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.

A STUDY OF THE ULTRASTRUCTURE OF THE SURFACE OF THE TRANSPLANTABLE LINE VERO CELLS INFECTED WITH THE RABIES VIRUS (RABV, LISSAVIRUS, RHABDOVIRIDAE).

Characteristics of the effect of attenuated rabies virus strain «Moscow 3253¼ on morphological parameters of transplantable line Vero cells were studied by atomic force microscopy (AFM). Methods based on phase contrast microscopy and immunofluorescence were used to confirm the specificity of interaction and to identify the infectious activity of the rabies virus. Images of intact Vero cells and Vero cells infected with rabies virus were obtained at different periods of cultivation. The character of changes in the cell dimensions (length, width, height) and the cell membrane roughness depending on the rabies virus cultivation time was determined. During the observation period both increases and decreases in the size of the cells were recorded. The size of the infected cells exceeded that of the intact. An increase in the membrane roughness in cells exposed to rabies occurred during the entire period of observation, since the first hours of the interaction of the virus with the cell, while the intact Vero cells exhibited only minor changes in the membrane surface roughness, which were not dependent on the age of the culture. The dependence of the increase in the cell membrane roughness on the infecting dose of the rabies virus was determined. The obtained results open up the prospect of developing a methodological approach to the quantitative in vitro evaluation of the rabies virus using AFM. Changes in the cell membrane roughness appear to be the most indicative parameter for such evaluation.

[The development of biochip to detect anti-cholera antibodies in human blood serum].

The full-scaled agglutinating immunoassay is commonly applied to detect content of antibodies to cholera agent Vibrio cholerae human in blood serum under application of serological diagnostic. The time of analysis implementation amounts to 18 hours. To shorten time of detection of antibodies a biological microchip (biochip) was developed. The biochip represents an activated slide with immobilized corpuscle and soluble antigen cholera agent (O-antigens, cholera toxin). The experimental work resulted in development of scheme of biochip and selection of optimal conditions of sorption and implementation of immunologic analysis using biochip. The application of biochip facilitated to detect specific antibodies to antigens of cholera agent in commercial experimental animal serums and blood serums of ill patients. The time of analysis implementation amounted to 2-3 hours. The results are substantiated by bacteriological and serological methods.

[New approaches to detection of legionellosis causative agents in clinical samples and environment objects by PCR].

AIM: Selection of perspective targets as a base for development of test systems for the detection of legionella DNA in study material by PCR with electrophoresis and hybridization-fluorescent accounting of the results. MATERIALS AND METHODS: 22 Legionella pneumophila, 3 Legionella spp. strains and 30 cultures ofheterologic microorganisms, clinical material and environmental object samples were used in the study. Genome analysis was carried out by using Mega 3.1 program. Primer selection was conducted by using Primer Express program and BLAST algorithm and TaqMan format probes on the website www.genscript.com. RESULTS: Analysis of 712 legionella nucleotide sequences for the presence of novel species-specific and conservative for L. pneumophila loci was carried out. Fragments of life-support genes were selected for the analysis: fliC, mompS, ftsZ, dotA, dnaX, trpS, rpoB, rnp, proA, gspA. The most perspective DNA targets were established to be ftsZ and mompS genes. Based on the selected loci, PCR test systems were constructed for the detection of DNA oflegionella causative agent in biological material and environment objects, their diagnostic value was characterized. CONCLUSION: The studies carried out have shown the perspective of use of life-support ftsZ and mompS genes for the construction of novel preparations for legionellosis genetic diagnostics.

[Development and use of the assay for Legionella pneumophila detection based on fluorescent real-time/endpoint polymerase-chain reaction].

The aim of the work was to develop a PCR-based assay for detection of L. pneumophila and L. micdadei in environmental samples as well as in clinical samples from low respiratory tract and to assess its analytic characteristics. The assay was used during investigation of the outbreak developed in July 2007 in town Verkhnyaya Pyshma (Sverdlovsk region). Polymerase-chain reaction (PCR)with fluorescent detection,sequencing and cloning of DNA fragments were used. Developed assay based on the PCR with fluorescent real-time/ endpointdetection is able to detect L. pneumophila in clinical and environmental samples and to quantify amount of bacterial DNA in water. Specificity of analysis (100%) was assessed using the panel of bacterial strains and samples from healthy individuals. Analytic sensitivity of assay and quantitation limit was 1000 GU in 1 ml. Sensitivity of the assay of artificially contaminated biological samples was 1000 bacteria in 1 ml. During outbreak investigation L. pneumophila DNAwas detected in 4 lung samples from 4 fatal cases, from 1 of 2 sputum samples, 1 of 2 bronchoalveolar lavage samples with X-ray confirmed pneumonia. Legionella's DNA was found in samples from cooling towers, central hot water supply as well as from showerheads in apartments of 3 patients. Fountain and drinking water samples were PCR-negative. Specificity of PCR-positive results was confirmed by sequencing. Use of the assay during outbreak in- vestigation allowed to confirm the diagnosis in fatal cases and quickly identify the possible source of infection.

[The blood serum systems in patients with tuberculosis]. / Syvorotochnye sistemy krovi u bol'nykh tuberkulezom.

Phenotypes of haptoglobin (Hp), group-specific proteins (Gc), transferrins and ceruloplasmin were studied by means of polyacrylamide gel disk electrophoresis of serum proteins in 411 tuberculosis patients and 283 apparently healthy individuals living in the North-West and Extreme North. Regardless of their ethnic affiliation, tuberculosis patients tend to accumulate Hp 2-2, Gc 1-1 carriers and Hp 2-2 + Gc 1-1 combinations. The persons with the above types of individual proteins usually have a serious clinical picture of the disease and lower activity of the immune T-system, as confirmed by different methods of multidimensional statistical analysis. Hp 2-2, Gc 1-1 carriers and Hp 2-2 and Gc 1-1 combinations among the Extreme North's healthy aboriginal population are more widespread than among the healthy inhabitants of the North-West.

[Indices of humoral immunity in inhabitants of the Far North infected and sick with tuberculosis]. / Pokazateli gumoral'nogo immuniteta u infitsirovannykh i bol'nykh tuberkulezom zhitelei Krainego Severa.

As a result of a complex study of the humoral immunity indices in tuberculosis infected and affected inhabitants of the Extreme North with reference to the immunoglobulin A, G, M levels, titers of heterohemagglutinins (HHA) and specific antibodies defined by means of indirect hemagglutination test (IHAT), complement fixation test with tuberculin and IHAT with phosphatidic antigen (IHAT-Ph), two simple and quite informative tests, i.e. HHA and IHAT-Ph, are recommended for the practical use in health system. HHA level greater than 1/4 indicates the absence of immunodeficiency in system B. The results of IHAT-Ph, being adequate to the main clinical manifestations of tuberculosis (including bacillary excretion and lung tissue destruction), can be used as an additional diagnostic and evaluation method of tuberculous activity when the subjects from specific infection foci are examined or during their inpatient and dispensary observation and management.