JVI Accepts, published online ahead of print on 20 August 2014 J. Virol. doi:10.1128/JVI.00692-14 Copyright © 2014, American Society for Microbiology. All Rights Reserved.

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Immunodeficient Mouse Models with Different Disease Profiles by in vivo Infection with the Same

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Clinical Isolate of Enterovirus 71

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Running Title: NOD/SCID, stat-1 KO, and ifngr KO mice for EV71

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Authors: Chun-Che Liao1,2,#, An-Ting Liou2,3,# , Ya-Shu Chang2, Szu-Yao Wu2,4 ,

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Chih-Shin Chang2,4, Chien-Kuo Lee5, John T. Kung6, Pang-Hsien Tu2, Ya-Yen Yu7, Chi-Yung

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Lin7, Jen-Shiou Lin7,and Chiaho Shih1, 2,3 *

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Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan

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Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan

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Taiwan International Graduate Program (TIGP) in Molecular Medicine, National Yang-Ming University and Academia Sinica, Taipei, Taiwan

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Institute of Microbiology and Immunology, National Yang-Ming University, Taipei, Taiwan Institute of Immunology, National Taiwan University, Taipei 100, Taiwan

Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan Section of Clinical Virology and Molecular Diagnosis, Department of Laboratory Medicine, Changhua Christian Hospital, Changhua, Taiwan

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*To whom correspondence should be addressed

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#The first two authors made equal contribution to this paper.

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Mailing address: Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan

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Tel: 886-2-2652-3996; Fax: 8862-2652-3597; E-mail: [email protected] 1

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ABSTRACT

Like poliovirus, severe infection with enterovirus 71 (EV71) can cause neuropathology.

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Unlike poliovirus, EV71 is often associated with hand, foot, and mouth disease (HFMD). Here, we

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established three mouse models for experimental infection with the same clinical isolate of EV71. The

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NOD/SCID mouse model is unique in developing skin rash, an HFMD-like symptom. While the

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NOD/SCID model developed limb paralysis and death at near 100% efficiency, the interferon-gamma

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receptor knockout (ifngr KO) and the stat-1 knockout mice exhibited paralysis and death rates near 78%

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and 30%, respectively. Productive infection with EV71 depends on viral dose, host age, and inoculation

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routes. Infectious EV71, and VP1-specific RNA and protein in muscle, brain, and spinal cord, were

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compared side-by-side between NOD/SCID and stat-1 models before, during, and after disease onset.

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Spleen fibrosis and muscle degeneration are common in NOD/SCID and stat-1models. Main differences

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between these two models include their disease manifestations and cytokine/chemokine profiles. Pathology

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of the NOD/SCID model includes 1) inflammation and expression of viral VP1 antigen in muscle; 2)

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increased neutrophils and decreased eosinophils and lymphocytes; 3) hair loss and skin rash. Characteristic

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pathology of the stat-1 model includes 1) a strong tropism of EV71 for central nervous system; 2) detection

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of VP1 protein in the Purkinje layer of cerebellar cortex, pons, brainstem, and spinal cord; 3) amplification

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of microglial cells; 4) dystrophy of intestinal villi. Our comparative studies on these new models by oral or

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intraperitoneal (i.p.) infection underscored the contribution of host immunity, including interferon-gamma

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receptor, to EV71 pathogenesis.

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IMPORTANCE

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Asia-Pacific regions. Disease manifestations include subclinical infection, common cold-like syndromes,

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hand-foot-and-mouth disease (HFMD), uncomplicated brainstem encephalitis, severe dysregulated

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autonomic nerve system, fatal pulmonary edema, and cardiopulmonary collapse. To date, no effective

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vaccine or treatment is available. A user-friendly and widely accessible animal model for researching EV71

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infection and pathogenesis is urgently needed by the global community, both academia and industry.

In the past decade, enterovirus 71 (EV71) has emerged as a major threat to the public health in the

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INTRODUCTION

In the past decade, enterovirus 71 (EV71) has emerged as a major threat to the

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public health in the Asia-Pacific regions (1, 2). As a member of the Picornaviridae, EV71 is related to

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poliovirus (3), hepatitis A virus (EV72) (4), and coxsackieviruses (5). It is likely that both viral and host

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factors could contribute to the diverse pathogenesis profiles, ranging from subclinical infection, common

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cold-like syndromes, hand-foot-and-mouth disease (HFMD), to uncomplicated brainstem encephalitis,

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severe dysregulated autonomic nerve system, fatal pulmonary edema, and cardiopulmonary collapse. To

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date, no effective vaccine or treatment is available (6, 7). A user-friendly and widely accessible animal

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model for EV71 infection will be most convenient to the research community.

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By serial passages through mouse brain, Wang et al. (8) reported experimental infection of newborn ICR

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mice with an EV71 mouse-adapted strain MP4. In general, newborn mice (1-2 day old) are difficult to

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handle technically. A further adapted strain M2 can infect 12-14 day old ICR mice (9). However, it remains

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unclear if adaptive mutations associated with the various mouse-adapted strains can be found in human

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natural infection. For example, a G145E adaptive mutation at the VP1 of a mouse-adapted EV71 strain is

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essential for the hind limb paralysis in 3- to 4-week-old NOD/SCID mice (10). Cynomolgus monkeys could

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also serve as an animal model for studying EV71 pathogenesis (11). However, monkey is not a convenient

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model, and not easily accessible to most laboratories. AG129 mice are known to lack type I and II

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interferon receptors (IFNR), and therefore are severely defective in innate immunity. Khong et al. (12)

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reported that AG129 mice were susceptible to non-mouse-adapted EV71 infection via i.p. and oral routes.

