Biology subjects

Maya, S.

Publications and source records attributed to Maya, S..

7 recordsLinked to original sources

bioRxiv2025

Strain-Specific Tropism and Transcriptional Responses of Enterovirus D68 Infection in Human Spinal Cord Organoids

The mechanisms by which Enterovirus D-68 (EV-D68) infection leads to acute flaccid myelitis (AFM), a severe neurological condition characterized by sudden muscle weakness and paralysis, remain poorly understood. To investigate the cellular tropism and infection dynamics of EV-D68, we profiled naive and EV-D68-infected human spinal cord organoids (hSCOs) derived from induced pluripotent stem cells (iPSCs) using single-cell RNA sequencing (scRNA-seq). Examining the cellular composition of healthy hSCOs, we found that hSCOs comprise diverse cell types, including neurons, astrocytes, oligodendrocyte progenitor cells (OPCs), and multipotent glial progenitor cells (mGPCs). Upon infection with two EV-D68 strains, US/IL/14-18952 (a B2 strain) and US/MA/18-23089 (a B3 strain), we observed distinct viral tropism and host transcriptional responses. Notably, US/IL/14-18952 showed a significant preference for neurons, while US/MA/18-23089 exhibited higher rates of infection in cycling astrocytes and OPCs. These findings provide novel insights into the host cell tropism of EV-D68 in the spinal cord, offering insight into the potential mechanisms underlying AFM pathogenesis. Understanding the dynamics of infection at single-cell resolution will inform future therapeutic strategies aimed at mitigating the neurological impact of enteroviral infections.

microbiology↗

bioRxiv2025

α-Synuclein aggregates induce mitochondrial damage and trigger innate immunity to drive neuron-microglia communication

Tunneling nanotubes (TNTs) enable direct intercellular transfer of macromolecules, organelles, and pathogenic protein aggregates. While -synuclein (-Syn) aggregates are known to promote TNT formation, the underlying mechanisms remain poorly defined. Here, using human neuronal and microglial cell lines, as well as iPSC-derived dopaminergic neurons and microglia, we show that -Syn aggregates induce severe mitochondrial damage, leading to cytosolic release of mitochondrial DNA (mtDNA) and activation of the cGAS-STING-NF- {kappa}B-IRF3 pathway. This innate immune response drives actin cytoskeleton remodeling and the formation of TNT-like structures, promoting intercellular transfer of -Syn from neurons to microglia. Additionally, neuronal cells transfer damaged mitochondria to microglia, where they undergo lysosome-mediated degradation. Neuron-to-microglia communication under -Syn- induced stress also triggers a bystander inflammatory response in microglia, suggesting a neuroimmune activation. Our findings identify mitochondrial damage and STING-mediated inflammation as key drivers of TNT formation and -Syn propagation, highlighting new potential targets to modulate disease progression in Synucleinopathies.

cell biology↗

bioRxiv2024

Recombinant B3 clade enterovirus D68 strains are efficiently rescued in 293T cells and infect human spinal cord organoids.

