The effect of enterovirus 71 immunization on neuropathogenesis and protein expression profiles in the thalamus of infected rhesus neonates

Virology 432 (2012) 417–426

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The effect of enterovirus 71 immunization on neuropathogenesis and protein expression profiles in the thalamus of infected rhesus neonates Huicheng Chen a,b,1, Ying Zhang a,1, Erxia Yang a, Longding Liu a, Yanchun Che a, Jingjing Wang a, Hongling Zhao a, Donghong Tang a, Chenghong Dong a, Lixian Yang a, Dong Shen a, Xi Wang a, Yun Liao a, Lichun Wang a, Ruixiong Na a, Yan Liang a, Qihan Li a,n a Institute of Medical Biology, Chinese Academy of Medical Science and Peking Union Medical College, Yunnan Key Laboratory of Vaccine Research and Development on Severe Infectious Diseases, Kunming, Yunnan 650118, PR China b School of Life Sciences, Yunnan University, Kunming 650091, PR China

a r t i c l e i n f o

abstract

Article history: Received 29 March 2012 Returned to author for revisions 22 May 2012 Accepted 29 June 2012 Available online 21 July 2012

Enterovirus 71 (EV71) is a major pathogen that causes hand–foot–mouth disease (HFMD). Our previous studies have demonstrated that the complete process of pathogenesis, which may include tissue damage induced by host inflammatory responses and direct tissue damage caused by viral infection, can be observed in the central nervous system (CNS) of animals infected in the laboratory with EV71. Based on these observations, the neuropathogenesis and protein expression profiles in the thalamic tissues of EV71-infected animals were further analyzed in the present study. Changes in protein expression profiles following immunization with the inactivated EV71 vaccine followed by virus challenge were observed and evaluated, and their physiological roles in viral pathogenesis are discussed. Taken together, the results of these experiments provide evidence regarding the neuropathogenesis and molecular mechanisms associated with EV71 infection and identify several protein indicators of pathogenic changes during viral infection. Crown Copyright & 2012 Published by Elsevier Inc. All rights reserved.

Keywords: Enterovirus 71 (EV71) Expression profiles Inactivated virus vaccines Neuropathogenesis Neonatal rhesus monkeys Thalamus

Introduction In recent years, studies on infectious diseases in children have called attention to enterovirus 71 (EV71), which is the major pathogen that causes hand–foot–mouth disease (HFMD) throughout the world, especially in regions of Asia (McMinn, 2002; Ooi et al., 2007). This focus is not only because of the recent HFMD epidemics in mainland China and other locations in Asia (Chan et al., 2000; Shimizu et al., 1999; Yang et al., 2009) but also because of the fatal cases associated with EV71-induced neuropathogenesis during these epidemics (Chen et al., 2007; Ho et al., 1999; McMinn, 2002). At present, based on a preliminary pathological study of clinical cases of EV71 infection (Wong et al., 2008), a consensus has been reached that this viral infection might cause serious pathological changes in the central nervous system (CNS) of a small number of individuals. Infants are especially at risk for developing CNS pathology that causes neurogenic pulmonary edema, which is the major cause of death

n Correspondence to: Institute of Medical Biology, Chinese Academy of Medicine Science and Peking Union Medical College, 935 Jiaoling Road, Kunming, Yunnan 650118, PR China. Fax: þ 86 871 8334483. E-mail address: [email protected] (Q. Li). 1 Huicheng Chen and Ying Zhang contributed equally to this work.

associated with HFMD (Hsueh et al., 2000; Lin et al., 2006; Wong et al., 2008). However, due to a lack of adequate human clinical pathological data, the pathological processes and mechanisms of EV71 infection remain elusive (Huang et al., 2009). Therefore, it is important to conduct further analyses of the pathogenesis of this disease, especially its neuropathogenesis, from the histopathology of infected organs to the molecular mechanisms of viral infection. In previous studies by our group and others, pathological analyses of EV71 infection in primates have revealed that the complete process of EV71 pathogenesis can be observed in the CNS of animals, such as cynomolgus monkeys and rhesus monkey infants, that are infected with the virus in the laboratory (Liu et al., 2011; Nagata et al., 2004; Zhang et al., 2011). Additionally, some pathological and etiological indicators have demonstrated that this infectious process can be validated (Liu et al., 2011; Zhang et al., 2011). These results provide an understanding of the pathological mechanisms underlying EV71 infection from a histological perspective. Based on the data obtained in these primate studies, it might be possible to use these established animal models to evaluate EV71 vaccines or therapeutic drugs with respect to histopathology, immunology and pharmacology (Liu et al., 2011). However, the indicators obtained in previous studies are a general index and only show changes in the histopathology, etiology and immunology associated

0042-6822/$ - see front matter Crown Copyright & 2012 Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.virol.2012.06.026

