Biology subjects

Kumar, N.

Publications and source records attributed to Kumar, N..

At least 145 records · Page 8Linked to original sources

bioRxiv2021

Influenza virus replication in cardiomyocytes drives heart dysfunction and fibrosis

Cardiac dysfunction is a common extrapulmonary complication of severe influenza virus infection. Prevailing models propose that influenza-associated heart dysfunction is indirectly triggered by cytokine mediated cardiotoxicity downstream of the inflamed lung, rather than by direct infection of cardiac tissue. To test the etiology of cardiac dysfunction resulting from influenza virus infection, we generated a novel recombinant H1N1 influenza A virus that was attenuated in cardiomyocytes by incorporation of target sequences for miRNAs expressed specifically in that cell type (miR133b and miR206). Compared with control virus, mice infected with the miR-targeted virus had significantly reduced heart viral titers, confirming cardiac attenuation of viral replication. The miR-targeted virus, however, was fully replicative and inflammatory in lungs when compared to control virus, and induced similar systemic weight loss. The miR-targeted virus induced considerably lower levels of cardiac arrhythmia, fibrosis, and inflammation, compared with control virus, in mice lacking interferon induced transmembrane protein 3 (IFITM3), which serve as the only available model for severe influenza-associated cardiac pathology. We conclude that robust replication of virus in the heart is required for pathology even when lung inflammation is severe. Indeed, we show that human stem cell-derived cardiomyocytes are susceptible to influenza virus infection. This work establishes a fundamental new paradigm in which influenza virus damages the heart through direct infection of cardiomyocytes.

immunology↗

bioRxiv2021

Adeno-associated virus (AAV) reduces cortical dendritic complexity in a TLR9-dependent manner

Recombinant adeno-associated viruses (AAVs) allow rapid and efficient gene delivery in the nervous system. AAVs are widely used in research and are the basis of multiple FDA-approved gene therapies. Here, we find that the immune response to AAVs genome reduces dendritic complexity in mammalian cortex. Dendritic loss associated with AAV-mediated gene delivery occurs at experimentally-relevant titers, cannot be explained by responses to transgene expression or surgery, and is not restricted to a particular capsid serotype, encoded transgene, promoter, or production facility. AAV-associated dendritic loss is accompanied by a decrease in the frequency and amplitude of miniature excitatory postsynaptic currents (mEPSCs) and upregulation of immune molecules that can limit dendritic complexity and synaptic transmission. Blocking detection of unmethylated CpG-rich DNA via Toll-like receptor 9 (TLR9) protects dendritic complexity, suggesting that immunodetection of a core feature of the AAV genome triggers dendritic loss. These results reveal previously unsuspected impacts of AAV on neuronal structure and function and identify TLR9 inhibitors as important tools to improve the safety and efficacy of AAV-mediated gene delivery in the nervous system.

neuroscience↗

bioRxiv2021

Rapid, multianalyte detection of opioid metabolites in wastewater

By monitoring opioid metabolites, wastewater-based epidemiology (WBE) could be an excellent tool for real-time information on consumption of illicit drugs. A key limitation of WBE is the reliance on costly laboratory-based techniques that require substantial infrastructure and trained personnel, resulting in long turnaround times. Here, we present an aptamer-based graphene field effect transistor (AptG-FET) platform for simultaneous detection of three different opioid metabolites. This platform provides a reliable, rapid, and inexpensive method for quantitative analysis of opioid metabolites in wastewater (WW). The platform delivers a limit of detection (LOD) 2-3 orders of magnitude lower than previous reports, but in line with the concentrations range (pg/ml to ng/ml) of these opioid metabolites present in real samples. To enable multianalyte detection we developed a facile, reproducible, and high yield fabrication process producing twenty G-FETs with integrated side gate platinum (Pt) electrodes on a single chip. Our devices achieved the simultaneous and selective multianalyte detection of three different metabolites: Noroxycodone (NX), 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), and Norfentanyl (NF) in wastewater.

