An amplicon-based approach for fast and scalable genome sequencing of lumpy skin disease virus on benchtop next-generation sequencers (preprint)

Lumpy Skin Disease (LSD) is a viral disease predominantly affecting cattle and caused by a poxvirus belonging to the capripoxvirus genus. LSD is characterized by extensive cutaneous lesions, with severe consequent morbidity and sometimes mortality of the affected animal. Timely diagnosis and control of the spread of infections through measures including vaccination are therefore of great importance in preventing social and economic consequences of the disease. Genomic studies from outbreaks in the past have provided unique insights including the identification of recombinant variants in recent years. Genome sequencing and genomic surveillance of the disease therefore could provide useful insights into the evolution and epidemiology of the virus and could potentially also contribute to the development of diagnostic tools. Previous approaches for genome sequencing of the virus used a variety of approaches including amplicon-based sequencing as well as metagenomic approaches, which are tedious, time-consuming as well as costly. The wide availability of benchtop next-generation sequencing equipment and the application of sequencing-based approaches to enable genomic epidemiology of SARS-CoV-2 at scale, motivated us to create an amplicon-based approach based on the Illumina COVIDSeq assay1. for fast, scalable and cost-effective sequencing of the Lumpy Skin Disease Virus. This protocol is a modification of the previously published COVIDSeq assay 1 and can be adapted to any Illumina sequencing platform as an accelerated and scalable system for quick detection as well as genomic surveillance of LSD. For complete details on the use and execution of this protocol, please refer to Bhatt et al. (2023).2

A case of SARS-CoV-2 Omicron variant co-infection with Influenza A-H3N2 from India (preprint)

We report a case of SARS-CoV-2 Omicron variant co-infection with influenza A H3N2 detected from Kerala, India. The patient, a 10-year-old girl, had symptoms of low-grade fever, cough, and cold. As part of the ongoing surveillance, a throat swab was taken and sent for testing, and the influenza A virus isolated from the patient was identified as subtype H3N2. Whole-genome sequencing and analysis of the viral isolates suggested that the SARS-CoV-2 isolate belonged to BA.4.1 sublineage of Omicron while the influenza A isolate belonged to the 2a.3 clade of H3N2 and clustered with other H3N2 genomes from Maldives, India, Bangladesh, and the United Arab Emirates. The report highlights the importance of genomic surveillance of SARS-CoV-2 co-infections with other respiratory illnesses for understanding the prevalence of co-infections and their rapid detection and prevention.

Anti-COVID-19 Activity of FDA Approved Drugs through RNA G-quadruplex Binding (preprint)

The COVID-19 pandemic caused by SARS-CoV-2 has caused millions of infections and deaths worldwide. Limited treatment options and the threat from emerging variants underline the need for novel and widely accessible therapeutics. G-quadruplexes (G4s) are nucleic acid secondary structures known to affect many cellular processes including viral replication and transcription. We identified heretofore not reported G4s with remarkably low mutation frequency across >4 million SARS-CoV-2 genomes. The G4 structure was targeted using FDA approved drugs that can bind G4s - Chlorpromazine (CPZ) and Prochlorperazine (PCZ). We found significant inhibition in lung pathology and lung viral load of SARS-CoV-2 challenged hamsters when treated with CPZ, PCZ that was comparable to the widely used antiviral drug Remdesivir. In support, in vitro G4 binding, inhibition of reverse transcription from RNA isolated from COVID-infected humans, and attenuated viral replication and infectivity in vero cell cultures were clear in case of both CPZ/PCZ. Apart from the wide accessibility of CPZ/PCZ, targeting relatively invariant nucleic acid structures poses an attractive strategy against fast mutating viruses like SARS-CoV-2.

Clinical characteristics of AY.4 infections are similar to B.1.617.2 infections: a preliminary study

Background: The SARS-CoV-2 Delta variant (B.1.617.2) was first detected in India in late 2020 and soon became the predominant lineage owing to its high transmissibility. Over time, the virus has acquired mutations and has evolved into many new sub-lineages. AY.4 is one such sub-lineage that grew in frequency globally. Therefore, we aimed to compare the severity of infection due to Delta sub-lineages to Delta infections in Pune, Maharashtra, India. Material and Methods: Whole-genome sequencing and analysis of 255 SARS-CoV-2 positive samples, collected between 1st August to 1st September 2021, by BJ Government Medical College, Pune, was carried out at the Indian Institute of Science Education and Research (IISER), Pune and the Council of Scientific and Industrial Research-Institute of Genomics and Integrative Biology (CSIR-IGIB), New Delhi. Individual-level data on these patients were collected from ICMR COVID-19 Data Portal. Additional information regarding the presence of any symptoms, comorbidities, hospitalization, international travel history within 14 days and vaccination status was collected by telephonic interview with each patient by the BJGMC Sequencing Team.