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with EV71 infection, which may not reflect the physical changes and related molecular mechanisms involved in the process of infection. The results from recent studies of severe HFMD cases caused by EV71 infection indicate that in addition to tissue damage directly caused by viral infection of the CNS, tissue damage may also occur due to host inflammatory responses (Lin et al., 2003; Wang et al., 2003; Wong et al., 2008). In our previous studies, experiments on EV71-infected rhesus monkey neonates demonstrated that the virus can infect animals via the respiratory tract and cause common clinical manifestations, including blister-like lesions on the oral mucosa and skin (Liu et al., 2011). Additionally, an increase in the body temperature of these animals was accompanied by the emergence of viremia and pathological changes in the CNS, among other effects (Liu et al., 2011; Zhang et al., 2011). These pathological features have been used to specifically evaluate the effectiveness of and the protection provided by the inactivated EV71 vaccine (Dong et al., 2011,2010). Based on the dynamic analysis of these features, 2D protein electrophoresis was used to evaluate the changes in protein expression in the thalamic tissues of rhesus monkey neonates that were immunized with the inactivated EV71 vaccine prior to viral challenge. Dynamic changes in protein expression were observed in these animals in comparison with the thalamic tissues from unimmunized animals that were directly infected with the virus. Based on our interpretation of the changes in the expression of these identified proteins, further evaluation of the response of immunized animals to the challenge with live virus was conducted in this study.

during days 7–10 p.i. in the main organs and tissues of the monkeys (Liu et al., 2011), which might indicate that viral loads in the organs and tissues may be used as indicators of EV71 infection. In this study, the viral loads in tissues of the CNS, including cerebrospinal fluid (CSF) collected from the VI and UI groups, were measured to evaluate the ability of the inactivated EV71 vaccine to protect the CNS of infected monkeys. The qRTPCR data and direct virus titers in tissue culture were similar. No virus was detected in the CNS tissue samples collected from the VI group (Fig. 2a). In contrast, peak viral titers were observed 10 day p.i. in most tissues of the CNS, including the cerebrum, thalamus and cerebellum, except in the CSF that was collected from the UI group. The peak viral titer in the spinal cord was observed at day 7 p.i. and was maintained until day 10 p.i. (Fig. 2a). Furthermore, qRT-PCR amplification demonstrated that the viral RNA copies in the samples from the VI group were lower than 100 (Fig. 2b). The medulla oblongata from the UI group had higher than 106 viral RNA copies (Fig. 2b), and the viral peak coincided with the viral titers that were determined in tissue culture. These results and previous reports (Liu et al., 2011; Nagata et al., 2004) indicate that the CNS is a primary target organ of EV71 viral proliferation in neonatal monkeys. Observations made using immunohistochemical detection with EV71-specific antibodies in the various CNS tissues further support these findings (Fig. S2). The viral loads detected in the CNS of the samples from the VI group, which had been immunized with the inactivated EV71 vaccine, clearly indicate that the immune response induced by vaccination has the capacity to inhibit viral proliferation in the CNS.

Results

The detection of antibodies and inflammatory factors in the cerebrospinal fluid of VI and UI monkeys

Pathological analysis of CNS tissues from neonatal monkeys in the VI and UI groups As previously demonstrated by pathological data from the fatal human cases and animals infected with EV71, the majority of the pathological lesions in the CNS induced by EV71 infection are found in the thalamus, pons and spinal cord (Liu et al., 2011; Wong et al., 2008; Zhang et al., 2011). The lesions observed in these regions could contribute to neurogenic pulmonary edema (Lum et al., 1998). Therefore, most of our work focused on the pathological features of the tissues, specifically the thalamus, pons and spinal cord, to observe the efficacy of the inactivated EV71 vaccine and to ultimately define these three sites as those potentially critical for the evaluation of candidate vaccines (Dong et al., 2011). In our comparative study, no significant pathological changes were observed in the VI group, except for a few cases of inflammatory cell infiltration in some parts of the thalamus, pons and spinal cord. However, changes were observed in the UI group, such as typical inflammatory cell aggregation, vascular cuffing and a small amount of neuronal cell degeneration and necrosis (Fig. 1). Similar pathological changes to those observed in the thalamus, pons and spinal cord were also observed during synchronized observations of other tissues of the CNS and major organs (Fig. S1). These results are in accordance with the previous work on other target organs (Dong et al., 2011). Furthermore, the pathological changes were found to be most pronounced on day 10 post-infection (p.i.) and began to disappear slightly on day 14 p.i. (Fig. 1). These pathological changes might therefore be used as defined pathological markers of encephalitis. Virus distribution in the CNS tissues of neonatal monkeys in the VI and UI groups Our previous studies on EV71-infected neonatal rhesus monkeys demonstrated that the highest viral loads were detectable

