Prevalence of Crimean-Congo hemorrhagic fever virus in rural areas of Guinea.

This article presents the results of a comprehensive survey of Guinea with the aim of assessing the burden of Crimean-Congo hemorrhagic fever virus (CCHFV) in rural areas of the country. Human serum samples (n = 2207) were studied using enzyme-linked immunosorbent assay (ELISA) for the presence of specific IgG against CCHFV. In addition, 4273 samples of partially- or fully-engorged ticks from several sources (cattle, domestic and roving dogs, and small mammals) were collected and studied using ELISA and RT-qPCR to detect CCHFV antigen and specific RNA. The data obtained show that 3.0 % of the population in rural Guinea was seropositive, without significant geographical or sexual differences. Seropositive individuals, however, were mainly in the 'active age' group (16-45 years old). Among ticks studied, the estimated prevalence of CCHFV was 1.3 ± 0.4 %. Five out of eight tick species studied were identified as CCHFV carriers in Guinea. Therefore, it can be assumed that the territory of Guinea is a single, continuous, natural focus of CCHFV. This identified medium intensity focus merits further study.

[Genome structure and origin of nontoxigenic strains of Vibrio cholerae of El Tor biovar with different epidemiological significance].

Intraspecies genetic differentiation of nontoxigenic strains of Vibrio cholerae of El Tor biovar containing one of the key pathogenicity genes, tcpA, is studied along with the phylogenetic relationships between these strains and toxigenic isolates. Comparative analysis of the whole genome nucleotide sequences demonstrates for the first time that ctxA ­ tcpA + strains vary considerably and can be clustered into two separate groups, the CTXφ­RS1φ +VPI+VSP+/CTXφ­RS1φ­VPI+VSP+ isolates and the CTXφ­RS1φ­VPI+VSP­ isolates, differing in their epidemiological significance. In the course of model experiments, it is established that nontoxigenic potentially epidemic CTXφ­RS1φ +VPI+VSP+/CTXφ­RS1φ­VPI+VSP+ isolates are derivatives of toxigenic strains. The results of whole genome SNP analysis of 35 Vibrio cholerae strains confirm these data and indicate genetic remoteness of nontoxigenic CTXφ­RS1φ­VPI+VSP­ strains both from the potentially epidemic strains and from the toxigenic isolates. It is found that the genomes of the CTXφ­RS1φ­VPI+VSP­ strains contain unique SNPs which are characteristic of them alone. The new data on the structure of the genome of nontoxigenic strains with different epidemiological significance may be further used for their genetic differentiation.

[Comparative resistance of typical and genetically altered Vibrio cholerae biovar El Tor strains to the effect of unfavorable environmental factors].

AIM: Carry out comparative analysis of survival of typical strains and genovariants of V. cholerae biovar El Tor imported in different years to the territory of Russian Federation, in the absence of nutrients and under the conditions of temperature stress. MATERIALS AND METHODS: 24 V. cholerae biovar El Tor strains isolated in 1970 - 2011 were studied, 8 of those were typical isolates and 16--genetically altered variants. Strain survival was studied in 0.9% sodium chloride solution and autoclaved river water at various temperature modes (5, 25, 37 and 42 degrees C). Protein composition and exopolysaccharide production were determined by electrophoresis method by U.K. Laemmli. RESULTS: Genovariants as well as typical strains were shown to be able to exist for a long time (up to 5 months) in the absence of nutrients at the temperature of 25 degrees C. However, unlike typical eltor vibrios, genovariants were more resistant to temperature stress. As a result of adaptation to high temperature (42 degrees C) biosynthesis of porin proteins of outer membrane OmpU and/or OmpT is increased in genovariant cells, and at lower temperatures (5 degrees C)--exopolysaccharide. CONCLUSION: V. cholerae biovar El Tor genovariants are able to adapt to temperature change better, that may facilitate their higher survival in the environment.

[Algorithm of toxigenic genetically altered Vibrio cholerae El Tor biovar strain identification].

