4-Aminotetrahydroquinazoline N-oxides form a prominent chemotype of orthoflavivirus reproduction inhibitors, attractive for the search of lead compounds for development as direct-action antiviral agents. Here we explored the relationship between the structure and antiviral activity within an extended series of adamantyl-containing 4-aminotetrahydroquinazolines, synthesized via SNAr reactions of 4-chlorotetrahydroquinazolines with adamantyl-containing amines. Most of the obtained compounds inhibited the reproduction of tick-borne encephalitis, yellow fever, and West Nile viruses in phenotypic assays with micromolar EC50s. The most favorable substituents at 4-amino group were 2-adamant-(1/2)-ylethyls, while the position 2 of heterocyclic core served as a toxicity switch, 2-chlorotetrahydroquinazolines being much less toxic than other analogues. Time-of-addition experiments for hit compounds against West Nile virus revealed a possibility for multi-factorial mechanism of action in the 4-aminotetrahydroquinazoline series.

Introduction. Cases of hemorrhagic fever with renal syndrome (HFRS) have increased over the past ten years, including in regions previously non-endemic for HFRS. Approximately 98% of all cases in Russia are associated with Puumala hantavirus, while only about 2% of sporadic cases are caused by Hantaan, Amur, Seoul, Kurkino and Sochi hantaviruses. The main HFRS foci are located in the European part of Russia. In endemic areas, the risk of infection exists for all population groups, especially military personnel and workers in the forestry and agricultural sectors. The etiotropic therapy absence can be solved only through vaccination. However, licensed vaccines based on Hantaan or Seoul hantaviruses do not provide protection against Puumala hantavirus, for which no vaccines currently exist worldwide. An experimental vaccine (EV) for the prevention of HFRS, based on Puumala and Hantaan hantaviruses, has been developed at the Chumakov Federal Scientific Center for Research and Development of Immune-and-Biological Products of the Russian Academy of Sciences (Institute of Poliomyelitis). The aim of the study is to determine the EV immunogenicity against HFRS in a guinea pig model using various immunisation schedules. Materials and methods. The induction of neutralising antibodies (NAb) was assessed in the blood serum of guinea pigs following immunisation with mono- and bivalent EV by means of a neutralisation assay based on 50% suppression of focus-forming units in Vero E6 cell culture. The results indicate the formation of a humoral immune response following EV immunisations. Immunogen administration induced the NAb production up to peak values followed by a decrease after certain intervals. Repeated EV administration showed similar NAb dynamics with prolonged circulation. Three variants involving double immunisation and a subsequent booster after one year were prioritised for clinical trials (0, 150, 300; 0, 14, 182, 364; 0, 30, 364). Conclusion. This study indicates the development of a persistent and intense immune response, the strength of which depended on the EV immunisation schedule. The optimal timing for immunogen administration has been determined. The results of this study will be the basis for subsequent clinical trials of hantavirus vaccines.

Background: Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal cancers, with a five-year survival rate of less than 12%. Oncolytic viruses are considered a promising immunotherapeutic approach, but their effectiveness is often limited by the innate interferon response, which is the main antiviral barrier in resistant tumors. A combination of an oncolytic virus with JAK/STAT pathway inhibitors has been proposed to overcome this resistance. Methods: In this study, we investigated the oncolytic and immunotherapeutic potential of the attenuated yellow fever vaccine strain YFV 17D in combination with the JAK1/JAK2 inhibitor ruxolitinib in a panel of six PDAC cell lines with diverse genetic profiles and JAK/STAT signaling activities and in a syngeneic immunocompetent PAN02 mouse model. Results: In vitro, we identified three distinct response patterns: synergistic enhancement of viral replication and cytopathic effect, lack of synergism due to absent interferon signaling, and increased viral replication without cytopathic effect. Despite different cell mutation profiles, the cell response patterns were determined by the basal JAK/STAT activity of each cell line rather than by specific KRAS or TP53 mutation. In vivo, the combination therapy significantly extended median survival compared to YFV 17D monotherapy and the control group and induced tumor regression in 62.5% of animals. Since no difference in intratumoral viral RNA was detected between the combination and monotherapy groups, we suggest that the antitumor effect in vivo was mediated not by enhanced viral replication, but by activation of adaptive immunity. This is indirectly suggested by increased intratumoral IL-12, a fourfold increase in CD8+ T cell infiltration, a reduction in CD4+ cells, and the generation of virus-neutralizing antibodies. No systemic cytokine surge, neurovirulence, or viral dissemination was observed, suggesting the safety of the proposed regimen. Conclusions: These findings clarify the immune mechanisms determining the efficacy of YFV 17D combined with ruxolitinib and establish the basis for personalized patient stratification by tumor interferon signaling status.