Clinical descriptions of fatal cases of EV71 infection have suggested that the pathological lesions in the CNS can progress rapidly to pulmonary edema and respiratory failure (Liu et al., 2000; Nolan et al., 2003). This type of acute pathogenic progress might be caused by a severe inflammatory reaction mechanism, such as a ‘‘cytokine storm’’ (Lin et al., 2002; Wang et al., 2003). In studies evaluating the efficacy and safety of candidate EV71 vaccines, the levels of inflammatory factors are commonly considered to be one type of potential indicator, especially in the CSF (Dong et al., 2011; Liu et al., 2011). However, based on reports about antibody-dependent enhanced infection (ADE) by EV71 (Han et al., 2011; Wang et al., 2010) and antibody-mediated cytotoxicity (Ch’ng et al., 2011), levels of specific antibodies against EV71 in the cerebrospinal fluid were measured, and the titer was compared to that in peripheral blood. The results demonstrated that the levels of the IL-2, IL-4, IL-5, IL-6, TNF-a and IFN-g in the cerebrospinal fluid of the monkeys in the VI group did not increase significantly or fluctuate and were maintained within the normal range (Fig. 3a). In contrast, the levels of the majority of these inflammatory factors, especially IL-6 and TNF-a, tended to increase in the cerebrospinal fluid of the monkeys in the UI group, which was demonstrated by higher observed levels of IL-6 during disease progression (Fig. 3). These factors were sustained at levels that were significantly higher than normal from days 7 to 14 p.i. The highest values were observed on day 10 and then gradually declined. However, measurement of anti-EV71 antibodies using the neutralizing test failed to detect antibodies in the cerebrospinal fluid of either group (Fig. S3). In contrast, an extensive increase in antibodies in the peripheral blood of the VI group and a slight increase in the UI group at the same time point were observed (Fig. S3). The higher levels of cytokines in the cerebrospinal fluid are similar to those reported from the studies of other animals infected with EV71 as

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well as the test results from some EV71-infected patients (Chang et al., 2006; Lin et al., 2002,2003).

2D electrophoresis of the thalamic tissues of VI and UI monkeys To better understand the molecular biological features of the pathological changes in the CNS induced by EV71 infection and to link them to the mechanisms underlying the protective effect generated by the inactivated EV71 vaccine, 2D electrophoresis was performed to compare the thalamic tissues of the 3 neonatal monkey groups. Before the first phase of isoelectric focusing (IEF), the loaded gel samples from these 3 groups were subjected to total protein quantification using the PlusOne 2D Quant kit. An antibody to GAPDH was used for Western blotting as a

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reference internal control and it displayed equal intensities when analyzed using conventional processes (Fig. S4a). The 2D gels were then analyzed using an image scanner (Epson EU-88) (Fig. S4b). The spots of upregulated or downregulated protein molecules were identified in the 2D gels of the samples taken from the VI and UI groups based on the background of the UU group. After the identification of the protein spots by MALDI-TOF/TOF mass spectrometry analysis, peptide scores less than 80 were regarded as significantly differentially expressed molecules (p o0.05). In total, the profiles of 47 regular protein molecules, not including viral proteins, were obtained (Table S2). We identified changes in the expression of 9 molecules involved in signal transduction, 8 cellular structure proteins, 13 molecules involved in cellular metabolism, 4 macromolecular biosynthesis-related molecules, 10 molecules involved in energy metabolism and bio-oxidation, 2 molecules involved in the ubiquitin-proteasome pathway and

Fig. 1. Pathological changes in the thalamus, pons and spinal cord of vaccine-immunized or unimmunized neonatal monkeys infected with EV71. In the vaccinated group (VI), monkeys immunized with 160 EU of the inactivated vaccine were inoculated with 104.5 TCID50/100 ml of EV71 (FY-23 strain). In the unvaccinated group (UI), the monkeys were injected with PBS and inoculated with 104.5 TCID50/100 ml of EV71 (FY-23 strain). The control group monkeys were uninfected and unimmunized (UU). The tissue (thalamus, pons and spinal cord) sections were stained with hematoxylin and eosin (H&E) and imaged using an optical microscope with original magnifications of 100x (thalamus and spinal cord; bar, 60 mm) and 200x (pons; bar, 30 mm). The infiltration of inflammatory cells (black arrow), vascular cuffing (green arrow) and neuron retrogression (yellow arrow) are shown. Samples were collected at day 10 and day 14 p.i. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article).

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1 stress response protein in the thalamic tissues of monkeys from the VI and UI groups (Table S2).

The protein expression profile in the thalami of the VI and UI monkeys Prior to investigating the specific pathological features in the thalami of neonatal monkeys infected with EV71, we further explored the expression profiles of proteins identified in several functional groups that may have been potentially generated by these pathological features. The results demonstrated a 1- to 5-fold fluctuation in the levels of the majority of the molecules, and values greater than 5-fold were observed for some individual molecules (Fig. 4a). Among the signal transmission molecules that are closely correlated with viral infection, the expression level of neuronal protein NP2 (TAGLN3, a member of the calmodulin protein family) was upregulated in the thalami of UI monkeys at day 10 p.i. and returned to normal levels at day 14 p.i. (Fig. 4b). Conversely, the expression profile of this protein was unchanged in the VI group (Fig. 4b). The expression profile of another protein, galectin-1 (LGALS1, a member of the galactosidase-binding protein family), was normal in the thalami of UI monkeys at day 10 p.i. and was downregulated at day 14 p.i. However, galectin-1 levels were downregulated in the VI group at day 10 and day 14 p.i. (Fig. 4b). With respect to the proteins associated with cellular structures, the expression profile of the stathmin-like protein (STMN1, a member of the microtubule-associated protein family) was upregulated in the UI group at day 10 p.i. and downregulated at day 14 p.i., whereas it was downregulated in the VI group day 10 and day 14 p.i. (Fig. 4c). Interestingly, the expression level of ubiquitin carboxyl terminal hydrolase (UCHL1) doubled in the UI group at day 10 and day 14 p.i. but increased slightly in the VI

