Natural killer-like B cells are a distinct but infrequent innate immune cell subset modulated by SIV infection of rhesus macaques.

Natural killer-like B (NKB) cells are unique innate immune cells expressing both natural killer (NK) and B cell receptors. As first responders to infection, they secrete IL-18 to induce a critical cascade of innate and adaptive immune cell infiltration and activation. However, limited research exists on the role of NKB cells in homeostasis and infection, largely due to incomplete and erroneous evaluations. To fill this knowledge gap, we investigated the expression of signaling and trafficking proteins, and the in situ localization and transcriptome of naïve NKB cells compared to conventionally-defined NK and B cells, as well as modulations of these cells in SIV infection. Intracellular signaling proteins and trafficking markers were expressed differentially on naïve NKB cells, with high expression of CD62L and Syk, and low expression of CD69, α4ß7, FcRg, Zap70, and CD3z, findings which were more similar to B cells than NK cells. CD20+NKG2a/c+ NKB cells were identified in spleen, mesenteric lymph nodes (MLN), colon, jejunum, and liver of naïve rhesus macaques (RM) via tissue imaging, with NKB cell counts concentrated in spleen and MLN. For the first time, single cell RNA sequencing (scRNAseq), including B cell receptor (BCR) sequencing, of sorted NKB cells confirmed that NKB cells are unique. Transcriptomic analysis of naïve splenic NKB cells by scRNAseq showed that NKB cells undergo somatic hypermutation and express Ig receptors, similar to B cells. While only 15% of sorted NKB cells showed transcript expression of both KLRC1 (NKG2A) and MS4A1 (CD20) genes, only 5% of cells expressed KLRC1, MS4A1, and IgH/IgL transcripts. We observed expanded NKB frequencies in RM gut and buccal mucosa as early as 14 and 35 days post-SIV infection, respectively. Further, mucosal and peripheral NKB cells were associated with colorectal cytokine milieu and oral microbiome changes, respectively. Our studies indicate that NKB cells gated on CD3-CD14-CD20+NKG2A/C+ cells were inclusive of transcriptomically conventional B and NK cells in addition to true NKB cells, confounding accurate phenotyping and frequency recordings that could only be resolved using genomic techniques. Although NKB cells were clearly elevated during SIV infection and associated with inflammatory changes during infection, further interrogation is necessary to acurately identify the true phenotype and significance of NKB cells in infection and inflammation.

Tunability in 3-Site Electronic Excitation Transfer Dynamics: Insights into the Role of Perturbative Coupling.

Electronic excitation transfer dynamics in photosynthetic systems, including the Fenna-Matthews-Olson complex, are often modeled using the interaction picture of three two-level systems, also known as the 3-site system. Among the two possible configurations, uphill and downhill, a recent publication reported an intriguing correlation between population dynamics and the intersite coupling. Specifically, the uphill configuration has been shown to have a pronounced dependence on perturbations in the intersite coupling, whereas the downhill configuration displays negligible dependence. In this study, we consider a generic 3-site configuration and model site interactions through the Markovian master equation. Through this approach, we derive succinct analytical expressions for the population dynamics between the sites, shedding light on the differences in behavior between the two configurations. Using these analytical expressions, we demonstrate the range of tunability in population dynamics achievable with minimal changes in intersite coupling, and we validate these findings through simulation results. These insights into the population dynamics of a 3-site system are expected to play a crucial role in facilitating the design of efficient energy-transfer pathways in molecular systems.

Frequency-potency analysis of IgG+ memory B cells delineates neutralizing antibody responses at single-cell resolution.

Identifying individual functional B cell receptors (BCRs) is common, but two-dimensional analysis of B cell frequency versus BCR potency would delineate both quantity and quality of antigen-specific memory B cells. We efficiently determine quantitative BCR neutralizing activities using a single-cell-derived antibody supernatant analysis (SCAN) workflow and develop a frequency-potency algorithm to estimate B cell frequencies at various neutralizing activity or binding affinity cutoffs. In an HIV-1 fusion peptide (FP) immunization study, frequency-potency curves elucidate the quantity and quality of FP-specific immunoglobulin G (IgG)+ memory B cells for different animals, time points, and antibody lineages at single-cell resolution. The BCR neutralizing activities are mainly determined by their affinities to soluble envelope trimer. Frequency analysis definitively demonstrates dominant neutralizing antibody lineages. These findings establish SCAN and frequency-potency analyses as promising approaches for general B cell analysis and monoclonal antibody (mAb) discovery. They also provide specific rationales for HIV-1 FP-directed vaccine optimization.

