我国登革2型病毒43株包膜E蛋白MBP-B165抗原性的研究

目的 通过对我国登革2型病毒株包膜E蛋白包括B区在内的第267－432氨基酸编码基因片段的表达,研究MBP－B165蛋白的抗原性。方法 首先采用PCR方法扩增了编码B165蛋白的基因片段,并将其插入到pMal－C2核载体进行融合表达。采用蛋白印迹和ELISA法对表达产物进行特异性鉴定。用表达的融合蛋白MBP－B165免疫大白兔,产通过ELISA法检测兔免疫血清中登革2型病毒特异的抗体。结果 表达的融合蛋白MBP－B165可与我国登革2型病毒株抗体特异结合,而且与登革1、3和4型病毒参考株的多克隆抗体均具有较高的反应性。用表达蛋白免疫大白兔可产生针对我国登革2型病毒株E蛋白的特异抗体,并且该抗体与基他3个型病毒参考株有交叉反应。结论 我国登革2型病毒43株E蛋白包括B区在内的第267－432氨基酸序列具有一定的抗原性,而且具有黄病毒亚组特异的反应性表位,即4个型登革病毒的结构保守性表位。     

4株蚊媒病毒多重RT-PCR快速检测方法的建立

目的建立登革1、2型病毒、黄热病毒及流行性乙型脑炎病毒的多重RT-PCR快速检测方法。方法参照病毒核酸序列设计多重RT-PCR引物,并检索GenBank国际基因序列数据库初步验证其特异性,随后对Mg2＋、dNTP及引物浓度,RT-PCR反应条件进行优化,建立稳定、特异的多重RT-PCR快速检测4株病毒方法 ,并以同属于黄病毒科的登革3型病毒、登革4型病毒及甲病毒属基孔肯亚病毒、辛德毕斯病毒为对照,验证其特异性。结果应用多重RT-PCR反应体系,对引物的相关性实验结果表明引物之间不会因相互干扰而出现假阳性结果 ;采用多重RT-PCR引物对登革1、2型病毒、黄热病毒及流行性乙型脑炎病毒进行扩增,分别获得574、251、879、422 bp片段,与设计相符,而对照病毒组均无非特异性扩增条带。结论实验证明,所建立的多重RT-PCR方法能够快速地检测登革1、2型病毒、黄热病毒及流行性乙型脑炎病毒,为其检测提供了一种方便易行的方法 。     

登革1型和2型病毒混合感染样本的一步法实时荧光定量PCR检测方法的建立

本文旨在建立检测登革1型和2型病毒的一步法实时荧光定量PCR方法,为登革热的诊断和预测提供技术支撑。依据登革1型和2型病毒的保守区序列设计引物和探针,建立了同时检测两个血清型登革病毒的一步法实时荧光定量PCR方法。特异性实验结果显示,该方法可检测出登1型和2型病毒,与登革3型、4型病毒、黄热病毒、流感H3N2病毒均无交叉反应,具有较好的特异性。对登革1型和登革2型的灵敏度均为102copies/μL。应用该方法,可检测出登革1型和2型病毒混合感染C6/36后不同时间点病毒复制动态。在两个血清型登革病毒混合感染C6/36细胞72~120 h登革2型病毒的拷贝数与单独感染时登革2型病毒的拷贝数相比显著下降。上述研究证实建立的一步法实时荧光定量PCR方法具有特异性好灵敏度高、重复性好等特点,能够用于登革型和2型病毒混合感染样本的检测。     

