福建省白纹伊蚊中登革病毒的分离与鉴定

近年来福建省登革热(Dengue fever,DF)输入性病例持续存在,且登革热的主要传播媒介白纹伊蚊在全省内广泛分布,为了解福建省福州市登革热的媒介白纹伊蚊携带登革病毒(Dengue virus,DENV)状况,2017年10月7日在福州市台江区元一花园小区内开展伊蚊监测,采用双层叠帐法捕获255只白纹伊蚊蚊体研磨液上清提取核酸后用实时荧光RT-PCR法检测DENV特异性核酸,将检测阳性的蚊体研磨液上清接种C6/36细胞进行病毒分离,成功分离到1株DENV病毒株mosquito13/Fujian/2017;经实时荧光RT-PCR法鉴定所分离病毒株的血清型为I型;利用型特异性引物通过RT-PCR扩增病毒E基因并测序进行分子遗传特性分析;E基因核苷酸和氨基酸同源性分析显示,该毒株与2017年10月17日同小区本地登革热病例血清中分离得到的登革毒株E基因序列完全一致,与越南2014年分离株KT825033/Vietnam/2014核苷酸(99.7%)和氨基酸(99.8%)同源性最高;系统进化树分析表明所分离登革病毒毒株的基因型为I型,与东南亚地区的越南,泰国,柬埔寨等国家进化关系相近,可能输入来源于东南亚国家。本研究证实了登革热外潜伏期的存在以及白纹伊蚊在登革热疫情传播过程中的媒介作用,提示在登革热的防控工作中媒介登革病毒监测、检测的重要性,也提示福建省需要加强输入来源监测,特别是东南亚入境人员的监测。     

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     

五株登革病毒的分离鉴定及E基因序列分析

登革病毒作为一种重要的蚊媒传播病毒,威胁全球人类健康,及时甚至实时了解病毒的变异和流行病学规律,对科学防控登革热具有重要意义.因此,本研究利用广东省深圳市发现的登革热患者血清进行病毒分离、鉴定及囊膜蛋白E基因的扩增、测序及生物信息学分析病毒E基因进化特点.结果显示,应用Vero和C6/36细胞培养法从登革热病患血清中分离获得5株登革病毒,利用MEGA7软件对囊膜蛋白E的核苷酸和氨基酸序列进行分析显示,5株均属血清Ⅱ型登革病毒,其中输入性SZ926株和本地株SZ38株同源性为99.5％,均与泰国16681经典毒株关系密切,可能衍生于同一祖先;SZ868株和SZ871株为境外输入性病患分离株,两株病毒核苷酸同源性达到99.9％,说明两株病毒可能源于同一毒株;SZ29株与新几内亚的New Guinea C毒株同源性为99.8％,显示出进化保守、稳定传播的特点.基因型分析显示,SZ926和SZ38为基因型Ⅲ型,SZ868和SZ871为基因型Ⅳ型,SZ29为基因型Ⅰ型.本研究成功分离获得5株血清Ⅱ型登革病毒,分属于3个基因型,且不同基因型的E基因整体变异较低,而且我国存在多种基因型感染的潜在威胁,必须加强防范意识和研究力度.     

DENV2海南分离株NS1全序列测定和生物信息学分析

目的:对登革病毒2型海南分离株NS1全序列进行测定和生物信息学分析,了解其系统 进化和分子流行病学特征.方法:采用RT-PCR法扩增D2-Hainan全长NS1基因,将其克隆入载 体pPIcZáB,测序,采用相关软件进行生物信息学分析.结果:获得D2-H ainan株NS1全长基 因1056bp,其序列与登革病毒2型NGC株的同源性为95%.系统进化树分析显示该毒株与登革 病毒2型China04株的亲缘关系最近.初步分析了NS1蛋白的氨基酸序列特征和二级结构.结论:登革病毒流行株存在地域性和复杂性,对D2-Hainan株NS1基因及其编 码蛋白信息特征的了解,有助于进一步研究其基因特征与病毒毒力的关系.     

