Immune profile and responses of a novel dengue DNA vaccine encoding an EDIII-NS1 consensus design based on Indo-African sequences.

The ongoing COVID-19 pandemic highlights the need to tackle viral variants, expand the number of antigens, and assess diverse delivery systems for vaccines against emerging viruses. In the present study, a DNA vaccine candidate was generated by combining in tandem envelope protein domain III (EDIII) of dengue virus serotypes 1-4 and a dengue virus (DENV)-2 non-structural protein 1 (NS1) protein-coding region. Each domain was designed as a serotype-specific consensus coding sequence derived from different genotypes based on the whole genome sequencing of clinical isolates in India and complemented with data from Africa. This sequence was further optimized for protein expression. In silico structural analysis of the EDIII consensus sequence revealed that epitopes are structurally conserved and immunogenic. The vaccination of mice with this construct induced pan-serotype neutralizing antibodies and antigen-specific T cell responses. Assaying intracellular interferon (IFN)-γ staining, immunoglobulin IgG2(a/c)/IgG1 ratios, and immune gene profiling suggests a strong Th1-dominant immune response. Finally, the passive transfer of immune sera protected AG129 mice challenged with a virulent, non-mouse-adapted DENV-2 strain. Our findings collectively suggest an alternative strategy for dengue vaccine design by offering a novel vaccine candidate with a possible broad-spectrum protection and a successful clinical translation either as a stand alone or in a mix and match strategy.

Proteomic Investigation of COVID-19 Severity During the Tsunamic Second Wave in Mumbai.

Maharashtra was severely affected during the noxious second wave of COVID-19, with the highest number of cases recorded across India. The emergence of new symptoms and dysregulation of multiple organs resulted in high disease severity during the second wave which led to increased difficulties in understanding the molecular mechanisms behind the disease pathology. Exploring the underlying factors can help to relieve the burden on the medical communities to some extent by prioritizing the patients and, at the same time, opening avenues for improved treatments. In the current study, we have performed a mass-spectrometry-based proteomic analysis to investigate the disease pathology using nasopharyngeal swab samples collected from the COVID-19 patients in the Mumbai region of Maharashtra over the period of March-June 2021, the peak of the second wave. A total of 59 patients, including 32 non-severe and 27 severe cases, were considered for this proteomic study. We identified 23 differentially regulated proteins in severe patients as a host response to infection. In addition to the previously identified innate mechanisms of neutrophil and platelet degranulation, this study revealed significant alterations of anti-microbial peptide pathways in severe conditions, illustrating its role in the severity of the infectious strain of COVID-19 during the second wave. Furthermore, myeloperoxidase, cathepsin G, and profilin-1 were identified as potential therapeutic targets of the FDA-approved drugs dabrafenib, ZINC4097343, and ritonavir. This study has enlightened the role of the anti-microbial peptide pathway associated with the second wave in India and proposed its importance in potential therapeutics for COVID-19.

Detection and isolation of SARS-CoV-2 Eta variant from the international travelers and local residents of India.

International travel has been the major source for the rapid spread of new SARS-CoV-2 variants across the globe. During SARS-CoV-2 genomic surveillance, a total of 212 SARS-CoV-2 positive clinical specimens were sequenced using next-generation sequencing. A complete SARS-CoV-2 genome could be retrieved from 90 clinical specimens. Of them, 14 sequences belonged to the Eta variant from clinical specimens of international travelers (n = 12) and local residents (n = 2) of India, and 76 belonged to other SARS-CoV-2 variants. Of all the Eta-positive specimens, the virus isolates were obtained from the clinical specimens of six international travelers. Many variants of interest have been found to cause substantial community transmission or cluster infections. The detection of this variant with lethal E484K mutation across the globe and India necessitates persistent genomic surveillance of the SARS-CoV-2 variants, which would aid in taking preventive action.

Development of a PNA-DiSc2 based portable absorbance platform for the detection of pathogen nucleic acids.

