Enveloped viruses pseudotyped with mammalian myogenic cell fusogens target skeletal muscle for gene delivery.

Entry of enveloped viruses into cells is mediated by viral fusogenic proteins that drive membrane rearrangements needed for fusion between viral and target membranes. Skeletal muscle development also requires membrane fusion events between progenitor cells to form multinucleated myofibers. Myomaker and Myomerger are muscle-specific cell fusogens but do not structurally or functionally resemble classical viral fusogens. We asked whether the muscle fusogens could functionally substitute for viral fusogens, despite their structural distinctiveness, and fuse viruses to cells. We report that engineering of Myomaker and Myomerger on the membrane of enveloped viruses leads to specific transduction of skeletal muscle. We also demonstrate that locally and systemically injected virions pseudotyped with the muscle fusogens can deliver µDystrophin to skeletal muscle of a mouse model of Duchenne muscular dystrophy and alleviate pathology. Through harnessing the intrinsic properties of myogenic membranes, we establish a platform for delivery of therapeutic material to skeletal muscle.

Comment on Kopanska et al. Disorders of the Cholinergic System in COVID-19 Era-A Review of the Latest Research. Int. J. Mol. Sci. 2022, 23, 672.

We read the recent review article by Marta Kopanska et al. [...].

Pathological Features of Echovirus-11-Associated Brain Damage in Mice Based on RNA-Seq Analysis.

Echovirus 11 (E11) is a neurotropic virus that occasionally causes fatal neurological diseases in infected children. However, the molecular mechanism underlying the disease and pathological spectrum of E11 infection remains unclear. Therefore, we modelled E11 infection in 2-day-old type I interferon receptor knockout (IFNAR-/-) mice, which are susceptible to enteroviruses, with E11, and identified symptoms consistent with the clinical signs observed in human cases. All organs of infected suckling mice were found to show viral replication and pathological changes; the muscle tissue showed the highest viral replication, whereas the brain and muscle tissues showed the most obvious pathological changes. Brain tissues showed oedema and a large number of dead nerve cells; RNA-Seq analysis of the brain and hindlimb muscle tissues revealed differentially expressed genes to be abundantly enriched in immune response-related pathways, with changes in the Guanylate-binding protein (GBP) and MHC class genes, causing aseptic meningitis-related symptoms. Furthermore, human glioma U251 cell was identified as sensitive target cells for E11 infection. Overall, these results provide new insights into the pathogenesis and progress of aseptic meningitis caused by E11.

Muscle Regeneration and RNA: New Perspectives for Ancient Molecules.

The ability of the ribonucleic acid (RNA) to self-replicate, combined with a unique cocktail of chemical properties, suggested the existence of an RNA world at the origin of life. Nowadays, this hypothesis is supported by innovative high-throughput and biochemical approaches, which definitively revealed the essential contribution of RNA-mediated mechanisms to the regulation of fundamental processes of life. With the recent development of SARS-CoV-2 mRNA-based vaccines, the potential of RNA as a therapeutic tool has received public attention. Due to its intrinsic single-stranded nature and the ease with which it is synthesized in vitro, RNA indeed represents the most suitable tool for the development of drugs encompassing every type of human pathology. The maximum effectiveness and biochemical versatility is achieved in the guise of non-coding RNAs (ncRNAs), which are emerging as multifaceted regulators of tissue specification and homeostasis. Here, we report examples of coding and ncRNAs involved in muscle regeneration and discuss their potential as therapeutic tools. Small ncRNAs, such as miRNA and siRNA, have been successfully applied in the treatment of several diseases. The use of longer molecules, such as lncRNA and circRNA, is less advanced. However, based on the peculiar properties discussed below, they represent an innovative pool of RNA biomarkers and possible targets of clinical value.

Dynamics of Polarized Macrophages and Activated CD8+ Cells in Heart Tissue of Atlantic Salmon Infected With Piscine Orthoreovirus-1.

