Application of a synchronous integrator in phase fluorometry

It is demonstrated, that a luminescence spectrometer, in which signal detection is performed on the principle of synchronous integration, can be used as a phase fluorometer. For enhancing the accuracy, luminescence lifetime is measured by the zero method: the temporal shift of the synchrointegrator's reference signal is measured relative to the excitation wherein the output signal equals to zero. Besides lifetime measurement, the phase zero procedure, unlike the stroboscopic ones, allows to resolve not only phosphorescence spectra, but fluorescence ones as well

Development of DNA aptamer selection approach based on membrane ultrafiltration of aptamer/target complex

Background. Aptamers are small single-stranded DNA or RNA molecules that have an affinity for a specific target molecule. The main method of aptamers construction is the technology of systematic evolution of ligands with exponential enrichment (SELEX). However, the exact approach depends on the nature of target molecules, and is selected and optimized by each researcher independently. The article describes the technique of production of aptamers to the tick-borne encephalitis virus (TBEV) using membrane ultrafiltration with a molecular weight cut-off of 100 kDa. As a result, the pool of aptamers with observable affinity for TBEV is successfully selected and enriched.The aim. To develop the technique suitable for selection of specific DNA aptamers to a live, crude TBEV suspension directly in cell culture supernatant.Materials and methods. The selection of aptamers was carried out using a modified SELEX DNA aptamer technology in combination with semipermeable membrane ultrafiltration using Vivaspin 6 (Sartorius, Germany) concentrators of molecular weight cut-off of 100 kDa. Enrichment of a specific pool of aptamers was performed using real time polymerase chain reaction. Aptamers were sequenced with automated Sanger sequencing method. The direct virucidal effect of the aptamers was determined by the decrease in the titer of the infectious virus after incubation with the aptamer.Results. The pool of aptamers to TBEV was selected and enriched. This aptamer pool expressed affinity both to the infectious TBEV and to the TBEV antigen. Sixteen aptamers were sequenced from this pool and four of them were synthesized and tested for antiviral activity against TBEV. No antiviral activity was observed.Conclusions. The technique developed that can be successfully used to select aptamers to a live virus culture for the viruses comparable in size to TBEV or larger

Antiviral activity of herbal extracts against the tick borne encephalitis virus (literature review)

Tick-borne encephalitis (TBE) is a widely known viral disease that is transmitted through the bites of ixodid ticks. In severe cases, it leads to disability or death of the patient. The causative agent of the TBE is the tick-borne encephalitis virus (TBEV), which belongs to the Flaviviridae family. Despite the great importance of TBE in human pathology, currently the only way to prevent and treat this disease is a donor immunoglobulin, which has a number of significant disadvantages. In this paper, we analyzed modern literature on the detection of antiviral properties of herbal preparations and assessed the possibility of using them to search for new drugs against TBE. In general, the published works can be divided into two areas of research - the study of whole plant extracts and the study of the biological activity of the purified extract components. The most informative results can be obtained under certain conditions; for example, the study of both water extracts of plants and extracts with organic solvents, fractionation of the initial preparations with the detected antiviral activity followed by the concentration of the target substance; identification of an antiviral agent to obtain standardized drugs and preclinical testing in laboratory animals and cell cultures. This approach made it possible to identify three substances - hebulinic acid, panicalagin and baikalein - possessing inhibitory activity against Dengue flavivirus. The article discusses the possibility of using these substances against TBEV and the optimal algorithm for searching for antiviral agents in plant preparations

LONG-TERM TRENDS OF CHANGES IN INFESTATION RATE OF THE BAIKAL REGION POPULATION BY IXODIC TICKS

Ixodid ticks are the vectors of many pathogens including tick-borne encephalitis virus and the Lyme disease agent Borrelia burgdorferi sensu lato. In Pribaikalye, Ixodes persulcatus, Dermacentor nuttalli, D. silvarum and Haemaphysalis concinna are regarded as main vectors of the diseases. Recently, significant changes in world tick fauna have been reported and this affects the spread of tick-borne pathogens. We studied the current species diversity, population structure and the number of attacks of hard ticks (Acari:Ixodidae) on humans in Irkutsk region and nearby territories during the season 2015. Long-term trends of changes in attack rate were analyzed as well. In total, 4268 individual ticks were identified and analyzed during the season 2015. The majority (86 %) of victims was bitten by I. persulcatus; 13.55 % of attacks on humans were caused by D. nuttalli and D. silvarum; whereas H. concinna was documented only in 2 cases (~0.05 %). No non-endemic or invasive tick species were documented in 2015. The seasonal activity and the age/gender structure of tick population that bite human hosts were described as well. The comparison of the results of 2015 with previously reported datasets on tick bite ratesfrom 1992-2001 andfrom 2007-2014 indicates that, after the burst of tick bites in 1992-2001, there are no significant increase of attack rate since 2007. Possible implications to human health and epidemiology of tick-borne infections are discussed

Hantavirus infection in patients with fever of unknown origin and in small mammals in the Baikal region

