[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.

[Restriction endonuclease Asi256I recognizing the nucleotide sequence 5'-GATC-3'].

A strain producing a restriction endonuclease was isolated from soil samples and identified as the Arthrobacter sp. strain Ck256. The enzyme produced by this strain was termed Asi2561. The isolation procedure for this enzyme was described, and the optimal conditions for its function were determined. It was shown that the restriction endonuclease Asi256I is a true isoschizomer of MboI, it has a temperature optimum of 6 degrees C, and can be used in molecular-biological and genetic-engineering studies performed at low temperatures.

[Genomic S segment of Crimean-Congo hemorrhagic fever virus circulating in Russia and Bulgaria].

S-segment nucleotide sequences for two Crimean-Congo hemorrhagic fever (CCHF) virus strains isolated in the Rostov Region of Russia and in Bulgaria have been determined. Analysis of complete S-segment nucleotide sequences in the viral strains from different regions of the world has established that the CCHF virus strains isolated from ticks and human beings in different southern Russian regions in 1967 and 2000 are very closely genetically and they form an individual subgroup in the basic European genetic group. By the S-segment structure, the CCHF virus strain isolated in Bulgaria in 1978 belongs to the same genetic group as a representative of its second subgroup. Analysis of the S-segment 3'-noncoding region suggests that the CCHF virus circulating in Europe, Central Asia, and China may have originated from one global focus of infection, including several CCHF virus genovariants. During evolution, fragmental exchange apparently occurred in the S-segment 3'-noncoding region as a result of homological recombination.

[Genetic monitoring of the Crimean-Congo Hemorrhagic Fever virus in Kazakhstan and Tajikistan in 2001-2003].

Blood specimens obtained from 32 CCHF patients were tested for the presence of CCHF virus markers. In addition, 3210 ticks of the genera Hyalomma asiaticum, Hyalomma anatolicum, and Dermacentor niveus were examined to identify the CCHF virus antigen and RNA. This material was obtained during the 2001-2003 local outbreaks of CCHF in Kazakhstan and Tajikistan. The nucleotide sequence in the region 983-1282 of S segment of the CCHF virus for 12 wild type strains was determined. The phylogenetic relationships among the established biovariants of CCHF virus, and also between these biovariants and those from other regions of the world were identified. We were the first to demonstrate the presence of an African-like genotype of CCHF virus in the territory of Kazakhstan. The conclusion was made that two genotypes of CCHF virus were in circulation in Kazakhstan. It was also demonstrated that CCHF virus, circulating in the territories of Kazakhstan and Tajikistan, was genetically heterogeneous.

Genetic analysis of the M RNA segment of Crimean-Congo hemorrhagic fever virus strains involved in the recent outbreaks in Russia.

Crimean-Congo hemorrhagic fever (CCHF) is a severe zoonosis with a high fatality rate. In Russia, local CCHF outbreaks have occurred in the Stavropol Territory, and the Volgograd and Astrakhan Regions during 2000 and 2001. Seven strains of CCHF virus (CCHFV) were isolated from infected patients and collected ticks. Two fragments of the CCHF virus M genome segment were PCR amplified and their nucleotide sequences were determined. All these virus strains appear to be closely related (up to 5.8% nucleotide sequence differences) and form a distinct clade on the CCHFV phylogenetic tree. Within this clade, CCHFV strains from Stavropol and Astrakhan cluster together, whereas those from Volgograd form a separate subgroup.

[Genetic characteristics of the S-segment of RNA from two strains of the Crimean-Congo hemorrhagic fever virus isolated in the south of Russia and in Uzbekistan]. / Geneticheskaia kharakteristika S-segmenta RNK dvukh shtammov virusa Krymskoi-Kongo gemorragicheskoi likhoradki, izolirovannykh na ioge Rossii i v Uzbekistane.

Complete S-segment nucleotide sequences of genomic RNA were determined for two Crimea-Congo hemorrhagic fever (CCHF) virus strains, i.e. LEIV 10145 Uz isolated from ticks in Uzbekistan, 1985, and LEIV 29223 Stv isolated from a patient in Stavropol region, 2000. It was established that the S-segment length is 1672 and 1674 nucleotides. Therefore, the initiating codon (for methionine) is located at positions 56-58; the length of translation frames for the nucleocapsid protein is 482 amino acid residues. Distinctions in the length of S-segment, as compared to other strains, are related only with the 5' and 3' non-coding regions. A comparison of the nucleotide and amino-acid sequences of S-segments of genome of the mentioned strains with the early published data showed that the CCHF virus strain isolated in Uzbekistan is mostly close to strains isolated in China, and that the strain isolated in Stavropol region forms, jointly with Drozdov strain isolated in the Astrakhan region, a separate branch in the phylogenetic tree.

[Characteristics of Crimean-Congo hemorrhagic fever virus circulating in Russia and Central Asia]. / Kharakteristika virusa Krymskoi-Kongo gemorragicheskoi likhoradki, tsirkuliruiushchego v Rossii i respublikakh Srednei Azii.

Five antigen-positive samples isolated from patients with Crimean-Congo hemorrhagic fever (CCHF) and from Hyalomma marginatum ticks collected in the European part of Russia and three laboratory strains of CCHF isolated in Russia, Uzbekistan, and Tadjikistan were analyzed by reverse transcription-polymerase chain reaction (RT-PCR) and sequencing. Comparison of nucleotide sequences of fragments of CCHF virus genome S segment and phylogenetic analysis of Russian strains showed that all CCHF strains isolated from humans and H. marginatum circulating in Russia were closely related and differed essentially from CCHF variants from other regions. Strains isolated in Uzbekistan and Tadjikistan were most closely related to CCHF strains from China.

