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Skeletal muscle wasting is a clinically proven pathology associated with Japanese encephalitis virus (JEV) infection; however, underlying factors that govern skeletal muscle damage are yet to be explored. The current study aims to investigate the pathobiology of skeletal muscle damage using a mouse model of JEV infection. Our study reveals a significant increment in viral copy number in skeletal muscle post-JEV infection, which is associated with enhanced skeletal muscle cell death. Molecular and biochemical analysis confirms NOX2-dependent generation of reactive oxygen species, leading to autophagy flux inhibition and cell apoptosis. Along with this, an alteration in mitochondrial dynamics (change in fusion and fission process) and a decrease in the total number of mitochondria copies were found during JEV disease progression. The study represents the initial evidence of skeletal muscle damage caused by JEV and provides insights into potential avenues for therapeutic advancement., (© 2024 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

Chikungunya (CHIKV) and Mayaro (MAYV) viruses are arthritogenic alphaviruses that promote an incapacitating and long-lasting inflammatory muscle-articular disease. Despite studies pointing out the importance of skeletal muscle (SkM) in viral pathogenesis, the long-term consequences on its physiology and the mechanism of persistence of symptoms are still poorly understood. Combining molecular, morphological, nuclear magnetic resonance imaging, and histological analysis, we conduct a temporal investigation of CHIKV and MAYV replication in a wild-type mice model, focusing on the impact on SkM composition, structure, and repair in the acute and late phases of infection. We found that viral replication and induced inflammation promote a rapid loss of muscle mass and reduction in fiber cross-sectional area by upregulation of muscle-specific E3 ubiquitin ligases MuRF1 and Atrogin-1 expression, both key regulators of SkM fibers atrophy. Despite a reduction in inflammation and clearance of infectious viral particles, SkM atrophy persists until 30 days post-infection. The genomic CHIKV and MAYV RNAs were still detected in SkM in the late phase, along with the upregulation of chemokines and anti-inflammatory cytokine expression. In agreement with the involvement of inflammatory mediators on induced atrophy, the neutralization of TNF and a reduction in oxidative stress using monomethyl fumarate, an agonist of Nrf2, decreases atrogen expression and atrophic fibers while increasing weight gain in treated mice. These data indicate that arthritogenic alphavirus infection could chronically impact body SkM composition and also harm repair machinery, contributing to a better understanding of mechanisms of arthritogenic alphavirus pathogenesis and with a description of potentially new targets of therapeutic intervention.

The recent outbreak of monkeypox virus (MPXV) was unprecedented in its size and distribution. Those living with uncontrolled HIV and low CD4 T cell counts might develop a fulminant clinical mpox course with increased mortality, secondary infections, and necrotizing lesions. Fatal cases display a high and widespread MPXV tissue burden. The underlying pathomechanisms are not fully understood. We report here the pathological findings of an MPXV-driven abscess in gastrocnemius muscle requiring surgery in an immunocompromised patient with severe mpox. Presence of virus particles and infectivity were confirmed by electron microscopy, expansion microscopy, and virus culture, respectively. MPXV tissue distribution by immunohistochemistry (IHC) showed a necrotic core with infection of different cell types. In contrast, at the lesion rim fibroblasts were mainly infected. Immune cells were almost absent in the necrotic core, but were abundant at the infection rim and predominantly macrophages. Further, we detected high amounts of alternatively activated GPNMB + -macrophages at the lesion border. Of note, macrophages only rarely colocalized with virus-infected cells. Insufficient clearance of infected cells and infection of lesion-associated fibroblasts sustained by the abundance of profibrotic macrophages might lead to the coalescing of lesions and the severe and persistent clinical mpox course observed in immunocompromised patients., (© 2024 The Author(s). Journal of Medical Virology published by Wiley Periodicals LLC.)

Fillet discoloration by red and melanized focal changes (RFCs and MFCs) is common in farmed Atlantic salmon Salmo salar. In farmed rainbow trout Oncorhynchus mykiss, similar changes have been noted, but their prevalence and histological characteristics have not been investigated. Thus, we conducted a study encompassing 1293 rainbow trout from 3 different farm sites in Norway, all examined at the time of slaughter. Both macroscopic and histological assessments of the changes were performed. Reverse transcription (RT)-qPCR analyses and in situ hybridization (ISH) were used to detect the presence and location, respectively, of potential viruses. Only 1 RFC was detected in a single fillet, while the prevalence of MFCs ranged from 1.46 to 6.47% between populations. The changes were predominantly localized in the cranioventral region of the fillet. Histological examinations unveiled necrotic myocytes, fibrosis, and regeneration of myocytes. Melano-macrophages were found in the affected areas and in myoseptal adipose tissue. Organized granulomas were observed in only 1 fish. Notably, the presence of inflammatory cells, including melano-macrophages, appeared lower compared to what has been previously documented in Atlantic salmon MFCs. Instead, fibrosis and regeneration dominated. RT-qPCR and ISH revealed the presence of piscine orthoreovirus 1 (PRV-1) and salmonid alphavirus (SAV) in skeletal muscle. However, these viruses were not consistently associated with lesioned areas, contrasting previous findings in Atlantic salmon. In conclusion, rainbow trout develop MFCs of a different character than farmed Atlantic salmon, and we speculate whether the observed pathological differences are contributing to their reduced occurrence in farmed rainbow trout.