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Infected mice developed limb paralysis and death. An adapted strain of EV71 which can infect adult AG129

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mice has been reported (13). Yamayoshi et al. (14) reported that human Scavenger receptor B2 (SCARB2)

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can function as a receptor for EV71 entry in vitro. Similarly, Nishimura et al. (15) reported that human

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P-selectin glycoprotein ligand-1(PSGL-1) can also facilitate EV71 infection in vitro. Recently, it was 4

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reported that human SCARB2 transgenic (Tg) mice can serve as an infectious animal model of EV71 (16,

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17). While these receptor Tg mice can result in limb paralysis upon i.p. injection with EV71, no typical

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symptoms of HFMD with skin rash were observed. In fact, acute flaccid paralysis clinically is a rare event

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(0.31%) in human EV71 natural infection (1, 18). Furthermore, limb paralysis is not a unique phenotype of

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EV71, many other viruses can also cause limb paralysis in animal models (19, 20). Another important issue

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in the current SCARB2 receptor Tg model is its lack of oral infection (17). While oral infection was

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observed in another SCARB2 Tg model, the efficiency appeared to be very low using a so-called Isehara

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strain of EV71 (success rate only 2/50 in three experiments) (16).Therefore, it is urgent to develop an

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animal model that can closely mimick human natural infection, such as producing the HFMD-like

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phenotype via a more efficient oral infection.

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In literature, the same animal model infected with different genotypes of EV71 was conducted (16, 17). In

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contrast, using the same viral strain for infection of different animal models remains to be explored.

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Considering the outbred human population, the effect of host genetic factors on viral pathogenesis needs to

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be investigated.

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In this study, by using the same clinical isolate of EV71 in three newly established animal models

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susceptible to EV71 infection, we demonstrated that these models with different genetic backgrounds

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exhibited different disease profiles. Interestingly, in the non-obese diabetic mice with severe combined

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immunodeficiency disease (NOD/SCID), the infection and disease manifestations appeared to be more

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systemic in multiple tissues than neurotropic. In particular, spleen fibrosis, muscle inflammation and

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destruction, as well as abnormal hematological features, were evident in the NOD/SCID model. Notably, no

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productive infection and disease were observed when UV-treated EV71 was inoculated. A robust oral 5

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infection system was established using the NOD/SCID mouse model. In contrast, in the stat-1 KO model,

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EV71 infection was highly neurotropic, with striking VP1 expression and massive microglial cell

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amplification in CNS. Both Pons and cerebellar Purkinje cells were strongly targeted by EV71, suggesting a

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retrograde axonal transport mechanism. While the strong neurotropism of EV71 in the stat-1 KO model

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resembles what has been described in patients, our studies on the NOD/SCID model suggests that EV71

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might also cause a systemic infection-like symptoms. Most likely, both muscle and CNS infections could

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contribute to limb paralysis. We report here that skin rash in NOD/SCID mice can be observed by i.p. or

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oral route of inoculation. Furthermore, by using an interferon-gamma receptor knockout mouse model, we

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demonstrated that IFN-gamma receptor is likely to be critical for the protection from EV71 infection and

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pathogenesis. Our new models provide a novel tool for both basic science research and development on

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antivirals and vaccine candidates.

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MATERIALS AND METHODS

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Ethics Statement. All animal experiments were conducted under protocols approved by Academia Sinica

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Institutional Animal Care & Utilization Committee (ASIACUC Protocol number 2010003). Research was

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conducted in compliance with the principles stated in the Guide for the Care and Use of Laboratory Animals,

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National Research Council, 1996.

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Cells and virus preparation. Human rhabdomyosarcoma (RD) cells (ATCC CCL-136) were grown in

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DMEM (Gibco) with 10% of FBS (Hyclone) and 1% of penicillin/streptomycin (Gibco). EV71 clinical

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isolates were kindly provided by Section of Clincial Virology and Molecular Diagnosis, Department of

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Laboratory Medicine, Changhua Christian Hospital. For virus preparation, RD cells were infected with

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EV71 clinical isolate F23 with 0.01 M.O.I and maintained in DMEM with 0.2 % FBS. Virus was harvested

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by three free-thaw cycles and clarified by centrifugation at 3000 ×g for 30 minutes at 4 °C. Virus in

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clarified medium was concentrated by ultracentrifugation in Beckman SW28 rotors at 121896 ×g (26000

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rpm) for 4 hours at 4 °C with 30% sucrose cushion and resuspended in PBS. The virus titer was determined

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by plaque assay before animal infection. In brief, RD cell monolayers in 6-well plates (SPL Life Science)

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were infected with 10-fold serial diluted virus in DMEM. After incubation at 37 °C with 5 % CO2 for 1 hour,

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virus was removed and cells were covered 4ml of 0.3% agarose (Lonza) in DMEM with 0.2% FBS. After

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incubation in 37 °C with 5 % CO2 for 3 days, the cells were fixed by 3.7% formalin (Merk) for 1 hour and

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stained with 0.5% crystal violet. For UV-inactivation experiment, purified EV71 was irradiated by UV at

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750 mJ/cm2 using a CL-1000 Ultraviolet Crosslinker (UVP). The residual viral titer after UV-treatment was

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determined by plaque assay.

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Experimental infection and antibody treatment.NOD/SCID mice were purchased from LASCO Co., Ltd

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(Taiwan). The stat-1 KO mice and ifngr KO (129/Sv/Ev inbred) mice were provided by Dr. Chien-Kuo

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Lee and Dr. John Kung, respectively. Mice were housed under specific pathogen-free condition in the 7

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individual-ventilated cage. One-week-old NOD/SCID or stat-1 KO mice were infected via an

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intraperitoneal (i.p.) or subcutaneous (s.c.) route using an EV71 clinical strain F23 at a dose of 105 to 108

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pfu/mouse. For oral infection, 108 pfu of EV71 was injected into each NOD/SCID mouse (3-day-old) with a

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24-guage feeding tube after 6 hours of fasting. Neutralizing antibodies against IFN-α receptor or

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IFN-γ (Bio X Cell) were i.p. injected at a dose of 150 μg/mouse into one-week old wild type C57BL/6

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mice (NAR Labs, Taiwan). EV71 infection (108 pfu/mouse) was via i.p. route. IFN-α receptor neutralizing

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antibody was injected one day before EV71 inoculation, followed by repetitive antibody treatments on 1

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and 3 dpi. For IFN-γ neutralizing antibody, it was coinjected with EV71, followed by repetitive antibody

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treatments on 1 and 2 dpi. Disease manifestation was monitored daily.

