Development of the cell-ELISA test for the subtype identification of circulating influenza A(H1) and A(H3) viruses

The sensitive version of cell-ELISA was developed for the subtype-specific differentiation of current influenza A(H1N1)pdm09 and A(H3N2) viruses that are circulating in the human population. This method is based on the estimation of virus reproduction in infected MDCK cells. The detection step of this method is an interaction of the subtype-specific monoclonal antibodies (mAbs) with the viral hemagglutinin (НА) molecule. The influenza A virus strains, isolated in the 2014 epidemic season, were used to validate this method.It was shown that when using mAbs # 1/ # 2 or # 4 at a concentration of 10-15 µg/ml, the developed variant of cell-ELISA was able to detect НА protein synthesized in the infected cells of influenza A(H3N2) and A(H1N1)pdm09 viruses, respectively.The developed method can be used for the identification of modern influenza A viruses with low hemagglutination activity, which is not possible by the conventional hemagglutination inhibition test.The sensitive version of cell-ELISA was developed for the subtype-specific differentiation of current influenza A(H1N1)pdm09 and A(H3N2) viruses that are circulating in the human population. This method is based on the estimation of virus reproduction in infected MDCK cells. The detection step of this method is an interaction of the subtype-specific monoclonal antibodies (mAbs) with the viral hemagglutinin (НА) molecule. The influenza A virus strains, isolated in the 2014 epidemic season, were used to validate this method. It was shown that when using mAbs # 1/ # 2 or # 4 at a concentration of 10-15 µg/ml, the developed variant of cell-ELISA was able to detect НА protein synthesized in the infected cells of influenza A(H3N2) and A(H1N1)pdm09 viruses, respectively. The developed method can be used for the identification of modern influenza A viruses with low hemagglutination activity, which is not possible by the conventional hemagglutination inhibition test

PHYSICOCHEMICAL CHARACTERIZATION OF RIBAVIRIN WITH SPECTROSCOPY AND MOLECULAR MODELING

This work was supported by Mendeleev University of Chemical Technology of Russia. Project Number K-003-2018

ЛАБОРАТОРНАЯ ДИАГНОСТИКА ОСТРЫХ РЕСПИРАТОРНЫХ ВИРУСНЫХ ИНФЕКЦИЙ В УСЛОВИЯХ ЭВОЛЮЦИОННОЙ ИЗМЕНЧИВОСТИ ВИРУСОВ ГРИППА

As a result of laboratory investigation 1651 patients at the age from 0 months till 18 years, hospitalized with ARI from 2008 to 2011, in ¾ cases the virus etiology of disease was found. At the same time in 2009-2011 was marked significant prevalence of pandemic influenza virus infection A(Н1N1)v in a kind mono- and mikst-infections (in 18,8 and 10,2 % of cases accordingly), and adenoviruses, espiratory sincitial viruses and parainfluenza were the most frequent concomitants (in 4,8, 3,2 and 2,4 % of cases). In a clinical picture of disease acute nasopharynx lesion (67,8 %) was predominated, complicating by croup in 12,2 % of cases, by acute bronchitis in 12,1 % of cases, and more rare by pneumonia (in 8,2 % of cases). The analysis of results of immunofluorescence test in comparison with the cumulative data of other laboratory methods (polymerase chain reaction, immuneenzyme analysis, virus isolation, serological examination) has shown possibility of discovery with its help the valuable information on etiology ARI, including influenza, at early stages of disease and in short terms.В результате лабораторного обследования 1651 пациента в возрасте от 0 мес. до 18 лет, госпитализированных с ОРВИ-инфекцией в период с 2008 по 2011 г., в 67,2–77,8% случаев выявлена вирусная этиология заболевания. При этом в 2009–2011 гг. отмечалось преобладание пандемического вируса гриппа А(Н1N1)v в виде моно- и микст-инфекций (в 18,8 и 10,2% случаев соответственно), а наиболее частыми ассоциантами были аденовирусы, РС-вирусы и вирусы парагриппа (в 4,8, 3,2 и 2,4% случаев). В клинической картине заболевания преобладал острый ринофарингит (67,8%), осложняющийся в 12,2% случаев острым стенозирующим ларинготрахеитом, в 12,1% случаев – острым бронхитом, реже – пневмонией (в 8,2% случаев). Анализ результатов иммунофлуоресцентного метода в сравнении с совокупными данными других лабораторных тестов (полимеразная цепная реакция, иммуноферментный анализ, вирусовыделение, серология) показал возможность получения с его помощью ценной информации об этиологии ОРВИ, включая грипп, на ранних стадиях заболевания и в короткие сроки

