登革病毒Ⅱ型对人脐静脉血管内皮细胞通透性的研究

目的 研究登革病毒Ⅱ型(DENV-2)对人脐静脉血管内皮细胞(HUVEC)通透性的影响.方法 DENV-2病毒滴定,DENV-2接种HUVEC单层细胞,用间接免疫荧光法动态观察DENV-2感染HUVEC的情况(30 min,l、3、6、12、24、30、36、42、48、72 h),实时定量荧光PCR方法检测病毒载量,transwell法检测DENV-2对HUVEC通透性的影响,透射电镜观察细胞超微结构的改变.结果 DENV-2对HUVEC通透性的影响30 min时作用最为明显,其次为42 h时.且病毒载量与HUVEC通透性改变呈正相关.透射电镜结果显示有细胞微绒毛脱落,胞质溶解,部分核膜间隙增宽,甚至是细胞核中也存在裂隙,部分线粒体嵴消失甚至空化,线粒体髓样变,包膜不完整.结论 DENV-2对HUVEC通透性有影响,为探究登革病毒的发病机制提供一定的理论依据.

Abstract：

Objective To research Dengue virus type 2 (DENV-2) to human umbilical vein endothelial cells (HUVEC) permeability. Methods The titration of DENV-2 was detected and HUVEC infection DENV-2 layer of cells. At different time points after infection (30 min, 1 h, 3 h, 6 h, 12 h, 30 h,36 h, 24 h, 42 h, 48 h,72 h), the permeability in HUVEC were detected after Dengue virus infection. The virus load in HUVEC was detected by quantitative real-time PCR. And ultrastructure in HUVEC was observed by electron microscope. Results The permeability in HUVEC was changed after Dengue virus infection by time lasting. The most permeability in HUVEC was changed after Dengue virus infection at 30 min and 42 h. And at the same time the virus load were most value in all of time. The results of the cell membrane were changed by electron microscope. The deciduous cell microvillus and dissd endochylema were founded. And some of nuclear membrane blank was wided by Dengue virus. Even the leakage was founded in cell nucleus. The other change included that disappearance mitochondrial and vacuolization crista, elder pith change mitochondria. In addition, the complete of cell membrane were dissolved. Conclusion The permeability of HUVEC was changed by DENV-2. To explore the pathogenesis of Dengue virus provide certain theoretical basis.     

登革Ⅱ型病毒对淋巴细胞和骨髓细胞SCE率影响的实验研究

报道３０例离体培养的人体外周血淋巴细胞及２０例离体培养的正常人骨髓细胞加入登革Ⅱ型病毒前后的ＳＣＥ率改变，结果表明：登革Ⅱ型病毒可以提高外周血淋巴细胞及骨髓细胞的ＳＣＥ率，说明该病毒对人体这两类细胞的染色体均具有损伤作用。实验还观察到不同个体的淋巴细胞及骨髓细胞在加入登革Ⅱ型病毒后，其ＳＣＥ率的升高有个体差异，说明了染色体或ＤＮＡ损伤程度不等。     

贵州白纹伊蚊对登革病毒易感性的研究

目的 用细胞、分子生物学技术进行贵州省白纹伊蚊不同地理株对登革病毒(DEN)易感性的研究。方法 采集贵州省9个地(州)市共计15个县(区)白纹伊蚊幼虫标本，饲养为成蚊；取羽化后3～5日龄期的贵州不同地理株白纹伊蚊，用不同型别的DEN分别经口连续感染3d，于首次感染后的4、7、10、14d收集感染成蚊标本；制备蚊悬液，碘化钠法提取RNA，用DENNS1基因区通用引物经逆转录．聚合酶链反应(RT-PCR)检测DEN核酸；蚊悬液接种C6／36细胞进行病毒分离，制作细胞抗原片，经间接免疫荧光法检测DEN抗原；同时感染白纹伊蚊海南株作为对照。结果 DEN1-4型国际参考株感染白纹伊蚊贵州省不同地方株，其感染比率分别为12／15、12／15、8／15和13／15。结论 白纹伊蚊贵州省不同地方株对DEN1-4型国际参考株普遍易感，表明贵州省具备引起登革热流行的条件。     

