哺乳动物细胞西尼罗病毒样颗粒表达、纯化及其鉴定

目的利用哺乳动物细胞表达含有西尼罗病毒(WNV)prM和E蛋白,形成病毒样颗粒(virus-like particles,VLPs),为西尼罗病毒感染的免疫诊断试剂的研制奠定基础。方法筛选典型西尼罗病毒株,构建重组质粒,转染293T细胞,表达并纯化西尼罗病毒prM-E蛋白,利用透射电镜、免疫印迹试验、间接免疫荧光实验(IFA)和酶链免疫吸附试验(ELISA)对表达产物进行鉴定。结果重组质粒转染细胞后产生病毒样颗粒(viruslike particles VLPs),转染细胞上清纯化物中透射电镜观察到重组蛋白形成的球型颗粒,免疫印迹试验和间接免疫荧光试验表明,表达的病毒样颗粒蛋白能够与抗西尼罗病毒抗体特异结合,具有良好的抗原性;间接ELISA证实,重组蛋白可以作为抗原用于检测患者血清特异性抗体。结论在哺乳动物细胞中表达的西尼罗病毒样颗粒具有良好的抗原性,为西尼罗病毒感染快速特异诊断试剂研制奠定了基础。     

SYBR GreenⅠ荧光定量PCR检测西尼罗病毒

目的建立快速、敏感和特异的检测西尼罗病毒的SYBRGreenⅠ荧光定量PCR法，用于西尼罗病毒（WNV）疾病的早期诊断。方法采用RT—PCR扩增WNV基因片段，将其克隆至T载体，重组质粒测序并进行同源性分析；阳性质粒用于替代WNV，以阳性质粒为模板，建立SYBRGreenⅠ荧光定量PCR检测方法，并进行敏感性和特异性检测。结果经测序证实扩增片段属于WNV，所建立的SYBRGreenⅠ荧光定量PCR法可检测到10拷贝的西尼罗病毒RNA，而对其他黄病毒科成员日本脑炎病毒及登革热病毒的检测则为阴性，表明该方法特异。结论SYBRGreenⅠ荧光定量PCR简便快速，具有较高的敏感性和特异性，可以成为一种早期检测西尼罗病毒的新方法。     

西尼罗病毒特异性抗原片段筛选及ELISA方法的研究

目的筛选西尼罗病毒特异抗原片段,并建立其ELISA快检方法。方法参考亲水性、抗原性、可塑性、表面可及性及二级结构信息,对西尼罗病毒抗原表位进行系统分析,预测得分值较高的抗原区域经原核表达、Western blot筛选及亲和层析纯化后,包被ELISA微孔板,对ELISA反应条件进行系统优化,建立其ELISA快检方法。结果预测获病毒特异性抗原表位29个,对其中11段进行了表达,经筛选,获得西尼罗病毒特异抗原片段及西尼罗与乙型脑炎病毒共同抗原片段各一段。利用西尼罗病毒特异抗原片段WnAg16建立了检测西尼罗病毒抗体的ELISA方法,与所试相近病毒无交叉反应,S/N比值均在23以上,对鼠抗西尼罗病毒多抗的检出下限达1∶204 800。结论利用特异性抗原初步建立了检测西尼罗病毒抗体的ELISA方法。     

TaqMan荧光定量RT—PCR快速检测西尼罗病毒的研究

目的建立特异、快速、灵敏的TaqMan实时荧光定量聚合酶链反应（TaqMan—based Real—timePCR）用于西尼罗病毒（WNV）核酸检测，为WNV的感染诊断和流行病学监测奠定技术基础。方法根据文献公开发表的WNV核酸检测引物和TaqMan探针，优选最佳引物探针。应用DNAWorks2．4在线软件设计8条寡核苷酸片段，基于PCR合成包含引物探针扩增区域的230个核苷酸作为WNV模拟核酸。并对TaqMan-based Real—timePCR条件进行优化，验证该方法的特异性、灵敏度。结果该方法对WNV核酸检测有高度特异性，与流行性乙型脑炎、登革热1～4型等虫媒病毒均无交叉反应，对构建的DNA检测灵敏度达100拷贝／反应。结论TaqMan—based Real—timePCR是一种快速检测WNV特异、敏感的方法，适用于WNV的早期感染诊断和流行病学监测。     

