Amplicon-based nanopore minion sequencing of patients with COVID-19 omicron variant from India

SARS-CoV-2 infection has been playing havoc with emerging omicron variants of concern (VoC). Here, we report sequencing of the omicron variant in 13 patients from India using Oxford Nanopore Technology (ONT) Minion, wherein a rapid amplicon based sequence analysis was performed to assess and compare with existing 34 mutations in spike glycoprotein. We highlight and discuss the nature of these mutations that are unique and common to other populations. This is perhaps the first report on omicron variants from India using a long read sequencing chemistry.

Clinicogenomic analysis of breakthrough infections by SARS CoV2 variants after ChAdOx1 nCoV- 19 vaccination in healthcare workers

BackgroundIndia saw a massive surge and emergence of SARS CoV2 variants. We elucidated clinical and humoral immune response and genomic analysis of vaccine breakthrough (VBT) infections after ChAdOx1 nCoV-19 vaccine in healthcare workers (HCWs). MethodsThe study was conducted on 1858 HCWs receiving two doses of ChAdOx1 nCoV-19 vaccine. Serial blood samples were collected to measure SARS CoV2 IgG and neutralizing antibodies. 46 RT-PCR positive samples from VBT infections were subjected to whole genome sequencing (WGS). ResultsInfection was confirmed in 219 (11.79%) HCWs of which 21.46% (47/219) were non-vaccinated, significantly more (p <0.001) than 9.52% (156/1639) vaccinated group. VBT infections were significantly higher in doctors and nurses compared to other hospital staff (p <0.001). Unvaccinated individuals had 1.57 times higher risk of infection compared to partially vaccinated (p 0.02) and 2.49 times than fully vaccinated (<0.001). Partially vaccinated were at higher risk of infection than fully vaccinated (RR 1.58,p 0.01). There were 3 (1.36%) severe cases and 1 death in unvaccinated group compared to none in the vaccinated. Non-response after 14 days of second dose was seen in 6.5% (3/46) and low antibody levels (1-4.62 S/CO) in 8.69% (4/46). Delta variant (B.1.617.2) was dominant (69.5%) and reinfection was documented in 4 (0.06%) HCWs. ConclusionsNearly one in ten vaccinated HCWs can get infected, more so with only single dose (13.65%) than two doses (8.62%). Fully vaccinated are better protected with higher humoral immune response. Genomic analysis revealed an alarming rise of Delta variant (B.1.617.2) in VBT infections.

Genomic characterization and Epidemiology of an emerging SARS-CoV-2 variant in Delhi, India

Delhi, the national capital of India, has experienced multiple SARS-CoV-2 outbreaks in 2020 and reached a population seropositivity of over 50% by 2021. During April 2021, the city became overwhelmed by COVID-19 cases and fatalities, as a new variant B.1.617.2 (Delta) replaced B.1.1.7 (Alpha). A Bayesian model explains the growth advantage of Delta through a combination of increased transmissibility and partial reduction of immunity elicited by prior infection (median estimates; x1.5-fold, 20% reduction). Seropositivity of an employee and family cohort increased from 42% to 86% between March and July 2021, with 27% reinfections, as judged by increased antibody concentration after previous decline. The likely high transmissibility and partial evasion of immunity by the Delta variant contributed to an overwhelming surge in Delhi. One-Sentence SummaryDelhi experienced an overwhelming surge of COVID-19 cases and fatalities peaking in May 2021 as the highly transmissible and immune evasive Delta variant replaced the Alpha variant.

