Biology subjects

Freeman, M. C.

Publications and source records attributed to Freeman, M. C..

5 recordsLinked to original sources

bioRxiv2026

Enterovirus D68 VP1 and VP3 determine neurotropism in human spinal cord organoids

Enterovirus D68 (EV-D68) is a non-polio enterovirus that can cause a polio-like paralysis condition, acute flaccid myelitis (AFM). EV-D68 associated AFM cases waned in the US after 2018 and the reasons for this are unknown. It has recently been demonstrated that EV-D68 containing point mutations in viral structural proteins VP1 and VP3 resulted in decreased paralysis in different neonatal mouse models. However, phenotypes of these mutations in a human multicellular central nervous system (CNS) model are unknown. We hypothesized that mutations in VP1 and VP3 will similarly direct neurotropism in human spinal cord organoids (hSCO). To investigate this, we recreated viruses with mutations in VP3 (I88V) or VP1 (L1I/N2D/T98A/E283K or L1P/V148A/K282R) and infected hSCOs. We found that VP3 I88V and VP1 L1I/N2D/T98A/E283K resulted in decreased titer and viral protein staining, consistent with attenuated neurovirulence in previously published murine models. When these mutations were combined, their effects on neurotropism were not additive. Sequence analysis of recently circulating EV-D68 strains reveals that VP3 I88 and VP1 E283 have remained the dominant amino acid residues since 2014, whereas VP1 sites 1, 2, and 98 have higher population diversity, indicating that these residues may be contributing to newly reduced neurovirulence after 2018.

microbiology↗

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↗

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↗

bioRxiv2021

Respiratory and intestinal epithelial cells exhibit differential susceptibility and innate immune responses to contemporary EV-D68 isolates

Enterovirus D68 (EV-D68) has been implicated in outbreaks of severe respiratory illness and acute flaccid myelitis (AFM) and is detected in patient respiratory samples and from stool and wastewater, suggesting both respiratory and enteric routes of transmission. Here, we used a panel of EV-D68 isolates, including a historical isolate and multiple contemporary isolates from AFM outbreak years, to define the dynamics of viral replication and the host response to infection in primary human airway cells and stem cell-derived enteroids. We show that some recent EV-D68 isolates have decreased sensitivity to acid and temperature compared with an earlier isolate and that the respiratory, but not intestinal, epithelium induces a robust type III interferon (IFN) response that restricts infection. Our findings define the differential responses of the respiratory and intestinal epithelium to contemporary EV-D68 isolates and suggest that some isolates have the potential to target both the human airway and gastrointestinal tracts.

microbiology↗

bioRxiv2020

Integrating tracking and resight data from breeding Painted Bunting populations enables unbiased inferences about migratory connectivity and winter range survival

Archival geolocators have transformed the study of small, migratory organisms but analysis of data from these devices requires bias correction because tags are only recovered from individuals that survive and are re-captured at their tagging location. We show that integrating geolocator recovery data and mark-resight data enables unbiased estimates of both migratory connectivity between breeding and non-breeding populations and region-specific survival probabilities for wintering locations. Using simulations, we first demonstrate that an integrated Bayesian model returns unbiased estimates of transition probabilities between seasonal ranges and to determine how different sampling designs influence estimability of transition probabilities. We then parameterize the model with tracking data and mark-resight data from declining Painted Bunting (Passerina ciris) populations breeding in the eastern United States, hypothesized to be threatened by the illegal pet trade in parts of their Caribbean, non-breeding range. Consistent with this hypothesis, we found that male buntings wintering in Cuba were 20% less likely to return to the breeding grounds than birds wintering elsewhere in their range. Improving inferences from archival tags through proper data collection and further development of integrated models will advance our understanding of full annual cycle ecology of migratory species.

ecology↗