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Mammalian orthoreovirus (reovirus) strains type 1 Lang (T1L) and type 3 Dearing-RV (T3D-RV) infect the intestine in mice but differ in the induction of inflammatory responses. T1L infection is associated with the blockade of oral immunological tolerance to newly introduced dietary antigens, whereas T3D-RV is not. T1L infection leads to an increase in infiltrating phagocytes, including macrophages, in gut-associated lymphoid tissues that are not observed in T3D-RV infection. However, the function of macrophages in reovirus intestinal infection is unknown. Using cells sorted from infected intestinal tissue and primary cultures of bone-marrow-derived macrophages (BMDMs), we discovered that T1L infects macrophages more efficiently than T3D-RV. Analysis of T1L × T3D-RV reassortant viruses revealed that the viral S4 gene segment, which encodes outer-capsid protein σ3, is responsible for strain-specific differences in infection of BMDMs. Differences in the binding of T1L and T3D-RV to BMDMs also segregated with the σ3-encoding S4 gene. Paired immunoglobulin-like receptor B (PirB), which serves as a receptor for reovirus, is expressed on macrophages and engages σ3. We found that PirB-specific antibody blocks T1L binding to BMDMs and that T1L binding to PirB -/- BMDMs is significantly diminished. Collectively, our data suggest that reovirus T1L infection of macrophages is dependent on engagement of PirB by viral outer-capsid protein σ3. These findings raise the possibility that macrophages function in the innate immune response to reovirus infection that blocks immunological tolerance to new food antigens.IMPORTANCEMammalian orthoreovirus (reovirus) infects humans throughout their lifespan and has been linked to celiac disease (CeD). CeD is caused by a loss of oral immunological tolerance (LOT) to dietary gluten and leads to intestinal inflammation following gluten ingestion, which worsens with prolonged exposure and can cause malnutrition. There are limited treatment options for CeD. While there are genetic risk factors associated with the illness, triggers for disease onset are not completely understood. Enteric viruses, including reovirus, have been linked to CeD induction. We found that a reovirus strain associated with oral immunological tolerance blockade infects macrophages by virtue of its capacity to bind macrophage receptor PirB. These data contribute to an understanding of the innate immune response elicited by reovirus, which may shed light on how viruses trigger LOT and inform the development of CeD vaccines and therapeutic agents., Competing Interests: The authors declare no conflict of interest.

Serotype 3 (T3) strains of mammalian orthoreovirus (reovirus) spread to the central nervous system to infect the brain and cause lethal encephalitis in newborn mice. Although reovirus targets several regions in the brain, susceptibility to infection is not uniformly distributed. The neuronal subtypes and anatomic sites targeted throughout the brain are not precisely known. Reovirus binds several attachment factors and entry receptors, including sialic acid (SA)-containing glycans and paired immunoglobulin-like receptor B (PirB). While these receptors are not required for infection of some types of neurons, reovirus engagement of these receptors can influence neuronal infection in certain contexts. To identify patterns of T3 neurotropism, we used microbial identification after passive tissue clearance and hybridization chain reaction to stain reovirus-infected cells throughout intact, optically transparent brains of newborn mice. Three-dimensional reconstructions revealed in detail the sites targeted by reovirus throughout the brain volume, including dense infection of the midbrain and hindbrain. Using reovirus mutants incapable of binding SA and mice lacking PirB expression, we found that neither SA nor PirB is required for the infection of various brain regions. However, SA may confer minor differences in infection that vary by region. Collectively, these studies indicate that many regions in the brain of newborn mice are susceptible to reovirus and that patterns of reovirus infection are not dependent on reovirus receptors SA and PirB.IMPORTANCENeurotropic viruses invade the central nervous system (CNS) and target various cell types to cause disease manifestations, such as meningitis, myelitis, or encephalitis. Infections of the CNS are often difficult to treat and can lead to lasting sequelae or death. Mammalian orthoreovirus (reovirus) causes age-dependent lethal encephalitis in many young mammals. Reovirus infects neurons in several different regions of the brain. However, the complete pattern of CNS infection is not understood. We found that reovirus targets almost all regions of the brain and that patterns of tropism are not dependent on receptors sialic acid and paired immunoglobulin-like receptor B. These studies confirm that two known reovirus receptors do not completely explain the cell types infected in brain tissue and establish strategies that can be used to understand complete patterns of viral tropism in an intact brain., Competing Interests: The authors declare no conflict of interest.

