Expression and purification of enterovirus type 71 polyprotein P1 using Pichia pastoris system

Enterovirus type 71(EV71) causes severe hand-foot-and-mouth disease (HFMD) resulting in hundreds of deaths of children every year; However, currently, there is no effective treatment for EV71. In this study, the EV71 poly-protein (EV71-P1 protein) gene was processed and cloned into the eukaryotic expression vector pPIC9k and then expressed in Pichia pastoris strain GS115. The EV71 P1 protein with a molecular weight of 100 kD was produced and secreted into the medium. The soluble EV71 P1 protein was purified by column chromatography with a recovery efficiency of 70%. The result of the immunological analysis showed that the EV71 P1 protein had excellent immunogenicity and could stimulate the production of EV71-VP1 IgG antibody in injected rabbits. We suggest that EV71-P1 protein is an ideal candidate for an EV71 vaccine to prevent EV71 infection.     

Cell surface sialylation affects binding of enterovirus 71 to rhabdomyosarcoma and neuroblastoma cells

ABSTRACT: BACKGROUND: Enterovirus 71 (EV71) is a major causative agent of hand-foot-and-mouth disease (HFMD), and infection of EV71 to central nerve system (CNS) may result in a high mortality in children less than 2 years old. Although there are two highly glycosylated membrane proteins, SCARB2 and PSGL-1, which have been identified as the cellular and functional receptors of EV71, the role of glycosylation in EV71 infection is still unclear. RESULTS: We demonstrated that the attachment of EV71 to RD and SK-N-SH cells was diminished after the removal of cell surface sialic acids by neuraminidase. Sialic acid specific lectins, MAA and SNA, could compete with EV71 and restrained the binding of EV71 significantly. Preincubation of RD cells with fetuin also reduced the binding of EV71. In addition, we found that SCARB2 was a sialylated glycoprotein and interaction between SCARB2 and EV71 was retarded after desialylation. CONCLUSIONS: In this study, we demonstrated that cell surface sialic acids assist in the attachment of EV71 to host cells. Cell surface sialylation should be a key regulator that facilitates the binding and infection of EV71 to RD and SK-N-SH cells.     

Immunity to avirulent enterovirus 71 and coxsackie A16 virus protects against enterovirus 71 infection in mice

In this study, we sought to determine whether intratypic and intertypic cross-reactivity protected against enterovirus 71 (EV71) infection in a murine infection model. We demonstrate that active immunization of 1-day-old mice with avirulent EV71 strain or coxsackie A16 virus (CA16) by the oral route developed anti-EV71 antibodies with neutralizing activity (1:16 and 1:2, respectively). Splenocytes from both EV71- and CA16-immunized mice proliferated upon EV71 or CA16, but not coxsackie B3 virus (CB3), antigen stimulation. Immunized mice became more resistant to virulent EV71 strain challenge than nonimmunized mice. There was an increase in the percentage of activated splenic T cells and B cells in the immunized mice 2 days after EV71 challenge. The CA16 immune serum reacted with EV71 antigens in an enzyme-linked immunosorbent assay and neutralized EV71 but not CB3 or poliovirus at a titer of 1:4. Passive immunization with the CA16 immune serum reduced the clinical score, diminished the organ viral load, and increased the survival rate of mice upon EV71 challenge. CB3 neither shared in vitro cross-reactivity with EV71 nor provided in vivo protection after both active and passive immunization. These results illustrated that live vaccine is feasible for EV71 and that intertypic cross-reactivity of enteroviruses may provide a way to determine the prevalence of EV71.     

细菌脂多糖对肠道病毒71型感染影响的初步研究

肠道病毒71型(enterovirus 71, EV71)感染常导致婴幼儿手足口病(hand, foot and mouth disease, HFMD),细菌脂多糖(lipopolysaccharide, LPS)在多种肠道病毒感染过程中起重要作用。本研究旨在探讨细菌LPS对EV71感染的影响。将EV71与LPS共孵育,测定病毒被热处理后残留病毒的活力,以及病毒感染过程中病毒基因拷贝数的变化。结果显示,热处理后病毒活力逐渐丧失,而经LPS处理的病毒活力丧失的速度减缓,且残留病毒活力与LPS浓度呈正相关;LPS处理后的病毒在黏附、侵入、胞内复制及释放过程中基因拷贝数较对照组均降低;免疫印迹分析表明LPS与抗VP1单克隆抗体可竞争性结合EV71,且粪便中的EV71可被抗大肠埃希菌抗体识别。上述结果提示,LPS可增强EV71热稳定性,抑制EV71感染过程,且LPS可能与EV71结合。     

