Detection of Ehrlichia spp. and Theileria spp. in Hyalomma anatolicum ticks collected in Tajikistan.

The objectives of our study were to survey the prevalence of genetic markers for Rickettsia spp., Ehrlichia spp., Anaplasma spp., Babesia spp., and Theileria spp. in Hyalomma anatolicum ticks collected in southwestern Tajikistan and to perform sequencing and phylogenetic analysis of fragments of the 16S rRNA gene and groESL operon from Ehrlichia spp. and fragments of the 18S rRNA gene of Theileria spp. detected in H. anatolicum ticks. Hyalomma anatolicum ticks collected in the Tursunzade and Rudaki districts of Tajikistan were tested for DNA of Rickettsia spp., Ehrlichia spp., Anaplasma spp., Babesia spp., and Theileria spp. by PCR with specific primers. The amplified fragments were sequenced and analyzed. DNA of Ehrlichia spp. (3.3 %) and Theileria spp. (3.3 %) was detected only in H. anatolicum ticks collected from the Rudaki district, and DNA of Ehrlichia spp. (0.7 %) was found in H. anatolicum ticks from the Tursunzade district. Sequence analysis of fragments of the 16S rRNA gene and groESL operon from Ehrlichia spp. revealed high similarity to Ehrlichia spp. The Tajik isolates of Theileria spp. were genotyped as Theileria annulata based on the analysis of 18S rRNA gene sequences. The phylogenetic analysis demonstrates that Ehrlichia spp. isolates are highly similar to Ehrlichia spp. circulating in China and Brazil. The isolate Tajikistan-5 is closely related to the putative novel species Ehrlichia mineirensis. The Tajik isolates of Theileria spp. were clustered with T. annulata isolates from Turkey, Iran, Pakistan, and China by phylogenetic analyses.

Concentration of viruses and electron microscopy.

Nearly all lethal viral outbreaks in the past two decades were caused by newly emerging viruses. Viruses are often studied by electron microscopy (EM), which provides new high-resolution data on the structure of viral particles relevant to both fundamental virology and practical pharmaceutical nanobiotechnology. Electron microscopy is also applied to ecological studies to detect viruses in the environment, to analysis of technological processes in the production of vaccines and other biotechnological components, and to diagnostics. Despite the advances in more sensitive methods, electron microscopy is still in active use for diagnostics. The main advantage of EM is the lack of specificity to any group of viruses, which allows working with unknown materials. However, the main limitation of the method is the relatively high detection limit (107 particles/mL), requiring viral material to be concentrated. There is no most effective universal method to concentrate viruses. Various combinations of methods and approaches are used depending on the virus and the goal. A modern virus concentration protocol involves precipitation, centrifugation, filtration, and chromatography. Here we describe the main concentrating techniques exemplified for different viruses. Effective elution techniques are required to disrupt the bonds between filter media and viruses in order to increase recovery. The paper reviews studies on unique traps, magnetic beads, and composite polyaniline and carbon nanotubes, including those of changeable size to concentrate viral particles. It also describes centrifugal concentrators to concentrate viruses on a polyethersulfone membrane. Our review suggests that the method to concentrate viruses and other nanoparticles should be chosen with regard to objectives of the study and the equipment status of the laboratory.

[Genetic variants of the Crimean-Congo hemorrhagic fever virus circulating in endemic areas of the southern Tajikistan in 2009].

506 Hyalomma anatolicum ticks were collected and assayed in two Crimean-Congo hemorrhagic fever (CCHF) endemic regions of Tajikistan. Antigen and RNA of CCHF virus were detected in 3.4% of tick pools from Rudaki district using ELISA and RT-PCR tests. As of Tursunzade district, viral antigen was identified in 9.0% of samples and viral RNA was identified in 8.1% of samples. The multiple alignment of the obtained nucleotide sequences of CCHF virus genome S-segment 287-nt region (996-1282) and multiple alignment of deduced amino acid sequences of the samples, carried out to compare with CCHF virus strains from the GenBank database, as well as phylogenetic analysis, enabled us to conclude that Asia 1 and Asia 2 genotypes of CCHF virus are circulating in Tajikistan. It is important to note that the genotype Asia 1 virus was detected for the first time in Tajikistan.

