In 2014, the United States experienced an epidemic of acute flaccid myelitis (AFM) cases in children coincident with a nationwide outbreak of enterovirus D68 (EV-D68) respiratory disease. Up to half of the 2014 AFM patients had EV-D68 RNA detected by RT-PCR in their respiratory secretions, although EV-D68 was only detected in cerebrospinal fluid (CSF) from one 2014 AFM patient. Given previously described molecular and epidemiologic associations between EV-D68 and AFM, we sought to develop an animal model by screening seven EV-D68 strains for the ability to induce neurological disease in neonatal mice. We found that four EV-D68 strains from the 2014 outbreak (out of five tested) produced a paralytic disease in mice resembling human AFM. The remaining 2014 strain, as well as 1962 prototype EV-D68 strains Fermon and Rhyne, did not produce, or rarely produced, paralysis in mice. In-depth examination of the paralysis caused by a representative 2014 strain, MO/14-18947, revealed infectious virus, virion particles, and viral genome in the spinal cords of paralyzed mice. Paralysis was elicited in mice following intramuscular, intracerebral, intraperitoneal, and intranasal infection, in descending frequency, and was associated with infection and loss of motor neurons in the anterior horns of spinal cord segments corresponding to paralyzed limbs. Virus isolated from spinal cords of infected mice transmitted disease when injected into naïve mice, fulfilling Koch’s postulates in this model. Finally, we found that EV-D68 immune sera, but not normal mouse sera, protected mice from development of paralysis and death when administered prior to viral challenge. These studies establish an experimental model to study EV-D68-induced myelitis and to better understand disease pathogenesis and develop potential therapies.

Insulin-dependent (type 1) diabetes mellitus (T1D) onset is mediated by individual human genetics as well as undefined environmental influences such as viral infections. The group B coxsackieviruses (CVB) are commonly named as putative T1D-inducing agents. We studied CVB replication in nonobese diabetic (NOD) mice to assess how infection by diverse CVB strains affected T1D incidence in a model of human T1D. Inoculation of 4-or 8-week-old NOD mice with any of nine different CVB strains significantly reduced the incidence of T1D by 2-to 10-fold over a 10-month period relative to T1D incidences in mock-infected control mice. Greater protection was conferred by more-pathogenic CVB strains relative to less-virulent or avirulent strains. Two CVB3 strains were employed to further explore the relationship of CVB virulence phenotypes to T1D onset and incidence: a pathogenic strain (CVB3/M) and a nonvirulent strain (CVB3/GA). CVB3/M replicated to four-to fivefold-higher titers than CVB3/GA in the pancreas and induced widespread pancreatitis, whereas CVB3/GA induced no pancreatitis. Apoptotic nuclei were detected by TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) assay in CVB3/M-infected pancreata but not in CVB3/GA-infected pancreata. In situ hybridization detected CVB3 RNA in acinar tissue but not in pancreatic islets. Although islets demonstrated inflammatory infiltrates in CVB3-protected mice, insulin remained detectable by immunohistochemistry in these islets but not in those from diabetic mice. Enzyme-linked immunosorbent assay-based examination of murine sera for immunoglobulin G1 (IgG1) and IgG2a immunoreactivity against diabetic autoantigens insulin and HSP60 revealed no statistically significant relationship between CVB3-protected mice or diabetic mice and specific autoimmunity. However, when pooled sera from CVB3/M-protected mice were used to probe a Western blot of pancreatic proteins, numerous proteins were detected, whereas only one band was detected by sera from CVB3/GA-protected mice. No proteins were detected by sera from diabetic or normal mice. Cumulatively, these data do not support the hypothesis that CVB are causative agents of T1D. To the contrary, CVB infections provide significant protection from T1D onset in NOD mice. Possible mechanisms by which this virus-induced protection may occur are discussed.The group B coxsackieviruses (CVB; family Picornaviridae, genus Enterovirus, species group B coxsackievirus; six serotypes, CVB1 to -6) are among the best studied of human enteroviruses (102). The CVB genome is a single strand of positive sense RNA 7,400 nucleotides in length that encodes 11 proteins in a single open reading frame (89). The CVB have been associated with diverse human diseases, among the more serious of which are myocarditis, pancreatitis, and aseptic meningitis. The CVB have been soundly implicated as causes of human myocarditis (1, 26, 42, 60-62, 73, 74, 108, 109) and pancreatitis (2,41,54,58,66,107) and, furthermore, cause these diseases readily i...