biophysics↗

bioRxiv2021

Balancing growth amidst salinity stress-lifestyle perspectives from the extremophyte model Schrenkiella parvula

Schrenkiella parvula, a leading extremophyte model in Brassicaceae, can grow and complete its life cycle under multiple environmental stresses, including high salinity. While foundational genomic resources have been created for S. parvula, a comprehensive physiological or structural characterization of its salt stress responses is absent. We aimed to identify the influential traits that lead to stress-resilient growth of this species. We examined salt-induced changes in the physiology and anatomy of S. parvula throughout its lifecycle across multiple tissues. We found that S. parvula maintains or even exhibits enhanced growth during various developmental stages at salt stress levels known to inhibit growth in Arabidopsis and most crops. The resilient growth of S. parvula was associated with key traits that synergistically allow continued primary root growth, expansion of xylem vessels across the root-shoot continuum, and a high capacity to maintain tissue water levels by developing larger and thicker leaves while facilitating continued photosynthesis during salt stress. These traits at the vegetative phase were followed by a successful transition to the reproductive phase via early flowering, development of larger siliques, and production of viable seeds during salt stress. Additionally, the success of self-fertilization during early flowering stages was dependent on salt-induced filament elongation in flowers that aborted in the absence of salt. Our results suggest that the maintenance of leaf water status and enhancement of selfing in early flowers to ensure reproductive success, are among the most influential traits that contribute to the extremophyte lifestyle of S. parvula in its natural habitat. One sentence summarySchrenkiella parvula salt-resilient growth is facilitated by uncompromised primary root growth, expansion of xylem vessels, maintenance of leaf water status and photosynthesis, and early flowering.

plant biology↗

bioRxiv2021

The genome trilogy of Anopheles stephensi, an urban malaria vector, reveals structure of a locus associated with adaptation to environmental heterogeneity

BackgroundAnopheles stephensi is the most menacing malaria vector to watch for in newly urbanizing parts of the world. The fitness is reported to be a direct consequence of the vector adapting to laying eggs in over-head water tanks with street-side water puddles polluted by oil and sewage. Large frequent inversions of malaria vectors are implicated in adaptation. ResultsWe report the assembly of a strain of An. stephensi of the type-form, collected from a construction site from Chennai (IndCh) in 2016. The genome completes the trilogy with respect to a 16 Mbp inversion (2Rb) in An. stephensi associated with adaptation to environmental heterogeneity. Comparative genome analysis revealed breakpoint structure and allowed extraction of 22,650 segregating SNPs for typing this inversion. Using whole genome sequencing of 82 individual mosquitoes, we conclude that one third of both wild and laboratory populations maintain heterozygous genotype of 2Rb. The large number of SNPs are tailored to assign inversion genotype directly from 1740 exonic SNPs 80% of which are expressed in various developmental stages. ConclusionsThe genome trilogy approach accelerates study of fine structure and typing of important inversions in malaria vectors putting the genome resources for the much understudied An. stephensi, on par with the extensively studied malaria vector, Anopheles gambiae. We argue that the IndCh genome is relevant for field translation work compared to those reported earlier by showing that individuals from diverse populations cluster with IndCh pointing to significant commerce between cities, perhaps, allowing for survival of the fittest strain.

genomics↗

bioRxiv2021

Performance of Core Genome Multilocus Sequence Typing Compared to Capillary-Electrophoresis PCR Ribotyping and SNP Analysis of Clostridioides difficile

