Elena V. Protopopova

Wild bird species contribute significantly to the rapid geographic dissemination of tick-borne encephalitis viruses (TBEV) and West Nile virus (WNV), facilitating the establishment of new natural foci of these orthoflaviviruses . However, the impact of TBEV and WNV population variability on shaping these foci, as well as the potential emergence of new human-pathogenic viral variants, remain underexplored. This study aimed to assess the genetic heterogeneity of TBEV (Siberian and Far Eastern genotypes) and WNV, isolated simultaneously from the tissues of a single garden reed warbler ( Acrocephalus dumetorum ) collected in the suburbs of Tomsk. The methods of viral strain isolation on various cell cultures were used in combination with a whole-genome analysis of isolates through traditional and high-throughput sequencing (NGS) methods. Consensus full-genome nucleotide sequences of the viruses were obtained by Sanger sequencing and compared with those obtained by NGS, with single nucleotide substitutions (single nucleotide variants, SNVs) accounting for 2 % or higher within the population under study. Our findings revealed single nucleotide polymorphisms (SNPs) associated with both synonymous and non-synonymous nucleotide substitutions, primarily located within the non-structural protein genes of TBEV and WNV. Notably, recombination events were not detected in the genomes of isolated orthoflaviviruses . The WNV isolate, Tomsk/bird/2006/A4, and the TBEV isolates, PT12 and PT122, obtained from A. dumetorum, exhibited heterogeneous viral populations, with SNVs ranging in frequency from 1.75 to 19.88 % for WNV and from 2.08 to 23.73 % for TBEV. Most identified SNPs shared similar nucleotide substitutions in the genomes of already known strains of TBEV and WNV, suggesting that these SNVs could play a crucial role in viral adaptation and underscore the genetic and phenotypic diversity of these viruses in nature.

West Nile fever is an infectious disease caused by the West Nile virus (WNV), which is transmitted by mosquitoes. Epidemiological surveillance confirms the potential risk of WNV infection in human populations. The lack of specific antiviral therapeutics and vaccines against WNV underscores the urgent need to develop effective therapeutic approaches. In this study, a recombinant chimeric monoclonal antibody (mAb) 900 was generated based on the broadly neutralizing and protective murine mAb 9E2. The antigen-binding regions of the murine mAb were fused with the constant domains (CH2–CH3) of human IgG1. Two key amino acid clusters, M252/S254/T256 and H433/N434, were introduced into the CH2–CH3 domains to enhance the affinity of mAb 900 for the neonatal Fc receptor (FcRn). The engineered mAb 900 was produced in CHO cells and purified to high homogeneity. Biophysical characterization confirmed its stability and correct dimeric assembly. Comparative analysis demonstrated that mAb 900 retained the high antigen-binding affinity and potent virus-neutralizing activity of its murine predecessor. Most importantly, mAb 900 demonstrated significant protective efficacy in a lethal mouse model of WNV infection. These results establish the proof of concept for mAb 900 as a promising candidate for further preclinical development against WNV infection.

Ixodes ticks are vectors of many infectious human and animal pathogens. During the biting process, the tick secretes specific proteins produced in its salivary glands. These proteins play key roles in various biological processes, including modulation of the host immune response, pathogen transmission, blood processing, and tick reproduction and development. Among these proteins, serpins have received special attention and are considered as promising candidates for the development of vaccines and methods to control infectious diseases transmitted by ticks. Research aim. To obtain and characterize in vitro recombinant RCL domain of serpin Ipis of ticks Ixodes persulcatus. Material and methods. PCR product was cloned into PCR 2.1 and pET-200 D/TOPO plasmids. Transfection was performed on chemically competent E. coli One Shot TOP10 and E. coli BL21(DE3) cells. The protein was purified from impurities by nickel chelate chromatography using Ni-NTA-Superflow resin. To analyze the immunogenicity of the obtained protein, mice of BALB/c line were immunized. The obtained sera of laboratory animals were tested by enzyme immunoassay. The structures of tick serpins were constructed by homology modeling using AlphaFold server (https://www.alphafold.com/). Results. The recombinant RCL domain of serpin RCL of Ipis serpin - ticks Ixodes persulcatus was obtained and characterized in this study. The immunogenicity of the recombinant protein was evaluated by immunization of laboratory animals and the specific activity of the obtained sera was assessed by ELISA and immunoblot methods. 3D models of tick serpins were constructed. Conclusion. Thus, in this study, the recombinant RCL domain of serpin Ipis of Ixodes persulcatus ticks was obtained and characterized. After additional studies, the obtained recombinant RCL domain of serpin Ipis is planned to be used as mono- and multi-antigenic protein vaccines.