AIM: Development of an algorithm of genetically altered Vibrio cholerae biovar El Tor strai identification that ensures determination of serogroup, serovar and biovar of the studied isolate based on pheno- and genotypic properties, detection of genetically altered cholera El Tor causative agents, their differentiation by epidemic potential as well as evaluation of variability of key pathogenicity genes. MATERIALS AND METHODS: Complex analysis of 28 natural V. cholerae strains was carried out by using traditional microbiological methods, PCR and fragmentary sequencing. RESULTS: An algorithm of toxigenic genetically altered V. cholerae biovar El Tor strain identification was developed that includes 4 stages: determination of serogroup, serovar and biovar based on phenotypic properties, confirmation of serogroup and biovar based on molecular-genetic properties determination of strains as genetically altered, differentiation of genetically altered strains by their epidemic potential and detection of ctxB and tcpA key pathogenicity gene polymorphism. The algorithm is based on the use of traditional microbiological methods, PCR and sequencing of gene fragments. CONCLUSION: The use of the developed algorithm will increase the effectiveness of detection of genetically altered variants of the cholera El Tor causative agent, their differentiation by epidemic potential and will ensure establishment of polymorphism of genes that code key pathogenicity factors for determination of origins of the strains and possible routes of introduction of the infection.

[Genovariants of the cholera agent biovar El Tor: construction, molecular-genetic, and proteomic analysis].

Experimental modeling of origination of the virulent Vibrio cholerae El Tor genovariants is presented. It was demonstrated that the genovariants obtained did not differ from the natural genetically modified strains emerged in a natural population of the agent, either in phenotypical or genotypic properties. Using the PCR assay and sequencing techniques it was proved that the constructed genovariants carried a CTX(Class phi) prophage genome region with ctxBl gene of the V. cholerae classical biovar in the chromosome. It is shown that the prophage structure alterations lead to the increase in the toxigenicity and virulence in the genovariants compared to the typical strain-recipient. Moreover, as regards proteomics, changes in the expression of 26 proteins that perform various functions in the cell, such as metabolism, energy exchange, transportation, etc., were demonstrated. The data are indicative of the impact that a new DNA region in the genome of the genovariants has on the expression level of different house-keeping genes. The results obtained testify to the fact that one of the mechanisms of the genovariant emergence in the natural populations of the agent can be horizontal gene transfer.

[Microevolution of cholera agent in the modern period].

AIM: To carry out comparative molecular genetic analysis of highly pathogenic atypical Vibrio cholerae strains biovar El Tor, isolated in the territory of RF, in order to determine micro-evolutionary alterations of cholera agent in the modern period. MATERIALS AND METHODS: 38 clinical strains have been examined by means of polymerase chain reaction, sequencing and MLVA-analysis. The selected strains were isolated at different periods of time during cholera epidemic complications and differed between each other in virulence. RESULTS: It is demonstrated that new variants have emerged in the course of short-term microevolution. Their genome structure and function differ from those of all previously known strains. The genome alterations have been caused by point mutations in ctxB u tcpA genes associated with virulence and located in CTXΦ prophage and pathogenicity island VPI-1 respectively, as well as by the extended deletion in pandemicity island VSP-II. Presented is the dynamics of genome structure and function alterations in modern strains. CONCLUSION: The discovered genomic alterations in the new variants of the agent evolved in the process of microevolution are indicative of their epidemic potential enhancement and probability of virulence potentiation.

[Comparative molecular-genetic analysis of mobile elements in natural strains of cholera agent].

The molecular-genetic peculiarities of the prophage CTXφ genome, the VPI-1 pathogenicity island, and the VSP-II pandemicity island in recently emerged, genetically altered Vibrio cholerae El Tor strains have been studied. The genome of the prophage CTXφ, which contains the ctxAB operon, which codes cholera toxin (CT), was shown to be unstable. A comparative analysis of the nucleotide sequences of the two phage genome regions (the ctxB gene and ctxAB operon promoter region) among 23 genovariant strains allowed us to reveal the presence of distinct ctxB gene alleles (ctxB1 or ctxB7) that differed in single-nucleotide substitutions and the polymorphism of the ctxAB operon promoter region. An analysis of the VPI-1 structure showed that tcpA gene that encodes the basic protein of the toxin-coregulated pilli (TCP) was represented in the genovariants by four alleles. The variability of the VSP-II was also revealed. It was manifested in the occurence of the deletions of varying length (2069 or 13 105 bp).