SARS-CoV、HCoV-229E和OC43核衣壳蛋白的克隆表达及抗原相关性分析

目的克隆表达SARS冠状病毒(SARS-CoV)和人冠状病毒(HCoV-229E、HCoV-OC43)核衣壳(N)蛋白,并对其重组蛋白的抗原相关性进行探讨。方法RT-PCR扩增SARS-CoV、HCoV-229E和HCoV-OC43的N基因全长序列,克隆到原核表达载体pQE30,IPTG诱导表达重组蛋白,用Westernblot和免疫荧光鉴定。结果获得SARS-CoV、HCoV-229E和HCoV-OC43His-N融合蛋白的相对分子质量(Mr)分别约为47×103、44×103、50×103,与相应预测值相符,Westernblot和免疫荧光证实,3种融合蛋白仅与相应的免疫血清特异性结合,3种蛋白的抗原性相互间无交叉反应。结论获得具有免疫原性的SARS-CoV、HCoV-229E和HCoV-OC43的核衣壳融合蛋白,其3种N蛋白抗原性在免疫动物血清中不存在交叉反应。     

SARS-CoV、229E和OC43的抗原相关性研究

目的：分析SARS冠状病毒（SARS—CoV）和人冠状病毒（229E和OCA3）的抗原相关性。方法：制备三株冠状病毒的免疫兔血清及其重组核衣壳（N）蛋白的免疫小鼠血清，分别采用SARS法、Western blot和免疫荧光法对免疫血清进行检测以分析三株冠状病毒的抗原相关性。结果：Westem blot结果显示重组N蛋白免疫小鼠血清仅与各自的冠状病毒或重组N蛋白有特异性反应，相互间无交叉反应，而SARS结果显示OC43重组N蛋白免疫小鼠血清与SARS—CoV、229E N蛋白出现了构象抗体的交叉反应。同时，免疫荧光结果显示SARS—CoV和OCA3免疫兔血清与229E感染细胞存在较明显的交叉反应，但在SARS、Westem blot结果中全病毒免疫兔血清均仅与各自N蛋白特异性反应，相互间无交叉反应。结论：SARS—CoV、229E和OCA3N蛋白抗原性在免疫动物血清中不存在交叉反应，而SARS—CoV、OC43全病毒免疫血清均和229E出现明显的交叉反应。另外，基因重组的OCA3 N蛋白与另外二种N蛋白出现重组构形表位的交叉反应，提示以基因重组的蛋白作为诊断试剂，可能会因为蛋白的空间构象发生改变而产生非特异性反应。     

新冠状病毒与SARS

冠状病毒可引起人和动物的多种疾病，人类冠状病毒HCoV-OC43和HCoV-229E主要引起人类的普通感冒。但是最近新分离出的冠状病毒可引起严重急性呼吸综合征(severe acute respiratory syndrome，SARS)，本文将就冠状病毒的生物学特性与致病性．新冠状病毒与SARS的关系，SARS冠状病毒的研究进展作一综述。     

2007年秋冬季北京地区成人发热就诊患者中人冠状病毒229E感染状况分析

目的 了解北京地区成人发热就诊患者中人冠状病毒HCoV-229E感染病原学、临床表现和流行病学特点.方法 2007年10月至12月从北京地区发热且有呼吸道症状的就诊成人中采集158份鼻咽拭子标本,利用荧光定量RT-PCR法进行HCoV-229E筛查;利用常规PCR方法扩增HCoV-229E基因片段测序;对感染阳性患者进行HCoV-NL63、HCoV-HKU1及HMPV的共感染筛查、临床表现描述和流行病学特点分析.结果 158份鼻咽拭子标本中荧光定量RT-PCR检测出103(65.2%)例HCoV-229E阳性;其中26例存在与HCoV-NL63(3例)、HCoV-HKU1(3例)及HMPV(20例)的混合感染;HCoV-229E阳性标本的临床表现以头痛(70.9%)、咽红(70.9%)、咽痛(69%)、寒颤(68%)、咳嗽(33%)、有痰(21.3%)、流涕(21.4%)和鼻塞(16.5%)为主,少量可见呕吐(6.8%)、呼吸困难(3.9%)和腹泻(1.9%);统计学分析表明其感染与性别、年龄无关.结论 人冠状病毒HCoV-229E感染在秋冬季成人发热就诊患者中为常见病原,可合并感染其他病毒.其临床表现呈上呼吸道感染特征.2007年秋冬季在北京地区成人中可能存在HCoV-229E局部流行.     

