A novel porcine deltacoronavirus (PdCV) was first discovered in Ohio and Indiana in February 2014, rapidly spread to other states in the United States and Canada, and caused significant economic loss in the swine industry. The origin and virulence of this novel porcine coronavirus are not known. Here, we characterized U.S. PdCV isolates and determined their virulence in gnotobiotic and conventional piglets. Genome analyses revealed that U.S. PdCV isolates possess unique genetic characteristics and share a close relationship with Hong Kong and South Korean PdCV strains and coronaviruses (CoVs) of Asian leopard cats and Chinese ferret-badgers. The PdCV-positive intestinal content (Ohio CVM1) and the cell culture-adapted PdCV Michigan (MI) strain were orally inoculated into gnotobiotic and/or conventional piglets. Within 1 to 3 days postinfection, profuse watery diarrhea, vomiting, and dehydration were observed. Clinical signs were associated with epithelial necrosis in the gastric pits and small intestine, the latter resulting in severe villous atrophy. Mild interstitial pneumonia was identified in the lungs of PdCV-infected piglets. High levels of viral RNA (8 to 11 log RNA copies/g) were detected in intestinal tissues/luminal contents and feces of infected piglets, whereas moderate RNA levels (2 to 5 log RNA copies/g) were detected in blood, lung, liver, and kidney, indicating multisystemic dissemination of the virus. Polyclonal immune serum against PdCV but not immune serum against porcine epidemic diarrhea virus (PEDV) reacted with PdCV-infected small-intestinal epithelial cells, indicating that PdCV is antigenically distinct from PEDV. Collectively, we demonstrate for the first time that PdCV caused severe gastrointestinal diseases in swine.

Measles virus is a negative-sense, single-stranded RNA virus within the Mononegavirales order, which includes several human pathogens, including rabies, Ebola, Nipah, and Hendra viruses. The measles virus nucleoprotein consists of a structured N-terminal domain, and of an intrinsically disordered C-terminal domain, N TAIL (aa 401-525), which undergoes induced folding in the presence of the C-terminal domain (XD, aa 459-507) of the viral phosphoprotein. Within N TAIL , an a-helical molecular recognition element (a-MoRE, aa 488-499) involved in binding to P and in induced folding was identified and then observed in the crystal structure of XD. Using small-angle X-ray scattering, we have derived a low-resolution structural model of the complex between XD and N TAIL , which shows that most of N TAIL remains disordered in the complex despite P-induced folding within the a-MoRE. The model consists of an extended shape accommodating the multiple conformations adopted by the disordered N-terminal region of N TAIL , and of a bulky globular region, corresponding to XD and to the C terminus of N TAIL (aa 486-525). Using surface plasmon resonance, circular dichroism, fluorescence spectroscopy, and heteronuclear magnetic resonance, we show that N TAIL has an additional site (aa 517-525) involved in binding to XD but not in the unstructured-to-structured transition. This work provides evidence that intrinsically disordered domains can establish complex interactions with their partners, and can contact them through multiple sites that do not all necessarily gain regular secondary structure.Keywords: measles virus; nucleoprotein; phosphoprotein; intrinsic disorder; induced folding; NMR; CD; SAXS Measles virus (MV) is an enveloped RNA virus within the Morbillivirus genus of the Paramyxoviridae family.Its nonsegmented, negative-sense, single-stranded RNA genome is encapsidated by the viral nucleoprotein (N) within a helical nucleocapsid. This N-RNA complex is used as a template for both transcription and replication. These latter activities are carried out by the viral polymerase complex, which consists of two components, the large protein (L) and the phosphoprotein (P) (for review, see Lamb and Kolakofsky 2001).

The Henipavirus genome is encapsidated by the nucleoprotein (N) within a helical nucleocapsid that recruits the polymerase complex via the phosphoprotein (P). In a previous study, we reported that in henipaviruses, the N-terminal domain of the phosphoprotein and the C-terminal domain of the nucleoprotein (N TAIL ) are both intrinsically disordered. Here we show that Henipavirus N TAIL domains are also disordered in the context of full-length nucleoproteins. We also report the cloning, purification, and characterization of the C-terminal X domains (P XD ) of Henipavirus phosphoproteins. Using isothermal titration calorimetry, we show that N TAIL and P XD form a 1:1 stoichiometric complex that is stable under NaCl concentrations as high as 1 M and has a K D in the M range. Using far-UV circular dichroism and nuclear magnetic resonance, we show that P XD triggers an increase in the ␣-helical content of N TAIL . Using fluorescence spectroscopy, we show that P XD has no impact on the chemical environment of a Trp residue introduced at position 527 of the Henipavirus N TAIL domain, thus arguing for the lack of stable contacts between the C termini of N TAIL and P XD . Finally, we present a tentative structural model of the N TAIL -P XD interaction in which a short, order-prone region of N TAIL (␣-MoRE; amino acids 473-493) adopts an ␣-helical conformation and is embedded between helices ␣2 and ␣3 of P XD , leading to a relatively small interface dominated by hydrophobic contacts. The present results provide the first detailed experimental characterization of the N-P interaction in henipaviruses and designate the N TAIL -P XD interaction as a valuable target for rational antiviral approaches.

