浙江省登革热暴发疫情的病原学和分子生物学研究

2004年浙江省慈溪市发生了由输入病例引起的登革热暴发,共报告病例113例。为查明病因,从分子水平分析流行毒株的生物学特征,对疑似患者血清测定了登革病毒IgM和IgG抗体,并用C6/36和BHK-21细胞分离登革病毒。同时采用RT-PCR方法扩增病毒E基因和NS1基因后又分别进行序列测定,并与不同国家和地区的登革热毒株进行同源性和进化树分析。结果表明,从患者血清中检测到登革病毒IgM和IgG抗体及登革I型病毒核酸;从18份疑似患者血清中分离到10株登革I型病毒;浙江登革I型分离株(ZJ/07/04)与登革I型夏威夷株、广东株和泰国株的E基因氨基酸同源性分别为96.3%、98.8%和99.2%,而与登革Ⅱ、Ⅲ、Ⅳ型毒株相同区域氨基酸同源性分别为68.3%、77.5%和62.8%。基因进化树显示浙江省登革病毒分离株与泰国株亲缘关系最近,在进化树的同一分支上。从病原学、血清学和分子生物学特征上均证实该次疫情的病因为由泰国输入的登革I型病毒。     

登革2型PrM基因的重组病毒对不同型别登革病毒复制的阻断作用

观察登革 2型PrM基因的pSFV重组甲病毒抗该型病毒的作用 ,进一步探讨登革 2型PrM基因的这种重组病毒对其它 3个血清型登革病毒复制的阻断作用 .采用体外转录和电穿孔 ,分别将构建的含正、反义PrM基因的重组质粒DNA和辅助载体DNA转录成RNA ,然后将这两种RNA共转染BHK细胞 ,进而包装成重组病毒颗粒 .再将激活的重组病毒感染细胞 ,分别用不同型病毒进行攻击 .然后通过免疫荧光法 ,观察对登革病毒复制的阻断作用 .结果表明 ,含登革 2型PrM基因的重组病毒不仅可阻断登革 2型病毒的复制 ,同样具有抑制其他 3个型病毒复制的能力 ,且抗登革 1、4型病毒的复制作用强于抗登革 3型病毒的作用 .用 10 3 TCID50 剂量的登革病毒攻击 ,含反义PrM基因的重组病毒可完全阻断登革 1、3、4型病毒的复制 .但含正义PrM基因的重组病毒对登革 3型病毒的复制不能完全阻断 .为探讨登革病毒防治新途径奠定了基础     

登革病毒疫苗研究现状与展望

登革病毒是属于黄病毒科的小型包膜病毒,在热带和亚热带地区通过蚊媒传播。其感染可引起临床症状轻微的登革热,甚至危及生命的登革出血热和登革休克综合征。登革病毒包含4种血清型,有效的登革病毒疫苗需对4种血清型的登革病毒均具有抗病毒保护作用。目前,尚未有针对登革病毒的特效药和成熟的疫苗产品。各类登革病毒疫苗均在研发中,其中一些已进入临床试验阶段。本文就登革病毒疫苗研究进展作一综述并对未来发展进行展望。     

001基于3种抗原以IgM捕获酶联免疫吸附试验对登革病毒血清分型

登革热是由黄病毒科的登革病毒引起，并影响全世界热带和亚热带城镇的地方性病毒流行病。近年来，登革热及更严重的登革出血热和登革休克综合征已成为分布更广的公共卫生问题和重点流行病。登革病毒有4个不同血清型，初次感染后，对同型登革病毒可导致终身保护性免疫，而对再次感染其他3型登革病毒，     

登革病毒感染的免疫与免疫病理的研究进展

登革病毒感染是世界上热带与亚热带地区一个严重的公众卫生健康问题,本文综述了登革病毒感染后机体免疫的改变,并从抗体、Ｔ细胞激活及细胞因子过量释放方面探讨了登革出血热（ＤＨＦ）／登革休克综合征（ＤＳＳ）的发病机理。     

一种前景广阔的单剂量、四价减毒登革热疫苗候选物

<正>登革病毒的迅速传播在全球范围内造成了巨大的健康威胁,居住在超过100个国家,约占全球40%的人口面临着登革病毒传播的风险,其中包括美国。2010年,全球共报告登革病毒感染案例3.9亿,其中0.96亿出现了感染症状。感染登革病毒会造成一系列临床症状,包括与其他疾病无明显差别的轻微发热、典型登革热或者重症登革热。重症登革热的特征是出血和/或血浆渗漏(登革出血热和登革休克     

