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Given the rapid spread of coronavirus disease 2019 (COVID-19) globally, test systems are needed for its diagnosis, timely treatment, and introduction of quarantine measures. In the shortest possible time, a diagnostic system based on real-time reverse-transcription polymerase chain reaction to detect the ribonucleic acid of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in nasopharyngeal and oropharyngeal smears was developed and registered. The method determines the nucleocapsid and small-membrane protein genes and the human PGK1 gene, acting as internal control reactions. The nucleotide sequences of SARS-CoV-2 were analyzed, and primers were selected. The conditions for carrying out real-time reverse-transcription polymerase chain reaction and the composition of a set of reagents were set. The diagnostic sensitivity and specificity of the kit were tested on biological samples, with the addition of inactivated SARSCoV-2. The high analytical characteristics of the developed set of reagents were demonstrated, with a sensitivity of at least 103 GE/mL and a specificity of 100%, and no false-positive or false-negative results were recorded. The high specificity of the test system was shown on a representative sample of genetic materials of respiratory viral pathogens. Clinical and laboratory tests of the diagnostic "SARS-CoV-2 test” were conducted in the N.F. Gamalei National Research Center for Epidemiology and Microbiology. A set of reagents for the detection of ribonucleic acid of SARS-CoV-2 through on real-time reverse-transcription polymerase chain reaction for in vitro diagnostics "SARS-CoV-2 test” was registered in the Russian Federation as a medical device (Registration certificate no. RZN 2020/10632, dated 06/03/2020). The article can be used under the CC BY-NC-ND 4.0 license © Authors, 2022.
ABSTRACT
The development of coronavirus infection outbreak into a pandemic, coupled with the lack of effective COVID-19 therapies, is a challenge for the entire pharmaceutical industry. This study aimed to assess the treatment and preventive efficacy of the amino acid-peptide complex (APC) in male Syrian hamsters infected with SARSCoV-2 (intranasal administration of 26 mul of the virus culture, titer of 4 x 104 TCD50/ml). In a modeled COVID-19 case, APC administered for treatment and preventive purposes reduced lung damage. Compared to the positive control group, test group had the lung weight factor 15.2% smaller (trend), which indicates a less pronounced edema. Microscopic examination revealed no alveolar edema, atypical hypertrophied forms of type II alveolocytes, pulmonary parenchyma fibrinization. The macrophage reaction intensified, which is probably a result of the APC-induced activation of regenerative processes in the lung tissues. Spleens of the animals that received APC for therapeutic and preventive purposes were less engorged and had fewer hemorrhages. The decrease of body weight of the test animals that received APC for treatment and prevention was insignificant (p < 0.05), which indicates a less severe course of COVID-19. Administered following a purely therapeutic protocol, APC proved ineffective against SARS-CoV-2 post-infection. Thus, APC-based drug used as a therapeutic and preventive agent reduces pulmonary edema and makes morphological signs of lung tissue damage less pronounced in male Syrian hamsters infected with SARS-CoV-2.Copyright © Extreme Medicine.All right reserved.
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INTRODUCTION: Convalescent COVID-19 patients have various signs of central nervous system damage, including those directly associated with SARS-CoV-2. Hence, studies of SARS-COV-2 related morphological changes in neocortex are particularly relevant for understanding the mechanisms of their formation and development of approaches to preclinical evaluation of the effectiveness of antiviral drugs. The purpose of the research is a longitudinal study of the ultrastructural alterations in Syrian hamsters neocortex after experimental SARS-CoV-2 infection. MATERIALS AND METHODS: Male Syrian hamsters weighing 80100 g, aged 4 to 6 weeks, were infected with 26 l SARS-CoV-2 intranasally with 4104 TCD50/ml of viral particles. The animals were euthanized on days 3, 7 or 28 post-infection, the brain was extracted with the cortex excision. The material analysis was performed using transmission electron microscopy. RESULTS AND DISCUSSION: On day 3 post-infection, the number of moderately hyperchromic neurons in neocortex increased, while by the day 7 the number of apoptotic cells significantly increased. Simultaneously, an increased signs of neuronophagy and representation of atypical glia were observed. Increased number of altered oligodendrocytes was observed on day 28 post-infection. Viral invasion was accompanied by changes in neocortical cells since day 3 post-infection, such as transformation of their nucleus, the rough endoplasmic reticulum and the Golgi vesicles as well as microvascular spasm with perivascular edema. CONCLUSION: As a result of electron microscopic study, the ultrastructural alterations in neocortex were described in an experimental model of SARS-CoV-2 infection. The findings can be used to identify the mechanisms of infection pathogenesis and to search for the new directions in development of medicines.