柯萨奇B组病毒IgM抗体蛋白芯片法检测分析

目的分析蛋白芯片法检测柯萨奇B组病毒(CVB)IgM抗体的临床应用价值。方法收集356例呼吸道病毒性感染者血清和85例非呼吸道感染患者及30例健康献血员的血清标本,同时采用蛋白芯片法和ELISA法检测,进行灵敏度和特异性的比较。结果蛋白芯片法和ELISA法的阳性检出率分别为27.0%和20.8%,两种方法检测检测柯萨奇B组病毒IgM抗体的差异有统计学意义(2χ=17.3,P<0.01);85例非呼吸道感染的病毒性肝炎患者的血清,ELISA法检出阳性7例,阴性78例,特异性91.8%;蛋白芯片法检出阳性6例,阴性79例,特异性92.4%;30例健康献血员的血清,两种方法均未检出CVB-IgM抗体,特异性100.0%。结论蛋白芯片法检测柯萨奇B组病毒IgM抗体具有灵敏度高、特异性强、标本用量少的特点,值得推广应用。     

复合检测5种重要发热病原悬浮芯片筛查方法的建立

目的建立同时检测甲型流感病毒、人禽流感病毒、鼠疫耶尔森菌、SARS冠状病毒、结核分枝杆菌5种发热病原抗体蛋白悬浮芯片方法。方法分别用各病原体的诊断抗原耦联不同编码的微球作为反应载体,采用间接反应模式,建立蛋白悬浮芯片方法检测引起发热体征的5种病原的特异性抗体,在同一反应体系中对人血清中的流感、禽流感、鼠疫、SARS、肺结核等特异性抗体进行复合检测。结果建立的蛋白悬浮芯片5种病原抗体快速筛查系统具有较高的特异性和敏感性,同时检测鼠抗流感NPIgG、兔抗禽流感H5N1IgG、兔抗鼠疫F1IgG、兔抗SARSNIgG、兔抗结核分枝杆菌IgG的检测灵敏度分别为968.80ng/ml、69.40ng/ml、6.30ng/ml、15.50ng/ml、1.60ng/ml。结论重要发热病原悬浮芯片快速筛查系统能快速、敏感、特异、同时检测人血清中的5种病原抗体,对口岸出入境人员是否携带病原的快速筛查具有广阔的应用前景。     

福建省发现1例登革3型病毒感染者

[目的]对福建省2006年发现的1例登革热病例进行实验室检测并鉴定病毒型别。[方法]应用免疫层析及ELISA方法检测感染者双份血清中病毒IgM和IgG抗体,逆转录-套式PCR方法扩增登革病毒特异性核酸片段,并与1～4型病毒特异性产物比较,判定其型别。[结果]双份血清标本均检出病毒IgM抗体,恢复期血清中IgG抗体出现阳转。使用通用引物和型特异性引物,在急性期标本中检出血清3型登革病毒特异性核酸,其他型别为阴性。流行病学调查表明该病人系从菲律宾回国,在当地有蚊虫叮咬史。[结论]该登革热病例为血清3型病毒感染。结合流行病学调查,其感染地可能为菲律宾,为防止病毒暴发流行,应对其进行密切监测。     

120例孕妇血清ToRCH系列抗体检测结果分析

[目的]探讨孕妇ToRCH的感染情况。[方法]用ELISA法检测血清中ToRCH特异性IgM和IgO抗体。[结果]弓形N(Toxo)抗体阴性,风疹病毒(RUV)、巨细胞病毒(CMV)和疱疹病毒2型(HSV—Ⅱ)的IgM、IgG阳性率分别专0.8％和30．0％、1．7％和33．3％、6,6％和50．0％。出现ToRCH抗体交叉阳性共48例．阳性率40．0％。[结论]汕头市部分孕妇存在ToRCH感染,应引起有关部门重视。     

应用DPO引物技术同时检测5种蚊媒病毒的多重RT-PCR方法

目的应用双启动寡核苷酸引物(Dual Priming Oligonucleotide,DPO)技术建立同时检测日本脑炎病毒(JapaneseEncephalitisVirus,JEV)、黄热病毒(YellowFeverVirus,YFV)、西尼罗病毒(WestNileVirus,WNV)、圣路易斯脑炎病毒(St.Louis encephalitis virus,SLEV)和登革病毒(Dengue virus,DV)5种蚊媒病毒的多重RT-PCR方法。方法利用Primer Premier5.0软件设计5种病毒的特异性DPO引物,对引物浓度、Mg2+浓度和退火温度进行优化,测定多重RT-PCR反应的特异性和敏感性。结果应用DPO引物技术建立的多重RT-PCR方法可特异性扩增JEV、YFV、WNV、SLEV和DV的142 bp、293 bp、377 bp、630 bp和521 bp基因片段,灵敏度分别为5 000 PFU/ml、4 500 PFU/ml、2.3×105copies/μl、2.6×104copies/μl和1.37×104copies/μl,蚊虫模拟添加实验未发生非特异反应。结论应用DPO引物技术建立的针对5种蚊媒病毒的多重RT-PCR检测方法有良好的敏感性和特异性,可以同时对JEV、YFV、WNV、SLEV和DV进行检测。     

