C4亚型人肠道病毒71型的小鼠肌肉适应株对小鼠具有致死毒力

目的人肠道病毒71型（EV71）感染婴幼儿可导致手足口病,偶尔伴随有严重的并发症。建立EV71的小鼠模型是深入研究病毒感染的致病机制、进行疫苗和药物评价的基础。方法将EV71的临床分离株在小鼠骨骼肌中进行系列传代,得到了小鼠的肌肉适应株MP10。使用MP10经腹腔注射感染10日龄ICR小鼠,对感染小鼠的症状、病毒复制和病理进行了监测。结果与临床分离株相比,MP10对人横纹肌瘤细胞（rhabdomyosarcoma cell,RD细胞）的感染力提高,并且对小鼠的毒力增强,MP10感染10日龄小鼠可导致其瘫痪和死亡。病毒主要在骨骼肌中复制,并引发大面积的坏死性肌炎,同时神经组织未观察到病变。病理分析发现：感染小鼠体内与呼吸运动相关的肌肉均发生严重的坏死性肌炎,推测此类肌肉的坏死会影响小鼠的呼吸功能,从而成为导致小鼠死亡的因素之一。结论建立了C4亚型EV71毒株感染10日龄ICR小鼠的模型,该模型可作为研究EV71感染的致病机制、以及疫苗和药物评价的工具。     

肠道病毒71型（EV71）对ICR小鼠的感染

目的研究肠道病毒71型经不同途径感染不同日龄ICR小鼠后的感染状况,了解肠道病毒71型的感染特点,为了解EV71小鼠感染机制和模型制备提供实验信息和技术支撑。方法分别通过口腔途径、颅腔途径、肌肉途径及腹腔途径感染1日龄、7日龄及3~4周龄SPF级ICR小鼠,定期安乐动物,采集各器官组织进行病原学诊断,确定EV71病毒感染情况;同时建立一步RT-PCR、病毒分离、IFA及IEA等方法。结果经腹腔途径感染成年鼠出现竖毛、弓背、消瘦症状,其他各途径感染小鼠感染后未见竖毛、弓背、觅食减少、体重减轻、精神呆滞及神经系统症状。颅腔注射3~4周龄ICR小鼠能在脑组织检测到病毒RNA,腹腔注射和肌肉注射1日龄乳鼠能在肌肉组织和肠道检测到病毒RNA,其中,肌肉组织病毒分离可检测到活病毒。本研究同时建立了分子生物学、血清学方法,为今后研究其它适合EV71的动物模型奠定了基础。结论临床分离的EV71毒株通过口腔接种、颅腔、肌肉、腹腔注射途径感染1日龄、7日龄及3~4周龄SPF级ICR小鼠的疾病程度和病毒检出不同,ICR乳鼠及成年鼠可作为该病毒感染机制、病毒体内分布等基础研究,但用作EV71动物模型应用,感染程度尚不十分理想。     

EV71不同临床株的新生小鼠毒力研究

目的探索不同EV71毒株在新生鼠的毒力。方法临床轻症、重症和死亡来源的EV71毒株,接种1日龄BALB/c乳鼠,观察14 d小鼠的症状,于感染后1、3、5、7 d和9 d,取小鼠组织RT-QPCR测病毒载量,HE染色观察病变。结果重症株与轻症株症状严重程度差异无显著性(P=0.693),且两组均较死亡株组重(P=0.000,P=0.000);轻症、重症和死亡株组小鼠生存率分别为77.2%、81.7%和97.8%,差异有显著性(P=0.0010,P=0.001,P=0.0004);轻症株组肺出血最重,脑、肌肉、脾和肠病理定性差异无显著性;轻症株组各组织病毒量最高,死亡株组最低。结论临床轻症毒株在乳鼠症状最重,死亡株最轻,与人的临床结局不一致,可能与病毒基因、宿主等因素相关。     