SARS-CoV核衣壳蛋白用于SARS血清学诊断的研究

目的　研究SARS患者血清中抗SARS冠状病毒 (SARS CoV)核衣壳 (N)蛋白特异性抗体的产生规律 ,评价N蛋白在SARS诊断中的作用。方法　采用间接ELISA法测定 2 5 0例临床确诊、1 88例临床疑似、6 2例临床排除SARS患者血清中针对N蛋白IgG抗体 ,并与其SARS CoVIgG总抗体水平进行比较。结果　临床确诊病例针对N蛋白和全病毒特异性抗体IgG的阳性率均随着病程的延长而增高。N IgG在发病第 1周检出阳性率为 3.4 5 %(4 1 1 6 ) ,第 2周为 6 1 .76 %(42 6 8) ,第 3周为81 .82 %(1 8 2 2 ) ,2 2～ 93d达到 1 0 0 %(44 4 4) ;SARS CoVIgG第 1周检出阳性率为 4 .31 %(5 1 1 6 ) ,第 2周为 5 5 .88%(38 6 8) ,第 3周为 77.2 7%(1 7 2 2 ) ,2 2～ 93d达到 1 0 0 %(44 4 4) ;临床疑似病例N IgG检出阳性率为 3.1 9%(6 1 88) ,SARS CoVIgG检出阳性率为 2 .6 6 %(5 1 88) ;临床排除病例N IgG和SARS CoVIgG检出阳性率均为 6 .4 5 %(4 6 2 )。两种方法检测阳性率差异无统计学意义 (χ2 =0 .1 80 ,P <0 .0 0 1 ) ;两者之间符合率为 98.2 0 %(491 5 0 0 ) ,正常人群SARS CoVN IgG阳性率为 1 .88%(1 4 74 5 )。结论　SARS CoVN重组蛋白对于SARS诊断试剂的研究具有重要价值。     

人冠状病毒HKU1血清学检测方法的建立及其初步应用

目的 表达HCoV-HKU1的壳蛋白（N蛋白）及棘突蛋白（S蛋白）,建立检测血清中相应抗体的方法.方法 使用原核表达系统表达纯化HCoV-HKU1的N蛋白,转膜制条建立基于蛋白印迹的N抗体检测方法.构建HCoV-HKU1 S蛋白的真核表达质粒,转染细胞后,用间接免疫荧光（IFA）建立S抗体检测方法.利用已建立的N抗体和S抗体检测方法,检测了100份正常成人血清.结果 经Western Blot检测,S和N蛋白表达正确;利用已建立的血清学检测方法分析100份正常成人血清,其中HCoV-HKU1 S抗体的阳性率为47%,N抗体的阳性率为48哂,S抗体和N抗体均阳性的占总数的21%,双抗体均阴性的占总数的22%.S抗体和N抗体共同检测可获得74%的阳性检出率.结论 所建立的方法可用于HCoV-HKU1血清学分析,共同检测HCoV-HKU1 S抗体和N抗体可获得较好的检测结果.在正常成年中HCoV-HKU1抗体阳性检出率较高.     