登革病毒促进人树突状细胞分泌TNF-α、IL-6、IFN-γ的动态观察

目的：研究登革病毒（DV）对人树突状细胞（DC）产生细胞因子的影响。 方法： 人外周新鲜血常规分离单核细胞，经细胞因子GM-CSF、IL-4诱导培养DC，形态学特征、细胞表型和淋巴细胞刺激能力鉴定。用登革病毒2型感染DC，于作用后6、12、24、48、72 h分别收集上清液和细胞，间接免疫荧光法检测细胞上病毒抗原表达，ELISA法检测登革病毒感染后细胞因子TNF-α、IL-6、IFN-γ水平的动态变化。 结果： 人外周血经GM-CSF、IL-4诱导培养1周即可得到典型树突状细胞。间接免疫荧光法证明感染的DC胞浆和胞膜上携带登革病毒抗原，DV感染使DC分泌TNF-α、IL-6能力显著大于对照组（P<0.01），但其分泌IFN-γ的能力无明显改变。 结论： 树突状细胞是登革病毒的靶细胞，登革病毒感染可促进树突状细胞分泌TNF-α、IL-6。树突状细胞可能参与机体抗登革病毒感染的免疫防御机制。     

重组登革病毒E蛋白结构域Ⅲ抑制登革病毒感染的初步研究

目的 了解原核表达的登革病毒(dengue virus, DV)的E蛋白结构域Ⅲ直接抑制登革病毒感染及其抗体的中和作用.方法 在大肠埃希菌中表达1~4型登革病毒E蛋白结构域Ⅲ(EⅢ).重组蛋白纯化后,进行阻断DV-2感染BHK~21细胞试验.用重组蛋白制备免疫血清,检测抗体中和作用.结果 在大肠埃希菌中成功表达了1-4型登革病毒E蛋白结构域埃希菌,4型重组E蛋白结构域Ⅲ均能够阻断2型DV感染,4型重组蛋白的免疫血清均能中和2型DV,但中和抗体效价不同.结论 原核表达的登革病毒结构域Ⅲ可以直接抑制病毒感染,所产生的抗体具有中和作用.直接抑制和中和抗体均对同型病毒作用较强.     

温度对白纹伊蚊传播登革2型病毒易感性影响的研究

目的 研究不同温度下白纹伊蚊对登革病毒的易感性差异.方法 登革2型病毒人工经口感染白纹伊蚊,冻麻挑出吸饱血的雌蚊放入新的蚊笼,分别置于18℃、21℃、26℃、31℃、33℃和36℃的人工气候箱中饲养,经外潜伏期后,解剖蚊虫,间接免疫荧光法分别检测蚊虫的头部、唾液腺和胸腹部内的登革2型病毒抗原,并计算蚊虫的播散感染率.结果 18℃白纹伊蚊感染率最低,胸腹部感染率为8％,头部和唾液腺未检出.31℃胸腹部感染率最高为82％,头部和唾液腺感染率33℃时最高.36℃各部位感染率均较33℃下降.18℃的播散感染率最低为0,36℃播散感染率最高,达到了100％.结论 白纹伊蚊感染率随温度的升高,呈现先升后降趋势.18℃可能是白纹伊蚊传播登革病毒的最低温度.播散感染率随温度的升高逐渐升高.     

贵州独山、兴义不明原因发热病人登革病毒的分离和鉴定的初步研究

目的 对贵州独山、兴义两地夏秋季不明原因发热病人血清进行登革病毒(DEN)分离及鉴定,从病原学角度证实贵州省人群DEN的感染情况.方法 于2005年6至10月份收集贵州独山县、兴义市两地不明原因发热病人血清356份,并接种于生长良好的单层C6/36细胞盲传3代,观察细胞病变,用抗DEN1～4型单克隆抗体通过间接免疫荧光法进行型别鉴定;用DEN NSl基因区特异性通用引物进行RT-PCR检测分离的DEN毒株核酸,经序列测定并作系统发生树分析.结果 3份病人血清标本可使C6/36发生细胞病变,用单克隆抗体、RT-PCR扩增和序列测定,鉴定为DEN2;系统发生树分析证明,分离的病毒与DEN2-43、DEN2-44株系统进化关系最近.结论 贵州省独山、兴义两地人群中存在DEN感染.     