Intra-host variability in global SARS-CoV-2 genomes as signatures of RNA editing: implications in viral and host response outcomes

During the course of the COVID-19 pandemic, large-scale genome sequencing of SARS-CoV-2 has been useful in tracking its spread and in identifying Variants Of Concern (VOC). Besides, viral and host factors could contribute to variability within a host that can be captured in next-generation sequencing reads as intra-host Single Nucleotide Variations (iSNVs). Analysing 1, 347 samples collected till June 2020, we recorded 18, 146 iSNV sites throughout the SARS-CoV-2 genome. Both, mutations in RdRp as well as APOBEC and ADAR mediated RNA editing seem to contribute to the differential prevalence of iSNVs in hosts. Noteworthy, 41% of all unique iSNVs were reported as SNVs by 30th September 2020 in samples submitted to GISAID, which increased to [~]80% by 30th June 2021. Following this, analysis of another set of 1, 798 samples sequenced in India between November 2020 and May 2021 revealed that majority of the Delta (B.1.617.2) and Kappa (B.1.617.1) variations appeared as iSNVs before getting fixed in the population. We also observe hyper-editing events at functionally critical residues in Spike protein that could alter the antigenicity and may contribute to immune escape. Thus, tracking and functional annotation of iSNVs in ongoing genome surveillance programs could be important for early identification of potential variants of concern and actionable interventions. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=177 SRC="FIGDIR/small/417519v3_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@12b6ac2org.highwire.dtl.DTLVardef@16df897org.highwire.dtl.DTLVardef@dbbec2org.highwire.dtl.DTLVardef@c8de14_HPS_FORMAT_FIGEXP M_FIG C_FIG

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 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.

Integrated genomic view of SARS-CoV-2 in India

India first detected SARS-CoV-2, causal agent of COVID-19 in late January-2020, imported from Wuhan, China. March-2020 onwards; importation of cases from rest of the countries followed by seeding of local transmission triggered further outbreaks in India. We used ARTIC protocol based tiling amplicon sequencing of SARS-CoV-2 (n=104) from different states of India using a combination of MinION and MinIT from Oxford Nanopore Technology to understand introduction and local transmission. The analyses revealed multiple introductions of SARS-CoV-2 from Europe and Asia following local transmission. The most prevalent genomes with patterns of variance (confined in a cluster) remain unclassified, here, proposed as A4-clade based on its divergence within A-cluster. The viral haplotypes may link their persistence to geo-climatic conditions and host response. Despite the effectiveness of non-therapeutic interventions in India, multipronged strategies including molecular surveillance based on real-time viral genomic data is of paramount importance for a timely management of the pandemic.

IL35 modulation altered survival, cytokine environment and histopathological consequences during malaria infection in mice.

BACKGROUND: The immune modulating potential of IL-35 in multiple human disorders has been reported. Consequent upon the recognition of inflammatory cytokine activation and its preponderance for mediating pathology during malaria infection, the study aimed to characterize the expression and functional contribution(s) of IL-35 in Plasmodium berghei (strain ANKA) infected mice. METHODS: Plasmodium berghei infection in male ICR mice was used as the rodent model of choice. The time course of IL-35 expression in the systemic circulation and tissues of P. berghei infected mice as well as their healthy control counterparts was assessed by enzyme linked immunosorbent assay and immunohistochemistry respectively. The effect of modulating IL-35 by recombinant IL-35 protein or neutralizing anti-Epstein-Barr virus-induced gene 3 antibody on the cytokine environment during P. berghei infection was assessed by flow cytometry. Furthermore, the influence of modulating IL-35 on histopathological hallmarks of malaria and disease progression was evaluated. RESULTS: Interleukin-35 was significantly up regulated in serum and tissues of P. berghei infected mice and correlated with parasitaemia. Neutralization of IL-35 significantly enhanced the release of IFN-γ, decreased the expression of IL-6 and decreased parasitaemia patency. Neutralization of IL-35 was also associated with a tendency towards increased survival as well as the absence of pathological features associated with malaria infection unlike recombinant IL-35 protein administration which sustained a normal course of infection and unfavourable malaria associated histological outcomes in P. berghei infected mice. CONCLUSION: These results indicate the involvement of IL-35 in P. berghei induced malaria infection. IL-35 neutralization strategies may represent viable therapeutic modalities beneficial for the resolution of malaria infection.

Repurposing Pathogenic Variants of DMD Gene and its Isoforms for DMD Exon Skipping Intervention.