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., Competing Interests: Declaration of interests The authors declare competing financial interests: S.M.H. and D.P.M. have filed patent applications on this work through Cincinnati Children’s Hospital Medical Center., (Copyright © 2023 Elsevier Inc. All rights reserved.)

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

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.

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.

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., Competing Interests: The 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 © 2021 Malik, Nyman, Wessel, Dahle and Rimstad.)

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)., (© 2021 American Academy of Neurology.)

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., (Copyright © 2020 Elsevier Inc. All rights reserved.)

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.

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.

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., Competing Interests: None

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.

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., (Copyright © 2019 IBRO. Published by Elsevier Ltd. All rights reserved.)

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-β (IFN-β knockout [IFN-β-KO] mice or mice treated with an IFN-β-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-β 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-β-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-β had increased neutrophil infiltration into musculoskeletal tissues, and depletion of neutrophils in IFN-β-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-β 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-β 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-β 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., (Copyright © 2019 American Society for Microbiology.)

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.

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., (© 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

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., Competing Interests: I have read the journal's policy and the authors of this manuscript have the following competing interests: MSD is a consultant for Inbios and on the Scientific Advisory Board of Moderna, and has filed a provisional patent on Mxra8 antibodies and related proteins.

Melanized focal changes in skeletal muscle of farmed Atlantic salmon (Salmo salar) are a major quality problem. The aetiology is unknown, but infection with Piscine orthoreovirus (PRV) has been associated with the condition. Here, we addressed the pathogenesis of red and melanized focal changes and their association with PRV. First, a population of farmed fish (PRV-negative prior to sea transfer) was sequentially investigated throughout the seawater period. The fish were autopsied and tested for PRV infection. Muscular changes were described by macroscopy and histology, and a classification system was established. Second, in an experimental infection trial, PRV was injected intramuscularly to induce changes. The farmed fish was gradually infected with PRV. Red focal changes occurred throughout the observation period with a low prevalence regardless of PRV status. Melanized changes were highly diverse and their prevalence increased during the trial. Changes of low macroscopic grade and histological category were more prevalent in PRV-negative fish. Diffuse granulomatous melanized changes only occurred after PRV infection. No muscular changes were observed in the experimentally challenged fish. Our studies do not indicate that PRV infection causes red focal changes, but seems important in the development of granulomatous melanized changes., (© 2019 The Authors. Journal of Fish Diseases Published by John Wiley & Sons Ltd.)

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.

Piscine orthoreovirus (PRV) is ubiquitous in farmed Atlantic salmon and sometimes associated with disease - most notably, Heart and Skeletal Muscle Inflammation (HSMI). However, PRV is also widespread in non-diseased fish, particularly in Pacific Canada, where few cases of severe heart inflammation have been documented. To better understand the mechanisms behind PRV-associated disease, this study investigated the infection dynamics of PRV from Pacific Canada and the potential for experimental passage of putatively associated heart inflammation in Pacific-adapted Mowi-McConnell Atlantic salmon. Regardless of the PRV source (fish with or without HSMI-like heart inflammation), infections led to high-load viremia that induced only minor focal heart inflammation without significant transcriptional induction of inflammatory cytokines. Repeated screening of PRV dsRNA/ssRNA along with histopathology and gene expression analysis of host blood and heart tissues identified three distinct phases of infection: (1) early systemic dissemination and replication without host recognition; (2) peak replication, erythrocyte inclusion body formation and load-dependent host recognition; (3) long-term, high-load viral persistence with limited replication or host recognition sometimes accompanied by minor heart inflammation. These findings contrast previous challenge trials with PRV from Norway that induced severe heart inflammation and indicate that strain and/or host specific factors are necessary to initiate PRV-associated disease.

