Phylogenetic analysis of variants of the Puumala virus (Hantaviridae: Orthohantavirus) circulating in the Saratov region.

The objective is to determine the complete nucleotide sequence and conduct a phylogenetic analysis of genome variants of the Puumala virus isolated in the Saratov region. MATERIALS AND METHODS: The samples for the study were field material collected in the Gagarinsky (formerly Saratovsky), Engelssky, Novoburassky and Khvalynsky districts of the Saratov region in the period from 2019 to 2022. To specifically enrich the Puumala virus genome in the samples, were used PCR and developed a specific primer panel. Next, the resulting PCR products were sequenced and the fragments were assembled into one sequence for each segment of the virus genome. To construct phylogenetic trees, the maximum parsimony algorithm was used. RESULTS: Genetic variants of the Puumala virus isolated in the Saratov region have a high degree of genome similarity to each other, which indicates their unity of origin. According to phylogenetic analysis, they all form a separate branch in the cluster formed by hantaviruses from other subjects of the Volga Federal District. The virus variants from the Republics of Udmurtia and Tatarstan, as well as from the Samara and Ulyanovsk regions, are closest to the samples from the Saratov region. CONCLUSION: The data obtained show the presence of a pronounced territorial confinement of strains to certain regions or areas that are the natural biotopes of their carriers. This makes it possible to fairly accurately determine the territory of possible infection of patients and/or the circulation of carriers of these virus variants based on the sequence of individual segments of their genome.

[Study of the prevalence of antibodies to some arboviruses in the population of the Republic of Guinea].

INTRODUCTION: Acute febrile diseases kill more than 250,000 people annually in West Africa. Malaria and typhoid fever traditionally occupy most of the total structure of registered fevers. However, these data do not fully reflect the true overall disease patterns in the West African region. This is due to the fact that diagnosis is mainly based on the clinical signs of the infectious process, suggesting that a certain number of diseases may be caused by arboviruses. The detection of specific antibodies (ABs) to infectious pathogens in the blood sera of residents of a particular area is a reliable indicator of the circulation of these pathogens in a particular territory.The aim of this study was to determine the prevalence of antibodies to a number of arboviruses: Dengue (DENV), West Nile (WNV) (family Flaviviridae), Crimean-Congo hemorrhagic fever (orthonairo)virus (CCHFV), Batai (Batai virus), Bhanja (BHAV) (order Bunyavirales), Chikungunya (CHIKV), and Sindbis (SINV) (family Togaviridae) in the population of the Republic of Guinea. MATERIAL AND METHODS: In total, a panel of 2,620 blood serum samples from people living in all landscape and geographical areas of Guinea was collected for the study. Detection of IgG antibodies was performed using an enzyme-linked immunoassay (ELISA). RESULTS: In total, ABs to Batai virus were detected in 144 samples (5.5%), BHAV in 58 (2.2%), WNV in 892 (34.0 %), DENV in 659 (25.2 %), CCHFV in 58 (2.2 %), CHIKV in 339 (12.9 %), and SINV in 52 samples (2.0 %). DISCUSSION: The obtained results indicate serological evidence of the spectrum of arboviruses in the population of all landscape and geographical zones of the Republic of Guinea, confirming their active circulation in this territory. CONCLUSION: Given the high epidemiological significance of arbovirus infectious diseases, it is an urgent task to continue studying its share in the structure of febrile diseases in the territory of the Republic of Guinea.

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.

Obtaining and Characteristic of Antibodies to Vibrio cholerae Protective Antigens Conjugated with Gold Nanoparticles.

Gold nanoparticle conjugates with Vibrio cholerae antigens were synthesized. The animals were immunized with the obtained conjugates. Rabbit polyclonal antibodies to the antigens were obtained, which showed high specific activity. On the model of white laboratory mice, the protective activity of conjugates of cholera antigens with nanoparticles during infection of vaccinated animals was evaluated using a commercial vaccine as a control. It was shown that in terms of immunogenicity, the created prototypes of cholera vaccine using gold nanoparticles as a carrier and adjuvant complied with the production regulations for the Russian national cholera chemical vaccine.

