Synthesis and In Vitro Study of Antiviral Activity of Glycyrrhizin Nicotinate Derivatives against HIV-1 Pseudoviruses and SARS-CoV-2 Viruses.

When developing drugs against SARS-CoV-2, it is important to consider the characteristics of patients with different co-morbidities. People infected with HIV-1 are a particularly vulnerable group, as they may be at a higher risk than the general population of contracting COVID-19 with clinical complications. For such patients, drugs with a broad spectrum of antiviral activity are of paramount importance. Glycyrrhizinic acid (Glyc) and its derivatives are promising biologically active compounds for the development of such broad-spectrum antiviral agents. In this work, derivatives of Glyc obtained by acylation with nicotinic acid were investigated. The resulting preparation, Glycyvir, is a multi-component mixture containing mainly mono-, di-, tri- and tetranicotinates. The composition of Glycyvir was characterized by HPLC-MS/MS and its toxicity assessed in cell culture. Antiviral activity against three strains of SARS-CoV-2 was tested in vitro on Vero E6 cells by MTT assay. Glycyvir was shown to inhibit SARS-CoV-2 replication in vitro (IC502-8 µM) with an antiviral activity comparable to the control drug Remdesivir. In addition, Glycyvir exhibited marked inhibitory activity against HIV pseudoviruses of subtypes B, A6 and the recombinant form CRF63_02A (IC50 range 3.9-27.5 µM). The time-dependence of Glycyvir inhibitory activity on HIV pseudovirus infection of TZM-bl cells suggested that the compound interfered with virus entry into the target cell. Glycyvir is a promising candidate as an agent with low toxicity and a broad spectrum of antiviral action.

New class of hantaan virus inhibitors based on conjugation of the isoindole fragment to (+)-camphor or (-)-fenchone hydrazonesv.

This work presents the design and synthesis of camphor, fenchone, and norcamphor N-acylhydrazone derivatives as a new class of inhibitors of the Hantaan virus, which causes haemorrhagic fever with renal syndrome (HFRS). A cytopathic model was developed for testing chemotherapeutics against the Hantaan virus, strain 76-118. In addition, a study of the antiviral activity was carried out using a pseudoviral system. It was found that the hit compound possesses significant activity (IC50 = 7.6 ± 2 µM) along with low toxicity (CC50 > 1000 µM). Using molecular docking procedures, the binding with Hantavirus nucleoprotein was evaluated and the correlation between the structure of the synthesised compounds and the antiviral activity was established.

Recombinant SARS-CoV-2 S Protein Binds to Glycans of the Lactosamine Family in vitro.

Many viruses, beside binding to their main cell target, interact with other molecules that promote virus adhesion to the cell; often, these additional targets are glycans. The main receptor for SARS-CoV-2 is a peptide motif in the ACE2 protein. We studied interaction of the recombinant SARS-CoV-2 spike (S) protein with an array of glycoconjugates, including various sialylated, sulfated, and other glycans, and found that the S protein binds some (but not all) glycans of the lactosamine family. We suggest that parallel influenza infection will promote SARS-CoV-2 adhesion to the respiratory epithelial cells due to the unmasking of lactosamine chains by the influenza virus neuraminidase.

Isolation of clade 2.3.4.4b A(H5N8), a highly pathogenic avian influenza virus, from a worker during an outbreak on a poultry farm, Russia, December 2020.

This study presents the isolation of influenza A(H5N8) virus clade 2.3.4.4b from a poultry worker during an outbreak of highly pathogenic avian influenza A(H5N8) among chickens at a poultry farm in Astrakhan, Russia in December 2020. Nasopharyngeal swabs collected from seven poultry workers were positive for influenza A(H5N8), as confirmed by RT-PCR and sequencing. The influenza A(H5N8) virus was isolated from one of the human specimens and characterised. Sporadic human influenza A(H5)2.3.4.4. infections represent a possible concern for public health.

