Study  of the biological properties  of attenuated variants of the virulent A/WSN/33 strain of influenza virus, obtained by the site-specific  mutagenesis of PB2-gene

The aim of this study was to generate dendritic cells from the bone marrow of mice (DC) in vitro and to assess the effect of virulent and attenuated variants of influenza virus on the maturation of DCs. Granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL-4) were used in combination, to induce differentiation of mouse bone marrow (BM) mononucleocytes into DCs. On the 5th day, distinct variants of influenza virus were added to the cell culture, and the cells were additionally incubated for 2 days. The morphological characteristics of DCs, immunophenotype of DCs and expression of some Toll-like receptors were evaluated. On the 5th day of incubation. the DCs acquired typical morphological characteristics. DCs were large in size with an eccentrically located nucleous, often irregular in shape, with numerous processes. On the 7th day of incubation with influenza virus variants, their cytoplasm was somewhat denser. DCs acquired more processes, necessary for intercellular contacts. Expression levels of CD11c, a specific marker of BM-derived DCs, and of co-stimulatory molecules such as CD40, CD80, CD86, and MHC-II were elevated in mature DCs. Virulent versus attenuated strains of the influenza virus induced special variants of DCs differentiation, with respect to expression rates of differentiation markers, as well as expression of Toll-like receptors and costimulatory molecules. Conclusions. The in vitro cultured murine mononucleocytes derived from bone marrow can produce a large number of n-DCs, that can mature in the presence of different variants.During evolution of the DC immunophenotype treated with variant influenza viruses, we have found distinct signs of immunosuppression.The attenuated U-2 and M-26 influenza variants obtained by site-specific mutagenesis upon development of DCs immunophenotype, exhibited a decreased immunosuppressive activity and were not inferior to the cold-adapted (CA) reassortant for the most positions, but exceeded it in some instances. These studies can help to assess the criteria for evaluation the efficiency of in vitro developed influenza vaccines.

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Effect of virulent and vaccine variants of influenza virus on the immunophenotype of dendritic cells generated from murine bone marrow

Ахматова

Ртищев

Markushin

et al. 2020

Med. immunol.

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Immunoregulatory functions of natural killer cells in avian influenza virus infection

Shatunova,

Gavrilova,

Rtishchev

et al. 2023

Med. immunol.

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The avian influenza virus (AIV) has a great pandemic potential both in animals, and in human population. For effective struggle against this virus, it is important to study the immune response against AIV, and, in particular, the features of T cell immunity. At the period of early immune response, when the activation of adaptive immunity factors has not yet occurred, the natural killers, which have both cytotoxic and immunoregulatory functions, are known to play a key role in the fight against the influenza virus. In this study, we investigated the interaction between natural killers and T helpers in the process of antiviral response. The aim of this study was to study immunoregulatory functions of NK cells in BALB/c mice vaccinated with different doses of avian influenza virus.We used a strain of mouse-adapted avian influenza virus (AIV) of the H5N2 serotype (A/duck/ Pennsylvania/10218/1984). The determination of the virus titer in the mice lung tissue was performed by infecting 10-day-old chicken embryos (SPF) with lung extracts at various decimal dilutions, followed by the immune agglutination test. Quantitative determination of natural killers and T helpers (Th) in the mice spleen tissue was carried out by flow cytometry. The amount of Th cells in mice spleens increased at low infectious dose (2.5 EID50) which is explained by active clonal expansion of this cell subpopulation. The infectious disease process ended upon complete virus elimination from the lungs. The amounts of Th cells were also increased in a group of mice infected with intermediate infectious dose (25 EID50), however, being accompanied by virus replication in lungs. When the mice were infected with a high dose of the virus (250 EID50), there was a decrease in the Th cells number versus control group of animals, whereas replication of AIV was noted throughout the entire observation period in the lung tissue.The amounts of helper T cells at high doses of viral infection could be decreased due to activation of NK cells. The activated NK cells may then eliminate exhausted helper T cells. Thus, NK cells immunoregulatory function represents an important immunological factor which keeps balance between the pathogen destruction and excessive inflammation of the lung tissue affected by the avian influenza virus.

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Determination of cold-adapted influenza virus (Orthomyxoviridae: <i>Alphainfluenzavirus</i>) polymerase activity by the minigenome method with a fluorescent protein

Ivanov,

Lyashko,

Kost

et al. 2023

Problems of Virology

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Введение. Ранее было показано, что полимеразные белки PB1 и PB2 определяют холодоадаптированный фенотип вируса гриппа А/Краснодар/101/35/59 (H2N2). Цель работы. Создать репортерные конструкции и определить активность вирусной полимеразы при 33 и 37 °С методом минигенома. Материалы и методы. Совместная трансфекция клеток Cos-1 плазмидами pHW2000, экспрессирующими белки вирусной полимеразы PB1, PB2, PA, NP (минигеном) и репортерную конструкцию. Результаты. На основе сегмента 8 созданы две репортерные конструкции, которые содержат прямую или инвертированную последовательность NS1-GFP-NS2 для экспрессии белков NS2 и NS1, трансляционно слитных с зелёным флуоресцентным белком (GFP), которые позволили оценить транскрипционную и/или репликативную функции вирусной полимеразы. Заключение. Полимераза вируса А/Краснодар/101/35/59 (H2N2) обладает более высокой репликативной и транскрипционной активностью при 33 ºС, чем при 37 °С. Её транскрипционная активность в большей степени зависит от температуры, чем репликативная. Репликативная и транскрипционная активности полимеразы вируса A/Puerto Rico/8/34 (H1N1, вариант Mount Sinai) не имеют существенных различий и не зависят от температуры.

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Study of the biological properties of attenuated variants of the virulent A/WSN/33 strain of influenza virus, obtained by the site-specific mutagenesis of PB2-gene

Cited by 3 publications

References 18 publications

Effect of virulent and vaccine variants of influenza virus on the immunophenotype of dendritic cells generated from murine bone marrow

Effect of virulent and vaccine variants of influenza virus on the immunophenotype of dendritic cells generated from murine bone marrow

Immunoregulatory functions of natural killer cells in avian influenza virus infection

Determination of cold-adapted influenza virus (Orthomyxoviridae: <i>Alphainfluenzavirus</i>) polymerase activity by the minigenome method with a fluorescent protein

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