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Enterovirus D68 (EV-D68) has been implicated in outbreaks of severe respiratory illness and is associated with acute flaccid myelitis (AFM). EV-D68 is often detected in patient respiratory samples but has also been detected in stool and wastewater, suggesting the potential for both respiratory and enteric routes of transmission. Here, we used a panel of EV-D68 isolates, including a historical pre-2014 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 earlier isolates and that the respiratory, but not intestinal, epithelium induces a robust type III interferon response that restricts infection. Our findings define the differential responses of the respiratory and intestinal epithelium to contemporary EV-D68 isolates and suggest that a subset of isolates have the potential to target both the human airway and gastrointestinal tracts., Competing Interests: MF, AW, JC, MM, LY, JK, CC No competing interests declared

Acute flaccid myelitis (AFM) is a serious neurological illness first recognized in the United States in 2014, with subsequent outbreaks every two years. Following extensive etiologic testing by multiple laboratories of hundreds of specimens collected from patients diagnosed with AFM, no consistent cause of AFM has been identified. However, viruses, including enteroviruses, have been implicated through detection in non-sterile site specimens and antibody studies. Cytokines and chemokines play important roles in the modulation of the innate and adaptive immune response to pathogens. In the current study, we measured levels of cytokines and chemokines in serum and CSF collected from confirmed AFM patients and non-AFM control patients, to identify unique biomarkers as potential hallmarks of AFM pathogenesis. Analysis of ratios of cytokines and chemokines in the CSF compared to the serum indicate that the pro-inflammatory cytokines/chemokines IP-10 and IL-6 were significantly elevated in AFM patients compared to non-AFM patients. These results may provide additional insight into potential etiologies, pathogenic mechanisms, and treatments for AFM., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Several barriers protect the central nervous system (CNS) from pathogen invasion. Yet viral infections of the CNS are common and often debilitating. Understanding how neurotropic viruses co-opt host machinery to overcome challenges to neuronal entry and transmission is important to combat these infections. Neurotropic reovirus disseminates through neural routes and invades the CNS to cause lethal encephalitis in newborn animals. To define mechanisms of reovirus neuronal entry and directional transport, we used primary neuron cultures, which reproduce in vivo infection patterns displayed by different reovirus serotypes. Treatment of neurons with small-molecule inhibitors of different endocytic uptake pathways allowed us to discover that the cellular machinery mediating macropinocytosis is required for reovirus neuronal entry. This mechanism of reovirus entry differs from clathrin-mediated endocytosis, which is used by reovirus to invade non-neuronal cells. Analysis of reovirus transport and release from isolated soma or axonal termini of neurons cultivated in microfluidic devices indicates that reovirus is capable of retrograde but only limited anterograde neuronal transmission. The dynamics of retrograde reovirus movement are consistent with fast axonal transport coordinated by dynein along microtubules. Further analysis of viral transport revealed that multiple virions are transported together in axons within non-acidified vesicles. Reovirus-containing vesicles acidify after reaching the soma, where disassembly of virions and release of the viral core into the cytoplasm initiates replication. These results define mechanisms of reovirus neuronal entry and transport and establish a foundation to identify common host factors used by neuroinvasive viruses. Furthermore, our findings emphasize consideration of cell type-specific entry mechanisms in the tailored design of neurotropic viruses as tracers, oncolytic agents, and delivery vectors., Competing Interests: The authors have declared that no competing interests exist.

Viruses in species Parechovirus A ( Picornaviridae ) are associated with a wide variety of clinical manifestations. Parechovirus A3 (PeV-A3) is known to cause sepsis-like illness, meningitis, and encephalitis in infants and young children. To date, no specific therapies are available to treat PeV-A3-infected children. We had previously identified two FDA-cleared antifungal drugs, itraconazole (ITC) and posaconazole (POS), with potent and specific antiviral activity against PeV-A3. Time-of-addition and synchronized infection assays revealed that POS targets an early stage of the PeV-A3 life cycle. POS exerts an antiviral effect, evidenced by a reduction in viral titer following the addition of POS to Vero-P cells before infection, coaddition of POS and PeV-A3 to Vero-P cells, incubation of POS and PeV-A3 prior to Vero-P infection, and at attachment. POS exerts less of an effect on virus entry. A PeV-A3 enzyme-linked immunosorbent assay inhibition experiment, using an anti-PeV-A3 monoclonal antibody, suggested that POS binds directly to the PeV-A3 capsid. POS-resistant PeV-A3 strains developed by serial passage in the presence of POS acquired substitutions in multiple regions of the genome, including the capsid. Reverse genetics confirmed substitutions in capsid proteins VP0, VP3, and VP1 and nonstructural proteins 2A and 3A. Single mutants VP0_K66R, VP0_A124T, VP3_N88S, VP1_Y224C, 2A_S78L, and 3A_T1I were 4-, 9-, 12-, 34-, 51-, and 119-fold more resistant to POS, respectively, than the susceptible prototype strain. Our studies demonstrate that POS may be a valuable tool in developing an antiviral therapy for PeV-A3.