Cell and Organism Technologies for Assessment of the SARS-CoV-2 Infectivity in Fluid Environment.

Under conditions of COVID-19 pandemic, considerable amounts of SARS-CoV-2 contained in household, municipal, and medical wastewaters inevitably reach natural water bodies. Possible preservation of virus infectivity in liquid environment is of a paramount epidemiological importance. Experiments demonstrated that SARS-CoV-2 is resistant to multiple freezing/thawing cycles and retains its infectivity in tap and river water for up to 2 days at 20°C and 7 days at 4°C. In natural milk, its viability is preserved in a refrigerator for 6 days. The exposure of aquarium fish to the virus-containing water fails to cause any infection.

Wild Animal Migration As a Potential Threat of Introduction of New Viruses into Russia.

The SARS-CoV-2 pandemic has shown how serious the problem of re-emerging zoonotic infections is for our existence. Migrations of animals, which are natural reservoirs of a particular virus, play a colossal role in the spread of pathogens to new territories. Examples are the migrations of both land animals (carnivores, rodents, and ungulates) and many marine mammals (pinnipeds and cetaceans). Yet the most interesting from the point of view of the speed and range of the spread of viral infections are migrations associated with flights. In nature, these can be migrations of insects, bats, and, of course, birds. Unfortunately, there are very few studies on the migration of these animals in Russia. Considering the problems related to climate change and other environmental factors, it is important to obtain up-to-date data on the changing animal migration routes and, as a consequence, to develop domestic equipment, particularly transmitters, to fix them.

Low incidence of human coronavirus among hospitalized children in Novosibirsk city, Russia during pre-pandemic period (2013-2020).

We investigated the incidence of 15 respiratory viruses among 2991 children with acute respiratory infections in Novosibirsk city, Russia, prior to the COVID-19 pandemic (2013-2020). Viral infections were detected in 72.5% cases. The incidence of human coronavirus was 2% (Alphacoronaviruses, 63%; Betacoronaviruses, 37%).

Development of a Purified Viral Preparation for Studies of COVID-19 (SARS-CoV-2) Biology.

Different methods for producing bulk quantities of concentrated and purified SARS-CoV-2 for the use as antigens and for the research into COVID-19 biology were tested.

Adaptation of the Newcastle Disease Virus to Cell Cultures for Enhancing Its Oncolytic Properties.

This study focuses on the adaptation of natural Newcastle disease virus (NDV) strains isolated from wild birds to human tumor cells. Many candidates for virotherapy are viruses pathogenic for human. During recombination of genetic material, there always exists a risk of getting a virus with an unstable genome. This problem can be solved by using natural apathogenic viruses as oncolytic agents. The Newcastle disease virus is the causative agent of contagious avian diseases. Its natural strains exhibit an antitumor effect and are considered safe for humans. As shown in earlier studies, the oncolytic properties of natural strains can be enhanced during adaptation to cell cultures, without interference in the virus genome. This study demonstrates that serial passaging increases the viral infectious titer in cancer cells. Moreover, the viability of tumor cells decreases post-infection when Newcastle disease virus strains are adapted to these cell cultures. The findings of this study complement the well-known data on the adaptation of the Newcastle disease virus to human cancer cells. Hence, it is possible to obtain a NDV strain with a more pronounced oncolytic potential during adaptation. This should be taken into account when choosing a strategy for designing anticancer drugs based on this virus.

Death Mechanisms of Pulmonary Alveolocytes in Mice Infected with Influenza Viruses A/H1N1/California/04/2009 and A/H5N1/Goose/Krasnoozerskoye/627/05.

In CBA mice infected with influenza viruses A/H1N1/California/04/2009 and A/H5N1/Goose/Krasnoozerskoye/627/05 in a dose of 10 MLD50, the mechanisms of death of pulmonary alveolocytes over 10 postinfection days were studied by light microscopy, immunohistochemistry, and morphometry. In mice infected with A/H1N1, alveolocytes died predominantly via necrosis, while apoptosis mostly employed the mitochondrial pathway. In mice infected with A/H5N1, apoptosis was the dominant mechanism of alveolocyte death proceeded via membrane receptor signaling followed by switching to FAS-mediated pathway via activation of FADD, the apoptotic signal transduction protein.

Changes in the Structure of Mouse Kidney in the Acute Period after Infection with Influenza Viruses A/H5N1 and A/H1N1.

Changes in the kidney structure in outbred and inbred male BALB/c mice were analyzed in the acute period after infection with influenza viruses A/H5N1 (10 MLD50; 10 days) and A/H1N1 (1 MLD50; 30 days). Antibodies to influenza viruses of both strains were most often expressed by endothelial cells of the glomeruli and arterioles and were rarely expressed by mesangiocytes and tubule epithelial cells. In the kidney, destructive processes induced by viruses and by ischemia due to massive blood vessel thrombosis. Mesangiocytes expressed factors, indicating that they could be qualified as M1 and M2 macrophages. Kidney destruction was more significant after infection of mice with the A/H5N1 virus, but in both experiments cell infiltrates were actually absent, probably due to blood vessel thrombosis and limited possibility of migration of mononuclear phagocytes and lymphocytes to the kidney.