Apigenin Inhibits Enterovirus-71 Infection by Disrupting Viral RNA Association with trans-Acting Factors

Flavonoids are widely distributed natural products with broad biological activities. Apigenin is a dietary flavonoid that has recently been demonstrated to interact with heterogeneous nuclear ribonucleoproteins (hnRNPs) and interferes with their RNA editing activity. We investigated whether apigenin possessed antiviral activity against enterovirus-71 (EV71) infection since EV71 infection requires of hnRNP proteins. We found that apigenin selectively blocks EV71 infection by disrupting viral RNA association with hnRNP A1 and A2 proteins. The estimated EC₅₀ value for apigenin to block EV71 infection was determined at 10.3 µM, while the CC₅₀ was estimated at 79.0 µM. The anti-EV71 activity was selective since no activity was detected against several DNA and RNA viruses. Although flavonoids in general share similar structural features, apigenin and kaempferol were among tested compounds with significant activity against EV71 infection. hnRNP proteins function as trans-acting factors regulating EV71 translation. We found that apigenin treatment did not affect EV71-induced nucleocytoplasmic redistribution of hnRNP A1 and A2 proteins. Instead, it prevented EV71 RNA association with hnRNP A1 and A2 proteins. Accordingly, suppression of hnRNP A1 and A2 expression markedly reduced EV71 infection. As a positive sense, single strand RNA virus, EV71 has a type I internal ribosome entry site (IRES) that cooperates with host factors and regulates EV71 translation. The effect of apigenin on EV71 infection was further demonstrated using a bicistronic vector that has the expression of a GFP protein under the control of EV71 5′-UTR. We found that apigenin treatment selectively suppressed the expression of GFP, but not a control gene. In addition to identification of apigenin as an antiviral agent against EV71 infection, this study also exemplifies the significance in antiviral agent discovery by targeting host factors essential for viral replication.     

Enterovirus71 (EV71) Utilise Host microRNAs to Mediate Host Immune System Enhancing Survival during Infection

Hand, Foot and Mouth Disease (HFMD) is a self-limiting viral disease that mainly affects infants and children. In contrast with other HFMD causing enteroviruses, Enterovirus71 (EV71) has commonly been associated with severe clinical manifestation leading to death. Currently, due to a lack in understanding of EV71 pathogenesis, there is no antiviral therapeutics for the treatment of HFMD patients. Therefore the need to better understand the mechanism of EV71 pathogenesis is warranted. We have previously reported a human colorectal adenocarcinoma cell line (HT29) based model to study the pathogenesis of EV71. Using this system, we showed that knockdown of DGCR8, an essential cofactor for microRNAs biogenesis resulted in a reduction of EV71 replication. We also demonstrated that there are miRNAs changes during EV71 pathogenesis and EV71 utilise host miRNAs to attenuate antiviral pathways during infection. Together, data from this study provide critical information on the role of miRNAs during EV71 infection.     

中国EV71病毒VP1蛋白生物信息学分析

以肠道病毒71型(Enterovirus71,EV71)VP1蛋白基因序列为基础,利用生物软件对EV71病毒中国分离株VP1蛋白进行进化树、N-糖基化位点、二级结构及抗原位点的预测和分析。结果显示国内分离株多为C4亚型,有3株湖南分离株为A型,提示疫苗的研发应着重于预防C4b亚型EV71疫苗的研发。     

Cyclophilin A Associates with Enterovirus-71 Virus Capsid and Plays an Essential Role in Viral Infection as an Uncoating Regulator