Analysis of the monkeypox virus genome.

Monkeypox virus (MPV) belongs to the orthopoxvirus genus of the family Poxviridae, is endemic in parts of Africa, and causes a human disease that resembles smallpox. The 196,858-bp MPV genome was analyzed with regard to structural features and open reading frames. Each end of the genome contains an identical but oppositely oriented 6379-bp terminal inverted repetition, which similar to that of other orthopoxviruses, includes a putative telomere resolution sequence and short tandem repeats. Computer-assisted analysis was used to identify 190 open reading frames containing >/=60 amino acid residues. Of these, four were present within the inverted terminal repetition. MPV contained the known essential orthopoxvirus genes but only a subset of the putative immunomodulatory and host range genes. Sequence comparisons confirmed the assignment of MPV as a distinct species of orthopoxvirus that is not a direct ancestor or a direct descendent of variola virus, the causative agent of smallpox.

[Restriction endonuclease Sst 12I from a strain of Streptomyces sp. recognizing the nucleotide sequence 5'-CTGCAG-3']. / Endonukleasa restriktsii Sst12I iz shtamma Streptomyces sp., uznaiushchaia posledovatel'nost' nukleotidov 5'-CTGCAG-3'.

A new restriction endonuclease Sst12I belonging to the II type and recognizing the sequence 5'-CTGCAG-3' was isolated from the bacterial strain Streptomyces sp. St-12. The enzyme hydrolyzes DNA between adenine and guanine residues; thus, it is a true isoschizomer of restrictase PstI. In contrast to PstI, the restriction endonuclease Sst12I hydrolyses DNA both at 37 degrees and 55 degrees C and remains active after long-term storage.

[Genetic identification of the Crimean-Congo hemorrhagic fever virus during epidemic outbreak in Kazakhstan in 2000]. / Geneticheskaia identifikatsiia virusa Krymskoi-Kongo gemorragicheskoi likhoradki vo vremia épidemicheskoi vspyshki v Kazakhstane v 2000 g.

Sera samples from patients suspected of Crimean-Congo hemorrhagic fever (CCHF) taken during epidemic outbreak at the territory of Sarysusky and Moiynkumsky districts of the Zhambyl region in Kazakhstan, in 2000, were analysed by means of reverse transcription-polymerase chain reaction (RT-PCR) and sequencing of virus genome fragments. Genome RNA of CCHF virus was found in 2 assays. Analysis of nucleotide sequences of fragments of S-segment of viral genome revealed in the Sarysusky districts circulation of CCHF virus, genetically resembled to close phylogenetically to CCHF virus strains from China.

Human monkeypox and smallpox viruses: genomic comparison.

Monkeypox virus (MPV) causes a human disease which resembles smallpox but with a lower person-to-person transmission rate. To determine the genetic relationship between the orthopoxviruses causing these two diseases, we sequenced the 197-kb genome of MPV isolated from a patient during a large human monkeypox outbreak in Zaire in 1996. The nucleotide sequence within the central region of the MPV genome, which encodes essential enzymes and structural proteins, was 96.3% identical with that of variola (smallpox) virus (VAR). In contrast, there were considerable differences between MPV and VAR in the regions encoding virulence and host-range factors near the ends of the genome. Our data indicate that MPV is not the direct ancestor of VAR and is unlikely to naturally acquire all properties of VAR.

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.

The genomic sequence analysis of the left and right species-specific terminal region of a cowpox virus strain reveals unique sequences and a cluster of intact ORFs for immunomodulatory and host range proteins.

Sequencing and computer analysis of the left (52,283 bp) and right (49,649 bp) variable DNA regions of the cowpox virus strain GRI-90 (CPV-GRI) has revealed 51 and 37 potential open reading frames (ORFs), respectively. Comparison of the structure-function organization of these DNA regions of CPV-GRI with those previously published for corresponding regions of genomes of vaccinia virus, strains Copenhagen (VAC-COP) and Western Reserve (VAC-WR); and variola major virus, strains India-1967 (VAR-IND), Bangladesh-1975 (VAR-BSH); and alastrim variola minor virus, strain Garcia-1966 (VAR-GAR), was performed. Within the left terminal region under study, an extended DNA sequence (14,171 bp), unique to CPV, has been found. Within the right region of the CPV-GRI genome two segments, which are unique to CPV DNA (1579 and 3585 bp) have been found. Numerous differences have been revealed in the genetic structure of CPV-GRI DNA regions, homologous to fragments of the genomes of the above-mentioned orthopoxvirus strains. A cluster of ORFs with structural similarity ot immunomodulatory and host range function of other poxviruses have also been detected. A comparison of the sequences of ORF B, crmA, crmB, crmC, IMP, and CHO hr genes of CPV Brighton strain (CPV-BRI) with the corresponding genes in strain GRI-90 have revealed an identity at the amino acid level ranging from 82 to 96% between the two strains. The findings are significant in light of the recent demonstration of CPV as an important poxvirus model system to probe the precise in vivo role(s) of the unique virally encoded immunomodulatory proteins. Also, the presence of a complete and intact repertoire of immunomodulatory proteins, ring canal proteins family, and host range genes indicates that CPV may have been the most ancient of all studied 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.