Piscine orthoreovirus (PRV-1) infection causes heart and skeletal muscle inflammation (HSMI) in farmed Atlantic salmon (Salmo salar). The virus is also associated with focal melanized changes in white skeletal muscle where PRV-1 infection of macrophages appears to be important. In this study, we studied the macrophage polarization into M1 (pro-inflammatory) and M2 (anti-inflammatory) phenotypes during experimentally induced HSMI. The immune response in heart with HSMI lesions was characterized by CD8+ and MHC-I expressing cells and not by polarized macrophages. Fluorescent in situ hybridization (FISH) assays revealed localization of PRV-1 in a few M1 macrophages in both heart and skeletal muscle. M2 type macrophages were widely scattered in the heart and were more abundant in heart compared to the skeletal muscle. However, the M2 macrophages did not co-stain for PRV-1. There was a strong cellular immune response to the infection in the heart compared to that of the skeletal muscle, seen as increased MHC-I expression, partly in cells also containing PRV-1 RNA, and a high number of cytotoxic CD8+ granzyme producing cells that targeted PRV-1. In skeletal muscle, MHC-I expressing cells and CD8+ cells were dispersed between myocytes, but these cells did not stain for PRV-1. Gene expression analysis by RT-qPCR complied with the FISH results and confirmed a drop in level of PRV-1 following the cell mediated immune response. Overall, the results indicated that M1 macrophages do not contribute to the initial development of HSMI. However, large numbers of M2 macrophages reside in the heart and may contribute to the subsequent fast recovery following clearance of PRV-1 infection.

Skeletal Muscle and Peripheral Nerve Histopathology in COVID-19.

OBJECTIVE: To explore the spectrum of skeletal muscle and nerve pathology of patients who died after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and to assess for direct viral invasion of these tissues. METHODS: Psoas muscle and femoral nerve sampled from 35 consecutive autopsies of patients who died after SARS-CoV-2 infection and 10 SARS-CoV-2-negative controls were examined under light microscopy. Clinical and laboratory data were obtained by chart review. RESULTS: In SARS-CoV-2-positive patients, mean age at death was 67.8 years (range 43-96 years), and the duration of symptom onset to death ranged from 1 to 49 days. Four patients had neuromuscular symptoms. Peak creatine kinase was elevated in 74% (mean 959 U/L, range 29-8,413 U/L). Muscle showed type 2 atrophy in 32 patients, necrotizing myopathy in 9, and myositis in 7. Neuritis was seen in 9. Major histocompatibility complex-1 (MHC-1) expression was observed in all cases of necrotizing myopathy and myositis and in 8 additional patients. Abnormal expression of myxovirus resistance protein A (MxA) was present on capillaries in muscle in 9 patients and in nerve in 7 patients. SARS-CoV-2 immunohistochemistry was negative in muscle and nerve in all patients. In the 10 controls, muscle showed type 2 atrophy in all patients, necrotic muscle fibers in 1, MHC-1 expression in nonnecrotic/nonregenerating fibers in 3, MxA expression on capillaries in 2, and inflammatory cells in none, and nerves showed no inflammatory cells or MxA expression. CONCLUSIONS: Muscle and nerve tissue demonstrated inflammatory/immune-mediated damage likely related to release of cytokines. There was no evidence of direct SARS-CoV-2 invasion of these tissues. CLASSIFICATION OF EVIDENCE: This study provides Class IV evidence that muscle and nerve biopsies document a variety of pathologic changes in patients dying of coronavirus disease 2019 (COVID-19).

Case Report: Iliopsoas Hematoma during the Clinical Course of Severe COVID-19 in Two Male Patients.

Anticoagulation plays a major role in reducing the risk of systematic thrombosis in patients with severe COVID-19. Serious hemorrhagic complications, such as intracranial hemorrhage, have also been recognized. However, intra-abdominal hemorrhage is under-recognized because of its rare occurrence, despite high mortality. Here, we discuss two cases of spontaneous iliopsoas hematoma (IPH) likely caused by anticoagulants during the clinical course of COVID-19. We also explored published case reports to identify clinical characteristics of IPH in COVID-19 patients. The use of anticoagulants may increase the risk of lethal IPH among COVID-19 patients becsuse of scarce data on optimal dosage and adequate monitoring of anticoagulant effects. Rapid diagnosis and timely intervention are crucial to ensure good patient outcomes.

