies of VZV genetic diversity to be carried out, amplifying and sequencing primers for individual VZV genes have been catalogued. Additionally, this report will facilitate the selection of VZV primers by which to distinguish clinical VZV isolates from vaccinia virus isolates.

Outbreak of human monkeypox, Democratic Republic of Congo, 1996 to 1997.

Human monkeypox is a zoonotic smallpox-like disease caused by an orthopoxvirus of interhuman transmissibility too low to sustain spread in susceptible populations. In February 1997, 88 cases of febrile pustular rash were identified for the previous 12 months in 12 villages of the Katako-Kombe Health Zone, Democratic Republic of Congo (attack rate = 22 per 1,000; case-fatality rate = 3.7%). Seven were active cases confirmed by virus isolation. Orthopoxvirus- neutralizing antibodies were detected in 54% of 72 patients who provided serum and 25% of 59 wild-caught animals, mainly squirrels. Hemagglutination-inhibition assays and Western blotting detected antibodies in 68% and 73% of patients, respectively. Vaccinia vaccination, which protects against monkeypox, ceased by 1983 after global smallpox eradication, leading to an increase in the proportion of susceptible people.

Detection and differentiation of old world orthopoxviruses: restriction fragment length polymorphism of the crmB gene region.

A restriction fragment length polymorphism (RFLP) assay was developed to identify and differentiate Old World, African-Eurasian orthopoxviruses (OPV): variola, vaccinia, cowpox, monkeypox, camelpox, ectromelia, and taterapox viruses. The test uses amplicons produced from virus genome DNA by PCR with a consensus primer pair designed from sequences determined for the cytokine response modifier B (crmB) gene of 43 different OPV strains of known taxonomic origin. The primer pair amplified a single specific product from each of the 115 OPV samples tested. Size-specific amplicons identified and differentiated ectromelia and vaccinia virus strains, which contain a truncated crmB gene, and enabled their differentiation from other OPV species. Restriction digests of amplified products allowed the identification and differentiation of variola, monkeypox, camelpox, vaccinia, and cowpox virus species and strains.

Rapid genotyping of varicella-zoster virus vaccine and wild-type strains with fluorophore-labeled hybridization probes.

We developed a single-tube rapid method for the detection and differentiation of varicella-zoster virus (VZV) vaccine and wild-type strains that combines rapid-cycle PCR with wild-type-specific fluorescent probe melting profiles for product genotyping. A region including the polymorphic site in VZV open reading frame (ORF) 62 was amplified in the presence of two fluorescence-labeled hybridization probes. During the annealing step of the thermal cycling, both probes bound to their complementary sequences in the amplicon, resulting in resonance energy transfer, thus providing real-time fluorescence monitoring of PCR. Continuous acquisition of fluorescence data during a melting curve analysis at the completion of PCR revealed that loss of fluorescence occurred in a strain-specific manner as the detection probe, which was fully complementary to the wild-type VZV ORF 62 region, melted off the template. Use of this method allowed genotyping of samples within minutes after the completion of PCR, eliminating the need for post-PCR sample manipulation. In addition to reducing the time required to produce a result, this method substantially reduces the risk of contamination of the final product as well as the risk of sample tracking errors. The genotypes of 79 VZV-positive samples determined by this fluorescent resonance energy transfer (FRET) method were identical to the genotypes obtained by conventional PCR and restriction fragment length polymorphism analysis. The genotyping of VZV strains by the FRET method is a rapid and reliable method that is suitable for typing and that is also practical for use for the processing of large numbers of specimens.

Improved identification and differentiation of varicella-zoster virus (VZV) wild-type strains and an attenuated varicella vaccine strain using a VZV open reading frame 62-based PCR.

A new method was developed to identify and differentiate varicella-zoster virus (VZV) wild-type strains from the attenuated varicella Oka vaccine strain. The PCR technique was used to amplify a VZV open reading frame (ORF) 62 region. A single specific amplicon of 268 bp was obtained from 71 VZV clinical isolates and several laboratory strains. Subsequent digestion of the VZV ORF 62 amplicons with SmaI enabled accurate strain differentiation (three SmaI sites were present in amplicons of vaccine strain VZV, compared with two enzyme cleavage sites for all other VZV strains tested). This method accurately differentiated the Oka vaccine strain from wild-type VZV strains circulating in countries representing all six populated continents. Moreover, the assay more reliably distinguished wild-type Japanese strains from the vaccine strain than did previously described methods.