[Entification of the Rubella virus genotype 1H in Western Siberia].

Molecular epidemiological study of novel strain of Rubella virus isolated during the outbreak in Western Siberia in 2004 was described. Detailed phylogenetic analysis performed based upon entire SP-region, which encodes all three Rubella structural proteins (C, E2, and E1), was implemented. This analysis provides characterization of this strain and classifies it as 1H genotype, thereby correcting previous classification of this strain based upon shorter nucleotide sequence, only encoding E1 protein. Therefore, this study identified the genotype of the Rubella virus not previously detected in Western Siberia (and even entire Russian Federation), which highlights the importance of more extensive characterization of genetic variability of the Rubella virus, especially with regard to potential influence of vaccination on the Rubella virus mutagenesis.

[Clinical and immunological characteristics of rubella in western Siberia].

Evaluations of immune system of 155 patients with rubella and 90 contacts with patients were examined. Detection of viral genetic material in blood, urine, and nasopharyngeal swabs has been performed using RT-PCR method. Clinical diagnosis has been confirmed by RT-PCR in 114 (73.5%) patients. Changes of laboratory tests for rubella without clinical signs of the infection were observed in 20% of contacts. Complex ELISA- and PCR-assisted examination of patients can help to determine the stage of disease and characteristics of immune response. For differential diagnostic of rubella and other infectious diseases with exanthema it is rational to perform complex examination of patients using immunologic and molecular biologic methods.

[Analysis of genomes of two rubella virus strains from the 2004-2005 outbreaks in West Siberia].

Two outbreaks of rubella infections notified in the Tomsk and Kemerovo Regions were investigated. Two rubella virus strains from one patient in each outbreak were isolated and genetically characterized. Reverse transcription polymerase chain reaction was used to reveal partial E1 gene sequence at a length of 915 nucleotides. Analysis indicated that the rubella virus strains circulating in the West-Siberian region belonged to international genetic 1g group, which had been first detected in Russia.

[Genotyping of rubella virus circulating in Western Siberia of Russia during 2004-2006 epidemic period].

Twenty one strains of rubella virus were isolated in the Western Siberia during 2004-2006 epidemic period. Genotyping of isolated strains was performed by partial sequencing of glycoprotein E1 gene. Phylogenetic analysis showed that 20 out of 21 isolated in the Western Siberia strains of rubella virus belonged to genotype 1g, and 1 strain (isolated in Altai region in 2006)--to genotype 1E.

[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.

[ELISA and RT-PCR-based research of viruses in the ticks collected in the foci of Crimean-Congo fever in Kazakhstan and Tajikistan in 2001-2002].

Different species of ticks were found, in the territories of Kazakhstan and Tajikistan, to be infected with the virus of Crimean-Congo hemorrhagic fever (CKHF). The virologic evaluation included determination of antigen and RNA of the CKHF virus by ELISA and RT-PCR, respectively. The below tick species were found to be involved in the epidemic process: Hyalomma asiaticum, Dermacentor niveus (Kazakhastan) and Hyalomma anatolicum (Tajikistan). The results testify to the fact that Hyalomma ticks are the main carrier of the above virus in the Middle Asia. At the same time, Dermacentor niveus ticks are infection carriers in Kazakhstan.

[Study of virus contamination of Ixodes ticks in the foci of Crimean-Congo hemorrhagic fever in Kazakhstan and Tajikistan].

The data on the contamination of different of ticks with Crimean-Congo hemorrhagic fever (CCHF) virus on the territory of Kazakhstan and Tajikistan were obtained. The methods of the evaluation of the virus contamination of ticks included the determination of the antigen and CCHF virus RNA by the methods of the enzyme immunoassay and the reverse transcription PCR respectively. Different tick species were found to be involved in the epidemic process: Hyalomma asiaticum, Dermatocentor niveus (Kazakhstan) and Hyalomma anatolicum (Tajikistan). The results obtained in this study confirmed that the main vector of CCHF virus in Central Asia were ticks of the genus Hyalomma, and in Kazakhstan the vectors of this virus also included ticks Dermatocentor niveus.

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.

[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.