2012年在上海市发现中国大陆首例输入性D8基因型麻疹病例

本文对中国首例输入性D8基因型麻疹病毒基因特征进行分析。用ELISA法检测血清麻疹病毒IgM抗体;用Vero/Slam细胞对采集的咽拭子标本进行病毒分离,分离到的麻疹毒株用RT-PCR方法扩增其核蛋白基因3′端的部分序列,并对扩增产物进行核苷酸序列测定和分析,以3′端456个核苷酸为目的片段进行基因亲缘性关系分析。结果表明,上海市2012年共报告1 105疑似麻疹病例,其中实验室确诊590例,临床符合病例2例,排除513例,报告发病率为2.52/10万;共采集到984份疑似麻疹病例咽拭子标本,分离到247株麻疹病毒,病毒分离阳性率为25.3%;除Shanghai12-239为D8基因型外,其他均为H1a基因亚型。Shanghai12-239与世界卫生组织(World Health Organization,WHO)参考株(Manchester.UNK30.94(D8)AF280803)核苷酸序列同源性为97.8%,氨基酸序列同源性为98.6%。与WHO其他基因型参考株核苷酸序列同源性为89.6%~94.5%,氨基酸序列同源性为88.7%~95.3%。     

浙江省麻疹病毒分离株的基因特性与免疫原性研究

为了研究浙江省1999～2003年麻疹病毒分离株的抗原性变化以及基因特性,阐明其基因特性与麻疹流行的关系,以及抗原性变化对疫苗免疫效果的影响.我们从麻疹患者含漱液中分离麻疹病毒株,制备大鼠免疫血清,与疫苗株沪191和Edmonston株等进行交叉中和试验,测定各毒株之间抗原比;并采集儿童麻疹疫苗免疫前后血清,分别测定其对不同毒株的中和抗体滴度;采用RT-PCR法扩增麻疹毒株血凝素蛋白(H)及核蛋白(N)基因,进行核苷酸序列测定.结果浙江99-1和浙江02-2株与沪191株之间的抗原比分别为3.0和7.3;儿童免疫后血清对浙江02-2株的麻疹中和抗体平均几何滴度(GMT)为15.03,明显低于沪191疫苗株(GMT为68.12);浙江省1999～2003年麻疹分离株之间,H基因氨基酸同源性为99.8%,N基因氨基酸同源性为98.5%～100.0%;与沪191疫苗株的H基因和N基因相比较,分离株与其在氨基酸水平上同源性分别为95.5%～95.7%和95.9%～96.3%.从基因进化树显示,1999～2003年浙江省分离到的麻疹毒株属于H1基因型,但与疫苗株沪191在抗原性和基因特性上已存在明显的差异.     

浙江省麻疹病毒分离株的基因特性与免疫原性研究

为了研究浙江省1999～2003年麻疹病毒分离株的抗原性变化以及基因特性,阐明其基因特性与麻疹流行的关系,以及抗原性变化对疫苗免疫效果的影响。我们从麻疹患者含漱液中分离麻疹病毒株,制备大鼠免疫血清,与疫苗株沪191和Edmonston株等进行交叉中和试验,测定各毒株之间抗原比;并采集儿童麻疹疫苗免疫前后血清,分别测定其对不同毒株的中和抗体滴度;采用RT-PCR法扩增麻疹毒株血凝素蛋白(H)及核蛋白(N)基因,进行核苷酸序列测定。结果浙江99-1和浙江02-2株与沪191株之间的抗原比分别为3.0和7.3;儿童免疫后血清对浙江02-2株的麻疹中和抗体平均几何滴度(GMT)为15.03,明显低于沪191疫苗株(GMT为68.12);浙江省1999～2003年麻疹分离株之间,H基因氨基酸同源性为99.8%,N基因氨基酸同源性为98.5%～100.0%;与沪191疫苗株的H基因和N基因相比较,分离株与其在氨基酸水平上同源性分别为95.5%～95.7%和95.9%～96.3%。从基因进化树显示,1999～2003年浙江省分离到的麻疹毒株属于H1基因型,但与疫苗株沪191在抗原性和基因特性上已存在明显的差异。     

浙江地区呼吸道合胞病毒G基因分子特征分析

收集2004年冬季浙江省儿童医院呼吸道感染患儿的鼻咽吸引物,进行呼吸道合胞病毒(RSV)分离,对分离株用特异性RT-PCR鉴定;对鉴定阳性的RSV毒株进行G蛋白全基因序列测定,并与RSV国际标准株及各地参考株的G蛋白基因序列进行同源性比较并构建基因进化树。结果表明,在4株RSV浙江分离株中,3株G蛋白基因ORF全长897bp,另1株为894bp;各分离株之间的核苷酸与氨基酸同源性分别为98.05%、97.06%;与A型RSV标准株的核酸同源性为95.69%,与B型RSV标准株的同源性为78.79%;其3’端基因高变区核苷酸序列与RSVGA2亚型的同源性最高。提示2004年冬季浙江省RSV流行株属于RSVGA2亚型。     

