Transgenic Mouse Models Establish a Protective Role of Type 1 IFN Response in SARS-CoV-2 infection-related Immunopathology

Type 1 interferon (IFN-I) response is the first line of host defense against invading viruses. In the absence of definite mouse models, the role of IFN-I in SARS-CoV-2 infections remained to be perplexing. Here, we developed two mouse models, one with constitutively high IFN-I response (hACE2; Irgm1-/-) and the other with dampened IFN-I response (hACE2; Ifnar1-/-) to comprehend the role of IFN-I response during SARS-CoV-2 invasion. We found that hACE2; Irgm1-/- mice were resistant to lethal SARS-CoV-2 infection with substantially reduced cytokine storm and immunopathology. In striking contrast, a severe SARS-CoV-2 infection along with immune cells infiltration, inflammatory response, and enhanced pathology was observed in the lungs of hACE2; Ifnar1-/- mice. Additionally, hACE2; Ifnar1-/- mice were highly susceptible to SARS-CoV-2 neuroinvasion in the brain accompanied by immune cell infiltration, microglia/astrocytes activation, cytokine response, and demyelination of neurons. The hACE2; Irgm1-/- Ifnar1-/- double knockout mice or hACE2; Irgm1-/- mice treated with STING or RIPK2 pharmacological inhibitors displayed loss of the protective phenotypes observed in hACE2; Irgm1-/- mice suggesting that heightened IFN-I response accounts for the observed immunity. Taken together, we explicitly demonstrate that IFN-I protects from lethal SARS-CoV-2 infection, and Irgm1 (IRGM) could be an excellent therapeutic target. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=133 SRC="FIGDIR/small/520843v1_ufig1.gif" ALT="Figure 1"> View larger version (51K): org.highwire.dtl.DTLVardef@1fda6daorg.highwire.dtl.DTLVardef@1d573dborg.highwire.dtl.DTLVardef@a96318org.highwire.dtl.DTLVardef@a8cd68_HPS_FORMAT_FIGEXP M_FIG C_FIG

Isolation and Characterization of SARS-CoV-2 strains circulating in Eastern India.

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.

Underlying co morbidity reveals unique immune signatures in Type II diabetes patients infected with SARS-CoV2

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.

Nanopore 16S rRNA sequencing reveals alterations in nasopharyngeal microbiome and enrichment of Mycobacterium and Mycoplasma in patients with COVID 19

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome corona virus 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 that respiratory infections alter nasopharyngeal (NP) microbiome diversity and enrich opportunistic pathogens. In this study, we have sequenced the 16S rRNA variable regions, V1 through V9, extracted from NP samples of control and COVID-19 (symptomatic and asymptomatic) participants using the Oxford Nanopore technology. Comprehensive bioinformatics analysis investigating the alpha/beta diversities, non-metric multidimensional scaling, correlation studies, canonical correspondence analysis, linear discriminate analysis, and dysbiosis index analysis revealed control and COVID-19-specific NP microbiomes. We observed significant dysbiosis in COVID-19 NP microbiome with abundance of opportunistic pathogens such as Cutibacterium, Corynebacterium, Oerskovia, and Cellulomonas in asymptomatic patients, and of Streptomyces and Mycobacteriaceae family in symptomatic patients. Furthermore, we observed sharp rise in enrichment of opportunistic pathogens in symptomatic patients, with abundance of Mycobacteria and Mycoplasma, which strongly correlated with the occurrences of chest pain and fever. Our findings contribute novel insights regarding emergence of opportunistic pathogens in COVID-19 patients and their relationship with symptoms, suggesting their potential role in coinfections.

Genomic profiles of Covishield and Covaxin breakthrough SARS-CoV-2 strains from Odisha, India

Vaccine breakthrough infections pose a vast challenge in the eradication of the COVID pandemic situation. Emerging SARS-CoV-2 variants of concern infecting the immunized individuals indicate an ongoing battle between host immunity and natural selection of the pathogen. Our report sheds light on the prominent SARS-CoV-2 variations observed in the isolates from AZD1222/Covishield and BBV152/Covaxin vaccinated subjects.

Clinical, immunological and genomic characterization of asymptomatic and symptomatic cases with SARS-CoV-2 infection, India

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.