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Histopathological and immunohistochemical (IHC) stain. The euthanatized mice were perfused

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transcardialiy with PBS followed by 10% neutral buffered formalin (CHIN I PAO CO.,LTD, Taiwan).

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Tissues were fixed in 10% neutral buffered formalin overnight. Fixed tissues were paraffin embedded,

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sliced, and stained with H&E or Masson's trichrome stain by Pathology Core Laboratory, IBMS,

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AcademiaSinica. Xylene and ethanol were used for deparaffinization and rehydration. Retrieval buffer pH

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6.0 (Dako) and endogenous enzyme blocker (Dako) were used for antigen retrieval and blocking steps. The

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slides were washed with PBS containing 0.1% Tween 20 (PBST) and incubated with anti-VP1 (Abnova),

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anti-F4/80 (Taiwan GeneTex), anti-Keratin 6, anti-Keratin 14 (Covance), anti-pan-collagen TypeⅠ(Millipore)

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or anti-IBA1 (Taiwan GeneTex) antibodies. These slides were washed with PBST and incubated in

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secondary antibody, anti-rabbit (Dako) or anti-rat (Dako) antibody. DAB system (Dako) was used to present

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signal of each antigen. Sections were counterstained with haematoxylin (J.T. Baker), and mounted with

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mounting reagent (MUTO Pure Chemicals).

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Tissue sampling and virus titration. After euthanasia, blood samples were collected by heart puncture

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prior to PBS perfusion. Organs and tissues were harvested, weighed and homogenized by a mechanical 8

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homogenizer (Next Advance) in PBS. Virus in supernatants of clarified homogenate and blood serum were

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routinely first amplified by passaging the tissue-derived virus through the RD cells for one round (24 hrs)

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before reinfection with RD cells for plaque assay. Viral titer was determined by plaque assay and expressed

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as PFU per gram (for tissue) or per ml (for sera).

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Real time RT-PCR. Total RNA of tissue homogenates were extracted by WelPrep cell/tissue RNA kit

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(Welgene), and used for reverse transcription by High Capacity cDNA Reverse Transcription kit (Applied

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Biosystems). The synthetic cDNA was subjected to real time quantitative PCR analysis by ABI 7500

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(Applied Biosystems) with Power SYBR Green PCR Master Kit (Applied Biosystems). The specific

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primers for VP1: forward: CTAGAGGGTACCACCAATCC; reverse: AACCTGGCCAGTAGGAGT.

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primer sequences of the internal control GAPDH: forward: GTTCCTACCCCCAATGTG; reverse:

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CAACCTGGTCCTCAGTGTAG.

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Cytokine quantification. Cytokines in tissue homogenates were measured by Procarta Immunoassay Kit

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(Affymetrix) according to the manufacturer’s instructions. Detection sensitivity of various cytokines was as

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described in manufacturer’s protocol.

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Complete blood count (CBC). Blood samples from infected mice were collected with 1.5 mg/ml of

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Na2EDTA. The complete blood count of mice were conducted by Abbott Cell-Dyn 3700.

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Immunoblotting. RIPA buffer was used to extract protein from tissue homogenates. VP1 protein and

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tubulin were detected by anti-VP1 antibody (Abnova) and anti-tubulin antibody (Taiwan GeneTex),

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respectively.

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Statistical analysis. All experimental results from two to six independent experiments were analyzed by

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Student’s t-test. Two Way ANOVA was used to analyze the differences of HFMD-like syndromes and limb

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paralysis between oral and non-oral routes of infection.

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The

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RESULTS

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Host immunity plays an important role in resistance and clearance of viral infection. For example, AG129

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mice deficient in interferon (IFN-α/β and -γ) signaling can be infected with EV71 in vivo (12). To address

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the issue of immunity and host defense against EV71, we tested several immunodeficient mouse strains for

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their susceptibility to infection with clinical isolates of EV71 (Fig. 1A). To our surprise, neither IFN

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receptor-alpha knockout mice (ifnar KO) nor the complement C5-deficient FVB mice could support EV71

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infection. Instead, we observed successful infection in stat-1 KO, NOD/SCID, and IFN receptor-gamma

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knockout mice (ifngr KO). Because we succeeded in the ifngr KO model only very recently, our more

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detailed characterizations focused primarily on the other two models. The stat-1 KO mice lacks the STAT1

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gene and is defective in IFN signaling (21), while NOD/SCID mice are known to be defective in innate

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immunity of NK cells as well as humoral immunity of T and B cells (22).

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We i.p. injected EV71 (genotype B5, strain F23, Materials & Methods) into 1-week old NOD/SCID mice,

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and monitored the time course of viral titer in the sera post-inoculation (Fig. 1B). On day 3 post inoculation

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(dpi), viral titer reached a peak, suggesting that EV71 was replicating in the inoculated mice. However, viral

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titer began to decline on day 4, and became undetectable on day 8. We noted occasional (36%) hair loss

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(Fig. 1C) on day 2-6, and skin rash (Fig. 1D, Fig. S1A) after 9 dpi. In contrast, near 100% limb paralysis

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was observed on 8-12 dpi (Fig. 1E, Fig. S1B). These phenomena were not detected in PBS-injected

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NOD/SCID control. To test whether the disease manifestation could be caused by input virus without viral

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proliferation in vivo, we performed UV-inactivation experiment (Materials and Methods). These

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UV-inactivated virus produced neither plaques in RD cells (data not shown) nor death and clinical score in

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NOD/SCID mice inoculated with UV-inactivated virus (Fig. 1F and 1G). This result strongly supports for

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the correlation between viral replication (Fig. 1B) and pathogenesis (Fig. 1F and 1 G). 10

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The survival rate and clinical score were dependent on viral dose (below 106 pfu/mouse vs. 107-108

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pfu/mouse) (Fig. 1H-1I), host age (1 wk vs. 2-4 wk) (Fig. 1J-1K), and injection route (oral, s.c., and i.p.)

