Comparison of Fragments in Human Hemostatic Proteins That Mimics Fragments in Proteins of A/H1N1 Viruses and Coronaviruses.

Objective: To compare the repertoire of proteins of the human hemostatic system and fragments mimicking these proteins in the proteins of influenza A/H1N1 viruses and coronaviruses. Material and methods. Influenza viruses A/H1N1 (A/Brevig Mission/1/18), A/St. Petersburg /RII04/2016 (H1N1)pdm09, coronaviruses SARS-CoV and SARS-CoV-2 (strain Wuhan-Hu-1) were used for comparative computer analysis. The sources of the primary structures of proteins of the analyzed viruses and 41 proteins of the human hemostatic system were publicly available Internet databases, respectively, www.ncbi.nlm.nih.gov and www.nextprot.org. The search for homologous sequences in the structure of viral proteins and hemostatic proteins was carried out by comparing fragments of 12 amino acids in length, taking as related those that showed identity at ≥8 positions. Results. Comparative analysis of the repertoire of cellular proteins of the hemostatic system and fragments mimicking these proteins in the structure of proteins of viruses A/H1N1 1918, A(H1N1)pdm09 isolated in 2016, SARS-CoV and SARS-CoV-2, showed a significant difference between SARS-CoV-2 and analyzed viruses. In the protein structure of the SARS-CoV-2 virus, mimicry was revealed for almost all analyzed hemostasis proteins. As for the comparison of viruses A/H1N1 1918, A(H1N1)pdm09 2016 and SARS-CoV, the influenza virus A/H1N1 1918 and SARS-CoV are the closest in the repertoire of hemostatic proteins. Conclusion. Obtained bioinformatic analysis data can serve as a basis for further study of the role of homologous fragments in the regulation of hemostasis of the host organism.

[Modulation of endothelial factors activity in human endothelial cells in influenza A(H1N1)pdm09 virus infection].

INTRODUCTION: Influenza A virus infection can lead to endothelial dysfunction (ED), including apoptosis of endothelial cells and modulation of endothelial factor activities. Affected biochemical factors may include those playing important roles in vascular homeostasis. However, the effect of this pathogen on the expression pattern of key endothelial factors is still unknown.The aim of this work was to study the expression of endothelial nitric oxide synthase (eNOS) and plasminogen activator inhibitor-1 (PAI-1, serpin E1) in the EA.hy926 endothelial cells. RESEARCH OBJECTIVES: to assess expression of eNOS and PAI-1 in endothelial cells infected with influenza virus A(H1N1)pdm09, and to identify homologous fragments in structure of viral proteins and endothelial factors. MATERIAL AND METHODS: Cells were infected with influenza virus A/St. Petersburg/48/16 (H1N1)pdm09 and analyzed in dynamics in 6, 12, 18, 24, 48, and 72 hrs post infection (hpi). Detection of endothelial factors expression levels was performed by immunocytochemical method (ICC) using antibodies for eNOS and PAI-1 while quantitative assessment of expression levels was carried out by program Nis-Elements F3.2 («Nikon¼, Japan). The search for homologous sequences between viral proteins and eNOS and PAI-1 was performed by computer comparison. Sequences were analyzed as fragments 12 amino acid residues (aar) in length. RESULTS AND DISCUSSION: eNOS expression in infected cells had decreased to 7.9% by 6 hpi (control was taken as 100%) to 3.3% at 72 hpi. PAI-1 expression varied significantly over the course of the experiment: by 6 hpi it had decreased to 49.6%, and to 43.2% by 12 hpi. Later PAI-1 levels were: 116.3% (18 hpi); 18.9% (24 hpi); 23.5% (48 hpi), and 35% (72 hpi). CONCLUSION: These results indicate that influenza A infection of endothelial cells causes a significant decrease in eNOS expression, while modulating PAI-1 one. The described phenomenon can be used in the further development of directions of pathogenetic therapy of vascular complications of infection caused by this pathogen.

[Modeling influenza virus infection in mature Wistar rats].