肠道病毒71型(EV-A71)感染ICR小鼠模型的建立

为研究肠道病毒71型(Enterovirus 71,EV-A71)经不同攻毒方式感染不同日龄ICR乳鼠的感染情况,了解EV-A71在小鼠体内的动态分布和感染机制,为建立EV-A71感染动物模型,本研究采用分离自重症手足口患儿的EV-A71毒株,分别通过肌肉注射(Intramuscular injection,IM)、腹腔注射(Intraperitoneal injection,IP)以及脑内注射(Intracerebral injection,IC)的方式感染3日、5日和9日龄ICR乳鼠,感染后定期采集血液和各组织,通过Realtime PCR追踪各组织中病毒载量变化,并且通过切片制作和免疫组化对感染乳鼠进行病原学和病理学分析。结果显示:对于低日龄(≤5日龄)乳鼠,注射剂量在104.5 TCID50/g·体重时IM、IP及IC均为较好的感染方式,都会出现神经症状,且有极高的致死率。随着日龄增长,感染后症状有所减轻,但IM和IP感染途径对大日龄乳鼠仍具有良好的致病性,且IM和IP死亡率显著高于IC死亡率。经IM、IP和IC注射感染病毒的3日龄乳鼠在6dpi体重相对于各对照组分别下降了1.54g(31.43%)、1.31g(15.06%)和2.52g(44.28%),而经IM和IP感染的5日龄乳鼠6dpi体重相对于各对照组分别下降了0.605g(8.95%)、0.886g(15.51%),经IC感染的5日龄乳鼠6dpi体重相对于对照组上升了0.904g(14.70%),感染组体重均显著低于对应的同期对照组(P0.05)。经IM、IP和IC感染病毒的3日龄乳鼠9dpi均死亡,而5日龄乳鼠9dpi存活率分别为42.8%、25%和87.5,14dpi存活率分别为0%、0%和25%,9日龄乳鼠9dpi存活率分别为70%、84.62%和100%。病理学及免疫组化检查显示EV-A71病毒具有强烈的嗜神经性及嗜骨骼肌的特性,可导致病毒血症、脑神经元及骨骼肌坏死、心肌间质水肿及多脏器炎症反应。我们系统地研究了不同攻毒方式感染不同日龄的ICR乳鼠后的病毒动态分布及免疫病理损伤,发现经肌肉注射或腹腔注射5日龄乳鼠能够建立理想的EV-A71感染动物模型。     

新甲型H1N1流感病毒小鼠致死模型的建立

建立新甲型H1N1流感病毒小鼠致死模型,为研究致病性、宿主适应性以及疫苗保护性提供动物模型,并寻找病毒在适应宿主过程中影响毒力和适应性的关键位点。将新甲型H1N1流感病毒A/四川/SWL1/2009 H1N1在小鼠中连续传15代,各代次毒株均在MDCK细胞上增殖后进行测序,根据序列分析结果选择6个传代毒株感染小鼠,连续监测14 d体重和死亡情况;并对第14代和15代病毒在噬斑实验纯化后克隆和测序分析。原代病毒不致死BABL/C小鼠,经动物体内连续传代适应宿主动物后,其毒力增强,具体表现为所选的6个传代毒株中第7、11、15代毒株可以100%致死试验小鼠;分析这6个传代毒株的全基因组表明这些毒株的部分氨基酸位点发生突变。新甲型H1N1流感病毒经小鼠体内连续传代后,建立了小鼠致死模型,病毒毒力增强可能与某些氨基酸位点的改变有关。     

