双黄连注射液抑制病毒性脑炎模型小鼠脑组织核因子κB的表达：具有时间和剂量的依赖性

有研究认为双黄连注射液的抗病毒作用可能与抑制核因子κB的活性有关。实验观察柯萨奇病毒B3病毒性脑炎模型小鼠脑组织中核因子κB的表达,发现腹腔注射双黄连注射液检测模型小鼠脑组织中核因子κB基因和蛋白表达明显降低,随着干预时间延长和治疗剂量增加,抑制效应更显著。证实双黄连注射液以剂量和时间依赖形式降低病毒性脑炎模型小鼠脑组织中核因子κB的表达。     

中药双黄连对柯萨奇病毒脑炎模型的药效观察

目的:观察中药双黄连对人胚脑柯萨奇B3病毒(CVB3)脑炎模型的药效观察。方法:采用人胚脑皮层细胞原代培养,感染CVB_3,用双黄连进行抑毒实验。抑毒指标为培养液肌酸激酶测定和CVB_3定量测定。结果:双黄连组对CVB_3感染后的细胞病变有明显抑制作用。结论:双黄连对脑神经细胞感染CVB_3有一定的抑制作用。     

MMP-9在单纯疱疹病毒性脑炎中的表达及对血脑屏障的影响

目的研究单纯疱疹病毒感染小鼠脑组织后MMP-9的变化及对血脑屏障通透性的影响。方法小鼠颅内注射单纯疱疹病毒1型(HSV-1)制造单纯疱疹病毒性脑炎模型,于感染后第3天、第7天,应用明胶酶谱分析检测脑组织中MMP-9活性,免疫荧光共聚焦检测MMP-9的细胞来源,通过检测渗出到脑血管外的伊文斯蓝(Evans Blue,EB)的含量以及脑组织水含量观察血脑屏障的通透性。结果 HSV-1感染后第3天、第7天病毒组中MMP-9活性较正常对照组明显增高,MMP-9活性增高与血脑屏障通透性增高相一致,给予MMP-9抑制剂BB-94后血脑屏障通透性降低,激活的小胶质细胞是MMP-9的主要来源。结论激活的小胶质细胞表达MMP-9破坏血脑屏障,在单纯疱疹病毒性脑炎的发病机制中起着重要作用。     

人胚脑神经细胞感染柯萨奇B族病毒的形态学研究

采用人胚脑皮质神经细胞原代培养，接种100TCID50柯萨奇B3病毒（CVB3）后用光镜、组织染色法及酶学测定观察病变全过程。人胚脑神经细胞受到病毒感染后48h逐渐出现细胞固缩、折光性增强、脱落、碎裂样改变。肌酸激酶增高。ＣＢＶ３感染所致的人胚脑皮质细胞病变，反映了人脑在自然状态下感染ＣＶＢ３后的神经细胞病变过程。     

γ-氨基丁酸在小鼠单纯疱疹病毒性脑炎中的表达变化

目的:观察γ-氨基丁酸(GABA)在小鼠单纯疱疹病毒性脑炎(HSE)病程中的表达变化,探讨其在病毒感染性脑损伤中的作用。方法:颅内病毒直接接种的方法建立小鼠疱疹病毒性脑炎模型,免疫组化的方法同时检测疱疹病毒(HSV)抗原和GABA在脑炎不同时间的表达变化,结果用图像分析系统进行分析。结果:病毒接种3d后脑组织颞叶、额叶、海马均出现HSV抗原阳性细胞,7-10dHSV抗原阳性细胞数目达高峰,阳性面积比最大,部分脑组织出现坏死,14d后抗原阳性表达细胞开始减少。GABA的表达变化与HSV的表达呈相反趋势,其阳性神经元数目则随病程而减少,在病程的7-10d表达降至最低,部分标本脑组织的坏死区几乎无表达,14d后表达开始增强。结论:GABA的表达变化与病毒性脑炎的严重程度有一定的相关性,可能对感染性脑损伤具有保护作用。     

柯萨奇B3病毒感染体外培养神经元和星形胶质细胞的研究

目的研究体外纯培养的神经元和星形胶质细胞对柯萨奇B3病毒(CoxsackievirusB3,CVB3)的敏感性.方法CVB3分别感染纯培养新生Balb/c鼠脑的神经元和星形胶质细胞,收集感染后6、24、48、72h的培养上清.显微镜下连续动态观察病变及检测感染后不同时间的培养上清接种于Hela细胞上的病毒滴度.结果CVB3在培养的神经元和星形胶质细胞内均有增殖,但两者发生病变的时间及程度不同.结论Balb/c鼠脑神经元上有CVB3的受体,而星形胶质细胞上CVB3的受体情况尚待进一步研究.     