海南岛125株登革病毒的分离与鉴定

1985年9月儋县发生一次由Ⅱ型登革病毒引起的登革热和登革出血热的爆发流行,至1986年10月疫情已由儋县波及本岛十县二市。根据记载,国外登革热流行期间,常伴多个型别登革病毒同时传播。为掌握本岛此次登革热流行时除Ⅱ型病毒外,是否尚有其他型别流行,于1986年1月至10月登革热流行期间,取十县二市278例急性期病人血清,接种C6/36白纹伊蚊细胞系,进行病毒分离,共获得125株病毒。均用抗登革     

浙江地区呼吸道合胞病毒G基因分子特征分析

收集2004年冬季浙江省儿童医院呼吸道感染患儿的鼻咽吸引物,进行呼吸道合胞病毒(RSV)分离,对分离株用特异性RT-PCR鉴定;对鉴定阳性的RSV毒株进行G蛋白全基因序列测定,并与RSV国际标准株及各地参考株的G蛋白基因序列进行同源性比较并构建基因进化树.结果表明,在4株RSV浙江分离株中,3株G蛋白基因ORF全长897bp,另1株为894bp;各分离株之间的核苷酸与氨基酸同源性分别为98.05%、97.06%;与A型RSV标准株的核酸同源性为95.69%,与B型RSV标准株的同源性为78.79%;其3'端基因高变区核苷酸序列与RSV GA2亚型的同源性最高.提示2004年冬季浙江省RSV流行株属于RSV GA2亚型.     

Evaluation of Internet-Based Dengue Query Data: Google Dengue Trends

Dengue is a common and growing problem worldwide, with an estimated 70–140 million cases per year. Traditional, healthcare-based, government-implemented dengue surveillance is resource intensive and slow. As global Internet use has increased, novel, Internet-based disease monitoring tools have emerged. Google Dengue Trends (GDT) uses near real-time search query data to create an index of dengue incidence that is a linear proxy for traditional surveillance. Studies have shown that GDT correlates highly with dengue incidence in multiple countries on a large spatial scale. This study addresses the heterogeneity of GDT at smaller spatial scales, assessing its accuracy at the state-level in Mexico and identifying factors that are associated with its accuracy. We used Pearson correlation to estimate the association between GDT and traditional dengue surveillance data for Mexico at the national level and for 17 Mexican states. Nationally, GDT captured approximately 83% of the variability in reported cases over the 9 study years. The correlation between GDT and reported cases varied from state to state, capturing anywhere from 1% of the variability in Baja California to 88% in Chiapas, with higher accuracy in states with higher dengue average annual incidence. A model including annual average maximum temperature, precipitation, and their interaction accounted for 81% of the variability in GDT accuracy between states. This climate model was the best indicator of GDT accuracy, suggesting that GDT works best in areas with intense transmission, particularly where local climate is well suited for transmission. Internet accessibility (average ∼36%) did not appear to affect GDT accuracy. While GDT seems to be a less robust indicator of local transmission in areas of low incidence and unfavorable climate, it may indicate cases among travelers in those areas. Identifying the strengths and limitations of novel surveillance is critical for these types of data to be used to make public health decisions and forecasting models.     

Phylogenetic Analysis of the Dengue Virus Strains Causing the 2019 Dengue Fever Outbreak in Hainan,China

Dengue virus is an arthropod-borne pathogen that is transmitted to humans primarily by Aedes spp.mosquitos,causing the acute infectious disease,dengue fever (DF).Until 2019,no dengue outbreak had been reported in Hainan Province for over20 years.However,in early September of 2019,an increasing number of infected cases appeared and the DF outbreak lasted for over one month in Haikou City,Hainan Province.In our study,we collected 97 plasma samples from DF patients at three hospitals,as well as 1585 mosquito larvae samples from puddles in different areas of Haikou.There were 49(50.5%) plasma samples found to be strongly positive and 9 (9.3%) plasma samples were weakly positive against the NS1 antigen.We discovered DENV both in the patient's plasma samples and mosquito larvae samples,and isolated the virus from C6/36 cells inoculated with the acute phase serum of patients.Phylogenetic analysis revealed that the new strains were the most closely related to the epidemic strain in the southern regions of China,belonging to lineage IV,genotype I,DENV-1.Compared to the seven closest strains from neighboring countries and provinces,a total of 18 amino acid mutations occurred in the coding sequences (CDS) of the new isolated strain,DENV1 HMU-HKU-2.Our data shows that dengue virus is re-emerged in Hainan,and pose new threats for public health.Thus regular molecular epidemiological surveillance is necessary for control and prevention of DENV transmission.     