Understanding the mechanisms underlying viral entry is crucial for controlling viral diseases. In this study, we investigated the interactions between reovirus and Nogo-receptor 1 (NgR1), a key mediator of reovirus entry into the host central nervous system. NgR1 exhibits a unique bivalent interaction with the reovirus capsid, specifically binding at the interface between adjacent heterohexamers arranged in a precise structural pattern on the curved virus surface. Using single-molecule techniques, we explored for the first time how the capsid molecular architecture and receptor polymorphism influence virus binding. We compared the binding affinities of human and mouse NgR1 to reovirus μ1/σ3 proteins in their isolated form, self-assembled in 2D capsid patches, and within the native 3D viral topology. Our results underscore the essential role of the concave side of NgR1 and emphasize that the spatial organization and curvature of the virus are critical determinants of the stability of the reovirus-NgR1 complex. This study highlights the importance of characterizing interactions in physiologically relevant spatial configurations, providing precise insights into virus-host interactions and opening new avenues for therapeutic interventions against viral infections.

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.

Many functions of viral attachment proteins are established, but less is known about the biological importance of viral attachment protein encapsidation efficiency. The mammalian orthoreovirus (reovirus) σ1 attachment protein forms filamentous trimers that incorporate into pentamers of the λ2 capsid protein. Reovirus strains vary in the efficiency of σ1 encapsidation onto progeny virions, which influences viral stability during entry into cells and the efficacy of tumor cell lysis. While the role of σ1 encapsidation has been evaluated in studies using cultured cells, the contribution of attachment protein encapsidation efficiency to viral infection in animals is less clear. Polymorphisms in reovirus σ1 at residues 22 and 249 have been implicated in viral dissemination in mice and susceptibility to proteolysis in the murine intestine, respectively. To determine whether these residues contribute to σ1 encapsidation efficiency, we engineered σ1 mutant viruses with single- and double-residue substitutions at sites 22 and 249. We found that substitutions at these sites alter the encapsidation of σ1 and that reoviruses encapsidating higher amounts of σ1 bind cells more avidly and have a modest replication advantage in a cell-type-specific manner relative to low σ1-encapsidating reoviruses. Furthermore, we found that a high σ1-encapsidating reovirus replicates and disseminates more efficiently in mice relative to a low σ1-encapsidating reovirus. These findings provide evidence of a relationship between viral attachment protein encapsidation efficiency and viral replication in cell culture and animal hosts., Importance: Viral attachment proteins can serve multiple functions during viral replication, including attachment to host cells, cell entry and disassembly, and modulation of host immune responses. The relationship between viral attachment protein encapsidation efficiency and viral replication in cells and animals is poorly understood. We engineered and characterized a panel of reoviruses that differ in the capacity to encapsidate the σ1 attachment protein. We found that strains encapsidating σ1 with higher efficiency bind cells more avidly and replicate and spread more efficiently in mice relative to those encapsidating σ1 with lower efficiency. These results highlight a function for σ1 attachment protein capsid abundance in viral replication in cells and animals, which may inform future use of reovirus as an oncolytic therapeutic., Competing Interests: The authors declare no conflict of interest.

Mammalian orthoreovirus (reovirus) is a nonenveloped virus that establishes primary infection in the intestine and disseminates to sites of secondary infection, including the CNS. Reovirus entry involves multiple engagement factors, but how the virus disseminates systemically and targets neurons remains unclear. In this study, we identified murine neuropilin 1 (mNRP1) as a receptor for reovirus. mNRP1 binds reovirus with nanomolar affinity using a unique mechanism of virus-receptor interaction, which is coordinated by multiple interactions between distinct reovirus capsid subunits and multiple NRP1 extracellular domains. By exchanging essential capsid protein-encoding gene segments, we determined that the multivalent interaction is mediated by outer-capsid protein σ3 and capsid turret protein λ2. Using capsid mutants incapable of binding NRP1, we found that NRP1 contributes to reovirus dissemination and neurovirulence in mice. Collectively, our results demonstrate that NRP1 is an entry receptor for reovirus and uncover mechanisms by which NRPs promote viral entry and pathogenesis., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The mammalian orthoreovirus (reovirus) σNS protein is required for formation of replication compartments that support viral genome replication and capsid assembly. Despite its functional importance, a mechanistic understanding of σNS is lacking. We conducted structural and biochemical analyses of a σNS mutant that forms dimers instead of the higher-order oligomers formed by wildtype (WT) σNS. The crystal structure shows that dimers interact with each other using N-terminal arms to form a helical assembly resembling WT σNS filaments in complex with RNA observed using cryo-EM. The interior of the helical assembly is of appropriate diameter to bind RNA. The helical assembly is disrupted by bile acids, which bind to the same site as the N-terminal arm. This finding suggests that the N-terminal arm functions in conferring context-dependent oligomeric states of σNS, which is supported by the structure of σNS lacking an N-terminal arm. We further observed that σNS has RNA chaperone activity likely essential for presenting mRNA to the viral polymerase for genome replication. This activity is reduced by bile acids and abolished by N-terminal arm deletion, suggesting that the activity requires formation of σNS oligomers. Our studies provide structural and mechanistic insights into the function of σNS in reovirus replication., (© 2024. The Author(s).)