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(Fig. 1L-1M).When mice were inoculated at a lower dose of EV71 (below 106 pfu), or at an older age (2-4

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wks), no death and clinical score were ever observed (Fig. 1H-1K).Oral infection appeared to be less

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efficient than infections via i.p. or s.c. route in a highly reproducible manner (Fig. 1N). Therefore, we

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established a reproducible protocol of in vivo infection by i.p., s.c., and oral injection with an EV71 clinical

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isolate (B5 genotype, strain F23) using the NOD/SCID mouse system. The i.p. protocol was used in most of

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the following NOD/SCID studies in this paper.

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Next, we examined the growth kinetics of the inoculated EV71 in different tissues at different time points

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post-inoculation (Fig. 2A-2H). Viral RNA extracted from various tissues was measured by RT-qPCR

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analysis using GAPDH as an internal control, and infectious virus titer was measured by standard plaque

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formation assay using human RD rhabdomyosarcoma cells (Materials and Methods). Interestingly, we

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observed two general phenomena in most tissues:

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tissues examined, and viral RNA tended to decline on day 3 in most tissues, except for kidney and heart. In

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kidney (Fig. 2C), the rapid drop of viral RNA occurred earlier on day 2, instead of day 3. In the heart tissue

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(Fig. 2D), we observed no decline of viral RNA throughout the entire time course. In general, the decline of

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infectivity of viral titer appeared to precede that of viral RNA. We also noted that in brain and muscle (Fig.

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2F and 2G), there were small peaks of viral RNA at 2 dpi. Unlike the tissues of intestine, spleen, and kidney,

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muscle and brain are anatomically most distal to the i.p. injection site. Therefore, the viral RNA titers in

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brain and muscle at 1 dpi were very low. In the first round of “tug of war” between the host defense

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mechanism and the replicating virus in brain and muscle, host defense of the immunodeficient NOD/SCID

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mice enjoyed a transient victory over the invading virus at 2-3 dpi. However, in the second round of “tug of

1) Viral titer tended to decline on day 2 in all eight

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war”, replicating virus began to take off after 3 dpi. Overall, such a “fall-and-rise” pattern of viral titer and

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viral RNA in most tissues at earlier time points post-inoculation, strongly suggests host-attempted clearance

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of the input virus (tissue culture-derived) at the initial phase of viral infection by an unknown host system.

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The following trend of a steady increase of viral titer and RNA post-infection, lends a strong support for in

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vivo EV71 replication in NOD/SCID mice. 2) At the later time points before death (8-11 dpi), infectious

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viral titer dropped to a very low or undetectable level in most tissues, except for spleen (Fig. 2B) and

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muscle (Fig. 2F). In sharp contrast, the viral RNA level at 8-11 dpi reduced only slightly in spleen and brain,

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or remained to be increasing in other tissues. This discrepancy between infectious viral titer and viral RNA

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seems to be paradoxical, and we interpret it as due to the significantly delayed clearance of non-infectious

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viral RNA relative to the more rapid clearance of viral infectivity, as was observed in the earlier phase at

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2-3 dpi. These non-infectious viral RNA could originate from infectious virus being inactivated by the

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released cytokines during inflammation (See Discussion). We speculate here that the amplification of viral

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titer before reaching the point of disease onset could explain for why the kinetics of clearance of viral RNA

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lagged far behind that of viral infectivity. In other words, clearance of non-infectious viral RNA could be

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observed, if the infected NOD/SCID mice could have survived for a longer period.

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We examined sectioned tissues between EV71 infected and mock infected tissues by H&E staining. Most

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apparent pathologies were noted in spleen, muscle, spinal cord and skin (Fig. 3A). In spinal cord, shrunken

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neurons with dark nuclei were consistent with degenerated neurons, as highlighted by black arrows. In

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contrast to the control, the infected muscle was atrophic and irregular in size. In skin section, we noted

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vacuoles (arrowheads in Fig. 3A) in the dermis of infected mice with skin lesions, suggesting inflammation

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related edema. The spleen of infected mice revealed extensive dense collagen bands on H&E stained

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sections, indicative of fibrosis, which was confirmed by the Masson’s trichrome staining as the blue colored 12

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deposits (Fig. 3B). Spleen atrophy was revealed by the reduced size (left panel, Fig. 3C) and weight (right

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panel, Fig. 3C) of the dissected spleens of EV71 infected NOD/SCID mice. EV71-encoded VP1 protein can

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be detected as brown color in spleen, muscle, and skin by immunohistochemical staining (IHC) using an

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anti-VP1 polyclonal antibody (Fig. 3D). We also detected a cell surface marker of infiltrating macrophages

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using anti-F4/80 monoclonal antibody (Fig. 3E). Using antibodies specific for keratin 6, keratin 14, and

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pan-collagen, we performed IHC staining of skin sections, and detected no apparent difference in the

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expression of keratin and collagen between infected mice and mock infected controls (Fig. S2).

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We compared the hematological characteristics between infected mice and mock control by complete blood

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counts (Materials and Methods), and detected no significant difference in platelets and monocytes (data not

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shown), minor difference in MCV (mean cell volume), MCH (mean cell hemoglobin), and RDW (Red

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blood cell volume distribution width), and more significant difference in the numbers of neutrophil,

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eosinophil, and lymphocytes (Fig. 4). This reduction in lymphocytes is consistent with the interpretation

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that EV71 in the NOD/SCID system could bias the differentiation of bone marrow cells toward the myeloid

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over the lymphoid lineages. The significantly increased neutrophil and reduced eosinophil also suggest a

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strong influence of EV71 on the myeloid differentiation pathways toward neutrophil over the eosinophil.

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In addition to the NOD/SCID model, we succeeded in establishing stat-1 KO mice as another model

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susceptible to in vivo infection with a clinical isolate of EV71 (genotype B5, strain F23). Although we did

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not succeed in productive infection using the subcutaneous (s.c.) route, we observed significant clinical

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score by i.p. injection of 108 pfu EV71 into 1-wk old stat-1 KO mice (Fig. 5A). Again, host age is a critical

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factor, since we have observed no productive infection and disease manifestation in 2-wk-old mice.