Synthesis and Antiviral Activity of Camphene Derivatives against Different Types of Viruses.

To date, the 'one bug-one drug' approach to antiviral drug development cannot effectively respond to the constant threat posed by an increasing diversity of viruses causing outbreaks of viral infections that turn out to be pathogenic for humans. Evidently, there is an urgent need for new strategies to develop efficient antiviral agents with broad-spectrum activities. In this paper, we identified camphene derivatives that showed broad antiviral activities in vitro against a panel of enveloped pathogenic viruses, including influenza virus A/PR/8/34 (H1N1), Ebola virus (EBOV), and the Hantaan virus. The lead-compound 2a, with pyrrolidine cycle in its structure, displayed antiviral activity against influenza virus (IC50 = 45.3 µM), Ebola pseudotype viruses (IC50 = 0.12 µM), and authentic EBOV (IC50 = 18.3 µM), as well as against pseudoviruses with Hantaan virus Gn-Gc glycoprotein (IC50 = 9.1 µM). The results of antiviral activity studies using pseudotype viruses and molecular modeling suggest that surface proteins of the viruses required for the fusion process between viral and cellular membranes are the likely target of compound 2a. The key structural fragments responsible for efficient binding are the bicyclic natural framework and the nitrogen atom. These data encourage us to conduct further investigations using bicyclic monoterpenoids as a scaffold for the rational design of membrane-fusion targeting inhibitors.

Severe cases of seasonal influenza and detection of seasonal A(H1N2) in Russia in 2018-2019.

Data obtained from monitoring cases of severe influenza, cases of vaccinated individuals, and unique cases were used to describe influenza viruses that circulated in Russia in the 2018-2019 epidemic season. A high proportion of the mutations D222G/N in A(H1N1)pdm09 HA was detected in fatal cases. Viruses of the B/Victoria lineage with deletions in HA were detected in Russia, and a reassortant seasonal influenza A(H1N2) virus was identified. A C-terminal truncation in the NS1 protein was detected in a substantial proportion of A(H3N2) viruses.

First cases of coronavirus disease 2019 (COVID-19) in the WHO European Region, 24 January to 21 February 2020.

In the WHO European Region, COVID-19 surveillance was implemented 27 January 2020. We detail the first European cases. As at 21 February, nine European countries reported 47 cases. Among 38 cases studied, 21 were linked to two clusters in Germany and France, 14 were infected in China. Median case age was 42 years; 25 were male. Late detection of the clusters' index cases delayed isolation of further local cases. As at 5 March, there were 4,250 cases.

Transfection of bone marrow derived cells with immunoregulatory proteins.

In vitro electroporation gene transfer was first performed in 1982. Today, this technology has become one of the major vehicles for non-viral transfection of cells. All non-viral transfections, such as calcium phosphate precipitation, lipofection, and magnetic transfection, have been shown to achieve a transfection efficiency of up to 70% in commonly used cell lines, but not in primary cells. Here we describe the use of electroporation to transfect primary mouse bone marrow-derived cells, such as macrophages (Mφ) and dendritic cells (DCs) with high efficiencies (45%-72%) and minimal cell death. The transfection efficiencies and cell death varied depending on the culture duration of the DCs and Mφ. Moreover, the electroporation efficiency was increased when conditioning medium was used for culturing the cells. Furthermore, we demonstrated that measuring the plasmid-encoded secreted proteins is a highly sensitive method for determining the transfection efficiency. In summary, electroporation with plasmid vectors is an efficient method for producing DCs and Mφ with transient expression of immunoregulatory proteins.

Humoral immunity to influenza in an at-risk population and severe influenza cases in Russia in 2016-2017.