Evaluation of the Diagnostic Accuracy of a New Dengue IgA Capture Assay (Platelia Dengue IgA Capture,Bio-Rad) for Dengue Infection Detection

Considering the short lifetime of IgA antibodies in serum and the key advantages of antibody detection ELISAs in terms of sensitivity and specificity, Bio-Rad has just developed a new ELISA test based on the detection of specific anti-dengue IgA. This study has been carried out to assess the performance of this Platelia Dengue IgA Capture assay for dengue infection detection. A total of 184 well-characterized samples provided by the French Guiana NRC sera collection (Laboratory of Virology, Institut Pasteur in French Guiana) were selected among samples collected between 2002 and 2013 from patients exhibiting a dengue-like syndrome. A first group included 134 sera from confirmed dengue-infected patients, and a second included 50 sera from non-dengue infected patients, all collected between day 3 and day 15 after the onset of fever. Dengue infection diagnoses were all confirmed using reference assays by direct virological identification using RT-PCR or virus culture on acute sera samples or on paired acute-phase sera samples of selected convalescent sera. This study revealed: i) a good overall sensitivity and specificity of the IgA index test, i.e., 93% and 88% respectively, indicating its good correlation to acute dengue diagnosis; and ii) a good concordance with the Panbio IgM capture ELISA. Because of the shorter persistence of dengue virus-specific IgA than IgM, these results underlined the relevance of this new test, which could significantly improve dengue diagnosis accuracy, especially in countries where dengue virus is (hyper-) endemic. It would allow for additional refinement of dengue diagnostic strategy.     

Serum Metabolomics Reveals Serotonin as a Predictor of Severe Dengue in the Early Phase of Dengue Fever

Effective triage of dengue patients early in the disease course for in- or out-patient management would be useful for optimal healthcare resource utilization while minimizing poor clinical outcome due to delayed intervention. Yet, early prognosis of severe dengue is hampered by the heterogeneity in clinical presentation and routine hematological and biochemical measurements in dengue patients that collectively correlates poorly with eventual clinical outcome. Herein, untargeted liquid-chromatography mass spectrometry metabolomics of serum from patients with dengue fever (DF) and dengue hemorrhagic fever (DHF) in the febrile phase (<96 h) was used to globally probe the serum metabolome to uncover early prognostic biomarkers of DHF. We identified 20 metabolites that are differentially enriched (p<0.05, fold change >1.5) in the serum, among which are two products of tryptophan metabolism–serotonin and kynurenine. Serotonin, involved in platelet aggregation and activation decreased significantly, whereas kynurenine, an immunomodulator, increased significantly in patients with DHF, consistent with thrombocytopenia and immunopathology in severe dengue. To sensitively and accurately evaluate serotonin levels as prognostic biomarkers, we implemented stable-isotope dilution mass spectrometry and used convalescence samples as their own controls. DHF serotonin was significantly 1.98 fold lower in febrile compared to convalescence phase, and significantly 1.76 fold lower compared to DF in the febrile phase of illness. Thus, serotonin alone provided good prognostic utility (Area Under Curve, AUC of serotonin = 0.8). Additionally, immune mediators associated with DHF may further increase the predictive ability than just serotonin alone. Nine cytokines, including IFN-γ, IL-1β, IL-4, IL-8, G-CSF, MIP-1β, FGF basic, TNFα and RANTES were significantly different between DF and DHF, among which IFN-γ ranked top by multivariate statistics. Combining serotonin and IFN-γ improved the prognosis performance (AUC = 0.92, sensitivity = 77.8%, specificity = 95.8%), suggesting this duplex panel as accurate metrics for the early prognosis of DHF.     

The Ethnoecology of Dengue Fever

This article employs an ethnoecological analysis to link indigenous, ethnomedical, and Western biomedical ideas of infectious disease causation/ prevention. The ethnoecological analysis is expanded to include the cultural and historical context of political will and community participation in dengue fever control activities in an urban neighborhood in the Dominican Republic. Findings indicate that a key source of dengue fever transmission has been overlooked because it falls between established gender-role boundaries, and that mala union, an explanatory concept central to the failure of previous community-based interventions, emerges from local views of national political history. Data were generated through a neighborhood household survey, key respondent interviews, and participant-observation. [Ethnoecology, Caribbean, gender roles, dengue fever, Dominican Republic]