Clostridioides difficile is the most common cause of antibiotic-associated gastrointestinal infections. Capillary-electrophoresis (CE)-PCR ribotyping is currently the gold standard for C. difficile typing but lacks discriminatory power to study transmission and outbreaks in detail. New molecular methods have the capacity to differentiate better, but backward compatibility with CE-PCR ribotyping must be assessed. Using a well-characterized collection of diverse strains (N=630; 100 unique ribotypes [RTs]), we aimed to investigate PCR ribotyping prediction from core genome multilocus sequence typing (cgMLST). Additionally, we compared the discriminatory power of cgMLST (SeqSphere & EnteroBase) and whole genome MLST (wgMLST) (EnteroBase) with single nucleotide polymorphism (SNP) analysis). A unique cgMLST profile (>6 allele differences) was observed in 82/100 ribotypes, indicating sufficient backward compatibility. Intra-RT allele difference varied per ribotype and MLST clade. Application of cg/wgMLST and SNP analysis in two outbreak settings with ribotypes RT078 and RT181 (known with a low intra-ribotype allele difference) showed no distinction between outbreak- and non-outbreak strains, in contrast to wgMLST and SNP analysis. We conclude that cgMLST has the potential to be an alternative to CE-PCR ribotyping. The method is reproducible, easy to standardize and offers higher discrimination. However, in some ribotype complexes adjusted cut-off thresholds and epidemiological data are necessary to recognize outbreaks. We propose to decrease the current threshold of 6 to 3 alleles to better identify outbreaks.

molecular biology↗

bioRxiv2021

Spatial organisation and homeostasis of epithelial lineages at the gastroesophageal junction is regulated by the divergent Wnt mucosal microenvironment

The gastroesophageal junction (GEJ), where squamous and columnar epithelia meet, is a hotspot for Barretts metaplasia development, dysbiosis and carcinogenesis. However, the mechanisms regulating GEJ homeostasis remain unclear. Here, by employing organoids, bulk and single-cell transcriptomics, single-molecule RNA in situ hybridisations and lineage tracing, we identified the spatial organisation of the epithelial, stromal compartment and the regulators that maintain the normal GEJ homeostasis. During development, common KRT8 progenitors generate committed unilineage p63/KRT5-squamous and KRT8-columnar stem cells responsible for the regeneration of postnatal esophagus and gastric epithelium that meet at GEJ. A unique spatial distribution of Wnt regulators in the underlying stromal compartment of these stem cells creates diverging Wnt microenvironments at GEJ and supports their differential regeneration. Further, we show that these tissue-resident stem cells do not possess the plasticity to transdifferentiate to the other lineage with the altered Wnt signals. Our study provides invaluable insights into the fundamental process of GEJ homeostasis and is crucial for understanding disease development.

developmental biology↗

bioRxiv2021

Mechanism of tissue expansion in the early stage of the migration

The collective cell migration is observed in many biological processes such as wound healing, embryogenesis, and cancer metastasis. Despite extensive theoretical and experimental studies on collective cell motion, there is no unified mechanism to explain it. In this work, we experimentally report the collectively growing cell colonies in the sub-marginal region of a freely expanding cell monolayer. These colonies could be responsible for the highly aligned collective cell migration observed in front cell rows. Our results provide a basic framework to understand the physical mechanism responsible for collective cell migration in the freely expanding monolayer.

biophysics↗

bioRxiv2021

hnRNPA1 regulates early translation to replication switch in SARS-CoV-2 life cycle

Our study suggests that methylation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA is essential for its optimal replication in the target cells. Heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1, an RNA-binding protein) was shown to mediate deposition of N6-methyladenosine (m6A) in internal SARS-CoV-2 RNA. The levels of hNRNPA1 expression and extent of methylation varied, depending on the course of SARS-CoV-2 life cycle. The recruitment of eIF4E (translational initiation factor) facilitated viral RNA translation at 1 hour post infection (1 hpi). However, at 2 hpi, methylation of internal SARS-CoV-2 RNA recruited hNRNPA1 which facilitated viral RNA transcription but resulted in translational repression, a phenomenon contributing in understanding the early translation to replication switch in the viral life cycle. Besides, the abrogation of methylation also produced a defective 5 cap of viral RNA which failed to interact with eIF4E, thereby resulting in a decreased synthesis of viral proteins. To conclude, methylation of the internal and 5 cap of SARS-CoV-2 RNA was shown to regulate transcription and translation of SARS-CoV-2 in a time dependent manner. IMPORTANCERNA modifications are found in all life forms and have been linked to development, health and diseases. Our study reveals that internal SARS-CoV-2 RNA methylation (m6A) is essential for interaction with hNRNPA1 to effectively synthesize viral genome. Besides, m6A-marked RNA and hRNPA1 interaction was also shown to regulate early translation to replication switch in SARS-CoV-2 life cycle. Blocking SARS-CoV-2 RNA methylation resulted in reduced virus yield, suggesting epitranscriptomic machinery (methylation) facilitates SARS-CoV-2 replication and might represent potential target for new antiviral drugs against COVID-19.