This work aimed to analyze the herd immunity to influenza among a Russian population living in regions with an increased risk of emergence of viruses with pandemic potential, and to isolate and investigate virus strains from severe influenza cases, including fatal cases, during the 2016-2017 epidemic season. In November 2016 - March 2017 highly pathogenic influenza outbreaks were registered in Russia among wild birds and poultry. No cases of human infection were registered. Analysis of 760 sera from people who had contact with infected or perished birds revealed the presence of antibodies to A(H5N1) virus of clade 2.3.2.1c and A(H5N8) virus of clade 2.3.4.4. The 2016-2017 influenza epidemic season in Russia began in weeks 46-47 of 2016 with predominant circulation of influenza A(H3N2) viruses. Strains isolated from severe influenza cases mainly belonged to 3C.2a.2 and 3C.2a.3 genetic groups. Up to the 8th week of 2017 severe influenza cases were often caused by influenza B viruses which belonged to 1A genetic group with antigenic properties similar to B/Brisbane/60/2008. All influenza A and B virus strains isolated in the 2016-2017 epidemic season were sensitive to oseltamivir and zanamivir.

Genotyping of hepatitis B and C virus Russian isolates for reference serum panel construction.

Approximately 2% and 5% of the world human population is estimated to be infected with HCV and HBV, respectively. Reference panels of HCV and HBV serum samples with defined genotypes and serotypes is necessary for monitoring of the specificity and sensitivity of diagnostic test kits. The aim of this study was to determine genotypes/serotypes of HBV and HCV circulating in Russia in order to construct a panel of reference sera containing these HCV genotypes and HBV serotypes. A total of 343 HBsAg-positive and 207 anti-HCV positive serum samples were collected from patients with HBV and HCV infection from different cities between years 2002 and 2010 in St. Petersburg, Krasnodar, Nizhny Novgorod, Novosibirsk, Barnaul, Gorno-Altaisk, and Khabarovsk. HBV DNA was found in 76.4% of HBsAg positive samples by PCR for the S gene and HCV RNA was found in 71.5, 70.0, and 64.7% of anti-HCV positive samples in the 5'UTR, Core, and NS5B regions, respectively. The prevalence and proportion of HBV genotype/serotype associations were as follows: A/adw2, 2.1%; D/ayw2, 54.0%; D/ayw3, 43.1%; D/adw2, 0.7%. A new combination of genotype D and adw2 serotype was discovered. The distribution of HCV genotypes was the following: 43.6%, b; 3.8%, 2a; and 52.6%, 3a. Russian National reference panels of HBV and HCV lyophilized sera were developed to monitor specificity and sensitivity of approved kits and for the certification of newly developed assays.

Human Adenovirus and Influenza A Virus Exacerbate SARS-CoV-2 Infection in Animal Models.

In this study, we investigated the features of the infectious process by simulating co-infection with SARS-CoV-2 and human adenovirus type 5 (HAdV-5) or influenza A virus (IAV) in vitro and in vivo. The determination of infectious activity of viruses and digital PCR demonstrated that during simultaneous and sequential HAdV-5 followed by SARS-CoV-2 infection in vitro and in vivo, the HAdV-5 infection does not interfere with replication of SARS-CoV-2. The hamsters co-infected and mono-infected with SARS-CoV-2 exhibited nearly identical viral titers and viral loads of SARS-CoV-2 in the lungs. The hamsters and ferrets co-infected by SARS-CoV-2- and IAV demonstrated more pronounced clinical manifestations than mono-infected animals. Additionally, the lung histological data illustrate that HAdV-5 or IAV and SARS-CoV-2 co-infection induces more severe pathological changes in the lungs than mono-infection. The expression of several genes specific to interferon and cytokine signaling pathways in the lungs of co-infected hamsters was more upregulated compared to single infected with SARS-CoV-2 animals. Thus, co-infection with HAdV-5 or IAV and SARS-CoV-2 leads to more severe pulmonary disease in animals.

Safety and Pharmacokinetics of the Substance of the Anti-Smallpox Drug NIOCH-14 after Oral Administration to Laboratory Animals.