High throughput detection and genetic epidemiology of SARS-CoV-2 using COVIDSeq next-generation sequencing

The rapid emergence of coronavirus disease 2019 (COVID-19) as a global pandemic affecting millions of individuals globally has necessitated sensitive and high-throughput approaches for the diagnosis, surveillance, and determining the genetic epidemiology of SARS-CoV-2. In the present study, we used the COVIDSeq protocol, which involves multiplex-PCR, barcoding, and sequencing of samples for high-throughput detection and deciphering the genetic epidemiology of SARS-CoV-2. We used the approach on 752 clinical samples in duplicates, amounting to a total of 1536 samples which could be sequenced on a single S4 sequencing flow cell on NovaSeq 6000. Our analysis suggests a high concordance between technical duplicates and a high concordance of detection of SARS-CoV-2 between the COVIDSeq as well as RT-PCR approaches. An in-depth analysis revealed a total of six samples in which COVIDSeq detected SARS-CoV-2 in high confidence which were negative in RT-PCR. Additionally, the assay could detect SARS-CoV-2 in 21 samples and 16 samples which were classified inconclusive and pan-sarbeco positive respectively suggesting that COVIDSeq could be used as a confirmatory test. The sequencing approach also enabled insights into the evolution and genetic epidemiology of the SARS-CoV-2 samples. The samples were classified into a total of 3 clades. This study reports two lineages B.1.112 and B.1.99 for the first time in India. This study also revealed 1,143 unique single nucleotide variants and added a total of 73 novel variants identified for the first time. To the best of our knowledge, this is the first report of the COVIDSeq approach for detection and genetic epidemiology of SARS-CoV-2. Our analysis suggests that COVIDSeq could be a potential high sensitivity assay for the detection of SARS-CoV-2, with an additional advantage of enabling the genetic epidemiology of SARS-CoV-2.

Human embryonic stem cell-derived cardiomyocytes express SARS-CoV-2 host entry proteins: screen to identify inhibitors of infection (preprint)

Patients with cardiovascular comorbidities are more susceptible to severe infection with SARS-CoV-2, known to directly cause pathological damage to cardiovascular tissue. We outline a screening platform using human embryonic stem cell-derived cardiomyocytes, confirmed to express the protein machinery critical for SARS-CoV-2 infection, and a pseudotyped virus system. The method has allowed us to identify benztropine and DX600 as novel inhibitors of SARS-CoV-2 infection.

Bispecific antibody prevents SARS-CoV-2 escape and protects mice from disease (preprint)

Neutralizing antibodies targeting the receptor binding domain (RBD) of the SARS-CoV-2 Spike (S) are among the most promising approaches against coronavirus disease 2019 (COVID-19). We developed a bispecific, IgG1-like molecule based on two antibodies derived from COVID-19 convalescent donors, C121 and C135. CoV-X2 simultaneously binds two independent sites on the RBD and, unlike its parental antibodies, completely prevents S binding to Angiotensin-Converting Enzyme 2 (ACE2), the virus cellular receptor. Furthermore, CoV-X2 recognizes a broad panel of RBD variants and neutralizes SARS-CoV-2 and the escape mutants generated by the single monoclonals at sub-nanomolar concentrations. In a novel model of SARS-CoV-2 infection with lung inflammation, CoV-X2 protects mice from disease and suppresses viral escape. Thus, simultaneous targeting of non-overlapping RBD epitopes by IgG-like bispecific antibodies is feasible and effective, combining into a single molecule the advantages of antibody cocktails.