HCoV-OC43 逃避人树突状细胞免疫清除机制的初步研究

目的:了解人冠状病毒OC43(Human coronavirus OC43,HCoV-OC43)逃避人树突状细胞(Dendritic cell,DC)免疫监视作用的初步机制。方法:利用HCoV-OC43 阳性病人的标本感染BSC-1 细胞分离OC43 病毒,相差显微镜观察细胞病变(Cytopathic effect,CPE),实时荧光定量聚合酶链反应(Real-time PCR)进行鉴定;利用人细胞因子GM-CSF 和IL-4 联合体外诱导DC 分化,于诱导7 d 后用HCoV-OC43 病毒感染DC。采用透射电镜观察DC 感染后的形态,Real-time PCR 检测DC 功能相关细胞因子的表达水平;流式细胞术检测DC 比例及其功能相关共刺激分子的表达。结果:成功建立HCoV-OC43 体外感染DC 的体系。HCoV-OC43 能感染DC 并刺激其产生免疫应答,但培养上清中不能检测到病毒核酸;HCoV-OC43 感染会导致DC细胞表达IFN-α、IFN-β、CCL3 和CCL5 的量显著下调,但其共刺激分子HLA-DR、CD1c 和CD86 的表达不受抑制。结论:HCoV-OC43 可感染人DC 细胞并刺激其产生免疫应答,但不能产生活的子代病毒;HCoV-OC43 可通过抑制宿主DC 细胞IFN-β等相关炎症因子和趋化因子的分泌,来实现免疫逃逸。     

探讨载脂蛋白E和超敏C反应蛋白与冠心病的相关性

目的 探讨载脂蛋白E(apolipoprotein E,ApoE)和超敏C反应蛋白(hypersensitive C-reactive protein,hs-CRP)与冠心病的相关性.方法 采用免疫比浊方法测定334例患者(疾病组)及100名体检健康者(对照组)ApoE、hs-CRP水平.采用统计学t检验,P<0.05为差异有统计学意义.作为冠心病危险因素,采用Logistic回归分析对两个检测项目进行评估,并绘制ApoE、hs-CRP单独及联合诊断冠心病的ROC曲线.结果(1)患病组血清ApoE和hs-CRP水平均高于对照组(P<0.01),差异存在统计学意义;(2)Logistic回归分析显示,ApoE及hs-CRP均是冠心病发生的危险因子,ApoE:OR=0.824,95％可信区间(CI)为0.774～0.8790;hs-CRP:OR=0.106,95％CI为0.054～0.209;(3)联合检测诊断曲线下面积(AUC)大于ApoE、hs-CRP单独检测AUC,分别为0.986、0.883、0.945.结论 免疫比浊法测定血清中的ApoE水平与冠心病的发生存在相关性,并且与hs-CRP同时存在于冠心病的发生和发展过程中,二者联合检测对于疾病的临床诊治具有非常重要的价值.     

载脂蛋白E基因多态性与中国人阿尔茨海默病和颈动脉粥样硬化相关性研究

为了探讨中国散发性阿尔茨海默病（SAD)颈动脉粥样硬化（CAS）与ApoE基因多态性的关系,应用聚合酶链式反应－限制性片段长度多态性（PCR－RFLP）技术检测46名SAD患者、31名CAS患者及50名正常老年人的ApoE基因多态性分布特征。结果显示SAD组及CAS组ApoE ε4等位基因频率均高于对照组（P＜0．05）;SAD组为15．2％,CAS组为14．5％,对照组为3．0％,但CAS组ApoEε2等位基因频率明显高于SAD组（P＜0．05）;CAS组为41．9％,SAD组为21．7％本研究提示,ApoE4等位基因是SAD和CAS的共同危险因素,但ApoE2等位基因对CAS病人患AD具有保护作用。     