We report the development of a portable absorption (PortAbs)-based pathogen nucleic acid detection system using peptide nucleic acid (PNA) and a cyanine dye, DiSc2(5). When the dye binds to the PNA-DNA hybrid, it results in a characteristic ∼110 nm shift in the dye absorbance, which we measure using PortAbs. The protocol involves amplification of the target DNA, PNA-DNA hybridization and dye complexing steps followed by absorption measurement. The system is built using a broad-spectrum photodiode whose output is amplified and then measured by a high resolution (24 or 32 bit) analog-to-digital converter. The excitation pulses of light are delivered by a color-changing LED. The sequence of excitation, measurement and display of results are all controlled by an embedded Raspberry-Pi board (or alternatively a laptop). At higher concentrations of the target amplicon (∼200 ng), the color change can be detected visually. At lower concentrations, PortAbs outperforms a plate reader and can detect target DNA as low as 30 ng or approximately 10 nM which is at least 10 fold better than previously reported studies. We validate the methodology using SARS-CoV-2 clinical samples containing about 1000 copies of the viral RNA and show that the entire workflow takes about 90 min. The cost of the complete standalone system is less than INR 40 000 (approx. 500 USD).

A Multi-omics Longitudinal Study Reveals Alteration of the Leukocyte Activation Pathway in COVID-19 Patients.

Severe coronavirus disease 2019 (COVID-19) infection may lead to lung injury, multi-organ failure, and eventually death. Cytokine storm due to excess cytokine production has been associated with fatality in severe infections. However, the specific molecular signatures associated with the elevated immune response are yet to be elucidated. We performed a mass-spectrometry-based proteomic and metabolomic analysis of COVID-19 plasma samples collected at two time points. Using Orbitrap Fusion LC-MS/MS-based label-free proteomic analysis, we identified around 10 significant proteins, 32 significant peptides, and 5 metabolites that were dysregulated at the severe time points. Few of these proteins identified by quantitative proteomics were validated using the multiple reaction monitoring (MRM) assay. Integrated pathway analysis using distinct proteomic and metabolomic signatures revealed alterations in complement and coagulation cascade, platelet aggregation, myeloid leukocyte activation pathway, and arginine metabolism. Further, we highlight the role of leukocyte activation and arginine metabolism in COVID-19 pathogenesis and targeting these pathways for COVID-19 therapeutics.

Rapid Classification of COVID-19 Severity by ATR-FTIR Spectroscopy of Plasma Samples.

The coronavirus disease 2019 (COVID-19) pandemic continues to ravage the world, with many hospitals overwhelmed by the large number of patients presenting during major outbreaks. A rapid triage for COVID-19 patient requiring hospitalization and intensive care is urgently needed. Age and comorbidities have been associated with a higher risk of severe COVID-19 but are not sufficient to triage patients. Here, we investigated the potential of attenuated total reflectance Fourier-transform infrared (ATR-FTIR) spectroscopy as a rapid blood test for classification of COVID-19 disease severity using a cohort of 160 COVID-19 patients. A simple plasma processing and ATR-FTIR data acquisition procedure was established using 75% ethanol for viral inactivation. Next, partial least-squares-discriminant analysis (PLS-DA) models were developed and tested using data from 130 and 30 patients, respectively. Addition of the ATR-FTIR spectra to the clinical parameters (age, sex, diabetes mellitus, and hypertension) increased the area under the ROC curve (C-statistics) for both the training and test data sets, from 69.3% (95% CI 59.8-78.9%) to 85.7% (78.6-92.8%) and 77.8% (61.3-94.4%) to 85.1% (71.3-98.8%), respectively. The independent test set achieved 69.2% specificity (42.4-87.3%) and 94.1% sensitivity (73.0-99.0%). Diabetes mellitus was the strongest predictor in the model, followed by FTIR regions 1020-1090 and 1588-1592 cm-1. In summary, this study demonstrates the potential of ATR-FTIR spectroscopy as a rapid, low-cost COVID-19 severity triage tool to facilitate COVID-19 patient management during an outbreak.

Phylogenetic classification of the whole-genome sequences of SARS-CoV-2 from India & evolutionary trends.