BACKGROUND: Since most of the modern human population has no anti-smallpox immunity, it is extremely important to develop and implement effective drugs for the treatment of smallpox and other orthopoxvirus infections. The objective of this study is to determine the main characteristics of the chemical substance NIOCH-14 and its safety and bioavailability in the body of laboratory animals. METHODS: The safety of NIOCH-14 upon single- or multiple-dose intragastric administration was assessed according to its effect on the main hematological and pathomorphological parameters of laboratory mice and rats. In order to evaluate the pharmacokinetic parameters of NIOCH-14 administered orally, a concentration of ST-246, the active metabolite of NIOCH-14, in mouse blood and organs was determined by tandem mass spectrometry and liquid chromatography. RESULTS: The intragastric administration of NIOCH-14 at a dose of 5 g/kg body weight caused neither death nor signs of intoxication in mice. The intragastric administration of NIOCH-14 to mice and rats at doses of 50 and 150 µg/g body weight either as a single dose or once daily during 30 days did not cause animal death or critical changes in hematological parameters and the microstructure of internal organs. The tissue availability of NIOCH-14 administered orally to the mice at a dose of 50 µg/g body weight, which was calculated according to concentrations of its active metabolite ST-246 for the lungs, liver, kidney, brain, and spleen, was 100, 69.6, 63.3, 26.8 and 20.3%, respectively. The absolute bioavailability of the NIOCH-14 administered orally to mice at a dose of 50 µg/g body weight was 22.8%. CONCLUSION: Along with the previously determined efficacy against orthopoxviruses, including the smallpox virus, the substance NIOCH-14 was shown to be safe and bioavailable in laboratory animal experiments.

Epidemiology of Zoonotic Coxiella burnetii in The Republic of Guinea.

BACKGROUND: Q fever is a zoonotic infectious disease characterized by fever, malaise, chills, significant weakness, and muscle pain. In some cases, the disease can become chronic and affect the inner membranes of the heart, such as the valves, leading to endocarditis and a high risk of death. Coxiella burnetii (C. burnetii) is the primary causative agent of Q fever in humans. This study aims to monitor the presence of C. burnetii in ticks collected from small mammals and cattle in the Republic of Guinea (RG). METHODS: Rodents were trapped in the Kindia region of RG during 2019-2020, and ticks were collected from cattle in six regions of RG. Total DNA was extracted using a commercial kit (RIBO-prep, InterLabService, Russia) following the manufacturer's instructions. Real-time PCR amplification was conducted using the kit (AmpliSens Coxiella burnetii-FL, InterLabService, Russia) to detect C. burnetii DNA. RESULTS AND CONCLUSIONS: Bacterial DNA was detected in 11 out of 750 (1.4%) small mammals and 695 out of 9620 (7.2%) tick samples. The high number of infected ticks (7.2%) suggests that they are the main transmitters of C. burnetii in RG. The DNA was detected in the liver and spleen of a Guinea multimammate mouse, Mastomys erythroleucus. These findings demonstrate that C. burnetii is zoonotic in RG, and measures should be taken to monitor the bacteria's dynamics and tick prevalence in the rodent population.

Assessment of Safety and Prophylactic Efficacy of the EpiVacCorona Peptide Vaccine for COVID-19 Prevention (Phase III).

The State Research Center of Virology and Biotechnology "VECTOR" of the Federal Service for the Oversight of Consumer Protection and Welfare (Rospotrebnadzor) has developed the peptide-based EpiVacCorona vaccine, which is the first synthetic peptide-based antiviral vaccine for mass immunization in international vaccinology. An early clinical trial (Phase I-II) demonstrated that the EpiVacCorona vaccine is a safe product. The "Multicenter double-blind, placebo-controlled, comparative, randomized trial to assess the tolerability, safety, immunogenicity and prophylactic efficacy of the EpiVacCorona COVID-19 vaccine based on peptide antigens in 3000 volunteers aged 18 years and older" was performed regarding vaccine safety. The key objectives of the study were to evaluate the safety and prophylactic efficacy of the two-dose EpiVacCorona vaccine administered via the intramuscular route. The results of the clinical study (Phase III) demonstrated the safety of the EpiVacCorona vaccine. Vaccine administration was accompanied by mild local reactions in ≤27% of cases and mild systemic reactions in ≤14% of cases. The prophylactic efficacy of the EpiVacCorona COVID-19 vaccine after the completion of the vaccination series was 82.5% (CI95 = 75.3-87.6%). The high safety and efficacy of the vaccine give grounds for recommending this vaccine for regular seasonal prevention of COVID-19 as a safe and effective medicinal product.