Sarcopenia: An underlying treatment target during the COVID-19 pandemic.

The role of skeletal muscle mass in modulating immune response and supporting metabolic stress has been increasingly confirmed. Patients with sarcopenia, characterized by reduced muscle mass and muscle strength, were reported to have poor immune response and metabolic stress when facing acute infection, major surgeries, and other attacks. Based on empirical data, patients with sarcopenia are speculated to have increased infection rates and dismal prognoses amid the current 2019 novel coronavirus disease (COVID-19) epidemic. COVID-19 infection also aggravates sarcopenia because of the increased muscle wasting caused by systematic inflammation and the reduced physical activity and inadequate nutrient intake caused by social isolation. Notably, the interventions targeting skeletal muscle are anticipated to break the vicious circle and benefit the treatment of both conditions. We recommend sarcopenia assessment for populations with advanced age, inactivity, chronic disease, cancers, and nutritional deficiency. Patients with sarcopenia and COVID-19 infection need intensive care and aggressive treatments. The provision of at-home physical activities together with protein supplementation is anticipated to reverse sarcopenia and promote the prevention and treatment of COVID-19. The recommended protocols on nutritional support and physical activities are provided in detail.

Histological findings in skeletal muscle of SARS-CoV2 infected patient.

Autopsies represent medical procedures through which the causes of patients' deaths are determined or, through tissue sampling and microscopic examination of slides in usual stains or special tests, one can offer the basis for understanding the physiopathological mechanisms that contribute to the patients' death Histological findings of tissue samples from patients who have died of COVID-19 have been mainly orientated to lung, heart, liver, kidney damage with a small percent of them following other organs, but none has, to our knowledge, studied skeletal muscle.

Neurological manifestations of COVID-19: A brief review.

The COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has been predominantly a respiratory manifestation. Currently, with evolving literature, neurological signs are being increasingly recognized. Studies have reported that SARS-CoV-2 affects all aspects of the nervous system including the central nervous system (CNS), peripheral nervous system (PNS) and the muscular system as well. Not all patients have reverse transcription-polymerase chain reaction positive for the virus in the cerebrospinal fluid, and diagnosing the association of the virus with the myriad of neurological manifestations can be a challenge. It is important that clinicians have a high-index of suspicion for COVID-19 in patients presenting with new-onset neurological symptoms. This will lead to early diagnosis and specific management. Further studies are desired to unravel the varied neurological manifestations, treatment, outcome and long-term sequel in COVID-19 patients.

The activation of antiviral RNA interference not only exists in neural progenitor cells but also in somatic cells in mammals.

The RNA interference (RNAi) pathway directs an important antiviral immunity mechanism in plants and invertebrates. Recently, we and others have demonstrated that the antiviral RNAi response is also conserved in mammals, at least to five distinct RNA viruses, including Zika virus (ZIKV). ZIKV may preferentially infect neuronal progenitor cells (NPCs) in the developing foetal brain. Ex vivo ZIKV infection induces RNAi-mediated antiviral response in human NPCs, but not in the more differentiated NPCs or somatic cells. However, litter is known about the in vivo property or function of the virus-derived small-interfering RNAs (vsiRNAs) targeting ZIKV. Here we report a surprising observation: different from ex vivo observations, viral small RNAs (vsRNAs) targeting ZIKV were produced in vivo upon infection in both central neuron system (CNS) and muscle tissues. In addition, our findings demonstrate the production of canonical vsiRNAs in murine CNS upon antiviral RNAi activation by Sindbis virus (SINV), suggesting the possibility of antiviral immune strategy applied by mammals in the CNS.

Chikungunya virus replication in skeletal muscle cells is required for disease development.