Although hepatitis E virus (HEV) transmission has been demonstrated after consumption of products containing infected pig liver, human cases can be also associated with other pig meat products, such as sausages. Data on HEV viremia and dissemination in muscle meat of infected animals are still sparse, especially during long-term infection. Previously, we have shown that experimental co-infection of pigs with HEV and porcine reproductive and respiratory syndrome virus (PRRSV) lengthens HEV infection up to 49 days and increases the likelihood of the presence of HEV RNA in the liver of the pig at a later stage of infection. In the present study, we show that during experimental HEV-PRRSV co-infection, prolonged HEV viremia, up to 49 days post-inoculation (dpi), is detected. The long-term viremia observed was statistically associated with the absence of HEV seroconversion. HEV RNA was also frequently detected, at a late stage of infection (49 dpi), in the three different types of muscle tested: femoral biceps, psoas major or diaphragm pillar. The HEV RNA load could reach up to 1 · 10 6 genome copies per gram of muscle. Detection of HEV in muscle meat was statistically associated with high HEV loads in corresponding liver and fecal samples. The presence of HEV in pig blood, femoral biceps and major psoas, corresponding to ham and tenderloin muscles respectively, is of concern for the food industry. Hence, these results indicate new potential risks for consumers and public health regarding pork products., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Enterovirus 71 (EV71) is the main pathogen of severe hand-foot-mouth disease (HFMD). Long non-coding RNAs (lncRNAs) are recognized as pivotal factors during the pathogenesis of viral infection. However, the critical functions of lncRNAs in EV71⁻host interactions have not been characterized. Here, for the first time, we performed global transcriptome analysis of lncRNA and mRNA expression profiles in EV71-infected human rhabdomyosarcoma (RD) cells and skeletal muscle of mice using second-generation sequencing. In our study, a total of 3801 novel lncRNAs were identified. In addition, 23 lncRNAs and 372 mRNAs exhibited remarkable differences in expression levels between infected and uninfected RD cells, while 104 lncRNAs and 2647 mRNAs were differentially expressed in infected skeletal muscle from neonatal mice. Comprehensive bioinformatics analysis included target gene prediction, lncRNA‑mRNA co-expression network construction, as well as gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis mainly focused on differentially-expressed genes (DEGs). Our results suggest that lncRNAs may participate in EV71 infection-induced pathogenesis through regulating immune responses, protein binding, cellular component biogenesis and metabolism. The present study provides novel insights into the functions of lncRNAs and the possible pathogenic mechanism following EV71 infection.

Enterovirus (EV) 71 infection has been widely acknowledged as the leading cause of severe hand, foot and mouth disease (HFMD), which may rapidly lead to fatal pulmonary edema. In this study, we established a mouse model for EV71 infection exhibiting high incidence of severe symptoms with pulmonary edema. Mast cells (MCs) accumulation, activation and allergic inflammation were found in the brains, lungs and skeletal muscle of mice after EV71 infection, especially in the lungs of mice. Levels of histamine, platelet-activating factor (PAF), interleukin (IL)-4, IL-5, IL-13, tumor necrosis factor-α (TNF-α), nitric oxide (NO), endocrine gland-derived vascular endothelial growth factor (EG-VEGF) and noradrenaline (NA) were increased in EV71-infected lungs. In addition, EV71 infection reduced the number of pulmonary T cells, dendritic cells (DCs) and monocytes, and increased the number of lung eosinophils, Tregs and MCs. MCs number and tryptase expression in target organs or tissues posed a trend towards an increase from control to severe mice. There were positive correlations between MCs number in the brains (r = 0.701, P = 0.003), lungs (r = 0.802, P < 0.0001), skeletal muscles (r = 0.737, P = 0.001) and mean clinical score. Thus, our results suggested that MCs contributed to the pulmonary edema during EV71 infection.

Vaccination remains the most cost-effective biomedical approach for controlling influenza disease. In times of pandemics, however, these vaccines cannot be produced in sufficient quantities for worldwide use by the current manufacturing capacities and practices. What is needed is the development of adjuvanted vaccines capable of inducing an adequate or better immune response at a decreased antigen dose. Previously we showed that the protein adjuvant rOv-ASP-1 augments influenza-specific antibody titers and survival after virus challenge in both young adult and old-age mice when administered with the trivalent inactivated influenza vaccine (IIV3). In this study we show that a reduced amount of rOv-ASP-1, with 40-times less IIV3 can also induce protection. Apparently the potency of the rOv-ASP-1 adjuvanted IIV3 vaccine is independent of the IIV3-specific Th1/Th2 associated antibody responses, and independent of the presence of HAI antibodies. However, CD4 + T helper cells were indispensable for the protection. Further, rOv-ASP-1 with or without IIV3 elicited the increased level of various chemokines, which are known chemoattractant for immune cells, into the muscle 4 h after immunization, and significantly induced the recruitment of monocytes, macrophages and neutrophils into the muscles. The recruited monocytes had higher expression of the activation marker MHCII on their surface as well as CXCR3 and CCR2; receptors for IP-10 and MCP-1, respectively. These results show that the rOv-ASP-1 adjuvant allows substantial antigen sparing of IIV3 by stimulating at the site of injection the accumulation of chemokines and the recruitment of immune cells that can augment the activation of CD4 + T cell immune responses, essential for the production of antibody responses. Protection elicited by the rOv-ASP-1 adjuvanted IIV3 vaccine also appears to function in the absence of MyD88-signaling. Future studies will attempt to delineate the precise mechanisms by which the rOv-ASP-1 adjuvanted IIV3 vaccine works., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Background: Human immunodeficiency virus (HIV)-infected individuals are at increased risk of age-associated functional impairment, even with effective antiretroviral therapy (ART). A concurrent characterization of skeletal muscle, physical function, and immune phenotype in aviremic middle-aged HIV-infected adults represents a knowledge gap in prognostic biomarker discovery., Methods: We undertook a prospective observational study of 170 middle-aged, HIV-infected ambulatory men and women with CD4+ T-cell counts of at least 350/µL and undetectable plasma viremia while on effective ART, and uninfected control participants. We measured biomarkers for inflammation and immune activation, fatigue, the Veterans Aging Cohort Study mortality index, and physical function. A subset also received a skeletal muscle biopsy and computed tomography scan., Results: Compared to the uninfected participants, HIV-infected participants displayed increased immune activation (P < .001), inflammation (P = .001), and fatigue (P = .010), and in a regression model adjusting for age and sex displayed deficits in stair-climb power (P < .001), gait speed (P = .036), and predicted metabolic equivalents (P = .019). Skeletal muscle displayed reduced nuclear peroxisome proliferator-activated receptor-γ coactivator 1α-positive myonuclei (P = .006), and increased internalized myonuclei (P < .001) that correlated with immune activation (P = .003) and leukocyte infiltration (P < .001). Internalized myonuclei improved a model for HIV discrimination, increasing the C-statistic from 0.84 to 0.90., Conclusions: Asymptomatic HIV-infected middle-aged adults display atypical skeletal muscle profiles, subclinical deficits in physical function, and persistent inflammation and immune activation. Identifying biomarker profiles for muscle dysregulation and risk for future functional decline in the HIV-infected population will be key to developing and monitoring preventive interventions., Clinical Trials Registration: NCT03011957.

