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.

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.

Genetic Characterization of Avian Influenza A(H5N6) Virus Clade 2.3.4.4, Russia, 2018.

Timely identification of pandemic influenza threats depends on monitoring for highly pathogenic avian influenza viruses. We isolated highly pathogenic avian influenza A(H5N6) virus clade 2.3.4.4, genotype G1.1, in samples from a bird in southwest Russia. The virus has high homology to human H5N6 influenza strains isolated from southeast China.

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.

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.

Evaluation of the Immunosafety of Cucurbit[n]uril In Vivo.

Cucurbiturils are a family of macrocyclic oligomers capable of forming host-guest complexes with various molecules. Due to noncovalent binding to drug molecules and low toxicity, cucurbiturils has been extensively investigated as potential carriers for drug delivery. However, the immune system's interactions with different drug carriers, including cucurbiturils, are still under investigation. In this study, we focused on cucurbiturils' immunosafety and immunomodulation properties in vivo. We measured blood counts and lymphocyte subpopulations in blood, spleen, and bone marrow, and assessed the in vivo toxicity to spleen and bone marrow cells after intraperitoneal administration to BALB/c mice. When assessing the effect of cucurbit[6]uril on blood parameters after three intraperitoneal injections within a week in laboratory animals, a decrease in white blood cells was found in mice after injections of cucurbit[6]util, but the observed decrease in the number of white blood cells was within the normal range. At the same time, cucurbit[7]uril and cucurbit[8]uril did not affect the leukocyte counts of mice after three injections. Changes in the number of platelets, erythrocytes, and monocytes, as well as in several other indicators, such as hematocrit or erythrocyte volumetric dispersion, were not detected. We show that cucurbiturils do not have immunotoxicity in vivo, with the exception of a cytotoxic effect on spleen cells after сucurbit[7]uril administration at a high dosage. We also evaluated the effect of cucurbiturils on cellular and humoral immune responses. We founded that cucurbiturils in high concentrations affect the immune system in vivo, and the action of various cucurbiturils differs in different homologues, which is apparently associated with different interactions in the internal environment of the body.

Fraction of plasma exomeres and low-density lipoprotein cholesterol as a predictor of fatal outcome of COVID-19.

Transcriptomic analysis conducted by us previously revealed upregulation of genes involved in low-density lipoprotein particle receptor (LDLR) activity pathway in lethal COVID-19 caused by SARS-CoV-2 virus (severe acute respiratory syndrome coronavirus 2). Last data suggested the possible role of extracellular vesicles in COVID-19 pathogenesis. The aim of the present study was to retrospectively evaluate parameters of cholesterol metabolism and newly identified EVs, exomeres, as possible predictors of fatal outcome of COVID-19 patients infected by the Alpha and the Delta variants of SARS-CoV-2 virus. Blood from 67 patients with severe COVID-19 were collected at the time of admission to the intensive care unit (ICU) and 7 days after admission to the ICU. After 30 days patients were divided into two subgroups according to outcome-34 non-survivors and 33 survivors. This study demonstrated that plasma low- and high-density lipoprotein cholesterol levels (LDL-C and HDL-C) were decreased in non-survivors compared to controls at the time of admission to the ICU. The conjoint fraction of exomeres and LDL particles measured by dynamic light scattering (DLS) was decreased in non-survivors infected by the Alpha and the Delta variants compared to survivors at the time of admission to the ICU. We first showed that reduction of exomeres fraction may be critical in fatal outcome of COVID-19.

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.

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.

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.

A Comprehensive Examination of Factors Impacting Collegiate Athletes' Utilization of Psychological Assessment and Intervention Services.

Although collegiate athletes underutilize mental health programming, investigators have rarely examined factors that may influence their participation in such programs. The current study examined how structured interviews and demographic factors influence collegiate athletes to use psychological programming. Two-hundred and eighty-nine collegiate athletes were referred to the study. They were screened for mental health and randomly assigned to one of two semi-structured interviews based on experimental phase. Participants in Phase I received standard engagement (SE; N = 35) or SE+discussion of mental health (DMH; N = 44). Phase II participants received SE+DMH (N = 82) or SE+DMH + discussion of personal ambitions (DPA; N = 66). Phase III participants received SE+DMH+discussion about their culture of choice (DCC) (N = 25) or SE+DMH+discussion of sport culture (DSC) (N = 37). After receiving the respective interview participants were offered psychological assessment and intervention. Chi squared analyses revealed class standing, mental health symptom severity, referral type, and type of engagement interview influenced program commitment/utilization. Logistic regression analyses indicated SE+DMH+DPA and SE+DMH+DSC uniquely improved assessment attendance whereas referrals from the athletic department and coaches/teammates, participation in sport performance workshops, and senior status uniquely improved assessment and intervention attendance.