European Consensus Guidelines on the Management of Respiratory Distress Syndrome, 2022 : What is New?

We present a summary of the European Consensus guidelines on management of respiratory distress syndrome, which were released in 2022, and compare these with the current National Neonatology Forum of India guidelines, and discuss the feasibility of their application in the Indian settings.

High throughput analysis of B cell dynamics and neutralizing antibody development during immunization with a novel clade C HIV-1 envelope.

A protective HIV-1 vaccine has been hampered by a limited understanding of how B cells acquire neutralizing activity. Our previous vaccines expressing two different HIV-1 envelopes elicited robust antigen specific serum IgG titers in 20 rhesus macaques; yet serum from only two animals neutralized the autologous virus. Here, we used high throughput immunoglobulin receptor and single cell RNA sequencing to characterize the overall expansion, recall, and maturation of antigen specific B cells longitudinally over 90 weeks. Diversification and expansion of many B cell clonotypes occurred broadly in the absence of serum neutralization. However, in one animal that developed neutralization, two neutralizing B cell clonotypes arose from the same immunoglobulin germline and were tracked longitudinally. Early antibody variants with high identity to germline neutralized the autologous virus while later variants acquired somatic hypermutation and increased neutralization potency. The early engagement of precursors capable of neutralization with little to no SHM followed by prolonged affinity maturation allowed the two neutralizing lineages to successfully persist despite many other antigen specific B cells. The findings provide new insight into B cells responding to HIV-1 envelope during heterologous prime and boost immunization in rhesus macaques and the development of selected autologous neutralizing antibody lineages.

Intradermal but not intramuscular modified vaccinia Ankara immunizations protect against intravaginal tier2 simian-human immunodeficiency virus challenges in female macaques.

Route of immunization can markedly influence the quality of immune response. Here, we show that intradermal (ID) but not intramuscular (IM) modified vaccinia Ankara (MVA) vaccinations provide protection from acquisition of intravaginal tier2 simian-human immunodeficiency virus (SHIV) challenges in female macaques. Both routes of vaccination induce comparable levels of serum IgG with neutralizing and non-neutralizing activities. The protection in MVA-ID group correlates positively with serum neutralizing and antibody-dependent phagocytic activities, and envelope-specific vaginal IgA; while the limited protection in MVA-IM group correlates only with serum neutralizing activity. MVA-ID immunizations induce greater germinal center Tfh and B cell responses, reduced the ratio of Th1 to Tfh cells in blood and showed lower activation of intermediate monocytes and inflammasome compared to MVA-IM immunizations. This lower innate activation correlates negatively with induction of Tfh responses. These data demonstrate that the MVA-ID vaccinations protect against intravaginal SHIV challenges by modulating the innate and T helper responses.

Modulation of type I interferon responses potently inhibits SARS-CoV-2 replication and inflammation in rhesus macaques.

Type I interferons (IFN-I) are critical mediators of innate control of viral infections but also drive the recruitment of inflammatory cells to sites of infection, a key feature of severe coronavirus disease 2019. Here, IFN-I signaling was modulated in rhesus macaques (RMs) before and during acute SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) infection using a mutated IFN-α2 (IFN-modulator; IFNmod), which has previously been shown to reduce the binding and signaling of endogenous IFN-I. IFNmod treatment in uninfected RMs was observed to induce a modest up-regulation of only antiviral IFN-stimulated genes (ISGs); however, in SARS-CoV-2-infected RMs, IFNmod reduced both antiviral and inflammatory ISGs. IFNmod treatment resulted in a potent reduction in SARS-CoV-2 viral loads both in vitro in Calu-3 cells and in vivo in bronchoalveolar lavage (BAL), upper airways, lung, and hilar lymph nodes of RMs. Furthermore, in SARS-CoV-2-infected RMs, IFNmod treatment potently reduced inflammatory cytokines, chemokines, and CD163+ MRC1- inflammatory macrophages in BAL and expression of Siglec-1 on circulating monocytes. In the lung, IFNmod also reduced pathogenesis and attenuated pathways of inflammasome activation and stress response during acute SARS-CoV-2 infection. Using an intervention targeting both IFN-α and IFN-ß pathways, this study shows that, whereas early IFN-I restrains SARS-CoV-2 replication, uncontrolled IFN-I signaling critically contributes to SARS-CoV-2 inflammation and pathogenesis in the moderate disease model of RMs.