人、猪、禽戊型肝炎病毒血清学关系的研究

目的研究人、猪、禽戊型肝炎病毒（HEV）的血清学关系。方法应用ELISA分别以人、猪、禽HEVORF2重组蛋白p166human、p166swine、p268avian检测人、猪、鸡血清及其他标本中抗．HEVIs,G，用SAS软件进行统计学分析，同时进行序列同源性比较。结果p166human和p166swine对HEV实验感染动物血清、HEVORF2重组蛋白免疫血清和单克隆抗体均呈阳性反应，而p268avian均呈阴性反应。以p166human、p166swine、p268avian检测人、猪、鸡血清抗．HEVIgG，戊型肝炎患者血清检出率分别为98．5％、97．7％和1．5％，正常人血清为10．0％、10．0％和4．0％，猪血清为26．9％、25．6％和1．3％，鸡血清为4．3％、2．2％和33．3％。p268avian与p166human或p166swine的检出率差异有统计学意义（P〈0．001）。相关性分析表明，p166human和p166swine对不同样本的检测均呈直线正相关，而p268avitm与p166human或p166swine无直线相关性。人和猪HEVpORt2的序列同源性在88．2％。99．2％，而禽HEV与人、猪HEVpORt2的同源性仅为45．5％～46．1％，其中含有多个插入和缺失突变。结论人和猪HEV血清学关系密切，而禽HEV与人、猪HEV抗原性差异有统计学意义，无血清学相关性。因此禽HEV与人、猪HEV的关系应予进一步考证。     

Development of a recombinant truncated nucleocapsid protein based immunoassay for detection of antibodies against human coronavirus OC43

Human coronaviruses are one of the main causes of upper respiratory tract infections in humans. While more often responsible for mild illness, they have been associated with illnesses that require hospitalization. In this study, an assay for one of the human coronaviruses, OC43, was developed using a truncated recombinant nucleocapsid (N) protein antigen in an enzyme immunosorbent assay (ELISA) and evaluated using serum collected from HCoV-OC43-infected patients, healthy adults, and patients with other respiratory virus infections. Results showed that the diagnostic sensitivity and specificity of the assay were 90.9% (10/11) and 82.9% (39/47), respectively. To evaluate the clinical utility of the ELISA, serum samples collected from patients during an outbreak of HCoV-OC43 infection and previously identified as positive by HCoV-OC43 whole N ELISA were screened resulting in 100% diagnosis agreement between the testing methods. These results suggest that this assay offers a reliable method to detect HCoV-OC43 infection and may be a useful tool in coronavirus seroepidemiological studies.     

Circulation of genetically distinct contemporary human coronavirus OC43 strains

In this study, we report the complete genome sequence of two contemporary human coronavirus OC43 (HCoV-OC43) strains detected in 2003 and 2004, respectively. Comparative genetic analyses of the circulating strains and the prototype HCoV-OC43 strain (ATCC VR759) were performed. Remarkably, a lower than expected similarity is found between the complete genomes and more in particular between the spike genes of the BE03 and BE04 strains. This finding suggests the existence of two genetically distinct HCoV-OC43 strains, circulating in Belgium in 2003 and 2004. Spike gene sequencing of three additional 2003 and two additional 2004 HCoV-OC43 strains, and subsequent phylogenetic analysis confirm this assumption. Compared to the ATCC prototype HCoV-OC43 strain, an important amino acid substitution is present in the potential cleavage site sequence of the spike protein of all contemporary strains, restoring the N-RRXRR-C motif, associated with increased spike protein cleavability in bovine coronaviruses. We here describe specific characteristics associated with circulating HCoV-OC43 strains, and we provide substantial evidence for the genetic variability of HCoV-OC43.     

Determination of coronavirus 229E antibody by an immune-adherence hemagglutination method.

An immune-adherence hemagglutination (IAHA) method for coronavirus 229E antibody determination has been developed both for diagnosis of recent infections and for detection of long-past infections. Results have been compared with those obtained by complement fixation (CF), neutralization (Nt), and indirect hemagglutination (IHA) tests. The IAHA method has been shown to be as sensitive as the CF, Nt, and IHA tests in detecting cases of acute 229E infection. However, in a seroepidemiological survey of 343 healthy people of all ages, IAHA detected 229E antibody in 254 individuals (74.0%), Nt in 166 (48.3%), IHA in 89 (25.9%), and CF in 30 (8.7%). A study of the prevalence of coronavirus 229E IAHA antibody in the different age groups has shown that during the second decade of life nearly 100% of the population acquire this type of antibody, whereas only 50% are positive at the end of the first decade. In the older age groups, the high frequency of CF antibody (“marker” of recent infection) indirectly confirms the high rate of 229E reinfections and the nonprotective nature of IAHA antibody. CF titer ? 1:8 in 90% of cases corresponded to IAHA titers ? 1:64. However, sera with IAHA titers of ? 1:128 were often CF-negative. Recent 229E infections (or reinfections), as determined by the presence of CF antibody, were more frequent in April-May than in October-November. Three cases of acute infection showing 229E seroconversion (two adults and one child) were observed during the winter-spring season. IAHA appears to be the test of choice for seroepidemiological surveys.     

