Spatio-temporal dynamics of intra-host variability in SARS-CoV-2 genomes.

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). 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 1347 samples collected till June 2020, we recorded 16 410 iSNV sites throughout the SARS-CoV-2 genome. We found ∼42% of the iSNV sites to be reported as SNVs by 30 September 2020 in consensus sequences submitted to GISAID, which increased to ∼80% by 30th June 2021. Following this, analysis of another set of 1774 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) lineage-defining variations appeared as iSNVs before getting fixed in the population. Besides, mutations in RdRp as well as RNA-editing by APOBEC and ADAR deaminases seem to contribute to the differential prevalence of iSNVs in hosts. We also observe hyper-variability 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.

SARS-CoV-2 B.1.617.2 Delta variant replication and immune evasion.

The B.1.617.2 (Delta) variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first identified in the state of Maharashtra in late 2020 and spread throughout India, outcompeting pre-existing lineages including B.1.617.1 (Kappa) and B.1.1.7 (Alpha)1. In vitro, B.1.617.2 is sixfold less sensitive to serum neutralizing antibodies from recovered individuals, and eightfold less sensitive to vaccine-elicited antibodies, compared with wild-type Wuhan-1 bearing D614G. Serum neutralizing titres against B.1.617.2 were lower in ChAdOx1 vaccinees than in BNT162b2 vaccinees. B.1.617.2 spike pseudotyped viruses exhibited compromised sensitivity to monoclonal antibodies to the receptor-binding domain and the amino-terminal domain. B.1.617.2 demonstrated higher replication efficiency than B.1.1.7 in both airway organoid and human airway epithelial systems, associated with B.1.617.2 spike being in a predominantly cleaved state compared with B.1.1.7 spike. The B.1.617.2 spike protein was able to mediate highly efficient syncytium formation that was less sensitive to inhibition by neutralizing antibody, compared with that of wild-type spike. We also observed that B.1.617.2 had higher replication and spike-mediated entry than B.1.617.1, potentially explaining the B.1.617.2 dominance. In an analysis of more than 130 SARS-CoV-2-infected health care workers across three centres in India during a period of mixed lineage circulation, we observed reduced ChAdOx1 vaccine effectiveness against B.1.617.2 relative to non-B.1.617.2, with the caveat of possible residual confounding. Compromised vaccine efficacy against the highly fit and immune-evasive B.1.617.2 Delta variant warrants continued infection control measures in the post-vaccination era.

Detection of Mycobacterium tuberculosis purified ESAT-6 (Rv3875) by magnetic bead-coupled gold nanoparticle-based immuno-PCR assay.

PURPOSE: Immuno-PCR (I-PCR), an ultrasensitive method, combines the versatility of ELISA with the exponential amplification capacity of PCR. Coupling of detection antibodies with the reporter DNA is a critical step of I-PCR. Gold nanoparticles (GNPs) and magnetic beads (MBs) are relatively easy to attach with the antibodies and DNA. Therefore, we designed MB-coupled GNP-based I-PCR (MB-GNP-I-PCR) assay for the detection of Mycobacterium tuberculosis antigen. METHODS: GNPs were synthesized by chemical reduction and seed-mediated synthesis. Functionalized GNPs were prepared by coupling GNPs with the detection antibodies and reporter DNA and were characterized. Detection limit of M. tuberculosis-specific purified early secreted antigenic target-6 (ESAT-6) (Rv3875) was determined by MB-GNP-I-PCR. RESULTS: Transmission electron microscopy revealed spherical and slightly polydispersed GNPs of ~20 and ~60 nm size. Coupling of antibodies to GNPs was indicated by a shift in absorption maxima from 524 to 534 nm, which was confirmed by transmission electron microscopy. A color reaction with ELISA and the presence of 76 bp product by PCR further validated the coupling of detection antibodies and signal DNA to the functionalized GNPs. Also, attachment of capture antibodies with MBs was confirmed by magneto-ELISA. Detection limit of purified ESAT-6 by MB-GNP-I-PCR was determined to be 10 fg/mL, 105-fold lower than analogous ELISA. Notably, no sample matrix effect was observed in the saliva samples of healthy individuals spiked with the purified ESAT-6. CONCLUSION: Unlike conventional I-PCR (solid format), MB-GNP-I-PCR (liquid format) is relatively simple with the reduced background signals, which can be further exploited for the clinical diagnosis of tuberculosis.

