Whole genome analysis of Human Mastadenovirus D causing Keratoconjunctivitis in India - A multicentre study.

INTRODUCTION: Human mastadenovirus (HAdV) types 8, 37, 64 have been considered the major contributors in Epidemic keratoconjunctivitis (EKC) epidemics, but recent surveillance data have shown the involvement of emerging recombinants, including HAdV-53, HAdV-54, and HAdV-56. In our initial work, positive samples for adenovirus revealed that our strains were closer to HAdV-54 than HAdV-8. Hence, the current study aimed to use whole genome technology to identify the HAdV strain correctly. METHODOLOGY: Oxford Nanopore technique was used, wherein a Targeted sequencing approach using long-range PCR amplification was performed. Primers were designed using HAdV-54 (AB448770.2) and HAdV-8 (AB897885.1) as reference sequences. Amplicons were sequenced on the GridION sequencer. Sequences were annotated using Gatu software, and similarities with standard reference sequence was calculated using Bioedit software. The phylogenetic tree was built after alignment in MEGA v7.0 using Neighbour joining method for each of the genes: Penton, Hexon, and Fiber. The effect of novel amino acid changes was evaluated using the PROVEAN tool. The Recombination Detection Program (RDP) package Beta 4.1 was used to identify recombinant sequences. RESULTS: Of the five samples sequenced, OL450401, OL540403, and OL540406 showed nucleotide similarity to HAdV-54 in the penton region. Additionally, OL450401 showed a statistically significant recombination event with HAdV-54 as minor and HAdV-8 as major parents. This was further supported by phylogenetic analysis as well. CONCLUSIONS: In the present study, we have found evidence of a shift from HAdV-8 towards HAdV-54, thus stressing the need for surveillance of HAdVs and to stay updated on the rise of new recombinants.

Cytokine profile, neutralisation potential and viral replication dynamics in sera of chikungunya patients in India: a cross-sectional study.

Background: Chikungunya disease (CHIKD) is a threat to global health, as it impairs the quality of life of an infected individual ranging from months to years. A systematic evaluation of the serological, virological, and immunological aspects of the circulating viruses and their impact on the host response is imperative for better understanding of the evolving disease dynamics. Methods: Serum samples were collected from 196 acute CHIKD patients from ten tertiary care hospitals across India during 2016-2021. Out of 196 patients, paired convalescent samples were collected from 51 patients (one-month post-onset of symptoms). The serum samples were profiled for cytokines and neutralisation capacity. Further, chikungunya virus (CHIKV) was isolated from the acute sera and the replication kinetics of the clinical isolates was evaluated. Findings: Serological analysis indicated that neutralisation could be correlated to seroconversion in the convalescent phase but not found significant in acute phase. In the acute phase samples, there was a correlation between elevated serum levels of IFN-γ, IP-10, MCP-1 and MIG and disease severity. During convalescent phase, pro-inflammatory markers such as IL-6, IL-1ß, IL-9 and IP-10 were found to be elevated with a corresponding decline in the secretion of anti-inflammatory cytokines such as IL-4 and IL-10, which correlated with persistent arthralgia. Analysis of replication of the clinical isolates revealed that 68.4% of viruses were fast-growing in the Vero cells (cytopathic effect [CPE] observed within 24 h post-infection), and their corresponding acute serum samples showed an elevated secretion of IFN-α, IL-1RA, IL-17F, IL-9, MCP-1 and MIP-1α. Interpretation: This study provides an important overview of neutralisation capabilities and cytokine responses along with virus pathogenesis associated with CHIKV infections in India. Funding: Biotechnology Industry Research Assistance Council (BIRAC).

First detection of Varicella Zoster Virus clade 9 cases in India during mpox surveillance.