Использование микрокультурального иммуноферментного анализа для субтиповой идентификации циркулирующих вирусов гриппа А(Н1) и А(Н3)

The sensitive version of cell-ELISA was developed for the subtype-specific differentiation of current influenza A(H1N1)pdm09 and A(H3N2) viruses that are circulating in the human population. This method is based on the estimation of virus reproduction in the infected MDCK cells. The detection step of this method is an interaction of the subtype-specific monoclonal antibodies (mAbs) with the viral hemagglutinin (НА). The influenza A virus strains, isolated in 2014 epidemic season, were used to validate this method. It was shown that by using mAb # 1/ # 2 or # 4 at a concentration of 10-15 µg / ml the developed variant of cell-ELISA allows the detection of НА protein, synthesized in the cells infected with influenza A(H3N2) or A(H1N1)pdm09 virus, respectively. The developed method can be used for identification of HA subtype of modern influenza A viruses with low HA activity, which is not possible by the conventional hemagglutination inhibition test.Разработан чувствительный вариант микрокультурального ИФА (cell-ELISA) для субтиповой дифференциации современных вирусов гриппа А(Н1N1)pdm09 и А(Н3N2), циркулирующих в человеческой популяции. Метод основан на оценке репродукции вируса в инфицированной культуре клеток MDCK c использованием на стадии детекции cубтип-специфичных моноклональных антител (mAb), взаимодействующих с гемагглютинином (HA) вируса гриппа. При отработке метода использованы штаммы вирусов гриппа A, выделенные из клинических образцов в эпидемический сезон 2014 года. Показано, что при использовании mAb #1/#2 или #4 в концентрации 10–15 мкг/мл разработанный вариант cell-ELISA позволяет детектировать синтезируемый в зараженных клетках белок HA соответственно вирусов гриппа А(H3N2) или A(H1N1)pdm09. Разработанный метод может быть использован для идентификации современных штаммов вируса гриппа А в процессе их репродукции в клеточной культуре MDCK, что позволяет проводить субтипирование вирусов с низкой гемагглютинирующей активностью, когда невозможно использовать общепринятый метод – реакцию торможения гемагглютинации (РТГА)

Роль респираторных инфекций в обострениях бронхиальной астмы

Nineteen patients aged 18–65 years with moderate and severe exacerbations of atopic asthma were examined for respiratory viruses, Mycoplasma pneumoniae, and Chlamydophila pneumoniae. Interferon system, IL-4 and γ-IFN serum levels were also investigated. Viral infections (RS-virus, adenovirus, influenza types A (H1N1, H3N2) and B viruses, parainfluenza types 1 and 3 viruses) were diagnosed serologically or using PCR with direct detection of viral nucleic acids in 73.6 % of the patients. Diagnostic level of Mycoplasma pneumoniae antigen was found in 78.9 % of the patients, anti-Chlamydophila pneumoniae antibodies were detected in 31.6 %. Leukocyte interferon-producing function was decreased in all the patients.У 19 пациентов в возрасте 18–65 лет с атопической бронхиальной астмой во время тяжелых и среднетяжелых обострений проведено обследование на наличие респираторных вирусов, Mycoplasma pneumoniae и Chlamydophila pneumoniae, оценены состояние системы интерферона, уровни IL-4 и γ-IFN в сыворотке крови. У 73,6 % пациентов серологически или путем прямого выявления вирусных нуклеиновых кислот методом ПЦР подтверждено наличие вирусной инфекции (респираторно-синцитиальный вирус — РС-вирус, аденовирус, грипп А (H1N1, H3N2) и В, парагрипп 1-го и 3-го типа). У 78,9 % пациентов в сыворотке крови обнаружен антиген Mycoplasma pneumoniae в диагностически значимом титре, у 31,6 % пациентов — антитела к Chlamydophila pneumoniae. У всех пациентов отмечено выраженное снижение интерферон-продуцирующей способности лейкоцитов