Serological responses in patients with severe acute respiratory syndrome coronavirus infection and cross-reactivity with human coronaviruses 229E, OC43, and NL63

The serological response profile of severe acute respiratory syndrome (SARS) coronavirus (CoV) infection was defined by neutralization tests and subclass-specific immunofluorescent (IF) tests using serial sera from 20 patients. SARS CoV total immunoglobulin (Ig) (IgG, IgA, and IgM [IgGAM]) was the first antibody to be detectable. There was no difference in time to seroconversion between the patients who survived (n = 14) and those who died (n = 6). Although SARS CoV IgM was still detectable by IF tests with 8 of 11 patients at 7 months postinfection, the geometric mean titers dropped from 282 at 1 month postinfection to 19 at 7 months (P = 0.001). In contrast, neutralizing antibody and SARS CoV IgGAM and IgG antibody titers remained stable over this period. The SARS CoV antibody response was sometimes associated with an increase in preexisting IF IgG antibody titers for human coronaviruses OC43, 229E, and NL63. There was no change in IF IgG titer for virus capsid antigen from the herpesvirus that was used as an unrelated control, Epstein-Barr virus. In contrast, patients who had OC43 infections, and probably also 229E infections, without prior exposure to SARS CoV had increases of antibodies specific for the infecting virus but not for SARS CoV. There is a need for awareness of cross-reactive antibody responses between coronaviruses when interpreting IF serology.     

A human coronavirus OC43 variant harboring persistence-associated mutations in the S glycoprotein differentially induces the unfolded protein response in human neurons as compared to wild-type virus

We have reported that human respiratory coronavirus OC43 (HCoV-OC43) is neurotropic and neuroinvasive in humans and mice, and that neurons are the primary target of infection in mice, leading to neurodegenerative disabilities. We now report that an HCoV-OC43 mutant harboring two persistence-associated S glycoprotein point mutations (H183R and Y241H), induced a stronger unfolded protein response (UPR) and translation attenuation in infected human neurons. There was a major contribution of the IRE1/XBP1 pathway, followed by caspase-3 activation and nuclear fragmentation, with no significant role of the ATF6 and eIF2-alpha/ATF4 pathways. Our results show the importance of discrete molecular viral S determinants in virus-neuronal cell interactions that lead to increased production of viral proteins and infectious particles, enhanced UPR activation, and increased cytotoxicity and cell death. As this mutant virus is more neurovirulent in mice, our results also suggest that two mutations in the S glycoprotein could eventually modulate viral neuropathogenesis.     

Vacuolating encephalitis in mice infected by human coronavirus OC43

Involvement of viruses in human neurodegenerative diseases and the underlying pathologic mechanisms remain generally unclear. Human respiratory coronaviruses (HCoV) can infect neural cells, persist in human brain, and activate myelin-reactive T cells. As a means of understanding the human infection, we characterized in vivo the neurotropic and neuroinvasive properties of HCoV-OC43 through the development of an experimental animal model. Virus inoculation of 21-day postnatal C57BL/6 and BALB/c mice led to a generalized infection of the whole CNS, demonstrating HCoV-OC43 neuroinvasiveness and neurovirulence. This acute infection targeted neurons, which underwent vacuolation and degeneration while infected regions presented strong microglial reactivity and inflammatory reactions. Damage to the CNS was not immunologically mediated and microglial reactivity was instead a consequence of direct virus-mediated neuronal injury. Although this acute encephalitis appears generally similar to that induced by murine coronaviruses, an important difference rests in the prominent spongiform-like degeneration that could trigger neuropathology in surviving animals.