During rabies virus infections, the minor salivary glands are one of the important organs for virus replication and excretion into the oral cavity. However, details of pathological findings and viral antigen distribution in the minor salivary glands remain poorly understood. In this study, we conducted pathological tests on the tongues of 71 rabid dogs in the Philippines; the minor salivary glands (von Ebner's glands, lingual glands), circumvallate papilla, autonomic ganglia, and skeletal muscles were evaluated. Inflammatory changes were observed in the von Ebner's glands of 20/71 dogs, in the circumvallate papilla of 10/71, and in the tongue muscle of 1/71. Conversely, no morphological changes were observed in the lingual glands and autonomic ganglia. Viral antigens were detected via immunohistochemistry-based methods in the cytoplasm of the acinar epithelium in the von Ebner's glands of all 71 dogs. Virus particles were confirmed in the intercellular canaliculi and acinar lumen via electron microscopy. In the autonomic ganglia, viral antigens were detected in 67/71 rabid dogs. Viral antigens were detected in the taste buds of all 71 dogs, and were distributed mainly in type II and III taste bud cells. In tongue muscle fibers, viral antigens were detected in 11/71 dogs. No virus antigens were detected in lingual glands. These findings suggest that rabies virus descends in the tongue along the glossopharyngeal nerve after proliferation in the brain, and von Ebner's glands and taste buds are one of the portals of virus excretion into the saliva in rabid dogs.

Highly pathogenic avian influenza viruses (HPAIV) of H5N1 subtype are a major global threat to poultry and public health. Export of poultry products, such as chicken and duck meat, is a known source for the cross-boundary spread of HPAI H5N1 viruses. Humans get infected with HPAI H5N1 viruses either by close contact with infected poultry or through consumption of fresh/undercooked poultry meat. Skeletal muscle is the largest soft tissue in chicken that has been shown to contain virus during systemic HPAIV infection and supports productive virus infection. However, the time between infection of a chicken with H5N1 virus and presence of virus in muscle tissue is not yet known. Further, it is also not clear whether chicken infected with low doses of H5N1 virus that cause non-fatal subclinical infections continue to accumulate virus in skeletal muscle. We investigated the amount and duration of virus detection in skeletal muscle of chicken experimentally infected with different doses (10 2 , 10 3 and 10 4 EID 50 ) of a HPAI H5N1 virus. Influenza viral antigen could be detected as early as 6 hr after infection and live virus was recovered from 48 hr after infection. Notably, chicken infected with lower levels of HPAI H5N1 virus (i.e., 10 2 EID 50 ) did not die acutely, but continued to accumulate high levels of H5N1 virus in skeletal muscle until 6 days post-infection. Our data suggest that there is a potential risk of human exposure to H5N1 virus through meat from clinically healthy chicken infected with a low dose of virus. Our results highlight the need to implement rigorous monitoring systems to screen poultry meat from H5N1 endemic countries to limit the global spread of H5N1 viruses., (© 2017 Blackwell Verlag GmbH.)

We report a case of spontaneous calf muscle haematoma, formed during the recovery phase of dengue haemorrhagic fever, which, to the best of our knowledge, has never been encountered before. A 45-year-old man presented with features of severe dengue and got admitted to our intensive care unit. He was treated with intravenous fluid therapy and supportive measures, and gradually improved, initially. However, during the recovery phase, he suddenly developed painful left calf, which was found tender, hot and swollen on physical examination. Colour Doppler ultrasound revealed left calf haematoma. As the patient rapidly developed local compartmental syndrome, surgical evacuation of the haematoma followed by urgent fasciotomy was performed. He recovered without further complication and was discharged home. At follow-up after 2 months, he remained well., Competing Interests: Competing interests: None declared., (© BMJ Publishing Group Ltd (unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.)