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Approximately 30% of the infected mice developed limb paralysis around 7 dpi (Fig. 5B), and no death was 13

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observed before sacrifice up to three weeks post-inoculation (data not shown). As shown in Fig. 5C, we

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observed evident pathology on H&E stained sections in EV71 infected mice, including loss of intestinal

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villi, dis-organized muscle structure, and spleen fibrosis. Although there was no significant pathology in

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brain stem and spinal cord by H&E staining (Fig. 5C), strong expression of VP1 protein was observed by

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IHC staining in pons of brain stem, cerebellar Purkinje cells, superior colliculus, and grey matter of spinal

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cord (Fig. 5D, 5E). Unlike the NOD/SCID model (Fig. 3D), we detected no VP1 protein and inflammation

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in muscle of the stat-1 KO model, albeit the muscle exhibited a certain degree of dystrophy by H&E (Fig.

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5C, 5E), which could be a consequence, rather than a cause, of limb paralysis. Injury of neuronal cells is

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often associated with amplification of adjacent microglial cells (23). Indeed, strong expression of IBA1, a

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microglial cell marker, was detected in brain and spinal cord by IHC using an anti-IBA1 antibody (Fig. 5F).

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Elevated cytokine levels are known to be associated with inflammation. Previously, increased levels of

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various combinations of IL-6, IL-10, IP-10, MCP-1, and TNF-αwere detected in serum or tissues in

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EV71-infected mice or humans (12, 17, 24, 25). We examined the tissue cytokine profiles in spleen, muscle,

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brain, and spinal cord in EV71-infected NOD/SCID model, and in brain and spinal cord in the stat-1 KO

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model (Table 1, Fig. S3, Fig. S4). Interestingly, the cytokine profiles, from brain and muscle of

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EV71-infected NOD/SCID mice, exhibited a high degree of similarity to the serum cytokine profile of

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EV71-infected patients with pulmonary edema, such as IFN-γ, IL1-β, IL-10, IP-10, MCP-1, TNF-α

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(muscle), and IL-6 (brain) (25). MCP-1 and IP-10 were reported to be associated with neuronal injury in

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EV71 infected patients with pulmonary edema (25). IP10 might have a certain degree of protective effect,

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since ip-10 KO mice infected with a mouse-adapted EV71 variant displayed more severe disease (26). The

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biological significance of specific cytokines from the NOD/SCID and stat-1 KO models will be discussed

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further in comparison with human patients and other mouse models. 14

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To compare the growth kinetics of EV71 in these two mouse models more closely, we performed EV71

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experimental infection of NOD/SCID and stat-1 KO mice side-by-side, and compared the viral RNA,

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infectivity, and viral protein expression in muscle, brain, and spinal cord, at different time points

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post-infection (before disease onset, onset, and after-onset) (Fig. 6). The viral RNA increased in copy

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number, reached a peak around disease onset, and declined after disease onset in both models (Fig. 6A). In

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contrast to the muscle and brain tissues, spinal cord of the stat-1 KO model contained a higher copy number

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of viral RNA at disease onset. The infectious titers of EV71 in these three tissues were also compared

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between these two models at different time points post-infection (Fig. 6B). Major differences in pfu activity

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between these two models include: 1) at disease onset, no infectious titers could be detected in brain and

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spinal cord in NOD/SCID mice; and 2) after-onset, while infectious EV71 could still be detected in the

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muscle of NOD/SCID mice, no infectivity was detected in the muscle of stat-1 KO mice. We also compared

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viral protein expression between these two models (Fig. 6C). At disease onset, while VP1 protein was

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detected in the muscle of NOD/SCID mice, it was not detectable in stat-1 KO mice (upper panel). In

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contrast, while VP1 protein was detected in the spinal cord of stat-1 KO mice, it was not detectable in

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NOD/SCID mice (bottom panel). No VP1 protein was detected in the brain tissue in both models at disease

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onset (middle panel).In summary, results in Figure 6 support the general notion that the same clinical isolate

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of EV71 exhibited different tissue tropism in two different mouse models –muscle tropism in the

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NOD/SCID model and neurotropism in the stat-1 KO model.

316 317

It is known that the stat-1 KO mice are defective in both IFN-alpha and IFN-gamma signaling (27, 28).

318

Since we did not observe any disease development by infecting the IFN-alpha receptor KO mice (ifnar KO)

319

with EV71 (Fig. 1A), we attempted to block the IFN signaling in vivo by administering antibodies specific 15

320

for both IFN-alpha receptor and IFN-gamma (Materials and Methods). One-week old wild type C57BL/6

321

mice were coinjected once with 150 ug/mouse of each anti-IFN-alpha receptor and anti-IFN-gamma

322

antibodies, one day before inoculation with EV71 (108 pfu/mouse). The antibody treatment was repeated

323

two more times after EV71 inoculation. We observed no disease development in this double antibody

324

experiment (data not shown). As an alternative approach, we tested the effect of IFN by using IFN-gamma

325

receptor knockout mice (ifngr KO) (29). As shown in Figure 7, we observed a more rapid onset of disease

326

and death in this model than those of NOD/SCID and stat-1 KO models (Fig. 7A and 7B). The death and

327

paralysis rates of the ifngr KO model are generally higher than that of the stat-1 KO model (78% vs. 31%).

328

Taken together, IFN-gamma appears to be important for protection against EV71 infection.

329 330 331

DISCUSSION

332

We established here three models for EV71 in vivo infection using NOD/SCID, stat-1 KO, and ifngr KO

333

mice. Since these mice are non-transgenic, and contain no putative human SCARB2 receptor for viral entry

334

(14, 15), it is puzzling that they could support EV71 infection and replication (Fig. 1B, 1F, 1G, Fig. 2). It is

335

possible that mouse SCARB2, which shares 99.9% amino acid sequence similarity with human SCARB2,

336

could serve as a surrogate receptor for viral entry, albeit at a much reduced efficiency (30). In addition, their

337

susceptibility to EV71 infection must be related to their immunodeficiency. NOD/SCID mice are deficient

338

not only in mature T and B cells, but also in NK cells (22). Unlike the stat-1 KO or AG129 mice,

339

NOD/SCID mice are not defective in IFN signaling. The pathogenesis profiles between NOD/SCID and

340

stat-1 KO mice, infected with the same EV71 clinical isolate F23, are very different and complementary to

341

each other (Table S1, Supplemental Material).

