Detection of SARS-CoV-2 RNA in nasopharyngeal swabs from COVID-19 patients and asymptomatic cases of infection by real-time and digital PCR.

In this work we tested two reagent kits developed by us for detecting SARS-CoV-2 RNA using a fragment of the ORF1ab gene in digital PCR and real-time PCR formats. Data were obtained on the detection of SARS-CoV-2 virus RNA in nasopharyngeal swabs of patients with COVID-19 and asymptomatic carriers. The developed reagent kits provided 100% sensitivity and a detection limit of 103 GE / ml for qPCR, and at least 200 copies / ml of viral RNA when performing digital PCR. These methods were tested using a panel of 1,328 samples collected from patients with suspected COVID-19 at the beginning of 2020 in the Russian Federation. It has been shown that dPCR is more sensitive and can be used to analyze samples with low viral load, including those from patients without clinical symptoms. dPCR significantly improves the accuracy of laboratory research and significantly reduces the number of false negative results in the diagnosis of SARS-CoV-2. Determination of the concentration of SARS-CoV-2 RNA in patients with different clinical course of the disease showed that the concentration of viral RNA can sharply decrease in the first days of the disease. A low concentration of viral RNA in samples from patients is also characteristic of asymptomatic disease. Digital PCR provides a higher detection rate for asymptomatic cases, which is approximately 75% of those infected, as opposed to 45% for real-time PCR. The results obtained on the use of the digital PCR method for detecting SARS-CoV-2 RNA showed that this method is especially suitable for detecting RNA in case of its low concentration in contacts, as well as for monitoring changes in viral load in convalescent patients.

Cowpox in a human, Russia, 2015.

We investigated the first laboratory-confirmed human case of cowpox virus infection in Russia since 1991. Phylogenetic studies of haemagglutinin, TNF-α receptor-like protein and thymidine kinase regions showed significant differences with known orthopoxviruses, including unique amino-acid substitutions and deletions. The described cowpox virus strain, taking into account differences, is genetically closely related to strains isolated years ago in the same geographical region (European part of Russia and Finland), which suggests circulation of viral strains with common origin in wild rodents without spread over long distances and appearance in other parts of the world.

DEVELOPMENT OF THE METHOD FOR IDENTIFICATION AND GENOTYPING OF THE BACTERIA PASTEURELLA MULTOCIDA AND MANNHEIMIA HAEMOLYTICA ON THE BASIS OF THE PCR AND PHYLOGENETIC ANALYSIS OF BACTERIAL CULTURES ISOLATED FROM CATTLE.

The results of development of a method for detection and genotyping of the bacteria Pasteurella multocida capsular five groups and Mannheimia haemolytica Al based on the multiplex polymerase chain reaction (PCR) with electrophoretic detection are submitted. Diagnostic sensitivity of the developed method was 103 CFU/ml in the study of the pure cultures and 105 CFU/g in the study of biological material. A study of 260 samples of biological material from infected animals revealed Pasteurella multocida in 50.0%, and Mannheimia haemolytica in 11.2% of the investigated samples. Circulation among the tested livestock of capsular groups B and E of Pasteurella multocida was not revealed. The majority of the tested samples contained group A, in some cases, group D, and, in one case, group F. On the basis of the phylogenetic analysis circulation of two different genetic types of Pasteurella multocida of the capsular group A was revealed.

[EVALUATION OF THE HUMAN SENSITIVITY TO SMALLPOX VIRUS BY THE PRIMARY CULTURES OF THE MONOCYTE-MACROPHAGES].

Studies of the primary cultures of granulocytes, mononuclear, and monocyte-macrophage cells derived from human blood were performed using variola virus (VARV) in the doses of 0.001-0.021 PFU/cell (plaques-forming units per cell). Positive dynamics of the virus accumulation was observed only in the monocyte-macrophages with maximum values of virus concentration (5.0-5.5 Ig PFU/ml) mainly within six days after the infection. The fact of VARV replication in the monocyte-macrophages was confirmed by the data of electron microscopy. At the same time, virus vaccines when tested in doses 3.3 and 4.2 Ig PFU/ml did not show the ability to reproduce in these human cells. The people sensitivity to VARV as assessed from the data obtained on human monocyte-macrophages corresponded to -1 PFU (taking into account the smooth interaction of the virus in the body to the cells of this type), which is consistent to previously found theoretical data on the virus sensitivity. The human susceptibility to VARV assessed experimentally can be used to predict the adequacy of developed smallpox models (in vivo) based on susceptible animals. This is necessary for reliable assessment of the efficiency of development of drugs for treatment and prophylaxis of the smallpox.