[SUBSPECIES DIFFERENTIATION OF YERSINIA PESTIS STRAINS BY PCR WITH HYBRIDIZATION-FLUORESCENT DETECTION].

AIM: Develop a method of differentiation of Y.pestis strains of different subspecies based on PCR with hybridization-fluorescent detection in real-time. MATERIALS AND METHODS: DNA target search for differentiation of subspecies of plague causative agent was carried out by Mauve 2.3.1, Mega 5.0 and BLAST algorithm based on comparison of full-genome sequenc- es of Y.pestis strains. Primers and TAqMan probes were calculated for the DNA targets found, conditions of PCR with hybridization-fluorescent detection - optimized. RESULTS: DNA targets carrying marker mutations for the caucasus, altai, gissar, ulegei subspecies, strains from Talass alpine plague reservoir were detected. The effectiveness of the DNA targets found and the developed approach of subspecies differentiation is confirmed on 101 Ypestis strains of different subspecies, isolated from natural foci of Russia, near and far abroad. CONCLUSION: The developed approach based on PCR with real-time detection allows for a rapid and effective differentiation of Ypestis strains of various subspecies.

WEST NILE FEVER IN THE SARATOV REGION IN 2013-2015.

West Nile virus (WNV) circulation in the territory of Saratov region and its role in the infectious pathology were investigated. For this purpose, in studies conducted in 2013-2015, suspensions of bloodsucking arthropods, organs of birds and small mammals were analyzed for the presence of WNV markers (antigens and/or RNA). The seroprevalence level in live-stock animals and population of the region was evaluated; clinical samples from patients with symptoms compatible with West Nile fever (WNF) were analyzed. As a result of the investigations, WNV markers were detected in field samples gathered in natural biotopes and in the city of Saratov. Immunity to WNV was detected in horses. A stable domain of persons with immunity to this agent was revealed among regional population. Patients with WNF have been annually registered in the region since 2012. The obtained results confirm active circulation of WNF in the Saratov region, as well as formation of stable natural and anthropourgic foci.

MOLECULAR-GENETIC PROPERTIES OF VIBRIO CHOLERAE EL TOR STRAINS CIRCULATING IN AFRICA.

The review contains some brief information on cholera epidemics in Africa. Based on the results of the whole genome sequencing of 30 clinical strains isolated in Africa in different periods of the 7th cholera pandemic (1985-2012), extensive genetic diversity has been revealed. It is demonstrated that at present cholera epidemics in Africa are caused by new variants of the agent, which emerged in South- Eastern Asia in consequence of not only new genes acquisition, but also genome alterations of pandemicity and pathogenicity islands. SNP analysis of 53 strains circulating at different times in the territory of the continent, as well as isolated in South-Eastern Asia, has been carried out. Phylogenetic relations between the majority of the African and Asian strains have been established. In addition, strains were shown to exist that are, apparently, endemic to the African region. Identified genetic diversity of the strains with varying virulence and drug resistance points out the necessity of continuous molecular monitoring of the cholera agent in Africa.

[ACTUAL PROBLEMS OF EPIDEMIOLOGIC CONTROL, LABORATORY DIAGNOSTICS AND PROPHYLAXIS OF CHOLERA IN RUSSIAN FEDERATION].