We previously selected muscle binding peptides 12.51 and 12.52 from "context-specific" phage display libraries for introduction into adenovirus (Ad) vectors. In this work, these peptides were inserted into the hypervariable region (HVR) 5 loop of the Ad5 hexon protein to display 720 peptides per virions. HVR-12.51 and 12.52 increased transduction of C2C12 cells up to 20-fold when compared to unmodified Ad5. 12.51 increased in vivo muscle transduction 2 to 7-fold over unmodified Ad after intramuscular injection in mice and hamsters. 12.52 did not increase muscle transduction. Notably, insertion of 12.51 into the hexon reduced liver transduction 80-fold when compared to unmodified Ad5 after intravenous injection. Increased muscle transduction in mice translated into increased immune responses after gene-based vaccination. These data suggest there are merits to retargeting and detargeting benefits to modifying the hexons of Ads with peptide ligands., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Enterovirus 71 (EV71) can cause hand, foot, and mouth disease in children, and severe infections can induce neurological complications and even death. However, the pathogenesis of EV71 remains unknown. The 2A proteinase (2A pro ) of EV71 plays an important role in segmenting the precursor polyprotein during viral replication, inhibiting host protein synthesis, and evading innate immunity. This study was to determine the function of EV71 2A pro in replication and virulence. A chimeric strain (SDLY 107-2A-1) was recombined by replacing 2A pro of a severe strain (SDLY107) with that of a mild strain (SDLY1) based on an infectious cDNA clone. The replication kinetics of the chimeric strain in vitro and in vivo were determined by qRT-PCR, which showed that the chimeric strain replicated slower and generated less viral RNA than the severe strain. The pathological change and viral load of chimeric strain infected mice were intermediate between severe strain infected mice and mild strain infected mice. Cellular cytotoxicity assays revealed that 2A pro was associated with the neurotoxicity of EV71. Histopathological and immunohistochemical assays detected tissue pathological damage in the lungs, muscles, brain, and intestinal tissues. Together, these results suggest that 2A pro modulates replication and virulence of EV71. This provides a theoretical basis for virulence determination of EV71., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Chequa iflavirus and a bunya-like virus infect redclaw crayfish (Cherax quadricarinatus) and they may cause mortality reaching 20-40% after about three weeks after a stress event. To complete River's postulates for viruses, virus-free animals are needed. Due to a lack of chequa iflavirus and bunya-like virus-free crayfish (testing shows>85% infection rate) coupled with the facts that iflavirus and bunyaviruses are found in insects and that crickets had been successful alternate hosts for crustacean viruses before, Acheta domesticus was trialled asa bioassay animal. There was no significant difference (P>0.05) in mortality rates between uninfected control crickets and infected crickets. Reverse transcriptase polymerase chain reaction for both viruses failed to find any trace of the RNA viruses in fed or inoculated crickets after 30days. The search for an alternative bioassay host will have to be widened., (Copyright © 2017 Elsevier Inc. All rights reserved.)

A new disease in farmed rainbow trout (Onchorhyncus mykiss) was described in Norway in 2013. The disease mainly affected the heart and resembled heart and skeletal muscle inflammation (HSMI) in Atlantic salmon (Salmo salar L.). HSMI is associated with Piscine orthoreovirus (PRV), and a search for a similar virus in the diseased rainbow trout led to detection of a sequence with 85% similarity to PRV. This finding called for a targeted effort to assess the risk the new PRV-variant pose on farmed rainbow trout and Atlantic salmon by studying infection and disease pathogenesis, aiming to provide more diagnostic knowledge. Based on the genetic relationship to PRV, the novel virus is referred to as PRV-Oncorhynchus mykiss (PRV-Om) in contrast to PRV-Salmo salar (PRV-Ss). In experimental trials, intraperitoneally injected PRV-Om was shown to replicate in blood in both salmonid species, but more effectively in rainbow trout. In rainbow trout, the virus levels peaked in blood and heart of cohabitants 6 weeks post challenge, along with increased expression of antiviral genes (Mx and viperin) in the spleen, with 80-100% of the cohabitants infected. Heart inflammation was diagnosed in all cohabitants examined 8 weeks post challenge. In contrast, less than 50% of the Atlantic salmon cohabitants were infected between 8 and 16 weeks post challenge and the antiviral response in these fish was very low. From 12 weeks post challenge and onwards, mild focal myocarditis was demonstrated in a few virus-positive salmon. In conclusion, PRV-Om infects both salmonid species, but faster transmission, more notable antiviral response and more prominent heart pathology were observed in rainbow trout.