[THE USE OF THE MODEL MOUSE ICR--VARIOLA VIRUS FOR EVALUATION OF ANTIVIRAL DRUG EFFICACY].

Mice of the ICR outbred population were infected intranasally (i/n) with the variola virus (VARV, strain Ind-3a). Clinical signs of the disease did not appear even at the maximum possible dose of the virus 5.2 lg PFU/head (plaque-forming units per head). In this case, 50% infective dose (ID50) of VARV estimated by the presence or absence of the virus in the lungs three days after infection (p.i.) was equal to 2.7 ± 0.4 lg PFU/head. Taking into account the 10% application of the virus in the lungs during the intranasal infection of the mice, it was adequate to 1.7 lg PFU/lungs. This indicates a high infectivity of the VARV for mice comparable to its infectivity for humans. After the i/n infection of mice with the VARV at a dose 30 ID50/ head the highest concentration of the virus detected in the lungs (4.9 ± 0.0 lg PFU/ml of homogenate) and in nasal cavity tissues (4.8 ± 0.0 lg PFU/ml) were observed. The pathomorphological changes in the respiratory organs of the mice infected with the VARV appeared at 3-5 days p.i., and the VARV reproduction noted in the epithelial cells and macrophages were noticed. When the preparations ST-246 and NIOCH-14 were administered orally at a dose of 60 µg/g of mouse weight up to one day before infection, after 2 hours, 1 and 2 days p.i., the VARV reproduction in the lungs after 3 days p.i. decreased by an order of magnitude. Thus, outbred ICR mice infected with the VARV can be used as a laboratory model of the smallpox when evaluating the therapeutic and prophylactic efficacy of the antismallpox drugs.

[Development of the disease in marmot at the intranasal infection with the monkeypox virus].

In experimental study the sensitivity of the Marmota bobak species to the monkeypox virus (MPXV) with the intranasal (i/n) infection was tested. It was demonstrated that 50% of the infective dose (ID50) of the MPXV on external clinical signs of the disease was 2.2 Ig plaque forming units (PFU). The percentage of the marmot mortality is slightly dependent on the infecting dose of the MPXV, therefore it is not possible to correctly determine the value of 50 % fatal dose (FD50) for these animals. The most pronounced external clinical signs of the disease were obtained in the marmots: pox-like skin rash throughout the surface of the body and mucous membranes, purulent discharge from the nose, lymphadenitis, discoordination, tremor of the extremities, fever, increased aggression, and ruffled fur. In the course of experiments intended to determine the dynamics of the accumulation of the MPXV in various organs, tissues, and blood serum of marmot infected i/n with dose of 3.7 Ig PFU, it was found that the trachea, lungs, and the bifurcation lymph nodes are the primary target organs. The trachea, lungs, nasal mucosa membrane, and skin are the organs with maximal virus replication recorded at 5, 7, 9, and 12 days after the infection. The transfer of the MPXV into the secondary target organs (nasal mucosa membrane, brain, spleen, duodenum, adrenal glands, and skin) was carried out in marmots with lymphogenic and hematogenic ways of the dissemination of the infection.

[Evaluation of therapeutic-prophylactic effectiveness of chemical compound NIOC-14 against ectromelia virus in vivo].

AIM: Study pharmacodynamic parameters of anti-viral effectiveness of a chemical compound NIOC-14 in experiments in mice infected with ectromelia virus (EV). MATERIALS AND METHODS: EV (K-1 strain) was obtained from the State Collection of Viral Infections and Rickettsioses Causative Agents of the State Scientific Centre of Virology and Biotechnology "Vector". Outbred ICR mice were intranasally infected with EV at a dose of 10 LD50 per animal (10 x 50% lethal doses/animal) and per orally received NIOC-14 or ST-246 as a positive control. Chemical compound NIOC-14 (7-[N'-(4-trifluoromethylbenzoyl)-hidrazincarbonyl]-tricyclo[3.2.2.0(2,4)]non-8-en-6-carbonic acid) was synthesized in Novosibirsk Institute of Organic Chemistry (NIOC). Anti-pox preparation ST-246, developed by SIGA Technologies Inc. (USA), was synthesized in NIOC using the technique described by the authors. RESULTS: 50% effective doses against EV in vivo were shown not to differ significantly between the preparations NIOC-14 (3.59 µg/g mouse mass) and ST-246 (5.08 µg/g mouse mass). During determination of therapeutic window, administration of NIOC-14 to mice 1 day or 1 hour before EV infection, as well as 1, 2 and 4 days after EV infection and then for 9 days was found to ensure 100% animal survival. Administration of NIOC-14 as well as ST-246 resulted in the decrease relative to control of EV titers in lungs, nasal cavity, brains, liver, spleen, kidneys and pancreas. CONCLUSION: Anti-viral effectiveness of NIOC-14 against EV in vivo was thus comparable by all the studied pharmacodynamic parameters with anti-viral activity of anti-pox-virus preparation ST-246.