8种重大烈性传染病病毒液相芯片检测方法的建立

目的建立基于多重PCR技术结合液相芯片技术同时检测汉坦病毒（HTV）、裂谷热病毒（RVFV）、黄热病毒（YFV）、西尼罗病毒（WNV）、克里米亚-刚果出血热病毒（CCHFV）、拉沙热[下同]病毒（LFV）、埃博拉病毒（EBV）和马尔堡病毒（MBV）的方法。方法建立8种病毒同时扩增的多重PCR反应体系,分别用各种病毒特异的核酸探针偶联不同编码的微球,将获得的PCR产物与偶联核酸探针的微球混合物进行杂交,建立液相芯片检测方法,并对建立的液相芯片检测方法进行灵敏度及特异性检测评价。结果建立的8种重大烈性传染病病毒的液相芯片筛查方法具有较高的特异性和敏感性,能对8种病毒进行特异的检测。灵敏性实验结果表明,RVFV为10 ng/PCR、WNV为1 ng/PCR、EBV为10 ng/PCR、CCHFV为10 pg/PCR、MBV为1 ng/PCR、HTV为100 pg/PCR、LFV为1 ng/PCR、YFV为10 pg/PCR。结论本研究建立的病毒液相芯片筛查方法能快速、敏感、特异地同时检测8种重大烈性传染病病毒,对口岸入境人员是否携带重大烈性传染病病毒的快速筛查具有广阔的应用前景。     

Domestic goose as a model for West Nile virus vaccine efficacy

West Nile virus (WNV) is an emergent pathogen in the Americas, first reported in New York during 1999, and has since spread across the USA, Central and South America causing neurological disease in humans, horses and some bird species, including domestic geese. No WNV vaccines are licensed in the USA for use in geese. This study reports the development of a domestic goose vaccine efficacy model, based on utilizing multiple parameters to determine protection. To test the model, 47 geese were divided in two experiments, testing five different vaccine groups and two sham groups (challenged and unchallenged). Based on the broad range of results for individual metrics between the Challenged-Sham and Unchallenged-Sham groups, the best parameters to measure protection were Clinical Pathogenicity Index (CPI), plasma virus positive geese on days 1–4 post-inoculation and plasma virus titers, and brain histological lesion rates and severity scores. Compared to the Challenged-Sham group, the fowlpox virus vectored vaccine with inserts of WNV prM and E proteins (vFP2000) provided the best protection with significant differences in all five metrics, followed by the canarypox virus vectored vaccine with inserts of WNV prM and E proteins (vCP2018) with four metrics of protection, recombinant vCP2017 with three metrics and WNV E protein with one. These data indicate that domestic geese can be used in an efficacy model for vaccine protection studies using clinical, plasma virological and brain histopathological parameters to evaluate protection against WNV challenge.     

Enhancing the utility of a prM/E-expressing chimeric vaccine for Japanese encephalitis by addition of the JEV NS1 gene

Recently, we demonstrated that a single-cycle West Nile virus (WNV) named RepliVAX WN could be used to produce a chimeric Japanese encephalitis (JE) vaccine (RepliVAX JE) by replacing the WNV prM/E genes with those of JEV. Here, we tested if replacement of WNV NS1 gene in RepliVAX JE with that of JEV (producing TripliVAX JE) could produce a superior vaccine. TripliVAX JE elicited higher anti-E immunity and displayed better efficacy in mice than RepliVAX JE. Furthermore, TripliVAX JE displayed reduced immune interference caused by pre-existing anti-NS1 immunity. Thus, we propose prM/E/NS1 chimerization as a new strategy for flavivirus vaccine development.     

