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Enterovirus D68 (EV-D68) is predominantly associated with mild respiratory infections, but can also cause severe respiratory disease and extra-respiratory complications, including acute flaccid myelitis. Systemic dissemination of EV-D68 is crucial for the development of extra-respiratory diseases, but it is currently unclear how EV-D68 spreads systemically (viremia). We hypothesize that immune cells contribute to the systemic dissemination of EV-D68, as this is a mechanism commonly used by other enteroviruses. Therefore, we investigated the susceptibility and permissiveness of human primary immune cells for different EV-D68 isolates. In human peripheral blood mononuclear cells inoculated with EV-D68, only B cells were susceptible but virus replication was limited. However, in B cell-rich cultures, such as Epstein–Barr virus-transformed B-lymphoblastoid cell line (BLCL) and primary lentivirus-transduced B cells, which better represent lymphoid B cells, were productively infected. Subsequently, we showed that dendritic cells (DCs), particularly immature DCs, are susceptible and permissive for EV-D68 infection and that they can spread EV-D68 to autologous BLCL. Altogether, our findings suggest that immune cells, especially B cells and DCs, could play an important role in the pathogenesis of EV-D68 infection. Infection of these cells may contribute to systemic dissemination of EV-D68, which is an essential step toward the development of extra-respiratory complications.

Morbilliviruses are members of the family Paramyxoviridae and are known for their ability to cause systemic disease in a variety of mammalian hosts. The prototypic morbillivirus, measles virus (MeV), infects humans and still causes morbidity and mortality in unvaccinated children and young adults. Experimental infection studies in non-human primates have contributed to the understanding of measles pathogenesis. However, ethical restrictions call for the development of new animal models. Canine distemper virus (CDV) infects a wide range of animals, including ferrets, and its pathogenesis shares many features with measles. However, wild-type CDV infection is almost always lethal, while MeV infection is usually self-limiting. Here, we made five recombinant CDVs, predicted to be attenuated, and compared their pathogenesis to the non-attenuated recombinant CDV in a ferret model. Three viruses were insufficiently attenuated based on clinical signs, fatality, and systemic infection, while one virus was too attenuated. The last candidate virus caused a self-limiting infection associated with transient viremia and viral dissemination to all lymphoid tissues, was shed transiently from the upper respiratory tract, and did not result in acute neurological signs. Additionally, an in-depth phenotyping of the infected white blood cells showed lower infection percentages in all lymphocyte subsets when compared to the non-attenuated CDV. In conclusion, infection models using this candidate virus mimic measles and can be used to study pathogenesis-related questions and to test interventions for morbilliviruses in a natural host species.IMPORTANCEMorbilliviruses are transmitted via the respiratory route but cause systemic disease. The viruses use two cellular receptors to infect myeloid, lymphoid, and epithelial cells. Measles virus (MeV) remains an important cause of morbidity and mortality in humans, requiring animal models to study pathogenesis or intervention strategies. Experimen

Canine distemper virus (CDV) causes systemic infection resulting in severe and often fatal disease in a large spectrum of animal host species. The virus is closely related to measles virus and targets myeloid, lymphoid, and epithelial cells, but CDV is more virulent and the infection spreads more rapidly within the infected host. Here, we aimed to study the pathogenesis of wild-type CDV infection by experimentally inoculating ferrets with recombinant CDV (rCDV) based on an isolate directly obtained from a naturally infected raccoon. The recombinant virus was engineered to express a fluorescent reporter protein, facilitating assessment of viral tropism and virulence. In ferrets, this wild type-based rCDV infected myeloid, lymphoid, and epithelial cells, and the infection resulted in systemic dissemination to multiple tissues and organs, especially those of the lymphatic system. High infection percentages in immune cells resulted in depletion of these cells both from circulation and from lymphoid tissues. The majority of CDV-infected ferrets reached their humane endpoints within 20 d and had to be euthanized. In that period, the virus also reached the central nervous system in several ferrets, but we did not observe the development of neurological complications during the study period of 23 d. Two out of 14 ferrets survived CDV infection and developed neutralizing antibodies. We show for the first time the pathogenesis of a non-adapted wild type-based rCDV in ferrets. IMPORTANCE Infection of ferrets with recombinant canine distemper virus (rCDV) expressing a fluorescent reporter protein has been used as proxy to understand measles pathogenesis and immune suppression in humans. CDV and measles virus use the same cellular receptors, but CDV is more virulent, and infection is often associated with neurological complications. rCDV strains in current use have complicated passage histories, which may have affected their pathogenesis. Here, we studied the pathogenesis of the