BACKGROUND: The multi-country mpox outbreak across the globe has led to the systematic surveillance of mpox cases in India. During the surveillance of mpox, we encountered cases of Varicella Zoster Virus (VZV) in suspected mpox cases amongst children & adults. This study focused on the genomic characterization of VZV in India. METHODS: A total of 331 mpox suspected cases were tested for VZV through real-time PCR, and the positive samples were subjected to next-generation sequencing to retrieve the whole genome of VZV using CLC genomics software. Phylogenetic analysis has been done in MEGA 11.0 software to identify circulating clades. RESULT: Of the 331 suspected cases, 28 cases with vesicular rashes were found to be positive for VZV. The maximum genome could be retrieved from the clinical specimens of 16 cases with coverage greater than 98% when mapped with reference strain Dumas (NC 001348). The phylogenetic analyses of these sequences determined the circulation of clades 1, 5, and 9 in India. Further, the sequence analysis demonstrated non-synonymous single nucleotide polymorphism (SNPs) among specific ORF of VZV including ORF 14, ORF 22, ORF 36, ORF 37 and ORF 51. Although clade 1 and 5 has been reported earlier, the circulation of clade 9 of VZV has been determined for the first time in India. CONCLUSION: Although the circulation of different clades of VZV was reported from India, the presence of clade 9 was detected for the first time during the mpox surveillance.

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.

A comparative analysis of the second and third wave of the Covid-19 pandemic: an experience from a tertiary care hospital in Western India.

Introduction. As the world was still recovering from the 2020 pandemic, the devastating impact of Covid-19 driven by the Delta variant shook the world in 2021. As the second wave was declining, there was an unusual surge in Covid-19 positive cases by the end of 2021 which led to global concern about the change in virus characteristics.Hypothesis/gap statement. Whole genome sequencing is critical for understanding a rapidly progressing pandemic.Aim. To provide an insight into the major differences encountered in the changing characteristics between the second and third waves of the pandemic at a tertiary care hospital in India.Methods. A retrospective observational cohort analysis was conducted on Covid-positive patients during the second wave of the Covid-19 pandemic (from March 2021 to April 2021) and the third wave of the Covid-19 pandemic (from December 2021 to January 2022).Results. Out of 303 Covid-19 positive cases, 52 samples were tested by whole genome sequencing during the second wave and 108 during the third wave. A decline of 18.5 % was observed in the case fatality rate from the second wave to the third wave. There was a 5 % decline in the number of patients admitted with ARDS and a 16.3 % decline in the number of patients with co-morbidities.In total, 51.9 percent of cases were due to the Delta variant during the second wave and 95 percent due to the Omicron variant during the third wave. We found that 36.5 % of Covid-positive patients during the second wave had been vaccinated compared to 40 % in the third wave.Conclusion. Whole genome sequencing of clinical samples from a wide range of individuals during a viral epidemic will enable us to develop a more rapid public health response to new variants and identify the required vaccine modifications more quickly.

Genomic analysis unveils genome degradation events and gene flux in the emergence and persistence of S. Paratyphi A lineages.

Paratyphoid fever caused by S. Paratyphi A is endemic in parts of South Asia and Southeast Asia. The proportion of enteric fever cases caused by S. Paratyphi A has substantially increased, yet only limited data is available on the population structure and genetic diversity of this serovar. We examined the phylogenetic distribution and evolutionary trajectory of S. Paratyphi A isolates collected as part of the Indian enteric fever surveillance study "Surveillance of Enteric Fever in India (SEFI)." In the study period (2017-2020), S. Paratyphi A comprised 17.6% (441/2503) of total enteric fever cases in India, with the isolates highly susceptible to all the major antibiotics used for treatment except fluoroquinolones. Phylogenetic analysis clustered the global S. Paratyphi A collection into seven lineages (A-G), and the present study isolates were distributed in lineages A, C and F. Our analysis highlights that the genome degradation events and gene acquisitions or losses are key molecular events in the evolution of new S. Paratyphi A lineages/sub-lineages. A total of 10 hypothetically disrupted coding sequences (HDCS) or pseudogenes-forming mutations possibly associated with the emergence of lineages were identified. The pan-genome analysis identified the insertion of P2/PSP3 phage and acquisition of IncX1 plasmid during the selection in 2.3.2/2.3.3 and 1.2.2 genotypes, respectively. We have identified six characteristic missense mutations associated with lipopolysaccharide (LPS) biosynthesis genes of S. Paratyphi A, however, these mutations confer only a low structural impact and possibly have minimal impact on vaccine effectiveness. Since S. Paratyphi A is human-restricted, high levels of genetic drift are not expected unless these bacteria transmit to naive hosts. However, public-health investigation and monitoring by means of genomic surveillance would be constantly needed to avoid S. Paratyphi A serovar becoming a public health threat similar to the S. Typhi of today.