Silver nanoparticles induced alterations in multiple cellular targets, which are critical for drug susceptibilities and pathogenicity in fungal pathogen (Candida albicans).

PURPOSE: A significant increase in the incidence of fungal infections and drug resistance has been observed in the past decades due to limited availability of broad-spectrum antifungal drugs. Nanomedicines have shown significant antimicrobial potential against various drug-resistant microbes. Silver nanoparticles (AgNps) are known for their antimicrobial properties and lower host toxicity; however, for clinical applications, evaluation of their impact at cellular and molecular levels is essential. The present study aims to understand the cellular and molecular mechanisms of AgNp-induced toxicity in a common fungal pathogen, Candida albicans. METHODS: AgNps were synthesized by chemical reduction method and characterized using UV-visible spectroscopy, X-ray powder diffraction, transmission electron microscopy, scanning electron microscopy-energy dispersive X-ray spectroscopy, energy dispersive X-ray fluorescence, and zeta potential. The anti-Candida activity of AgNps was assessed by broth microdilution and spot assays. Effects of AgNps on cellular and molecular targets were assessed by monitoring the intracellular reactive oxygen species (ROS) production in the absence and presence of natural antioxidant, changes in surface morphology, cellular ultrastructure, membrane microenvironment, membrane fluidity, membrane ergosterol, and fatty acids. RESULTS: Spherical AgNps (10-30 nm) showed minimum inhibitory concentration (minimum concentration required to inhibit the growth of 90% of organisms) at 40 µg/mL. Our results demonstrated that AgNps induced dose-dependent intracellular ROS which exerted antifungal effects; however, even scavenging ROS by antioxidant could not offer protection from AgNp mediated killing. Treatment with AgNps altered surface morphology, cellular ultrastructure, membrane microenvironment, membrane fluidity, ergosterol content, and fatty acid composition, especially oleic acid. CONCLUSION: To summarize, AgNps affected multiple cellular targets crucial for drug resistance and pathogenicity in the fungal cells. The study revealed new cellular targets of AgNps which include fatty acids like oleic acid, vital for hyphal morphogenesis (a pathogenic trait of Candida). Yeast to hypha transition being pivotal for virulence and biofilm formation, targeting virulence might emerge as a new paradigm for developing nano silver-based therapy for clinical applications in fungal therapeutics.

In vitro studies on oxidative stress-independent, Ag nanoparticles-induced cell toxicity of Candida albicans, an opportunistic pathogen.

Silver nanoparticles (AgNps) have attracted maximal attention among all metal nanoparticles, and the study of their biological properties has gained impetus for further medical adoption. This study evaluated the cellular and molecular mechanisms associated with the action of AgNps against an opportunistic pathogen, Candida albicans. Spherical, stable AgNp (average size 21.6 nm) prepared by a chemical reduction method showed minimum inhibitory concentration (required to inhibit the growth of 90% of organisms) at 40 µg/mL. AgNps have been reported to induce oxidative stress-mediated programmed cell death through the accumulation of intracellular reactive oxygen species (ROS). However, this study demonstrated that intracellular levels of AgNp-induced ROS could be reversed by using antioxidant ascorbic acid, but the sensitivity of AgNp-treated Candida cells could not be completely reversed. Moreover, in addition to the generation of ROS, the AgNps were found to affect other cellular targets resulting in altered membrane fluidity, membrane microenvironment, ergosterol content, cellular morphology, and ultrastructure. Thus, the generation of ROS does not seem to be the sole major cause of AgNp-mediated cell toxicity in Candida. Rather, the multitargeted action of AgNps, generation of ROS, alterations in ergosterol content, and membrane fluidity together seem to have potentiated anti-Candida action. Thus, this "nano-based drug therapy" is likely to favor broad-spectrum activity, multiple cellular targets, and minimum host toxicity. AgNps, therefore, appear to have the potential to address the challenges in multidrug resistance and fungal therapeutics.