DINC-COVID: A webserver for ensemble docking with flexible SARS-CoV-2 proteins (preprint)

Motivation: Recent efforts to computationally identify inhibitors for SARS-CoV-2 proteins have largely ignored the issue of receptor flexibility. We have implemented a computational tool for ensemble docking with the SARS-CoV-2 proteins, including the main protease (Mpro), papain-like protease (PLpro) and RNA-dependent RNA polymerase (RdRp). Results: Ensembles of other SARS-CoV-2 proteins are being prepared and made available through a user-friendly docking interface. Plausible binding modes between conformations of a selected ensemble and an uploaded ligand are generated by DINC, our parallelized meta-docking tool. Binding modes are scored with three scoring functions, and account for the flexibility of both the ligand and receptor. Additional details on our methods are provided in the supplementary material. Availability: dinc-covid.kavrakilab.org . Supplementary information: Details on methods for ensemble generation and docking are provided as supplementary data online.

Insights from Genomes and Genetic Epidemiology of SARS-CoV-2 isolates from the state of Andhra Pradesh (preprint)

Coronavirus disease (COVID-19) emerged from a city in China and has now spread as a global pandemic affecting millions of individuals. The causative agent, SARS-CoV-2 is being extensively studied in terms of its genetic epidemiology using genomic approaches. Andhra Pradesh is one of the major states of India with the third-largest number of COVID-19 cases with limited understanding of its genetic epidemiology. In this study, we have sequenced 293 SARS-CoV-2 genome isolates from Andhra Pradesh with a mean coverage of 13,324X. We identified 564 high-quality SARS-CoV-2 variants, out of which 15 are novel. A total of 18 variants mapped to RT-PCR primer/probe sites, and 4 variants are known to be associated with an increase in infectivity. Phylogenetic analysis of the genomes revealed the circulating SARS-CoV-2 in Andhra Pradesh majorly clustered under the clade A2a (94%), while 6% fall under the I/A3i clade, a clade previously defined to be present in large numbers in India. To the best of our knowledge, this is the most comprehensive genetic epidemiological analysis performed for the state of Andhra Pradesh.

The SARS-CoV-2 S1 spike protein mutation N501Y alters the protein interactions with both hACE2 and human derived antibody: A Free energy of perturbation study (preprint)

The N501Y mutation in Covid-19 arise many question but a small amount of data are currently available. An urgent understanding of N501Y mechanism of action at molecular level is highly required. Here, we present the preliminary results of our Free energy perturbation (FEP) and Molecular dynamics (MD) calculations for the interaction of the spike S1 receptor binding domain (RBD) with both the ACE2 receptor and an antibody, STE90-C11, derived from COVID-19 patients. The results shown that the S1 RBD-ACE2 interaction was increased whereas those with the STE90-C11 antibody significantly decreased (over about 160 times). This may explain the observed in UK more spread of the virus but also emerge an important question about the possible human immune response and already available vaccines. Indeed, the latter may still act well but our data indicate some possible reduction of their effect. Further studies of N501Y mutation are need.

TCR meta-clonotypes for biomarker discovery with tcrdist3: quantification of public, HLA-restricted TCR biomarkers of SARS-CoV-2 infection (preprint)

As the mechanistic basis of adaptive cellular antigen recognition, T cell receptors (TCRs) encode clinically valuable information that reflects prior antigen exposure and potential future response. However, despite advances in deep repertoire sequencing, enormous TCR diversity complicates the use of TCR clonotypes as clinical biomarkers. We propose a new framework that leverages antigen-enriched repertoires to form meta-clonotypes -- groups of biochemically similar TCRs -- that can be used to robustly quantify functionally similar TCRs in bulk repertoires. We apply the framework to TCR data from COVID-19 patients, generating 1,915 public TCR meta-clonotypes from the 18 SARS-CoV-2 antigen-enriched repertoires with the strongest evidence of HLA-restriction. Applied to independent cohorts, meta-clonotypes targeting these specific epitopes were more frequently detected in bulk repertoires compared to exact amino acid matches, and 44% (845/1915) were significantly enriched among COVID-19 patients that expressed the putative restricting HLA allele, demonstrating the potential utility of meta-clonotypes as antigen-specific features for biomarker development. To enable further applications, we developed an open-source software package, tcrdist3, that implements this framework and facilitates workflows for distance-based TCR repertoire analysis.