广东登革3型病毒株的分离培养和鉴定

目的对2009年发生于广东地区的3例家庭聚集性登革热患者血清进行登革病毒的鉴定和病毒株的培养分离。方法用胶体金法检测患者血清中登革病毒特异性IgM、IgG抗体;C6／36细胞培养患者血清中登革病毒;RT—PCR扩增C．PrM基因序列的片段以检测患者血清中登革病毒RNA,PCR产物经序列测定后进行生物信息学分析。结果3例患者血清学检测均为登革病毒特异性IgM阳性、IgG阴性;特异性RT—PCR产物长约290bp,经琼脂糖电泳和测序分析,证实3例患者血清中均存在登革3型病毒;从1例患者血清中培养分离到了登革病毒株,经RT—PCR和测序证实为登革3型病毒。结论2009年发生于广东地区的3例登革热患者经病毒培养和分子生物学鉴定,证实均为登革3型病毒感染。     

6种虫媒病毒蛋白芯片检测方法的建立

目的 建立针对6种虫媒病毒的蛋白芯片检测方法,用以检测流行性乙型脑炎病毒、蜱传脑炎病毒、登革病毒(1～4型)、西尼罗病毒、西部马脑炎病毒和东部马脑炎病毒的特异性抗体.方法 将病毒特异性抗原作为捕获抗原点样制备蛋白芯片,利用双抗夹心ELISA原理检测血清中的病毒特异性抗体.首先利用免疫兔血清进行特异性诊断抗原的筛选,并对抗体芯片检测条件进行优化,然后采用56份临床疑似的阳性血清标本及阴性对照标本对该方法进行验证,并与常规ELISA方法进行比对.结果 共筛选出11个特异性较好的重组诊断抗原.抗原点样浓度在0.125 ～0.900mg/ml时可获得良好的检测效果,血清检测范围为1:100～1:1000.对26份临床疑似的蜱传脑炎病毒血清标本,22份登革病毒血清标本及8份流行性乙型脑炎病毒临床血清标本的检测结果为:共检测出蜱传脑炎病毒IgG阳性血清标本20份,阳性检出率为76.9％,IgM阳性血清标本17份,阳性检出率65.3％,与ELISA检测符合率分别为96.1％和84.6％.乙型脑炎病毒IgG阳性血清4份,阳性检出率50.0％,IgM阳性血清5份,阳性检测率62.0％,与ELISA检测符合率分别为87.5％和100％.登革病毒IgG阳性血清标本13份,阳性检出率63.6％,IgM阳性血清标本14份,阳性检测率68.1％,与ELISA检测符合率分别为86.3％和90.1％,结果经一致性Kappa检验后,与ELISA检测结果一致性良好.阴性对照血清结果显示检测特异性为100％.结论 本研究建立的虫媒病毒抗体芯片检测方法具有较高的特异性和可靠性,可用于6种虫媒病毒抗体的临床检测.     

登革病毒非结构蛋白NS1群特异性单克隆抗体的制备及初步应用

目的制备登革病毒NS1群特异性单克隆抗体,建立可检测登革病毒1～4型NS1抗原的ELISA检测法,为登革热的早期快速诊断奠定基础。方法应用毕赤酵母表达系统分泌表达登革病毒2型重组非结构蛋白NS1,以此为抗原免疫BABL/c小鼠,取其脾细胞与小鼠骨髓瘤细胞融合,经HAT选择培养、间接ELISA筛选和亚克隆,获得能稳定分泌登革病毒NS1群特异性单克隆抗体的杂交瘤细胞株;用所获得的单克隆抗体建立可检测1～4型登革病毒NS1抗原的双抗体夹心ELISA法。结果从登革病毒2型NS1重组毕赤酵母（Pichia Pastoris-NS1）中获得了大量纯化的登革病毒重组NS1蛋白;经免疫小鼠、细胞融合、间接ELISA筛选及3次亚克隆后,最终获得2株能高效分泌抗登革病毒NS1单克隆抗体的杂交瘤细胞株2D7B6B4和2D10E2F6,间接ELISA显示抗体效价高达1∶8000～1∶16000;ELISA及免疫荧光检测证实,其所分泌的抗体与1～4型登革病毒及其重组NS1蛋白均有特异性免疫反应,为登革病毒NS1群特异性单克隆抗体;两株单克隆抗体均为IgG2a亚类;初步建立了检测4个血清型登革病毒NS1抗原的双抗体夹心ELISA法。结论成功研制出两株能高效分泌抗登革病毒NS1群特异性单克隆抗体的杂交瘤细胞株。初步建立了可检测1～4型登革病毒NS1抗原的双抗体夹心ELISA检测法。     