342 16

343

In vivo viral replication and tissue tropism in the animal models. The rapid “fall-and-rise” pattern of

344

viral RNA and infectious titer in most tissues in the NOD/SCID model lends a strong support for an active

345

replication of EV71 in vivo (Fig. 2). The clearance of the non-infectious and infectious viral RNAs

346

appeared to lag far behind the clearance of viral infectivity. By comparing the growth curves of the

347

inoculated virus in NOD/SCID mice and stat-1 mice side-by-side, it became clear that the viral RNA titer

348

reached the peak at the point of disease onset in both models (Fig. 6A). In contrast to the active viral growth

349

in Fig. 2 and 6, UV-inactivated virus produced no disease and clinical score in the NOD/SCID mice (Fig.

350

1F and 1G). Taken together, these results strongly suggest in vivo synthesis of viral RNA genome and

351

infectious virus titer before the disease onset. The presence of infectious virus titer in the muscle at 11 dpi

352

(Fig. 2F) and the strong presence of VP1 antigen by Western blot at disease onset (8 dpi) (Fig. 6C), favors

353

the notion of muscle-tropism of EV71 in the NOD/SCID model. In contrast, the detection of VP1 in the

354

brain and spinal cord by IHC (Fig. 5D and 5E) and in the spinal cord by Western blot (Fig. 6C), favors the

355

notion of neurotropism of EV71 in the stat-1 model.

356 357

HFMD-like skin rash. As shown in Table 2, we compared the characteristics of several currently available

358

mouse models for EV71 infection, including ICR mice or IP-10 knockout mice, infected with

359

mouse-adapted EV71 variants (8, 26), SCARB2 Tg mice(16, 17), and interferon-deficient AG129(12). To

360

our knowledge, the NOD/SCID system is the only model that mimicks cutaneous lesion or skin rash of

361

human HFMD (Table2), which was found in about 70% of EV71 infected children with disease

362

manifestation (1, 2, 31, 32). No skin rash was observed in previous mouse studies (8, 16, 17, 33, 34), or the

363

cynomolgusmonkey model (11, 35).In one hSCARB2 Tg model, hair loss, but no skin rash, was observed

364

(17). In our current NOD/SCID model, both hair loss and skin rash were detected in 36 % of mice

365

inoculated i.p., and 14% of mice via oral infection (Fig. 1N).We observed VP1 expression by IHC in hair 17

366

follicles (Fig. 3D), and edema-like vacuoles in dermis (Fig. 3A) in EV71 i.p. infected NOD/SCID model.

367 368

Spleen fibrosis, identified by the Masson’s trichrome staining, is common in both NOD/SCID andstat-1

369

KO mouse models (Fig. 3B, Fig. 5C). However, only spleen atrophy (2, 12, 36, 37), but no spleen fibrosis,

370

has ever been reported in mouse models and humans. Whether spleen fibrosis can be detected in human

371

natural infection deserves further investigation in the future.

372 373

Limb paralysis due to myositis or CNS involvement? EV71 infection presents two major clinical

374

features: HFMD and neurological disorder (2). In AG129 mouse model and hSCARB2 Tg model, viral

375

protein and injury were found in both limb muscle and CNS (12, 17). It is therefore difficult to definitively

376

sort out whether limb paralysis is primarily caused by muscle or CNS injury.As discussed below, we

377

addressed this issueby using the same virus strain in two complementary models of NOD/SCID and stat-1

378

KO mice.

379 380

In the stat-1 KO model, we observed limb paralysis (30%), as well as strong expression of viral VP1

381

antigen (Fig. 5D, 5E) and inflammation (microglial IBA1 protein in Fig. 5F), only in the brain stem and

382

spinal cord, but not in the limb muscle (Fig. 5C and 5E). In this regard, EV71 in thestat-1 KO model is

383

highly neurotropic than muscle-tropic, and limb paralysis is primarily caused by neurological disease. In

384

contrast, in the NOD/SCID model, we obtained no direct evidence for neurotropism of EV71. Although

385

near 100% of infected NOD/SCID mice developed limb paralysis, we detected no VP1 in the CNS of

386

infected NOD/SCID mice by IHC on 4, 8, and 12 dpi (data not shown). In addition, infectious virus

387

appeared to have been cleared from CNS before 8 dpi (Fig. 2G, Fig. 2H). Given the massive destruction and

388

strong expression of VP1 in muscle (Fig. 3A, Fig. 3D), but not in the CNS (data not shown), we interpreted 18

389

here that limb paralysis in the NOD/SCID model could be caused mainly by a muscle, rather than

390

neurological problem. Taken together, limb paralysis could be caused by either muscle or neurological

391

injury, depending on the immunogenetic background of the host animals.

392 393

Viral spread pathways. Based on the IHC staining of VP1 in the stat-1 KO model, VP1 was strongly

394

expressed in all levels of spinal cord, including anterior horn and all areas of grey matter (Fig. 5E). In

395

addition, we detected VP1 in pons of brain stem (Fig. 5D), but not in the cerebral cortex (data not shown).

396

Taken together, this result is in support for the retrograde axonal transport from peripheral nerves to spinal

397

cord ascending to CNS, in a manner similar to other enterovirus systems, including EV71 mouse-adapted

398

strains(34, 38, 39), and poliovirus(40). On the other hand, we also detected VP1 expression in a small area

399

of superior colliculus of mid-brain (Fig. 5D), which is not anatomically or directly connected with spinal

400

cord. IL-17, as detected in Table 1, has been reported to influence the permeability of the blood brain barrier

401

(41). It cannot be excluded that EV71 could somehow pass through the blood brain barrier, perhaps at a

402

lower efficiency, in infected mice.

Future investigation is needed to further address this issue.