BACKGROUND & OBJECTIVES: Several phylogenetic classification systems have been devised to trace the viral lineages of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, inconsistency in the nomenclature limits uniformity in its epidemiological understanding. This study provides an integration of existing classifications and describes evolutionary trends of the SARS-CoV-2 strains circulating in India. METHODS: The whole genomes of 330 SARS-CoV-2 samples were sequenced using next-generation sequencing (NGS). Phylogenetic and sequence analysis of a total of 3014 Indian SARS-CoV-2 sequences from 20 different States/Union Territories (January to September 2020) from the Global Initiative on Sharing All Influenza Data (GISAID) database was performed to observe the clustering of Nextstrain and Phylogenetic Assignment of Named Global Outbreak LINeages (Pangolin) lineages with the GISAID clades. The identification of mutational sites under selection pressure was performed using Mixed Effects Model of Evolution and Single-Likelihood Ancestor Counting methods available in the Datamonkey server. RESULTS: Temporal data of the Indian SARS-CoV-2 genomes revealed that except for Uttarakhand, West Bengal and Haryana that showed the circulation of GISAID clade O even after July 2020, the rest of the States showed a complete switch to GR/GH clades. Pangolin lineages B.1.1.8 and B.1.113 identified within GR and GH clades, respectively, were noted to be indigenous evolutions. Sites identified to be under positive selection pressure within these clades were found to occur majorly in the non-structural proteins coded by ORF1a and ORF1b. INTERPRETATION & CONCLUSIONS: This study interpreted the geographical and temporal dominance of SARS-CoV-2 strains in India over a period of nine months based on the GISAID classification. An integration of the GISAID, Nextstrain and Pangolin classifications is also provided. The emergence of new lineages B.1.1.8 and B.1.113 was indicative of host-specific evolution of the SARS-CoV-2 strains in India. The hotspot mutations such as those driven by positive selection need to be further characterized.

Dengue and Chikungunya Virus Co-infections: The Inside Story.

INTRODUCTION: There have been various studies from India describing the acute presentation and the long-term sequalae of Chikungunya (CHIKV) infection. However, there are very few studies discussing the Chikungunya-Dengue (DENV) co-infection from Western India. The present project was undertaken to study the clinical features of Dengue and Chikungunya co-infection and compare with Chikungunya mono-infection; correlate the clinical findings with seroprevalence and molecular identification of Dengue and Chikungunya using IgM ELISA and RTPCR. MATERIAL AND METHODS: Three hundred suspected cases of Dengue and/or Chikungunya patients from out patients department and indoor wards, more than 12 years of age suffering from acute febrile illness, joint pains and rash for less than 10 days were included from June 2010 to April 2015. Proven cases of malaria, enteric fever, leptospirosis were excluded from the study. Leptospira IgM, Dengue IgM and PCR, Chikungunya IgM and PCR was done on all 300 samples. RESULTS: Sero-surveillance of the patients revealed that 59% (177) patients were positive for Dengue IgM alone, while 2% (6) tested positive for Chikungunya IgM alone. 6.7% (20) patients tested positive for Dengue and Chikungunya both. Ninty seven (32.3%) patients were negative for Dengue and Chikungunya. Of the 300 samples, 7% (21) were positive for DENV, 35% (105) were positive for CHIKV, 10% (30) were both DENV and CHIKV positive and 48% (144) were negative for both through RT-PCR. DISCUSSION: In our study, the patients of CHIKV mono-infection and DENV + CHIK co-infection had high VAS score, morning stiffness, arthralgias, restriction of joint movements as compared to patients with DENV mono infection. Patients of dengue mono infection had bone pains and myalgias in addition to joint pains; however there was restriction of joint movements in only 13.2% as compared with 100% of mono CHIKV or dual infection. These clinical features can be helpful in distinguishing DENV mono infection as compared to co-infection. The study highlights the diagnostic importance of RT-PCR in CHIKV and DENV co-infection, as 10% cases were identified using RT-PCR as compared to 6.7% cases by IgM antibodies.

SARS-CoV-2 severity classification from plasma sample using confocal Raman spectroscopy.

The world is on the brink of facing coronavirus's (COVID-19) fourth wave as the mutant forms of viruses are escaping neutralizing antibodies in spite of being vaccinated. As we have already witnessed that it has encumbered our health system, with hospitals swamped with infected patients observed during the viral outbreak. Rapid triage of patients infected with SARS-CoV-2 is required during hospitalization to prioritize and provide the best point of care. Traditional diagnostics techniques such as RT-PCR and clinical parameters such as symptoms, comorbidities, sex and age are not enough to identify the severity of patients. Here, we investigated the potential of confocal Raman microspectroscopy as a powerful tool to generate an expeditious blood-based test for the classification of COVID-19 disease severity using 65 patients plasma samples from cohorts infected with SARS-CoV-2. We designed an easy manageable blood test where we used a small volume (8 µl) of inactivated whole plasma samples from infected patients without any extra solvent usage in plasma processing. Raman spectra of plasma samples were acquired and multivariate exploratory analysis PC-LDA (principal component based linear discriminant analysis) was used to build a model, which segregated the severe from the non-severe COVID-19 group with a sensitivity of 83.87%, specificity of 70.60% and classification efficiency of 76.92%. Among the bands expressed in both the cohorts, the study led to the identification of Raman fingerprint regions corresponding to lipids (1661, 1742), proteins amide I and amide III (1555, 1247), proteins (Phe) (1006, 1034), and nucleic acids (760) to be differentially expressed in severe COVID-19 patient's samples. In summary, the current study exhibits the potential of confocal Raman to generate simple, rapid, and less expensive blood tests to triage the severity of patients infected with SARS-CoV-2.