Raccoons are naturally susceptible to canine distemper virus (CDV) infection and can be a potential source of spill-over events. CDV is a highly contagious morbillivirus that infects multiple species of carnivores and omnivores, resulting in severe and often fatal disease. Here, we used a recombinant CDV (rCDV) based on a full-genome sequence detected in a naturally infected raccoon to perform pathogenesis studies in raccoons. Five raccoons were inoculated intratracheally with a recombinant virus engineered to express a fluorescentreporter protein, and extensive virological, serological, histological, and immunohistochemical assessments were performed at differenttime points post inoculation. rCDV-infected white blood cells were detected as early as 4 days post inoculation (dpi). Raccoon necropsies at 6 and 8 dpi revealed replication in the lymphoid tissues, preceding spread into peripheral tissues observed during necropsies at 21 dpi. Whereas lymphocytes, and to a lesser extent myeloid cells, were the main target cells of CDV at early time points, CDV additionally targeted epithelia at 21 dpi. At this later time point, CDV-infected cells were observed throughout the host. We observed lymphopenia and lymphocyte depletion from lymphoid tissues after CDV infection, in the absence of detectable CDV neutralizing antibodies and an impaired ability to clear CDV, indicating that the animals were severely immunosuppressed. The use of a wild-type-based recombinant virus in a natural host species infection study allowed systematic and sensitive assessment of antigen detection by immunohistochemistry, enabling further comparative pathology studies of CDV infection in differentspecies.

Background Enterovirus-D68 (EV-D68) predominantly causes respiratory disease. However, EV-D68 infections also have been associated with central nervous system (CNS) complications, most specifically acute flaccid myelitis (AFM). Diagnosing EV-D68-associated CNS disease is challenging since viral RNA is rarely detected in cerebrospinal fluid (CSF). Objective In order to determine an EV antibody index (AI), we evaluated the value of a commercially available quantitative ELISA to detect EV-specific antibodies in paired CSF and blood. Study design Nine paired CSF and blood samples were obtained from patients with EV-D68-associated AFM or from patients with a confirmed EV-associated CNS disease. EV-specific antibodies were detected using a quantitative ELISA. A Reiber diagram analysis was performed, by which the AI was calculated. Subsequently, EV ELISA results were compared with an EV-D68 virus neutralization test. Results ELISA detected EV-specific antibodies in 1 out of the 3 patients with EV-D68-associated AFM and in 3 out of the 6 patients with confirmed EV-associated CNS disease. In these patients, the AI was indicative for intrathecal antibody production against enterovirus. Assay comparison showed that EV-D68 neutralizing antibody detection increased the sensitivity of EV-D68 antibody detection. Conclusions A quantitative EV IgG ELISA in combination with Reiber diagram analysis and AI-calculation can be used as a diagnostic tool for EV-associated CNS disease, including EV-D68. An EV-D68 specific ELISA will improve the sensitivity of the tool. With the growing awareness that the detection of non-polio enteroviruses needs to be improved, diagnostic laboratories should consider implementation of EV serology.

In 2014, enterovirus D68 (EV-D68) emerged causing outbreaks of severe respiratory disease in children worldwide. In a subset of patients, EV-D68 infection was associated with the development of central nervous system (CNS) complications, including acute flaccid myelitis (AFM). Since then, the number of reported outbreaks has risen biennially, which emphasizes the need to unravel the systemic pathogenesis in humans. We present here a comprehensive review on the different stages of the pathogenesis of EV-D68 infection–infection in the respiratory tract, systemic dissemination and infection of the CNS–based on observations in humans as well as experimental in vitro and in vivo studies. This review highlights the knowledge gaps on the mechanisms of systemic dissemination, routes of entry into the CNS and mechanisms to induce AFM or other CNS complications, as well as the role of virus and host factors in the pathogenesis of EV-D68.