Study of Antiviral Efficiency of Oxidized Dextrans In Vitro and In Vivo.

Antiviral efficiency of oxidized dextrans (OD) with different molecular weights and oxidation degree (OD40min, OD70min, OD40max, and OD70 max) was studied in vitro and in vivo. Dextrans OD40max and OD70max prevented the development of the cytopathic effect of influenza A(H1N1)pdm09 virus in more than 50% MDCK cells vs. control (no OD). Four intranasal doses of OD40min, OD40max, and OD70min and one intranasal dose of OD70max before infection of BALB/c mice with A(H1N1)pdm09 influenza virus significantly reduced mortality and prolonged life span in comparison with controls receiving saline. These and our previous data attest to clear-cut preventive effect of OD in influenza infection.

Preventive Effects of Oxidized Dextran on Functional Activity of Pulmonary Macrophages in Mice Infected with Influenza A Virus.

We analyzed cytokine profile of pulmonary macrophages in mice infected with highly pathogenic influenza A/H5N1 virus after preventive injections of oxidized dextran. Light microscopy, immunohistochemistry, and morphometric examinations showed that preventive injections of oxidized dextran led to more effective virus elimination, modulation of the proinflammatory cytokine response, and host antiviral response and reduce animal mortality. Our findings allow recommending oxidized dextran for further studies in order to create a vaccine with antiviral and adjuvant potencies.

Study of SMAD-Dependent Signal Pathway in the Development of Early Pulmonary Fibrosis in Mice Infected with Influenza A/H1N1 Virus.

Early fibrosis of the visceral organs is one of the main complications of infection caused by influenza A virus. Structural manifestations and molecular regulators of the epithelialmesenchymal transformation as a possible mechanism of fibrosis progression were studied in mice infected with influenza A/H1N1 A/Tomsk/13/2010 virus. We found early fibrosis of the lungs against the background of minor changes in fibroblast count. However, enhanced expression of TGF-ß and SMAD-2 by macrophages and alveolocytes attested to possible development of epithelial-mesenchymal transformation and its contribution to activation of fibrogenesis process in the lungs.

Pathogenicity assessment of wild-type and mouse-adapted influenza A(H1N1) pdm09 viruses in comparison with highly pathogenic influenza A(H5N1) virus.

Here we compare the results of pathological and virological examinations of mice experimentally infected with either wild-type or mouse-adapted pandemic A(H1N1) pdm09 viruses and highly pathogenic avian influenza (HPAI) virus A(H5N1). Mice were sacrificed on days 1, 3, 6, and 10 post infection or whenever morbidity was severe enough to justify euthanasia. Morbidity rates were calculated on the basis of clinical signs (weight loss, poor hair coat, hunched posture and paresis); virus-induced disease was characterised by the histopathology of lung; virus dissemination was determined by virus isolation on organ samples of lung, brain, liver, kidney and spleen. All mice infected with mouse-adapted A(H1N1) pdm09 died in the course of the experiment, whereas 20% of animals survived the infection with A(H5N1). Echinocyte formation changed the rheological properties of blood in animals infected with either mouse-adapted A(H1N1) pdm09 or A(H5N1). To sum up, the adaptation of pandemic A(H1N1) pdm09 virus can confer an enhanced virulence similar to or even exceeding that of HPAI A(H5N1) virus.

Serological Detection of Causative Agents of Infectious and Invasive Diseases in the Beluga Whale Delphinapterus leucas (Pallas, 1776) (Cetacea: Monodontidae) from Sakhalinsky Bay.

Serological detection of some pathogens in the beluga whale Delphinapterus leucas population from Sakhalinsky Bay of the Sea of Okhotsk (Sakhalin-Amur beluga whale stock) was performed in 2013-2014 after the largest recorded flood of the Amur River (among observations since 1896). The percent of this population that is immune to the causative agents of clonorchosis was 25.6%; toxoplasmosis, 11.5%; brucellosis, 38.5%; tuberculosis, 30.8%; cetacean morbillivirus infection, 16.7%; and to alpha and gamma herpes viruses each, 21.8%.

Studies of Influenza A/H1N1 A/Tomsk/13/2010 Virus Topology during Development of Infectious Process in Mammals.

Influenza A/H1N1 A/Tomsk/13/2010 virus registered in Siberia in 2010 proved to be an extremely pathogenic strain. Dynamic study of the topology of this influenza virus strain in the lungs, liver, kidneys, lymph nodes, and great vessels of infected mice was carried out. Influenza A virus was detected by immunohistochemical methods in cells of different histogenesis in all the studied organs throughout the observation period (days 1-30 postinfection), which indicated effective replication and long persistence of influenza A/H1N1 A/Tomsk/13/2010 virus in mammalian cells.

Adaptation of influenza A(H1N1)pdm09 virus in experimental mouse models.