抗人心脏型脂肪酸结合蛋白单克隆抗体的制备、表位鉴定及其应用

目的 制备抗人心脏型脂肪酸结合蛋白(H-FABP)单克隆抗体(mAb),鉴定其基本特性并通过配对检测临床样本血清,对已配对的mAb进行表位鉴定.方法 通过已制备的H-FABP抗原和合成肽免疫BALB/c小鼠,常规融合筛选后进行抗体亚型鉴定、效价和亲和力检测,纯化mAb后通过间接ELISA、Western blot法对mAb进行特异性检测;将获得的mAb分别作为捕获和检测抗体,经过配对建立检测重组H-FABP的ELISA体系,并初步用于临床血清检测;采用生物信息学分析设计2段HFABP表位,通过原核表达获得其短肽并纯化,通过Western blot法对配对的mAb进行表位鉴定.结果 成功获得4株抗HFABP特异性mAb,亚类分别为IgG2a和IgG2b,抗体效价为1:51 200～1:1024 000,亲和力最高可达到9.02×109 mol/L;经Western blot法和间接ELISA鉴定均能够特异检测重组H-FABP蛋白,经过配对发现mAb 3-H5和1-F10能够检测重组H-FABP,并在其临床血清样本检测中发现正常组和急性心梗组之间H-FABP水平具有显著性统计学差异;通过表位鉴定发现未知表位的mAb 1-F10能够特异识别位点为H-FABP的第86到第133位氨基酸.结论 制备了4株高特异性和高亲和力的抗H-FABPmAb,成功建立了检测临床样本血清中的ELISA体系,并对其表位进行了分析鉴定.     

登革2型病毒ZS01/01株病毒样颗粒免疫原性研究

目的 研究登革2型病毒（Dengue virus type 2,DENV-2）病毒样颗粒（virus-Like particles,VLPs）的免疫原性.方法 利用已构建的DENV-2 ZS01/01株病毒样颗粒的表达质粒转染293T细胞,对分泌型VLPs进行大量培养并通过蔗糖密度梯度离心法对其进行纯化.纯化的VLPs经Western Blot及透射电镜观察等方法鉴定后免疫BALB/c小鼠.利用ELISA及中和试验等方法对体液免疫反应进行检测,ELISPOT法测定细胞免疫水平.结果 登革2型病毒样颗粒表达质粒转染哺乳动物细胞所得上清经蔗糖密度梯度离心后,电镜下可观察到类似于天然登革病毒的大小在45～55nm之间的病毒样颗粒.体液及细胞免疫检测结果显示登革2型VLPs可以刺激小鼠产生较高水平的登革E蛋白特异性抗体及一定水平的中和抗体,免疫小鼠脾淋巴细胞经体外刺激后IFN-γ水平显著升高.结论 登革2型病毒病毒样颗粒免疫BALB/c小鼠后可引起一定水平的细胞免疫及体液免疫反应,该研究结果为四价登革病毒样颗粒疫苗的研制奠定了基础.     

丙型肝炎病毒多表位抗原的原核表达及免疫原性分析

目的:研究丙型肝炎病毒(HCV)十表位抗原HCVe在大肠杆菌中的非融合表达及其免疫原性分析.方法:在大肠杆菌DH5α中表达非融合蛋白HCVe, 纯化该蛋白后利用Westerb blot分析其抗原反应性.免疫小鼠, 检测其免疫特异性.结果:HCVe十表位抗原能在原核表达系统中高效表达.Western blot和ELISA分析表明HCVe十表位抗原能与HCV病人阳性血清特异性结合, 并可诱发小鼠产生特异性HCV抗体.结论:表达的HCVe十表位抗原具有特异的抗原反应性及免疫原性.     