国内首次自患者标本中同时分离的登革2型和3型病毒的鉴定

采用间接免疫荧光方法 ,检测患者血清标本中的抗登革病毒IgM和IgG抗体 ;同时将病人急性期血清接种C6 36细胞进行病毒分离。从分离的病毒悬液中提取RNA ,进行RT PCR扩增和序列测定。结果显示 ,该患者血清中存在抗登革病毒的IgM和IgG抗体。从病人血清中分离的病毒 ,经RT PCR和序列测定证实为登革 2型和 3型病毒的特异序列。表明该患者为登革 2型和 3型病毒混合感染     

登革四价疫苗研究进展

登革病毒感染引起的登革热和登革出血热是重要的蚊媒病毒病。近20年来,在全球范围内登革出血热的发病率和病死率急剧上升,但目前仍缺乏安全有效的疫苗。着重介绍目前已经或正在进入临床研究阶段的四价登革减毒活疫苗,以及灭活疫苗、亚单位疫苗、病毒载体疫苗和DNA疫苗的研究进展。     

登革病毒的系统发生树

登革病毒在除欧洲以外的所有大陆都有流行,弄清登革病毒在全世界的流行趋势及其致病机理对控制登革流行非常重要。本文就登革病毒系统发生树的构建方法及系统发生树的登革病毒分子流行病学、致病机理研究中的应用作一综述。     

登革病毒包膜蛋白的原核表达及免疫原性研究

登革病毒感染引起的登革热和登革出血热/登革休克综合征是目前流行最为广泛的虫媒病毒病,但其发病机制不明,也无疫苗和特异性抗病毒药物用于防治。登革病毒包膜蛋白(E蛋白)在病毒致病和免疫过程中发挥重要作用。本研究构建了登革病毒2型E蛋白基因的重组质粒E/pGEX-6P-1,并优化表达条件,获得登革病毒E蛋白的高效可溶性表达;用谷胱甘肽琼脂糖凝胶4B亲和层析柱纯化,获得纯度较高的蛋白。该蛋白具有较好的免疫原性,免疫小鼠后可获得高效价抗体。本研究为进一步了解登革病毒E蛋白的功能及其在相关疾病中的应用奠定了基础。     

登革病毒流行株的分离鉴定及其毒力位点变异研究

目的:从登革热患者血清中分离登革病毒,鉴定流行株的血清型及其毒力。对其中两分离株E基因进行序列测定,分析其可能的毒力位点变异。方法:采集临床诊断为登革热患者急性期血清91份,接种于C6/36细胞分离病毒,应用间接免疫荧光法鉴定及分型。并通过乳鼠脑内接种和空斑试验,测定分离株的毒力。扩增2株分离株E基因,克隆到pGEM-T载体进行序列测定,分析变异位点。结果:在91份血清中经2~3次传代分离出8株病毒,鉴定为登革1型病毒。在E蛋白影响毒力的3个区段中,两分离株有3处存在变异。结论:推测此次广州地区流行登革热可能由DEN 1型病毒感染引起,流行株的毒力较弱。毒力减弱可能和其基因位点变异有关。     

Recovery of Dengue Viruses from Tissues of Experimentally Infected Rhesus Monkeys

A tissue explant culture technique for the recovery of dengue virus from experimentally infected monkey tissue is described and compared with tissue culture assay of tissue triturates and co-cultivation of trypsinized cells in cell cultures. The most efficient technique was one in which minced tissue was explanted in co-culture with dengue virus-susceptible LLC-MK2 monkey kidney cells. This technique shows promise of being useful for detection of virus in autopsy material from fatal dengue hemorrhagic fever cases.     

Monoclonal antibody to dengue capsid protein: Its application in dengue studies

Dengue fever (DF) and dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) are considered the most important arthropod-borne viral diseases in terms of morbidity and mortality. The emergency and severity of dengue (Den) infections increase the necessity of an early, quick and effective dengue laboratory diagnostic. Viral isolation is considered a gold standard for diagnosis of dengue infection using monoclonal antibodies (mAbs) as a tool for determining serotype specificity. Alternatives have been used to improve sensitivity and time to dengue diagnosis. Based on the early expression of dengue C protein in the life cycle, we focused our study on the application of an anti-dengue 2 virus capsid protein mAb in dengue diagnosis. The kinetic expression of dengue-2 capsid in mosquito cells and its immuno-localization in experimentally infected suckling albin Swiss (OF-1) mice brain tissues was established. The results demonstrate the possible utility of this mAb in early dengue diagnosis versus traditional isolation. In addition, a preliminary study of an enzyme immunoassay method using 8H8 mAb for specific detection of dengue C protein antigen was performed, making possible recombinant C protein quantification. The results suggest that detection of dengue capsid protein could be useful in the diagnosis of early dengue infection.Key words: monoclonal antibodies, capsid protein, dengue virus, diagnosis, immunoassays     

Establishment of Murine Infection Models with Biological Clones of Dengue Viruses Derived from a Single Clinical Viral Isolate

Virologica Sinica - Dengue virus (DENV) is a single-stranded RNA virus transmitted by mosquitoes in tropical and subtropical regions. It causes dengue fever, dengue hemorrhagic fever and dengue...     