Transcriptomic Profiles Reveal Downregulation of Low-Density Lipoprotein Particle Receptor Pathway Activity in Patients Surviving Severe COVID-19.

To assess the biology of the lethal endpoint in patients with SARS-CoV-2 infection, we compared the transcriptional response to the virus in patients who survived or died during severe COVID-19. We applied gene expression profiling to generate transcriptional signatures for peripheral blood mononuclear cells (PBMCs) from patients with SARS-CoV-2 infection at the time when they were placed in the Intensive Care Unit of the Pavlov First State Medical University of St. Petersburg (Russia). Three different bioinformatics approaches to RNA-seq analysis identified a downregulation of three common pathways in survivors compared with nonsurvivors among patients with severe COVID-19, namely, low-density lipoprotein (LDL) particle receptor activity (GO:0005041), important for maintaining cholesterol homeostasis, leukocyte differentiation (GO:0002521), and cargo receptor activity (GO:0038024). Specifically, PBMCs from surviving patients were characterized by reduced expression of PPARG, CD36, STAB1, ITGAV, and ANXA2. Taken together, our findings suggest that LDL particle receptor pathway activity in patients with COVID-19 infection is associated with poor disease prognosis.

Evaluation of HA-D222G/N polymorphism using targeted NGS analysis in A(H1N1)pdm09 influenza virus in Russia in 2018-2019.

Outbreaks of influenza, which is a contagious respiratory disease, occur throughout the world annually, affecting millions of people with many fatal cases. The D222G/N mutations in the hemagglutinin (HA) gene of A(H1N1)pdm09 are associated with severe and fatal human influenza cases. These mutations lead to increased virus replication in the lower respiratory tract (LRT) and may result in life-threatening pneumonia. Targeted NGS analysis revealed the presence of mutations in major and minor variants in 57% of fatal cases, with the proportion of viral variants with mutations varying from 1% to 98% in each individual sample in the epidemic season 2018-2019 in Russia. Co-occurrence of the mutations D222G and D222N was detected in a substantial number of the studied fatal cases (41%). The D222G/N mutations were detected at a low frequency (less than 1%) in the rest of the studied samples from fatal and nonfatal cases of influenza. The presence of HA D222Y/V/A mutations was detected in a few fatal cases. The high rate of occurrence of HA D222G/N mutations in A(H1N1)pdm09 viruses, their increased ability to replicate in the LRT and their association with fatal outcomes points to the importance of monitoring the mutations in circulating A(H1N1)pdm09 viruses for the evaluation of their epidemiological significance and for the consideration of disease prevention and treatment options.

Detection of Influenza Virus Using a SOI-Nanoribbon Chip, Based on an N-Type Field-Effect Transistor.

The detection of influenza A virions with a nanoribbon detector (NR detector) has been demonstrated. Chips for the detector have been fabricated based on silicon-on-insulator nanoribbon structures (SOI nanoribbon chip), using a complementary metal-oxide-semiconductor (CMOS)-compatible technology-by means of gas-phase etching and standard optical photolithography. The surface of the SOI nanoribbon chip contains a matrix of 10 nanoribbon (NR) sensor elements. SOI nanoribbon chips of n-type conductance have been used for this study. For biospecific detection of target particles, antibodies against influenza virus have been covalently immobilized onto NRs. Influenza A virus detection was performed by real-time registration of the source-drain current through the NRs. The detection of the target viral particles was carried out in buffer solutions at the target particles concentration within the range from 107 to 103 viral particles per milliliter (VP/mL). The lowest detectable concentration of the target viral particles was 6 × 10-16 M (corresponding to 104 VP/mL). The use of solutions containing ~109 to 1010 VP/mL resulted in saturation of the sensor surface with the target virions. In the saturation mode, detection was impossible.

The Tissue Distribution of SARS-CoV-2 in Transgenic Mice With Inducible Ubiquitous Expression of hACE2.