Simulation of Molecular Dynamics of SARS-CoV-2 S-Protein in the Presence of Multiple Arbidol Molecules: Interactions and Binding Mode Insights.

In this work, we evaluated the antiviral activity of Arbidol (Umifenovir) against SARS-CoV-2 using a pseudoviral system with the glycoprotein S of the SARS-CoV-2 virus on its surface. In order to search for binding sites to protein S of the virus, we described alternative binding sites of Arbidol in RBD and in the ACE-2-RBD complex. As a result of our molecular dynamics simulations combined with molecular docking data, we note the following fact: wherever the molecules of Arbidol bind, the interaction of the latter affects the structural flexibility of the protein. This interaction may result both in a change in the shape of the domain-enzyme binding interface and simply in a change in the structural flexibility of the domain, which can subsequently affect its affinity to the enzyme. In addition, we examined the possibility of Arbidol binding in the stem part of the surface protein. The possibility of Arbidol binding in different parts of the protein is not excluded. This may explain the antiviral activity of Arbidol. Our results could be useful for researchers searching for effective SARS-CoV-2 virus inhibitors targeting the viral entry stage.

(+)-Usnic Acid and Its Derivatives as Inhibitors of a Wide Spectrum of SARS-CoV-2 Viruses.

In order to test the antiviral activity, a series of usnic acid derivatives were synthesized, including new, previously undescribed compounds. The activity of the derivatives against three strains of SARS-CoV-2 virus was studied. To understand the mechanism of antiviral action, the inhibitory activity of the main protease of SARS-CoV-2 virus was studied using the developed model as well as the antiviral activity against the pseudoviral system with glycoprotein S of SARS-CoV-2 virus on its surface. It was shown that usnic acid exhibits activity against three strains of SARS-CoV-2 virus: Wuhan, Delta, and Omicron. Compounds 10 and 13 also showed high activity against the three strains. The performed biological studies and molecular modeling allowed us to assume that the derivatives of usnic acid bind in the N-terminal domain of the surface glycoprotein S at the binding site of the hemoglobin decay metabolite.

Borneol Ester Derivatives as Entry Inhibitors of a Wide Spectrum of SARS-CoV-2 Viruses.

In the present work we studied the antiviral activity of the home library of monoterpenoid derivatives using the pseudoviral systems of our development, which have glycoproteins of the SARS-CoV-2 virus strains Wuhan and Delta on their surface. We found that borneol derivatives with a tertiary nitrogen atom can exhibit activity at the early stages of viral replication. In order to search for potential binding sites of ligands with glycoprotein, we carried out additional biological tests to study the inhibition of the re-receptor-binding domain of protein S. For the compounds that showed activity on the pseudoviral system, a study using three strains of the infectious SARS-CoV-2 virus was carried out. As a result, two leader compounds were found that showed activity on the Wuhan, Delta, and Omicron strains. Based on the biological results, we searched for the potential binding site of the leader compounds using molecular dynamics and molecular docking methods. We suggested that the compounds can bind in conserved regions of the central helices and/or heptad repeats of glycoprotein S of SARS-CoV-2 viruses.

Structure-Based Design, Synthesis, and Biological Evaluation of the Cage-Amide Derived Orthopox Virus Replication Inhibitors.