group at day 10 p.i. and was clearly downregulated below normal control levels at day 14 p.i. (Fig. 4g). Notably, the expression profile of the diazepam-binding inhibitory protein (DBI, also known as benzodiazepine receptor inhibitory peptide or acylcoenzyme A-binding protein) was upregulated by more than 3-fold in the UI group at day 10 and day 14 p.i. but was at normal levels in the VI group at the same time points (Fig. 4d). Similar expression profiles were found for the other functional groups (Fig. 4). Quantification of protein transcript levels by RT-PCR To quantify the transcript levels of the above proteins in the thalami of monkeys in the VI and UI groups, RT-PCR was performed using primers designed based on the mRNA sequences of 2–3 genes that encode proteins in each functional group. A total of 18 genes, including the PARK7 and TAGLN3 genes from the group of signal transmission variations and the GMFB, STMN1 and MAP1B genes from the cellular structure protein group, were selected for primer design. The RNA extracted from the thalamic tissues from each monkey group was used as template. The results are shown in Fig. 5. The transcript levels quantified by RT-PCR amplification had the same tendency as those observed in the thalami of the UU monkeys (Fig. 5), which confirms the results from our 2D electrophoresis analysis.

Discussion In studies analyzing fatal cases of human EV71 infection and viral animal models, the mechanisms underlying the pathogenic process, which is frequently characterized by pathological changes in the CNS and partial lung failure correlated with encephalitis, remain to be elucidated (Kung et al., 2007; Lin

Fig. 2. Dynamic viral distribution in CNS of vaccine-immunized or unimmunized monkeys that were infected with EV71. (a) Titration of the virus in the CNS tissues of the vaccinated (VI) and unvaccinated (UI) groups. Viral titer was measured using a microtitration assay according to the procedure described in the Materials and Methods section. (b) RT-PCR amplification of viral copies in the vaccinated (VI) and unvaccinated (UI) groups. Viral copies were quantified according to in vitro-synthesized RNA by spectrophotometry, and the quantity was expressed as a relative copy number, which was determined by the equation [(mg of RNA/ml)/(molecular weight)]  Avogadro’s number¼ viral copy number/ml. Tissue specimens were collected from organs of the animals in the groups described above on days 4, 7, 10 and 14 p.i. Values are the mean for 2 samples at each time point.

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Fig. 3. Inflammatory cytokines in the cerebrospinal fluid of the vaccine-immunized or unimmunized neonatal monkeys that were infected with EV71. In the vaccinated group (VI), monkeys immunized with 160 EU of inactivated vaccine were inoculated with 104.5 TCID50/100 ml of EV71 (FY-23 strain). In the unvaccinated group (UI), monkeys were injected with PBS and inoculated with 104.5 TCID50/100 ml of EV71 (FY-23 strain). The concentrations of the proinflammatory cytokines (IL-2, IL-4, IL-5, IL-6, TNF-a and IFN-g) were analyzed according to the procedure described in the Materials and Methods section. The normal ranges (data collected from two monkeys from the uninfected and unimmunized (UU) group) are shown as a dotted line. np o 0.05 indicates a significant difference between the UI group and VI group at the same time point. nnp o0.01 indicates remarkable significance.

et al., 2002). Infection-induced pathological changes remain the predominant cause of death associated with HFMD in infants and young children infected with EV71 (Huang et al., 2006; Zimmerman, 1999). Theoretically, the potential reasons include tissue damage resulting from viral proliferation, a pathological immune response or ‘‘cytokine storm’’ inflammatory reaction induced by viral infection, or other unknown reasons (Lin et al., 2002; Wang et al., 2003). Therefore, investigation and characterization of the mechanisms that cause pathological changes in the CNS during EV71 infection would not only provide an understanding of the pathogenesis of EV71 infection but, more importantly, would also provide detailed criteria for evaluating the efficacy of inactivated EV71 vaccine candidates based on an understanding of the characteristics of the pathological changes. Therefore, our studies highlight important pathological changes in the CNS of EV71-infected neonatal monkeys, including dynamic profiles of virus distribution and proliferation as well as host inflammatory reaction status. Additionally, we determined whether the immune response induced by the inactivated EV71 vaccine has the capacity to protect tissues in the CNS from damage caused by viral infection and inflammatory reactions. Additionally, to determine the molecular mechanisms underlying neuropathological changes induced by EV71 infection and the potential for evaluating candidate inactivated EV71 vaccines, the protein expression profiles in the thalami from EV71-infected and vaccine-immunized monkeys were analyzed using a 2D protein assay. The molecular features of EV71 pathogenesis that are described here represent preliminary data that provide insight into the details of the protection conferred by immunization. The clinical pathological examination of fatal cases of EV71 infection shows the features typical of an inflammatory reaction (Wong et al., 2008). In our previous work, we described similar histopathological changes, which are frequently found in the CNS of EV71-infected neonatal monkeys in the thalamus, pons and medulla oblongata as well as part of the cerebrum and spinal cord