Background. Hantavirus infections are one of the most important zoonotic diseases in the Russian Federation. However, the pathogenicity of Hantaviruses circulating in Siberia is still unknown. The aim of the study was to assess the incidence of Hantavirus infection in patients with fever of unknown origin from Irkutsk region and adjacent territories. Materials and methods. Serum samples collected in 2016 from individuals with symptoms of health deterioration were screened by ELISA to investigate possible Hantavirus infection, in addition to tick-borne infections. Depending on date of tick bite specific immunoglobulin G (IgG) and immunoglobulin M (IgM) antibodies to tick-borne encephalitis, borreliosis and Hantavirus were tested by ELISA or screened by PCR for tick-borne encephalitis, borreliosis, human granulocytic anaplasmosis and monocytic erlychiosis RNA/DNA. Results. Out of 92 serum samples, 6.5 % were positive for hantavirus-specific antibodies, which is at least twice higher than the seroprevalence among healthy people in the Irkutsk region. IgM and IgM+IgG antibodies were found in 5.4 % of cases, while IgG antibodies - in 1.1 % of cases. The clinical symptoms among sero-positive patients were fever, skin lesion, fatigue syndrome, headache and lymphadenitis. IgM antibodies to borreliosis were found in two hantavirus sero-positive cases. Lung tissues from small mammals captured in 2016 were screened by ELISA for the presence of Hantavirus antigen. Hantaviral antigen was detected in 6.3 % of 48 samples. Conclusions. Thus, present and previous studies demonstrate existence of natural foci of Hantavirus infections in addition to tick-born infections in the territory of the Irkutsk region

NECESSITY TO IMPROVE THE EMERGENCY DIAGNOSTICS OF TICK-BORNE INFECTIONS IN PEOPLE BITTEN BY IXODID TICKS ABROAD OF THE RUSSIAN FEDERATION

Introduction. Annually, there are several patients attended the Center for Diagnosis and Prevention of Tick-borne Infections in  Irkutsk after bites of ticks that happened outside the Pribaikalye  region or abroad. In such cases, the attacking ticks do not belong to  convenient species that are usual for Eastern Siberia. Consequently,  the spectrum of pathogenic microorganisms transmitted by these  ticks may significantly differ from those that are detected by usual  laboratory tests. Thus, both physicians and laboratory personnel may  have difficulties in proper detection and identification of pathogens as well as in diagnosing and treating of such patients.The purpose of the study was the analysis of potential risks of human infection with the pathogens that are common in foreign countries outside the Russian Federation.Material and methods. The article uses information from electronic databases created by the authors during 2007-2017.Results and discussion. During 11 years of observations, 52 tick bites were registered in 20 countries, with 48 of them in the Eastern  Hemisphere (92.3 %), three (5.8 %) in the United States and one  (1.9 %) in the Republic of Cuba. The results indicate a real danger of infection by tick-borne pathogens of people traveling as the tourists  and with business purposes to the countries of Europe, Asia and America. Conclusion. It is necessary to improve the existing algorithm for diagnosis, prevention and treatment for people bitten by ixodid ticks  outside the Russian Federation, taking into account the possibility of  infection by inconvenient imported infections

ASSESSMENT OF NEUTRALIZING PROPERTIES OF DNA-APTAMERS AND EXTRACTS OF MEDICINAL HERBS AGAINST THE TICK-BORNE ENCEPHALITIS VIRUS

Tick borne encephalitis (TBE) is a dangerous neurological disease that is transmitted to humans through the bite of Ixodid ticks. The disease exhibits an estimated 16 000 cases recorded annually over 30 European and Asian countries. The agent of TBE is the tick borne encephalitis virus (TBEV), belonging to the family Flaviviridae, genus Flavivirus. In spite the significant impact of TBE on human health, there is a serious lack of specific treatment against this disease. The only specific drug available is the human anti-TBEV immunoglobulin from vaccinated blood donors. The drug is produced and used in Russia only, both to prevent and to cure the TBE. In this work, we evaluated the ability of TBEV-specific DNA-aptamers and extracts of traditional medicine plants to neutralize the TBEV Selection of aptamers was performed using SELEX approach. Extracts of the seeds of Momordica cochinchinensis and Terminalia chebula were produced by boiling the ground seeds in water, clarified by centrifugation and filtration steps and filter sterilized. The SELEX had produced two aptamers - My13 and My38. Neither of two was capable to neutralize TBEV in vitro. The ability to shield the antibody binding sites on the surface of TBEV virions was also absent. The extract of M. cochinchinensis exhibited no neutralizing activity as well. Surprisingly, the T. chebula extract completely neutralized the TBEV after 30 min of incubation at 37 °C. The possible explanations and further development of the project are discussed

Application of a synchronous integrator in phase fluorometry

It is demonstrated, that a luminescence spectrometer, in which signal detection is performed on the principle of synchronous integration, can be used as a phase fluorometer. For enhancing the accuracy, luminescence lifetime is measured by the zero method: the temporal shift of the synchrointegrator's reference signal is measured relative to the excitation wherein the output signal equals to zero. Besides lifetime measurement, the phase zero procedure, unlike the stroboscopic ones, allows to resolve not only phosphorescence spectra, but fluorescence ones as well