病毒性脑炎患者Borna病病毒P24基因片段的检测

目的探讨Borna病病毒(BDV)感染与人类病毒性脑炎的关系。方法采用荧光定量套式逆转录酶聚合酶链反应(FQ nRT PCR)检测了59例临床诊断的原因未明的病毒性脑炎及112名健康人外周血单个核细胞(PBMC)中BDVP24基因片段。结果59例原因未明的病毒性脑炎患者中有3例PBMC中检出BDVP24基因片段,而112名健康对照均未检出。病毒性脑炎患者BDV阳性率(5.08%)高于健康对照,差异有统计学意义(P<0.05),且BDVP24基因片段检测阳性病例的脑脊液中其他常见致脑炎病毒(单纯疱疹病毒、带状疱疹病毒、腮腺炎病毒、柯萨奇病毒和巨细胞病毒)检查均为阴性。结论中国的部分病毒性脑炎患者中存在BDV感染,BDV感染与病毒性脑炎的发病可能有关。     

柯萨奇B_3病毒感染体外培养神经元和星形胶质细胞的研究

目的 :研究体外纯培养的神经元和星形胶质细胞对柯萨奇B3 病毒 (CoxsackievirusB3 ,CVB3 )的敏感性。方法 :CVB3 分别感染纯培养新生Balb/c鼠脑的神经元和星形胶质细胞 ,收集感染后 6、2 4、48、72h的培养上清。显微镜下连续动态观察病变及检测感染后不同时间的培养上清接种于Hela细胞上的病毒滴度。结果 :CVB3 在培养的神经元和星形胶质细胞内均有增殖 ,但两者发生病变的时间及程度不同。结论 :Balb/c鼠脑神经元上有CVB3 的受体 ,而星形胶质细胞上CVB3 的受体情况尚待进一步研究     

肠道病毒脑炎病原学及临床分析

目的 探讨肠道病毒脑炎病原学及临床特点.方法 139例脑炎病人中25例早期病人的脑脊液行病原学检查,回顾性分析临床资料.结果 25例脑脊液PCR检验中20例为柯萨奇病毒B组5型阳性.结论 靖江市2006年脑炎流行病原学为柯萨奇病毒,预后相对较好.     

病毒性脑炎早期诊断及病原学探讨(附175例报道)

目的探讨病毒性脑炎的早期诊断方法及病原学诊断的临床价值。方法对我院2005年临床诊断病毒性脑炎的175份病例就其临床资料进行回顾性分析,并对部分患者的脑脊液标本应用ELISA方法检测脑脊液中单纯疱疹病毒,呼吸道合胞病毒,柯萨奇B组病毒的IgM。结果脑电图检查160例,异常136例(85.00%);头CT检查90例,异常15例(16.67%);头MRI检查65例,异常42例(64.62%);脑脊液细胞学检测175例,异常166例(94.86%);血清特异性病毒抗体IgM检测74例,阳性26例,其中单纯疱疹病毒IgM阳性的13例,柯萨奇B组病毒IgM阳性的6例,呼吸道合胞病毒IgM阳性4例,巨细胞病毒IgM阳性3例;脑脊液特异性病毒抗体IgM检测70例,结果均为阴性。结论脑脊液细胞学在病毒性脑炎早期诊断及鉴别诊断中具有重要的价值,病原学诊断是病毒性脑炎诊断研究的发展方向,但其方法学有待于进一步完善。     

A fatal case of coxsackievirus B4 meningoencephalitis

BACKGROUND: Coxsackieviruses and echoviruses are common causes of aseptic meningitis, but they rarely cause life-threatening illness. We report a fatal case of coxsackievirus B4 meningoencephalitis in a woman who developed extrapyramidal symptoms suggestive of encephalitis lethargica. The exact causative agent of encephalitis lethargica has rarely been found, but most cases of the syndrome are assumed to be of viral origin. CASE DESCRIPTION: A 33-year-old woman previously treated with methylprednisolone and cyclophosphamide for Henoch-Sch?nlein purpura was transferred from a referring hospital because of sore throat, fever, and chills. Her neurologic findings progressed from headache with mild photophobia to lethargy, cogwheeling, increased tone in all 4 limbs, and brisk reflexes. The patient was diagnosed as having coxsackievirus B4 meningoencephalitis and, despite treatment with the experimental antiviral agent pleconaril, died of an overwhelming central nervous system infection and myocarditis. Magnetic resonance imaging showed focal hyperintense lesions in the substantia nigra that corresponded to the location of pathological changes seen at autopsy. CONCLUSIONS: This patient had a fulminant coxsackievirus B4 viral meningoencephalitis with a clinical pattern reminiscent of encephalitis lethargica and striking focal abnormalities in the substantia nigra identified on magnetic resonance imaging. The magnetic resonance imaging findings correlated with pathological changes identified at autopsy that were similar to the pathological findings observed in patients with encephalitis lethargica and postencephalitic parkinsonism. It is likely that the patient's immunocompromised state led to an overwhelming infection from an otherwise relatively innocuous viral infection.     