Humoral Immune Response Profile of COVID-19 Reveals Severity and Variant-Specific Epitopes: Lessons from SARS-CoV-2 Peptide Microarray.

The amaranthine scale of the COVID-19 pandemic and unpredictable disease severity is of grave concern. Serological diagnostic aids are an excellent choice for clinicians for rapid and easy prognosis of the disease. To this end, we studied the humoral immune response to SARS-CoV-2 infection to map immunogenic regions in the SARS-CoV-2 proteome at amino acid resolution using a high-density SARS-CoV-2 proteome peptide microarray. The microarray has 4932 overlapping peptides printed in duplicates spanning the entire SARS-CoV-2 proteome. We found 204 and 676 immunogenic peptides against IgA and IgG, corresponding to 137 and 412 IgA and IgG epitopes, respectively. Of these, 6 and 307 epitopes could discriminate between disease severity. The emergence of variants has added to the complexity of the disease. Using the mutation panel available, we could detect 5 and 10 immunogenic peptides against IgA and IgG with mutations belonging to SAR-CoV-2 variants. The study revealed severity-based epitopes that could be presented as potential prognostic serological markers. Further, the mutant epitope immunogenicity could indicate the putative use of these markers for diagnosing variants responsible for the infection.

COVID-19 Serological Survey-3 Prior to Second Wave in Mumbai, India.

Background: Subsequent to serosurveys 1 and 2 for COVID-19 carried out in three wards of Mumbai in July and August 2020, Municipal Corporation of Greater Mumbai conducted serosurvey 3 in March 2021. This was to identify the extent of exposure by testing specific IgG antibodies against COVID-19. Material and Methods: A cross-sectional study was conducted to find the prevalence of seropositivity in Mumbai, which included 10,197 samples belonging to patients visiting public dispensaries (slum population, 6006) and private (nonslum population, 4191) laboratories of Aapli Chikitsa network for blood investigations for non-COVID illnesses. The ward-wise number of unlinked anonymous samples from 24 wards was predecided by using probability proportionate sampling. The samples were collected using quota sampling technique as per predecided sample for each ward. These samples collected from nonimmunized individuals were tested for IgG antibodies at the Molecular Biology Laboratory of Kasturba Hospital for Infectious Diseases by chemiluminescence assay (CLIA) method. Results: The overall seropositivity was found to be 36.3% (41.6% in slum and 28.59% in nonslum population). It was more in city wards (38.28%) followed by western suburb (36.47%) and then eastern suburb wards (34.86%), matching with the proportion of cases in these wards during the study period. There was no significant difference in seropositivity among males and females and in different age groups. Conclusions: Seropositivity is higher in slum areas than nonslum areas. It has reduced in slum areas and increased in nonslum areas as compared to findings of serosurveys 1 and 2. This explains the detection of a greater number of cases from nonslum areas in the second wave. The average seropositivity of 36.3% justifies the necessity of immunization on a wider scale in the city. Periodic serosurveys are required at fixed intervals to monitor the trend of infection and level of herd immunity.

SARS-CoV-2 variants: Impact on biological and clinical outcome.

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) that was first identified in December 2019, in Wuhan, China was found to be the etiological agent for a novel respiratory infection that led to a Coronavirus Induced Disease named COVID-19. The disease spread to pandemic magnitudes within a few weeks and since then we have been dealing with several waves across the world, due to the emergence of variants and novel mutations in this RNA virus. A direct outcome of these variants apart from the spike of cases is the diverse disease presentation and difficulty in employing effective diagnostic tools apart from confusing disease outcomes. Transmissibility rates of the variants, host response, and virus evolution are some of the features found to impact COVID-19 disease management. In this review, we will discuss the emerging variants of SARS-CoV-2, notable mutations in the viral genome, the possible impact of these mutations on detection, disease presentation, and management as well as the recent findings in the mechanisms that underlie virus-host interaction. Our aim is to invigorate a scientific debate on how pathogenic potential of the new pandemic viral strains contributes toward development in the field of virology in general and COVID-19 disease in particular.

