Polina Igorevna Elboeva

Hemorrhagic fever with renal syndrome (HFRS) remains one of the leading natural focal infections in Russia, characterized by pronounced clinical variability. Clinical manifestations of HFRS, in particular, may be determined by single nucleotide polymorphisms (SNPs) in early immune response genes in patients. We determined the allele and genotype frequencies of SNPs in the CASP1 (rs580253) and IL‑6 (rs1800795) genes. CASP1 encodes caspase‑1, an enzyme that is the product of the activated inflammasome, while IL‑6 encodes interleukin‑6, a cytokine of the acute immune response. It was found that the genotype frequencies in the control group conformed to the Hardy–Weinberg law, whereas in HFRS patients a statistically significant deviation from the expected equilibrium was detected, which suggests the existence of correlations between the genotypes of the studied SNPs and the clinical manifestations of the disease.

Мультиэпитопные вакцины представляют собой перспективный инструмент профилактики вирусных инфекций, способный индуцировать комплексный гуморальный и клеточный иммунный ответ. В статье рассмотрены современные подходы к проектированию in silico химерных вакцинных конструкций: критерии отбора эпитопов, методы прогнозирования перспективных Т- и В-клеточных эпитопов, выбор линкеров и адъювантов, структурное моделирование, молекулярный докинг и симуляция иммунного ответа. Интеграция иммунноинформатических методов и молекулярного моделирования существенно ускоряет разработку вакцинных кандидатов и повышает точность прогнозирования до начала экспериментальной проверки.

The aim of the work was to detect Orthohantavirus puumalaense (PUUV) reassortant strains in bank voles (Clethrionomys glareolus) of the Republic of Tatarstan (RT), which are closely related to strains previously isolated from patients with hemorrhagic fever with renal syndrome (HFRS) from the RT. Materials and methods . Viral RNA was detected by RT-PCR in the lung tissue of bank voles captured in the Vysokogorsky and Pestrechinsky districts of the RT in 2023–2024. cDNA was synthesized for RT-PCR-positive samples. PUUV genome fragments of S- and M-segments were amplified using PCR, followed by sequencing (using the Sanger method and Oxford Nanopore technology) and phylogenetic analysis. Results and discussion . It has been found that 14 PUUV strains detected in bank voles from four capture sites fall into 3 clusters. In two strains from the vicinity of the village of Kinderi (cluster 2), a reassortant genome has been identified: the S-segment is closely related to strains from the Vysokogorsky district (nucleotide sequence (NS) identity is 99.1–100 %), while the M-segment is closely related to strains from the Pestrechinsky district (96.3–97.0 %). These virus strains are genetically close to reassortants previously found in HFRS patients infected by PUUV in the Vysokogorsky district of the RT. Detected in the vicinity of the village Kinderi PUUV strains appear to be of reassortant origin.

Introduction. The Kurkino and Sochi viruses of species Orthohantavirus dobravaense (ODOB) are among the pathogens that cause hemorrhagic fever with renal syndrome in the European part of Russia. However, the current literature provides limited data on the distribution of genetic variants of the ODOB in Russia. The aim is to identify ODOB in several regions of Volga, Central and Ural Federal districts of the Russian Federation and analyze their genome. Materials and methods. Total RNA was isolated from lung tissue samples of striped field mouse (Apodemus agrarius) and yellow-necked mouse (A. flavicollis) captured in a number of areas of the Volga Federal District and neighboring regions in 2015–2023. Orthohantavirus RNA was detected by RT-PCR using specific primers to ODOB. The PCR amplification products were separated in agarose gel, purified, and subjected to Sanger sequencing. Comparative and phylogenetic analyses were performed for the sequenced genome fragments. Results. Orthohantavirus RNA was detected in one sample of A. flavicollis from the Ulyanovsk region. Based on the analysis of the nucleotide sequences of the sequenced PCR products, it was found that the highest values of similarity were obtained when comparing the identified strain with the reference Kurkino virus from the Tula region. The data from the phylogenetic analysis of the sequenced fragment of the S and M segments allowed us to establish that the identified isolate is closely related to the Kurkino virus found earlier in A. agrarius in the Tula region. Thus, the detected isolate was identified as variant of Kurkino virus, which is also widespread in the central regions of Russia, Wetern Siberia and close related to genome variants that are distributed in Central Europian countries. Conclusions. It is proved for the first time that: 1) the range of Orthohantavirus dobravaense (Kurkino virus) extends to a part of the territory of the Volga Federal District; 2) Orthohantavirus dobravaense (Kurkino virus) and Orthohantavirus puumalaense (Puumala virus) are co-circulating in the Ulyanovsk region.

(1) Background: Hemorrhagic fever with renal syndrome (HFRS) remains a prevalent zoonosis in Eurasia. Orthohantavirus puumalaense (PUUV), carried by bank voles (Myodes glareolus), is the principal zoonotic pathogen of HFRS in this region. Despite ongoing efforts to develop effective drugs and vaccines against PUUV, this challenge remains. (2) Aim: In this study, we aimed to express a large quantity of the PUUV recombinant N (rN) protein using E. coli. We also sought to develop a protocol for extracting the rN protein from pellets, solubilizing, and refolding it to restore its native form. This protocol is crucial for producing a large quantity of rN protein to develop vaccines and diagnostic tools for HFRS. (3) Methods; PUUV S segment open reading frame (ORF) coding for N protein was synthesized and cloned into the plasmid vector pET-28 (A+). The ORF was transformed, expressed and induced in BL21(DE3) pLysS E. coli strain. Subsequently, rN protein was purified using immobilized metal affinity and ion chromatography. Immune reactivity of rN protein was tested by employing in house and commercial VektoHanta-IgG kit ELISA methods (both in vitro and in vivo). (4) Results: The best conditions for scaling up the expression of the PUUV rN protein were an incubation temperature of 20 °C during a 20 h incubation period, followed by induction with 0.5 mM IPTG. The most significant protein yield was achieved when the pellets were incubated in denaturing buffer with 8M urea. The highest yield of refolded proteins was attained using non-denaturing buffer (50 mM Tris-HCl) supplemented with arginine. A final 50 μL of PUUV rN protein solution with a concentration of 7 mg/mL was recovered from 1 L of culture. The rN protein elicited an antibody response in vivo and reacted with serum taken from patients with HFRS by ELISA in vitro. (5) Conclusion: Therefore, the orthohantavirus N protein’s ability to elicit immune response in vivo suggests that it can be used to develop vaccines against PUUV after conducting in vitro and in vivo studies to ascertain neutralising antibodies.