Introduction. Mosquito-borne human diseases caused by Zika virus and West Nile virus (WNV) are widespread across multiple continents and cause major outbreaks. Their ranges overlap and the possibility of mixed infections is obvious. The information of such mixed infections is limited. The aim of the study is to investigate the features of mixed infection of WNV and Zika virus in vitro and in vivo in order to assess their possible interference and/or enhancement of viral infection. Materials and methods. The study used West Nile virus and Zika virus strains Vlg27924 and MR766, respectively. The infectious activity of viruses during mono- and co-infection was determined on Vero E6 cell culture using RT-PCR, as well as on BALB/c mice using various administration schemes. Results. In vitro studies of co-infection with WNV and Zika virus showed that co-infection leads to interference, with the degree of competitive inhibition of replication being more pronounced for Zika virus, reaching 1000 times or more when compared to mono-infection. During simultaneous infection in mice, Zika virus does not affect the development of lethal infection caused by WNV. However, preliminary (4 and 20 days) infection with a sublethal dose of Zika virus reliably protects animals from subsequent administration of 10 and 100 LD50 WNV, respectively. In pre-infected and co-infected animals with Zika virus, the development of WNV-specific viral neutralizing antibodies was recorded in higher titers than in WNV monoinfected animals. Conclusion. The presence of in vitro interference between the studied orthoflaviviruses was shown, most pronounced in relation to the Zika virus. No significant effect was observed with simultaneous co-infection in vivo. However, pre-infection of mice with Zika virus provides protection to animals from lethal WNV infection due to the induction of high levels of antibodies that specifically neutralize its infectious activity.

Ixodes ticks are vectors of many infectious human and animal pathogens. During the process of biting, the tick secretes specific proteins produced in its s

The annual number of reported human cases of flavivirus infections continues to increase. Measures taken by local healthcare systems and international organizations are not fully successful. In this regard, new approaches to treatment and prevention of flavivirus infections are relevant. One promising approach is to use monoclonal antibody preparations. The mouse mAb 10H10 is capable of interacting with viruses belonging to the genus Orthoflavivirus which are pathogenic to humans. ELISA and molecular modeling data can indicate that mAb 10H10 recognizes the fusion loop region of E protein. The KD of interaction between the mAb 10H10 and recombinant analogs of the E protein of the tick-borne encephalitis (TBEV), Zika (ZIKV) and dengue (DENV) viruses range from 1.5 to 4 nM. The aim of this study was to map the epitope of this antibody using phage display technology. After three rounds of biopanning, 60 individual phage clones were chosen. The amino acid sequences of the selected peptides were conveniently divided into five groups. Based on the selected peptides, bacteriophages were obtained carrying peptides on the surfaces of the pIII and pVIII proteins, which were tested for binding to the antibody in ELISA. Thus, the epitope of the mAb 10H10 is the highly conserved region 98-DRGWGNXXGLFGK-110 of the flavivirus E protein. The structures of the complexes of the identified peptides with the antibody paratope are proposed using the molecular docking and dynamics methods.

In this study, we investigated the features of co-infection with SARS-CoV-2 and the enterovirus vaccine strain LEV8 of coxsackievirus A7 or enterovirus A71 for Vero E6 cells and Syrian hamsters. The investigation of co-infection with SARS-CoV-2 and LEV-8 or EV-A71 in the cell model showed that a competitive inhibitory effect for these viruses was especially significant against SARS-CoV-2. Pre-infection with enteroviruses in the animals caused more than a 100-fold decrease in the levels of SARS-CoV-2 virus replication in the respiratory tract and more rapid clearance of infectious SARS-CoV-2 from the lower respiratory tract. Co-infection with SARS-CoV-2 and LEV-8 or EV-A71 also reduced the severity of clinical manifestations of the SARS-CoV-2 infection in the animals. Additionally, the histological data illustrated that co-infection with strain LEV8 of coxsackievirus A7 decreased the level of pathological changes induced by SARS-CoV-2 in the lungs. Research into the chemokine/cytokine profile demonstrated that the studied enteroviruses efficiently triggered this part of the antiviral immune response, which is associated with the significant inhibition of SARS-CoV-2 infection. These results demonstrate that there is significant viral interference between the studied strain LEV-8 of coxsackievirus A7 or enterovirus A71 and SARS-CoV-2 in vitro and in vivo.