Chikungunya virus (CHIKV) is an arbovirus capable of causing a severe and often debilitating rheumatic syndrome in humans. CHIKV replicates in a wide variety of cell types in mammals, which has made attributing pathologic outcomes to replication at specific sites difficult. To assess the contribution of CHIKV replication in skeletal muscle cells to pathogenesis, we engineered a CHIKV strain exhibiting restricted replication in these cells via incorporation of target sequences for skeletal muscle cell-specific miR-206. This virus, which we term SKE, displayed diminished replication in skeletal muscle cells in a mouse model of CHIKV disease. Mice infected with SKE developed less severe disease signs, including diminished swelling in the inoculated foot and less necrosis and inflammation in the interosseous muscles. SKE infection was associated with diminished infiltration of T cells into the interosseous muscle as well as decreased production of Il1b, Il6, Ip10, and Tnfa transcripts. Importantly, blockade of the IL-6 receptor led to diminished swelling of a control CHIKV strain capable of replication in skeletal muscle, reducing swelling to levels observed in mice infected with SKE. These data implicate replication in skeletal muscle cells and release of IL-6 as important mediators of CHIKV disease.

Propriospinal Neurons of L3-L4 Segments Involved in Control of the Rat External Urethral Sphincter.

Coordination of activity of external urethral sphincter (EUS) striated muscle and bladder (BL) smooth muscle is essential for efficient voiding. In this study we examined the morphological and electrophysiological properties of neurons in the L3/L4 spinal cord (SC) that are likely to have an important role in EUS-BL coordination in rats. EUS-related SC neurons were identified by retrograde transsynaptic tracing following injection of pseudorabies virus (PRV) co-expressing fluorescent markers into the EUS of P18-P20 male rats. Tracing revealed not only EUS motoneurons in L6/S1 but also interneurons in lamina X of the L6/S1 and L3/L4 SC. Physiological properties of fluorescently labeled neurons were assessed during whole-cell recordings in SC slices followed by reconstruction of biocytin-filled neurons. Reconstructions of neuronal processes from transverse or longitudinal slices showed that some L3/L4 neurons have axons projecting toward and into the ventro-medial funiculus (VMf) where axons extended caudally. Other neurons had axons projecting within laminae X and VII. Dendrites of L3/L4 neurons were distributed within laminae X and VII. The majority of L3/L4 neurons exhibited tonic firing in response to depolarizing currents. In transverse slices focal electrical stimulation (FES) in the VMf or in laminae X and VII elicited antidromic axonal spikes and/or excitatory synaptic responses in L3/L4 neurons; while in longitudinal slices FES elicited excitatory synaptic inputs from sites up to 400 µm along the central canal. Inhibitory inputs were rarely observed. These data suggest that L3/L4 EUS-related circuitry consists of at least two neuronal populations: segmental interneurons and propriospinal neurons projecting to L6/S1.

Generation of Yellow Fever virus vaccine in skeletal muscle cells of chicken embryos.

BACKGROUND: The Yellow Fever (YF) vaccine is produced by the inoculation of embryonated chicken eggs with YF17DD virus on the ninth day of development. Full embryos are collected on the twelfth day of development for vaccine formulation. Skeletal muscle tissue is the main site where biosynthesis of viral particles occurs. OBJECTIVES: This study aimed to analyse the experimental infection of skeletal muscle cells of chicken embryos by the 17DD Yellow Fever virus (YFV) in vivo and in vitro. METHODS: Chicken embryos infected with YF17DD virus were analysed by immunofluorescence using confocal and super-resolution microscopes. Primary cultures of skeletal muscle cells of non-infected chicken embryos were evaluated for susceptibility and permissiveness to YF17DD virus using different protocols. This evaluation was performed based on morphological, viral titration, molecular biology, and colorimetric techniques. FINDINGS: The present work phenotypically characterises embryonic chicken skeletal muscle cells as myogenic precursors expressing the Pax7 transcription factor in some cases. We demonstrated that these cells are susceptible to in vitro infection at different multiplicities of infection (MOIs), reproducing the same infection pattern observed in vivo. Furthermore, myogenic precursors and myoblasts are preferred infection targets, but establishment of infection does not depend on the presence of these cells. The peak of viral production occurred at 48 hpi, with decay occurring 72 hpi, when the cytopathic effect can be observed. MAIN CONCLUSIONS: In conclusion, the primary culture of chicken skeletal muscle cells is a good model for studying muscle cells infected with YF17DD virus. This culture system displays satisfactory emulation of the in vitro phenomenon observed, contributing to our understanding of virus infection dynamics and leading to the development of alternative methods of vaccine production.