Viruses utilize host factors for their efficient proliferation. By evaluating the inhibitory effects of compounds in our library, we identified inhibitors of cyclophilin A (CypA), a known immunosuppressor with peptidyl-prolyl cis-trans isomerase activity, can significantly attenuate EV71 proliferation. We demonstrated that CypA played an essential role in EV71 entry and that the RNA interference-mediated reduction of endogenous CypA expression led to decreased EV71 multiplication. We further revealed that CypA directly interacted with and modified the conformation of H-I loop of the VP1 protein in EV71 capsid, and thus regulated the uncoating process of EV71 entry step in a pH-dependent manner. Our results aid in the understanding of how host factors influence EV71 life cycle and provide new potential targets for developing antiviral agents against EV71 infection.     

miR-545 promoted enterovirus 71 replication via directly targeting phosphatase and tensin homolog and tumor necrosis factor receptor-associated factor 6

Enterovirus 71 (EV71) is a small, nonenveloped icosahedral RNA virus and is the predominant causative pathogen of hand-foot-and-mouth disease. Recently, microRNAs (miRNAs) are reported to play important roles in the pathogenesis of EV71 replication. This study investigated the role of miR-545 in the EV71 replication and explored the underlying molecular mechanisms. We showed that miR-545 was upregulated in the EV71-infected human embryonic kidney (HEK) 293 cells and rhabdomyosarcoma (RD) cells. Overexpression of miR-545 promoted the viral replication of EV71 and attenuated the inhibitory effects of EV71 on cell viability in HEK293 and RD cells; while knockdown of miR-545 significantly suppressed the EV71 replication in these two cell lines. Bioinformatics analysis and luciferase reporter assay showed that miR-545 directly targeted the 3′untranslated region of phosphatase and tensin homolog (PTEN) and tumor necrosis factor receptor-associated factor 6 (TRAF6) in HEK293 cells. Furthermore, miR-545 negatively regulated the messenger RNA (mRNA) and protein expression of PTEN and TRAF6. The mRNA and protein expression of PTEN and TRAF6 was also suppressed by EV71 infection, which was attenuated by miR-545 knockdown in HEK293 cells. Overexpression of PTEN and TRAF6 both suppressed the EV71 replication in HKE293 cells, and also attenuated the enhanced effects of miR-545 overexpression on the EV71 replication in HEK293 cells. Collectively, our study for the first time showed that miR-545 had an enhanced effect on the EV71 replication in HEK293 and RD cells. Further mechanistic results indicated that miR-545 promoted EV71 replication at least partly via targeting PTEN and TRAF6.     

Enterovirus 71 disrupts interferon signaling by reducing the level of interferon receptor 1

The recent outbreak of enterovirus 71 (EV71) infected millions of children and caused over 1,000 deaths. To date, neither an effective vaccine nor antiviral treatment is available for EV71 infection. Interferons (IFNs) have been successfully applied to treat patients with hepatitis B and C viral infections for decades but have failed to treat EV71 infections. Here, we provide the evidence that EV71 antagonizes type I IFN signaling by reducing the level of interferon receptor 1 (IFNAR1). We show that the host cells could sense EV71 infection and stimulate IFN-β production. However, the induction of downstream IFN-stimulated genes is inhibited by EV71. Also, only a slight interferon response and antiviral effects could be detected in cells treated with recombinant type I IFNs after EV71 infection. Further studies reveal that EV71 blocks the IFN-mediated phosphorylation of STAT1, STAT2, Jak1, and Tyk2 by reducing IFNAR1. Finally, we identified the 2A protease encoded by EV71 as an antagonist of IFNs and show that the protease activity is required for reducing IFNAR1 levels. Taken together, our study for the first time uncovers a mechanism used by EV71 to antagonize type I IFN signaling and provides new targets for future antiviral strategies.     

Enterovirus 71-induced autophagy increases viral replication and pathogenesis in a suckling mouse model

Background

We previously reported that Enterovirus 71 (EV71) infection activates autophagy, which promotes viral replication both in vitro and in vivo. In the present study we further investigated whether EV71 infection of neuronal SK-N-SH cells induces an autophagic flux. Furthermore, the effects of autophagy on EV71-related pathogenesis and viral load were evaluated after intracranial inoculation of mouse-adapted EV71 (MP4 strain) into 6-day-old ICR suckling mice.