Community transmission of SARS-CoV-2 with B.1.1.7 lineage in Mumbai, India.

The B.1.1.7 (Alpha) variant has been detected in Mumbai, India during February 2021. Subsequently, we retrieved 43 sequences from specimens of 51 COVID-19 cases from Mumbai. The sequence analysis revealed that the cases were mainly affected with Alpha variant which suggests its role in community transmission of SARS-CoV-2 in Mumbai, India.

Opposing roles for sMAdCAM and IL-15 in COVID-19 associated cellular immune pathology.

Immune cell dysregulation and lymphopenia characterize COVID-19 pathology in moderate to severe disease. While underlying inflammatory factors have been extensively studied, homeostatic and mucosal migratory signatures remain largely unexplored as causative factors. In this study, we evaluated the association of circulating IL-6, soluble mucosal addressin cell adhesion molecule (sMAdCAM), and IL-15 with cellular dysfunction characterizing mild and hypoxemic stages of COVID-19. A cohort of SARS-CoV-2 infected individuals (n = 130) at various stages of disease progression together with healthy controls (n = 16) were recruited from COVID Care Centres (CCCs) across Mumbai, India. Multiparametric flow cytometry was used to perform in-depth immune subset characterization and to measure plasma IL-6 levels. sMAdCAM, IL-15 levels were quantified using ELISA. Distinct depletion profiles, with relative sparing of CD8 effector memory and CD4+ regulatory T cells, were observed in hypoxemic disease within the lymphocyte compartment. An apparent increase in the frequency of intermediate monocytes characterized both mild as well as hypoxemic disease. IL-6 levels inversely correlated with those of sMAdCAM and both markers showed converse associations with observed lympho-depletion suggesting opposing roles in pathogenesis. Interestingly, IL-15, a key cytokine involved in lymphocyte activation and homeostasis, was detected in symptomatic individuals but not in healthy controls or asymptomatic cases. Further, plasma IL-15 levels negatively correlated with T, B, and NK count suggesting a compensatory production of this cytokine in response to the profound lymphopenia. Finally, higher levels of plasma IL-15 and IL-6, but not sMAdCAM, were associated with a longer duration of hospitalization.

Frequency of Effector Memory Cells Expressing Integrin α4ß7 Is Associated With TGF-ß1 Levels in Therapy Naïve HIV Infected Women With Low CD4+ T Cell Count.

Integrin α4ß7 expressing CD4+ T cells are preferred targets for HIV infection and are thought to be predictors of disease progression. Concurrent analysis of integrin α4ß7 expressing innate and adaptive immune cells was carried out in antiretroviral (ART) therapy naïve HIV infected women in order to determine its contribution to HIV induced immune dysfunction. Our results demonstrate a HIV infection associated decrease in the frequency of integrin α4ß7 expressing endocervical T cells along with an increase in the frequency of integrin α4ß7 expressing peripheral monocytes and central memory CD4+ T cells, which are considered to be viral reservoirs. We report for the first time an increase in levels of soluble MAdCAM-1 (sMAdCAM-1) in HIV infected individuals as well as an increased frequency and count of integrin ß7Hi CD8+ memory T cells. Correlation analysis indicates that the frequency of effector memory CD8+ T cells expressing integrin α4ß7 is associated with levels of both sMAdCAM-1 and TGF-ß1. The results of this study also suggest HIV induced alterations in T cell homeostasis to be on account of disparate actions of sMAdCAM-1 and TGF-ß1 on integrin α4ß7 expressing T cells. The immune correlates identified in this study warrant further investigation to determine their utility in monitoring disease progression.

Severe SARS-CoV-2 Breakthrough Reinfection With Delta Variant After Recovery From Breakthrough Infection by Alpha Variant in a Fully Vaccinated Health Worker.

