Maria P. Fedina

Introduction. Powassan virus (POWV) is a tick-borne orthoflavivirus capable of causing neurological diseases. In the Russian Far East, the area affected by POWV overlaps with that of another neurotropic orthoflavivirus, tick-borne encephalitis virus (TBEV). Currently, there are no differentiated test systems for specific detection of POWV in the presence of TBEV. The aim of this work is to create differentiating test systems for the detection of POWV in a mixture with TBEV and other orthoflaviviruses. Materials and methods. We constructed a genome alignment of eight POWV and five TBEV strains to select differentiating primers using MEGA X. To assess the specificity of the assay, we used POWV, several TBEV strains, and other orthoflaviviruses. PCR and qPCR were performed with the selected oligonucleotides, and the specificity of amplification was verified by electrophoresis in agarose gel and Sanger sequencing. cDNA was obtained from virus-containing material by isolating RNA and performing a reverse transcription reaction. Results. The selected oligonucleotides for the qPCR-based differentiation system ensured specific detection of POWV without detecting signals from other orthoflaviviruses. The assay’s limit of detection was 102 copies/sample. Analysis of mixed samples containing POWV and TBEV demonstrated no distortion of the measurement results. Furthermore, differentiating oligonucleotides were selected and tested to amplify extended regions of the POWV and TBEV genomes for subsequent sequencing and phylogenetic analysis. Conclusion. A laboratory technique based on PCR has been developed that allows to detect the POWV in samples containing other orthoflaviviruses.

Background: West Nile virus (WNV) is a rapidly growing problem worldwide. The lack of emergency treatment and a safe licensed vaccine against WNV allows the virus to cause sporadic outbreaks of human disease, including fatal cases. Formalin-inactivated vaccines have been used for a long time and have been shown to be very safe and effective, especially in susceptible populations. Methods: By adapting tick-borne encephalitis vaccine production technology, we produced a laboratory-inactivated vaccine against WNV based on the strain SHUA, isolated from humans with a lethal WNV infection in the year 2021. Results: The potential vaccine was tested for safety in vitro and in vivo in outbred SHK mice of different ages, including PCR analysis of the brains of these mice to test for the absence of viral RNA after intracerebral injection. Conclusions: The inactivated whole-virion laboratory vaccine showed 100% seroconversion and immunogenicity against WNV strain SHUA-1, isolated from a lethal human case, and provided the mice with 100% protection from disease and death.

Non-human primates, due to their similarities in immune response to humans, are the preferred model for studying infectious processes and any associated cognitive impairments. Behavioral tests are indispensable for investigating pathogenesis in neuroinfections, especially those that do not manifest with noticeable clinical symptoms, as well as in the transition to a chronic form of the disease. Modeling viral infection requires specialized experimental conditions. Our work describes techniques for investigating mnemonic functions, tiredness, attentional focus, quick-wittedness, and basic behavioral responses in primates under the assumed conditions for infections with viruses that do not have an airborne route of transmission. It also outlines approaches to the training and selection of primates for virological research, as well as analyzing gender differences in learning abilities, the impact of housing conditions on the results, and the correlation between training success and behavioral test scores. These methods will allow a more detailed study of non-human primates as a model for researching cognitive and behavioral impairments under infectious and immune stress, as well as the design of less energy-intensive experiments for evaluating the efficacy and safety of therapeutic and prophylactic strategies at early stages of infection.