The major inducible 70-kDa heat shock protein (hsp72) binds measles virus (MV) nucleocapsids and increases MV gene expression. The cytoplasmic tail of the MV N protein (N(TAIL)) contains three hydrophobic domains (Box-1-3) that are potential targets of hsp72 interaction. Low affinity binding to Box-3 is correlated to hsp72-dependent stimulation of MV minireplicon reporter gene expression whereas interactions between hsp72 and Box-1 and/or -2 have not been documented. The present work showed that virus deficient in Box-3/hsp72 interaction retains the ability to form nucleocapsid/hsp72 complexes, identifying Box-2 but not Box-1 as a mediator of high affinity hsp72 binding. Box-2 is the binding site for the viral P protein X domain (XD), where P tethers the viral polymerase to nucleocapsid in support of transcription and genome replication, and competitive inhibition of XD binding to N(TAIL) by hsp72 was shown. Recognition of a common binding site by P and hsp72 represents a potential mechanism for host cell modulation of viral gene expression.

Measles virus (MV) is a negative-strand RNA virus within the Morbillivirus genus of the Paramyxoviridae family. The MV transcriptional complex consists of the virus-encoded RNAdependent RNA polymerase (L), the polymerase cofactor (P), and a ribonucleoprotein template consisting of the singlestranded RNA genome and the nucleocapsid protein (N protein). N protein monomers assemble on the RNA genome during replication to form a single-start left-handed helix, protecting the genome from nuclease degradation. Encapsidation is initiated on specific sequences found within the leader RNA, drawing upon pools of soluble N-P heterodimers, whereas elongation of encapsidation occurs in an RNA sequence-independent manner (7, 24). Since genomic replication presumably is dependent upon concurrent encapsidation, functional motifs in the N protein required for genomic replication must include an RNA binding domain for the initiation of encapsidation, a binding site for P to form an N-P encapsidation complex, and N-N interaction sites required to drive nucleocapsid elongation and maintain nucleocapsid structural integrity. Additionally, N protein must contain a P binding site that is exposed on the nucleocapsid, thereby permitting the viral polymerase complex to interact with formed ribonucleoprotein templates during both transcription and genomic replication.The expression of MV N protein deletion mutants demonstrates that only the amino-terminal three-fourths of the molecule (i.e., amino acids 1 to 398) is required for the formation of organized nucleocapsid-like particles, localizing the N-N interaction domain to this highly conserved portion of the protein (1, 16). This conclusion is supported by the observation that selective proteolysis of the MV nucleocapsid can remove the C-terminal 15 kDa of the N protein while leaving nucleocapsid structural integrity and the amino-terminal 45-kDa N protein fragment intact (12). Also identified within the latter region are two sites necessary for the formation of soluble N-P complexes, localized to amino acids 4 to 188 and 304 to 373 (1). Enhanced sensitivity of the C terminus of the N protein to proteolysis is consistent with its exposure on the surface of the formed nucleocapsid. Within this exposed hypervariable domain is a third binding site for P, tentatively localized between amino acids 457 and 525 (1). For Sendai virus, the C-terminal domain of the N protein is required for template function in RNA replication assays (5) and contains a P binding site (4). Given the essential role of P in supporting viral polymerase function, a model for paramyxoviruses emerges in which P provides the link between L and N protein domains that are exposed on the nucleocapsid and that are necessary for both transcription and replication.The putative template function of the MV N protein C

Background: Adult dogs with degenerative myelopathy (DM) have progressive ataxia and paresis of the pelvic limbs, leading to paraplegia and euthanasia. Although most commonly reported in German Shepherd dogs, high disease prevalence exists in other breeds.Objective: Our aim was the clinical and histopathologic characterization of familial degenerative myelopathy (FDM) in Pembroke Welsh Corgi (PWC) dogs.Animals: Twenty-one PWCs were prospectively studied from initial diagnosis until euthanasia. Methods: Neurologic examination, blood tests, cerebrospinal fluid (CSF) analysis, electrodiagnostic testing, and spinal imaging were performed. Concentrations of 8-iso-prostaglandin F 2 a (8-isoprostane) were measured in CSF. Routine histochemistry was used for neuropathology. Deoxyribonucleic acid and pedigrees were collected from 110 dogs.Results: Median duration of clinical signs before euthanasia was 19 months. Median age at euthanasia was 13 years. All dogs were nonambulatory paraparetic or paraplegic, and 15 dogs had thoracic limb weakness at euthanasia. Electrodiagnostic testing and spinal imaging were consistent with noncompressive myelopathy. No significant difference was detected in 8-isoprostane concentrations between normal and FDM-affected dogs. Axonal and myelin degeneration of the spinal cord was most severe in the dorsal portion of the lateral funiculus. Pedigree analysis suggested a familial disease.Conclusions and Clinical Importance: Clinical progression of FDM in PWC dogs was similar to that observed in other breeds but characterized by a longer duration. Spinal cord pathology predominates as noninflammatory axonal degeneration. Oxidative stress injury associated with 8-isoprostane production is not involved in the pathogenesis of FDM-affected PWC dogs. A familial disease is suspected.

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The knock-out analyses of neuregulin and its receptors have indicated that they play essential roles in Schwann cell development. However, the role they play in oligodendrocyte development in vivo has remained unclear, because such knock-out animals die before CNS myelination begins. We examined the role of neuregulin signaling in the CNS by generating transgenic mice that express a dominant-negative mutant of the ErbB2 receptor among oligodendrocytes, using an MBP promoter. The transgenic mice exhibited widespread hypomyelination, resulting from a reduction in oligodendrocyte differentiation. The number of progenitors was conversely increased in the transgenic mice. We report that a reduction in oligodendrocyte differentiation is attributed in part to apoptosis of oligodendrocyte progenitors as they exit the cell cycle. A significant reduction in the number of p27+ oligodendrocyte precursors in the transgenic mice supports this conclusion. Taken together, these data suggest that for oligodendrocyte progenitors, ErbB2 signaling plays a role in governing a properly timed exit from the cell cycle during development into myelinating oligodendrocytes.