403 404

Pulmonary edema. Pulmonary edema is associated with high mortality rate in EV71-infected patients with

405

brainstem encephalitis (31, 42-44). In cases of bulbar poliomyelitis, fatal pulmonary edema was also

406

reported (45). So far, no pulmonary edema has ever been observed in any EV71 animal models (12, 16, 17,

407

33), including our own NOD/SCID and stat-1 KO mice (data not shown). While pulmonary edema was

408

generally believed to be neurogenic, it cannot be excluded whether pulmonary failure could be contributed

409

in part by myositis involved in lung respiration. As discussed earlier, we observed no apparent injury or

410

viral protein in the CNS of our NOD/SCID model (data not shown); however, massive muscle damage is

411

evident (Fig. 3A), a phenomenon similar to a recent report on pulmonary hypoventilation in 2-week-old 19

412

ICR mice infected with a mouse-adapted strain of EV71 (46).

413 414

Oral infection via a natural enteric route. Inoculation via the oral route with mouse-adapted strains of

415

EV71 can result in productive infection and disease in mice (8, 33). In the human SCARB2 Tg mouse

416

model, neurological signs can be obtained when inoculated via the i.p. route. However, when inoculated via

417

the intragastric (i.g.) route at 107 TCID50, only one out of twenty (1/20) mice showed neurological signs

418

(16) . In a separate experiment using 108 TCID50, no incidence of neurological signs (0/10) was observed.

419

The reason for the very low efficiency of oral infection with EV71 in the hSCARB2 Tg model (16) remains

420

unclear. Using the IFN-α receptor KO model, we obtained no productive infection via the oral route either

421

(Fig. 1A). However, recently, we succeeded reproducibly in oral infection in the NOD/SCID mouse model

422

(Fig. 1L, 1M, 1N, Table 2).Similarly, AG129 mice defective in both IFN-α/β and IFN-γ signaling, can be

423

infected with EV71 via the oral route (12). While we succeeded in establishing an efficient in vivo infection

424

via the i.p. route using the ifngr KO model (Fig. 7), it remains unclear if we can establish oral infection

425

using this ifngr KO model. The potential role of IFN signaling in EV71 clearance and pathogenesis remains

426

to be elucidated in the future (47).

427 428

Cytokine profiles and infiltrating macrophages. We noted the striking similarity of cytokine profiles

429

between the NOD/SCID model (muscle, brain, and spinal cord) and patients (serum and cerebrospinal

430

fluid), including IFN-γ, IL-10, IP-10, IL-6, MCP-1, IL-1β, and TNF-α (25) (Table 1). Therefore, the

431

NOD/SCID model, despite its immunodeficiency, could serve as an animal model resembling human

432

EV71 infection, at least as far as cytokine profiles are concerned. In the AG129 model (12), serum IL-1β

433

was induced by EV71 only via the oral route, but not the i.p. route. In contrast, in our studies via the i.p.

434

route, we detected IL-1β in brain and muscle of the NOD/SCID model. Furthermore, it is interesting to 20

435

note that IL-17F was detected in the spinal cord of both EV71-infected NOD/SCID and stat-1 KO models

436

(Table 1). IL-17 cytokines, including IL-17F, are known to be involved in neuroinflammation in

437

experimental autoimmune encephalomyelitis (EAE) (48). We also detected IL-23 in the spinal cord of the

438

NOD/SCID model (Table 1). IL-23, a member of the IL-12 family of cytokines, can promote Th17 cell

439

development. The IL-23/IL-17 axis is well known to play an important role in various inflammatory

440

diseases (49). Finally, the fact that we detected macrophage inflammation protein 2 (MIP2) in CNS and

441

muscle (Table 1), as well as F4/80 in the muscle of NOD/SCID mice (Fig. 3E), suggests that infiltrating

442

macrophages could contribute to inflammation and cytokine release in this model.

443 444

Interferon-gamma signaling and viral infection. IFN-gamma is known to exercise pleiotropic effects on

445

the immune system. In addition to its antiviral effect, IFN-gamma plays an immunomodulatory role in

446

macrophages, dendritic cells, and lymphoid cells (50-52). Mice without IFN-gamma receptor exhibited

447

increased susceptibility to infection with Listeria monocytogenes, vaccinia virus (29), lymphocytic

448

choriomeningitis virus (LCMV) (53-55), herpes simplex virus type 1 (HSV-1) (51), and dengue

449

infection-induced paralysis (56). However, IFN-gamma and its receptor do not seem to be important for

450

infection with vesicular stomatitis virus (VSV) (55, 57, 58). In the case of EV71, we demonstrated here a

451

protective role of IFN-gamma receptor to EV71-induced paralysis and death (Fig. 7).

452 453

As shown in Table 1, we detected IFN-gamma in muscle, brain, and spinal cord of NOD/SCID mice as well

454

as in the muscle of the stat-1 mice. The absence of IFN-gamma in the spinal cord of the stat-1 mice is

455

consistent with the strong presence of VP1 antigen in the spinal cord by Western blot analysis (lower panel,

456

Fig. 6C). Similarly, the presence of IFN-gamma in the brain and spinal cord of the NOD/SCID mice (Table

457

1) could explain its absence of detectable viral antigen and pathology in the brain and spinal cord (Fig. 6C). 21

458

Although IFN-gamma can be detected in muscle of the NOD/SCID mice, viral RNA, viral protein, and

459

infectious titer can be detected in the muscle of NOD/SCID mice at disease onset (Fig. 6).It is possible that

460

the existing level of IFN-gamma is too low to clear the virus effectively.

461 462

It remains somewhat puzzling that the stat-1KO mice, defective in both IFN-alpha/beta and IFN-gamma

463

signalings, had a paralysis rate around 30% and no death; while the ifngr KO mice, defective in only the

464

IFN-gamma, but not IFN-alpha, displayed a paralysis and death rate near 78%.Because of the difference in

465

their respective genetic backgrounds (C57BL/6 for ifnar KO vs. 129/Sv/Ev for ifngr KO), it is impossible to

466

compare these two mouse systems at present. Another issue that may be worth discussion here is that, we

467

did not succeed in inducing paralysis in our anti-IFN-gamma antibody experiment (data not shown).