SARS-CoV-2, which causes COVID-19, continues to circulate around the world, making it necessary to study the impact of rapidly emerging mutations on escape from neutralizing antibodies and pathogenesis. While RBD mutations are well characterized, mutations in the N-terminal domain (NTD) of the spike protein remain comparatively understudied despite their relevance to antibody recognition. This study investigates two phenotypically distinct SARS-CoV-2 mutants, which exhibited differences in plaque morphology on Vero cells. Whole-genome sequencing via Illumina identified a novel 12-nucleotide insertion in the spike NTD. This insertion induced a frameshift, introducing five new amino acids potentially altering viral behavior, receptor interactions, and antibody detection in ELISAs. The study further explores the pathogenicity of these variants in a hamster model. These findings underscore the importance of monitoring NTD mutations, which may contribute to immune evasion and influence therapeutic antibody efficacy, highlighting gaps in current research on SARS-CoV-2 evolution.

Relevance . Acute respiratory infections (ARI) are a leading cause of morbidity and mortality in children worldwide. The COVID-19 pandemic has significantly influenced the circulation of other respiratory viruses. Understanding the interactions between SARSCoV-2 and other respiratory pathogens is crucial for predicting disease severity, improving diagnostic and treatment methods, and developing effective prevention strategies. Aims. This study aims to analyze changes in the circulation of respiratory viruses at different stages of the COVID-19 pandemic, investigate the prevalence and characteristics of viral and bacterial co-infections in children, and assess their impact on disease progression. Conclusions. At the initial stage of the COVID-19 pandemic, the incidence of respiratory viral infections declined, followed by a rapid resurgence after the lifting of restrictive measures. In children, COVID19 presents with milder symptoms than in adults and has a high proportion of asymptomatic cases. Mixed SARS-CoV-2 infections with other respiratory viruses are more common in younger children and do not significantly increase disease severity. However, secondary bacterial infections in children with COVID-19 substantially elevate the risk of severe disease progression.

Objective: We evaluate the immunotherapeutic potential of the yellow fever virus vaccine strain 17D (YFV 17D) for intratumoral therapy of pancreatic cancer in mice. Methods: The cytopathic effect of YFV 17D on mouse syngeneic pancreatic cancers cells were studied both in vitro and in vivo and on human pancreatic cancers cells in vitro. Results: YFV 17D demonstrated a strong cytopathic effect against human cancer cells in vitro. Although YFV 17D did not exhibit a lytic effect against Pan02 mouse cells in vitro, a single intratumoral administration of 17D caused a delay in tumor growth and an increase in median survival by 30%. Multiple injections of 17D did not further improve the effect on tumor growth; however, it notably extended the median survival. Furthermore, preliminary immunization with 17D enhanced its oncotherapeutic effect. Conclusions: Intratumoral administration of yellow fever virus vaccine strain 17D delayed tumor in a murine model of pancreatic cancer. The fact that YFV 17D in vitro affected human cancer cells much more strongly than mouse cancer cells appears promising. Hence, we anticipate that the in vivo efficacy of YFV-17D-based oncolytic therapy will also be higher against human pancreatic carcinomas compared to its effect on the mouse pancreatic tumor.

Objectives: Most antiviral vaccines are created by inactivating the virus using chemical methods. The inactivation and production of viral vaccine preparations after the irradiation of viruses with accelerated electrons has a number of significant advantages. Determining the integrity of the genome of the resulting viral particles is necessary to assess the quality and degree of inactivation after irradiation. Methods: This work was performed on the Sabin 2 model polio virus. To determine the most sensitive and most radiation-resistant part, the polio virus genome was divided into 20 segments. After irradiation at temperatures of 25 °C, 2–8 °C, −20 °C, or −70 °C, the amplification intensity of these segments was measured in real time. Results: The best correlation between the amplification cycle and the irradiation dose at all temperatures was observed for segment 3D, left. Consequently, this section of the poliovirus genome is the least resistant to the action of accelerated electrons and is the most representative for determining genome integrity. The worst dependence was observed for the VP1 right section, which, therefore, cannot be used to determine genome integrity during inactivation. The electrochemical approach was also employed for a comparative assessment of viral RNA integrity before and after irradiation. An increase in the irradiation dose was accompanied by an increase in signals indicating the electrooxidation of RNA heterocyclic bases. The increase in peak current intensity of viral RNA electrochemical signals confirmed the breaking of viral RNA strands during irradiation. The shorter the RNA fragments, the greater the peak current intensities. In turn, this made the heterocyclic bases more accessible to electrooxidation on the electrode. Conclusions: These results are necessary for characterizing the integrity of the viral genome for the purpose of creating of antiviral vaccines.