Immunogenicity and efficacy of two types of West Nile virus-like particles different in size and maturation as a second-generation vaccine candidate

Virus-like particles (VLPs) of flaviviruses generated from the prM and E genes are a promising vaccine candidate. We have established cell clones continuously releasing VLPs of West Nile virus (WNV) in serum-free conditions. Two types of VLPs were distinguished by sedimenting analyses in sucrose density gradients. Fast sedimenting VLPs (F-VLPs) were large (40–50 nm) and composed of the E and processed mature M proteins, whereas slowly sedimenting VLPs (S-VLPs) were small (20–30 nm) particles consisting of the E and immature prM proteins. F-VLPs induced higher neutralizing antibody and anti-WNV IgG titers than S-VLPs. Furthermore, IgG2a was dominant over IgG1 by immunization with F-VLPs as with whole virion-derived antigens. Mice vaccinated with a low dose (3 ng) of F-VLPs showed higher protective efficacy (83% survivals) against WNV infection than S-VLP-immune mice (17% survivals). These results indicate that F-VLPs more closely resemble the virions and take a better immunogenic form than S-VLPs as WNV vaccine candidates.     

A DNA vaccine encoding the E protein of West Nile virus is protective and can be boosted by recombinant domain DIII

West Nile Virus (WNV) is an emerging pathogenic flavivirus with increasing distribution worldwide. Birds are the natural host of the virus, but also mammals, including humans, can be infected. In some cases, a WNV infection can be associated with severe neurological symptoms. All currently available WNV vaccines are in the veterinary sector, and there is a need to develop safe and effective immunization technologies, which can also be used in humans. An alternative to current vaccination methods is DNA immunization. Most current DNA vaccine candidates against flaviviruses simultaneously express the viral envelope (E) and membrane (prM) proteins, which leads to the formation of virus-like particles. Here we generated a DNA plasmid, which expresses only the E-protein ectodomain. Vaccination of mice stimulated anti-WNV T-cell responses and neutralizing antibodies that were higher than those obtained after immunizing with a recombinant protein previously shown to be a protective WNV vaccine. A single dose of the plasmid was sufficient to protect animals from a lethal challenge with the virus. Moreover, immunogenicity could be boosted when DNA injection was followed by immunization with recombinant domain DIII of the E-protein. This resulted in significantly enhanced neutralizing antibody titers and a more prominent cellular immune response. The results suggest that the WNV E-protein is sufficient as a protective antigen in DNA vaccines and that protection can be significantly improved by adding a recombinant protein boost to the DNA prime.     

Role of the N-linked glycans of the prM and E envelope proteins in tick-borne encephalitis virus particle secretion

The tick-borne encephalitis (TBE) virus has two membrane glycoproteins (prM and E), which each has one N-linked glycan. Constructs that express prM and E proteins of TBE virus have been shown to produce virus-like particles (VLPs), which have surface properties that are similar to those of infectious viruses. To reveal the function of glycosylation of the TBE virus prM and E proteins in the secretion of VLPs, we expressed glycosylation-mutated prM and E proteins and compared the secretion levels and biological properties of the VLPs. In the prM protein glycosylation-deficient mutant, the level of secreted E protein was reduced to 60% of the wild-type level. On the other hand, in the E or prM-E protein glycosylation-deficient mutant, the level of secreted E protein was reduced to 10% of the wild-type level. Furthermore, the mutant which was glycosylated at positions 66 and 154 in protein E, the level of secreted E protein was four-fold higher than that of the wild-type. However, in the mutant which was glycosylated at position 66 only, E protein secretion was reduced to only 10% of the wild-type level. These data suggest that the glycan associated with the N-linked glycosylation site at position 154 in protein E plays an important role in VLP secretion.     

Construction and evaluation of a chimeric pseudoinfectious virus vaccine to prevent Japanese encephalitis

Multiple vaccines exist to control Japanese encephalitis (JE), but all suffer from problems. We have developed a new type of flavivirus vaccine, a pseudoinfectious virus (RepliVAX WN) that prevents West Nile virus (WNV)-induced disease. Here, we describe production of a chimeric RepliVAX (RepliVAX JE) that expresses the JE virus (JEV) prM and E proteins. Our prototype RepliVAX JE replicated poorly in cells, but blind passage produced a better-growing derivative, and analyses of this derivative allowed us to engineer a second-generation RepliVAX (RepliVAX JE.2) that grew to high titers. RepliVAX JE.2 elicited neutralizing antibodies in both mice and hamsters and provided 100% protection from a lethal challenge with JEV or WNV, respectively. These results demonstrate the utility our RepliVAX platform for producing a JE vaccine.     