Main problems of system of epidemiologic control for cholera active in Russian Federation, as well as laboratory diagnostics and vaccine prophylaxis of this especially dangerous infection, that had emerged in the contemporary period of the ongoing 7th pandemic of cholera, are discussed. Features of the genome of natural strains of Vibrio cholerae of El Tor biovar, that possess a poten- tial epidemic threat, as well as problems, that have emerged during isolation of these strains from samples of water of surface water bodies during their monitoring, are also examined. The main direction of enhancement of the system of epidemiologic control for cholera consist in develop- ment of a new algorithm of differentiation of administrative territories of Russian Federation by types of epidemic manifestations, as well as optimization of monitoring of environment objects. Integration of modern highly informative technologies into practice, as well as development of new generation diagnostic preparations based on DNA-chips and immunechips is necessary to increase effectiveness of the conducted operative and retrospective diagnostics in the contemporary period. Creation of national cholera vaccine, ensuring simultaneous protection from cholera causative agents of both O1 and O139 serogroups, is also required.

[IMPACT OF CASPIAN SEA LEVEL FLUCTUATIONS ON THE EPIZOOTIC ACTIVITY OF THE CASPIAN SANDY NATURAL PLAGUE FOCUS].

There is evidence that in 1923-2014 the sharp aggravations of the epizootic situation of plague in the area of its Caspian sandy natural focus after long interepizootic periods are in time with the ups of the Caspian Sea in the extrema of 11-year solar cycles. There were cases of multiple manifestations of plague in the same areas in the epizootic cycles of 1946-1954, 1979-1996, 2001, and 2013-2014. The paper considers the possible role of amebae of the genus Acanthamoeba and nematodes, the representatives of the orders Rhabditida and Tylenchida in the microfocal pattern of plague manifestations.

STRUCTURAL ORGANIZATION OF PORIN GENES (INTACT AND DISRUPTED BY IS 100) BORDERING THE PGM LOCUS OF THE YERSINIA PESTIS AND YERSINIA PSEUDOTUBERCULOSIS STRAINS.

In highly virulent strains of Yersinia pestis, the porin gene border- ing pigmentation (pgm) locus was observed to be usually broken by IS100. In this case, the pgm locus that carries virulence genes (high pathogenicity island) and biofilm formation genes (hms operon) is flanked by direct copies of IS100, which can cause its destabilization. We studied the prevalence of the intact and dis- rupted porin genes among 240 strains of Y. pestis from 39 natural centers in Russia and abroad, and 68 strains of Yersinia pseudotuberculosis from different geographical regions. The majority of the highly virulent Y. pestis strains and some phylogenetic lines of Y. pseudotuberculosis 0:1 serotype contain disrupted porin genes. At the same time, deletion of the pgm locus by flanking IS100 in Y pseudotuberculosis is impossible, because IS100 is integrated in the porin gene in the reverse orientation as compared to Y pestis. The porin genes are intact in all Y pestis strains with low epidemic importance and some phylogenetic lines of highly virulent Y pestis strains from some desert foci and Caspian sandy focus, as well as most strains of Y pseudotuberculosis 0:1 serotype. Less virulent strains of Y pseudotuberculosis 0:3 serotype revealed extensive deletion, which included the porin gene and a portion of the gene astE. The nucleotide sequence of the porin genes in Y pestis and Y pseudotuberculosis strains from different geographical regions are identical. Three alleles of the porin gene differ solely by the site of integration and orientation of IS 100 or by the lack of integration. The nucleotide sequence of IS 100, embedded in the porin gene of Yersinia, has minor differences only in two Y pestis strains isolated in America. Low frequency of Hms- mutations correlates with the intact condition of the porin gene in Y pestis. This correlation is absent in Y pseudotuberculosis.

[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.

[Analysis of the Nucleotide Sequence of a Cryptic Plasmid from Yersinia pestis Strains in the Central Caucasian High-Mountain Plague Focus].