Influence of HLA class II polymorphism on predicted cellular immunity against SARS-CoV-2 at the population and individual level (preprint)

Development of effective adaptive immune responses after coronavirus disease 2019 (COVID-19) and after vaccination against SARS-CoV-2 is predicated on recognition of viral peptides, presented in the context of HLA class II molecules, by CD4+ T-cells. We capitalised on extensive high resolution HLA data deposited in the National Marrow Donor Program registry to obtain detailed information on human HLA haplotype frequencies of twenty five human populations and used a bioinformatics approach to investigate the role of HLA polymorphism on SARS-CoV-2 immunogenicity at the population and at the individual level. Within populations, we identify wide inter-individual variability in predicted CD4+ T-cell reactivity against structural, non-structural and accessory SARS-CoV-2 proteins, according to expressed HLA genotype. However, we find similar potential for anti-SARS-CoV-2 cellular immunity at the population level, across all ethnic groups examined, suggesting that HLA polymorphism is unlikely to account for observed disparities in clinical outcomes after COVID-19 among different race and ethnic groups. We predict robust immune reactivity against SARS-CoV-2 Spike protein, the basis for the majority of current vaccination efforts, both at the population and individual level, despite significant variation in Spike-derived peptide presentation by individual HLA genotypes. Finally, we provide comprehensive maps of SARS-CoV-2 proteome immunogenicity accounting for population coverage in major ethnic groups. Our findings provide important insight on the potential role of HLA polymorphism on development of protective immunity after SARS-CoV-2 infection and after vaccination and a firm basis for further experimental studies in this field.

Genetic epidemiology of variants associated with immune escape from global SARS-CoV-2 genomes (preprint)

Many antibody and immune escape variants in SARS-CoV-2 are now documented in literature. The availability of SARS-CoV-2 genome sequences enabled us to investigate the occurrence and genetic epidemiology of the variants globally. Our analysis suggests that a number of genetic variants associated with immune escape have emerged in global populations.

Differential Dynamic Behavior of Prefusion Spike Proteins of SARS Coronaviruses 1 and 2 (preprint)

Within the last two decades, severe acute respiratory syndrome (SARS) coronaviruses 1 and 2 (SARS-CoV-1 and SARS-CoV-2) have caused two major outbreaks. For reasons yet to be fully understood the COVID-19 outbreak caused by SARS-CoV-2 has been significantly more widespread than the 2003 SARS epidemic caused by SARS-CoV-1, despite striking similarities between the two viruses. One of the most variable genes differentiating SARS-CoV-1 and SARS-CoV-2 is the S gene that encodes the spike glycoprotein. This protein mediates a crucial step in the infection, i.e., host cell recognition and viral entry, which starts with binding to the host cell angiotensin converting enzyme 2 (ACE2) protein for both viruses. Recent structural and functional studies have shed light on the differential binding behavior of the SARS-CoV-1 and SARS-CoV-2 spike proteins. In particular, cryogenic electron microscopy (cryo-EM) studies show that ACE2 binding is preceded by a large-scale conformational change in the spike protein to expose the receptor binding domain (RBD) to its binding partner. Unfortunately, these studies do not provide detailed information on the dynamics of this activation process. Here, we have used an extensive set of unbiased and biased microsecond-timescale all-atom molecular dynamics (MD) simulations of SARS-CoV-1 and SARS-CoV-2 spike protein ectodomains in explicit solvent to determine the differential behavior of spike protein activation in the two viruses. Our results based on nearly 50 microseconds of equilibrium and nonequilibrium MD simulations indicate that the active form of the SARS-CoV-2 spike protein is considerably more stable than the active SARS-CoV-1 spike protein. Unlike the active SARS-CoV-2 spike, the active SARS-CoV-1 spike spontaneously undergoes a large-scale conformational transition to a pseudo-inactive state, which occurs in part due to interactions between the N-terminal domain (NTD) and RBD that are absent in the SARS-CoV-2 spike protein. Steered MD (SMD) simulations indicate that the energy barriers between active and inactive states are comparatively lower for the SARS-CoV-1 spike protein. Based on these results, we hypothesize that the greater propensity of the SARS-CoV-2 spike protein to remain in the active conformation contributes to the higher transmissibility of SARS-CoV-2 in comparison to SARS-CoV-1. These results strongly suggest that the differential binding behavior of the active SARS-CoV-1 and 2 spike proteins is not merely due to differences in their RBDs as other domains of the spike protein such as the NTD could play a crucial role in the effective binding process, which involves the pre-binding activation. Therefore, our hypothesis predicts that mutations in regions such as the NTD, which is not directly involved in binding, may lead to a change in the effective binding behavior of the coronavirus.