(Fig. 1). The distribution and variety of such pathological changes may correlate with virus distribution and proliferation (Fig. 2) (Liu et al., 2011). Such a correlation suggests that a biological feature of viral infection may elicit direct pathological changes in the CNS. In parallel, the fact that we failed to detect the typical pathological changes and an absence of virus in the CNS of inactivated EV71 vaccine-immunized monkeys (Figs. 1 and 2) supports the above conclusion and demonstrates the efficacy of our candidate vaccine. Additionally, the analysis of the cerebrospinal fluid from EV71-infected monkeys indicates that the levels of the associated cytokines, such as IL-6, IFN-g and TNF-a, tend to increase throughout the infectious process (Fig. 3), which is consistent with the reports on EV71 infection in humans (Lin et al., 2002). However, the cause of the pathological changes in the CNS requires further clarification. The normal levels of cytokines in the cerebrospinal fluid of vaccine-immunized monkeys support the efficacy of our candidate vaccine. In addition, this study failed to detect EV71-neutralizing antibodies in the cerebrospinal fluid of EV71-infected monkeys and vaccine-immunized monkeys (Fig. S3), which implies a lack of ADE in the CNS of EV71-infected and vaccine-immunized monkeys. Nevertheless, these results are not sufficient to determine the cause of EV71mediated pathogenesis and the molecular mechanism of the candidate vaccine. Our study further compared the protein expression profiles of thalamic tissues of EV71-infected and vaccine-immunized monkeys using a 2D protein assay. The results demonstrated that the pathogenic process was associated with altered expression profiles of at least 47 proteins (Table S2), and the profiles of these proteins might have specific significance. For example, the highly conserved PARK7 protein, which interacts with PIAS (protein inhibitor of activated STAT, a member of the SUMO-1 ligase family), is encoded by the dj-1 gene and was identified in the group of molecules that regulate transcription (Takahashi et al., 2001). PARK7, in association with other proteins such as p54nrb,

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Fig. 4. Protein expression profile in the thalami of vaccine-immunized or unimmunized neonatal monkeys infected with EV71. (a) Proteins exhibiting a change in expression levels in the thalamic tissues of vaccine-immunized (VI), unimmunized (UI) and normal (UU, uninfected and unimmunized) neonatal monkeys. Monkeys in the VI group were immunized with 160 EU of inactivated vaccine and inoculated with 104.5 TCID50/100 ml of EV71 (FY-23 strain). Monkeys in the UI group were injected with PBS and inoculated with 104.5 TCID50/100 ml of EV71 (FY-23 strain). A matrix of 47 proteins at days 10 and 14 p.i, as analyzed using the Resolver analysis system (Rosetta Biosoftware, Seattle, WA, USA), is shown. The fold change in expression levels is relative to the vaccinated/unvaccinated/normal samples and is displayed in red (increased expression) or green (decreased expression). The number in the top left corner indicates the representative cluster. (b–h) The fold change in the expression of proteins within individual clusters. The dotted line indicates no change in expression (Y¼0). (b) Cluster 1, signal transduction proteins. (c) Cluster 2, cellular structural proteins. (d) Cluster 3, cellular metabolism-related proteins. (e) Cluster 4, macromolecular biosynthesis-related proteins. (f) Cluster 5, energy metabolism and bio-oxidation-related proteins. (g) Cluster 6, ubiquitin-proteasome pathway-related proteins. (h) Cluster 7, stress response proteins. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article).

has also been found to concomitantly defend against oxidative stress and necrocytosis resulting from mutations in a-synuclein (Abou-Sleiman et al., 2006). Mutation of this protein is associated with Parkinson’s disease, which suggests that the protein may exert functional protection on the neuronal cells in the CNS in response to viral infection (Bandopadhyay et al., 2004; Inden et al., 2006; Yanagida et al., 2006). This conclusion is supported by the observed upregulation of this protein in the thalamic tissues of EV71-infected animals. In contrast, the slight upregulation of PARK7 in the thalami of VI animals most likely indicates that the vaccine can effectively prevent pathological damage to neuronal cells upon viral infection (Fig. 4). This result is consistent with the data regarding NP25 (TAGLN3) and galectin-1 (LGALS1), which function positively in the transcriptional regulation of neuronal cell proliferation, differentiation and restoration (Camby et al., 2006; Ren et al., 1994) (Fig. 4). Additionally, the expression profiles of other proteins associated with the maintenance and recovery of neuronal cell function were found to have a similar tendency. For example, ubiquitin carboxyl-terminal hydrolase 1