In the present study, three mouse-adapted variants of influenza A(H1N1)pdm09 virus were obtained by lung-to-lung passages of BALB/c, C57BL/6z and CD1 mice. The significantly increased virulence and pathogenicity of all of the mouse-adapted variants induced 100% mortality in the adapted mice. Genetic analysis indicated that the increased virulence of all of the mouse-adapted variants reflected the incremental acquisition of several mutations in PB2, PB1, HA, NP, NA, and NS2 proteins. Identical amino acid substitutions were also detected in all of the mouse-adapted variants of A(H1N1)pdm09 virus, including PB2 (K251R), PB1 (V652A), NP (I353V), NA (I106V, N248D) and NS1 (G159E). Apparently, influenza A(H1N1)pdm09 virus easily adapted to the host after serial passages in the lungs, inducing 100% lethality in the last experimental group. However, cross-challenge revealed that not all adapted variants are pathogenic for different laboratory mice. Such important results should be considered when using the influenza mice model.

Evolution of influ- 245 enza A/H5N1 virus (1996-2016).

Twenty years ago in the South Chinese province of Guangdong the epizooty of highly pathogenic avian influenza (HPAI) H5N1 virus, which has laid the foundation of the largest epizooty in the contemporary history, has flashed. Hemagglutinin of prototype A/goose/Guangdong/1/1996 (H5N1) changing many times and generating new genetic subgroups participated in various reassortations; it still exists today. The present review is devoted to the retrospective analysis of HPAI/H5N1evolution for the last twenty years in the territory of Eurasia, Africa and America. The basis for the discussion is ecological model according to which new genetic variants are formed in the migration pathways with close contacts between different bird populations and in the overwintering areas where the maximum values of the immune layer occur; amplification of virus variants occurs in nesting areas among juvenile populations. The updated system of designations of genetic groups introduced by WHO/OIE/FAO H5 Evolution Working Group in 2015 is used.

Effects of preventive administration of oxidized dextran on liver injury and reparative regeneration in mice infected with influenza A/H5N1 virus.

Intranasal infection of outbred male mice with influenza A/H5N1 A/goose/Krasnoozerskoye/627/05 virus led to high (85%) mortality of animals. Morphological studies of liver specimens showed destructive changes in the parenchyma (93.5% hepatocytes), caused by long persistence of the virus in the liver. The virus persistence was conjugated with activation of cellular immunity, manifesting by an increase in the counts of cells with high expression of proinflammatory cytokines (TNF-α) and lysosomal enzymes (lysozyme, cathepsin D). Injections of oxidized dextran 3 and 1 days before infection reduced mortality and 2-fold attenuated destructive changes in the liver, presumably due to prevention of virus penetration into the target cells, modulation of immune reactions, and stimulation of reparative plastic processes.

Experimental study of the efficiency of oxidized dextran for prevention of influenza A/H5N1.

Oxidized dextran is suggested for prevention of infection induced by influenza A/H5N1 viruses, methods of its use and doses are determined. Two intravenous injections of dextran 3 and 1 days before experimental infection of outbred mice by influenza A/H5N1 A/goose/Krasnoozerskoye/627/05 virus resulted in a high preventive dose-dependent effect: the mean lifespan was 25% prolonged, the mortality decreased 3-fold.

Expression of profibrotic growth factors and their receptors by mouse lung macrophages and fibroblasts under conditions of acute viral inflammation in influenza A/H5N1 virus.

Morphological signs of early interstitial fibrosis, developing under conditions of acute viral inflammation (postinfection days 1-14), were observed in C57Bl/6 mice infected with influenza A/H5N1 A/goose/Krasnoozerskoye/627/05 virus. The development of fibrosis was confirmed by an increase in the number of lung cells expressing TNF-α. These changes were recorded in the presence of a many-fold increase in the counts of macrophages and fibroblasts expressing FGF, EGF, and their receptors.

Biological characteristics of influenza A(H1N1)pdm09 virus circulating in West Siberia during pandemic and post-pandemic periods.

We studied biological characteristics of influenza A(H1N1)pdm09 virus circulating in Siberia during the 2009 pandemic and the post-pandemic period of 2011. BALB/c mice were chosen as the experimental model. Virus titers in the lungs were evaluated on days 1, 3, 6 and blood serum titers on day 15 after infection with different strains. Blood sera of convalescents after influenza of 2010-2011 epidemic season were analyzed. Influenza A(H1N1)pdm09 virus strains isolated during the post-pandemic period of 2011 were characterized by low epidemic activity and virulence in comparison with the strains isolated during 2009 pandemic period, which indicates completion of the pandemic cycle.

Role of matrix metalloproteinases and their inhibitor in the development of early pulmonary fibrosis in mice infected with influenza A/H5N1 A/goose/Krasnoozerskoye/627/05 virus.

High levels of macrophages and fibroblasts expressing MMP-2, MMP-9, and MMP-10 against the background of progressing early fibrosis of the lungs (manifesting in an increase in volume density of type I, III, IV, and VI collagens) were found in C57Bl/6 mice infected with influenza A/H5N1 A/goose/Krasnoozerskoye/627/05 virus. Progressing fibrosis of the lungs in infected mice was associated with imbalance of collagen synthesis and degradation processes conjugated with high levels of macrophages and fibroblasts expressing TIMP-2.