[Development and validation of the real-time PCR assay for the identification of viral agents causing acute respiratory infections in humans].

The real time PCR assay targeting influenza A and B virus, 5 subtypes of influenza A virus (seasonal H1N1, pandemic H1N1 (2009), seasonal H3N2, pathogenic for human subtypes of avian influenza H5 and H7), respiratory syncytial virus, and adenovirus was developed. The analytical sensitivity of the developed assay was 1 x 10(3) genome equivalents per ml. The diagnostic sensitivity of the method was 1 x l0(3)-10(4) viral particles per ml. Experiments with human DNA/cDNA and viral cDNA showed a markedly high diagnostic specificity of the developed PCR assay. In the assay of the developed PCR test, 50 nasopharyngeal swab specimens were tested. The etiology was identified in 33 samples.

[Etiological structure of acute respiratory viral infections morbidity in Novosibirsk and Novosibirsk region in epidemic season 2011-2012].

INTRODUCTION: ARI occupying the first place in the structure of total human morbidity. The aim of the study was to investigate the species diversity of the viruses causing AR among residents of the Novosibirsk region during epidemic season (October to April). MATERIALS AND METHODS: 164 nasopharyngeal swabs were collected and analyzed. Viral RNA/DNA, cDNA synthesis and PCR were carried out employing "RIBO-prep" "eReverta-L", "AmpliSens Influenza virus A/B-FL" and "AmpliSens ARI-screen-FL" kits (CRI of Epidemiology). RESULTS: Etiological agent of the disease was found in 69(43%) samples. Monoinfection was found in 58 (35%). In 14 (9%) samples were detected serogroup I coronaviruses, in 13 (8%) rhinoviruses, in 7 (4%) respiratory syncytial virus, in 6 (4%) parainfluenza virus type 1, in 5 (3%) parainfluenza virus type 3. Adenoviruses and bocavirus were identified in 3 (2%) samples. Parainfluenza virus type 2 and 4, metapneumovirus, serogroup Il coronaviruses (HKU1 and OC43) were presented in 2 (1%) samples. In 11 (7%) samples was found mixed infection. CONCLUSION: The majority of common colds were caused by serogroup I coronaviruses (NL63 and 229E), rhinoviruses and mixed infections. The peak of species variability of viruses caused acute respiratory infections was determined in age group of children 2-4 years old. In older age groups the species variability of analyzed viruses was decreased, rhinovirus infection becomes prevalent.

[A comparative study of the antiviral activity of chemical compounds concerning the orthopoxviruses experiments in vivo].

In the experiments using intranasal (i/n) infection of mice with the ectromelia virus (EV) in a dose 10 LD50/head (10 x 50% lethal doselhead) or with the monkaypox virus (MPXV) in a dose 10 ID50/head (10 x 50% infective dose/ head) it was demonstrated that the antiviral efficiency of chemical compounds - the condensed derivatives of pyrrolidin-2,5-dion, as well as their predecessors and the nearest analogues, synthesized in Novosibirsk Institute of Organic Chemistry of the Siberian Branch of the Russian Academy of Sciences (NIOCH SB RAS) was observed. As a positive control we used the antipoxvirus chemical preparation ST-246 available from SIGA Technologies Inc. (USA), synthesized in NIOCH SB RAS by the technique suggested by the authors. It was demonstrated that the compound NIOCH-14 (7-[N'-(4-Trifluoromethylbenzoil)-hydrazidecarbonil]-tricyclo[3.2.2.02,4]non-8-en-6-carbonic acid) possessed comparable with ST-246 antiviral activity concerning EV and MPXV on all indicators used. Therefore, at infection of mice with EV (strain K-1) and peroral administration of NIOCH-14 and ST-246 in a dose 50 mkg/g of mouse weight (12-14 g) within 10 days the survival rate and average life expectancy of mice authentically exceeded the control levels. EV titers in lungs through 6 days after infection in the same groups were lower than in the control. In addition to that, after 7 days of infection of mice with MPXV (strain V79-1-005) and daily peroral administration of NIOCH-14 and ST-246 in a dose 60 mkg/g of mouse weight (9-11 g) authentic decrease in a part of infected animals and MPXV titers in lungs was observed.

[Outbreak of acute enterovirus intestinal infection in Sakhalin region in August 2010].