Hand, foot, and mouth disease (HFMD) is a global health concern. Family Picornaviridae members, particularly enterovirus A71 (EVA71) and coxsackievirus A16 (CVA16), are the primary etiological agents of HFMD; however, a third enterovirus A species, CVA6, has been recently associated with epidemic outbreaks. Study of the pathogenesis of CVA6 infection and development of antivirals and vaccines are hindered by a lack of appropriate animal models. We have developed and characterized a murine model of CVA6 infection that was employed to evaluate the antiviral activities of different drugs and the protective efficacies of CVA6-inactivated vaccines. Neonatal mice were susceptible to CVA6 infection via intramuscular inoculation, and the susceptibility of mice to CVA6 infection was age and dose dependent. Five-day-old mice infected with 10 5.5 50% tissue culture infective doses of the CVA6 WF057R strain consistently exhibited clinical signs, including reduced mobility, lower weight gain, and quadriplegia with significant pathology in the brain, hind limb skeletal muscles, and lungs of the infected mice in the moribund state. Immunohistochemical analysis and quantitative reverse transcription-PCR (qRT-PCR) analyses showed high viral loads (11 log 10 /mg) in skeletal muscle, and elevated levels of interleukin-6 (IL-6; >2,000 pg/ml) were associated with severe viral pneumonia and encephalitis. Ribavirin and gamma interferon administered prophylactically diminished CVA6-associated pathology in vivo , and treatment with IL-6 accelerated the death of neonatal mice. Both specific anti-CVA6 serum and maternal antibody play important roles in controlling CVA6 infection and viral replication. Collectively, these findings indicate that this neonatal murine model will be invaluable in future studies to develop CVA6-specific antivirals and vaccines. IMPORTANCE Although coxsackievirus A6 (CVA6) infections are commonly mild and self-limiting, a small proportion of children may have serious complications, such as encephalitis, acute flaccid paralysis, and neurorespiratory syndrome, leading to fatalities. We have established a mouse model of CVA6 infection by inoculation of neonatal mice with a CVA6 clinical isolate that produced consistent pathological outcomes. Here, using this model of CVA6 infection, we found that high levels of IL-6 were associated with severe viral pneumonia and encephalitis, as in an evaluation of antiviral efficacy in vivo , IL-6 had no protective effect and instead accelerated death in neonatal mice. We demonstrated that, as antiviral drugs, both gamma interferon and ribavirin played important protective roles in the early stages of infection, with increased survival in treated neonatal mice challenged with CVA6. Moreover, active and passive immunization with the inactivated vaccines and anti-CVA6 serum also protected mice against homologous challenge infections., (Copyright © 2017 American Society for Microbiology.)

Infectious bursal disease virus (IBDV), a member of the Birnaviridae family, is responsible for a devastating immunosuppressive disease affecting juvenile domestic chickens. IBDV particles are naked icosahedrons enclosing a bipartite double-stranded RNA genome harboring three open reading frames (ORF). One of these ORFs codes for VP5, a non-structural polypeptide dispensable for virus replication in tissue culture but essential for IBDV pathogenesis. Using two previously described recombinant viruses, whose genomes differ in a single nucleotide, expressing or not the VP5 polypeptide, we have analyzed the role of this polypeptide during the IBDV replication process. Here, we show that VP5 is not involved in house-keeping steps of the virus replication cycle; i.e. genome transcription/replication, protein translation and virus assembly. Although infection with the VP5 expressing and non-expressing viruses rendered similar intracellular infective progeny yields, striking differences were detected on the ability of their progenies to exiting infected cells. Experimental data shows that the bulk of the VP5-expressing virus progeny efficiently egresses infected cells during the early phase of the infection, when viral metabolism is peaking and virus-induced cell death rates are as yet minimal, as determined by qPCR, radioactive protein labeling and quantitative real-time cell death analyses. In contrast, the release of the VP5-deficient virus progeny is significantly abridged and associated to cell death. Taken together, data presented in this report show that IBDV uses a previously undescribed VP5-dependent non-lytic egress mechanism significantly enhancing the virus dissemination speed. Ultrastructural analyses revealed that newly assembled IBDV virions associate to a vesicular network apparently facilitating their trafficking from virus assembly factories to the extracellular milieu, and that this association requires the expression of the VP5 polypeptide., Competing Interests: The authors have declared that no competing interests exist.

Cancer patients often show a wasting syndrome for which there are little therapeutic options. Dietary polyphenols have been proposed for treating this syndrome, but their usefulness in cases associated with human papillomavirus (HPV)-induced cancers is unknown. We characterized HPV16-transgenic mice as a model of cancer cachexia and tested the efficacy of long-term oral supplementation with polyphenols curcumin and rutin. Both compounds were orally administered to six weeks-old HPV16-transgenic mice showing characteristic multi-step skin carcinogenesis, for 24weeks. Skin lesions and blood, liver and spleen inflammatory changes were characterized histologically and hematologically. Hepatic oxidative stress, skeletal muscle mass and the levels of muscle pro-inflammatory transcription factor NF-κB were also assessed. Skin carcinogenesis was associated with progressive, severe, systemic inflammation (leukocytosis, hepatitis, splenitis), significant mortality and cachexia. Curcumin and rutin totally suppressed mortality while reducing white blood cells and the incidence of splenitis and hepatitis. Rutin prevented muscle wasting more effectively than curcumin. Preservation of muscle mass and reduced hepatic inflammation were associated with down-regulation of the NF-κB canonical pathway and with reduced oxidative stress, respectively. These results point out HPV16-transgenic mice as a useful model for studying the wasting syndrome associated with HPV-induced cancers. Dietary NF-κB inhibitors may be useful resources for treating this syndrome., (Copyright © 2016 Elsevier Inc. All rights reserved.)