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 that resembles human COVID-19 patients. In this study, efforts were made to check the infectivity of a local SARS-CoV-2 isolate in 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 shows a similar type of pathophysiological manifestation of SARS-CoV-2 infection as reported earlier in COVID-19 patients and hamsters infected with other isolates. The lung-associated pathological changes were very prominent on the 4th day post-infection (dpi), mostly resolved by 14dpi. Here, we carried out quantitative proteomic analysis of the lung tissues from SARS-CoV-2-infected hamsters at day 4 and day 14 post infection. This resulted in the identification of 1,585 differentially expressed proteins of which 68 proteins were significantly altered among 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 the identification of candidate biomarkers and understanding the mechanism(s) involved in SARS-CoV-2 pathogenesis. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=143 HEIGHT=200 SRC="FIGDIR/small/434371v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@1930556org.highwire.dtl.DTLVardef@14376d6org.highwire.dtl.DTLVardef@2f064eorg.highwire.dtl.DTLVardef@1472572_HPS_FORMAT_FIGEXP M_FIG C_FIG

SARS-CoV-2 specific immune-signature in direct contacts of COVID-19 cases protect them from contracting disease: A Retrospective Study

The response to SARS-CoV-2 is largely impacted by the level of exposure and the status of immunity. The nature of protection shown by direct contacts of COVID-19 positive patients is quite intriguing to note. We aimed to study the immune differences reinforcing contact individuals in circumventing the disease. Our observation showed direct contacts of PCR positive patients developed elevated neutralizing antibody titres and cytokine levels. On the other hand, single cell data revealed differential usage of V(D)J genes and unique BCR clonotypes imparting protective immune signatures. Topicsserologic tests, immunoglobulin a, immunoglobulin g, immunoglobulin m, antibody titre; cytokine levels; virus neutralization; V(D)J sequencing; BCR clonotypes

Molecular basis of the logical evolution of the novel coronavirus SARS-CoV-2: A comparative analysis

A novel disease, COVID-19, is sweeping the world since end of 2019. While in many countries, the first wave is over, but the pandemic is going through its next phase with a significantly higher infectability. COVID-19 is caused by the novel Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) that seems to be more infectious than any other previous human coronaviruses. To understand any unique traits of the virus that facilitate its entry into the host, we compared the published structures of the viral spike protein of SARS-CoV-2 with other known coronaviruses to determine the possible evolutionary pathway leading to the higher infectivity. The current report presents unique information regarding the amino acid residues that were a) conserved to maintain the binding with ACE2 (Angiotensin-converting enzyme 2), and b) substituted to confer an enhanced binding affinity and conformational flexibility to the SARS-CoV-2 spike protein. The present study provides novel insights into the evolutionary nature and molecular basis of higher infectability and perhaps the virulence of SARS-CoV-2.

SARS-CoV2 genome analysis of Indian isolates and molecular modelling of D614G mutated spike protein with TMPRSS2 depicted its enhanced interaction and virus infectivity

COVID-19 that emerged as a global pandemic is caused by SARS-CoV-2 virus. The virus genome analysis during disease spread reveals about its evolution and transmission. We did whole genome sequencing of 225 clinical strains from the state of Odisha in eastern India using ARTIC protocol-based amplicon sequencing. Phylogenetic analysis identified the presence of all five reported clades 19A, 19B, 20A, 20B and 20C in the population. The analyses revealed two major routes for the introduction of the disease in India i.e. Europe and South-east Asia followed by local transmission. Interestingly, 19B clade was found to be much more prevalent in our sequenced genomes (17%) as compared to other genomes reported so far from India. The haplogroup analysis for clades showed evolution of 19A and 19B in parallel whereas the 20B and 20C appeared to evolve from 20A. Majority of the 19A and 19B clades were present in cases that migrated from Gujarat state in India suggesting it to be one of the major initial points of disease transmission in India during month of March and April. We found that with the time 20A and 20B clades evolved drastically that originated from central Europe. At the same time, it has been observed that 20A and 20B clades depicted selection of four common mutations i.e. 241 C>T (5UTR), P323L in RdRP, F942F in NSP3 and D614G in the spike protein. We found an increase in the concordance of G614 mutation evolution with the viral load in clinical samples as evident from decreased Ct value of spike and Orf1ab gene in qPCR. Molecular modelling and docking analysis identified that D614G mutation enhanced interaction of spike with TMPRSS2 protease, which could impact the shedding of S1 domain and infectivity of the virus in host cells.

A pilot study to investigate the fecal dissemination of SARS-CoV-2 virus genome in COVID-19 patients in Odisha, India

In infectious diseases, the routes of transmission play major roles in determining the rate and extent of disease spread. Though fomites and aerosol droplets are major sources of SARS-CoV-2 human to human transmission, studies have also reported possible involvement of other routes of transmission like fecal-oral. Multiple studies around the world have reported shedding of the SARS-CoV-2 viral genome in certain COVID-19 patient fecal samples. Hence, the major objective of this study was to get the experimental evidence whether in Indian COVID-19 patients fecal dissemination of the SARS-CoV-2 genome occurs or not. Information obtained from twelve number of patients from a COVID-19 hospital of Odisha has demonstrated that both symptomatic and asymptomatic Indian patients could be positive for the SARS-CoV-2 genome in their fecal component. The findings have also established a protocol to collect and extract viral RNA for SARS-CoV-2 detection in fecal samples. Together, the study supports the hypothesis of possible fecal-oral transmission of SARS-CoV-2 virus and provides a rationale to extend this study in a larger cohort of patient samples and correlate the significance of the SARS-CoV-2 virus genome detection in fecal samples with disease severity and transmission.