468

However, ifngr KO mice displayed paralysis upon infection with EV71. It is possible that the blockage of a

469

cytokine ligand may not have the same phenotype as the blockage of a cytokine receptor. For example,

470

there appeared to be no difference in the infection with LCMV between IFN-gamma KO mice and wild

471

type mice (59). However, increased susceptibility to LCMV infection was reported by using ifngr KO mice

472

(55).

473 474

Age-dependent susceptibility. In humans, young age is a major risk factor for natural infection with EV71

475

(1, 2).

476

and death. For example, in our NOD/SCID and stat-1 KO models, we routinely i.p. inoculated

477

one-week-old mice for most studies. In other models, mice younger than two or three weeks old were

478

susceptible to EV71 infection via various routes and viral strains (12, 16, 17). Similarly, in one poliovirus

479

receptor (PVR) transgenic model, 2-week-old mice were four orders of magnitude more susceptible to

480

paralysis than adult mice (60). It remains to be investigated what kind of viral or host factors could

In animal models, younger age is also a crucial determinant for productive infection, pathogenesis,

22

481

contribute to the age dependency, such as the internal ribosome entry site (61), and the neonatal

482

development of the nerve, gut, and/or immune systems.

483 484

Potential for translational research. One last question to be addressed here is whether these

485

immunodeficient mice are adequate for EV71 vaccine research? Our answer is positive since these models

486

can still provide an in vivo platform for evaluation of vaccine efficacy by passive transfer of neutralizing

487

antibodies. Such antibodies can be induced by vaccination in immuno-competent animals or humans.

488

Finally, these user-friendly mouse models can be infected with clinical isolates of EV71, and thus provide

489

an opportunity for in vivo testing of anti-virals.

490 491

Acknowledgments.We thank the service by the Pathology Core Laboratory, Institute of Biomedical

492

Sciences (IBMS), Academia Sinica, Taiwan. This work is supported by Academia Sinica and National

493

Science Council, Taiwan.

494

23

495

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FIGURE LEGENDS

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Figure. 1

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mice infected with a clinical isolate of EV71 strain F23 (genotype B5). (A) Comparisons among various

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immune-deficient mouse models infected with EV71 via different routes (*limb paralysis rate about 78%;

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** limb paralysis rate about 31.3%). (B) One-week-old NOD/SCID mice were infected with 108 pfu of

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EV71 strain F23. Serum samples were collected from a group of five mice on day 1, 2, 3, 4, 8, and 11,

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respectively. Virus titer in serum was measured by plaque assay. One-week-old NOD/SCID mice were

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infected with EV71 strain F23 (left panel) or normal saline (right panel). Disease manifestations, including

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hair loss (C), skin rash (D), limb paralysis (E), and death, were monitored daily. Comparisons were made

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between different parameters of infection, including with or without UV treatment (F and G), different viral

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dose (105~108 pfu) (Hand I), host age (1~4weeks) (J and K), and inoculation route (i.p., s.c., or oral) (L and

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M). (N) A summary of disease manifestations in NOD/SCID mice infected with EV71 through different

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viral doses and routes. Clinical scores were defined as follows: 0: Healthy; 1: Hair loss, wasting, or ruffled

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hair; 2: Limb weakness; 3: Only one-limb paralysis; 4: 2-4 limb paralysis; 5: Death. Error bars indicate the

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standard deviations (sd) in 6 mice of each group.

Studies on various parameters which affect disease manifestations of immunodeficient

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Figure 2. A “fall-and-rise” pattern of viral RNA and infectious titer of EV71 at earlier time points

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post-inoculation in various tissues of EV71-infected NOD/SCID mice. One-week-old NOD/SCID mice

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were infected i.p. with 108 pfu of EV71 strain F23. On different days post-infection, mice were sacrificed.

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Viral RNA (left panel) and virus titer (right panel) of intestine(A), spleen (B), kidney (C), heart (D), lung

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(E), muscle (F), brain (G), and spinal cord (H) were determined by quantitative RT-PCR and plaque assay.

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Viral RNA was detected by VP1 primers and normalized by GAPDH primers. Virus titer in each tissue was

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normalized by the gram weight of dissected tissues. These dash lines in various graphs in (A) to (H) were 32

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drawn by connecting the average points of data at each time point. (see text for further discussion) *:

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indicating the time point of disease onset.

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Figure 3. Pathological changes of EV71-infected NOD/SCID mice. One-week-old NOD/SCID mice

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were infected i.p. with 108 pfu of EV71 (left panel) or normal saline (right panel). Moribund mice were

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sacrificed on day 12 post-infection. The paraffin sections were examined at 200X with H&E stain (A),

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Masson’s stain (B), anti-EV71 VP1 (D), and anti-F4/80 (E). (A) Multiple blue stained cells in muscle could

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represent infiltrated macrophages (see Fig. 3E below). The black arrows in spinal cord showed degenerated

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neurons. The arrowheads in skin and hair follicle highlight vacuous edema at 400X magnification. (B) The

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blue color showed collagen deposits and spleen fibrosis. (C) Spleen atrophy was observed on day 11

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post-infection (left panel). The weights of each dissected spleen from infected mice were measured and

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normalized with hearts (right panel). (D) IHC staining detected VP1 protein in spleen, muscle, and skin hair

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follicles. (E) Muscle-infiltrated macrophages can be visualized using anti-F4/80 antibody by IHC. The

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statistics was presented by mean ± standard deviation (sd) in 6 mice of each group. **p<0.01 by Student’s

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t-test compared with saline control.

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Figure 4. Hematological changes of NOD/SCID mice i.p. infected with EV71 (108 pfu) or normal

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saline. Blood was collected on day 11 post-infection. Complete blood counts were recorded by Abbott

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Cell-Dyn 3700. (MCV: Mean cell volume; MCH: Mean cell hemoglobin; RDW: Red blood cell volume

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distribution width). The statistics was presented by mean ± standard deviation (sd) in 6 mice of each group.

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*p < 0.05; **p

Immunodeficient mouse models with different disease profiles by in vivo infection with the same clinical isolate of enterovirus 71.

Like poliovirus infection, severe infection with enterovirus 71 (EV71) can cause neuropathology. Unlike poliovirus, EV71 is often associated with hand...
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