To determine animal hepatitis E virus (HEV) reservoirs, we analyzed serologic and molecular markers of HEV infection among wild animals in Germany. We detected HEV genotype 3 strains in inner organs and muscle tissues of a high percentage of wild boars and a lower percentage of deer, indicating a risk for foodborne infection of humans.

Apart from the genetically engineered, modified, strains of rabies virus (RABV), unmodified 'fixed' virus strains of RABV, such as the 'French' subtype of CVS11, are used to examine synaptically connected networks in the brain. This technique has been shown to have all the prerequisite characteristics for ideal tracing as it does not metabolically affect infected neurons within the time span of the experiment, it is transferred transneuronally in one direction only and to all types of neurons presynaptic to the infected neuron, number of transneuronal steps can be precisely controlled by survival time and it is easily detectable with a sensitive technique. Here, using the 'French' CVS 11 subtype of RABV in Wistar rats, we show that some of these characteristics may not be as perfect as previously indicated. Using injection of RABV in hind limb muscles, we show that RABV-infected spinal motoneurons may already show lysis 1 or 2 days after infection. Using longer survival times we were able to establish that Purkinje cells may succumb approximately 3 days after infection. In addition, some neurons seem to resist infection, as we noted that the number of RABV-infected inferior olivary neurons did not progress in the same rate as other infected neurons. Furthermore, in our hands, we noted that infection of Purkinje cells did not result in expected transneuronal labeling of cell types that are presynaptic to Purkinje cells such as molecular layer interneurons and granule cells. However, these cell types were readily infected when RABV was injected directly in the cerebellar cortex. Conversely, neurons in the cerebellar nuclei that project to the inferior olive did not take up RABV when this was injected in the inferior olive, whereas these cells could be infected with RABV via a transneuronal route. These results suggest that viral entry from the extracellular space depends on other factors or mechanisms than those used for retrograde transneuronal transfer. We conclude that transneuronal tracing with RABV may result in unexpected results, as not all properties of RABV seem to be ubiquitously valid.

Background: Recurrence of hand, foot and mouth disease (HFMD) pandemics continues to threaten public health. Despite increasing awareness and efforts, effective vaccine and drug treatment have yet to be available. Probiotics have gained recognition in the field of healthcare worldwide, and have been extensively prescribed to babies and young children to relieve gastrointestinal (GI) disturbances and diseases, associated or not with microbial infections. Since the faecal-oral axis represents the major route of HFMD transmission, transient persistence of probiotic bacteria in the GI tract may confer some protection against HFMD and limit transmission among children., Methods: In this work, the antiviral activity of two commercially available probiotics, namely Lactobacillus reuteri Protectis (L. reuteri Protectis) and Lactobacillus casei Shirota (L. casei Shirota), was assayed against Coxsackieviruses and Enterovirus 71 (EV71), the main agents responsible for HFMD. In vitro infection set-ups using human skeletal muscle and colon cell lines were designed to assess the antiviral effect of the probiotic bacteria during entry and post-entry steps of the infection cycle., Results: Our findings indicate that L. reuteri Protectis displays a significant dose-dependent antiviral activity against Coxsackievirus type A (CA) strain 6 (CA6), CA16 and EV71, but not against Coxsackievirus type B strain 2. Our data support that the antiviral effect is likely achieved through direct physical interaction between bacteria and virus particles, which impairs virus entry into its mammalian host cell. In contrast, no significant antiviral effect was observed with L. casei Shirota., Conclusions: Should the antiviral activity of L. reuteri Protectis observed in vitro be translated in vivo, such probiotics-based therapeutic approach may have the potential to address the urgent need for a safe and effective means to protect against HFMD and limit its transmission among children.

Background: The main goal of oral vaccination of foxes is eradication of rabies in the red fox population as rabies reservoirs. To evaluate the success of vaccination a serological testing is conducted as a part of monitoring program. Two different methods are used regarding rabies serology: virus neutralisation test and ELISA., Methods: In this study the reliability of BioPro ELISA was evaluated for testing haemolytic thoracic liquids and muscle extracts originated from 147 foxes in comparison to mFAVN. Also, the influence of heat treatment of samples on test results was investigated., Results: The specificity of the test for not-heat treated samples was 92.98% and sensitivity 79.20%. Diagnostic validity of the ELISA compared to the mFAVN test when not-heat treated samples were used was 89.16%. The specificity of the test for heat treated samples was 79.10% and sensitivity 96.36%. Diagnostic validity of the BioPro ELISA compared to the mFAVN test for heat treated samples was 94.30%., Conclusion: According to this study, the BioPro ELISA is reliable tool for detection of rabies specific antibodies in the context of evaluation of oral vaccination of foxes from poor quality samples as a substitution for virus neutralisation tests.