肠道病毒A71型VP1-E98K突变对hSCARB2-KI小鼠致病性的影响

目的 探究肠道病毒A71型(enterovirus A71, EV-A71)VP1-E98K突变对人清道夫受体B2敲入(human scavenger receptor class B member 2 knock-in, hSCARB2-KI)小鼠致病性的影响。方法 利用pSVA-EV-A71-Isehara感染性克隆重组质粒获取拯救的EV-A71 Isehara株,分别在人恶性胚胎横纹肌瘤(human rhabdomyosarcoma, RD)细胞和转染hSCARB2的RD细胞(RD-hSCARB2)中传代并收获病毒,感染小鼠后建立hSCARB2-KI小鼠感染模型。研究中还采用了感染小鼠的临床评分指标、RT-qPCR、HE染色和免疫荧光技术、ELISA、病毒滴定等方法研究病毒的致病性。结果 用RD细胞中连续传代获得的EV-A71 Isehara株攻击hSCARB2-KI小鼠,未观察到预期的体质量减少、行动迟缓、肢体瘫痪等临床表现;经测序确定该病毒株衣壳蛋白发生了VP1-E98K突变,故称之为VP1-98K突变株。而野毒型EV-A71 Isehara株(VP1-98E野毒株)感染的h...     

EV71感染BABL/c乳鼠模型构建

目的 构建中重症EV71型手足口病的BABL/c乳鼠模型。方法 1日龄BALB/c乳鼠连续3 d腹腔注射EV71病毒液，监测小鼠体质量、生存率和临床疾病评分；采用HE染色检测脑、肺和骨骼肌组织病理变化；光镜下观察感染乳鼠脑、肺和骨骼肌组织匀浆作用RD细胞的细胞病变反应；采用蛋白质免疫印迹和免疫荧光法检测VP1和3D蛋白水平。结果 与正常对照组相比，EV71感染乳鼠体质量从第3天开始下降，2组比较差异有统计学意义（t=8.421 0,P<0.05）；生存率随感染时间延长逐渐下降；疾病评分显著增加；脑、肺及骨骼肌组织中VP1和3D蛋白表达与正常对照组比较显著升高（脑组织：t=6.355 7、3.423 2，肺组织：t=14.864 8、4.469 0，骨骼肌：t=7.908 5、4.923 7，均P<0.01）。结论 成功构建了中重症EV71型手足口病BALB/c乳鼠模型。     

我国新分离乙型脑炎病毒株毒力特征研究

将分离自不同年代的17株乙脑毒株在小鼠脑内传代,然后将病毒在BHK21细胞单层上观察不同毒株的空斑形成大小形态,小鼠脑内和皮下途径接种观察病毒的毒力,结果显示不同毒株在BHK21细胞上形成的噬斑大小不尽相同,减毒株形成的噬斑最小。所有毒株对小鼠的脑内毒力都很强,病毒滴度高达lg8.0/mL以上,毒株间无明显差异。毒株对9～11g小日龄小鼠的皮下毒力有一定差异但不明显,但对14～16g较大日龄小鼠则差别明显。PFU/LD50的对数值差异除一株(M47株)为8.44外,其余各株差别在3.94～0.45间。本研究结果证明自然界乙脑毒株存在明显的神经外毒力差异,毒力差异与分离年代和病毒基因型无关,但从人体分离到的毒株毒力表现较强。     

季节性流感病毒H1N1 BALB/c鼠肺适应株的建立及其分子机制

目的建立季节性流感病毒H1N1的鼠肺适应株,并对适应的分子机理进行研究。方法以病毒滴鼻感染小鼠,通过在BALB/c小鼠肺组织中连续传代,观察小鼠存活情况及肺病理改变,来获得季节性流感病毒H1N1的鼠肺适应株。结果季节性流感H1N1 A/Brisbane/59/2007病毒野生型毒株,经过在小鼠体内进行8次传代后,毒力逐渐增强,从无致病力到致死率达到100%,对鼠肺适应株与野生型毒株进行基因比对,发现适应株HA基因发生了3个有义突变。结论野生季节性低致病力H1N1流感病毒可经在小鼠中经过多次传代而获得高致病力H1N1鼠肺适应株,HA蛋白89位Thr至Ile的突变对毒力的增强起决定性作用。     

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.     