Mosquito and mammalian cells grown on microcarriers for four-serotype dengue virus production: variations in virus titer, plaque morphology, and replication rate

Dengue (DEN) viruses consisting of four distinct serotypes cause diseases such as dengue fever, dengue hemorrhagic fever, and dengue shock syndrome in humans. Most of the dengue viruses can be effectively propagated in some mosquito and mammalian cell lines. In this study, we applied microcarrier cell culture technology to study two relevant aspects involving dengue virus, one on biotechnology of cell growth and virus production, and the other on virus biology concerning genetic variation of a virus population. We investigated the growth of C6/36 mosquito cells and Vero cells grown on Cytodex 1 microcarriers. High-titer DEN virus production can be achieved in C6/36 and Vero cells infected at low cell inoculation density, in the lag-phase cell stage, and at low multiplicity of infection (MOI). The maximum titers produced for DEN-1, DEN-3, and DEN-4 viruses were approximately 10- to 10,000-fold lower than for DEN-2 virus produced in C6/36 and Vero cells grown on microcarriers. The DEN-2 virus produced in C6/36 cells displayed far more extensive plaque heterogeneity than in Vero cells. Microcarrier C6/36 mosquito cell culture appeared to be the most effective system for four-serotype DEN virus production. Interestingly, some selected variants of DEN virus may outgrow in Vero cells when using a T-flask culture. These results may provide useful information for DEN vaccine development.     

Selection for virulent dengue viruses occurs in humans and mosquitoes

Dengue is the most common mosquito-borne viral disease in humans. The spread of both mosquito vectors and viruses has led to the resurgence of epidemic dengue fever (a self-limited flu-like syndrome) and the emergence of dengue hemorrhagic fever (severe dengue with bleeding abnormalities) in urban centers of the tropics. There are no animal or laboratory models of dengue disease; indirect evidence suggests that dengue viruses differ in virulence, including their pathogenicities for humans and epidemic potential. We developed two assay systems (using human dendritic cells and Aedes aegypti mosquitoes) for measuring differences in virus replication that correlate with the potential to cause hemorrhagic dengue and increased virus transmission. Infection and growth experiments showed that dengue serotype 2 viruses causing dengue hemorrhagic fever epidemics (Southeast Asian genotype) can outcompete viruses that cause dengue fever only (American genotype). This fact implies that Southeast Asian genotype viruses will continue to displace other viruses, causing more hemorrhagic dengue epidemics.     

Serotyping of dengue viruses by an enzyme-linked immunosorbent assay

We have developed a sandwich enzyme-linked immunosorbent assay for serotyping dengue viruses. In this assay, we used antibody from dengue hemorrhagic fever patients for detection of flavivirus common antigens to confirm virus isolation in C6/36 cells and that from hyperimmune mouse ascitic fluids for serotyping. The anti-dengue antibody was immobilized on microplate wells for capturing of dengue antigens, which were then sandwiched with the same biotinylated antibody. Then the biotin in the solid phase was detected with peroxidase-conjugated streptavidin. We found that all the dengue strains tested were unequivocally identified by this method.     

Enhanced passive surveillance dengue infection among febrile children: Prevalence,co-infections and associated factors in Cameroon

Dengue virus (DENV) causes a spectrum of diseases ranging from asymptomatic, mild febrile to a life-threatening illness: dengue hemorrhagic fever. The main clinical symptom of dengue is fever, similar to that of malaria. The prevalence of dengue virus infection, alone or in association with other endemic infectious diseases in children in Cameroon is unknown. The aim of this study was to determine the prevalence of dengue, malaria and HIV in children presenting with fever and associated risk factors.Dengue overall prevalence was 20.2%, Malaria cases were 52.7% and HIV cases represented 12.6%. The prevalence of dengue-HIV co-infection was 6.0% and that of Malaria-dengue co-infection was 19.5%. Triple infection prevalence was 4.3%. Dengue virus infection is present in children and HIV-Dengue or Dengue- Malaria co-infections are common. Dengue peak prevalence was between August and October. Sex and age were not associated with dengue and dengue co-infections. However, malaria as well as HIV were significantly associated with dengue (P = 0.001 and 0.028 respectively). The diagnosis of dengue and Malaria should be carried out routinely for better management of fever.