In the United States (US), biosafety and biosecurity oversight of research on viruses is being reappraised. Safety in virology research is paramount and oversight frameworks should be reviewed periodically. Changes should be made with care, however, to avoid impeding science that is essential for rapidly reducing and responding to pandemic threats as well as addressing more common challenges caused by infectious diseases. Decades of research uniquely positioned the US to be able to respond to the COVID-19 crisis with astounding speed, delivering life-saving vaccines within a year of identifying the virus. We should embolden and empower this strength, which is a vital part of protecting the health, economy, and security of US citizens. Herein, we offer our perspectives on priorities for revised rules governing virology research in the US.

Lymph nodes (LNs) are critical sites for shaping tissue-specific adaptive immunity. However, the impact of LN sharing between multiple organs on such tailoring is less understood. Here, we describe the drainage hierarchy of the pancreas, liver, and the upper small intestine (duodenum) into three murine LNs. Migratory dendritic cells (migDCs), key in instructing adaptive immune outcome, exhibited stronger pro-inflammatory signatures when originating from the pancreas or liver than from the duodenum. Qualitatively different migDC mixing in each shared LN influenced pancreatic β-cell-reactive T cells to acquire gut-homing and tolerogenic phenotypes proportional to duodenal co-drainage. However, duodenal viral infections rendered non-intestinal migDCs and β-cell-reactive T cells more pro-inflammatory in all shared LNs, resulting in elevated pancreatic islet lymphocyte infiltration. Our study uncovers immune crosstalk through LN co-drainage as a powerful force regulating pancreatic autoimmunity., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Enterovirus D68 (EV-D68) is a nonpolio enterovirus associated with severe respiratory illness and acute flaccid myelitis (AFM), a polio-like illness causing paralysis in children. AFM outbreaks have been associated with increased circulation and genetic diversity of EV-D68 since 2014, although the virus was discovered in the 1960s. The mechanisms by which EV-D68 targets the central nervous system are unknown. Since enteroviruses are human pathogens that do not routinely infect other animal species, establishment of a human model of the central nervous system is essential for understanding pathogenesis. Here, we describe two human spinal cord organoid (hSCO)-based models for EV-D68 infection derived from induced, pluripotent stem cell (iPSC) lines. One hSCO model consists primarily of spinal motor neurons, while the another model comprises multiple neuronal cell lineages, including motor neurons, interneurons, and glial cells. These hSCOs can be productively infected with contemporary strains, but not a historic strain, of EV-D68 and produce extracellular virus for at least 2 weeks without appreciable cytopathic effect. By comparison, infection with hSCO with another enterovirus, echovirus 11, causes significant structural destruction and apoptosis. Together, these findings suggest that EV-D68 infection is not the sole mediator of neuronal cell death in the spinal cord in those with AFM and that secondary injury from the immune response likely contributes to pathogenesis. IMPORTANCE AFM is a rare condition that causes significant morbidity in affected children, often contributing to life-long sequelae. It is unknown how EV-D68 causes paralysis in children, and effective therapeutic and preventative strategies are not available. Mice are not native hosts for EV-D68, and thus, existing mouse models use immunosuppressed or neonatal mice, mouse-adapted viruses, or intracranial inoculations. To complement existing models, we report two hSCO models for EV-D68 infection. These three-dimensional, multicellular models comprised human cells and include multiple neural lineages, including motor neurons, interneurons, and glial cells. These new hSCO models for EV-D68 infection will contribute to understanding how EV-D68 damages the human spinal cord, which could lead to new therapeutic and prophylactic strategies for this virus., Competing Interests: J.V.W serves on advisory committees for GlaxoSmithKline and Quidel, which did not impact the work contained in this paper. Other authors are without conflicts.