Results

We demonstrated that in EV71-infected SK-N-SH cells, EV71 structural protein VP1 and nonstructural protein 2C co-localized with LC3 and mannose-6-phosphate receptor (MPR, endosome marker) proteins by immunofluorescence staining, indicating amphisome formation. Together with amphisome formation, EV71 induced an autophagic flux, which could be blocked by NH₄Cl (inhibitor of acidification) and vinblastine (inhibitor of fusion), as demonstrated by Western blotting. Suckling mice intracranially inoculated with EV71 showed EV71 VP1 protein expression (representing EV71 infection) in the cerebellum, medulla, and pons by immunohistochemical staining. Accompanied with these infected brain tissues, increased expression of LC3-II protein as well as formation of LC3 aggregates, autophagosomes and amphisomes were detected. Amphisome formation, which was confirmed by colocalization of EV71-VP1 protein or LC3 puncta and the endosome marker protein MPR. Thus, EV71-infected suckling mice (similar to EV71-infected SK-N-SH cells) also show an autophagic flux. The physiopathological parameters of EV71-MP4 infected mice, including body weight loss, disease symptoms, and mortality were increased compared to those of the uninfected mice. We further blocked EV71-induced autophagy with the inhibitor 3-methyladenine (3-MA), which attenuated the disease symptoms and decreased the viral load in the brain tissues of the infected mice.

Conclusions

In this study, we reveal that EV71 infection of suckling mice induces an amphisome formation accompanied with the autophagic flux in the brain tissues. Autophagy induced by EV71 promotes viral replication and EV71-related pathogenesis.     

Characterization of full-length enterovirus 71 strains from severe and mild disease patients in northeastern China

Human enterovirus 71 (EV71)-associated hand, foot, and mouth disease (HFMD) has been a leading cause of childhood infection in China since 2008. Epidemic and molecular characteristics of HFMD have been examined in many areas of China, including the central and southern regions. However, clinical and genetic characterization of EV71 in the northeastern region of China is scarce. In this study, a series of analyses were performed on seven full-length EV71 sequences from HFMD patients who had either severe or mild disease. We have determined that these seven circulating EV71 viruses from Changchun, China are actually complex recombinant viruses involving multiple type A human enterovirus (HEV). Classified as EV71 subtype C4 (EV71 C4), these Changchun EV71 viruses contain genetic recombination events between the CA4, CA5, EV71B4 and EV71C1 strains. Most of the structural protein region (P1) of these viruses resembled that of the prototype EV71 C1 strains. The non-structural protein domains (P2 and P3) showed a high degree of similarity with CA4, CA5 and EV71 B4 in different regions. The 5'UTR had unclassified recombination,while partial 3D region of these viruses showed a high degree of similarity to CA16. Phylogenetic analysis of full-length or partial sequences of isolates from severe or mild disease patients in Changchun always formed a single cluster in various phylogenetic analyses of different genomic regions, suggesting that all seven strains originated from one single common ancestor. There was no correlation between viral genomic sequence and virulence. Thus, we found that circulating recombinant forms of EV71 are prevalent among HFMD patients in Northeastern China. The existence of a unique cluster of EV71 related viruses in Northeast China has important implications for vaccine development that would address the increasing prevalence of HFMD.     

EV71诱导人神经细胞SH-SY5Y自噬的分子机制

【背景】EV71感染所致的重症手足口病易导致神经系统并发症,使患儿预后较差,甚至死亡。【目的】从EV71可诱导神经细胞自噬这一现象出发,探索该病毒诱导神经细胞自噬的miRNA机制,探讨EV71损伤神经细胞可能的分子机制。【方法】通过RT-PCR及Westernblot技术,在感染EV71病毒的人神经母细胞瘤细胞SH-SY5Y中检测细胞自噬变化;通过芯片分析细胞感染前后差异表达的miRNA分子,再使用miRNA mimics调节工具明确与EV71诱导神经细胞自噬有关的miRNA分子。【结果】EV71可诱导SH-SY5Y细胞自噬增加,下调细胞内miRNA29b(miR29b)分子的表达水平;当上调细胞内miR29b的表达后,EV71诱导细胞自噬增加的现象可被逆转,病毒复制水平下降。【结论】EV71诱导神经细胞自噬是通过下调miR29b分子的表达水平实现;miR29b不仅与自噬相关,它与EV71病毒复制也存在密切关系。因此,该研究不仅有助于阐明EV71导致神经系统损伤的具体分子机制,还为miR29b成为治疗EV71感染可能的新药物靶点奠定了理论基础。     