TREM2+ and interstitial-like macrophages orchestrate airway inflammation in SARS-CoV-2 infection in rhesus macaques.

The immunopathological mechanisms driving the development of severe COVID-19 remain poorly defined. Here, we utilize a rhesus macaque model of acute SARS-CoV-2 infection to delineate perturbations in the innate immune system. SARS-CoV-2 initiates a rapid infiltration of plasmacytoid dendritic cells into the lower airway, commensurate with IFNA production, natural killer cell activation, and a significant increase of blood CD14-CD16+ monocytes. To dissect the contribution of lung myeloid subsets to airway inflammation, we generate a longitudinal scRNA-Seq dataset of airway cells, and map these subsets to corresponding populations in the human lung. SARS-CoV-2 infection elicits a rapid recruitment of two macrophage subsets: CD163+MRC1-, and TREM2+ populations that are the predominant source of inflammatory cytokines. Treatment with baricitinib (Olumiant®), a JAK1/2 inhibitor is effective in eliminating the influx of non-alveolar macrophages, with a reduction of inflammatory cytokines. This study delineates the major lung macrophage subsets driving airway inflammation during SARS-CoV-2 infection.

Modulation of type I interferon responses potently inhibits SARS-CoV-2 replication and inflammation in rhesus macaques.

Type-I interferons (IFN-I) are critical mediators of innate control of viral infections, but also drive recruitment of inflammatory cells to sites of infection, a key feature of severe COVID-19. Here, and for the first time, IFN-I signaling was modulated in rhesus macaques (RMs) prior to and during acute SARS-CoV-2 infection using a mutated IFNα2 (IFN-modulator; IFNmod), which has previously been shown to reduce the binding and signaling of endogenous IFN-I. In SARS-CoV-2-infected RMs, IFNmod reduced both antiviral and inflammatory ISGs. Notably, IFNmod treatment resulted in a potent reduction in (i) SARS-CoV-2 viral load in Bronchoalveolar lavage (BAL), upper airways, lung, and hilar lymph nodes; (ii) inflammatory cytokines, chemokines, and CD163+MRC1-inflammatory macrophages in BAL; and (iii) expression of Siglec-1, which enhances SARS-CoV-2 infection and predicts disease severity, on circulating monocytes. In the lung, IFNmod also reduced pathogenesis and attenuated pathways of inflammasome activation and stress response during acute SARS-CoV-2 infection. This study, using an intervention targeting both IFN-α and IFN-ß pathways, shows that excessive inflammation driven by type 1 IFN critically contributes to SARS-CoV-2 pathogenesis in RMs, and demonstrates the potential of IFNmod to limit viral replication, SARS-CoV-2 induced inflammation, and COVID-19 severity.

Comparative effect of 1.2% atorvastatin gel and 1.2% rosuvastatin as a local drug delivery in treatment of intra-bony defects in chronic periodontitis.

Background: The present study was aimed to evaluate the efficacy of 1.2% Atorvastatin (ATV) with 1.2% Rosuvastatin (RSV) as local drug delivery for treatment of Chronic Periodontitis (CP). Materials and Methods: Forty patients were equally divided into two groups. Group A underwent scaling and root debridement and 1.2% ATV gel (1.2 mg/0.1 mL) was placed, whereas group B received scaling and root debridement and RSV (1.2 mg/0.1 ml) was placed. Results: The results showed that both the groups had improvement in all the recorded parameters, and the results obtained were statistically significant. When comparison was made between the groups, no significant difference was obtained between atorvastatin and rosuvastatin at baseline in all recorded parameters. However, after 6 months significant improvement was recorded in CAL (Clinical attachment level) and PD (Probing depth). The plaque index (PI) and gingival index (GI) score however showed improvement, but it did not attain the level of significance. Conclusion: The present study showed improvement in clinical parameters with the use of ATV and RSV gel when used in combination with scaling and root planing (SRP) in CP patients. Patients with RSV gel showed up significantly better than the ones in which ATV gel was placed.

A neutralizing antibody target in early HIV-1 infection was recapitulated in rhesus macaques immunized with the transmitted/founder envelope sequence.