Enterovirus D68 (EV-D68) is associated with respiratory disease in children. Between 2014 and 2018, biennial EV-D68 outbreaks coincided with peaks of a polio-like neurologic condition called acute flaccid myelitis (AFM). We hypothesized that specific mutations within the currently circulating B3 clade of EV-D68 impacted neurovirulence. However, recovery of these strains from infectious clones by published methods proved unsuccessful. Therefore, we tested different cell lines, reagents, and conditions to enhance efficiency of recombinant (r)EV-D68 rescue. In this study, we present a tractable rescue system to define virulence determinants in B3 clade strains. Using this approach, we successfully rescued historic and contemporary rEV-D68 strains to high titer after limited cell culture passage. All strains in our study replicated efficiently in the parental RD cell line and in human respiratory epithelial cell lines, with BEAS-2B cells exhibiting greater permissivity than A549 cells. While B2 and B3 clade strains could infect the SH-SY5Y neuroblastoma cell line, the neurovirulent B2 clade strain rUSA/IL/2014-18952 was more dependent on neuron differentiation than B3 clade strains and replication was not sustained under multi-cycle growth conditions. Conversely, replication of rUSA/IL/2014-18952 was more efficient in human spinal cord organoids, which model the cellular heterogeneity of the spinal cord, while replication of B3 clade strains was more modest. Immunofluorescence staining confirmed infection, with viral antigen colocalized with neurons. These findings suggest shifting dynamics of EV-D68 neurovirulence and provide a critical platform for further assessment of viral determinants of neurovirulence in B3 clade strains. IMPORTANCEEnterovirus D68 (EV-D68) can cause acute flaccid myelitis (AFM), a debilitating neurological condition of the spinal cord in children. Identifying viral determinants of EV- D68 neuropathogenesis is critical to understanding recent shifts in AFM prevalence; however, these investigations are limited to a small subset of infectious clones distantly related to currently circulating B3 clade strains. In this study, we leverage improved rescue strategies to characterize recombinant (r)EV-D68 strains from the dominant B3 clade. While all rEV-D68 replicate efficiently in the parental cell line and in human respiratory epithelial cells, B3 clade strains achieved greater titers in cultured neurons than a neurovirulent B2 clade strain, with less dependence on neuronal differentiation state. All B3 clade strains established infection in human spinal cord organoids, but replication varied between strains. Therefore, our study presents a tractable rescue system to begin to dissect viral determinants of shifting neurotropism within contemporary EV-D68 clades.

microbiology↗

bioRxiv2024

Immune cells promote paralytic disease in mice infected with enterovirus D68

Enterovirus D68 (EV-D68) is associated with acute flaccid myelitis (AFM), a poliomyelitis-like illness causing paralysis in young children. However, mechanisms of paralysis are unclear, and antiviral therapies are lacking. To better understand EV-D68 disease, we inoculated newborn mice intracranially to assess viral tropism, virulence, and immune responses. Wild-type (WT) mice inoculated intracranially with a neurovirulent strain of EV-D68 showed infection of spinal cord neurons and developed paralysis. Spinal tissue from infected mice revealed increased levels of chemokines, inflammatory monocytes, macrophages, and T cells relative to controls, suggesting that immune cell infiltration influences pathogenesis. To define the contribution of cytokine-mediated immune cell recruitment to disease, we inoculated mice lacking CCR2, a receptor for several EV-D68-upregulated cytokines, or RAG1, which is required for lymphocyte maturation. WT, Ccr2-/-, and Rag1-/- mice had comparable viral titers in spinal tissue. However, Ccr2-/- and Rag1-/- mice had significantly less paralysis relative to WT mice. Consistent with impaired T cell recruitment to sites of infection in Ccr2-/- and Rag1-/- mice, antibody-mediated depletion of CD4+ or CD8+ T cells from WT mice diminished paralysis. These results indicate that immune cell recruitment to the spinal cord promotes EV-D68-associated paralysis and illuminate new targets for therapeutic intervention. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=106 SRC="FIGDIR/small/618341v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@10b38b5org.highwire.dtl.DTLVardef@1b80d41org.highwire.dtl.DTLVardef@6e39bforg.highwire.dtl.DTLVardef@9156ed_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2024

Liver-specific Mettl14 deletion induces nuclear heterotypia and dysregulates RNA export machinery

Modification of RNA with N6-methyladenosine (m6A) has gained attention in recent years as a general mechanism of gene regulation. In the liver, m6A, along with its associated machinery, has been studied as a potential biomarker of disease and cancer, with impacts on metabolism, cell cycle regulation, and pro-cancer state signaling. However these observational data have yet to be causally examined in vivo. For example, neither perturbation of the key m6A writers Mettl3 and Mettl14, nor the m6A readers Ythdf1 and Ythdf2 have been thoroughly mechanistically characterized in vivo as they have been in vitro. To understand the functions of these machineries, we developed mouse models and found that deleting Mettl14 led to progressive liver injury characterized by nuclear heterotypia, with changes in mRNA splicing, processing and export leading to increases in mRNA surveillance and recycling.