Although the influenza virus only infects the respiratory system, myalgias are commonly experienced during infection. In addition to a greater risk of hospitalization and death, older adults are more likely to develop disability following influenza infection; however, this relationship is understudied. We hypothesized that upon challenge with influenza, aging would be associated with functional impairments, as well as upregulation of skeletal muscle inflammatory and atrophy genes. Infected young and aged mice demonstrated decreased mobility and altered gait kinetics. These declines were more prominent in hind limbs and in aged mice. Skeletal muscle expression of genes involved in inflammation, as well as muscle atrophy and proteolysis, increased during influenza infection with an elevated and prolonged peak in aged mice. Infection also decreased expression of positive regulators of muscle mass and myogenesis components to a greater degree in aged mice. Gene expression correlated to influenza-induced body mass loss, although evidence did not support direct muscle infection. Overall, influenza leads to mobility impairments with induction of inflammatory and muscle degradation genes and downregulation of positive regulators of muscle. These effects are augmented and prolonged with aging, providing a molecular link between influenza infection, decreased resilience and increased risk of disability in the elderly.

Heart and skeletal muscle inflammation (HSMI) is a significant and often fatal disease of cultured Atlantic salmon in Norway. The consistent presence of Piscine orthoreovirus (PRV) in HSMI diseased fish along with the correlation of viral load and antigen with development of lesions has supported the supposition that PRV is the etiologic agent of this condition; yet the absence of an in vitro culture system to demonstrate disease causation and the widespread prevalence of this virus in the absence of disease continues to obfuscate the etiological role of PRV with regard to HSMI. In this study, we explore the infectivity and disease causing potential of PRV from western North America-a region now considered endemic for PRV but without manifestation of HSMI-in challenge experiments modeled upon previous reports associating PRV with HSMI. We identified that western North American PRV is highly infective by intraperitoneal injection in Atlantic salmon as well as through cohabitation of both Atlantic and Sockeye salmon. High prevalence of viral RNA in peripheral blood of infected fish persisted for as long as 59 weeks post-challenge. Nevertheless, no microscopic lesions, disease, or mortality could be attributed to the presence of PRV, and only a minor transcriptional induction of the antiviral Mx gene occurred in blood and kidney samples during log-linear replication of viral RNA. Comparative analysis of the S1 segment of PRV identified high similarity between this North American sequence and previous sequences associated with HSMI, suggesting that factors such as viral co-infection, alternate PRV strains, host condition, or specific environmental circumstances may be required to cause this disease.

Melanised focal changes (black spots) are common findings in the white skeletal muscle of seawater-farmed Atlantic salmon (Salmo salar). Fillets with melanised focal changes are considered as lower quality and cause large economic losses. It has been suggested that red focal changes (red spots) precede the melanised focal changes. In the present work, we examined different populations of captive and wild salmon for the occurrence of both types of changes, which were investigated for the presence of different viruses by immunohistochemistry and RT-qPCR. The occurrence of red or melanised foci varied significantly between the populations, from none in wild fish control group, low prevalence of small foci in fish kept in in-house tanks, to high prevalence of large foci in farm-raised salmon. Large amounts of Piscine orthoreovirus (PRV) antigen were detected in all foci. No other viruses were detected. Red focal changes contained significantly higher levels of PRV RNA than apparently non-affected areas in white muscle of the same individuals. Some changes displayed a transient form between a red and melanised pathotype, indicating a progression from an acute to a chronic manifestation. We conclude that PRV is associated with the focal pathological changes in the white muscle of farmed Atlantic salmon and is a premise for the development of focal melanised changes.

Enterovirus-71 (EV71) is a viral pathogen that causes severe cases of hand, foot and mouth disease (HFMD) among young children, with significant mortality. Effective vaccines against HFMD are urgently required. Several EV71 virus-like particle (VLP) vaccine candidates were found to be protective in the neonatal mouse EV71 challenge model. However, to what extent the VLP vaccine protects susceptible organs against EV71 infection in vivo has remained elusive. In the present study, the comprehensive immunogenicity of a potential EV71 vaccine candidate based on VLPs was evaluated in a neonatal mouse model. Despite lower levels of neutralizing antibodies to EV71 in the sera of VLP-immunized mice compared with those in mice vaccinated with inactivated EV71, the VLP-based vaccine was shown to be able to induce immunoglobulin (Ig)G and IgA memory-associated cellular immune responses to EV71. Of note, the EV71 VLP vaccine candidate was capable of inhibiting viral proliferation in cardiac muscle, skeletal muscle, lung and intestine of immunized mice and provided effective protection against the pathological damage caused by viral attack. In particular, the VLP vaccine was able to inhibit the transportation of EV71 from the central nervous system to the muscle tissue and greatly protected muscle tissue from infection, along with recovery from the viral infection. This led to nearly 100% immunoprotective efficacy, enabling neonatal mice delivered by VLP-immunized female adult mice to survive and grow with good health. The present study provided valuable additional knowledge of the specific protective efficacy of the EV71 VLP vaccine in vivo, which also indicated that it is a promising potential candidate for being developed into an EV71 vaccine.