Background: Post infection immunity and post vaccination immunity both confer protection against COVID-19. However, there have been many whole genome sequencing proven reinfections and breakthrough infections. Both are most often mild and caused by Variants of Concern (VOC). Methods: The patient in our study underwent serial COVID-19 RT-PCR, blood tests for serology, acute phase reactants, and chest imaging as part of clinical care. We interviewed the patient for clinical history and retrieved reports and case papers. We retrieved stored RT-PCR positive samples for whole genome sequencing (WGS) of SARS-CoV-2 from the patient's breakthrough infections and the presumed index case. Findings: The patient had three RT-PCR confirmed SARS-CoV-2 infections. Two breakthrough infections occurred in quick succession with the first over 3 weeks after complete vaccination with COVISHIELD and despite post-vaccination seroconversion. The first breakthrough infection was due to the Alpha variant and the second due to the Delta variant. The Delta variant infection resulted in hypoxia, hospitalization, and illness lasting seven weeks. Serial serology, acute phase reactants, and chest imaging supported WGS in establishing distinct episodes of infection. WGS established a fully vaccinated family member as the index case. Interpretation: The patient had an Alpha variant breakthrough infection despite past infection, complete vaccination, and seroconversion. Despite boosting after this infection, the patient subsequently had a severe Delta variant breakthrough infection. This was also a WGS proven reinfection and, therefore, a case of breakthrough reinfection. The patient acquired the infection from a fully vaccinated family member.

Mumbai mayhem of COVID-19 pandemic reveals important factors that influence susceptibility to infection.

BACKGROUND: COVID-19 severity is disproportionately high in the elderly and people with comorbidities. However, other factors that predispose individuals to increased chances of infection are unclear. METHODS: Data from 18,600 people screened for COVID-19 in Mumbai during the outbreak's initial phase, March 7 to June 30, 2020, were used to assess risk factors associated with COVID-19 using the odds ratio analysis. FINDINGS: Males aged ≥60 years having both diabetes and hypertension were at the highest risk of COVID-19 infection (M vs. F OR=2.5, 95% CI=1.34-4.67, p = 0.0049). People having both diabetes and hypertension in ≥20 years (OR=4.11, 95% CI=3.26-5.20, p <0.0001), diabetes and hypertension independently in 20-39 (OR=4.13, 95% CI=2.22-7.70, p <0.0001, OR=4.32, 95% CI=2.10-8.88, p = 0.0001) and ≥60 years (OR=2.69, 95% CI=1.87-3.87, p <0.0001, OR=2.03, 95% CI=1.46-2.82, p <0.0001), chronic renal disease in 20-39 years (OR=5.38, 95% CI=1.91-15.09, p = 0.0007) age groups had significantly higher risk of COVID-19 infection than those without comorbidity. Quarantined people had significantly lower positive odds (OR=0.59, 95% CI=0.53-0.66, p <0.001) than non-quarantined people. INTERPRETATION: Our research indicates that the risk of getting COVID-19 disease is not equal. When considering sex, age, and comorbidity together, we found that males aged ≥60 years and having both diabetes and hypertension had a significantly high risk of COVID-19 infection. Therefore, remedial measures such as vaccination programs should be prioritized for at-risk individuals. FUNDING: SERB, India: SB/S1/COVID-2/2020 and Seed grant RD/0520-IRCCHC0-006 from IRCC, IIT Bombay.

Clinical, Serological, Whole Genome Sequence Analyses to Confirm SARS-CoV-2 Reinfection in Patients From Mumbai, India.

Background: SARS-CoV-2 infection may not provide long lasting post-infection immunity. While hundreds of reinfections have reported only a few have been confirmed. Whole genome sequencing (WGS) of the viral isolates from the different episodes is mandatory to establish reinfection. Methods: Nasopharyngeal (NP), oropharyngeal (OP) and whole blood (WB) samples were collected from paired samples of four individuals who were suspected of SARS-CoV-2 reinfection based on distinct clinical episodes and RT-PCR tests. Details from their case record files and investigations were documented. RNA was extracted from the NP and OP samples and subjected to WGS, and the nucleotide and amino acid sequences were subjected to genome and protein-based functional annotation analyses. Serial serology was performed for Anti-N IgG, Anti- S1 RBD IgG, and sVNT (surrogate virus neutralizing test). Findings: Three patients were more symptomatic with lower Ct values and longer duration of illness. Seroconversion was detected soon after the second episode in three patients. WGS generated a genome coverage ranging from 80.07 to 99.7%. Phylogenetic analysis revealed sequences belonged to G, GR and "Other" clades. A total of 42mutations were identified in all the samples, consisting of 22 non-synonymous, 17 synonymous, two in upstream, and one in downstream regions of the SARS-CoV-2 genome. Comparative genomic and protein-based annotation analyses revealed differences in the presence and absence of specific mutations in the virus sequences from the two episodes in all four paired samples. Interpretation: Based on the criteria of genome variations identified by whole genome sequencing and supported by clinical presentation, molecular and serological tests, we were able to confirm reinfections in two patients, provide weak evidence of reinfection in the third patient and unable to rule out a prolonged infection in the fourth. This study emphasizes the importance of detailed analyses of clinical and serological information as well as the virus's genomic variations while assessing cases of SARS-CoV-2 reinfection.