Loss of oral tolerance (LOT) to gluten, driven by dendritic cell (DC) priming of gluten-specific T helper 1 (Th1) cell immune responses, is a hallmark of celiac disease (CeD) and can be triggered by enteric viral infections. Whether certain commensals can moderate virus-mediated LOT remains elusive. Here, using a mouse model of virus-mediated LOT, we discovered that the gut-colonizing protist Tritrichomonas (T.) arnold promotes oral tolerance and protects against reovirus- and murine norovirus-mediated LOT, independent of the microbiota. Protection was not attributable to antiviral host responses or T. arnold-mediated innate type 2 immunity. Mechanistically, T. arnold directly restrained the proinflammatory program in dietary antigen-presenting DCs, subsequently limiting Th1 and promoting regulatory T cell responses. Finally, analysis of fecal microbiomes showed that T. arnold-related Parabasalid strains are underrepresented in human CeD patients. Altogether, these findings will motivate further exploration of oral-tolerance-promoting protists in CeD and other immune-mediated food sensitivities., Competing Interests: Declaration of interests E.F.V. is a member of the Biocodex International and National (Canada) Scientific Review Boards, a member of the Center for Gut Microbiome Research and Education Scientific Advisory Board of the AGA, Secretary of the International Society of the Study of Celiac Disease, and holds grants from Kallyope and Codexis, unrelated to this study. H.J.G. holds a grant from Codexis, unrelated to this study., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Nogo-66 receptor 1 (NgR1) binds a variety of structurally dissimilar ligands in the adult central nervous system to inhibit axon extension. Disruption of ligand binding to NgR1 and subsequent signaling can improve neuron outgrowth, making NgR1 an important therapeutic target for diverse neurological conditions such as spinal crush injuries and Alzheimer's disease. Human NgR1 serves as a receptor for mammalian orthoreovirus (reovirus), but the mechanism of virus-receptor engagement is unknown. To elucidate how NgR1 mediates cell binding and entry of reovirus, we defined the affinity of interaction between virus and receptor, determined the structure of the virus-receptor complex, and identified residues in the receptor required for virus binding and infection. These studies revealed that central NgR1 surfaces form a bridge between two copies of viral capsid protein σ3, establishing that σ3 serves as a receptor ligand for reovirus. This unusual binding interface produces high-avidity interactions between virus and receptor to prime early entry steps. These studies refine models of reovirus cell-attachment and highlight the evolution of viruses to engage multiple receptors using distinct capsid components.

Mammalian orthoreoviruses (reoviruses) serve as potential triggers of celiac disease and have oncolytic properties, making these viruses potential cancer therapeutics. Primary attachment of reovirus to host cells is mainly mediated by the trimeric viral protein, σ1, which engages cell-surface glycans, followed by high-affinity binding to junctional adhesion molecule-A (JAM-A). This multistep process is thought to be accompanied by major conformational changes in σ1, but direct evidence is lacking. By combining biophysical, molecular, and simulation approaches, we define how viral capsid protein mechanics influence virus-binding capacity and infectivity. Single-virus force spectroscopy experiments corroborated by in silico simulations show that GM2 increases the affinity of σ1 for JAM-A by providing a more stable contact interface. We demonstrate that conformational changes in σ1 that lead to an extended rigid conformation also significantly increase avidity for JAM-A. Although its associated lower flexibility impairs multivalent cell attachment, our findings suggest that diminished σ1 flexibility enhances infectivity, indicating that fine-tuning of σ1 conformational changes is required to successfully initiate infection. Understanding properties underlying the nanomechanics of viral attachment proteins offers perspectives in the development of antiviral drugs and improved oncolytic vectors.

Mammalian orthoreovirus (reovirus) infects most mammals and is associated with celiac disease in humans. In mice, reovirus infects the intestine and disseminates systemically to cause serotype-specific patterns of disease in the brain. To identify receptors conferring reovirus serotype-dependent neuropathogenesis, we conducted a genome-wide CRISPRa screen and identified paired immunoglobulin-like receptor B (PirB) as a receptor candidate. Ectopic expression of PirB allowed reovirus binding and infection. PirB extracelluar D3D4 region is required for reovirus attachment and infectivity. Reovirus binds to PirB with nM affinity as determined by single molecule force spectroscopy. Efficient reovirus endocytosis requires PirB signaling motifs. In inoculated mice, PirB is required for maximal replication in the brain and full neuropathogenicity of neurotropic serotype 3 (T3) reovirus. In primary cortical neurons, PirB expression contributes to T3 reovirus infectivity. Thus, PirB is an entry receptor for reovirus and contributes to T3 reovirus replication and pathogenesis in the murine brain., (© 2023. The Author(s).)