West Nile virus (WNV) is a neurotropic flavivirus that causes

Flaviviruses, particularly Japanese encephalitis virus (JEV) and West Nile virus (WNV), are important causes of virus-induced central nervous system (CNS) disease in humans. We used microarray analysis to identify cellular genes that are differentially regulated following infection of the brain with JEV (P3) or WNV (New York 99). Gene expression data for these flaviviruses were compared to those obtained following infection of the brain with reovirus (type 3 Dearing), an unrelated neurotropic virus. We found that a large number of genes were up-regulated by all three viruses (using the criteria of a change of >2-fold and a P value of <0.001), including genes associated with interferon signaling, the immune system, inflammation, and cell death/survival signaling. In addition, genes associated with glutamate signaling were down-regulated in infections with all three viruses (criteria, a >2-fold change and a P value of <0.001). These genes may serve as broad-spectrum therapeutic targets for virus-induced CNS disease. A distinct set of genes were up-regulated following flavivirus infection but not following infection with reovirus. These genes were associated with tRNA charging and may serve as therapeutic targets for flavivirus-induced CNS disease.

Apoptosis is an important mechanism of West Nile virus (WNV) pathogenesis within the central nervous system (CNS).N euronal apoptosis is an important mechanism of virus-induced pathogenesis within the central nervous system (CNS) (1). During West Nile virus (WNV) encephalitis, the proapoptotic executioner caspase, caspase 3, is activated, and mice lacking caspase 3 have reduced neuronal death and tissue injury following West Nile virus infection (2). Despite the importance of apoptosis in WNV pathogenesis, the exact pathways involved in triggering apoptotic cell death in the CNS have not yet been defined.Activation of initiator caspases 8 (3, 4) and 9 (3) occurs in cultured neuronal cells infected with WNV, and inhibition of these initiator caspases leads to reduced cleavage of the caspase 3 substrate poly(ADP-ribose) polymerase (PARP) (3, 4). These studies indicate that both extrinsic and intrinsic apoptotic signaling pathways are activated in vitro following WNV infection and are consistent with in vitro studies demonstrating that mitochondrial apoptotic signaling protein Bax is upregulated in neuronal cells following WNV infection (5) and that cytochrome c is released from the mitochondria (3). However, it remains to be seen whether the same apoptotic pathways are also activated in the intact brain during WNV encephalitis.Innate and adaptive immune responses also influence WNV pathogenesis within the CNS. Toll-like receptors 3 and 7 (TLR3 and TLR7) and the cytoplasmic proteins encoded by retinoic acidinducible gene I (RIG-I) and melanoma-differentiation-associated gene 5 (MDA5) are important for detection of WNV within the CNS, and defects in these, or additional, components of the interferon (IFN) response show enhanced viral burden and increased lethality (6). Although IFN restricts infection, pathogenic WNV strains attenuate IFN function at several steps of the induction and signaling cascade, allowing the virus to establish infection (6). Studies performed in mice (7-11) and humans (12-14) have also highlighted the role of CD8 ϩ T cells and the associated importance of pathways involving Fas ligand (10) and tumor necrosis factor (TNF)-related apoptosis-inducing factor ligand (TRAIL) (15) effector mechanisms in containing WNV CNS infection.In this report, we demonstrate that genes involved in death receptor (DR) apoptotic signaling are upregulated in the mouse brain following WNV infection. We also show for the first time that the activity of the DR-associated initiator caspase, caspase 8, is increased in the brain following WNV infection. WNV-induced activation of caspase 8 in the CNS is associated with cleavage of the proapoptotic Bcl-2 family protein Bid and with activation of caspase 9, suggesting that the caspase 8-dependent cleavage of Bid promotes intrinsic apoptotic signaling within the brains of infected animals. Utilization of WNV-infected ex vivo brain slice cultures (BSC), a novel model of WNV encephalitis, revealed that inhibition of caspase 8 decreased virus-induced activation of caspase 3 an...