A novel ORF1a-based SARS-CoV-2 RT-PCR assay to resolve inconclusive samples.

BACKGROUND: India bears the second largest burden of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. A multitude of reverse transcription polymerase chain reaction (RT-PCR) detection assays with disparate gene targets, including automated high-throughput platforms, are available. Varying concordance and interpretation of diagnostic results in this setting can result in significant reporting delays, leading to suboptimal disease management. This article reports the development of a novel ORF1a-based SARS-CoV-2 RT-PCR assay - Viroselect - that shows high concordance with conventional assays and the ability to resolve inconclusive results generated during the peak of the epidemic in Mumbai, India. METHODS: A unique target region within SARS-CoV-2 ORF1a - the non-structural protein 3 (nsp3) region - was used to design and develop the assay. This hypervariable region (1923-3956) between SARS-CoV-2, SARS-CoV-1 and Middle East respiratory syndrome coronavirus was utilized to design the primers and probes for the RT-PCR assay. The concordance of this assay with commonly used emergency use authorization (US Food and Drug Administration) manual kits and an automated high-throughput testing platform was evaluated. Further, a retrospective analysis was carried out using Viroselect on samples reported as 'inconclusive' between April and October 2020. RESULTS: In total, 701 samples were tested. Concordance analysis of 477 samples demonstrated high overall agreement of Viroselect with both manual (87.6%) and automated (84.7%) assays. Also, in the retrospective analysis of 224 additional samples reported as 'inconclusive', Viroselect was able to resolve 100% (19/19) and 93.7% (192/205) of samples which had inconclusive results on manual and automated high-throughput platforms, respectively. CONCLUSION: Viroselect had high concordance with conventional assays, both manual and automated, and has potential to resolve inconclusive samples.

sMAdCAM: IL-6 Ratio Influences Disease Progression and Anti-Viral Responses in SARS-CoV-2 Infection.

The role of sMAdCAM, an important gut immune migratory marker, remains unexplored in COVID-19 pathogenesis considering recent studies positing the gut as a sanctuary site for SARS-CoV-2 persistence. Thus, assimilating profiles of systemic inflammatory mediators with sMAdCAM levels may provide insights into the progression of COVID-19 disease. Also, the role of these markers in governing virus specific immunity following infection remains largely unexplored. A cohort (n = 84) of SARS-C0V-2 infected individuals included a group of in-patients (n = 60) at various stages of disease progression together with convalescent individuals (n = 24) recruited between April and June 2020 from Mumbai, India. Follow-up of 35 in-patients at day 7 post diagnosis was carried out. Th1/Th2/Th17 cytokines along with soluble MAdCAM (sMAdCAM) levels in plasma were measured. Also, anti-viral humoral response as measured by rapid antibody test (IgG, IgM), Chemiluminescent Immunoassay (IgG), and antibodies binding to SARS-CoV-2 proteins were measured by Surface Plasmon Resonance (SPR) from plasma. IL-6 and sMAdCAM levels among in-patients inversely correlated with one another. When expressed as a novel integrated marker-sMIL index (sMAdCAM/IL-6 ratio)-these levels were incrementally and significantly higher in various disease states with convalescents exhibiting the highest values. Importantly, sMAdCAM levels as well as sMIL index (fold change) correlated with peak association response units of receptor binding domain and fold change in binding to spike respectively as measured by SPR. Our results highlight key systemic and gut homing parameters that need to be monitored and investigated further to optimally guide therapeutic and prophylactic interventions for COVID-19.