Candidate screening of host cell membrane proteins involved in SARS-CoV-2 entry (preprint)

COVID-19 represents a real threat to the global population, and understanding the biological features of the causative virus (SARS-CoV-2) is imperative to aid in mitigating this threat. Analyses of proteins such as primary receptors and co-receptors (co-factors) that are involved in SARS-CoV-2 entry into host cells will provide important clues to help control the virus. Here, we identified host cell membrane protein candidates that were present in proximity to the attachment sites of SARS-CoV-2 spike proteins through the use of proximity labeling and proteomics analysis. The identified proteins represent candidate key factors that may be required for viral entry. Our results indicated that a number of membrane proteins, including DPP4, Cadherin-17, and CD133, were identified to co-localize with cell membrane-bound SARS-CoV-2 spike proteins in Caco-2 cells that were used to expand the SARS-CoV-2 virion. We anticipate that the information regarding these protein candidates will be utilized for the future development of vaccines and antiviral agents against SARS-CoV-2.

Potential impact on coagulopathy of gene variants of coagulation related proteins that interact with SARS-CoV-2 (preprint)

Thrombosis has been one of the complications of the Coronavirus disease of 2019 (COVID-19), often associated with poor prognosis. There is a well-recognized link between coagulation and inflammation, however, the extent of thrombotic events associated with COVID-19 warrants further investigation. Poly(A) Binding Protein Cytoplasmic 4 (PABPC4), Serine/Cysteine Proteinase Inhibitor Clade G Member 1 (SERPING1) and Vitamin K epOxide Reductase Complex subunit 1 (VKORC1), which are all proteins linked to coagulation, have been shown to interact with SARS proteins. We computationally examined the interaction of these with SARS-CoV-2 proteins and, in the case of VKORC1, we describe its binding to ORF7a in detail. We examined the occurrence of variants of each of these proteins across populations and interrogated their potential contribution to COVID-19 severity. Potential mechanisms by which some of these variants may contribute to disease are proposed. Some of these variants are prevalent in minority groups that are disproportionally affected by severe COVID-19. Therefore, we are proposing that further investigation around these variants may lead to better understanding of disease pathogenesis in minority groups and more informed therapeutic approaches.

Initial insights into the genetic epidemiology of SARS-CoV-2 isolates from Kerala suggest local spread from limited introductions (preprint)

Coronavirus disease 2019 (COVID-19) rapidly spread from a city in China to almost every country in the world, affecting millions of individuals. Genomic approaches have been extensively used to understand the evolution and epidemiology of SARS-CoV-2 across the world. Kerala is a unique state in India well connected with the rest of the world through a large number of expatriates, trade, and tourism. The first case of COVID-19 in India was reported in Kerala in January 2020, during the initial days of the pandemic. The rapid increase in the COVID-19 cases in the state of Kerala has necessitated the understanding of the genetic epidemiology of circulating virus, evolution, and mutations in SARS-CoV-2. We sequenced a total of 200 samples from patients at a tertiary hospital in Kerala using COVIDSeq protocol at a mean coverage of 7,755X. The analysis identified 166 unique high-quality variants encompassing 4 novel variants and 89 new variants identified for the first time in SARS-CoV-2 samples isolated from India. Phylogenetic and haplotype analysis revealed that the circulating population of the virus was dominated (94.6% of genomes) by three distinct introductions followed by local spread, apart from identifying polytomies suggesting recent outbreaks. The genomes formed a monophyletic distribution exclusively mapping to the A2a clade. Further analysis of the functional variants revealed two variants in the S gene of the virus reportedly associated with increased infectivity and 5 variants that mapped to five primer/probe binding sites that could potentially compromise the efficacy of RT-PCR detection. To the best of our knowledge, this is the first and most comprehensive report of genetic epidemiology and evolution of SARS-CoV-2 isolates from Kerala.

