ABSTRACT
Oxidative stress by reactive oxygen species (ROS) has been hypothesized to be the major mediator of SARS-CoV-2-induced pathogenesis. During infection, the redox homeostasis of cells is altered as a consequence of virus-induced cellular stress and inflammation. In such scenario, high levels of ROS bring about the production of pro-inflammatory molecules like IL-6, IL-1ß, etc. that are believed to be the mediators of severe COVID-19 pathology. Based on the known antioxidant, anti-inflammatory, mucolytic and antiviral properties of NAC, it has been hypothesized that NAC will have beneficial effects in COVID-19 patients. In the current study efforts have been made to evaluate the protective effect of NAC in combination with remdesivir against SARS-CoV-2 induced lung damage in the hamster model. The SARS-CoV-2 infected animals were administered with high (500 mg/kg/day) and low (150 mg/kg/day) doses of NAC intraperitoneally with and without remdesivir. Lung viral load, pathology score and expression of inflammatory molecules were checked by using standard techniques. The findings of this study show that high doses of NAC alone can significantly suppress the SARS-CoV-2 mediated severe lung damage (2 fold), but on the contrary, it fails to restrict viral load. Moreover, high doses of NAC with and without remdesivir significantly suppressed the expression of pro-inflammatory genes including IL-6 (4.16 fold), IL-1ß (1.96 fold), and TNF-α (5.55 fold) in lung tissues. Together, results of this study may guide future preclinical and clinical attempts to evaluate the efficacy of different doses and routes of NAC administration with or without other drugs against SARS-CoV-2 infection.
ABSTRACT
The emergence of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) as a serious pandemic has altered the global socioeconomic dynamics. The wide prevalence, high death counts, and rapid emergence of new variants urge for the establishment of research infrastructure to facilitate the rapid development of efficient therapeutic modalities and preventive measures. In agreement with this, SARS-CoV-2 strains were isolated from patient swab samples collected during the first COVID-19 wave in Odisha, India. The viral isolates were adapted to in vitro cultures and further characterized to identify strain-specific variations in viral growth characteristics. The neutralization susceptibility of viral isolates to vaccine-induced antibodies was determined using sera from individuals vaccinated in the Government-run vaccine drive in India. The major goal was to isolate and adapt SARS-CoV-2 viruses in cell culture with minimum modifications to facilitate research activities involved in the understanding of the molecular virology, host-virus interactions, drug discovery, and animal challenge models that eventually contribute toward the development of reliable therapeutics.
ABSTRACT
Purpose: The current global pandemic of coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), led to the investigation with clinical, biochemical, immunological, and genomic characterization from patients to understand the pathophysiology of viral infection. Methods: Samples were collected from six asymptomatic and six symptomatic SARS-CoV-2-confirmed hospitalized patients in Bhubaneswar, Odisha, India. Clinical details, biochemical parameters, and treatment regimen were collected from a hospital; viral load was determined by RT-PCR; and the levels of cytokines and circulating antibodies in plasma were assessed by Bio-Plex and isotyping, respectively. In addition, whole-genome sequencing of viral strains and mutational analysis were carried out. Results: Analysis of the biochemical parameters highlighted the increased levels of C-reactive protein (CRP), lactate dehydrogenase (LDH), serum SGPT, serum SGOT, and ferritin in symptomatic patients. Symptomatic patients were mostly with one or more comorbidities, especially type 2 diabetes (66.6%). The virological estimation revealed that there was no significant difference in viral load of oropharyngeal (OP) samples between the two groups. On the other hand, viral load was higher in plasma and serum samples of symptomatic patients, and they develop sufficient amounts of antibodies (IgG, IgM, and IgA). The levels of seven cytokines (IL-6, IL-1α, IP-10, IL-8, IL-10, IFN-α2, IL-15) were found to be highly elevated in symptomatic patients, while three cytokines (soluble CD40L, GRO, and MDC) were remarkably higher in asymptomatic patients. The whole-genome sequence analysis revealed that the current isolates were clustered with 19B, 20A, and 20B clades; however, 11 additional changes in Orf1ab, spike, Orf3a, Orf8, and nucleocapsid proteins were acquired. The D614G mutation in spike protein is linked with higher virus replication efficiency and severe SARS-CoV-2 infection as three patients had higher viral load, and among them, two patients with this mutation passed away. Conclusions: This is the first comprehensive study of SARS-CoV-2 patients from India. This will contribute to a better understanding of the pathophysiology of SARS-CoV-2 infection and thereby advance the implementation of effective disease control strategies.
Subject(s)
COVID-19 , Diabetes Mellitus, Type 2 , Genomics , Humans , Pandemics , SARS-CoV-2ABSTRACT
The Syrian golden hamster (Mesocricetus auratus) has recently been demonstrated as a clinically relevant animal model for SARS-CoV-2 infection. However, lack of knowledge about the tissue-specific expression pattern of various proteins in these animals and the unavailability of reagents like antibodies against this species hampers these models' optimal use. The major objective of our current study was to analyze the tissue-specific expression pattern of angiotensin-converting enzyme 2, a proven functional receptor for SARS-CoV-2 in different organs of the hamster. Using two different antibodies (MA5-32307 and AF933), we have conducted immunoblotting, immunohistochemistry, and immunofluorescence analysis to evaluate the ACE2 expression in different tissues of the hamster. Further, at the mRNA level, the expression of Ace2 in tissues was evaluated through RT-qPCR analysis. Both the antibodies detected expression of ACE2 in kidney, small intestine, tongue, and liver. Epithelium of proximal tubules of kidney and surface epithelium of ileum expresses a very high amount of this protein. Surprisingly, analysis of stained tissue sections showed no detectable expression of ACE2 in the lung or tracheal epithelial cells. Similarly, all parts of the large intestine were negative for ACE2 expression. Analysis of tissues from different age groups and sex didn't show any obvious difference in ACE2 expression pattern or level. Together, our findings corroborate some of the earlier reports related to ACE2 expression patterns in human tissues and contradict others. We believe that this study's findings have provided evidence that demands further investigation to understand the predominant respiratory pathology of SARS-CoV-2 infection and disease.
ABSTRACT
Syrian golden hamsters (Mesocricetus auratus) infected by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) manifests lung pathology. In this study, efforts were made to check the infectivity of a local SARS-CoV-2 isolate in a self-limiting and non-lethal hamster model and evaluate the differential expression of lung proteins during acute infection and convalescence. The findings of this study confirm the infectivity of this isolate in vivo. Analysis of clinical parameters and tissue samples show the pathophysiological manifestation of SARS-CoV-2 infection similar to that reported earlier in COVID-19 patients and hamsters infected with other isolates. However, diffuse alveolar damage (DAD), a common histopathological feature of human COVID-19 was only occasionally noticed. The lung-associated pathological changes were very prominent on the 4th day post-infection (dpi), mostly resolved by 14 dpi. Here, we carried out the quantitative proteomic analysis of the lung tissues from SARS-CoV-2-infected hamsters on day 4 and day 14 post-infection. This resulted in the identification of 1585 proteins of which 68 proteins were significantly altered between both the infected groups. Pathway analysis revealed complement and coagulation cascade, platelet activation, ferroptosis, and focal adhesion as the top enriched pathways. In addition, we also identified altered expression of two pulmonary surfactant-associated proteins (Sftpd and Sftpb), known for their protective role in lung function. Together, these findings will aid in understanding the mechanism(s) involved in SARS-CoV-2 pathogenesis and progression of the disease.