microbiology↗

bioRxiv2021

Natural infection of SARS-CoV-2 delta variant in Asiatic lions (Panthera leo persica) in India

In May 2021, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was detected in nine Asiatic lions (Panthera leo persica) in Arignar Anna Zoological Park, Chennai, Tamil Nadu, India. Sequence and phylogenetic analysis showed that the SARS-CoV-2 viruses belong to a variant of concern (VOC, delta variant, B.1.617.2 lineage) and that these viruses clustered with B.1.617.2 lineage viruses of the same geographical region detected in the same month.

microbiology↗

bioRxiv2021

3D-Scaffold: Deep Learning Framework to Generate 3D Coordinates of Drug-like Molecules with Desired Scaffolds.

The prerequisite of therapeutic drug design is to identify novel molecules with desired biophysical and biochemical properties. Deep generative models have demonstrated their ability to find such molecules by exploring a huge chemical space efficiently. An effective way to obtain molecules with desired target properties is the preservation of critical scaffolds in the generation process. To this end, we propose a domain aware generative framework called 3D-Scaffold that takes 3D coordinates of the desired scaffold as an input and generates 3D coordinates of novel therapeutic candidates as an output while always preserving the desired scaffolds in generated structures. We show that our framework generates predominantly valid, unique, novel, and experimentally synthesizable molecules that have drug-like properties similar to the molecules in the training set. Using domain specific datasets, we generate covalent and non-covalent antiviral inhibitors. To measure the success of our framework in generating therapeutic candidates, generated structures were subjected to high throughput virtual screening via docking simulations, which shows favorable interaction against SARS-CoV-2 main protease and non-structural protein endoribonuclease (NSP15) targets. Most importantly, our model performs well with relatively small volumes of training data and generalizes to new scaffolds, making it applicable to other domains.

bioinformatics↗

bioRxiv2021

Cdk5 drives formation of heterogeneous pancreatic neuroendocrine tumors

Pancreatic neuroendocrine tumors (PanNETs) are a heterogeneous population of neoplasms that arise from hormone-secreting islet cells of the pancreas and have increased markedly in incidence over the past four decades. Non-functional PanNETs, which occur more frequently than hormone-secreting tumors, are often not diagnosed until later stages of tumor development and have poorer prognoses. Development of successful therapeutics for PanNETs has been slow, partially due to a lack of diverse animal models for pre-clinical testing. Here, we report development of an inducible, conditional mouse model of PanNETs by using a bitransgenic system for regulated expression of the aberrant activator of Cdk5, p25, specifically in {beta}-islet cells. This model produces a heterogeneous population of PanNETs that includes a subgroup of well-differentiated, non-functional tumors. The utility of this model is enhanced by ability to form tumor-derived allografts. Production of these tumors demonstrates the causative potential of aberrantly active Cdk5 for generation of PanNETs. Further, we show that human PanNETs express Cdk5 pathway components, are dependent on Cdk5 for growth, and share genetic and transcriptional overlap with the INS-p25OE model. This new model of PanNETs will facilitate molecular delineation of Cdk5-dependent PanNETs and the development of new targeted therapeutics.

cancer biology↗

bioRxiv2021

Two microbiota subtypes identified in Irritable Bowel Syndrome with distinct responses to the low-FODMAP diet