(UCHL1) plays a major role in the maintenance of the normal function of neurons at the cellular level via binding to monoubiquitin in neurons (Liu et al., 2002). The upregulated expression of UCHL1 and DBI observed in the thalamic tissues of UI monkeys may indicate a protective effect on neuronal cells in response to viral infection, whereas the normal expression of UCHL1 and DBI (Table S2) in the thalami of the VI monkeys further emphasized the protective effect induced by immunization. Additionally, the upregulation of enzymes associated with cellular metabolism in the UI monkeys and their downregulation in the VI monkeys indicate that the pathological and physiological effects observed in the CNS of EV71 infected animals should not be solely attributed to the histopathological lesions generated by viral replication. In fact, upregulated expression of another specific protein, cyclophilin A, in the UI monkeys (Table S2, Fig. 4) suggests that the inflammatory response to viral infection is likely directly linked to the histopathological lesions in the CNS. The primary drug target of phosphoamidase repression, cyclophilin A, is an essential molecule that generates granulocyte,

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Fig. 5. Gene expression in the thalami of vaccine-immunized or unimmunized neonatal monkeys following infection with EV71. Real-time RT-PCR analysis of 18 selected genes expressed in the thalami of the vaccine-immunized or unimmunized neonatal monkeys at days 10 and 14 p.i. with EV71 strain FY-23. The results were normalized to the level of endogenous GAPDH. The y-axis indicates the relative quantity of the specific mRNA in the samples compared with the normal samples (UU monkeys, y¼0). Error bars indicate the SD of the relative quantities. np o 0.05 indicates a significant difference between the UI group and VI group at the same time point. nnp o 0.01 indicates remarkable significance. Cluster 1, signal transduction proteins (PARKT, TAGLN3, DDAH2). Cluster 2, cellular structural proteins (GMFB, STMN1, MAP1B). Cluster 3, cellular metabolism-related proteins (PGAM1, FABP5, DBI, DPYSL3, PHPT1). Cluster 4, macromolecular biosynthesis-related proteins (APRT, GABARAPL2). Cluster 5, energy metabolism and bio-oxidation-related proteins (HBA1, HSD17B10). Cluster 6, ubiquitin-proteasome pathway-related proteins (UCHL1, UBE2N). Cluster 7, stress response proteins (CRYAB).

eosinocyte and monocyte chemotaxis in response to inflammatory reactions (Arora et al., 2005). Based on the above mentioned speculation, the specific biological features of cyclophilin A and the upregulated expression of inflammatory factors in the cerebrospinal fluid, especially IL-6 and TNF-a (Fig. 3), it is reasonable to conclude that the inflammatory reactions associated with EV71 infection might cause the histopathological lesions in the CNS to some extent. This model is further supported by the normal

expression levels of cyclophilin A in vaccine-immunized neonatal monkeys. In summary, based on the above data and, in particular, the molecular biological features of these differentially expressed proteins, we hypothesize that the immune response induced by the inactivated EV71 vaccine could repress the histopathological effects in the CNS and subsequently reduce the inflammatory damage to cells. Similarly, the data also indicate that the

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histopathological lesions in the CNS that were previously reported to be induced by EV71 infection are correlated with inflammatory reactions as well as direct viral infection.

Competing interests The authors declare that they have no competing interests.

Materials and methods Viruses and cells The EV71 virus strain FY-23 was used in this study; it was isolated from hematic secretions from the respiratory tract of an infected male child who presented with clinical symptoms of severe cardiopulmonary collapse during an epidemic in Fuyang, China, in May 2008 (GenBank accession number: EU812515.1) (Ma et al., 2009). Using RT-PCR, the strain was identified as an EV71 C4 sub-genotype virus prior to Vero cell inoculation (Zhang et al., 2011). The viruses that were grown in Vero cells were harvested and frozen at 20 1C when the typical cytopathic effects developed and when the virus reached a concentration of 107.0–107.5 cell culture infectious doses (CCID50)/ml. Human diploid cells (KMB-17, human embryo lung cell) were maintained in DMEM (Hyclone, Logan, UT, USA) with 5% foetal bovine serum (Gibco, Grand Island, NY, USA) and grown to a confluent monolayer (Dong et al., 2010; Guo et al., 1981; Yang et al., 2006). Vero cells were maintained in DMEM with 5% foetal bovine serum.

Virus titration and neutralisation assay EV71 virus that had been harvested from cell culture or isolated from different organs and tissues of infected newborn rhesus monkeys were analysed by performing a microtitration assay using a standard protocol (Arita et al., 2006). The neutralisation test for the virus was performed as previously described (Liu et al., 2011).