Despite the fact that the variola virus is considered eradicated, the search for new small molecules with activity against orthopoxviruses remains an important task, especially in the context of recent outbreaks of monkeypox. As a result of this work, a number of amides of benzoic acids containing an adamantane fragment were obtained. Most of the compounds demonstrated activity against vaccinia virus, with a selectivity index SI = 18,214 for the leader compound 18a. The obtained derivatives also demonstrated activity against murine pox (250 ≤ SI ≤ 6071) and cowpox (125 ≤ SI ≤ 3036). A correlation was obtained between the IC50 meanings and the binding energy to the assumed biological target, the p37 viral protein with R2 = 0.60.

Re-Emergence of Circulation of Seasonal Influenza during COVID-19 Pandemic in Russia and Receptor Specificity of New and Dominant Clade 3C.2a1b.2a.2 A(H3N2) Viruses in 2021-2022.

The circulation of seasonal influenza in 2020-2021 around the world was drastically reduced after the start of the COVID-19 pandemic and the implementation of mitigation strategies. The influenza virus circulation reemerged in 2021-2022 with the global spread of the new genetic clade 3C.2a1b.2a.2 of A(H3N2) viruses. The purpose of this study was to characterize influenza viruses in the 2021-2022 season in Russia and to analyze the receptor specificity properties of the 3C.2a1b.2a.2 A(H3N2) viruses. Clinical influenza samples were collected at the local Sanitary-and-Epidemiological Centers of Rospotrebnadzor. Whole genome sequencing was performed using NGS. The receptor specificity of hemagglutinin was evaluated using molecular modeling and bio-layer interferometry. Clinical samples from 854 cases of influenza A and B were studied; A(H3N2) viruses were in the majority of the samples. All genetically studied A(H3N2) viruses belonged to the new genetic clade 3C.2a1b.2a.2. Molecular modeling analysis suggested a higher affinity of hemagglutinin of 3C.2a1b.2a.2. A(H3N2) viruses to the α2,6 human receptor. In vitro analysis using a trisaccharide 6'-Sialyl-N-acetyllactosamine receptor analog did not resolve the differences in the receptor specificity of 3C.2a1b.2a.2 clade viruses from viruses belonging to the 3C.2a1b.2a.1 clade. Further investigation of the A(H3N2) viruses is required for the evaluation of their possible adaptive advantages. Constant monitoring and characterization of influenza are critical for epidemiological analysis.

Engineering of Recombinant Sheep Pox Viruses Expressing Foreign Antigens.

Capripoxviruses with a host range limited to ruminants have the great potential to be used as vaccine vectors. The aim of this work was to evaluate attenuated sheep pox virus (SPPV) vaccine strain NISKHI as a vector expressing several genes. Open reading frames SPPV020 (ribonucleotide kinase) and SPPV066 (thymidine kinase) were selected as sites for the insertion of foreign genes. Two integration plasmids with expression cassette were designed and constructed. Recombinant SPPVs expressing an enhanced green fluorescent protein (EGFP) (rSPPV(RRΔ)EGFP and rSPPV(TKΔ)EGFP), Foot-and-mouth disease virus capsid protein (VP1), and Brucella spp. outer membrane protein 25 (OMP25) (rSPPV(RRΔ)VP1A-(TKΔ)OMP25) were generated under the transient dominant selection method. The insertion of foreign genes into the SPPV020 and SPPV066 open reading frames did not influence the replication of the recombinant viruses in the cells. Successful foreign gene expression in vitro was assessed by luminescent microscopy (EGFP) and Western blot (VP1 and OMP25). Our results have shown that foreign genes were expressed by rSPPV both in permissive (lamb testicles) and non-permissive (bovine kidney, saiga kidney, porcine kidney) cells. Mice immunized with rSPPV(RRΔ)VP1A-(TKΔ)OMP25 elicited specific antibodies to both SPPV and foreign genes VP1 and OMP25. Thus, SPPV NISKHI may be used as a potential safe immunogenic viral vector for the development of polyvalent vaccines.