Transmitted/founder (T/F) HIV-1 envelope proteins (Envs) from infected individuals that developed neutralization breadth are likely to possess inherent features desirable for vaccine immunogen design. To explore this premise, we conducted an immunization study in rhesus macaques (RM) using T/F Env sequences from two human subjects, one of whom developed potent and broad neutralizing antibodies (Z1800M) while the other developed little to no neutralizing antibody responses (R66M) during HIV-1 infection. Using a DNA/MVA/protein immunization protocol, 10 RM were immunized with each T/F Env. Within each T/F Env group, the protein boosts were administered as either monomeric gp120 or stabilized trimeric gp140 protein. All vaccination regimens elicited high titers of antigen-specific IgG, and two animals that received monomeric Z1800M Env gp120 developed autologous neutralizing activity. Using early Env escape variants isolated from subject Z1800M as guides, the serum neutralizing activity of the two immunized RM was found to be dependent on the gp120 V5 region. Interestingly, the exact same residues of V5 were also targeted by a neutralizing monoclonal antibody (nmAb) isolated from the subject Z1800M early in infection. Glycan profiling and computational modeling of the Z1800M Env gp120 immunogen provided further evidence that the V5 loop is exposed in this T/F Env and was a dominant feature that drove neutralizing antibody targeting during infection and immunization. An expanded B cell clonotype was isolated from one of the neutralization-positive RM and nmAbs corresponding to this group demonstrated V5-dependent neutralization similar to both the RM serum and the human Z1800M nmAb. The results demonstrate that neutralizing antibody responses elicited by the Z1800M T/F Env in RM converged with those in the HIV-1 infected human subject, illustrating the potential of using immunogens based on this or other T/F Envs with well-defined immunogenicity as a starting point to drive breadth.

Infiltration of inflammatory macrophages and neutrophils and widespread pyroptosis in lung drive influenza lethality in nonhuman primates.

Severe influenza kills tens of thousands of individuals each year, yet the mechanisms driving lethality in humans are poorly understood. Here we used a unique translational model of lethal H5N1 influenza in cynomolgus macaques that utilizes inhalation of small-particle virus aerosols to define mechanisms driving lethal disease. RNA sequencing of lung tissue revealed an intense interferon response within two days of infection that resulted in widespread expression of interferon-stimulated genes, including inflammatory cytokines and chemokines. Macaques with lethal disease had rapid and profound loss of alveolar macrophages (AMs) and infiltration of activated CCR2+ CX3CR1+ interstitial macrophages (IMs) and neutrophils into lungs. Parallel changes of AMs and neutrophils in bronchoalveolar lavage (BAL) correlated with virus load when compared to macaques with mild influenza. Both AMs and IMs in lethal influenza were M1-type inflammatory macrophages which expressed neutrophil chemotactic factors, while neutrophils expressed genes associated with activation and generation of neutrophil extracellular traps (NETs). NETs were prominent in lung and were found in alveolar spaces as well as lung parenchyma. Genes associated with pyroptosis but not apoptosis were increased in lung, and activated inflammatory caspases, IL-1ß and cleaved gasdermin D (GSDMD) were present in bronchoalveolar lavage fluid and lung homogenates. Cleaved GSDMD was expressed by lung macrophages and alveolar epithelial cells which were present in large numbers in alveolar spaces, consistent with loss of epithelial integrity. Cleaved GSDMD colocalized with viral NP-expressing cells in alveoli, reflecting pyroptosis of infected cells. These novel findings reveal that a potent interferon and inflammatory cascade in lung associated with infiltration of inflammatory macrophages and neutrophils, elaboration of NETs and cell death by pyroptosis mediates lethal H5N1 influenza in nonhuman primates, and by extension humans. These innate pathways represent promising therapeutic targets to prevent severe influenza and potentially other primary viral pneumonias in humans.

From structure to sequence: Antibody discovery using cryoEM.

One of the rate-limiting steps in analyzing immune responses to vaccines or infections is the isolation and characterization of monoclonal antibodies. Here, we present a hybrid structural and bioinformatic approach to directly assign the heavy and light chains, identify complementarity-determining regions, and discover sequences from cryoEM density maps of serum-derived polyclonal antibodies bound to an antigen. When combined with next-generation sequencing of immune repertoires, we were able to specifically identify clonal family members, synthesize the monoclonal antibodies, and confirm that they interact with the antigen in a manner equivalent to the corresponding polyclonal antibodies. This structure-based approach for identification of monoclonal antibodies from polyclonal sera opens new avenues for analysis of immune responses and iterative vaccine design.