The novel coronavirus disease COVID-19 has become one of the most socially significant infections. One of the main models for COVID-19 pathogenesis study and anti-COVID-19 drug development is laboratory animals sensitive to the virus. Herein, we report SARS-CoV-2 infection in novel transgenic mice conditionally expressing human ACE2 (hACE2), with a focus on viral distribution after intranasal inoculation. Transgenic mice carrying hACE2 under the floxed STOP cassette [(hACE2-LoxP(STOP)] were mated with two types of Cre-ERT2 strains (UBC-Cre and Rosa-Cre). The resulting offspring with temporal control of transgene expression were treated with tamoxifen to induce the removal of the floxed STOP cassette, which prevented hACE2 expression. Before and after intranasal inoculation, the mice were weighed and clinically examined. On Days 5 and 10, the mice were sacrificed for isolation of internal organs and the further assessment of SARS-CoV-2 distribution. Intranasal SARS-CoV-2 inoculation in hACE2-LoxP(STOP)×UBC-Cre offspring resulted in weight loss and death in 6 out of 8 mice. Immunostaining and focus formation assays revealed the most significant viral load in the lung, brain, heart and intestine samples. In contrast, hACE2-LoxP(STOP) × Rosa-Cre offspring easily tolerated the infection, and SARS-CoV-2 was detected only in the brain and lungs, whereas other studied tissues had null or negligible levels of the virus. Histological examination revealed severe alterations in the lungs, and mild changes were observed in the brain tissues. Notably, no changes were observed in mice without tamoxifen treatment. Thus, this novel murine model with the Cre-dependent activation of hACE2 provides a useful and safe tool for COVID-19 studies.

Deletion of BST2 Cytoplasmic and Transmembrane N-Terminal Domains Results in SARS-CoV, SARS-CoV-2, and Influenza Virus Production Suppression in a Vero Cell Line.

SARS-CoV-2, which emerged in Wuhan (China), has become a great worldwide problem in 2020 and has led to more than 1,000,000 deaths worldwide. Many laboratories are searching for ways to fight this pandemic. We studied the action of the cellular antiviral protein tetherin, which is encoded by the BST2 gene. We deleted the transmembrane domain-encoding part of the gene in the Vero cell line. The transmembrane domain is a target for virus-antagonizing proteins. We showed a decrease in SARS-CoV-2 in cells with deleted transmembrane BST2 domains compared to the initial Vero cell line. Similar results were obtained for SARS-CoV and avian influenza virus. This finding may help the development of antiviral therapies competitively targeting the transmembrane domain of tetherin with viral-antagonizing proteins.

Severe cases of seasonal influenza in Russia in 2017-2018.

The 2017-2018 influenza epidemic season in Russia was characterized by a relatively low morbidity and mortality. We evaluated herd immunity prior to the 2017-2018 influenza season in hemagglutination inhibition assay, and performed characterization of influenza viruses isolated from severe or fatal influenza cases and from influenza cases in people vaccinated in the fall of 2017. During the 2017-2018 epidemic season, 87 influenza A and B viruses were isolated and viruses of the 75 influenza cases, including selected viral isolates and viruses analyzed directly from the original clinical material, were genetically characterized. The analyzed A(H1N1)pdm09 viruses belonged to clade 6B.1, B/Yamagata-like viruses belonged to clade 3, and B/Victoria-like viruses belonged to clade 1A and they were antigenically similar to the corresponding vaccine strains. A(H3N2) viruses belonged to clade 3C.2a and were difficult to characterize antigenically and the analysis indicated antigenic differences from the corresponding egg-grown vaccine strain. The next generation sequencing revealed the presence of D222/G/N polymorphism in the hemagglutinin gene in 32% of the analyzed A(H1N1)pdm09 lethal cases. This study demonstrated the importance of monitoring D222G/N polymorphism, including detection of minor viral variants with the mutations, in the hemagglutinin gene of A(H1N1)pdm09 for epidemiological surveillance. One strain of influenza virus A(H1N1)pdm09 was resistant to oseltamivir and had the H275Y amino acid substitution in the NA protein. All other isolates were susceptible to NA inhibitors. Prior to the 2017-2018 epidemic season, 67.4 million people were vaccinated, which accounted for 46.6% of the country's population. Just before the epidemic season 33-47% and 24-30% of blood sera samples collected within the territory of Russia showed the presence of protective antibody titers against vaccine strains of influenza A and influenza B/Victoria-like, respectively. Mass vaccination of the population had evidently reduced the severity of the flu epidemic during the 2017-2018 influenza epidemic season in Russia.

Correction: Severe cases of seasonal influenza in Russia in 2017-2018.

[This corrects the article DOI: 10.1371/journal.pone.0220401.].