Conserved surface-exposed K/R-X-K/R motifs and net positive charge on poxvirus complement control proteins serve as putative heparin binding sites and contribute to inhibition of molecular interactions with human endothelial cells: a novel mechanism for evasion of host defense.

Vaccinia virus complement control protein (VCP) has been shown to possess the ability to inhibit both classical and alternative complement pathway activation. The newly found ability of this protein to bind to heparin has been shown in previous studies to result in uptake by mast cells, possibly promoting tissue persistence. It has also been shown to reduce chemotactic migration of leukocytes by blocking chemokine binding. In addition, this study shows that VCP-through its ability to bind to glycosaminoglycans (heparin-like molecules) on the surface of human endothelial cells-is able to block antibody binding to surface major histocompatibility complex class I molecules. Since heparin binding is critical for many functions of this protein, we have attempted to characterize the molecular basis for this interaction. Segments of this protein, generated by genetic engineering of the DNA encoding VCP into the Pichia pastoris expression system, were used to localize the regions with heparin binding activity. These regions were then analyzed to more specifically define their properties for binding. It was found that the number of putative binding sites (K/R-X-K/R), the overall positive charge, and the percentage of positively charged amino acids within the protein were responsible for this interaction.

Alastrim smallpox variola minor virus genome DNA sequences.

Alastrim variola minor virus, which causes mild smallpox, was first recognized in Florida and South America in the late 19th century. Genome linear double-stranded DNA sequences (186,986 bp) of the alastrim virus Garcia-1966, a laboratory reference strain from an outbreak associated with 0.8% case fatalities in Brazil in 1966, were determined except for a 530-bp fragment of hairpin-loop sequences at each terminus. The DNA sequences (EMBL Accession No. Y16780) showed 206 potential open reading frames for proteins containing >/=60 amino acids. The amino acid sequences of the putative proteins were compared with those reported for vaccinia virus strain Copenhagen and the Asian variola major strains India-1967 and Bangladesh-1975. About one-third of the alastrim viral proteins were 100% identical to correlates in the variola major strains and the remainder were >/=95% identical. Compared with variola major virus DNA, alastrim virus DNA has additional segments of 898 and 627 bp, respectively, within the left and right terminal regions. The former segment aligns well with sequences in other orthopoxviruses, particularly cowpox and vaccinia viruses, and the latter is apparently alastrim-specific.

A third distinct tumor necrosis factor receptor of orthopoxviruses.

Cowpox virus Brighton red strain (CPV) contains a gene, crmD, which encodes a 320-aa tumor necrosis factor receptor (TNFR) of 44% and 22% identity, respectively, to the CPV TNFR-like proteins, cytokine response modifiers (crm) CrmB and CrmC. The crmD gene was interrupted in three other cowpox strains examined and absent in various other orthopoxviruses; however, four strains of ectromelia virus (ECT) examined contained an intact crmD (97% identity to CPV crmD) and lacked cognates of crmB and crmC. The protein, CrmD, contains a transport signal; a 151-aa cysteine-rich region with 21 cysteines that align with human TNFRII ligand-binding region cysteines; and C-terminal region sequences that are highly diverged from cellular TNFR C-terminal region sequences involved in signal transduction. Bacterial maltose-binding proteins containing the CPV or ECT CrmD cysteine-rich region bound TNF and lymphotoxin-alpha (LTalpha) and blocked their in vitro cytolytic activity. Secreted viral CrmD bound TNF and LTalpha and was detectable after the early stage of replication, using nonreducing conditions, as 60- to 70-kDa predominant and 90- to 250-kDa minor disulfide-linked complexes that were able to be reduced to a 46-kDa form and deglycosylated to a 38-kDa protein. Cells infected with CPV produced extremely low amounts of CrmD compared with ECT. Possessing up to three TNFRs, including CrmD, which is secreted as disulfide-linked complexes in varied amounts by CPV and ECT, likely enhances the dynamics of the immune modulating mechanisms of orthopoxviruses.

Terminal region sequence variations in variola virus DNA.