In this article we discuss characteristics of fusion protein-based SARS-CoV-2 vaccines. We focus on recombinant vaccine antigens comprising fusion proteins consisting of combinations of SARS-CoV-2-derived antigens or peptides or combinations of SARS-CoV-2 antigens/peptides with SARS-CoV-2-unrelated proteins/peptides. These fusion proteins are made to increase the immunogenicity of the vaccine antigens and/or to enable special targeting of the immune system. The protein-based vaccine approach is exemplified solely in a proof of concept study by using W-PreS-O, a chimeric vaccine based on a single fusion protein (W-PreS-O), combining RBDs from Wuhan hu-1 wild-type and Omicron BA.1 with the hepatitis B virus (HBV)-derived PreS surface antigen adsorbed to aluminum hydroxide. The W-PreS-O vaccine was evaluated in Syrian hamsters which were immunized three times at three-week intervals with W-PreS-O or with aluminum hydroxide (placebo) before they were infected with Omicron BA.1. Neutralizing antibody (nAB) titers, weight, lung symptoms, and viral loads, as measured using RT-PCR in the upper and lower respiratory tracts, were determined. In addition, infectious virus titers from the lungs were measured using a plaque-forming assay. We found that W-PreS-O-vaccinated hamsters developed robust nABs against Omicron BA.1, showed almost no development of pneumonia, and had significantly reduced infectious virus titers in the lungs. Importantly, the viral loads in the nasal cavities of W-PreS-O-vaccinated hamsters were close to or above the PCR cycle threshold considered to be non-infectious. The data of our proof-of-concept study provides compelling evidence that the W-PreS-O vaccine has protective effect against Omicron BA.1 in a Syrian hamster in vivo infection model and thus support the promising results obtained also for other fusion protein-based SARS-CoV-2 vaccines.

IntroductionOn the background of kaleidoscopic changes of SARS-CoV-2 circulating variants, constant presence of SARS-CoV-2 in the human population hampers the dissection of native long-term immunogenicity of COVID-19 vaccines.MethodsFor this purpose, we performed a more than two-year-long evaluation of parameters of the humoral immune response elicited by intramuscularly (IM) and intranasally (IN) delivered adenovirus vector-based Sputnik V vaccine in nonhuman primates (NHP, Common marmosets), which are naturally nonsusceptible for SARS-CoV-2 infection.ResultsAlthough both immunization routes elicited prominent humoral immune responses in a short-term perspective, the long-term kinetics significantly differed between the IM and IN groups. While the titers of local and systemic antigen-specific antibodies (both IgA and IgG) nearly disappeared within two years upon IN vaccination, IM vaccination led to the highest IgG values in nasal swabs as well as IgA and IgG in serum specimens from NHPs by the end of observation period (day 764). Unlike IN vaccination, IM vaccination also resulted in a continuous long-term increase in serum maturation parameters such as antibody avidity, neutralization potency and breadth.DiscussionThe present study provides valuable information about distinct features of the long-term postvaccination humoral immune response in nonhuman primates induced by adenoviral COVID-19 vaccine administered by the intramuscular and intranasal routes commonly used in clinical practice.

Hemorrhagic fever with renal syndrome (HFRS) is the result of acute, zoonotic, natural foci hantavirus infections. It has serious social and medical importance due to its widespread distribution and the disease’s severity. There is a lack of effective etiotropic therapy and specific prophylaxis available. The aim of this review is to observe the etiological, clinical, and epidemiological features of nosologic HFRS forms in Russia, as well as differences and similarities with hantavirus pulmonary syndrome (HPS). The various clinical HFRS manifestations characterized diseases associated with Puumala, Kurkino, and Sochi hantaviruses in the Russian European part, and with Hantaan, Amur, and Seoul hantaviruses in the Russian Far East. Differences were observed for HFRS foci types based on biological characteristics and natural host population dynamics. As a result of clinical and epidemiological analysis six nosological forms were established, all of which were classified as “hemorrhagic fever with renal syndrome” according to the WHO’s expert recommendation from 1983 year. The study showed comparable taxonomic characteristics and determined the mechanism of human infection course for HFRS and HPS. The accumulated knowledge of this study allows for the combination of HFRS and HPS names into a common logical disease name “Hantavirus fever”.