Distinct Roles of Interferon Alpha and Beta in Controlling Chikungunya Virus Replication and Modulating Neutrophil-Mediated Inflammation.

Type I interferons (IFNs) are key mediators of the innate immune response. Although members of this family of cytokines signal through a single shared receptor, biochemical and functional variation exists in response to different IFN subtypes. While previous work has demonstrated that type I IFNs are essential to control infection by chikungunya virus (CHIKV), a globally emerging alphavirus, the contributions of individual IFN subtypes remain undefined. To address this question, we evaluated CHIKV pathogenesis in mice lacking IFN-ß (IFN-ß knockout [IFN-ß-KO] mice or mice treated with an IFN-ß-blocking antibody) or IFN-α (IFN regulatory factor 7 knockout [IRF7-KO] mice or mice treated with a pan-IFN-α-blocking antibody). Mice lacking either IFN-α or IFN-ß developed severe clinical disease following infection with CHIKV, with a marked increase in foot swelling compared to wild-type mice. Virological analysis revealed that mice lacking IFN-α sustained elevated infection in the infected ankle and in distant tissues. In contrast, IFN-ß-KO mice displayed minimal differences in viral burdens within the ankle or at distal sites and instead had an altered cellular immune response. Mice lacking IFN-ß had increased neutrophil infiltration into musculoskeletal tissues, and depletion of neutrophils in IFN-ß-KO but not IRF7-KO mice mitigated musculoskeletal disease caused by CHIKV. Our findings suggest disparate roles for the IFN subtypes during CHIKV infection, with IFN-α limiting early viral replication and dissemination and IFN-ß modulating neutrophil-mediated inflammation.IMPORTANCE Type I interferons (IFNs) possess a range of biological activity and protect against a number of viruses, including alphaviruses. Despite signaling through a shared receptor, there are established biochemical and functional differences among the IFN subtypes. The significance of our research is in demonstrating that IFN-α and IFN-ß both have protective roles during acute chikungunya virus (CHIKV) infection but do so by distinct mechanisms. IFN-α limits CHIKV replication and dissemination, whereas IFN-ß protects from CHIKV pathogenesis by limiting inflammation mediated by neutrophils. Our findings support the premise that the IFN subtypes have distinct biological activities in the antiviral response.

Dermal and muscle fibroblasts and skeletal myofibers survive chikungunya virus infection and harbor persistent RNA.