AUTOREACTIVE ANTIBODIES IN A HEALTHY HUMAN AND IN PATIENTS WITH VIRAL INFECTIONS

Abstract. This brief review presents the data obtained during the last two decades which allow to create a new view on autoimmunity. Regulatory and protective characteristics of autoreactive natural antibodies and their role in development of effective adaptive antiviral immune response are discussed. The article considers the problem of possible autoimmune complications due to some viral infections and antiviral vaccination

Boronic Acids as Prospective Inhibitors of Metallo-β-Lactamases: Efficient Chemical Reaction in the Enzymatic Active Site Revealed by Molecular Modeling

Boronic acids are prospective compounds in inhibition of metallo-β-lactamases as they form covalent adducts with the catalytic hydroxide anion in the enzymatic active site upon binding. We compare this chemical reaction in the active site of the New Delhi metallo-β-lactamase (NDM-1) with the hydrolysis of the antibacterial drug imipenem. The nucleophilic attack occurs with the energy barrier of 14 kcal/mol for imipenem and simultaneously upon binding a boronic acid inhibitor. A boron atom of an inhibitor exhibits stronger electrophilic properties than the carbonyl carbon atom of imipenem in a solution that is quantified by atomic Fukui indices. Upon forming the prereaction complex between NDM-1 and inhibitor, the lone electron pair of the nucleophile interacts with the vacant p-orbital of boron that facilitates the chemical reaction. We analyze a set of boronic acid compounds with the benzo[b]thiophene core complexed with the NDM-1 and propose quantitative structure-sroperty relationship (QSPR) equations that can predict IC50 values from the calculated descriptors of electron density. These relations are applied to classify other boronic acids with the same core found in the database of chemical compounds, PubChem, and proposed ourselves. We demonstrate that the IC50 values for all considered benzo[b]thiophene-containing boronic acid inhibitors are 30-70 μM

Evolution of Ceftriaxone Resistance of Penicillin-Binding Proteins 2 Revealed by Molecular Modeling

Penicillin-binding proteins 2 (PBP2) are critically important enzymes in the formation of the bacterial cell wall. Inhibition of PBP2 is utilized in the treatment of various diseases, including gonorrhea. Ceftriaxone is the only drug used to treat gonorrhea currently, and recent growth in PBP2 resistance to this antibiotic is a serious threat to human health. Our study reveals mechanistic aspects of the inhibition reaction of PBP2 from the wild-type FA19 strain and mutant 35/02 and H041 strains of Neisseria Gonorrhoeae by ceftriaxone. QM(PBE0-D3/6-31G**)/MM MD simulations show that the reaction mechanism for the wild-type PBP2 consists of three elementary steps including nucleophilic attack, C–N bond cleavage in the β-lactam ring and elimination of the leaving group in ceftriaxone. In PBP2 from the mutant strains, the second and third steps occur simultaneously. For all considered systems, the acylation rate is determined by the energy barrier of the first step that increases in the order of PBP2 from FA19, 35/02 and H041 strains. Dynamic behavior of ES complexes is analyzed using geometry and electron density features including Fukui electrophilicity index and Laplacian of electron density maps. It reveals that more efficient activation of the carbonyl group of the antibiotic leads to the lower energy barrier of nucleophilic attack and larger stabilization of the first reaction intermediate. Dynamical network analysis of MD trajectories explains the differences in ceftriaxone binding affinity: in PBP2 from the wild-type strain, the β3-β4 loop conformation facilitates substrate binding, whereas in PBP2 from the mutant strains, it exists in the conformation that is unfavorable for complex formation. Thus, we clarify that the experimentally observed decrease in the second-order rate constant of acylation (k2/KS) in PBP2 from the mutant strains is due to both a decrease in the acylation rate constant k2 and an increase in the dissociation constant KS