The traditional method used in the production of inactivated vaccines is chemical inactivation using beta-propiolactone or formaldehyde. An alternative method is inactivation by irradiation. Virus inactivation is often accompanied by a change in particle shape, which can negatively affect the preservation of antigens and immunogenicity. Therefore, determining the shape and structure of the viral particle after inactivation is an important step in the development of antiviral vaccines. The poliovirus strain Sabin 2 was inactivated with a dose of 30.5 ± 0.5 kGy. in a pulsed linear electron accelerator with a power of 15 kW and electron energy of 10 MeV. Samples inactivated with beta-propiolactone or formaldehyde were used for comparison. All types of inactivation resulted in D-antigen recovery as determined by enzyme-linked immunosorbent assay. There was no statistical difference between D-antigen recovery in irradiated samples and those inactivated chemically. The shape and structure of the inactivated poliovirus particles were studied using atomic force and electron microscopy. After inactivation with beta-propiolactone or formaldehyde, a change in the native icosahedral shape was observed, with many particles appearing flattened. Specific sorption of antibodies showed that the antigen is mainly preserved in intact capsids for all type of inactivation. However, in the case of inactivation with formaldehyde and accelerated electrons, a significant number of fragments measuring 10–20 nm in height were present. Their proportion was 38 ± 2% and 17 ± 2% for inactivation with accelerated electrons and formaldehyde, respectively. The proportion of bound fragments during inactivation with beta-propiolactone was less than 1%.

Relevance. COVID-19, caused by the SARS-CoV-2 virus, remains a global public health threat despite the end of the pandemic. In the four years since the onset of the pandemic, the SARS-CoV-2 genome has undergone significant changes, particularly in the gene encoding the spike (S) protein. These changes resulted from the accumulation of immune responses in the human population, allowing the virus to evade the immune response. A significant proportion of the population was infected early in the pandemic or vaccinated with vaccines based on earlier variants of the virus. The emergence of new mutant variants raises concerns about the potential for severe COVID-19 in previously infected or vaccinated individuals.Аim. To examine the specifics of antibody formation, as well as the spectrum and functional activity of these antibodies in patients with COVID-19.Conclusions. Antibodies produced in response to infection or vaccination show diversity in spectrum and functional activity. Changes in the viral genome may reduce antibody effectiveness, highlighting the importance of monitoring new SARS-CoV-2 variants and developing adapted vaccines. These data will be key in shaping COVID-19 vaccination and treatment strategies in a changing epidemiological situation.

The COVID-19 pandemic has altered respiratory infection patterns in pediatric populations. The emergence of the SARS-CoV-2 Omicron variant and relaxation of public health measures have increased the likelihood of coinfections. Previous studies show conflicting results regarding the impact of viral and bacterial coinfections with SARS-CoV-2 on severity of pediatric disease. This study investigated the prevalence and clinical impact of coinfections among children hospitalized with COVID-19 during the Omicron wave. A retrospective analysis was conducted on 574 hospitalized patients aged under 18 years in Russia, from January 2022 to March 2023. Samples from patients were tested for SARS-CoV-2 and other respiratory pathogens using qRT-PCR, bacterial culture tests and mass spectrometry, and ELISA. Approximately one-third of COVID-19 cases had coinfections, with viral and bacterial coinfections occurring at similar rates. Adenovirus and Staphylococcus aureus were the most common viral and bacterial coinfections, respectively. Viral coinfections were associated with higher fevers and increased bronchitis, while bacterial coinfections correlated with longer duration of illness and higher pneumonia rates. Non-SARS-CoV-2 respiratory viruses were linked to more severe lower respiratory tract complications than SARS-CoV-2 monoinfection. These findings suggest that during the Omicron wave, seasonal respiratory viruses may have posed a greater threat to children’s health than SARS-CoV-2.

Relevance. Lyme disease (LD) remains an important public health problem, especially in Russia, where the incidence is consistently high. To date, there is still no available vaccine against LD, and prevention involves non-specific measures. Aim: to review the literature and summarise data on progress, approaches and strategies for LD vaccine development. Conclusions. The first LD vaccines were developed in the 1990s. An OspA-based vaccine (LYMErix) was commercially available in the early 2000s but not widely distributed. An important milestone in the development of LD vaccines was the shift from the development of monovalent vaccines based on a single type of outer surface protein to the development of multivalent combinations that provide protection against different Borrelia genospecies. A multivalent OspA-based vaccine (VLA15) is in phase III clinical trials and is likely to be the next LD vaccine available on the market. New genetic strategies for vaccine development, identification of new immunogens, and development of vaccines targeting different parts of the LD transmission cycle are of broad interest for further development of LD vaccines.