Subject(s)
COVID-19 , Coronaviridae , Neocortex , Severe acute respiratory syndrome-related coronavirus , Cricetinae , Animals , Male , SARS-CoV-2 , Mesocricetus , Longitudinal Studies , Electrons , Disease Models, AnimalABSTRACT
INTRODUCTION: Verification of histological changes in respiratory system using Syrian (golden) hamsters (Mesocricetus auratus) as experimental model is an important task for preclinical studies of drugs intended for prevention and treatment of the novel coronavirus infection COVID-19.The aim of this work was to study pathological changes of pulmonary tissue in SARS-CoV-2 (Coronaviridae: Coronavirinae: Betacoronavirus; Sarbecovirus) experimental infection in Syrian hamsters. MATERIAL AND METHODS: Male Syrian hamsters weighting 80-100 g were infected by intranasal administration of culture SARS-CoV-2 at dose 4 × 104 TCID50/ml (TCID is tissue culture infectious dose). Animals were euthanatized on 3, 7 and 14 days after infection, with gravimetric registration. The viral load in lungs was measured using the polymerase chain reaction (PCR). Right lung and trachea tissues were stained with hematoxylin-eosin and according to Mallory. RESULTS AND DISCUSSION: The highest viral replicative activity in lungs was determined 3 days after the infection. After 7 days, on a background of the decrease of the viral load in lungs, a pathologically significant increase of the organ's gravimetric parameters was observed. Within 3 to 14 days post-infection, the lung histologic pattern had been showing the development of inflammation with a succession of infiltrative-proliferative, edematousmacrophagal and fibroblastic changes. It was found that initial changes in respiratory epithelium can proceed without paranecrotic interstitial inflammation, while in the formation of multiple lung parenchyma lesions, damage to the epithelium of bronchioles and acinar ducts can be secondary. The appearance of epithelioid large-cell metaplastic epithelium, forming pseudoacinar structures, was noted as a pathomorphological feature specific to SARS-CoV-2 infection in Syrian hamsters. CONCLUSION: As a result of the study, the specific features of the pathology of the respiratory system in SARSCoV-2 infected Syrian hamsters were described. These findings are of practical importance as reference data that can be used for preclinical studies to assess the effectiveness of vaccines and potential drugs.
Subject(s)
COVID-19 , Lung/pathology , Lung/virology , Mesocricetus , Animals , Coronaviridae , Disease Models, Animal , Inflammation , Male , Mesocricetus/immunology , Mesocricetus/virology , SARS-CoV-2ABSTRACT
Introduction. Amid the pandemic of the new coronavirus infection COVID-19, experimental models for screening both newly developed vaccines and drugs, and the already registered active pharmaceutical ingredients tested for new indications are in high demand. The registration of changes in biometric and morphological parameters that are significant for the investigative pathology can be an optimal screening tool for antiviral drugs and vaccines to treat COVID-19. In this regard, the purpose of this study was to describe the changes over time of organ and tissue changes using a biological model of a SARS-CoV-2-associated infection in the golden hamsters Mesocricetus auratus. Materials and methods. We performed the study in golden hamsters Mesocricetus auratus weighing 80–100 g. The animals were infected through intranasal administration of a culture of SARS-CoV-2 virus containing 4 × 104 TCID50 /mL. We recorded the animals’ weight before the infection and during the next 14 days and measured the specific gravity of the internal organs and the degree of their moisture saturation on days 3, 7, and 14 after the infection. Additionally, tissue samples were fixed in 10% neutral formalin;the preparation of histological specimens was performed according to the standard procedure. Statistical data processing was carried out using non-parametric tests. Results. In the model of SARS-CoV-2 infection in Mesocricetus auratus, we observed the manifestation of the infectious disease after 3 days. The most pronounced pathological changes in the overall health status of the animals and in the histology of internal organs were seen 7 days after the infection. We determined the weight loss and significant deviations in gravimetric coefficients of lungs, heart, spleen, and kidneys to be the indicators revealing the infectious disease course changes over time. Histologic evaluation showed typical changes in the SARS-CoV-2-associated visceral damage: the formation of polymorphonuclear cell infiltrates in the lung interalveolar septa and patchy dystrophic changes in the neurocytes of the brain screen-type centers with the demyelination of the commissural nerve guides. The detected pathological manifestations corresponded in time to an increase in the virus replicative activity in the lungs. Conclusion. The experimental model of the SARS-CoV-2-associated infection in golden hamsters Mesocricetus auratus can be recommended for screening experimental (preclinical) studies of the promising drugs’ efficacy for pathogenetic and etiotropic therapy of COVID-19. © 2021, MDV Group. All rights reserved.
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The characteristics of the biology of influenza viruses and coronavirus that determine the implementation of the infectious process are presented. With provision for pathogenesis of infection possible effects of serine proteinase inhibitors, heparin, and inhibitors of heparan sulfate receptors in the prevention of cell contamination by viruses are examined. It has been determined that chelators of metals of variable valency and antioxidants should be used for the reduction of replicative activity of viruses and anti-inflammatory therapy. The possibility of a pH-dependent impairment of glycosylation of cellular and viral proteins was traced for chloroquine and its derivatives. The use of low-toxicity drugs as part of adjunct therapy increases the effectiveness of synthetic antiviral drugs and interferons and ensures the safety of baseline therapy.
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The biology features of influenza viruses and coronaviruses that determine the implementation of the infectious process are described. Taking into account the pathogenesis of infection, the possible effects of serine proteinase blockers, heparin and heparan sulfate receptor blockers in the prevention of cell contamination by viruses are considered. The necessity of using chelators of metals of variable valency and antioxidants to reduce the replicative activity of viruses and anti-inflammatory therapy is determined. The possibility of a pH-dependent violation of the glycosylation of cellular and viral proteins is traced for chloroquine and its derivatives. The use of low-toxic registered drugs as part of adjuvant therapy increases the effectiveness of antiviral synthetic drugs and interferons, ensures the safety of the use of basic therapy. Изложены особенности биологии вирусов гриппа и коронавирусов, определяющие реализацию инфекционного процесса. С учетом патогенеза инфекции рассмотрены возможные эффекты блокаторов сериновых протеиназ, гепарина и блокаторов гепарансульфатных рецепторов в профилактике контаминации клеток вирусами. Определена необходимость применения хелаторов металлов переменной валентности и антиоксидантов для снижения репликативной активности вирусов и противовоспалительной терапии. Возможность pH-зависимого нарушения гликозилирования клеточных и вирусных белков прослежена для хлорохина и его производных. Применение низкотоксичных лекарственных средств в составе вспомогательной терапии повышает эффективность противовирусных синтетических препаратов и интерферонов, обеспечивает безопасность применения средств базисной терапии.