EV71小鼠适应株制备及体内外感染特点

目的用1日龄ICR小鼠传代制备EV71小鼠适应株,研究EV71亲代株与小鼠适应株的体内外感染特点,建立EV71感染ICR小鼠动物模型,为病毒疫苗和抗病毒药物的研究提供实用的动物评价工具。方法用1日龄ICR小鼠进行EV71病毒(Fuyang-0805)的传代,得到小鼠传代株。以一定浓度亲代株和传代株病毒分别接种RD、Vero、SY5Y、Caco-2四种细胞,定量方法检测各时间点不同毒株在四种细胞上的复制数量,CCK8方法测定各时间点细胞的存活率;同时,两毒株分别腹腔注射感染1日龄小鼠,定期安乐死动物,采集肺、小肠、骨骼肌、大脑四种器官组织,进行动物体内病毒半定量和定量分析,同时进行各器官组织病理学观察、免疫组织化学鉴定。结果与亲代毒株相比较,小鼠传代株(EV71-MMP4)表现出更强的肌肉来源细胞嗜性与毒性;同时,两毒株腹腔注射感染1日龄小鼠后,EV71-MMP4感染的小鼠体重增长较正常小鼠体重增长缓慢;半定量和定量RT-PCR显示,在小鼠肌肉中的病毒载量于感染后1d和5d达到高峰。EV71-MMP4感染组感染率较高、病毒组织分布较广、感染持续性较好、病毒载量较高,高剂量病毒感染后小鼠小肠、心肌和骨骼肌可观察到细胞空泡变性、淋巴细胞浸润等病理变化。免疫组织化学显示感染后小鼠骨骼肌有EV71病毒特异分布。结论阜阳EV71小鼠适应株表现出较亲代毒株更好的小鼠易感性、细胞毒性,所建立的动物模型可用于EV71病毒致病机制、感染特点的研究和病毒疫苗及药物的评价。     

Enterovirus 71-Induced Neurological Disorders in Young Gerbils,Meriones unguiculatus: Development and Application of a Neurological Disease Model

A reliable disease model mimicking Enterovirus 71 (EV71) infection in humans is essential for understanding pathogenesis and for developing a safe and effective vaccine. Commonly used rodent models including mouse or rat models are not suitable for vaccine evaluation because the rodents are resistant to EV71 infection after they reach the age of 6 days. In this study, 21-day-old gerbils inoculated intraperitoneally (IP) with a non mouse-adapted EV71 strain developed neurological lesion-related signs including hind limb paralysis, slowness, ataxia and lethargy similar to those of central nervous system (CNS) infection of EV71 in humans. The infected gerbils eventually died of the neurological lesions and EV71 could be isolated from lung, liver, spleen, kidney, heart, spinal cord, brain cortex, brainstem and skeletal muscle. Significantly high virus replication was detected in spinal cord, brainstem and skeletal muscle by cellular analysis, real-time quantitative PCR (RT-PCR) and immunohistochemical staining. Histopathologic changes such as neuronal degeneration, neuronal loss and neuronophagia were observed in spinal cord, brain cortex, brainstem, and skeletal muscle along with necrotizing myositis and splenic atrophy. Gerbils that received two doses of inactive whole-virus vaccine showed no EV71-specific symptoms after challenged with EV71. In contrast, gerbils that received mock vaccination died of EV71-induced neuropathology after challenged with EV71. The result indicates that gerbils can serve as a reliable disease model for evaluating safety and efficacy of EV71 vaccine.     