High throughput detection and genetic epidemiology of SARS-CoV-2 using COVIDSeq next generation sequencing (preprint)

The rapid emergence of coronavirus disease 2019 (COVID-19) as a global pandemic affecting millions of individuals globally has necessitated sensitive and high-throughput approaches for the diagnosis, surveillance and for determining the genetic epidemiology of SARS-CoV-2. In the present study, we used the COVIDSeq protocol, which involves multiplex-PCR, barcoding and sequencing of samples for high-throughput detection and deciphering the genetic epidemiology of SARS-CoV-2. We used the approach on 752 clinical samples in duplicates, amounting to a total of 1536 samples which could be sequenced on a single S4 sequencing flow cell on NovaSeq 6000. Our analysis suggests a high concordance between technical duplicates and a high concordance of detection of SARS-CoV-2 between the COVIDSeq as well as RT-PCR approaches. An in-depth analysis revealed a total of six samples in which COVIDSeq detected SARS-CoV-2 in high confidence which were negative in RT-PCR. Additionally, the assay could detect SARS-CoV-2 in 21 samples and 16 samples which were classified inconclusive and pan-sarbeco positive respectively suggesting that COVIDSeq could be used as a confirmatory test. The sequencing approach also enabled insights into the evolution and genetic epidemiology of the SARS-CoV-2 samples. The samples were classified into a total of 3 clades. This study reports two lineages B.1.112 and B.1.99 for the first time in India. This study also revealed 1,143 unique single nucleotide variants and added a total of 73 novel variants identified for the first time. To the best of our knowledge, this is the first report of the COVIDSeq approach for detection and genetic epidemiology of SARS-CoV-2. Our analysis suggests that COVIDSeq could be a potential high sensitivity assay for detection of SARS-CoV-2, with an additional advantage of enabling genetic epidemiology of SARS-CoV-2.

Analysis of the potential impact of genomic variants in SARS-CoV-2 genomes from India on molecular diagnostic assays (preprint)

An isolated epidemic of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) causing Coronavirus Diseases (COVID-19) originating in Wuhan, China has now rapidly emerged into a global pandemic affecting millions of people worldwide. Molecular detection of SARS-CoV-2 using reverse transcription polymerase chain reaction (RT-PCR) forms the mainstay in screening, diagnosis and epidemiology of disease. The virus has been evolving through base substitutions. The recent availability of genomes of SARS-CoV-2 isolates from different countries including India motivated us to assess the presence and potential impact of variations in target sites for the oligonucleotide primers and probes used in molecular diagnosis. We catalogued a total of 132 primers or probes sequences from the literature and the public domain. Our analysis revealed a total of 125 unique genetic variants in 80 either primers or probes binding sites. A total of 13 unique variants had allele frequency of [≥] 1% in Indian SARS-CoV-2 genomes mapped to the primers or probes binding sites. A total of 15 primers or probes binding sites had cumulative variant frequency of [≥] 1% in the SARS-CoV-2 genomes. These included primers or probes sites which are widely used in India and across the world for molecular diagnosis as well as approved by national and international agencies. This highlights the need for sequencing genomes of emerging pathogens to make evidence based policies for development and approval of diagnostics. To the best of our knowledge, ours is the most comprehensive analysis of genomic variants in genomes of SARS-CoV-2 isolates from India and their potential impact on efficacy of molecular diagnostics.

A rapid and sensitive method to detect SARS-CoV-2 virus using targeted-mass spectrometry (preprint)

In the last few months, there has been a global catastrophic outbreak of severe acute respiratory syndrome disease caused by the novel corona virus SARS-CoV-2 affecting millions of people worldwide. Early diagnosis and isolation is key to contain the rapid spread of the virus. Towards this goal, we report a simple, sensitive and rapid method to detect the virus using a targeted mass spectrometric approach, which can directly detect the presence of virus from naso-oropharyngeal swabs. Using a multiple reaction monitoring we can detect the presence of two peptides specific to SARS-CoV-2 in a 2.3 minute gradient run with 100% specificity and 90.4 % sensitivity when compared to RT-PCR. Importantly, we further show that these peptides could be detected even in the patients who have recovered from the symptoms and have tested negative for the virus by RT-PCR highlighting the sensitivity of the technique. This method has the translational potential of in terms of the rapid diagnostics of symptomatic and asymptomatic COVID-19 and can augment current methods available for diagnosis of SARS-CoV-2.