ObjectiveReducing FODMAPs can be clinically beneficial in IBS but the mechanism is poorly understood. We aimed to detect microbial signatures that might predict response to the low FODMAP diet and assess whether microbiota compositional and functional shifts could provide insights into its mode of action. DesignWe used metagenomics to determine high-resolution taxonomic and functional profiles of the stool microbiota from IBS cases and household controls (n=56 pairs) on their usual diet. Clinical response and microbiota changes were studied in 41 pairs after 4 weeks on a low FODMAP diet. ResultsUnsupervised analysis of baseline IBS cases pre-diet identified two distinct microbiota profiles, which we refer to as IBSP (pathogenic-like) and IBSH (health-like) subtypes. IBSP microbiomes were enriched in Firmicutes and genes for amino acid and carbohydrate metabolism, but depleted in Bacteroidetes species. IBSH microbiomes were similar to controls. On the low FODMAP diet IBSH and control microbiota were unaffected, but the IBSP signature shifted towards a health-associated microbiome with an increase in Bacteroidetes (p=0.009), a decrease in Firmicutes species (p=0.004) and normalization of primary metabolic genes. The clinical response to the low FODMAP diet was greater in IBSP subjects compared to IBSH (p = 0.02). Conclusion50% of IBS cases manifested a pathogenic gut microbial signature. This shifted towards the healthy profile on the low FODMAP diet; and IBSP cases showed an enhanced clinical responsiveness to the dietary therapy. The effectiveness of FODMAP exclusion in IBSP may result from the alterations in gut microbiota and metabolites produced. Microbiota signatures could be useful as biomarkers to guide IBS treatment; and investigating IBSP species and metabolic pathways might yield insights regarding IBS pathogenic mechanisms. O_TEXTBOXSignificance of this studyO_ST_ABSWhat is already known on this subject?C_ST_ABSO_LIIBS subjects often respond to a low FODMAP diet. C_LIO_LIThe gut microbiota has been implicated in IBS. C_LIO_LIThe microbiota in IBS subjects may change with diet. C_LI What are the new findings?O_LIWe were able to stratify patients with IBS according to their gut microbiota species and metabolic gene signatures. C_LIO_LIWe identified a distinct gut microbiota subtype with an enhanced clinical response to a low FODMAP diet compared to other IBS subjects. C_LI How might it impact on clinical practice in the foreseeable future?O_LIThe potential development of a microbiota signature as a biomarker to manage IBS cases with a low FODMAP diet recommendation. C_LIO_LIIf the bacteria represented in the IBSP subtype are shown to play a pathogenic role in IBS, perhaps through the metabolic activity this provides a target for new therapies and an intermediate phenotype by which to assess them. C_LI C_TEXTBOX

microbiology↗

bioRxiv2021

High Throughput Virtual Screening and Validation of a SARS-CoV-2 Main Protease Non-Covalent Inhibitor

Despite the recent availability of vaccines against the acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the search for inhibitory therapeutic agents has assumed importance especially in the context of emerging new viral variants. In this paper, we describe the discovery of a novel non-covalent small-molecule inhibitor, MCULE-5948770040, that binds to and inhibits the SARS-Cov-2 main protease (Mpro) by employing a scalable high throughput virtual screening (HTVS) framework and a targeted compound library of over 6.5 million molecules that could be readily ordered and purchased. Our HTVS framework leverages the U.S. supercomputing infrastructure achieving nearly 91% resource utilization and nearly 126 million docking calculations per hour. Downstream biochemical assays validate this Mpro inhibitor with an inhibition constant (Ki) of 2.9 {micro}M [95% CI 2.2, 4.0]. Further, using room-temperature X-ray crystallography, we show that MCULE-5948770040 binds to a cleft in the primary binding site of Mpro forming stable hydrogen bond and hydrophobic interactions. We then used multiple {micro}s-timescale molecular dynamics (MD) simulations, and machine learning (ML) techniques to elucidate how the bound ligand alters the conformational states accessed by Mpro, involving motions both proximal and distal to the binding site. Together, our results demonstrate how MCULE-5948770040 inhibits Mpro and offers a springboard for further therapeutic design. O_TEXTBOXSignificance StatementThe ongoing novel coronavirus pandemic (COVID-19) has prompted a global race towards finding effective therapeutics that can target the various viral proteins. Despite many virtual screening campaigns in development, the discovery of validated inhibitors for SARS-CoV-2 protein targets has been limited. We discover a novel inhibitor against the SARS-CoV-2 main protease. Our integrated platform applies downstream biochemical assays, X-ray crystallography, and atomistic simulations to obtain a comprehensive characterization of its inhibitory mechanism. Inhibiting Mpro can lead to significant biomedical advances in targeting SARS-CoV-2 treatment, as it plays a crucial role in viral replication. C_TEXTBOX