Background: Chikungunya virus (CHIKV) is an arbovirus that periodically emerges to cause large epidemics of arthritic disease. Although the robust immunity elicited by live-attenuated virus (LAV) vaccine candidates makes them attractive, CHIKV vaccine development has been hampered by a high threshold for acceptable adverse events., Methods: We evaluated the vaccine potential of a recently described LAV, skeletal muscle-restricted virus (SKE), that exhibits diminished replication in skeletal muscle due to insertion of target sequences for skeletal muscle-specific miR-206. We also evaluated whether these target sequences could augment safety of an LAV encoding a known attenuating mutation, E2 G82R. Attenuation of viruses containing these mutations was compared with a double mutant, SKE G82R., Results: SKE was attenuated in both immunodeficient and immunocompetent mice and induced a robust neutralizing antibody response, indicating its vaccine potential. However, only SKE G82R elicited diminished swelling in immunocompetent mice at early time points postinoculation, indicating that these mutations synergistically enhance safety of the vaccine candidate., Conclusions: These data suggest that restriction of LAV replication in skeletal muscle enhances tolerability of reactogenic vaccine candidates and may improve the rational design of CHIKV vaccines., (© The Author(s) 2022. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Pathological effects of apoptosis associated with viral infections of the central nervous system are an important cause of morbidity and mortality. Reovirus is a neurotropic virus that causes apoptosis in neurons, leading to lethal encephalitis in newborn mice. Reovirus-induced encephalitis is diminished in mice with germ line ablation of NF-κB subunit p50. It is not known whether the proapoptotic function of NF-κB is mediated by neural-cell-intrinsic (neural-intrinsic) processes, NF-κB-regulated cytokine production by inflammatory cells, or a combination of both. To determine the contribution of cell type-specific NF-κB signaling in reovirus-induced neuronal injury, we established mice that lack NF-κB p65 expression in neural cells using the Cre/loxP recombination system. Following intracranial inoculation of reovirus, 50% of wild-type (WT) mice succumbed to infection, whereas more than 90% of mice lacking neural cell NF-κB p65 (Nsp65 -/- ) survived. While viral loads in brains of WT and Nsp65 -/- mice were comparable, histological analysis revealed that reovirus antigen-positive areas in the brains of WT mice displayed increased immunoreactivity for cleaved caspase-3, a marker of apoptosis, relative to Nsp65 -/- mice. These data suggest that neural-intrinsic NF-κB-dependent factors are essential mediators of reovirus neurovirulence. RNA sequencing analysis of reovirus-infected brain cortices of WT and Nsp65 -/- mice suggests that NF-κB activation in neuronal cells upregulates genes involved in innate immunity, inflammation, and cell death following reovirus infection. A better understanding of the contribution of cell type-specific NF-κB-dependent signaling to viral neuropathogenesis could inform development of new therapeutics that target and protect highly vulnerable cell populations. IMPORTANCE Viral encephalitis contributes to illness and death in children and adults worldwide and has limited treatment options. Identifying common host factors upregulated by neurotropic viruses can enhance an understanding of virus-induced neuropathogenesis and aid in development of therapeutics. Although many neurotropic viruses activate NF-κB during infection, mechanisms by which NF-κB regulates viral neuropathogenesis and contributes to viral encephalitis are not well understood. We established mice in which NF-κB expression is ablated in neural tissue to study the function of NF-κB in reovirus neurovirulence and identify genes activated by NF-κB in response to reovirus infection in the central nervous system. Encephalitis following reovirus infection was dampened in mice lacking neural cell NF-κB. Reovirus induced a chemokine profile in the brain that was dependent on NF-κB signaling and was similar to chemokine profiles elicited by other neurotropic viruses. These data suggest common underlying mechanisms of encephalitis caused by neurotropic viruses and potentially shared therapeutic targets.

Spinareoviridae is a large family of icosahedral viruses that are usually regarded as non-enveloped with segmented (9-12 linear segments) dsRNA genomes of 23-29 kbp. Spinareovirids have a broad host range, infecting animals, fungi and plants. Some have important pathogenic potential for humans (e.g. Colorado tick fever virus), livestock (e.g. avian orthoreoviruses), fish (e.g. aquareoviruses) and plants (e.g. rice ragged stunt virus and rice black streaked dwarf virus). This is a summary of the ICTV Report on the family Spinareoviridae , which is available at ictv.global/report/spinareoviridae.

Celiac disease is an immune-mediated intestinal disorder that results from loss of oral tolerance (LOT) to dietary gluten. Reovirus elicits inflammatory Th1 cells and suppresses Treg responses to dietary antigen in a strain-dependent manner. Strain type 1 Lang (T1L) breaks oral tolerance, while strain type 3 Dearing reassortant virus (T3D-RV) does not. We discovered that intestinal infection by T1L in mice leads to the recruitment and activation of NK cells in mesenteric lymph nodes (MLNs) in a type I IFN-dependent manner. Once activated following infection, NK cells produce type II IFN and contribute to IFN-stimulated gene expression in the MLNs, which in turn induces inflammatory DC and T cell responses. Immune depletion of NK cells impairs T1L-induced LOT to newly introduced food antigen. These studies indicate that NK cells modulate the response to dietary antigen in the presence of a viral infection.

SARS-CoV-2 vaccines BNT162b2, mRNA-1273, and Ad26.COV2.S received emergency use authorization by the U.S. Food and Drug Administration in 2020/2021. Individuals being vaccinated were invited to participate in a prospective longitudinal comparative study of immune responses elicited by the three vaccines. In this observational cohort study, immune responses were evaluated using a SARS-CoV-2 spike protein receptor-binding domain ELISA, SARS-CoV-2 virus neutralization assays and an IFN- γ ELISPOT assay at various times over six months following initial vaccination. mRNA-based vaccines elicited higher magnitude humoral responses than Ad26.COV2.S; mRNA-1273 elicited the most durable humoral response, and all humoral responses waned over time. Neutralizing antibodies against the Delta variant were of lower magnitude than the wild-type strain for all three vaccines. mRNA-1273 initially elicited the greatest magnitude of T cell response, but this declined by 6 months. Declining immunity over time supports the use of booster dosing, especially in the setting of emerging variants., (© 2022. The Author(s).)