Chikungunya virus (CHIKV) is an arthritogenic alphavirus that acutely causes fever as well as severe joint and muscle pain. Chronic musculoskeletal pain persists in a substantial fraction of patients for months to years after the initial infection, yet we still have a poor understanding of what promotes chronic disease. While replicating virus has not been detected in joint-associated tissues of patients with persistent arthritis nor in various animal models at convalescent time points, viral RNA is detected months after acute infection. To identify the cells that might contribute to pathogenesis during this chronic phase, we developed a recombinant CHIKV that expresses Cre recombinase (CHIKV-3'-Cre). CHIKV-3'-Cre replicated in myoblasts and fibroblasts, and it induced arthritis during the acute phase in mice. Importantly, it also induced chronic disease, including persistent viral RNA and chronic myositis and synovitis similar to wild-type virus. CHIKV-3'-Cre infection of tdTomato reporter mice resulted in a population of tdTomato+ cells that persisted for at least 112 days. Immunofluorescence and flow cytometric profiling revealed that these tdTomato+ cells predominantly were myofibers and dermal and muscle fibroblasts. Treatment with an antibody against Mxra8, a recently defined host receptor for CHIKV, reduced the number of tdTomato+ cells in the chronic phase and diminished the levels of chronic viral RNA, implicating these tdTomato+ cells as the reservoir of chronic viral RNA. Finally, isolation and flow cytometry-based sorting of the tdTomato+ fibroblasts from the skin and ankle and analysis for viral RNA revealed that the tdTomato+ cells harbor most of the persistent CHIKV RNA at chronic time points. Therefore, this CHIKV-3'-Cre and tdTomato reporter mouse system identifies the cells that survive CHIKV infection in vivo and are enriched for persistent CHIKV RNA. This model represents a useful tool for studying CHIKV pathogenesis in the acute and chronic stages of disease.

Evolution of the Piscine orthoreovirus Genome Linked to Emergence of Heart and Skeletal Muscle Inflammation in Farmed Atlantic Salmon (Salmo salar).

Heart and skeletal muscle inflammation (HSMI) in farmed Atlantic salmon (Salmo salar) was first diagnosed in Norway in 1999. The disease is caused by Piscine orthoreovirus-1 (PRV-1). The virus is prevalent in farmed Atlantic salmon, but not always associated with disease. Phylogeny and sequence analyses of 31 PRV-1 genomes collected over a 30-year period from fish with or without HSMI, grouped the viral sequences into two main monophylogenetic clusters, one associated with HSMI and the other with low virulent PRV-1 isolates. A PRV-1 strain from Norway sampled in 1988, a decade before the emergence of HSMI, grouped with the low virulent HSMI cluster. The two distinct monophylogenetic clusters were particularly evident for segments S1 and M2. Only a limited number of amino acids were unique to the association with HSMI, and they all located to S1 and M2 encoded proteins. The observed co-evolution of the S1-M2 pair coincided in time with the emergence of HSMI in Norway, and may have evolved through accumulation of mutations and/or segment reassortment. Sequences of S1-M2 suggest selection of the HSMI associated pair, and that this segment pair has remained almost unchanged in Norwegian salmon aquaculture since 1997. PRV-1 strains from the North American Pacific Coast and Faroe Islands have not undergone this evolution, and are more closely related to the PRV-1 precursor strains not associated with clinical HSMI.

Mitochondrial dysfunction and role of harakiri in the pathogenesis of myositis.

The etiology of myositis is unknown. Although attempts to identify viruses in myositis skeletal muscle have failed, several studies have identified the presence of a viral signature in myositis patients. Here we postulate that in individuals with susceptible genetic backgrounds, viral infection alters the epigenome to activate the pathological pathways leading to disease onset. To identify epigenetic changes, methylation profiling of Coxsackie B infected human myotubes and muscle biopsies from polymyositis (PM) and dermatomyositis (DM) patients were compared to changes in global transcript expression induced by in vitro Coxsackie B infection. Gene and protein expression analysis and live cell imaging were performed to examine the mechanisms. Analysis of methylation and gene expression changes identified that a mitochondria-localized activator of apoptosis - harakiri (HRK) - is upregulated in myositis skeletal muscle cells. Muscle cells with higher HRK expression have reduced mitochondrial potential and poor ability to repair from injury as compared to controls. In cells from myositis patient toll-like receptor 7 (TLR7) activates and sustains high HRK expression. Forced over expression of HRK in healthy muscle cells is sufficient to compromise their membrane repair ability. Endurance exercise that is associated with improved muscle and mitochondrial function in PM and DM patients decreased TLR7 and HRK expression identifying these as therapeutic targets. Increased HRK and TLR7 expression causes mitochondrial damage leading to poor myofiber repair, myofiber death and muscle weakness in myositis patients and exercise induced reduction of HRK and TLR7 expression in patients is associated with disease amelioration. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.