An analysis of a 5.4-kbp cryptic plasmid detected in the course of whole-genome sequencing of the Yersinia pestis medieval biovar isolated in the Russian Central Caucasian high-mountain plague focus was performed. The identification of the nucleotide sequence of this cryptic plasmid and its structural and functional analysis revealed that it contained eight open reading frames, among which the following genes were identified: the rep gene of a replication protein, the virB6 gene of a type-IV secretion system inner membrane protein, the virB5gene of the type-IV secretion system minor pilin, and a number of genes probably associated with secretion and transport. A general analysis of the pCKF plasmid DNA showed that the adenine content was 28.34%, the cytosine content was 20.5%, the guanine content was 17.87%, and that of thymine was 33.28%, while the total G+C content appeared to be 38.38%. The G+C content of the chromosome of the Y pestis strain C-627 is 47.6%, which indicates that the pCKF plasmid was obtained from a microorganism equally-phylogenetically distant from the Yersinia bacteria andany other bacteria from the Enterobacteriaceae family. A comparison of the amino acid sequences.of hypothetical proteins encoded by pCKF plasmid with analogous proteins encoded by other bacteria was carried out. The possible contribution of the pCKF plasmid to the maintenance of the most ancient known phylogenetic line of Y. pestis medieval biovar, 2.MEDO, was discussed.

[CONTEMPORARY TENDENCIES IN CONSTRUCTING RECOMBINANT VACCINES FOR SPECIFIC PROPHYLAXIS OF PLAGUE].

An importance place in the system of prophylaxis measures against plague is allotted to vaccination of population contingents, that belong to risk groups for infection. The whole arsenal of accumulated knowledge on structure, properties, molecular nature, genetic determination, synthesis pathways, regulation and mechanisms of interaction with macroorganism of pathogenicity factors and immunogenicity of the infectious disease causative agent is used in the creation of new generation of vaccines. Contemporary technologies--genomics, proteomics, reverse vaccinology facilitate detection of protective antigens and help determine rational design of the vaccines. Main tendencies in development of recombinant live and chemical vaccines for specific prophylaxis of plague are presented in the review. Constructive approaches, that allow to produce highly effective and safe preparations are isolated.

[Analysis of diversity and identification of the genovariants of plague agent strains from Mongolian foci].

The genetic diversity of Yersinia pestis strains from the Mongolian natural plague foci has been investigated. A total of 32 strains isolated from western, eastern, and central aimaks, as well as from the territory of the Gobi region, have been studied. Twenty-four strains belong to the main Y. pestis subspecies, while eight belong to other subspecies. There is only one strain of biovar medievalis (genovariant 2.MED1) among the strains of the main subspecies, while the rest of the subspecies belong to the biovar antiqua. Biovar antiqua strains are split into three groups. Strains from the eastern part of the country were classified as genovariant 2.ANT3, and those from the western and central regions were classified as genovariant 3.ANT2, which was endemic for Mongolia. One strain from the Bayan-Ulegeiskii aimak had the rare genovariant 4.ANT. None of the strains of the biovar antiqua belonged to its ancient 0.ANT branch, which is inconsistent with the commonly accepted idea that ancient marmot's plague agent race originates from Mongolia. Six out of eight strains of the minor subspecies belonged to the ulegeica subspecies, which are endemic to Mongolia, one strain belonged to the microtus group, and the last belonged to a previously uncharacterized variant of the minor subspecies.

[A study on the taxonomy of soil amoebas from Caspian plague foci based on an analysis of ribosomal operon sequences].

The results of a study on the taxonomy and quantitative abundance of free-living amoebas in soil samples from the Russian plague foci of the northwestern Caspian steppe, the Caspian sand, and the Volga-Ural steppe are presented. Amoebas of the Willaertia and Hartmanella genera, as well as representatives of myxomycetes, were isolated from samples. From these, amoebas of the Acanthamoeba genus predominated and could be as abundantas 300000 cells per 1 g of soil. Sequencing of the 18S rRNA gene region showed that Acanthamoeba from the Volga-Ural steppe focus belonged to the A. castellanii species. Phylogenetic analysis confirmed that amoebas from two other Caspian foci belonged to the species of Acanthamoeba spp.