It has now been established that blood vessels are target for influenza, but the mechanism by which the influenza virus affects the cardiovascular system is unknown. The aim - adaptation of influenza virus A/St. Petersburg/48/16 H1N1(pdm09) to mature Wistar rats, as these animals are the main experimental model for studying the pathology of the cardiovascular system. MATERIAL AND METHODS: Passage of influenza A virus (IAV) in embryonated chicken eggs, intranasal inoculation of rats with virus-containing material s, production of pulmonary homogenate, determination of IAV titer in embryonated chicken eggs, detection of histological changes in lung and pulmonary vessels. RESULTS: The article presents the results of the adaptation of influenza virus A/St. Petersburg/48/16 H1N1(pdm09) to mature Wistar rats. The infectious titer of the virus in the homogenates of infected rats lungs at the last stage of adaptation was 7.0 lg EID50/ml. Histological studies revealed pronounced changes in the respiratory tract (spasm of bronchioles, submucosal edema, desquamation of ciliated epithelium of bronchioles) and pulmonary vessels (spasm, desquamation and swelling of endotheliocytes, dissociation and swelling of the elastic membrane and media). In order to identify IAV in blood vessels and lung tissues, an immunohistochemical study was performed using monoclonal antibodies to NP antigen of IAV. CONCLUSION: The data obtained allow us to conclude that the strain of influenza virus A/St. Petersburg/48/16 H1N1(pdm09) was adapted to mature Wistar rats maintaining virulent properties. The infectious titer of the virus at the last stage of adaptation was 7.0 lg EID50/ml. IAV identification is confirmed by immunohistochemical examination.

[Dynamics of the cell cycle in human endothelial cell culture infected with influenza virus].

Cell cycle in a culture of endothelial cells EAhy 926 infected with influenza virus was investigated. Cytometric analysis of culture, synchronized using contact inhibition, has shown that the exposure to the influenza virus in cells EAhy 926 lengthened S-phase of the cell cycle. This result has been tested and proven on culture EAhy 926 treated with nocodazole. Compared with lung carcinoma cells A549, in which influenza virus provokes the arrest of G0/G1 phase of the cycle, elongation of S-phase of cycle at a similar infection of endothelial culture EAhy 926 indicates that the influenza virus differently affects the dynamics of the cell cycle according to the origin of the infected culture.

Alterations in cell cycle dynamics in human endothelium cell culture infected with influenza virus.

The cell cycle of endothelium EAhy 926 cell culture infected with influenza virus has been studied. Cytometric analysis of cell culture synchronized by contact inhibition revealed the elongation of the S phase of the cell cycle in EAhy 926 cells under the influence of influenza virus. This result was shown in an EAhy 926 culture infected with influenza virus and treated with nocodazole. Comparison of a lung carcinoma A549 cell line in which influenza virus causes G0/G1 arrest and of an endothelial EAhy 926 cell line in which the same infection leads to S-phase elongation allows it to be suggested that different effects of influenza virus on cell cycle dynamics depend on the origin of infected cells.

Analysis of the genome structure of influenza A virus strains isolated during the epidemics of 1977-1980.

The genome structure of different A(H1N1) and A(H3N2) influenza virus strains isolated from 1977 to 1980 was analysed by electrophoresis of single-stranded RNA and of the double-stranded RNA obtained by hybridization with complementary RNA of the A/Stavropol 188/79 (H3N2) strain. The differences in genome structure are insignificant within the A(H1N1) subtype, and considerable (involving almost all the 8 RNA fragments) between the two A(H3N2) strains (A/Texas 1/77 and A/Stavropol 188/79). The great differences between the genomes of A(H1N1) and A(H3N2) strains do not exclude certain genetic relationships between these viruses.

Comparative characterization on the segmented RNA structure of influenza viruses A/Singapore 1/57 and A/Hong Kong 1/68.

A comparative characterization of the segmented RNA structure of A/Singapore 1/57 and A/Hong Kong 1/68 influenza viruses was done by polyacrylamide gel electrophoresis. The genomes of the viruses studied consist of 6 segment classes, with a total molecular weight of approximately 4 million daltons. The mathematical method suggested allows to define the number of RNA fragments, which is equal to 8-9, with a total molecular weight of about 6 million daltons. Electrophoretic patterns of the viruses investigated have individual features.