Localization of viral antigens in various tissues in animals (n = 21) orally-infected with various CCID₅₀^* doses.

<p>Localization of viral antigens in various tissues in animals (n = 21) orally-infected with various CCID₅₀^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0147463#t001fn001" target="_blank">*</a>} doses.</p

A Consistent Orally-Infected Hamster Model for Enterovirus A71 Encephalomyelitis Demonstrates Squamous Lesions in the Paws, Skin and Oral Cavity Reminiscent of Hand-Foot-and-Mouth Disease

<div><p>Enterovirus A71 (EV-A71) causes self-limiting, hand-foot-and-mouth disease (HFMD) that may rarely be complicated by encephalomyelitis. Person-to-person transmission is usually by fecal-oral or oral-oral routes. To study viral replication sites in the oral cavity and other tissues, and to gain further insights into virus shedding and neuropathogenesis, we developed a consistent, orally-infected, 2-week-old hamster model of HFMD and EV-A71 encephalomyelitis. Tissues from orally-infected, 2-week-old hamsters were studied by light microscopy, immunohistochemistry and <i>in situ</i> hybridization to detect viral antigens and RNA, respectively, and by virus titration. Hamsters developed the disease and died after 4–8 days post infection; LD₅₀ was 25 CCID₅₀. Macroscopic cutaneous lesions around the oral cavity and paws were observed. Squamous epithelium in the lip, oral cavity, paw, skin, and esophagus, showed multiple small inflammatory foci around squamous cells that demonstrated viral antigens/RNA. Neurons (brainstem, spinal cord, sensory ganglia), acinar cells (salivary gland, lacrimal gland), lymphoid cells (lymph node, spleen), and muscle fibres (skeletal, cardiac and smooth muscles), liver and gastric epithelium also showed varying amounts of viral antigens/RNA. Intestinal epithelium, Peyer’s patches, thymus, pancreas, lung and kidney were negative. Virus was isolated from oral washes, feces, brain, spinal cord, skeletal muscle, serum, and other tissues. Our animal model should be useful to study squamous epitheliotropism, neuropathogenesis, oral/fecal shedding in EV-A71 infection, person-to-person transmission, and to test anti-viral drugs and vaccines.</p></div

Pathological findings in CNS and non-CNS tissues from EV-A71 infected hamsters at day 4 post infection.

<p>Vacuolation and degeneration in brainstem neurons (A, arrows) that also demonstrated viral antigens (B) and RNA (C, arrows). Viral antigens were also detected in dorsal root ganglion (D, arrows) and spinal cord anterior horn cells (E). Viral antigens in the spleen (F, arrows), salivary gland acinar cells (G, arrows), and lymph node (I and inset, arrows), and viral RNA in the salivary gland acinar cell (H, arrows) were observed. Stains: Hematoxylin and eosin (A), immunohistochemistry with 3, 3’ diaminobenzidinetetrahydrochloride chromogen/hematoxylin (B, D, E, F, G, I), and in situ hybridization with nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate/hematoxylin (C, H). Original magnification: 10x objective (I), 20x objective (B-E), 40x objective (A, F, G, H, I inset). Scale bars: 50μm (I), 30μm (B-E), 15μm (A, F, G, H, I inset).</p

Viral titers in harvested tissues from EV-A71 infected hamsters (experiment 2; 10⁴ CCID₅₀ dose).

<p>Virus titer is expressed as the mean log₁₀ CCID₅₀ ± standard error of mean per 10% tissue homogenates derived from 4 hamsters at day 4 post infection. The highest titters were obtained from serum, hind limb muscle and the CNS. Oral wash viral titer was significantly higher than feces (<i>P</i> = 0.0001).</p

Infected hamster showing hind limb paralysis at day 4 post infection (A, arrow).

<p>Lesions on the lip (B, arrows) and paw (C, arrow) were observed in some animals.</p

Expression of CXCL10 and NiV nucleoprotein (N) genes in organs obtained from NiV-infected hamsters during the course of infection.

<p>Hamsters were sacrificed at different time points after infection and RNA was isolated from different organs and analyzed by RT-qPCR. Grey bars correspond to CXCL10 and black bars to NiV-N, calculated as described in Methods. Significant correlation was found between expression of CXCL10 and expression of NiV N (R² = 0,989, p<0,001, Pearson test).</p

Analysis of expression of interferon-related genes in NiV-infected HUVECs.

<p>A, RT-qPCR analysis of the expression of MXA, OAS1, IFN beta, CXCL11 and CXCL10. B, ELISA analysis of the production of CXCL10 protein in mock-infected (white bars) and NiV-infected (black bars) HUVC cultures. Results are expressed as the average of 2–3 individual experiments and bars represent standard deviation. *p<0.05, **p<0.01, Mann-Whitney U-test.</p

Primary human endothelial cells are highly permissive to NiV-infection.

<p>A, Mock-infected and B, NiV-infected HUVECs (MOI = 1) observed 24 h after infection show extensively developed syncytium formation. C, RT-qPCR analysis of the expression of NiV receptors ephrinB2 (EFNB2) and ephrinB3 (EFNB3) in HUVECs (black bars) and U373 astroglioma cells (white bars). D, Production of RNA specific for NiV genes: nucleoprotein (N), matrix (M), fusion protein (F), glycoprotein (G) and polymerase (L) in HUVECs during infection, determined by RT-qPCR. Fold change is relative to the number of copies of viral mRNAs 8 h or 24 h post infection compared to the number of copies obtained after 1 h of contact with the virus. E, Production of infectious NiV particles during the course of infection; supernatants taken at different time points were titrated on a Vero cell monolayer.</p

Immunohistochemical analysis of CXCL10 production in the brain durng NiV infection.

<p>Non-infected patients (A, ×100) and patients autopsied after lethal acute NiV-infection (B–H) were analysed for the expression of CXCL10 in endothelial cells in cerebral cortex (B, D, ×1000, arrows) and cells with neuronal morphology (E, F). CXCL10 immunoreactivity in perivascular inflammatory cells (C, G, H, ×400).</p

Impact of NiV infection on interferon pathway in HUVEC cells, determined using Ingenuity Pathway Analysis.

<p>NiV-induced upregulated genes are presented grouped, depending the cell compartment in which the corresponding proteins a localized (nucleus, cytoplasm, plasma membrane and extracellular space, for secreted proteins). Level of FC increase is indicated bellow each gene and intensity of red color corresponds to the FC.</p