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Genetic Analysis of Crimean-Congo Hemorrhagic Fever Virus in Russia

ABSTRACT Genetic analysis of wild-type Crimean-Congo hemorrhagic fever (CCHF) virus strains recovered in the European part of Russia was performed. Reverse transcriptase PCR followed by direct sequencing was used to recover partial sequences of the CCHF virus medium (M) genome segment (M segment) from four pools of Hyalomma marginatum ticks and six human patients. Phylogenetic analysis of the M-segment sequences from Russian strains revealed a close relatedness of the strains (nucleotide sequence diversity, ≤5.0%). The strains differed significantly from CCHF viruses from other regions of the world (nucleotide sequence diversity, 10.3 to 20.4%), suggesting that CCHF virus strains recovered in the European part of Russia form a distinct group.

Genetic characterization of the M RNA segment of Crimean-Congo hemorrhagic fever virus strains isolated in Russia and Tajikistan.

The data on the structure of the M genome segment of CCHF virus strains from Russia and Central Asia (Tajikistan) are presented. Data obtained have been compared with other available published sequences of the middle segment of strains from China, Nigeria, and Pakistan. It has been found that all the known strains can be divided into four genetic groups, based on the nucleotide sequence of the M genome segment and an amino acid sequence of the glycoprotein precursor it encodes, whereas VLG/TI29414 and STV/HU29223 strains from Russia form a separate group. The CCHF virus strain from Tajikistan, TADJ/HU8966, was genetically related to strains 7803 and 75024 from China, and together with these and the Nigerian IbAr 10200 strain, it forms another group.

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

Summary.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.

Rational design based synthetic polyepitope DNA vaccine for eliciting HIV-specific CD8+ T cell responses

Advances in defining HIV-1 CD8+ T cell epitopes and understanding endogenous MHC class I antigen processing enable the rational design of polyepitope vaccines for eliciting broadly targeted CD8+ T cell responses to HIV-1. Here we describe the construction and comparison of experimental DNA vaccines consisting of ten selected HLA-A2 epitopes from the major HIV-1 antigens Env, Gag, Pol, Nef, and Vpr. The immunogenicity of designed gene constructs was assessed after double DNA prime, single vaccinia virus boost immunization of HLA-A2 transgenic mice. We compared a number of parameters including different strategies for fusing ubiquitin to the polyepitope and including spacer sequences between epitopes to optimize proteasome liberation and TAP transport. It was demonstrated that the vaccine construct that induced in vitro the largest number of [peptide-MHC class I] complexes was also the most immunogenic in the animal experiments. This most immunogenic vaccine construct contained the N-terminal ubiquitin for targeting the polyepitope to the proteasome and included both proteasome liberation and TAP transport optimized spacer sequences that flanked the epitopes within the polyepitope construct. The immunogenicity of determinants was strictly related to their affinities for HLA-A2. Our finding supports the concept of rational vaccine design based on detailed knowledge of antigen processing.

Comparative studies of gamma-interferon receptor-like proteins of variola major and variola minor viruses

To study specific properties of the human gamma‐interferon (γ‐IFN) receptor‐like proteins of the highly virulent and low virulent strains of variola (smallpox) virus (VAR) recombinant plasmids determining synthesis of these proteins in E. coli cells have been constructed. The recombinant viral γ‐IFN receptor‐like proteins have been found to have high interferon‐neutralising activity with regards to human γ‐IFN but not murine γ‐IFN and human α‐IFN. The variola major and variola minor proteins under study do not differ in the efficiency of human γ‐IFN antiviral activity inhibition.

The Complete Genomic Sequence of Strain ROS/HUVLV-100, A Representative Russian Crimean Congo Hemorrhagic Fever Virus Strain

The complete genomic sequence (minus primer-generated ends) of the laboratory-adapted Crimean Congo hemorrhagic fever virus (CCHFV) strain ROS/HUVLV-100, isolated in 2003 from the blood of a deceased female from the Rostov region of southern European Russia, was determined by direct sequencing of overlapping reverse transcription/polymerase chain reaction amplified products. The size of the ROS/HUVLV-100 genome is 19.2 kilobases—individual genome segments are similar in size and sequence features to previously reported “Europe-1” group CCHFV strains. The low-passage ROS/HUVLV-100 strain is the first Russian Crimean Congo hemorrhagic fever virus isolate for which complete sequence information is available, and this work reports the first complete genomic CCHFV sequence determined from a single viral RNA preparation in the same laboratory.

Complete L segment coding-region sequences of Crimean Congo hemorrhagic fever virus strains from the Russian Federation and Tajikistan

Summary.The large (L) RNA segment of Crimean Congo hemorrhagic fever (CCHF) virus strain AST/TI30908, isolated from pooled Hyalomma marginatum ticks collected in 2002 from the Astrakhan region of European Russia, was amplified piecemeal using reverse-transcription/polymerase chain reaction, followed by direct sequencing of gel-purified amplicons. After removal of 5′ and 3′ primer-generated termini, the assembled AST/TI30908 L segment sequence is 12112 nucleotides long, with 41.3% G + C content, and is greater than 87% and 96% identical at the nucleotide and translated amino acid levels, respectively, to partial or full-length CCHF virus L segment sequences deposited in GenBank. A complete L segment coding-region sequence for CCHF virus strain TAJ/HU8966, isolated from a patient in Tajikistan in 1990, was determined in a similar fashion. This L segment (12133 nucleotides long, 41.1% G + C content) shares 88% nucleotide identity with the full-length strain Matin from Pakistan, and 97% nucleotide identity with a partial L segment sequence of strain Khodzha from Uzbekistan. Strain TAJ/HU8966 shares at least 96% identity at the translated amino acid level with all other CCHF virus L segment sequences. Although, for the most part, CCHF virus L polyprotein primary sequences are uniformly well conserved, a region of marked variability was identified in the N-terminal half of the RNA-dependent RNA polymerase. This region, approximately 50 amino acids in length, is flanked by previously-reported arenavirus and bunyavirus-conserved regions, and may prove useful in CCHF diagnosis and viral taxonomy.