TREM2+ and interstitial macrophages orchestrate airway inflammation in SARS-CoV-2 infection in rhesus macaques.

The COVID-19 pandemic remains a global health crisis, yet, the immunopathological mechanisms driving the development of severe disease remain poorly defined. Here, we utilize a rhesus macaque (RM) model of SARS-CoV-2 infection to delineate perturbations in the innate immune system during acute infection using an integrated systems analysis. We found that SARS-CoV-2 initiated a rapid infiltration (two days post infection) of plasmacytoid dendritic cells into the lower airway, commensurate with IFNA production, natural killer cell activation, and induction of interferon-stimulated genes. At this early interval, we also observed a significant increase of blood CD14-CD16+ monocytes. To dissect the contribution of lung myeloid subsets to airway inflammation, we generated a novel compendium of RM-specific lung macrophage gene expression using a combination of sc-RNA-Seq data and bulk RNA-Seq of purified populations under steady state conditions. Using these tools, we generated a longitudinal sc-RNA-seq dataset of airway cells in SARS-CoV-2-infected RMs. We identified that SARS-CoV-2 infection elicited a rapid recruitment of two subsets of macrophages into the airway: a C206+MRC1-population resembling murine interstitial macrophages, and a TREM2+ population consistent with CCR2+ infiltrating monocytes, into the alveolar space. These subsets were the predominant source of inflammatory cytokines, accounting for ~75% of IL6 and TNF production, and >90% of IL10 production, whereas the contribution of CD206+MRC+ alveolar macrophages was significantly lower. Treatment of SARS-CoV-2 infected RMs with baricitinib (Olumiant ® ), a novel JAK1/2 inhibitor that recently received Emergency Use Authorization for the treatment of hospitalized COVID-19 patients, was remarkably effective in eliminating the influx of infiltrating, non-alveolar macrophages in the alveolar space, with a concomitant reduction of inflammatory cytokines. This study has delineated the major subsets of lung macrophages driving inflammatory and anti-inflammatory cytokine production within the alveolar space during SARS-CoV-2 infection. ONE SENTENCE SUMMARY: Multi-omic analyses of hyperacute SARS-CoV-2 infection in rhesus macaques identified two population of infiltrating macrophages, as the primary orchestrators of inflammation in the lower airway that can be successfully treated with baricitinib.

Tissue-specific transcriptional profiling of plasmacytoid dendritic cells reveals a hyperactivated state in chronic SIV infection.

HIV associated immune activation (IA) is associated with increased morbidity in people living with HIV (PLWH) on antiretroviral therapy, and remains a barrier for strategies aimed at reducing the HIV reservoir. The underlying mechanisms of IA have not been definitively elucidated, however, persistent production of Type I IFNs and expression of ISGs is considered to be one of the primary factors. Plasmacytoid DCs (pDCs) are a major producer of Type I IFN during viral infections, and are highly immunomodulatory in acute HIV and SIV infection, however their role in chronic HIV/SIV infection has not been firmly established. Here, we performed a detailed transcriptomic characterization of pDCs in chronic SIV infection in rhesus macaques, and in sooty mangabeys, a natural host non-human primate (NHP) species that undergoes non-pathogenic SIV infection. We also investigated the immunostimulatory capacity of lymph node homing pDCs in chronic SIV infection by contrasting gene expression of pDCs isolated from lymph nodes with those from blood. We observed that pDCs in LNs, but not blood, produced high levels of IFNα transcripts, and upregulated gene expression programs consistent with T cell activation and exhaustion. We apply a novel strategy to catalogue uncharacterized surface molecules on pDCs, and identified the lymphoid exhaustion markers TIGIT and LAIR1 as highly expressed in SIV infection. pDCs from SIV-infected sooty mangabeys lacked the activation profile of ISG signatures observed in infected macaques. These data demonstrate that pDCs are a primary producer of Type I IFN in chronic SIV infection. Further, this study demonstrated that pDCs trafficking to LNs persist in a highly activated state well into chronic infection. Collectively, these data identify pDCs as a highly immunomodulatory cell population in chronic SIV infection, and a putative therapeutic target to reduce immune activation.

IL-21 and IFNα therapy rescues terminally differentiated NK cells and limits SIV reservoir in ART-treated macaques.