molecular biology↗

bioRxiv2023

Hepatitis delta virus RNA decline post inoculation in human NTCP transgenic mice is biphasic

Background and AimsChronic infection with hepatitis B and hepatitis delta viruses (HDV) is considered the most serious form of viral hepatitis due to more severe manifestations of and accelerated progression to liver fibrosis, cirrhosis, and hepatocellular carcinoma. There is no FDA-approved treatment for HDV and current interferon-alpha treatment is suboptimal. We characterized early HDV kinetics post inoculation and incorporated mathematical modeling to provide insights into host-HDV dynamics. MethodsWe analyzed HDV RNA serum viremia in 192 immunocompetent (C57BL/6) and immunodeficient (NRG) mice that did or did not transgenically express the HDV receptor - human sodium taurocholate co-transporting peptide (hNTCP). ResultsKinetic analysis indicates an unanticipated biphasic decline consisting of a sharp first-phase and slower second-phase decline regardless of immunocompetence. HDV decline after re-inoculation again followed a biphasic decline; however, a steeper second-phase HDV decline was observed in NRG-hNTCP mice compared to NRG mice. HDV-entry inhibitor bulevirtide administration and HDV re-inoculation indicated that viral entry and receptor saturation are not major contributors to clearance, respectively. The biphasic kinetics can be mathematically modeled by assuming the existence of a non-specific binding compartment with a constant on/off-rate and the steeper second-phase decline by a loss of bound virus that cannot be returned as free virus to circulation. The model predicts that free HDV is cleared with a half-life of 18 minutes (standard error, SE: 2.4), binds to non-specific cells with a rate of 0.06 hour-1 (SE: 0.03), and returns as free virus with a rate of 0.23 hour-1 (SE: 0.03). ConclusionsUnderstanding early HDV-host kinetics will inform pre-clinical therapeutic kinetic studies on how the efficacy of anti-HDV therapeutics can be affected by early kinetics of viral decline. LAY SUMMARYThe persistence phase of HDV infection has been studied in some animal models, however, the early kinetics of HDV in vivo is incompletely understood. In this study, we characterize an unexpectedly HDV biphasic decline post inoculation in immunocompetent and immunodeficient mouse models and use mathematical modeling to provide insights into HDV-host dynamics. Understanding the kinetics of viral clearance in the blood can aid pre-clinical development and testing models for anti-HDV therapeutics.

microbiology↗

bioRxiv2022

Structural features stabilized by divalent cation coordination within hepatitis E virus ORF1 are critical for viral replication

Hepatitis E virus (HEV) is an RNA virus responsible for over 20 million infections annually. HEVs open reading frame (ORF)1 polyprotein is essential for genome replication, though it is unknown how the different subdomains function within a structural context. Our data show that ORF1 operates as a multifunctional protein, which is not subject to proteolytic processing. Supporting this model, scanning mutagenesis performed on the putative papain-like cysteine protease (pPCP) domain revealed six cysteines essential for viral replication. Our data are consistent with their role in divalent metal ion coordination, which governs local and interdomain interactions that are critical for the overall structure of ORF1; further, the "pPCP" domain can only rescue viral genome replication in trans when expressed in the context of the full-length ORF1 protein but not as an individual subdomain. Taken together our work provides a comprehensive model of the structure and function of HEV ORF1. Significance StatementThe development of non-teratogenic and potent antiviral therapies against HEV have been hindered by an incomplete understanding of the viral replication cycle. Our work provides a mechanistic insight into the complex replicative cycle of this understudied human pathogen and identifies a novel domain-domain interaction that is vital for replicative fitness.

microbiology↗