柯萨奇病毒B1接种豚鼠建立多发性肌炎模型

目的探讨柯萨奇病毒Ｂ１（ＣＶＢ１）感染与多发性肌炎（ＰＭ）发病的关系，利用ＣＶＢ１接种豚鼠建立ＰＭ模型。方法分别用０．１ｍｌ（Ａ组）和０．３ｍｌ（Ｂ组）毒力（ＴＣＩＤ５０，即５０％细胞感染剂量）为１０－５的ＣＶＢ１分别一次性接种２４只和３６只豚鼠，并用３ｍｇ（阈下剂量）兔肌匀浆加完全弗氏佐剂每周免疫１次；另有１０只豚鼠，仅用３ｍｇ兔肌匀浆加完全弗氏佐剂每周免疫１次为对照组（Ｃ组），共５周。结果Ａ、Ｂ组３周后均有豚鼠出现四肢肌无力等症状，肌酶谱明显升高，豚鼠股肌病理呈局灶性炎性改变，Ｃ组无异常。Ａ、Ｂ组５周后的肌酶谱升高阳性率存在显著差异（Ａ组为１１．８％，Ｂ组为３８．２％）。结论ＣＶＢ１感染是ＰＭ发病诱因之一，其毒力及接种量与ＰＭ的发病相关。     

Roles of PAR1 and PAR2 in viral myocarditis

Viral myocarditis is estimated to cause ~ 20% of sudden death in people under the age of 40. A variety of viruses have been found to cause myocarditis including coxsackievirus B3 (CVB3). Many studies have been performed with CVB3 because there is a mouse model of CVB3-induced myocarditis. Studies have shown that the TLR3-IFNβ pathway plays a central role in the innate immune response to CVB3 infection. Our laboratory studies the role of protease activated receptors (PAR) in different biological responses including viral infection. We examined the effect of a deficiency in either PAR1 or PAR2 on CVB3-induced myocarditis. Interestingly, we found that PAR1 knockout mice had increased cardiac injury whereas PAR2 knockout mice had decreased cardiac injury. Our studies support the notion that PARs modulate the innate immune response and can have both positive and negative effects on TLR-dependent responses.     

Establishment of a panel of in‐house polyclonal antibodies for the diagnosis of enterovirus infections

The aim of this study was to establish a reliable method of virus detection for the diagnosis of critical enterovirus infections such as acute infective encephalitis, encephalomyelitis and myocarditis. Because histopathological and immunohistochemical analyses of paraffin‐embedded tissues play an important role in recognizing infectious agents in tissue samples, six in‐house polyclonal antibodies raised against three representative enteroviruses using an indirect immunofluorescence assay and immunohistochemistry were examined. This panel of polyclonal antibodies recognized three serotypes of enterovirus. Two of the polyclonal antibodies were raised against denatured virus particles from enterovirus A71, one was raised against the recombinant VP1 protein of coxsackievirus B3, and the other for poliovirus type 1 were raised against denatured virus particles, the recombinant VP1 protein and peptide 2C. Western blot analysis revealed that each of these antibodies recognized the corresponding viral antigen and none cross‐reacted with non‐enteroviruses within the family Picornaviridae. However, all cross‐reacted to some extent with the antigens derived from other serotypes of enterovirus. Indirect immunofluorescence assay and immunohistochemistry revealed that the virus capsid and non‐structural proteins were localized in the cytoplasm of affected culture cells, and skeletal muscles and neurons in neonatal mice experimentally‐infected with human enterovirus. The antibodies also recognized antigens derived from recent clinical isolates of enterovirus A71, coxsackievirus B3 and poliovirus. In addition, immunohistochemistry revealed that representative antibodies tested showed the same recognition pattern according to each serotype. Thus, the panel of in‐house anti‐enterovirus polyclonal antibodies described herein will be an important tool for the screening and pathological diagnosis for enterovirus infections, and may be useful for the classification of different enterovirus serotypes, including coxsackieviruses A and B, echoviruses, enterovirus A71 and poliovirus.     