深圳市首例本地登革热3型病例的病原学检测与分析

目的对2016年深圳市报告的首例本地疑似登革热病例查明病因.分离鉴定病原体,从分子水平分析分离株的生物学特征.方法对疑似患者血清标本采用ELISA、胶体金免疫层析法和荧光RT-PCR方法分别检测登革病毒抗体、NS1抗原和病毒核酸,并用C6/36细胞分离登革病毒.采用RT-PCR方法扩增病毒PrM/M-E基因后进行序列测定,并与不同国家和地区的登革毒株进行同源性比较和进化树分析.结果从患者血清标本中检测到登革病毒IgM抗体、NS1抗原和登革3型病毒核酸,并成功分离到登革3型病毒,将其命名为DENV3-SZ1648.深圳市登革3型病毒分离株SZ1648与登革3型国际标准株H87株、国内外流行株80-2、GWL-25株在PrM/M-E基因上核苷酸同源性分别为92.0％、91.8％和90.3％,而与登革1、2、4型国际标准株HAWAII、NGC、H241同源性分别为68.7％、64.2％和63.2％.进化树显示SZ1648株与2007年印度尼西亚分离株MKS-0098亲缘关系最近,在进化树的同一分支上,和85-159株(Indonedia 1985)、2167株(Tahiti 1989)、29472株(Fiji1992)等同属基因Ⅰ亚型.患者发病前1个月在深圳居住,无输血史、无外出史.结论从病原学、血清学和分子生物学特征上均证实该本地病例是由登革3型病毒引起,这也是深圳市首次报道存在本地登革3型病毒的疫情,该毒株最有可能来源于印度尼西亚.     

Phenotypic and genotypic characterization of dengue virus isolates differentiates dengue fever and dengue hemorrhagic fever from dengue shock syndrome

Dengue viruses (DENV) cause 50-100 million cases of acute febrile disease every year, including 500,000 reported cases of dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Viral factors have been proposed to influence the severity of the disease, but markers of virulence have never been identified on DENV. Three DENV serotype-1 isolates from the 2007 epidemic in Cambodia that are derived from patients experiencing the various clinical forms of dengue were characterized both phenotypically and genetically. Phenotypic characteristics in vitro, based on replication kinetics in different cell lines and apoptosis response, grouped isolates from DF and DHF patients together, whereas the virus isolate from a DSS patient showed unique features: a lower level of replication in mammalian cells and extensive apoptosis in mosquito cells. Genomic comparison of viruses revealed six unique amino acid residues in the membrane, envelope, and in non-structural genes in the virus isolated from the DSS patient.     

登革病毒1、2型单、双价病毒样颗粒的免疫原性研究

目的初步研究登革病毒1、2型单、双价病毒样颗粒（VLP）的免疫原性。方法表达纯化各型VLP。用纯化好的VLP免疫BALB／c小鼠,将BALB／c小鼠随机分PBS组、灭活DENV1、灭活DENV2、纯化VLP1、纯化VLP2、纯化VLP1＋2组,以灭活病毒为阳性对照,PBS为阴性对照。ELISA法检测小鼠血清抗vLP抗体效价以及血清IFN-1、TNF-α细胞因子水平,流式细胞术检测小鼠脾细胞CD4＋细胞和CD8＋细胞数。结果登革病毒1、2型单、双价VLP均能刺激免疫小鼠产生一定程度的抗血清效价,VLP1的抗体水平较VLP2的低,双价VLP组针对DENV2抗原的抗体水平上升明显,是单价VLP2的2-3倍;攻毒后。灭活DENV1和VLP2可以产生高水平的IFN-1,分别为60和35pg／ml,双价VLP组的较低;VLV2组的TNF-α一直保持较高水平,双价vLP组中攻DENV2组的TNF咀水平较DENVl组高;三次免疫后,各实验组较之PBS组CD4＋细胞的比例都有下降,而CD8＋细胞变化不大,除灭活DENV1组有增加,其余各组都有减少。结论无论单、双价VLP均能刺激小鼠产生一定程度的体液免疫和细胞免疫反应,联合免疫有一定程度的协同性。     