BACKGROUND: Duchenne Muscular Dystrophy (DMD) is a progressive, fatal neuromuscular disorder caused by mutations in the DMD gene. Emerging antisense oligomer based exon skipping therapy provides hope for the restoration of the reading frame. OBJECTIVES: Population-based DMD mutation database may enable exon skipping to be used for the benefit of patients. Hence, we planned this study to identify DMD gene variants in North Indian DMD cases. METHODS: A total of 100 DMD cases were recruited and Multiplex ligation-dependent probe amplification (MLPA) analysis was performed to obtain the deletion and duplication profile. RESULTS: Copy number variations (deletion/duplication) were found in 80.85% of unrelated DMD cases. Sixty-eight percent of cases were found to have variations in the distal hotspot region (Exon 45-55) of the DMD gene. Exon 44/45 variations were found to be the most prominent among single exon variations, whereas exon 49/50 was found to be the most frequently mutated locations in single/multiple exon variations. As per Leiden databases, 86.84% cases harboured out-of-frame mutations. Domain wise investigation revealed that 68% of mutations were localized in the region of spectrin repeats. Dp140 isoform was predicted to be absent in 62/76 (81.57%) cases. A total of 45/80 (56.25%) and 23/80 (28.70%) DMD subjects were predicted to be amenable to exon 51 and exon 45 skipping trials, respectively. CONCLUSION: A major proportion of DMD subjects (80%) could be diagnosed by the MLPA technique. The data generated from our study may be beneficial for strengthening of mutation database in the North Indian population.

Manganism without Parkinsonism: Isolated Unilateral Upper Limb Tremor in a Welder

No abstract available.

Applications of lipid based formulation technologies in the delivery of biotechnology-based therapeutics.

In the last decades several new biotechnologically-based therapeutics have been developed due to progress in genetic engineering. A growing challenge facing pharmaceutical scientists is formulating these compounds into oral dosage forms with adequate bioavailability. An increasingly popular approach to formulate biotechnology-based therapeutics is the use of lipid based formulation technologies. This review highlights the importance of lipid based drug delivery systems in the formulation of oral biotechnology based therapeutics including peptides, proteins, DNA, siRNA and vaccines. The different production procedures used to achieve high encapsulation efficiencies of the bioactives are discussed, as well as the factors influencing the choice of excipient. Lipid based colloidal drug delivery systems including liposomes and solid lipid nanoparticles are reviewed with a focus on recent advances and updates. We further describe microemulsions and self-emulsifying drug delivery systems and recent findings on bioactive delivery. We conclude the review with a few examples on novel lipid based formulation technologies.

Lack of association of transthyretin variations with spinocerebellar ataxia in north Indian population

Background & Objective: Transthyretin (TTR) has been associated with spinocerebellar ataxia (SCA) by several independent case reports. Coexistence of TTR and SCA mutations, overlapping clinical symptoms as well as altered levels of TTR in SCA patients suggest a correlation between TTR and SCA. To our knowledge, no large cohort based study has been attempted to examine the association of SCA with polymorphism in TTR gene. Here, we chose to investigate TTR variations in SCA patients (n=266) and controls (n=192) of North Indian ethnicity. Methods: We sequenced the exons including exon-intron boundaries of TTR gene in 55 patients and 55 controls. We observed four variations which were further genotyped by single base extension method (SNaPshot) in a larger cohort (SCA patients n=211 and controls n=137). Results: A novel synonymous variation c.372 C>G in exon 4 was detected in heterozygous condition in one control sample. We found nominal association for rs1800458 (Gly6Ser), with SCA (p-value < 0.05) which did not remain after Bonferroni correction for multiple tests. Pairwise linkage disequilibrium (LD) analysis revealed no LD between studied SNPs. Further, we employed two-marker sliding window analysis and observed a weak association of haplotype AT of rs1800458 and rs1667251 with SCA patients (p-value <0.05) which was not retained after Bonferroni correction. Conclusion: Our data suggests no association of genetic variations of TTR in SCA pathology.