The SARS-CoV-2 pandemic has underscored the necessity for functional transgenic animal models for testing. Mouse lines with overexpression of the human receptor ACE2 serve as the primary animal model to study COVID-19 infection. Overexpression of ACE2 under a strong ubiquitous promoter facilitates convenient and sensitive testing of COVID-19 pathology. We performed pronuclear microinjections using a 5 kb CAG-ACE2 linear transgene construct and identified three founder lines with 140, 72, and 73 copies, respectively. Two of these lines were further analyzed for ACE2 expression profiles and sensitivity to SARS-CoV-2 infection. Both lines expressed ACE2 in all organs analyzed. Embryonic fibroblast cell lines derived from transgenic embryos demonstrated severe cytopathic effects following infection, even at low doses of SARS-CoV-2 (0,1-1.0 TCID ₅₀ ). Infected mice from the two lines began to show COVID-19 symptoms three days post-infection and succumbed between days 4 and 7. Histological examination of lung tissues from terminally ill mice revealed severe pathological alterations. To further characterize the integration site in one of the lines, we applied Nanopore sequencing combined with Cas9 enrichment to examine the internal transgene concatemer structure. Oxford Nanopore sequencing (ONT) is becoming the gold standard for transgene insert characterization, but it is relatively inefficient without targeted region enrichment. We digested genomic DNA with Cas9 and gRNA against the ACE2 transgene to create ends suitable for ONT adapter ligation. ONT data analysis revealed that most of the transgene copies were arranged in a head-to-tail configuration, with palindromic junctions being rare. We also detected occasional plasmid backbone fragments within the concatemer, likely co-purified during transgene gel extraction, which is a common occurrence in pronuclear microinjections.

In this study, we investigated the features of co-infection with SARS-CoV-2 and nonpathogenic strain LEV8 of coxsackievirus A7 or enterovirus A71 for Vero E6 cells and Syrian hamsters. The investigation of co-infection with SARS-СoV-2 and LEV-8 or EV-A71 in the cell model was shown that a competitive inhibitory effect for these viruses were especially significant against SARS-CoV-2. Pre-infection with enteroviruses in animals caused more than a 100-fold decrease in the levels of SARS-CoV-2 virus replication in the respiratory tracts and more rapid clearance of the lower respiratory tract from infectious SARS-CoV-2. Co-infection with SARS-CoV-2 and LEV-8 or EV-A71 also reduced the severity of clinical manifestations of the SARS-CoV-2 infection for animals. Additionally, the histological data illustrated that co-infection with nonpathogenic coxsackievirus decreased the level of pathological changes by SARS-CoV-2 in the lungs. Research into the chemokine/cytokine profile demonstrated that the studied enteroviruses efficiently triggered this part of antiviral immune response, which can be associated with significant inhibition of SARS-CoV-2 infection. These results demonstrate that there exists a strong viral interference between the studied nonpathogenic strain of coxsackievirus A7 or enteroviruses A71 and SARS-CoV-2 pathogenic for humans in vitro and in vivo.

Tick-borne encephalitis virus (TBEV) causes a severe disease, tick-borne encephalitis (TBE), that has a substantial epidemiological importance for Northern Eurasia. Between 10,000 and 15,000 TBE cases are registered annually despite the availability of effective formaldehyde-inactivated full-virion vaccines due to insufficient vaccination coverage, as well as sporadic cases of vaccine breakthrough. The development of improved vaccines would benefit from the atomic resolution structure of the antigen. Here we report the refined single-particle cryo-electron microscopy (cryo-EM) structure of the inactivated mature TBEV vaccine strain Sofjin-Chumakov (Far-Eastern subtype) at a resolution of 3.0 Å. The increase of the resolution with respect to the previously published structures of TBEV strains Hypr and Kuutsalo-14 (European subtype) was reached due to improvement of the virus sample quality achieved by the optimized preparation methods. All the surface epitopes of TBEV were structurally conserved in the inactivated virions. ELISA studies with monoclonal antibodies supported the hypothesis of TBEV protein shell cross-linking upon inactivation with formaldehyde.