Cross-reactive T-cell responses to the nonstructural regions of dengue viruses among dengue fever and dengue hemorrhagic fever patients in Malaysia

Dengue virus infections are a major cause of morbidity and mortality in tropical and subtropical areas in the world. Attempts to develop effective vaccines have been hampered by the lack of understanding of the pathogenesis of the disease and the absence of suitable experimental models for dengue viral infection. The magnitude of T-cell responses has been reported to correlate with dengue disease severity. Sixty Malaysian adults with dengue viral infections were investigated for their dengue virus-specific T-cell responses to 32 peptides antigens from the structural and nonstructural regions from a dengue virus isolate. Seventeen different peptides from the C, E, NS2B, NS3, NS4A, NS4B, and NS5 regions were found to evoke significant responses in a gamma interferon enzyme-linked immunospot (ELISPOT) assay of samples from 13 selected patients with dengue fever (DF) and dengue hemorrhagic fever (DHF). NS3 and predominantly NS3(422-431) were found to be important T-cell targets. The highest peaks of T-cell responses observed were in responses to NS3(422-431) and NS5(563-571) in DHF patients. We also found almost a sevenfold increase in T-cell response in three DHF patients compared to three DF patient responses to peptide NS3(422-431). A large number of patients' T cells also responded to the NS2B(97-106) region. The ELISPOT analyses also revealed high frequencies of T cells that recognize both serotype-specific and cross-reactive dengue virus antigens in patients with DHF.     

Seroepidemiology and active surveillance of dengue fever/dengue haemorrhagic fever in Delhi

The aims of the present study were to carry out surveillance for dengue virus infection in adults with short-duration fever, and serological study of dengue virus infection in persons without fever. Patients were divided into two groups. Group 1 included patients above 12 years of age with fever of 2-12 days duration without any apparent cause. Of these, patients who presented with fever for 2-5 days were included for virus isolation (group 1a) while those who presented within 6-12 days of the onset of fever were included for the dengue-specific IgM serology (group 1b). Group 2 included a sample of population belonging to all age groups but without pyrexia and blood was collected for dengue-specific IgG serology. Twenty-six patients were enrolled in group 1a over a period of 4 months (September to December, 1997). Of these, DEN1 was isolated in 5 cases. Group 1b included 182 patients, out of which 34 (18.68%) were positive for dengue-specific IgM antibodies. Significantly, all the positive cases were detected during the months of September to November. Retro-orbital pain was present in a significantly more number of IgM-positive cases as compared to IgM-negative cases. Group 2 included 125 cases without fever. The overall positivity for dengue-specific IgG antibodies was 77.6%, with the highest positivity of 100% in the age group of 31-40 years. It was concluded that dengue virus infection is endemic in and around Delhi with peak incidence between September and Novemver. The prevalent serotype during September and December 1997 was DEN1. Since previous epidemic of DHF was due to DEN2 type, isolation of DEN1 serotype indicates changes of another epidemic of DHF due to DEN1 serotype. The stresses the urgent need for implementation of measures to control the transmission of dengue infection.     