MA104 Cell line presents characteristics suitable for enterovirus A71 isolation and proliferation

Enterovirus A71 (EV‐A71), one of the most important causative agents of hand, foot and mouth disease (HFMD) in children, can lead to severe clinical outcomes, even death. However, the infection spectrum of EV‐A71 in different cell lines remains unknown. Therefore, in this study, the biological characteristics of EV‐A71 Subgroup C4 in different cell lines were investigated. To this end, the infectivity of EV‐A71Jinan1002 isolated from children with severe HFMD was assessed in 18 different host cell lines. It was found that the MA104 cell line displayed biological characteristics suitable for EV‐A71 Subgroup C4 strain isolation and proliferation; indeed, it was found that a broad spectrum of cell lines can be infected by EV‐A71Jinan1002. Among the screened cells, four cell lines (HEK293, RD, MA104 and Marc145) produced high 50% tissue culture infective dose (TCID₅₀) values calculated in viral proliferations (ranged from 10^7.6 to 10^7.8); the TCID₅₀ being negatively associated with the time to appearance of CPE. Proliferation curves demonstrated that EV‐A71Jinan1002 amplifies more efficiently in MA104, Hep‐2 and RD cells. Remarkably, the virus isolation rate was much higher in MA104 cells than in RD cells. Thus this study, to our knowledge, is for the first to explore the infection spectrum of EV‐A71 subgroup C4 in such a large number of different cell lines. Our data provide useful reference data for facilitating further study of EV‐A71.     

Studies on Inhibition of Proliferation of Enterovirus-71 by Compound YZ-LY-0

In recent years, hand-foot-and-mouth disease (HFMD), which is caused by Enteroviruses, has emerged as a serious illness. It affects mainly children under the age of five and results in high fatality rates. Enterovirus 71 (EV71) is the main causative agent of HFMD in China and currently there are no effective anti-viral drugs available to treat HFMD. In the present study, we screened compounds for inhibition of proliferation of EV71. Compound YZ-LY-0 stalled the life cycle of EV71. The inhibitor exhibited EC₅₀ value of 0.29 μm against SK-EV006 strain of EV71. Notably, YZ-LY-0 had low cytotoxicity (CC₅₀ > 100 μM) and a high selectivity index (over 300) in Vero and RD cells. YZ-LY-0 in combination with an EV71 RdRp inhibitor or an entry inhibitor showed an antagonistic effect at very low concentrations. However, at higher concentrations the inhibitors exhibited a synergistic effect in inhibiting viral replication. Preliminary results on investigation of the mechanism of inhibition indicate that YZ-LY-0 does not block the entry of the virus in the host cell, but instead inhibits an early stage of EV71 replication. Our studies provide a potential clinical therapeutic option against EV71 infections and suggest that a combined application of YZ-LY-0 with other inhibitors could be more effective in the treatment 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。     

内标多重荧光RT-PCR同时检测肠道病毒71型和柯萨奇病毒A16型

【目的】为对当前爆发的手足口病进行快速准确的检测, 【方法】本研究建立了含内标的同时检测EV71和CA16的多重荧光RT-PCR方法,对该方法的特异性、灵敏度等进行评估,并对400多份临床样品进行了检测。【结果】实验结果表明,该检测方法特异性强,对10株EV71病毒、8株CA16病毒和25株其他人类病毒进行了检测,特异性为100%;该检测方法对EV71和CA16的检测灵敏度分别达到0.1 TCID50和1 TCID50;将0.1-104TCID50/ml EV71和CA16样本进行重复性实验,其变异系数分别为0.9-2.0%和0.9-2.3%。对400多份临床样品分别进行荧光RT-PCR检测和传统方法检测,结果显示,荧光RT-PCR对EV71和CA16的阳性检出率平均为46.1%和14.2%,比传统方法（34.5%和12.8%）的阳性检测率高。另外,实验数据显示,在粪便、直肠拭子、咽喉拭子样本中,PCR抑制物存在的比例为1.8%-3.4%,表明内标对监控PCR抑制物的存在具有重要作用。【结论】本方法能同时对EV71和CA16进行快速检测,并且灵敏度高,特异性好,由于加入了内标,能有效地监控假阴性的出现,适合于手足口病的临床检测。     

