Ambiguous Interpretation of the Results of TBEV prevalence in Ticks Evaluation Using Different Methods
According to data of Federal Service on Customers’ Rights Protection and Human Well-Being Surveillance tick-borne encephalitis virus (TBEV) prevalence in ticks, determined by enzyme-linked immunosorbent assay (ELISA), is several times higher than those determined by polymerase chain reaction (PCR). We assumed that this fact can be explained by: 1) serological cross-reaction with known viruses of the genus Flavivirus; 2) circulation of unknown flavivirus, antigenically close to TBEV; 3) serological cross-reaction with microorganisms that inhabit ticks; 4) violation of ELISA protocol. In this study we tried to find out factors affecting specificity and sensitivity of these methods. We used three TBEV strains representing the main subtypes, Omsk hemorrhagic fever virus, Langat virus, and Louping ill virus to evaluate possible cross-reactions with viruses of the genus Flavivirus. A commercial ELISA-based kit (VectoTBE-antigen, JSC «Vector-Best») detected all studied viruses of the genus Flavivirus, but in various concentrations. A commercial kit based on Real-Time PCR (AmpliSens® TBEV, B. burgdorferi s.l., A. phagocytophilum, E. chaffeensis/E. muris-FL, InterLabService) detected three TBEV subtypes and Louping ill virus. Analysis of experimentally TBEV infected ticks from laboratory line has shown that Real-Time PCR and PCR with electrophoretic detection of the product (RT-PCR) are significantly more sensitive than ELISA. Moreover, violation of sample preparation protocol of ELISA kit reduced sensitivity of this method in more than 2 times. Investigation of possible cross-reactions with microorganisms that inhabit ticks using ELISA and RT-PCR was performed on field-collected ticks from Moscow region and Republic of Tatarstan. All tick suspensions showed negative results in RT-PCR. When examined by ELISA, 34% ticks showed positive results on the presence of TBEV antigen. We isolated pure cultures of microorganisms from ELISApositive tick suspensions, equilibrated them by total protein using Bradford’s method and tested in commercial ELISA-based kit on TBEV antigen presence. Positive results showed 4 suspensions of 15 tested. We made attempts to identify non-TBEV flavivirus in ticks using RT-PCR with genus-specific primers. We didn’t reveal any viral RNA in ticks from Moscow region and Republic of Tatarstan. We also studied 128 ticks from Republic of Tuva. Our attempts to identify flavivirus failed. However, there were four samples containing PCR product, which length was close to the one expected. For those samples we determined nucleotide sequence, which corresponded to the genome fragment of Pseudomonas spp. bacteria. Similar species of this bacterium showed positive result in ELISA. Thus, according to our data all methods can show false-positive results during analysis of ticks from nature on the presence of TBEV due to interactions with other flaviviruses or microorganisms that inhabit ticks. RT-PCR and Real-Time PCR are more sensitive and specific than ELISA. Sequencing of the PCR product to identify infectious agent is obligatory to establish TBEV circulation on new territories and in controversial cases.