Background. Early detection of tick-borne pathogens in blood samples of infected people prevents the disease before clinical manifestations using antibiotic and immunoglobulin treatment. As far as tick-borne diseases are associated with significant activation of innate immune response, the changes in the cytokine profile soon after a tick bite could be used as an indicator of early tick-borne infection. Aims. The goal of this study was to characterize the production of cytokines during first 2-4 days after a tick bite in asymptomatic people infected with tick-borne pathogens. Materials and methods. The infection of tick-borne encephalitis virus, B. burgdorferi sensu lato, A. phagocytophilum and E. chaffeensis / E. muris was detected in blood of bitten humans using appropriate commercial real time RT-PCR and ELISA assays. Concentration of 14 cytokines in plasma of 89 infected and 45 non-infected people was established using quantitative ELISA kits. Results. Between 2 nd and 4 th days after tick bite, the prevalence and concentration of certain cytokines in blood of infected people significantly differed from those of the control group. Bacterial pathogens exhibited very similar patterns of cytokine profiles with induction of IL-1α, IL-8 и IFN-α and suppression of SOD, IL-1Ra и IL-17A. For virus infection the increased concentrations of IL-1α, IL-8, IL-10, IL-17A, IFN-γ and SOD were detected. The only suppressed cytokine was IL-1Ra receptor antagonist. Conclusions. The results suggest that evaluation of innate immune response between 2 nd and 4 th days after the tick bite could be a useful tool for evaluation of the risk of tick-borne disease for the humans bitten by ticks.
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.
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