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 angstrom. 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.

Hemorrhagic fever with renal syndrome (HFRS) and tick-borne encephalitis (TBE) are the most common viral diseases in Russia. HFRS is caused by six different types of hantaviruses: Hantaan, Amur, Seoul, Puumala, Kurkino, and Sochi, which are transmitted to humans through small mammals of the Muridae and Cricetidae families. TBE is caused by viruses belonging to five different phylogenetic subtypes. The similarities in the ecology of HFRS and TBE pathogens is presented here. Hantavirus-infected small mammals can transmit the virus to uninfected animals, and ticks can also transmit hantavirus to other ticks and mammals. Hantavirus transmission from ticks to humans is possible only hypothetically based on indirect data. Over the past 23 years, 164,582 cases of HFRS (4.9 per 105 people) and 71,579 cases of TBE (2.5 per 105 people) were registered in Russia. The mortality rate was 0.4% (668 cases) in HFRS and 1.6% deaths (1136 cases) in TBE. There were 4030 HFRS (2.5%) and 9414 TBE (13%) cases in children under 14 years old. HFRS and TBE cases were registered in 42 out of 85 Russian regions; in 18—only HFRS, in 13—only TBE, and 12 had no reported cases. The prospects of applying a combined vaccine for HFRS and TBE prevention are shown in this paper.

X-ray imaging of virus particles at the European XFEL could eventually allow their complete structures to be solved, potentially approaching the resolution of other structural virology methods. To achieve this ambitious goal with today's technologies, about 1 ml of purified virus suspension containing at least 10 12 particles per millilitre is required. Such large amounts of concentrated suspension have never before been obtained for enveloped viruses. Tick-borne encephalitis virus (TBEV) represents an attractive model system for the development of enveloped virus purification and concentration protocols, given the availability of large amounts of inactivated virus material provided by vaccine-manufacturing facilities. Here, the development of a TBEV vaccine purification and concentration scheme is presented combined with a quality-control protocol that allows substantial amounts of highly concentrated non-aggregated suspension to be obtained. Preliminary single-particle imaging experiments were performed for this sample at the European XFEL, showing distinct diffraction patterns.

Evolutionary potential of viruses can result in outbreaks of well-known viruses and emergence of novel ones. Pharmacological methods of intervening the reproduction of various less popular, but not less important viruses are not available, as well as the spectrum of antiviral activity for most known compounds. In the framework of chemical biology paradigm, characterization of antiviral activity spectrum of new compounds allows to extend the antiviral chemical space and provides new important structure-activity relationships for data-driven drug discovery. Here we present a primary assessment of antiviral activity of spiro-annulated derivatives of seven-membered heterocycles, oxepane and azepane, in phenotypic assays against viruses with different genomes, virion structures, and genome realization schemes: orthoflavivirus (tick-borne encephalitis virus, TBEV), enteroviruses (poliovirus, enterovirus A71, echovirus 30), adenovirus (human adenovirus C5), hantavirus (Puumala virus). Hit compounds inhibited reproduction of adenovirus C5, the only DNA virus in the studied set, in the yield reduction assay, and did not inhibit reproduction of RNA viruses.

Relevance. Hemorrhagic fever with renal syndrome (HFRS) and tick-borne encephalitis (TBE) are the most common natural focal diseases of viral etiology in Russia. The medical and social significance of these two infections is determined by the extensive foci of their spread, high annual morbidity rates, and the presence of severe forms of the disease can lead to permanent disability and even death. Aim. To assess the current epidemiological situation of HFRS and TBE in the Russian Federation. Conclusions. Over 23 years (from 2000 to 2022), 164,582 cases of HFRS were identified in Russia with an average annual rate of 4.9 cases per 100 thousand population, as well as 71,579 cases of TBE with an average annual rate of 2.5 cases per 100 thousand population; 668 (0.4%) and 1136 (1.6%) deaths from HFRS and TBE, respectively; 4030 (2.5%) and 9414 (13%) children under the age of 14 years among patients with HFRS and TBE, respectively. The incidence of HFRS and TBE per 100 thousand population of Russia was higher among rural residents than among urban residents. Most cases of HFRS were registered in the autumn-winter period, and TBE – at the end of June – the first half of July. Of the 85 administrative regions of Russia, cases of HFRS and TBE are registered in 42, in 18 – only HFRS, in 13 – only TBE, and in 12 regions no clinically diagnosed cases of HFRS and TBE have been identified.