Objectives: Health systems must adapt to an increased consumer-centric environment to remain relevant in an ever-growing competitive health care landscape in which convenience is a key driver of patient satisfaction and loyalty. To adapt to this new environment, health systems must redesign processes to transform the delivery of ambulatory care and provide near real-time access to specialty care., Study Design: A pediatric academic medical center in western Pennsylvania used a process-improvement approach to enhance timely access to specialty care and deliver a consumer-centric patient experience., Methods: Critical factors in this process included engagement of key stakeholders, implementation of scheduling best practices, development of a set of scheduling guidelines, increased use of advanced practice providers, and use of data analytics to measure and benchmark performance., Results: The time to schedule a new patient appointment decreased from 42 to 4 days and the patient satisfaction access domain increased by 57 percentile points., Conclusions: These factors should scale to other institutions, thereby enabling generalizable results.

The COVID-19 pandemic has highlighted structural inequities that are barriers to thriving for children in neighborhoods with concentrated disadvantage. Health systems are increasingly addressing health-related social needs. The "Pittsburgh Study" is a longitudinal, community-partnered study focused on child and adolescent thriving and racial equity. This initiative will elucidate critical influences on childhood health and thriving, evaluate developmentally appropriate interventions to improve outcomes from birth to high school, and establish a child health data hub. Integration of community members into scientific inquiry, rapid data-to-action cycles, and workforce development are strategies health systems may consider to enhance child health equity., Competing Interests: The authors have no conflicts of interest relevant to this article to disclose. Dr. Miller receives royalties for writing content for Up To Date, Wolters Kluwer, unrelated to the content of this article., (© Terence S. Dermody et al., 2022; Published by Mary Ann Liebert, Inc.)

Engagement of host receptors is essential for viruses to enter target cells and initiate infection. Expression patterns of receptors in turn dictate host range, tissue tropism, and disease pathogenesis during infection. Mammalian orthoreovirus (reovirus) displays serotype-dependent patterns of tropism in the murine central nervous system (CNS) that are dictated by the viral attachment protein σ1. However, the receptor that mediates reovirus CNS tropism is unknown. Two proteinaceous receptors have been identified for reovirus, junctional adhesion molecule A (JAM-A) and Nogo-66 receptor 1 (NgR1). Engagement of JAM-A is required for reovirus hematogenous dissemination but is dispensable for neural spread and infection of the CNS. To determine whether NgR1 functions in reovirus neuropathogenesis, we compared virus replication and disease in wild-type (WT) and NgR1 -/- mice. Genetic ablation of NgR1 did not alter reovirus replication in the intestine or transmission to the brain following peroral inoculation. Viral titers in neural tissues following intramuscular inoculation, which provides access to neural dissemination routes, also were comparable in WT and NgR1 -/- mice, suggesting that NgR1 is dispensable for reovirus neural spread to the CNS. The absence of NgR1 also did not alter reovirus replication, neural tropism, and virulence following direct intracranial inoculation. In agreement with these findings, we found that the human but not the murine homolog of NgR1 functions as a receptor and confers efficient reovirus binding and infection of nonsusceptible cells in vitro . Thus, neither JAM-A nor NgR1 is required for reovirus CNS tropism in mice, suggesting that other unidentified receptors support this function. IMPORTANCE Viruses engage diverse molecules on host cell surfaces to navigate barriers, gain cell entry, and establish infection. Despite discovery of several reovirus receptors, host factors responsible for reovirus neurotropism are unknown. Human NgR1 functions as a reovirus receptor in vitro and is expressed in CNS neurons in a pattern overlapping reovirus tropism. We used mice lacking NgR1 to test whether NgR1 functions as a reovirus neural receptor. Following different routes of inoculation, we found that murine NgR1 is dispensable for reovirus dissemination to the CNS, tropism and replication in the brain, and resultant disease. Concordantly, expression of human but not murine NgR1 confers reovirus binding and infection of nonsusceptible cells in vitro . These results highlight species-specific use of alternate receptors by reovirus. A detailed understanding of species- and tissue-specific factors that dictate viral tropism will inform development of antiviral interventions and targeted gene delivery and therapeutic viral vectors.

