Therapeutic role of N-acetyl cysteine (NAC) for the treatment and/or management of SARS-CoV-2-induced lung damage in hamster model.

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.

Clinical, Virological, Immunological, and Genomic Characterization of Asymptomatic and Symptomatic Cases With SARS-CoV-2 Infection in India.

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.

Long-read 16S-seq reveals nasopharynx microbial dysbiosis and enrichment of Mycobacterium and Mycoplasma in COVID-19 patients: a potential source of co-infection.

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a major global health concern. This virus infects the upper respiratory tract and causes pneumonia-like symptoms. So far, few studies have shown alterations in nasopharyngeal (NP) microbial diversity, enrichment of opportunistic pathogens and their role in co-infections during respiratory infections. Therefore, we hypothesized that microbial diversity changes, with increase in the population of opportunistic pathogens, during SARS-CoV2 infection in the nasopharynx, which may be involved in co-infection in COVID-19 patients. The 16S rRNA variable regions, V1-V9, of NP samples of control and COVID-19 (symptomatic and asymptomatic) patients were sequenced using the Oxford Nanopore™ technology. Comprehensive bioinformatics analysis for determining alpha/beta diversities, non-metric multidimensional scaling, correlation studies, canonical correspondence analysis, linear discriminate analysis, and dysbiosis index were used to analyze the control and COVID-19-specific NP microbiomes. We observed significant dysbiosis in the COVID-19 NP microbiome with an increase in the abundance of opportunistic pathogens at genus and species levels in asymptomatic/symptomatic patients. The significant abundance of Mycobacteria spp. and Mycoplasma spp. in symptomatic patients suggests their association and role in co-infections in COVID-19 patients. Furthermore, we found strong correlation of enrichment of Mycobacteria and Mycoplasma with the occurrences of chest pain and fever in symptomatic COVID-19 patients. This is the first study from India to show the abundance of Mycobacteria and Mycoplasma opportunistic pathogens in non-hospitalized COVID-19 patients and their relationship with symptoms, indicating the possibility of co-infections.

Quantitative proteomics of hamster lung tissues infected with SARS-CoV-2 reveal host factors having implication in the disease pathogenesis and severity.

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.

Identification of multipotent drugs for COVID-19 therapeutics with the evaluation of their SARS-CoV2 inhibitory activity.

The SARS-CoV2 is a highly contagious pathogen that causes COVID-19 disease. It has affected millions of people globally with an average lethality of ~3%. There is an urgent need of drugs for the treatment of COVID-19. In the current studies, we have used bioinformatics techniques to screen the FDA approved drugs against nine SARS-CoV2 proteins to identify drugs for repurposing. Additionally, we analyzed if the identified molecules can also affect the human proteins whose expression in lung changed during SARS-CoV2 infection. Targeting such genes may also be a beneficial strategy to curb disease manifestation. We have identified 74 molecules that can bind to various SARS-CoV2 and human host proteins. We experimentally validated our in-silico predictions using vero E6 cells infected with SARS-CoV2 virus. Interestingly, many of our predicted molecules viz. capreomycin, celecoxib, mefloquine, montelukast, and nebivolol showed good activity (IC50) against SARS-CoV2. We hope that these studies may help in the development of new therapeutic options for the treatment of COVID-19.

Analysis of Indian SARS-CoV-2 Genomes Reveals Prevalence of D614G Mutation in Spike Protein Predicting an Increase in Interaction With TMPRSS2 and Virus Infectivity.

Coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus, has emerged as a global pandemic worldwide. In this study, we used ARTIC primers-based amplicon sequencing to profile 225 SARS-CoV-2 genomes from India. Phylogenetic analysis of 202 high-quality assemblies identified the presence of all the five reported clades 19A, 19B, 20A, 20B, and 20C in the population. The analyses revealed Europe and Southeast Asia as two major routes for introduction of the disease in India followed by local transmission. Interestingly, the19B clade was found to be more prevalent in our sequenced genomes (17%) compared to other genomes reported so far from India. Haplotype network analysis showed evolution of 19A and 19B clades in parallel from predominantly Gujarat state in India, suggesting it to be one of the major routes of disease transmission in India during the months of March and April, whereas 20B and 20C appeared to evolve from 20A. At the same time, 20A and 20B clades depicted prevalence of four common mutations 241 C > T in 5' UTR, P4715L, F942F along with D614G in the Spike protein. D614G mutation has been reported to increase virus shedding and infectivity. Our molecular modeling and docking analysis identified that D614G mutation resulted in enhanced affinity of Spike S1-S2 hinge region with TMPRSS2 protease, possibly the reason for increased shedding of S1 domain in G614 as compared to D614. Moreover, we also observed an increased concordance of G614 mutation with the viral load, as evident from decreased Ct value of Spike and the ORF1ab gene.