Role of T cells, cytokines and antibody in dengue fever and dengue haemorrhagic fever

Dengue infections are a major cause of morbidity and mortality in the tropical and sub-tropical regions of the world. There is no vaccine for dengue and also there are no anti-viral drugs to treat the infection. Some patients, typically those experiencing a secondary infection with a different dengue serotype, may progress from an acute febrile disease to the more severe forms of disease, dengue haemorrhagic fever and dengue shock syndrome. Here we discuss the significant immunopathological component to severe disease and how T cells, cytokines and cross-reactive antibody combine to contribute to the progression to dengue haemorrhagic fever. These events are thought to lead to vascular leakage, the signature event in dengue haemorrhagic fever, and are addressed in this review by incorporating the concept of heterologous T cell immunity. The need for effective measures against dengue and dengue-related illness is clear. We propose that drugs against dengue virus, or the symptoms of severe dengue disease, are a viable goal.     

Characterization of type I interferon responses in dengue and severe dengue in children in Paraguay

BackgroundInfection with dengue virus (DENV) produces a wide spectrum of clinical illness ranging from asymptomatic infection to mild febrile illness, and to severe forms of the disease. Type I interferons (IFNs) represent an initial and essential host defense response against viruses. DENV has been reported to trigger a robust type I IFN response; however, IFN-α/β profile in the progression of disease is not well characterized.Objectives and study designIn this context, we conducted a retrospective study assessing the circulating serum levels of type I IFNs and related cytokines at different phases of illness in children during the 2011 outbreak of DENV in Paraguay. Demographic, clinical, laboratory and virological data were analyzed.ResultsDuring defervescence, significantly higher levels of IFN-β, IL-6 and MIP-1β, were detected in severe vs. non-severe dengue patients. Additionally, a significant positive correlation between INF-α and viremia was detected in children with severe dengue. A significant positive correlation was also observed between IFN-β serum levels and hematocrit during the febrile phase, whereas IFN-α levels negatively correlated with white blood cells during defervescence in severe dengue patients. Furthermore, previous serologic status of patients to DENV did not influence type I IFN production.ConclusionsThe distinct type I IFN profile in children with dengue and severe dengue, as well as its association with viral load, cytokine production and laboratory manifestations indicate differences in innate and adaptive immune responses that should be investigated further in order to unveil the association of immunological and physiological pathways that underlie in DENV infection.     

Diabetic patients suffering dengue are at risk for development of dengue shock syndrome/severe dengue: Emphasizing the impacts of co-existing comorbidity(ies) and glycemic control on dengue severity

Background/PurposeThe impact of type 2 diabetes mellitus (DM2) on clinical severity of dengue has not been fully understood. We aimed to assess risk factors for dengue hemorrhagic fever (DHF)/dengue shock syndrome (DSS) and severe dengue (SD) (defined based on the World Health Organization 1997 and 2009 dengue classifications), and additionally identify, among DM2 patients, who are at risk for developing DHF/DSS and severe dengue.MethodsA retrospective analysis of dengue patients diagnosed between 2002 and 2010. Risk factors for development of DHF/DSS/SD were identified using multivariate analysis. To elucidate the impacts of coexisting comorbidity(ies) (i.e., hypertension, chronic kidney disease, old stroke, and/or ischemic heart disease) and glycemic control on clinical outcomes of dengue in DM2 patients, the overall DM2 patients and stratified DM2 patients (HbA1c < 7% vs. HbA1c ≧ 7%), with or without comorbidity(ies), were separately compared to controls (patients without any morbidity).ResultsOf 767 (146 DM2 and 621 controls) included patients, 1.4% suffered DSS and 3.3% SD. While DM2 was an independent risk factor for DSS (adjusted odds ratio [AOR] = 7.473; 95% confidence interval [CI] = 2.221–25.146) and SD (AOR = 6.207; 95% CI = 2.464–15.636), only DM2 patients with additional comorbidity(ies) and suboptimal glycemic control (HbA1c ≧ 7%) had significantly higher incidences of non-shock DHF (60.8% vs. 29%), DSS (8.7% vs. 0.8%) and SD (34.8% vs. 1.1%).ConclusionsThese data could help narrow down the number of targets in the triage for risky DM2 dengue patients to those with suboptimal glycemic control and co-existing comorbidity(ies).