Unlike HIV infection, which progresses to AIDS absent suppressive anti-retroviral therapy, nonpathogenic infections in natural hosts, such African green monkeys, are characterized by a lack of gut microbial translocation and robust secondary lymphoid natural killer cell responses resulting in an absence of chronic inflammation and limited SIV dissemination in lymph node B-cell follicles. Here we report, using the pathogenic model of antiretroviral therapy-treated, SIV-infected rhesus macaques that sequential interleukin-21 and interferon alpha therapy generate terminally differentiated blood natural killer cells (NKG2a/clowCD16+) with potent human leukocyte antigen-E-restricted activity in response to SIV envelope peptides. This is in contrast to control macaques, where less differentiated, interferon gamma-producing natural killer cells predominate. The frequency and activity of terminally differentiated NKG2a/clowCD16+ natural killer cells correlates with a reduction of replication-competent SIV in lymph node during antiretroviral therapy and time to viral rebound following analytical treatment interruption. These data demonstrate that African green monkey-like natural killer cell differentiation profiles can be rescued in rhesus macaques to promote viral clearance in tissues.

A modified vaccinia Ankara vector-based vaccine protects macaques from SARS-CoV-2 infection, immune pathology, and dysfunction in the lungs.

A combination of vaccination approaches will likely be necessary to fully control the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. Here, we show that modified vaccinia Ankara (MVA) vectors expressing membrane-anchored pre-fusion stabilized spike (MVA/S) but not secreted S1 induced strong neutralizing antibody responses against SARS-CoV-2 in mice. In macaques, the MVA/S vaccination induced strong neutralizing antibodies and CD8+ T cell responses, and conferred protection from SARS-CoV-2 infection and virus replication in the lungs as early as day 2 following intranasal and intratracheal challenge. Single-cell RNA sequencing analysis of lung cells on day 4 after infection revealed that MVA/S vaccination also protected macaques from infection-induced inflammation and B cell abnormalities and lowered induction of interferon-stimulated genes. These results demonstrate that MVA/S vaccination induces neutralizing antibodies and CD8+ T cells in the blood and lungs and is a potential vaccine candidate for SARS-CoV-2.

Unilateral Hemilaminectomy for the Excision of Intradural Extramedullary Spinal Tumors: A Beginner's Challenges.

AIM: To analyze retrospectively our experience of unilateral hemilaminectomy in intradural extramedullary (IDEM) tumors. MATERIAL AND METHODS: This study included 28 patients with IDEM tumors who underwent unilateral hemilaminectomies during the period 2011?2016. The preoperative clinicoradiological data, intraoperative findings, and postoperative outcomes were analyzed using a Microsoft Excel database. RESULTS: The mean age of the patients was 49.14 ± 12.23 years, and the average duration of symptoms was 11.82 weeks. The tumor distribution in the spine was as follows: cervical spine (14.2%), cervicodorsal spine (3.6%), dorsal spine (39.3%), dorsolumbar spine (10.7%), and lumbar spine (32.1%). Twenty patients (71%) underwent two-level laminectomies, and three-level laminectomies were performed on eight patients (29%). The mean operative time was 4 hours and 40 minutes. CONCLUSION: Unilateral hemilaminectomy is a safe, minimally invasive procedure for the excision of IDEM tumors. Beginners may face difficulties with this procedure and should ideally take more time than with a conventional total laminectomy because of the narrow operating corridor. As more experience is gained, this procedure will become easier in selected cases.

Clinical and whole genome characterization of SARS-CoV-2 in India.

We report clinical profile of hundred and nine patients with SARS CoV-2 infection, and whole genome sequences (WGS) of seven virus isolates from the first reported cases in India, with various international travel histories. Comorbidities such as diabetes, hypertension, and cardiovascular disease were frequently associated with severity of the disease. WBC and neutrophil counts showed an increase, while lymphocyte counts decreased in patients with severe infection suggesting a possible neutrophil mediated organ damage, while immune activity may be diminished with decrease in lymphocytes leading to disease severity. Increase in SGOT, SGPT and blood urea suggests the functional deficiencies of liver, heart, and kidney in patients who succumbed to the disease when compared to the group of recovered patients. The WGS analysis showed that these isolates were classified into two clades: I/A3i, and A2a (four according to GISAID: O, L, GR, and GH). Further, WGS phylogeny and travel history together indicate possible transmission from Middle East and Europe. Three S protein variants: Wuhan reference, D614G, and Y28H were identified predicted to possess different binding affinities to host ACE2.