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.

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.

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.

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).

Delta variant SARS-CoV-2 infections in pediatric cases during the second wave in India.

BACKGROUND: During October 2020, Delta variant was detected for the first time in India and rampantly spread across the globe. It also led to second wave of pandemic in India which affected millions of people. However, there is limited information pertaining to the SARS-CoV-2 strain infecting the children in India. METHODS: Here, we assessed the SARS-CoV-2 lineages circulating in the pediatric population of India during the second wave of the pandemic. Clinical and demographic details linked with the nasopharyngeal/oropharyngeal swabs (NPS/OPS) collected from SARS-CoV-2 cases (n = 583) aged 0-18 year and tested positive by real-time RT-PCR were retrieved from March to June 2021. RESULTS: Symptoms were reported among 37.2% of patients and 14.8% reported to be hospitalized. The E gene CT value had significant statistical difference at the point of sample collection when compared to that observed in the sequencing laboratory. Out of these 512 sequences 372 were VOCs, 51 were VOIs. Most common lineages observed were Delta, followed by Kappa, Alpha and B.1.36, seen in 65.82%, 9.96%, 6.83% and 4.68%, respectively in the study population. CONCLUSION: Overall, it was observed that Delta strain was the leading cause of SARS-CoV-2 infection in Indian children during the second wave of the pandemic. We emphasize on the need of continuous genomic surveillance in SARS-CoV-2 infection even amongst children.

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.

Isolation and Genomic Characterization of SARS-CoV-2 Omicron Variant Obtained from Human Clinical Specimens.

Due to the failure of virus isolation of the Omicron variant in Vero CCL-81 from the clinical specimens of COVID-19 cases, an initial in vivo and subsequent in vitro approach was utilized for the isolation of the virus. A total of 74 oropharyngeal/nasopharyngeal specimens were collected from SARS-CoV-2 positive international travellers and a contact case at Delhi and Mumbai, India. All the specimens were sequenced using next-generation sequencing and simultaneously inoculated onto Vero CCL-81 cells for virus isolation. Subsequently, two omicron positive specimens were inoculated into Syrian hamsters for two passages. The initial passage of the positive hamster specimens was inoculated onto Vero CCL-81 cells. The clinical specimens, hamster specimens, and Vero CCL-81 passages were sequenced to assess the mutational changes in different host species. The replication of the Omicron variant in hamsters was confirmed with the presence of a high viral load in nasal turbinate and lung specimens of both passages. The successful isolation of the virus from hamster specimens with Vero CCL-81 was observed with cytopathic effect in infected cells and high viral load in the cell suspension. The genome analysis revealed the presence of L212C mutation, Tyrosine 69 deletion, and C25000T nucleotide change in spike gene of hamster passage sequences and an absence of V17I mutation in E gene in hamster passage sequences, unlike human clinical specimen and Vero CCL-81 passages. No change was observed in the furin cleavage site in any of the specimen sequences, suggesting intact pathogenicity of the virus isolate. Our data demonstrated successful isolation of the Omicron variant with the in vivo method first followed by in vitro method. The virus isolate could be used in the future to explore different aspects of the Omicron variant.

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.

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.

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.

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.