Caveolar Endocytosis Is Required for Human PSGL-1-Mediated Enterovirus 71 Infection

Enterovirus 71 (EV71) causes hand, foot, and mouth disease and severe neurological disorders in children. Human scavenger receptor class B member 2 (hSCARB2) and P-selectin glycoprotein ligand-1 (PSGL-1) are identified as receptors for EV71. The underling mechanism of PSGL-1-mediated EV71 entry remains unclear. The endocytosis required for EV71 entry were investigated in Jurkat T and mouse L929 cells constitutively expressing human PSGL-1 (PSGL-1-L929) or human rhabdomyosarcoma (RD) cells displaying high SCARB2 but no PSGL-1 by treatment of specific inhibitors or siRNA. We found that disruption of clathrin-dependent endocytosis prevented EV71 infection in RD cells, while there was no influence in Jurkat T and PSGL-1-L929 cells. Disturbing caveolar endocytosis by specific inhibitor or caveolin-1 siRNA in Jurkat T and PSGL-1-L929 cells significantly blocked EV71 infection, whereas it had no effect on EV71 infection in RD cells. Confocal immunofluorescence demonstrated caveola, and EV71 was directly colocalized. pH-dependent endosomal acidification and intact membrane cholesterol were important for EV71 infection, as judged by the pretreatment of inhibitors that abrogated the infection. A receptor-dominated endocytosis of EV71 infection was observed: PSGL-1 initiates caveola-dependent endocytosis and hSCARB2 activates clathrin-dependent endocytosis.     

Enterovirus-71 Virus-Like Particles Induce the Activation and Maturation of Human Monocyte-Derived Dendritic Cells through TLR4 Signaling

Enterovirus 71 (EV71) causes seasonal epidemics of hand-foot-and-mouth disease and has a high mortality rate among young children. We recently demonstrated potent induction of the humoral and cell-mediated immune response in monkeys immunized with EV71 virus-like particles (VLPs), with a morphology resembling that of infectious EV71 virions but not containing a viral genome, which could potentially be safe as a vaccine for EV71. To elucidate the mechanisms through which EV71 VLPs induce cell-mediated immunity, we studied the immunomodulatory effects of EV71 VLPs on human monocyte-derived dendritic cells (DCs), which bind to and incorporate EV71 VLPs. DC treatment with EV71 VLPs enhanced the expression of CD80, CD86, CD83, CD40, CD54, and HLA-DR on the cell surface; increased the production of interleukin (IL)-12 p40, IL-12 p70, and IL-10 by DCs; and suppressed the capacity of DCs for endocytosis. Treatment with EV71 VLPs also enhanced the ability of DCs to stimulate naïve T cells and induced secretion of interferon (IFN)-γ by T cells and Th1 cell responses. Neutralization with antibodies against Toll-like receptor (TLR) 4 suppressed the capacity of EV71 VLPs to induce the production of IL-12 p40, IL-12 p70, and IL-10 by DCs and inhibited EV71 VLPs binding to DCs. Our study findings clarified the important role for TLR4 signaling in DCs in response to EV71 VLPs and showed that EV71 VLPs induced inhibitor of kappaB alpha (IκBα) degradation and nuclear factor of kappaB (NF-κB) activation.     

Enterovirus 71 Virion-Associated Galectin-1 Facilitates Viral Replication and Stability

Enterovirus 71 (EV71) infection causes a myriad of diseases from mild hand-foot-and-mouth disease or herpangina to fatal brain stem encephalitis complicated with pulmonary edema. Several severe EV71 endemics have occurred in Asia-Pacific region, including Taiwan, and have become a serious threat to children’s health. EV71 infection is initiated by the attachment of the virion to the target cell surface. Although this process relies primarily upon interaction between viruses and cell surface receptors, soluble factors may also influence the binding of EV71 to host cells.Galectin-1 has been reported to participate in several virus infections, but is not addressed in EV71. In this study, we found that the serum levels of galectin-1 in EV71-infected children were higher than those in non-infected people. In EV71 infected cells, galectin-1 was found to be associated with the EV71 VP1 and VP3 via carbohydrate residues and subsequently released and bound to another cell surface along with the virus. EV71 propagated from galectin-1 knockdown SK-N-SH cells exhibited lower infectivity in cultured cells and less pathogenicity in mice than the virus propagated from parental cells. In addition, this galectin-1-free EV71 virus was sensitive to high temperature and lost its viability after long-term storage, which could be restored following supplement of recombinant galectin-1. Taken together, our findings uncover a new role of galectin-1 in facilitating EV71 virus infection.