The investigation of cases of acute intestinal infections in the Sakhalin region of Russia in August, 2010 is described. Epidemiological and molecular biological studies were conducted. After initial PCR screening and determining the nucleotide sequences of the positive samples the following enteroviruses were found: Coxsackie A2 - 42 samples (45%), Coxsackie A4--31 sample (34%), Enterovirus 71--6 samples (6,5%), Coxsackievirus B5--6 samples (6,5%), Coxsackie B3--4 samples (4%) and Coxsackie B1--4 samples (4%). The phylogenetic analysis of sequences showed that the closest analogues for the nucleotide sequences of these genotypes were previously identified in Japan, Korea and China in 2000-2010.

Reporter Transgenes for Monitoring the Antitumor Efficacy of Recombinant Oncolytic Viruses.

Accurate measurement of tumor size and margins is crucial for successful oncotherapy. In the last decade, non-invasive imaging modalities, including optical imaging using non-radioactive substrates, deep-tissue imaging with radioactive substrates, and magnetic resonance imaging have been developed. Reporter genes play the most important role among visualization tools; their expression in tumors and metastases makes it possible to track changes in the tumor growth and gauge therapy effectiveness. Oncolytic viruses are often chosen as a vector for delivering reporter genes into tumor cells, since oncolytic viruses are tumor-specific, meaning that they infect and lyse tumor cells without damaging normal cells. The choice of reporter transgenes for genetic modification of oncolytic viruses depends on the study objectives and imaging methods used. Optical imaging techniques are suitable for in vitro studies and small animal models, while deep-tissue imaging techniques are used to evaluate virotherapy in large animals and humans. For optical imaging, transgenes of fluorescent proteins, luciferases, and tyrosinases are used; for deep-tissue imaging, the most promising transgene is the sodium/iodide symporter (NIS), which ensures an accumulation of radioactive isotopes in virus-infected tumor cells. Currently, NIS is the only reporter transgene that has been shown to be effective in monitoring tumor virotherapy not only in preclinical but also in clinical studies.

[Microarray for diagnostics of human pathogenic influenza A viruses subtypes].

An oligonucleotide microchip was developed for diagnostics of human pathogenic Influenza A viruses subtypes. It contains discriminating probes for H1-, H2-, H3-, H5-, H7- and H9-subtypes of hemagglutinin and for N1-, N2-, and N7-subtypes of neuraminidase. The additional set of probes was used for M-gene of Influenza A viruses definition. Microchip was tested on samples pathogenic H5N1 avian influenza viruses, pandemic H1N1 swine influenza viruses and seasonal H1N1 and H3N2 influenza viruses. The microchip can be used for the analysis of both cultured strains and clinical specimens.

[Genetic variability of isolates of pandemic influenza A virus H1N1 isolated in Russia in 2009].

Complete nucleotide sequence of the genome segments encoding the surface glycoproteins, hemagglutinin, and neuraminidase of influenza A virus H1N1 derived from the patients with influenza in the context of pandemic (H1N1) 2009 was determined out of 14 isolates of pandemic influenza. The philogenetic analysis of these sequences demonstrated their genetic similarity to the corresponding genes of the pandemic influenza virus A (H1N1) 2009 isolates obtained in other countries; each gene homology was greater than 99%. Neuraminidase mutations causing virus resistance to oseltamivir and other neuraminidase inhibitors, known from the literature, were not detected. The hemagglutinin gene mutation D222G was found in 4 isolates from autopsy material. In the hemagglutinin of pandemic A/Salekhard/01/2009(H1N1) isolate a mutation G155E leading to the increase in viral replication in developing chick embryos was detected. The nature and frequency of nucleotides substitutions within HA and NA genes were determined in the current research.

[Studies of properties of pandemic influenza virus A/H1N1 circulated in Russian Federation in 2009 - 2010].

AIM: Studies of cultural, virologic, antigenic properties of 89 samples of pandemic influenza A(H1N1) 2009 virus isolated in Russian Federation from May 2009 to March 2010. MATERIALS AND METHODS: Properties of isolated samples were compared with those of the reference strain A/ California/04/2009 (H1N1). RESULTS: Studies of biological properties and analysis of genome nucleotide sequences of the isolated samples showed that those strains are closely related to the reference strain. CONCLUSION: Monitoring of genetic, virologic and antigenic properties of pandemic influenza A(H1N1) 2009 virus isolates carried out from May 2009 to March 2010 did not reveal significant changes in the abovementioned properties of the virus or emergence of mutations that can lead to such changes.

[The application of xMAP technology in genetic typing of measles virus].

