ABSTRACT
BackgroundSARS-CoV2 infection in patients with comorbidities, particularly T2DM has been a major challenge globally. Here, we did whole blood immunophenotyping along with plasma cytokine, chemokine, antibody isotyping and viral load determination from oropharyngeal swab to understand the immune pathology in the T2DM patients infected with SARS-CoV2. MethodsBlood samples from 25 Covid-19 positive patients having T2DM, 10 Covid-19 positive patients not having T2DM and 10 Covid-19 negative, non-diabetic healthy controls were assessed for various immune cells by analyzing for their signature surface proteins in mass cytometry. Circulating cytokines, chemokines and antibody isotypes were determined from plasma. Viral copy number was determined from oropharyngeal swabs. All our representative data corroborated with laboratory findings. ResultsOur observations encompass T2DM patients having elevated levels of both type I and type II cytokines and higher levels of circulating IgA, IgM, IgG1 and IgG2 as compared to NDM and healthy volunteers. They also displayed higher percentages of granulocytes, mDCs, plasmablasts, Th2-like cells, CD4+ EM cells, CD8+ TE cells as compared to healthy volunteers. T2DM patients also displayed lower percentages of pDCs, lymphocytes, CD8+ TE cells, CD4+, CD8+ EM. ConclusionOur study demonstrated that patients with T2DM displayed higher inflammatory markers and a dysregulated anti-viral and anti-inflammatory response when compared to NDM and healthy controls. Contribution to the fieldCovid-19 infection in people with comorbidities, particularly T2DM has been a cause of mortality in several nations and they represent an extremely vulnerable population to Covid-19. This study is one of the most comprehensive study from India, to understand the interplay between immune response and viremia occurring in these T2DM patients infected with SARS-CoV2 and will help in designing public health response and vaccination priorities.
ABSTRACT
Emergence of 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 establishment of research infrastructure to facilitate rapid development of efficient therapeutic modalities and preventive measures. In agreement with this, five SARS-CoV2 strains (ILS01, ILS02, ILS03, ILS15 and ILS24) of four different clades (19A, 19B, 20A and 20B) were isolated from patient swab samples collected during the 1st 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. All the five isolates showed substantial amount of virus induced CPE however ILS03 belonging to 20A clade displayed highest level of CPE. Time kinetics experiment revealed spike protein expression was evident after 16th hours post infection in all five isolates. ILS03 induced around 90% of cytotoxicity. Further, the susceptibility of various cell lines (human hepatoma cell line (Huh-7), CaCo2 cell line, HEK-293T cells, Vero, Vero-E6, BHK-21, THP-1 cell line and RAW 264.7 cells) were assessed. Surprisingly, it was found that the human monocyte cells THP-1 and murine macrophage cell line RAW 264.7 were permissive to all the SARS-CoV-2 isolates. 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 micro-neutralization assay suggested that both Covaxin and Covishield vaccines were equally effective (100% neutralization) against all of the isolates. The whole genome sequencing of culture adapted viral isolates and viral genome from patient oropharyngeal swab sample suggested that repetitive passaging of SARS-CoV2 virus in Vero-E6 cells did not lead to emergence of many mutations during the adaptation in cell culture. Phylogenetic analyses revealed that the five isolates clustered to respective clades. The major goal was to isolate and adapt SARS-CoV-2 viruses in in-vitro cell culture with minimal modification to facilitate research activities involved in understanding the molecular virology, host-virus interactions, application of these strains for drug discovery and animal challenge models development which eventually will contribute towards the development of effective and reliable therapeutics.
ABSTRACT
BackgroundThe current global pandemic of Coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2 led to the investigation with clinical, biochemical, immunological and genomic characterization from the patients to understand the pathophysiology of viral infection. MethodsSamples were collected from six asymptomatic and six symptomatic SARS-CoV-2 confirmed hospitalized patients in Bhubaneswar, Odisha, India. Clinical details, biochemical parameters, treatment regime were collected from hospital, viral load was determined by RT-PCR, levels of cytokines and circulating antibodies in plasma were assessed by Bioplex and isotyping respectively. In addition, the whole genome sequencing of viral strains and mutational analysis were carried out. FindingsAnalysis 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 indicating that patients with higher levels of few biochemical parameters might experience severe pathophysiological complications after SARS-CoV-2 infection. This was also observed that symptomatic patients were mostly with one or more comorbidities, especially diabetes (66.6%). Surprisingly the virological estimation revealed that there was no significant difference in viral load of oropharyngeal (OP) samples between the two groups. This suggests that the viral load in OP sample does not correlate with the disease severity and both asymptomatic and symptomatic patients are equally capable of transmitting the virus. Whereas, viral load was higher in plasma and serum samples of symptomatic patients suggesting that the development of clinical complications is mostly associated to high viral load in plasma and serum. This also demonstrated that the patients with high viral load in plasma and serum samples were found to develop sufficient amounts of antibodies (IgG, IgM and IgA). Interestingly, the levels of 7 cytokines (IL-6, IL-.1, IP-10, IL-8, IL-10, IFN-2, 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. Therefore, this data suggest that cytokines and chemokines may serve as "predictive indicator" of SARS-CoV-2 infection and contribute to understand the pathogenesis of COVID-19. The whole genome sequence analysis revealed that the current isolates were clustered with 19B, 20A and 20B clades, however acquired 11 additional changes in Orf1ab, spike, Orf3a, Orf8 and nucleocapsid proteins. The data also confirmed that the D614G mutation in spike protein is mostly 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. InterpretationThis 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 advance in the implementation of effective disease control strategies. FundingThis study was supported by the core funding of Institute of Life Sciences, Bhubaneswar, Dept of Biotechnology, India. Research in contextO_ST_ABSEvidence before this studyC_ST_ABSAsymptomatic patients are a source of concern as measures taken to control the spread of the virus are severely impacted by their undetectability. Presently, there is an inadequate information about the characteristics of the asymptomatic and symptomatic patients. The association between SARS-CoV-2 viral load, cytokines and risk of disease progression remains unclear in COVID-19 in Indian scenario. PubMed was searched for articles published up to May, 2021, using the keywords "SARS CoV-2 patients in India", or "2019 novel coronavirus patients in India". No published work about the patients data on SARS CoV-2 in Indian scenario could be identified. Added value of this studyThis investigation highlights the ability of both asymptomatic and symptomatic patients to transmit the virus equally. This study also demonstrates that the D614G mutation in the spike protein is associated with severe SARS-CoV-2 infection and enhance levels of inflammatory markers such as CRP and ferritin which can be predictive biomarkers for critical condition of patients. This is the first comprehensive study of SARS CoV-2 patients from India and will contribute to a better understanding of the pathophysiology of SARS-CoV-2 infection by advancing the implementation of effective disease control strategies. Implications of all the available evidenceThe current global pandemic of Coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2 led to the investigation with clinical, biochemical, immunological and viral genome sequencing to understand the pathophysiology of this virus infection. Samples were collected from six asymptomatic and six symptomatic SARS-CoV-2 confirmed hospitalized patients in Bhubaneswar, Odisha, India. This investigation highlights the ability of both asymptomatic and symptomatic patients to transmit the virus equally. This also demonstrated that the D614G mutation is mostly associated with higher virus replication capacity and severe SARS-CoV-2 infection and enhanced levels of inflammatory markers such as CRP and ferritin which are associated with critical conditions of patients. This is the first comprehensive study of SARS CoV-2 patients from India and will contribute to a better understanding of the pathophysiology of SARS-CoV-2 infection by advancing the implementation of competent disease control strategies.