biophysics↗

bioRxiv2021

Functional and taxonomic comparison of mouse and human gut microbiotas using extensive culturing and metagenomics

Mouse models are essential for biomedical science and drug discovery, yet it is not known how the bacteria in the mouse microbiota - important determinants of phenotypes of health and disease -affect their relevance to human disease. To interrogate the taxonomic and functional differences between the human and mouse gut microbiotas, we developed the Mouse Microbial Genome Collection (MMGC), a compilation of 276 genomes from cultured isolates and 45,218 metagenome-assembled genomes (MAGs) from 1,960 publicly available mouse metagenomes. The MMGC reveals that while only 2.65% of bacterial species are shared between mouse and human, over 80% of annotatable functions are present in both microbiomes. Using drug metabolism and butyrate synthesis as examples, we illustrate that although the species harbouring these key functions can differ between hosts, the MMGC enables identification of functionally equivalent taxa in the mouse and human microbiotas. The MMGC thereby facilitates the informed use of mice in biomedical research by providing access to the conservation and taxonomic locations of bacterial functions of interest.

microbiology↗

bioRxiv2021

Novel gut pathobionts confound results in a widely used mouse model of human inflammatory disease

The mammalian gut microbiota consists of hundreds of anaerobic bacterial species that shape intestinal homeostasis and influence host immune responses. Although the causal roles of specific human gut bacterial species in health and disease are emerging, the role of indigenous gut bacteria in driving immunophenotypic variability in mouse models of human disease remains poorly understood. We performed a large-scale experiment using 579 laboratory mice designed to identify and validate the causes of disease variability in the widely used dextran sulphate sodium (DSS) mouse model of inflammatory bowel disease. Using microbiome analysis, coupled with machine learning and targeted anaerobic culturing, we identified and isolated the novel gut pathobiont species Duncaniella muricolitica and Alistipes okayasuensis and fulfilled Kochs postulates in mice to show that each pathobiont exerts dominant effects in the DSS model leading to variable treatment responses. We show these pathobiont species are common, but not ubiquitous, in mouse facilities around the world, raising experimental design opportunities for improved mouse models of human intestinal diseases.

microbiology↗

bioRxiv2021

High-throughput screening of the ReFRAME, Pandemic Box, and COVID Box drug repurposing libraries against SARS-CoV2 nsp15 endoribonuclease to identify small-molecule inhibitors of viral activity.