Type I interferon signaling limits reoviral tropism within the brain and prevents lethal systemic infection

In vivo and ex vivo models of reoviral encephalitis were utilized to delineate the contribution of type I interferon (IFN) to the host’s defense against local central nervous system (CNS) viral infection and systemic viral spread. Following intracranial (i.c.) inoculation with either serotype 3 (T3) or serotype 1 (T1) reovirus, increased expression of IFN-α, IFN-β, and myxovirus-resistance protein (Mx1; a prototypical IFN stimulated gene) was observed in mouse brain tissue. Type I IFN receptor deficient mice (IFNAR^−/−) had accelerated lethality, compared to wildtype (B6wt) controls, following i.c. T1 or T3 challenge. Although viral titers in the brain and eyes of reovirus infected IFNAR^−/− mice were significantly increased, these mice did not develop neurologic signs or brain injury. In contrast, increased reovirus titers in peripheral tissues (liver, spleen, kidney, heart, and blood) of IFNAR^−/− mice were associated with severe intestinal and liver injury. These results suggest that reovirus-infected IFNAR^−/− mice succumb to peripheral disease rather than encephalitis per se. To investigate the potential role of type I IFN in brain tissue, brain slice cultures (BSCs) were prepared from IFNAR^−/− mice and B6wt controls for ex vivo T3 reovirus infection. Compared to B6wt controls, reoviral replication and virus-induced apoptosis were enhanced in IFNAR^−/− BSCs indicating that a type I IFN response, initiated by resident CNS cells, mediates innate viral immunity within the brain. T3 reovirus tropism was extended in IFNAR^−/− brains to include dentate neurons, ependymal cells, and meningeal cells indicating that reovirus tropism within the CNS is dependent upon type I interferon signaling.     

A brain slice culture model of viral encephalitis reveals an innate CNS cytokine response profile and the therapeutic potential of caspase inhibition

Viral encephalitis is a significant cause of human morbidity and mortality in large part due to suboptimal diagnosis and treatment. Murine reovirus infection serves as a classic experimental model of viral encephalitis. Infection of neonatal mice with T3 reoviruses results in lethal encephalitis associated with neuronal infection, apoptosis, and CNS tissue injury. We have developed an ex vivo brain slice culture (BSC) system that recapitulates the basic pathological features and kinetics of viral replication seen in vivo. We utilize the BSC model to identify an innate, brain-tissue specific inflammatory cytokine response to reoviral infection, which is characterized by the release of IL6, CXCL10, RANTES, and murine IL8 analog (KC). Additionally, we demonstrate the potential utility of this system as a pharmaceutical screening platform by inhibiting reovirus-induced apoptosis and CNS tissue injury with the pan-caspase inhibitor, Q-VD-OPh. Cultured brain slices not only serve to model events occurring during viral encephalitis, but can also be utilized to investigate aspects of pathogenesis and therapy that are not experimentally accessible in vivo.     

Minocycline delays disease onset and mortality in reovirus encephalitis

Minocycline is neuroprotective in many experimental models of neurodegenerative diseases and central nervous system (CNS) injury but has not previously been tested in a model of viral encephalitis. Experimental infection of neonatal mice with neurotropic reoviruses is a classic model for studying the pathogenesis of viral encephalitis. Intracerebral inoculation of serotype 3 reovirus strain Dearing (T3D) in neonatal mice results in lethal encephalitis caused by neuronal apoptosis throughout the CNS. Minocycline significantly delayed death in mice to 11.6 +/- 0.9 days post-infection vs. 8.6 +/- 0.7 days post-infection in controls (P < 0.01). Virus-induced CNS injury, apoptosis, viral titer and antigen expression were significantly decreased in the brains of minocycline-treated mice on 6 and 8 days post-infection compared to controls. Virus-induced injury and viral titer in minocycline-treated infected mice at 11 days post-infection were similar to those seen in untreated T3D-infected mice at 8 days post-infection. Little microglial or astrocytic invasion of brain regions with viral injury was found at any time-point in untreated or minocycline-treated mice, suggesting that in this model system the neuroprotective effect exerted by minocycline is more likely due to its anti-apoptotic properties rather than its capacity to inhibit microglial activation and limit gliosis. These findings, similar to those reported for neurodegenerative diseases, indicate that minocycline does not prevent development of fatal reovirus encephalitis but delays disease onset and progression, suggesting that minocycline treatment may provide a useful adjunctive therapy in viral CNS infections.