EV71 infection correlates with viral IgG preexisting at pharyngolaryngeal mucosa in children

Enterovirus 71(EV71) infection causes severe central nervous system damage, particularly for children under the age of 5 years old, which remains a major public health burden worldwide. Clinical data released that children may be repeatedly infected by different members in enterovirus and get even worsen. Mucosa, especially epithelium of alimentary canal, was considered the primary site of EV71 infection. It has been elusive whether the preexsiting viral antibody in mucosa plays a role in EV71 infection. To answer this question, we respectively measured viral antibody response and EV71 RNA copy number of one hundred throat swab specimens from clinically confirmed EV71-infected children. The results released that low-level of mucosal Ig G antibody against EV71 broadly existed in young population. More importantly, it further elucidated that the children with mucosal preexsiting EV71 Ig G were prone to be infected, which suggested a former viral Ig G mediated enhancement of viral infection in vivo.     

Monoclonal neutralizing antibodies against EV71 screened from mice immunized with yeast-produced virus-like particles

Periodic outbreaks of hand, foot and mouth disease(HFMD) occur in children under 5 years old, and can cause death in some cases. The C4 strain of enterovirus 71(EV71) is the main pathogen that causes HFMD in China. Although no drugs against EV71 are available, some studies have shown that candidate vaccines or viral capsid proteins can produce anti-EV71 immunity. In this study, female BABL/c mice(6–8 weeks old) were immunized with virus-like particles(VLPs) of EV71 produced in yeast to screen for anti-EV71 antibodies. Two hybridomas that could produce neutralizing antibodies against EV71 were obtained. Both neutralizing m Abs(D4 and G12) were confirmed to bind the VP1 capsid protein of EV71, and could protect 95% cells from 100 TCID50 EV71 infection at 25 μg/m L solution(lowest concentration). Those two neutralizing m Abs identified in the study may be promising candidates in development for m Abs to treat EV71 infection, and utilized as suitable reagents for use in diagnostic tests and biological studies.     

Exogenous interleukin-6, interleukin-13, and interferon-gamma provoke pulmonary abnormality with mild edema in enterovirus 71-infected mice

Background

Neonatal mice developed neurological disease and pulmonary dysfunction after an infection with a mouse-adapted human Enterovirus 71 (EV71) strain MP4. However, the hallmark of severe human EV71 infection, pulmonary edema (PE), was not evident.

Methods

To test whether EV71-induced PE required a proinflammatory cytokine response, exogenous pro-inflammatory cytokines were administered to EV71-infected mice during the late stage of infection.

Results

After intracranial infection of EV71/MP4, 7-day-old mice developed hind-limb paralysis, pulmonary dysfunction, and emphysema. A transient increase was observed in serum IL-6, IL-10, IL-13, and IFN-γ, but not noradrenaline. At day 3 post infection, treatment with IL-6, IL-13, and IFN-γ provoked mild PE and severe emphysema that were accompanied by pulmonary dysfunction in EV71-infected, but not herpes simplex virus-1 (HSV-1)-infected control mice. Adult mice did not develop PE after an intracerebral microinjection of EV71 into the nucleus tractus solitarii (NTS). While viral antigen accumulated in the ventral medulla and the NTS of intracerebrally injected mice, neuronal loss was observed in the ventral medulla only.

Conclusions

Exogenous IL-6, IL-13, and IFN-γ treatment could induce mild PE and exacerbate pulmonary abnormality of EV71-infected mice. However, other factors such as over-activation of the sympathetic nervous system may also be required for the development of classic PE symptoms.