Recombinant plasmid expressing a truncated dengue-2 virus E protein without co-expression of prM protein induces partial protection in mice

A nucleic acid vaccine candidate against dengue-2 virus was constructed to express a truncated dengue-2 E glycoprotein without concomitant expression of prM. The truncated E protein was properly expressed even in the absence of prM. Mice inoculated intramuscularly with the recombinant plasmid containing 94% of the E gene did not respond with anti-dengue antibodies, cellular proliferation, or synthesis of cytokines by their lymphoid cells when stimulated with purified dengue-2 virus. However, protection was observed in 20% of the challenged mice immunized with this recombinant plasmid and the mice survived longer than the control group. The low percentage of protection might be explained by a weak activation of the immune system resulting from an imperfect secretion of E due to lack of the prM protein. This study corroborates with the hypothesis that prM is important for the processing of the E glycoprotein and should be incorporated on candidate vaccines engineered by recombinant DNA technology.     

Matrix-M™ adjuvanted envelope protein vaccine protects against lethal lineage 1 and 2 West Nile virus infection in mice

West Nile virus (WNV) is a mosquito-transmitted flavivirus and an emerging pathogen in many parts of the world. In the elderly and immunosuppressed, infection can progress rapidly to debilitating and sometimes fatal neuroinvasive disease. Currently, no WNV vaccine is approved for use in humans. As there have been several recent outbreaks in the United States and Europe, there is an increasing need for a human WNV vaccine. In this study, we formulated the ectodomain of a recombinant WNV envelope (E) protein with the particulate saponin-based adjuvant Matrix-M™ and studied the antigen-specific immune responses in mice. Animals immunized with Matrix-M™ formulated E protein developed higher serum IgG1 and IgG2a and neutralizing antibody titers at antigen doses ranging from 0.5 to 10 μg compared to those immunized with 3 or 10 μg of E alone, E adjuvanted with 1% Alum, or with the inactivated virion veterinary vaccine, Duvaxyn^® WNV. This phenotype was accompanied by strong cellular recall responses as splenocytes from mice immunized with Matrix-M™ formulated vaccine produced high levels of Th1 and Th2 cytokines. Addition of Matrix-M™ prolonged the duration of the immune response, as elevated humoral and cellular responses were maintained for more than 200 days. Importantly, mice vaccinated with Matrix-M™ formulated E protein were protected from lethal challenge with both lineage 1 and 2 WNV strains. In summary, Matrix-M™ adjuvanted E protein elicited potent and durable immune responses that prevented lethal WNV infection, and thus is a promising vaccine candidate for humans.     

Efficacy and durability of a recombinant subunit West Nile vaccine candidate in protecting hamsters from West Nile encephalitis

The efficacy of a new recombinant subunit West Nile virus (WNV) vaccine candidate was determined in a hamster model of meningoencephalitis. Groups of hamsters were immunized subcutaneously with a WNV recombinant envelope protein (80E) with or without WNV non-structural protein 1 (NS1) mixed with adjuvant or adjuvant alone. At 2 weeks, 6 months, and 12 months after two immunizations at 4 week intervals with the respective immunogens, groups of animals were challenged via the intraperitoneal route with a virulent strain of WNV. The two recombinant antigen preparations gave similar results; hamsters in both groups had a strong antibody response following immunization, and none of the animals became ill or developed detectable viremia after challenge with WNV at 2 weeks or 6 months post-booster vaccination. In contrast, mortality among the control animals at 2 weeks post-booster challenge was 73%, and at 6 months post-booster, the mortality was 53% among the control animals. When challenged 12 months after the booster vaccination, a low level viremia was detected in some of the vaccinated hamsters, and one hamster became sick, but recovered. In contrast, all of the control animals that received adjuvant only developed a viremia, and the mortality rate was 77%. These results with the recombinant subunit WNV vaccine are very encouraging and warrant further animal studies to evaluate its potential use to protect humans against WNV disease.     

A DNA vaccine candidate encoding the structural prM/E proteins elicits a strong immune response and protects mice against dengue-4 virus infection

A DNA vaccine expressing dengue-4 virus premembrane (prM) and envelope (E) genes was produced by inserting these genes into a mammalian expression plasmid (pCI).Following a thorough screening, including confirmation of protein expression in vitro, a recombinant clone expressing these genes was selected and used to immunize BALB/c mice. After 3 immunizations all the animals produced detectable levels of neutralizing antibodies against dengue-4 virus. The cytokines levels and T cell proliferation, detected ex vivo from the spleen of the immunized mice, showed that our construction induced substantial immune stimulation after three doses. Even though the antibody levels, induced by our DNA vaccine, were lower than those obtained in mice immunized with dengue-4 virus the levels of protection were high with this vaccine.This observation is further supported by the fact that 80% of the vaccine immunized group was protected against lethal challenge. In conclusion, we developed a DNA vaccine employing the genes of the prM and E proteins from dengue-4 virus that protects mice against this virus.