SARS-CoV-2 has caused a global pandemic, and has taken over 1.7 million lives as of mid-December, 2020. Although great progress has been made in the development of effective countermeasures, with several pharmaceutical companies approved or poised to deliver vaccines to market, there is still an unmet need of essential antiviral drugs with therapeutic impact for the treatment of moderate-to-severe COVID-19. Towards this goal, a high-throughput assay was used to screen SARS-CoV-2 nsp15 uracil-dependent endonuclease (endoU) function against 13 thousand compounds from drug and lead repurposing compound libraries. While over 80% of initial hit compounds were pan-assay inhibitory compounds, three hits were confirmed as nsp15 endoU inhibitors in the 1-20 M range in vitro. Furthermore, Exebryl-1, a {beta}-amyloid anti-aggregation molecule for Alzheimers therapy, was shown to have antiviral activity between 10 to 66 M, in VERO, Caco-2, and Calu-3 cells. Although the inhibitory concentrations determined for Exebryl-1 exceed those recommended for therapeutic intervention, our findings show great promise for further optimization of Exebryl-1 as an nsp15 endoU inhibitor and as a SARS-CoV-2 antiviral. Author summaryDrugs to treat COVID-19 are urgently needed. To address this, we searched libraries of drugs and drug-like molecules for inhibitors of an essential enzyme of the virus that causes COVID-19, SARS-CoV-2 nonstructural protein (nsp)15. We found several molecules that inhibited the nsp15 enzyme function and one was shown to be active in inhibiting the SARS-CoV-2 virus. This demonstrates that searching for SARS-CoV-2 nsp15 inhibitors can lead inhibitors of SARS-CoV-2, and thus therapeutics for COVID-19. We are currently working to see if these inhibitors could be turned into a drug to treat COVID-19.

microbiology↗

bioRxiv2021

Molecular mechanisms underlying attenuation of live attenuated Japanese encephalitis virus vaccine SA14-14-2

The live attenuated Japanese encephalitis virus vaccine SA14-14-2 demonstrated [≥] 95 % efficacy and is today the vaccine of choice against JEV globally. Relative to its parent strain SA14, SA14-14-2 carries 46 nucleotide and 24 amino acid alterations, with 8 of the latter located within the envelope glycoprotein. The vaccine strain also fails to synthesize the nonstructural protein NS1 owing to a silent mutation that abrogates a-1-frameshifting event close to the 5 end of the NS2A coding sequence. Previous studies employing reverse genetics and mouse models implicated both absence of NS1 and mutated E, in attenuation of SA14-14-2. We demonstrate progressive reduction in ER stress sensor PERK levels and increased expression of CEBP-homologous protein (CHOP), accompanied by dephosphorylation of eIF2, inhibition of autophagy maturation and necroptosis following infection of cultured cells with wild-type JEV strain P20778. Autonomous expression of NS1 caused constitutive up-regulation of CHOP and loss of PERK. Conversely, infection with SA14-14-2 led to significantly increased IRE-1 activation, ER chaperone levels and autophagy. We report labile conformational epitopes accompanied by drastically reduced folding kinetics of intracellular SA14-14-2 envelope protein engendered by sluggish oxidation of cysteine sulfhydryl groups to form disulfide bonds within the endoplasmic reticulum along with altered envelope epitopes in extracellular SA14-14-2 viral particles. We also demonstrate near total conversion of prM to pr and M in SA14-14-2 virus particles. These alterations were accompanied by enhanced activation of mouse and human antigen presenting cells by SA14-14-2 along with superior CD8+ recall T cell responses to viral structural proteins in volunteers vaccinated with SA14-14-2. Author SummaryThe random process of cell culture passage adopted in generation of most live attenuated virus vaccines leads to fixation of multiple nucleotide changes in their genomes and renders it difficult if not impossible to pinpoint those mutations primarily responsible for their attenuated phenotype. Identifying the precise attenuating mutations and their modi operandi should aid in developing rationally attenuated vaccines for other viruses. We discovered that wild type (WT) JEV uses the nonstructural protein NS1 to take over the host protein synthesis machinery to produce viral proteins. Loss of NS1 in SA14-14-2 deprives the vaccine strain of this ability. Viruses uniformly target host death pathways to avoid generating potent antiviral immune responses. WT JEV prevents autophagy maturation. Conversely the SA14-14-2 vaccine activates autophagy due to unresolved ER stress caused by inability of its envelope glycoprotein to fold promptly post synthesis. Combined with enhanced proteolytic cleavage of the viral prM protein in SA14-14-2, this resulted in altered envelope epitopes on extracellular SA14-14-2 virus particles. These changes culminated in enhanced activation of innate and adaptive immune responses by SA14-14-2.

cell biology↗