Neonatal rhesus monkeys The animal work was conducted according to relevant national and international guidelines. In accordance with the recommendations of the Weatherall report, ‘‘The use of non-human primates in research’’, the following section includes the details of the animals’ welfare and the steps that were taken to ameliorate suffering in all work involving non-human primates. The Office of Laboratory Animal Management of Yunnan Province, China, approved the experimental procedures that were performed on the animals (approval number: SCXK (Dian) 2005-0005). A total of 10 1.5-month-old neonatal rhesus monkeys, 5 males and 5 females, were divided into the following 3 groups: 4 monkeys were given the vaccine-immunisation followed by a virus challenge (immunised and infected, VI), 4 monkeys were directly challenged with the virus as a control group (unimmunised and infected, UI), and 2 monkeys were used as normal control group (unimmunised and uninfected, UU). Each newborn monkey was maintained in a single cage with its mother and fed according to the guidelines established by the Committee of Experimental Animals at the Institute of Medical Biology, Chinese Academy of Medical Sciences (CAMS) (Gorska, 2000). All animals were held in isolation for 2 weeks before the initiation of the study. The inactivated EV71 vaccine (prepared from KMB-17 cells; 160 EU/ dose, EU: ELISA unit (Xie et al., 2010)) was inoculated intramuscularly into the right hind leg of neonatal rhesus monkeys at weeks 0 (to immunise) and 4 (to boost). The infection of neonatal rhesus monkeys The monkeys in the VI and UI groups were challenged with the live virus from the FY-23 strain (104.5 CCID50/dose) with an aerosol formulation via the respiratory tract 4 weeks after boosting (Liu et al., 2011). The monkeys in the UU group were treated with phosphate-buffered saline (PBS). The monkeys were examined daily for clinical manifestations. We collected 0.5 ml of cerebrospinal fluid from the anesthetised monkeys on days 0, 4, 7, 10 and 14. Two monkeys in each group were euthanised on days 10 and 14 for histopathological, biochemical and etiological examinations of various organs and tissues (Liu et al., 2011). Fresh thalamus tissues were obtained and washed with PBS to remove the blood. Biochemical tests of the cerebrospinal fluids

Inactivated EV71 virus vaccines The FY-23KB viral clone was obtained from the FY-23 strain through passaging 5 times and twice plaque selecting in human diploid cell (KMB-17 strain) (Dong et al., 2010). After identification and characterisation of the strain using biological, genetic and immunological analyses, aliquots of EV71 and FY-23KB were inoculated into KMB-17 monolayer cells as previously described (Dong et al., 2011). Briefly, the harvested viruses were centrifuged and inactivated with 1/4000 formalin before incubation at 37 1C for 96 h. The viruses were subsequently sampled at 4-h intervals. After inactivation, the virus was concentrated 200-fold by ultrafiltration (QuixStand Benchtop System, GE Healthcare, Milwaukee, WI, USA). Purification was performed on a Sepharose 6-FF column (GE Healthcare, Milwaukee, WI, USA). The purified inactivated virus was used to measure antigen content via an enzyme-linked immunosorbent assay (ELISA) (Xie et al., 2010). A solution of the inactivated virus was then emulsified in 1 mg/ ml Al(OH)3 adjuvant, which constituted the final vaccine. All of the vaccine samples were released for follow-up investigations after a quality control evaluation had been performed. The evaluation included an examination of the efficiency of viral inactivation and the vaccine antigen content.

To obtain CSF samples, the monkeys were anesthetised with ketamine hydrochloride (15 mg/kg), and a 0.5-ml sample of CSF was collected from each monkey by lumbar puncture within 25 min of the ketamine injection using a 1-cc syringe. The samples were centrifuged at 1000 g at 4 1C within 1 h to remove the cells and stored at 80 1C until further use. The concentrations of the proinflammatory cytokines (IL-2, IL-4, IL-5, IL-6, TNFa and IFN-g) were analysed using an ELISA kit (BD Biosciences, San Diego, CA, USA) according to the manufacturer’s instructions. The absorbance was measured with a microplate reader (BioTek Synergy 4, BioTek Instruments, Winooski, VT, USA). Histopathological and immunohistochemical analysis The tissue samples from the infected monkeys were fixed in 10% formalin, dehydrated through ethanol gradients and embedded in paraffin, as previously described (Liu et al., 2011). For histopathological analysis, 4-mm sections were cut for hematoxylin and eosin (H&E) staining. For the immunohistochemical analysis, 4-mm sections were cut for the antigen–antibody reaction, as described previously (Liu et al., 2011). The EV71 antigen was detected by mouse anti-enterovirus 71 monoclonal Antibody