The genetic typing of measles virus in clinical samples using xMAP technology was applied. The study provided the calculation and application of specific oligonucleotide probes of genotypes D4, D6 and D7 of measles virus. The strain HobO96 genotype A of measles virus as a check sample was used. The technical approaches to the optimization of preparatory work organization and to the process of identification of measles virus genotypes are described. The presence of genotypes D4, D6 and D7 in clinical samples is proved by the sequence analysis. The genetic typing effectiveness of technique of DNA hybridization using xMAP technology on the instrumental base BioPlex (BioRad, USA) is demonstrated.

[In vivo efficacy of Ingavirin against pandemic A (H1N1/09)v influenza virus].

Ingavirin was shown to be efficient in inhibition of the pandemic influenza virus strains A/California/04/2009 (H1N1)v, A/California/07/2009 (H1N1)v, A/Moscow/225/2009 (H1N1)v and A/Moscow/226/2009 (H1N1)v. as well as the influenza virus strain A/Aichi/2/68 (H3N2) in the lungs of the infected mice. After oral administration of Ingavirin the titers of the influenza virus strains in the lung homogenates lowered.

[Investigation of therapeutic and protective effect of Reaferon-ES Lipint on mice infected with avian influenza virus (subtype H5N1)].

The efficacy of the therapeutic, prophylactic and urgent prophylactic schemes for the use of Reaferon-ES lipint, a liposomal human recombinant alpha-interferon for oral use, was studied on mice infected with the avian influenza virus. Strain A/Chicken/Kurgan /05/2005 (subtype H5N1) of the avian influenza virus showed high virulence with respect to mice ICR. Theoretically-based calculations allowed to design an optimal therapeutic and prophylactic dose of the drug for the mice (1000 units/animal). It was observed that only after prophylactic use of Reaferon-ES lipint it was effective in protection of the mice infected with 10 LD50 of the avian influenza virus (A/Chicken/Kurgan/05/2005, H5N1). The protection coefficient was 0.35. Under such conditions the drug efficacy was comparable with that of Tamiflu. Therefore, Reaferon-ES lipint could be recommended for prophylaxis of the infection due not only to the season strains of the influenza virus, but also to the strains of the avian influenza virus.

[Molecular characteristic of influenza virus A H5N1 Strains isolated from poultry in Kurgan Region in 2005].

During the latter half of 2005 a widespread outbreak caused by influenza highly pathogenic H5N1 virus among wild and domestic birds occurred in Russia. As pathogenicity level is a polygenic feature and majority of individual genes of influenza A viruses contribute to pathogenicity of influenza viruses to birds, animals and humans. Nucleotide sequencing of the entire genome of influenza H5N1 virus isolates obtained in Kurgan region (Western Siberia) was performed. Structure of viral proteins was analyzed according to the predicted amino acid sequences. HA receptor-binding site of A/chicken/Kurgan/05/2005 and A/duck/Kurgan/08/2005 strains was typical for avian influenza viruses and contained Glu and Gly at positions 226 and 228, respectively. Structure of the cluster of positively charged amino acid residues at the cleavage site was identical for all isolates: QGERRRKKR. According to the data of neuraminidase structure analysis NA of the H5N1 isolates tested was suggested to belong to Z genotype. Amino acid residues typical for birds were revealed in 30 out of 32 positions of M1, M2, NP, PA and PB2 proteins determining host range specificity. One strain isolated in Kurgan contained lysine in position 627 of PB2 protein. Kurgan isolates was shown to have remantadine-sensitive genotype. Glutamic acid was found at position 92 of NS1 protein in both strains indicating virus resistance to interferon. Phylogenetic analyses allowed relating Kurgan isolates to subclade II of clade II of highly pathogenic H5N1 influenza viruses.

[4647-cell culture for preparation of recombinant bivaccine against smallpox and hepatitis B].

The seeding and working banks of a 4647-cell culture have been created. The 4647-cell culture in these banks has a high proliferative activity, as well as the morphology, typical of this line, and the karyotype and the enzymogram, which are characteristic for the cells of an African talapoin (Cercopithecus aethiops). The culture is not contaminated with bacteria, fungi, Mycoplasma, and viruses, including oncoviruses. The deposited 4647 cells have high viral productive properties for the accumulation of the recombinant virus strain b7,5S2-S vaccine and keep the stability of all biological properties during a long-term cultivation. The continuous 4647 cell line was tested at the L. A. Tarasevich State Institute of SK. The seeding and working banks of 4647-cell culture at passages 108 and 128 are recommended as a substrate for cultivation of the strain b7,5S2-S vaccinia, used to prepare a bivaccine against smallpox and hepatitis B.