Influence of the Active Site Flexibility on the Efficiency of Substrate Activation in the Active Sites of Bi-Zinc Metallo-β-Lactamases

The influence of the active site flexibility on the efficiency of catalytic reaction is studied by taking two members of metallo-β-lactamases, L1 and NDM-1, with the same substrate, imipenem. Active sites of these proteins are covered by L10 loops, and differences in their amino acid compositions affect their rigidity. A more flexible loop in the NDM-1 brings additional flexibility to the active site in the ES complex. This is pronounced in wider distributions of key interatomic distances, such as the distance of the nucleophilic attack, coordination bond lengths, and covalent bond lengths in the substrate. Substrate activation, quantified by Fukui electrophilicity index of the carbonyl carbon atom of the substrate, is also sensitive to the active site flexibility. In the tighter and more rigid L1 enzyme-substrate complex, the substrate is activated more efficiently. In the NDM-1 containing system, only one third of the states are activated to the same extent. Other fractions demonstrate lower substrate activation. Efficiency of the substrate activation and rigidity of the ES complex influence the following chemical reaction. In the more rigid L1-containing system, the reaction barrier of the first step of the reaction is lower, and the first intermediate is more stabilized compared to the NDM-1 containing system

Evolution of Ceftriaxone Resistance of Penicillin-Binding Proteins 2 Revealed by Molecular Modeling

Penicillin-binding proteins 2 (PBP2) are critically important enzymes in the formation of the bacterial cell wall. Inhibition of PBP2 is utilized in the treatment of various diseases, including gonorrhea. Ceftriaxone is the only drug used to treat gonorrhea currently, and recent growth in PBP2 resistance to this antibiotic is a serious threat to human health. Our study reveals mechanistic aspects of the inhibition reaction of PBP2 from the wild-type FA19 strain and mutant 35/02 and H041 strains of Neisseria Gonorrhoeae by ceftriaxone. QM(PBE0-D3/6-31G**)/MM MD simulations show that the reaction mechanism for the wild-type PBP2 consists of three elementary steps including nucleophilic attack, C–N bond cleavage in the β-lactam ring and elimination of the leaving group in ceftriaxone. In PBP2 from the mutant strains, the second and third steps occur simultaneously. For all considered systems, the acylation rate is determined by the energy barrier of the first step that increases in the order of PBP2 from FA19, 35/02 and H041 strains. Dynamic behavior of ES complexes is analyzed using geometry and electron density features including Fukui electrophilicity index and Laplacian of electron density maps. It reveals that more efficient activation of the carbonyl group of the antibiotic leads to the lower energy barrier of nucleophilic attack and larger stabilization of the first reaction intermediate. Dynamical network analysis of MD trajectories explains the differences in ceftriaxone binding affinity: in PBP2 from the wild-type strain, the β3-β4 loop conformation facilitates substrate binding, whereas in PBP2 from the mutant strains, it exists in the conformation that is unfavorable for complex formation. Thus, we clarify that the experimentally observed decrease in the second-order rate constant of acylation (k2/KS) in PBP2 from the mutant strains is due to both a decrease in the acylation rate constant k2 and an increase in the dissociation constant KS