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(Clone 10F0, Abcam, U.S.) and horseradish peroxidase (HRP)conjugated anti-mouse IgG antibodies (Sigma, Deisenhofen, Germany). The analysis of the tissue sections was performed using an optical microscope (Nikon, Tokyo, Japan). 2D protein electrophoresis analysis The tissue samples were subjected to repeated freeze–thaw cycles and then resuspended in 2D lysis buffer (8 M urea, 2 M thiourea, 4% CHAPS, 100 mM DTT, 2% IPG buffer). Quantification of total protein was performed using the PlusOne 2D Quant kit (GE Healthcare Europe GmbH, Freiburg, Germany) according to the manufacturer’s instructions. Approximately 200 mg of protein ¨ was precipitated according to the Wessel–Flugge method (Wessel and Flugge, 1984) and resuspended in 200 ml rehydration buffer (8 M urea, 2 M thiourea, 2% CHAPS, 100 mM DTT, 2% IPG buffer). The resuspended proteins were first separated based on their pI using 24 cm immobilised linear gradient strips (ImmobilineTM DryStrip, Amersham Biosciences Europe GmbH, Freiburg, Germany) covering a pH range of 3–10. The strips were rehydrated for 12 h and subsequently subjected to the following high voltages at 20 1C for electric focusing: 100 V for 5 h, a 500 V gradient for 1 h, a 1000 V gradient for 1 h, an 8000 V gradient for 2 h and an 8000 V to a total of 60,000 Vh. After equilibrating for 20 min in equilibration buffer (6 M urea, 30% glycerin, 2% SDS, 50 mM Tris pH 8.8, 100 mM DTT) and alkylation for an additional 20 min in the same solution containing 250 mM iodoacetamide, the proteins were separated by 12% SDS-PAGE. The spots were visualised using mass spec-compatible silver staining. To confirm the reproducibility and exclude the experimental variations in the 2D-PAGE assays and to detect the qualitative and quantitative changes in the obtained protein spots, the experiments were repeated three times.

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Western blotting The protein samples were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes (PVDF; Millipore Corporation, Bedford, MA, USA). Western blot analysis was performed using standard procedures (Shaw et al., 1992). Monoclonal antibodies recognising GAPDH were obtained from Sigma (Sigma, St. Louis, MO, USA). Extraction of total RNA and quantitative RT-PCR amplification Total RNA was extracted from fresh tissue homogenate from the experimental animals using a Qiagen RNeasy Mini Kit according to the manufacturer’s protocol (Qiagen, Hilden, Germany). The differential expression of 18 genes, which were selected based on the 2D gel electrophoresis analysis, was validated by real-time quantitative RT-PCR using SYBR Green-based detection (Table S1). The total RNA was eluted in a final volume of 20 ml. For quantification, a single-tube RT-PCR assay was performed using the TaqMan 1-step RT-PCR Master Mix in a 7500 Fast Real-time RT-PCR system (Applied Biosystems, Foster City, CA, USA). The following protocol was used for all PCR assays: 5 min at 42 1C and 10 s at 95 1C, followed by 40 cycles at 95 1C for 5 s and 60 1C for 30 s. The transcript of the conserved housekeeping gene GAPDH was used to normalise the samples because its expression level is relatively constant. Statistical analysis The data are expressed as the mean and the standard deviation. The statistical significance between the treatment groups was calculated using a two-way ANOVA (GraphPad Prism. GraphPad Software, San Diego, CA, USA), and a P value of o0.05 was considered to be significant.

Acknowledgments Mass spectrometry analysis The protein patterns on the gels were recorded as digitalised images using a high-resolution scanner (ImageScanner III EU-88, GE Healthcare, USA). The scanned images were analysed using Image Master 2D Platinum 7.0 software (GE Healthcare, USA). The differentially expressed proteins identified in each thalamus sample were compared to the normal thalamus samples (UU group). The protein spots of interest were excised from the silverstained 2D gels using an Ettan Spot Picker (GE Healthcare, Fairfield, USA). The proteins were destained with destaining solution containing 15 mM K3Fe(CN)6 and 50 mM NaS2O3. Subsequently, the proteins were digested with trypsin, which cleaves peptides on the carboxylic side of the arginine and lysine residues. Following digestion, the tryptic peptides were extracted from the gel pieces with 50% acetonitrile/0.1% TFA, concentrated nearly to dryness in a SpeedVac vacuum centrifuge and diluted to a total volume of 5 ml with 1% TFA. The 1 ml aliquots of the samples were analysed using the 4800 Plus MALDI-TOF/TOF system (matrixassisted laser desorption ionisation tandem time-of-flight; Applied Biosystems, Foster City, CA, USA). The positive ion mode was used to automatically collect data with a PMF scan extent of 800–3500 Da. The obtained MS and MS/MS spectra data were integrated and subjected to searches against the NCBInr-human database using the GPS 3.6 (Applied Biosystems, Foster City, CA, USA) and Mascot 2.1 (Matrix Science, http://www.matrixscience. com) databases. The algorithm was set to use trypsin as the enzyme, which permits a maximum of one missed cleavage site as well as assumes that carbamidomethyl is a fixed modification of cysteine and oxidised methionine and that the deamidation of asparagine and glutamine are variable modifications. The mass tolerance was set to 0.15 Da for MS and 0.25 Da for MS/MS.

This work was supported by the National Basic Research Program (2011CB504903 and 2012CB518901), National Natural Sciences Foundation of China (81171573 and 31100127), the Yunnan Natural Science Foundation (2011FB116) and the Grant of Basic Research of CASMS (2010IPB109).

Appendix A. Supplementary Information Supplementary data associated with this article can be found in the online version at doi:10.1016/j.virol.2012.06.026.

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