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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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

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

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.

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.

Species-specific differentiation of variola, monkeypox, and varicella-zoster viruses by multiplex real-time PCR assay.

A method of one-stage rapid detection and differentiation of epidemiologically important variola virus (VARV), monkeypox virus (MPXV), and varicella-zoster virus (VZV) utilizing multiplex real-time TaqMan PCR assay was developed. Four hybridization probes with various fluorescent dyes and the corresponding fluorescence quenchers were simultaneously used for the assay. The hybridization probes specific for the VARV sequence contained FAM/BHQ1 as a dye/quencher pair; MPXV-specific, JOE/BHQ1; VZV-specific, TAMRA/BHQ2; and internal control-specific, Cy5/BHQ3. The specificity and sensitivity of the developed method were assessed by analyzing DNA of 32 strains belonging to orthopoxvirus and herpesvirus species.

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.

Real-time PCR assay for specific detection of cowpox virus.

The species cowpox virus (CPXV), genus Orthopoxvirus (OPV), consists of isolates highly variable in their biological properties and their genotypes. A TaqMan PCR assay for the specific detection of CPXV DNA based on sequences of the ORF D11L has been developed recently. (Gavrilova et al., 2010; Shchelkunov et al., 2011); however, a rather limited panel of CPXV stains has been used. When a much larger panel of 47 CPXV DNAs has been tested, three strains could not be amplified at all because of large deletions in their respective ORF D11L. In addition, a deletion of 23bp led to low-efficiency detection of five other CPXV strains. To solve this problem a new primer/probe combinations was selected based on sequences of ORF D8L, and a new real-time PCR method for (i) a genus-specific detection of OPVs and (ii) a simultaneous CPXV-specific differentiation is described in this study. The specificity and sensitivity were assessed by analyzing DNA of 67 strains belonging to human-pathogenic OPV species, including variola virus, as well as specimens of CPXV-infected mice.

Species-specific identification of variola, monkeypox, cowpox, and vaccinia viruses by multiplex real-time PCR assay.

A method of one-stage rapid identification of variola (VARV), monkeypox (MPXV), cowpox (CPXV), and vaccinia (VACV) viruses, pathogenic for humans, utilizing multiplex real-time TaqMan PCR (MuRT-PCR) assay was developed. Four pairs of oligonucleotide primers and four hybridization probes with various fluorescent dyes and the corresponding fluorescence quenchers were concurrently used for MuRT-PCR assay. The hybridization probe specific for the VARV sequence contained FAM/BHQ1 as a dye/quencher pair; MPXV-specific, TAMRA/BHQ2; CPXV-specific, JOE/BHQ1; VACV-specific, Cy5/BHQ3. The specificity and sensitivity of the developed method were assessed by analyzing DNA of 29 strains belonging to six orthopoxvirus species as well as the DNA samples isolated from archive clinical specimens of human smallpox cases and experimental specimens isolated from CPXV-infected mice and MPXV-infected marmot.

Development of real-time PCR assay for specific detection of cowpox virus.

BACKGROUND: The number of recorded human cowpox cases are recently increasing. The symptoms caused by cowpox virus (CPXV) in a number of human cases are close to the symptoms characteristic of the orthopoxviral human infections caused by monkeypox or smallpox (variola) viruses. Any rapid and reliable real-time PCR method for distinguishing cowpox from smallpox and monkeypox is yet absent. OBJECTIVES: The aim of this study was to develop a quick and reliable real-time TaqMan PCR assay for specific detection of cowpox virus and to determine the sensitivity and specificity of this method. STUDY DESIGN: Based on aligned nucleotide sequences of orthopoxviruses, we found a virus-specific region in the CPXV genome and selected the oligonucleotide primers and hybridization probe within this region. The specificity of the developed method was tested using a panel of various orthopoxvirus (OPV) DNAs. The sensitivity was determined using the recombinant plasmid carrying a fragment of CPXV DNA and genomic DNA of the CPXV strain GRI-90. RESULTS: The analytical specificity of this method was determined using DNAs of 17 strains of four OPV species pathogenic for humans and amounted to 100%. The method allows 6 copies of plasmid DNA and 20 copies of CPXV DNA in the reaction mixture to be detected. CONCLUSION: A quick and reliable TaqMan PCR assay providing for a highly sensitive and specific detection of CPXV DNA was developed.