[A METHOD FOR DIFFERENTIATION OF BACILLUS ANTHRACIS STRAINS AND PHYLOGENETICALLY RELATED SPECIES BASED ON DETERMINATION OF THE STRUCTURAL DIFFERENCESBETWEEN CHROMOSOMAL GENES FOR BIOSYNTHESIS OF FLAGELLIN AND METHIONINE].

Nucleotide sequence analysis of several genes responsible for the anthrax pathogen definitive properties--motility and penicillinase activity--determined a chromosomal locus promising for interspecies differentiation. We demonstrated that the gene fliC encoding flagellin synthesis contains extended region, distinguishing B. anthracis strains from the majority of non-pathogenic and opportunistic bacilli. A novel method for the anthrax pathogen indication and identification based on determination of the differences in the chromosomal genes fliC and hom2 structure was suggested. A total of 60 strains of different Bacillus spp. (B. anthracis, B. cereus, B. thuringiensis, B. mycoides, B. megaterium, B. subtilis, etc.) were tested using two chromosomal DNA targets. The algorithm developed in this work permits to detect the pathogenic microorganism and reliably differentiate it from other Bacillus spp. representatives. The introduction of primers complementary to specific sequences of pXO1 and pXQ2 plasmids into the multiplex PCR makes it possible to receive additional information on proposed virulence of the isolate.

[Immunogenicity and safety of a prototype chemical anthrax vaccine in laboratory animal models].

AIM: Evaluation of immune stimulating and toxic effects of a vaccine prototype protein components. MATERIALS AND METHODS: Linear mice, guinea pigs and rabbits were immunized subcutaneously once or twice by recombinant protective antigen (rPA), S-layer protein (EA1) or their complex. Innate immunity structure activation was registered by changes in Toll-like receptor (TLR) expression. Adaptive immune response parameters were determined by established methods. Toxicity of the preparations was determined using flow cytofluorometry and densitomorphometry. RESULTS: The ability of rPA and EA1 to activate structures of innate immunity - TLR 2 and 6 - was established. Features of anti-PA antibody titer dynamics for each of the animal species was determined, a comparison with antibody formation during immunization with Bacillus anthracis STI- 1 was carried out. 2 immunizations ofbiomodels with a complex preparation combined with an adjuvant provides protection from infection by a test-strain that is comparable with protectivity of a live vaccine. Evidences regarding damaging effect of rPA and EAI on cells and tissues of macro organism were not detected throughout the study. CONCLUSION: Aprototype of a chemical anthrax vaccine under development has high immunogenicity and its protein components are not toxic for laboratory animals based on the results of complex testing.

[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.

Phylogenetic analysis of entomoparasitic nematodes, potential control agents of flea populations in natural foci of plague.

Entomoparasitic nematodes are natural control agents for many insect pests, including fleas that transmit Yersinia pestis, a causative agent of plague, in the natural foci of this extremely dangerous zoonosis. We examined the flea samples from the Volga-Ural natural focus of plague for their infestation with nematodes. Among the six flea species feeding on different rodent hosts (Citellus pygmaeus, Microtus socialis, and Allactaga major), the rate of infestation varied from 0 to 21%. The propagation rate of parasitic nematodes in the haemocoel of infected fleas was very high; in some cases, we observed up to 1,000 juveniles per flea specimen. Our study of morphology, life cycle, and rDNA sequences of these parasites revealed that they belong to three distinct species differing in the host specificity. On SSU and LSU rRNA phylogenies, these species representing three genera (Rubzovinema, Psyllotylenchus, and Spilotylenchus), constitute a monophyletic group close to Allantonema and Parasitylenchus, the type genera of the families Allantonematidae and Parasitylenchidae (Nematoda: Tylenchida). We discuss the SSU-ITS1-5.8S-LSU rDNA phylogeny of the Tylenchida with a special emphasis on the suborder Hexatylina.

[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.