Genome DNA terminal region sequences were determined for a Brazilian alastrim variola minor virus strain Garcia-1966 that was associated with an 0.8% case-fatality rate and African smallpox strains Congo-1970 and Somalia-1977 associated with variola major (9.6%) and minor (0.4%) mortality rates, respectively. A base sequence identity of > or = 98.8% was determined after aligning 30 kb of the left- or right-end region sequences with cognate sequences previously determined for Asian variola major strains India-1967 (31% death rate) and Bangladesh-1975 (18.5% death rate). The deduced amino acid sequences of putative proteins of > or = 65 amino acids also showed relatively high identity, although the Asian and African viruses were clearly more related to each other than to alastrim virus. Alastrim virus contained only 10 of 70 proteins that were 100% identical to homologs in Asian strains, and 7 alastrim-specific proteins were noted.

Potential virulence determinants in terminal regions of variola smallpox virus genome.

Smallpox eradication culminated the most successful antimicrobial campaign in medical history. To characterize further the linear double-stranded DNA genome of the aetiological agent of smallpox, we have determined the entire nucleotide sequence of the highly virulent variola major virus, strain Bangladesh-1975 (VAR-BSH; 186,102 base pairs, 33.7% G + C; Genbank accession number, L22579). Here we highlight features of the molecule and focus on a few of the 187 putative proteins that probably contribute to pathogenicity and virus host-range properties. One hundred and fifty proteins were markedly similar to those of vaccinia virus (smallpox vaccine), for which a complete sequence has been reported for strain Copenhagen (VAC-CPN; 191,636 base pairs, 33.3% G + C). The remaining 37 proteins reflected variola-specific sequences or open reading frame divergences for variant proteins, which are often truncated or elongated compared with their vaccinia counterparts.

[The importance of the blood transfusion factor in the epidemic process in hepatitis C in the patients of chronic hemodialysis units studied using a computer simulation model]. / Issledovanie znachimosti faktora gemotransfuzii v épidemicheskom protsesse pri gepatitie C u patsientov otdelenii khronicheskogo gemodializa s pomoshch'iu imitatsionnoi komp'iuternoi modeli.

Examinations for the presence of antibody to hepatitis C virus (HCV) were carried out in 144 patients of chronic hemodialysis wards and 83 blood donors. The anti-HCV were found in 26.4% of the patients and only in 1.2% of the blood donors. A definite increase in the incidence of HCV infection in the patients of hemodialysis wards was established in relation to the duration of the treatment, namely from 17.4% in the patients treated for up to 1 year to 37.5% in those treated for 6 years or more. Significant differences were observed in the rate of anti-HCV findings in the patients with kidney transplantation and in those who had not experience this operation. The results obtained by an imitation computer model indicated that the hemotransfusion factor is not the only one determining the high rate of HCV infection in patients of chronic hemodialysis wards, however its influence on the intensity of the epidemic process in hepatitis C in these wards was sufficiently high.

[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.

An efficient and simple method of DNA extraction from whole blood and cell lines to identify infectious agents.

Routine methods of extraction of DNA from blood involve the enrichment of cells by Ficoll-Hypaque gradient centrifugation followed by lysis of the cells with extraction buffer, proteinase K digestion of the lysate, and phenol:chloroform-isoamyl alcohol extraction. These methods generally require large amounts of blood, which poses a problem with pediatric patients. To overcome this, we developed a new method of extracting DNA directly from whole blood. This method involves the treatment of whole blood with an equal volume of NaI (3 M final concentration) followed by chloroform:isoamyl alcohol extraction to clear hemoglobin and cell debris. The clear aqueous layer is then mixed with isopropanol to obtain DNA. A large number of samples can easily be handled by this extraction procedure, as it can be carried out in 30 min and requires only a microcentrifuge.

[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.

Smallpox diagnosed 400 years later: results of skin lesions examination of 16th century Italian mummy.

Results are presented on virological examination conducted on WHO request of a Naples infant mummy with smallpox-like lesions. Electron microscopy of lesions confirmed the findings of Italian researchers: well-preserved virus-like structures, whose size and morphology were identical to those of orthopoxviruses have been revealed. It was shown that the virus in mummy skin had lost its viability. Viral antigenic activity could not be detected in EIA or RPHA, nor was determined its DNA in the test of DNA molecular hybridization.

[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.