Problems of Virology

Investigating the mechanisms of viral attachment and entry into cells is crucial for understanding viral pathogenesis and developing therapeutic strategies. The aim of this review is to characterize pseudo-typed particles based on the vesicular stomatitis virus (VSV) as a convenient and effective tool for studying viral entry into cells, based on literature data (PubMed, Scopus, and Web of Science), and to determine the prospects for combining this method with genetic and protein-based approaches.

VSV, a member of the Rhabdoviridae family, has a remarkable capacity for pseudotyping, which involves the replacement of its native glycoprotein (G) with envelope proteins from other viruses. This feature enables the modeling of the cell entry process without the need for wild-type viruses. The VSV genome is modified by deleting the G gene and incorporating reporter genes (e.g., GFP or luciferase), thereby facilitating the quantitative assessment of infectivity.

The methodology for generating pseudoviruses involves a two-plasmid cotransfection system in cell lines (e.g., HEK293T), with plasmids encoding the VSV structural proteins and the target viral envelope proteins. The advantages of the VSV system include high particle titers, rapid reporter signal manifestation, and the feasibility of work under Biosafety Level 2 conditions. However, limitations are associated with differences in the distribution of viral proteins on the surface of pseudoviruses compared to native virions, necessitating additional data validation. Conclusion. Methods for analyzing virus-cell interactions were studied, such as Virus Overlay Protein Binding Assay (VOPBA), RNA interference, CRISPR/Cas9 knockout, and gene overexpression. These approaches allow for the identification of cellular receptors, investigation of specific protein functions, and assessment of the impact of mutations. Future prospects for the application of VSV pseudoviruses include screening viral entry inhibitors, analyzing antibody neutralization, and vaccine development. Despite technical limitations, pseudotyped particles remain an indispensable tool for studying highly pathogenic and fastidious viruses. For the present review, a literature search was conducted in the PubMed, Scopus, and Web of Science databases.

The aim of this study was to determine the level of humoral immunity to the West Nile virus (WNV) in the Moscow region population after the end of the outbreak in October 2021, as well as to confirm the specificity of antibodies to WNV by comparatively testing patient sera for antibodies to the antigenically related endemic tick-borne encephalitis orthoflavivirus (TBEV) using ELISA-IgM, ELISA-IgG, and 50% plaque reduction neutralization test.

Materials and methods. We analyzed 1,594 sera from outpatients-residents of Moscow and the surrounding region–who underwent outpatient examination in the winter of 2021 at Infectious Diseases Clinical Hospital No. 1 in Moscow (IKB No. 1) and medical institutions of the Moscow Regional Research Institute named after M.F. Vladimirsky Regional Research Institute (MONIKI) conducted a study without any connection to WNV infection in the summer-autumn of 2021.

Results. All samples were negative in ELISA-IgM test with WNV and TBEV antigens. In an ELISA-IgG test with the WNV antigen, antibodies were detected in 64 samples (4.0%). All samples were tested for IgG antibodies to TBEV in an ELISA and neutralizing antibodies to WNV and TBEV in a 50% plaque reduction neutralization test. Specific antibodies to WNV were detected in 44 samples (68.8%), to TBEV in eleven (17.2%), and group-specific antibodies in nine (14.0%). According to the total data from the test of 1,594 sera from residents of the Moscow region, specific antibodies to WNV were detected in 2.8% of cases, to TBEV in 0.7%, and group-specific antibodies in 0.6%. Ten of the 11 individuals with specific IgG antibodies to TBEV were undergoing outpatient examination at Moscow’s Infectious Diseases Clinical Hospital No. 1 for a history of tick-borne encephalitis or for post-vaccination immunity testing following vaccination against this infection. The detection rate of specific antibodies to WNV in similar studies conducted in the same region in 2013 was 0.2%, while in 2021 it was 2.8%. The difference between these rates is statistically significant (p < 0.01).

Conclusion. Based on these data, it can be concluded that sporadic undiagnosed cases of WNV infection occurred in Moscow and the surrounding region between 2013 and 2021.

Introduction. The unfavorable epidemiological situation with viral tick-borne infections can be characterized not only by an increase in the incidence of «classical», well-known diseases, but also by the discovery of new pathogens, the role of which remains poorly understood. Yezo virus (YEZV) belongs to the group of recently discovered tick-borne nairoviruses. YEZV circulation is characteristic of Japan, China, and a number of regions in Russia (Primorsky, Khabarovsk, and Zabaykalsky Krais; Tomsk Oblast).

The aim of the study was to screen and characterize the molecular genetics of YEZV isolated from ticks in the Amur region.

Materials and methods. In the study, 704 individual tick samples (463 I. persulcatus ticks and 241 H. concinna ticks) collected from vegetation in 15 locations in 5 districts of the Amur Region were analyzed for the presence of YEZV RNA. Full-length genomic sequencing of the identified YEZV genetic variant was performed by preliminary enrichment of YEZV cDNA using multiplex PCR with a primer panel developed by us.

Results. The YEZV infection rate in the studied sample of I. persulcatus ticks was 0.2% (1/463; 95% CI: 0.1–1.2).

Conclusion. The results of this study expand our understanding of the spread of YEZV in Russia. Up-to-date information on the circulation and genetic diversity of nairoviruses that pose a potential threat to humans and animals contributes to the regulation and adjustment of preventive and anti-epidemic measures, as well as the implementation of adequate region-oriented laboratory diagnostics of IPK in the Far East, and especially in areas bordering China.

Introduction. Secondary bacterial pneumonias are complications responsible for most fatalities following influenza infection, and antiviral drugs are used to prevent their development.

The aim of the study is to evaluate the efficacy of tilorone and its combination with antibacterial agents in a murine model of secondary bacterial pneumonia caused by Staphylococcus aureus after influenza infection.

Materials and methods. BALB/c mice were infected with influenza virus A/California/04/2009 (pdm H1N1 2009) virus, followed by S. aureus infection four days later. Treatments included the interferon inducer tilorone, comparator oseltamivir, antibacterial agents cefuroxime and amoxicillin, or combinations of antivirals with antibiotics. Efficacy was assessed by increased survival, reduced weight loss, and inhibition of pathogen proliferation in the lungs.

Results. The efficacy of tilorone, as indicated by increased survival, reduced weight loss, and decreased pathogen load in the lungs, in a mouse model of secondary bacterial pneumonia following influenza infection, increased with reducing viral dose, and intraperitoneal administration of the drug was more effective than oral administration. The combination of tilorone with the antibiotic cefuroxime, to which S. aureus was sensitive, was more effective than either agent alone and completely protected animals from death and from viral and bacterial proliferation in the lungs. In contrast, the combination with amoxicillin, to which S. aureus was resistant, had no effect on disease outcome.

Conclusion. The efficacy of tilorone in this model depended on viral dose and drug administration route. Combining tilorone with cefuroxime was more effective than monotherapy.