Cholesterol homeostasis is required for the replication of many viruses, including Ebola virus, hepatitis C virus, and human immunodeficiency virus-1. Niemann-Pick C1 (NPC1) is an endosomal-lysosomal membrane protein involved in cholesterol trafficking from late endosomes and lysosomes to the endoplasmic reticulum. We identified NPC1 in CRISPR and RNA interference screens as a putative host factor for infection by mammalian orthoreovirus (reovirus). Following internalization via clathrin-mediated endocytosis, the reovirus outer capsid is proteolytically removed, the endosomal membrane is disrupted, and the viral core is released into the cytoplasm where viral transcription, genome replication, and assembly take place. We found that reovirus infection is significantly impaired in cells lacking NPC1, but infection is restored by treatment of cells with hydroxypropyl-β-cyclodextrin, which binds and solubilizes cholesterol. Absence of NPC1 did not dampen infection by infectious subvirion particles, which are reovirus disassembly intermediates that bypass the endocytic pathway for infection of target cells. NPC1 is not required for reovirus attachment to the plasma membrane, internalization into cells, or uncoating within endosomes. Instead, NPC1 is required for delivery of transcriptionally active reovirus core particles from endosomes into the cytoplasm. These findings suggest that cholesterol homeostasis, ensured by NPC1 transport activity, is required for reovirus penetration into the cytoplasm, pointing to a new function for NPC1 and cholesterol homeostasis in viral infection., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: K.C. is a member of the scientific advisory boards of Integrum Scientific, LLC, Biovaxys Technology Corp, and the Pandemic Security Initiative of Celdara Medical, LLC.

Background: Given the profound inequities in maternal and child health along racial, ethnic, and socioeconomic lines, strength-based, community-partnered research is required to foster thriving children, families, and communities, where thriving is defined as optimal development across physical, mental, cognitive, and social domains. The Pittsburgh Study (TPS) is a community-partnered, multi-cohort study designed to understand and promote child and youth thriving, build health equity, and strengthen communities by integrating community partners in study design, implementation, and dissemination. TPS launched the Tracking Health, Relationships, Identity, EnVironment, and Equity (THRIVE) Study to evaluate children's developmental stages and contexts from birth through completion of high school and to inform a child health data hub accessible to advocates, community members, educators, health professionals, and policymakers., Methods and Analysis: TPS is rooted in community-partnered participatory research (CPPR), health equity, antiracism, and developmental science. Using our community-informed conceptual framework of child thriving, the THRIVE Study will assess cross-cutting measures of place, environment, health service use, and other social determinants of health to provide longitudinal associations with developmentally appropriate child and youth thriving outcomes across participants in six cohorts spanning from pregnancy through adolescence (child ages 0-18 years). Data from electronic health records, school records, and health and human services use are integrated to assess biological and social influences of thriving. We will examine changes over time using paired t -tests and adjusted linear regression models for continuous thriving scores and McNemar tests and adjusted logistic regression models for categorical outcomes (thriving/not thriving). Data analyses will include mixed models with a random intercept (in combination with the previously-specified types of regression models) to account for within-subject correlation., Discussion: By enhancing assessment of child and youth well-being, TPS will fill critical gaps in our understanding of the development of child and youth thriving over time and test strategies to support thriving in diverse communities and populations. Through CPPR and co-design, the study aims to improve child health inequities across multiple socioecological levels and developmental domains., Competing Interests: FS is employed by YogaRoots On Location, LLC. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Ettinger, Landsittel, Abebe, Bey, Chavis, Navratil, Savage Friedman, Dermody, Miller and the Pittsburgh Study Committee Co-Leads.)

Breast cancer is the most common cancer in women. Although current therapies have increased survival rates for some breast cancer types, other aggressive invasive breast cancers remain difficult to treat. As the onset of breast cancer is often associated with the appearance of extracellular markers, these could be used to better target therapeutic agents. Here, we demonstrated by nanobiophysical approaches that overexpression of α-sialylated glycans in breast cancer provides an opportunity to combat cancer cells with oncolytic reoviruses. Notably, a correlation between cellular glycan expression and the mechanical properties of reovirus attachment and infection is observed in a serotype-dependent manner. Furthermore, we enhance the infectivity of reoviruses in malignant cells by the coinjection of α-sialylated glycans. In conclusion, this study supports both the use of reoviruses as an oncolytic agent in nanomedicine and the role of α-sialylated glycans as adjuvants in oncolysis, offering new perspective in oncolytic cancer therapy.

Replication of rotavirus, an important cause of gastroenteritis in children, proceeds in large, easily discernible cytoplasmic structures, called viroplasms or viral factories, but mechanisms underlying their formation and function in infected cells have remained mysterious. In this issue, Geiger et al (2021) used a combination of in silico, in vitro, and cell-based approaches to define how two essential rotavirus nonstructural proteins, NSP2 and NSP5, form liquid-liquid phase-separated condensates as the structural foundation of rotavirus factories., (© 2021 The Authors.)

The function of the mammalian orthoreovirus (reovirus) σNS nonstructural protein is enigmatic. σNS is an RNA-binding protein that forms oligomers and enhances the stability of bound RNAs, but the mechanisms by which it contributes to reovirus replication are unknown. To determine the function of σNS-RNA binding in reovirus replication, we engineered σNS mutants deficient in RNA-binding capacity. We found that alanine substitutions of positively charged residues in a predicted RNA-binding domain decrease RNA-dependent oligomerization. To define steps in reovirus replication facilitated by the RNA-binding property of σNS, we established a complementation system in which wild-type or mutant forms of σNS could be tested for the capacity to overcome inhibition of σNS expression. Mutations in σNS that disrupt RNA binding also diminish viral replication and σNS distribution to viral factories. Moreover, viral mRNAs only incorporate into viral factories or factory-like structures (formed following expression of nonstructural protein μNS) when σNS is present and capable of binding RNA. Collectively, these findings indicate that σNS requires positively charged residues in a putative RNA-binding domain to recruit viral mRNAs to sites of viral replication and establish a function for σNS in reovirus replication. IMPORTANCE Viral replication requires the formation of neoorganelles in infected cells to concentrate essential viral and host components. However, for many viruses, it is unclear how these components coalesce into neoorganelles to form factories for viral replication. We discovered that two mammalian reovirus nonstructural proteins act in concert to form functioning viral factories. Reovirus μNS proteins assemble into exclusive factory scaffolds that require reovirus σNS proteins for efficient viral mRNA incorporation. Our results demonstrate a role for σNS in RNA recruitment to reovirus factories and, more broadly, show how a cytoplasmic non-membrane-enclosed factory is formed by an RNA virus. Understanding the mechanisms of viral factory formation will help identify new targets for antiviral therapeutics that disrupt assembly of these structures and inform the use of nonpathogenic viruses for biotechnological applications.

The mammalian orthoreovirus double-stranded (ds) RNA-binding protein σ3 is a multifunctional protein that promotes viral protein synthesis and facilitates viral entry and assembly. The dsRNA-binding capacity of σ3 correlates with its capacity to prevent dsRNA-mediated activation of protein kinase R (PKR). However, the effect of σ3 binding to dsRNA during viral infection is largely unknown. To identify functions of σ3 dsRNA-binding activity during reovirus infection, we engineered a panel of thirteen σ3 mutants and screened them for the capacity to bind dsRNA. Six mutants were defective in dsRNA binding, and mutations in these constructs cluster in a putative dsRNA-binding region on the surface of σ3. Two recombinant viruses expressing these σ3 dsRNA-binding mutants, K287T and R296T, display strikingly different phenotypes. In a cell-type dependent manner, K287T, but not R296T, replicates less efficiently than wild-type (WT) virus. In cells in which K287T virus demonstrates a replication deficit, PKR activation occurs and abundant stress granules (SGs) are formed at late times post-infection. In contrast, the R296T virus retains the capacity to suppress activation of PKR and does not mediate formation of SGs at late times post-infection. These findings indicate that σ3 inhibits PKR independently of its capacity to bind dsRNA. In infected mice, K287T produces lower viral titers in the spleen, liver, lungs, and heart relative to WT or R296T. Moreover, mice inoculated with WT or R296T viruses develop myocarditis, whereas those inoculated with K287T do not. Overall, our results indicate that σ3 functions to suppress PKR activation and subsequent SG formation during viral infection and that these functions correlate with virulence in mice., Competing Interests: The authors have declared that competing interests exist.

Mammalian orthoreoviruses (reoviruses) are nonenveloped, double-stranded RNA viruses that replicate and assemble in cytoplasmic membranous organelles called viral inclusions (VIs). To define the cellular compartments involved in nonlytic reovirus egress, we imaged viral egress in infected, nonpolarized human brain microvascular endothelial cells (HBMECs). Electron and confocal microscopy showed that reovirus mature virions are recruited from VIs to modified lysosomes termed sorting organelles (SOs). Later in infection, membranous carriers (MCs) emerge from SOs and transport new virions to the plasma membrane for nonlytic egress. Transmission electron microscopy (TEM) combined with electron tomography (ET) and three-dimensional (3D) reconstruction revealed that these compartments are connected and form the exit pathway. Connections are established by channels through which mature virions are transported from VIs to MCs. In the last step, MCs travel across the cytoplasm and fuse with the plasma membrane, which facilitates reovirus egress. This bio-protocol describes the combination of imaging approaches (TEM, ET, and 3D reconstruction) to analyze reovirus egress zones. The spatial information present in the 3D reconstructions, along with the higher resolution relative to 2D projections, allowed us to identify components of a new nonlytic viral egress pathway., (Copyright © 2021 The Authors; exclusive licensee Bio-protocol LLC.)