Evaluation of a commercial Dengue NS1 enzyme-linked immunosorbent assay for early diagnosis of dengue infection

Purpose: Dengue is one of the most serious mosquito-borne viral infections affecting tropical and subtropical countries in the world. Since there is no immunoprophylactic or specific antiviral therapy available, timely and rapid diagnosis plays a vital role in patient management and implementation of control measures. This paper evaluates a commercially available NS1 antigen capture ELISA vis-a-vis SD bioline Dengue NS1 antigen test for early detection of dengue virus. Materials and Methods: To evaluate a commercial NS1 antigen detection kit vis-a-vis SD bioline Dengue NS1 antigen test, a total of 91 clinical samples were tested. Virological investigations with regard to dengue virus, viz. NS1 antigen capture ELISA (Panbio, Australia), SD bioline Dengue NS1 antigen test, RT-PCR and virus isolation were performed. Results: Out of 91 samples, 24 (26%) were positive by NS1 antigen capture ELISA, 15 (16%) by SD bioline Dengue NS1 antigen test and 11(12%) positive by RT-PCR analysis. The RT-PCR-positive samples were further subjected to virus isolation and resulted in three isolates. The results of the Panbio NS1 antigen capture ELISA, SD bioline Dengue NS1 antigen test, RT-PCR and virus isolation were correlated among themselves. Conclusions: The present study comprehensively established the utility of NS1 antigen ELISA in early diagnosis of dengue infection.     

白纹伊蚊感染登革病毒后病毒特异性piRNAs分析

目的 筛选并分析白纹伊蚊感染登革病毒后病毒特异性vsRNAs与vpiRNAs.方法 白纹伊蚊羽化后2～4d雌性成蚊显微注射DENV-2病毒NGC株,对照组注射生理盐水.8d后提取样品总RNA,分离小RNA,通过illumina Hiseq 2000测序仪进行测序分析.以SOAP2软件与DENV-2基因组和互补序列进行比对.对病毒特异性的vsRNAs与vpiRNAs的长度、碱基与链偏好性、基因组分布进行分析.结果 对于感染蚊虫,我们共计获得3835个特异的DENV-2来源的vsRNAs,其中395个特异性的vpiRNAs.94.99％ vpiRNAs主要来源于病毒的正链基因组.vpiRNAs在DENV-2基因组上的分布也具有偏向性,最高峰位于病毒非结构蛋白NS5编码区域的3＆#39;末端.第10位碱基具有较强的腺嘌呤偏好性,但第1位碱基不具有尿嘧啶偏好性;也无典型“乒乓”扩增循环的特征.结论 白纹伊蚊感染登革病毒后,体内出现病毒特异性vsRNAs与vpiRNAs,对其鉴定与分析将为在白纹伊蚊抗病毒免疫研究提供理论基础.     

登革病毒反义寡核苷酸的抗病毒活性测定

测定了一段与登革病毒2型(DEN—2)基因5′端非编码区及相邻部份编码区20个核苷酸序列互补的反义寡核苷酸(ASON)的抗病毒活性。DEN—2感染的蚊细胞于感染前后不同时间给予不同剂量的ASON,在对照组出现明显细胞病变时,观察给药组的细胞出现病变(CPE)并测定病毒含量。另用中等剂量的ASON加入感染的细胞内,每24h动态观察CPE、病毒含量、抗原表达及核酸释放。结果显示,ASON对细胞的保护作用与用药时间及剂量明显相关,给药组的CPE、膜表面病毒抗原的表达及病毒核酸的检出时间均比对照组晚,两组培养液内病毒的TCID_(50)最大可相差10~5以上。实验表明,所用ASON可抑制DEN在蚊细胞中的增殖。     

2型登革病毒诱导RAW264.7细胞凋亡的机制及凋亡对病毒复制的影响

 目的：探讨2型登革病毒（DENV2）感染能否诱导RAW264.7细胞凋亡,并初步探讨凋亡对病毒复制的影响。方法：用DENV2感染RAW264.7细胞,MTT检测细胞活性,Hoechst 33342染色检测细胞核变化,Annexin V-FITC/PI双染流式细胞术检测细胞凋亡,Western blotting检测caspase-3和caspase-8活化片段的变化,比色法检测caspase-9活性变化,JC-1染色检测线粒体膜电位变化,Z-VAD-FMK抑制细胞凋亡后以TCID50检测感染细胞上清病毒滴度。结果：DENV2感染RAW264.7细胞24 h、36 h及48 h后细胞活性受到抑制,免疫荧光检测有核固缩现象,流式细胞术检测发现病毒感染诱导了细胞凋亡,Western blotting检测发现活化caspase-3和caspase-8的表达增加,caspase-9活性也增加,JC-1染色发现病毒感染诱导RAW264.7细胞线粒体膜电位降低,用Z-VAD-FMK抑制凋亡后感染细胞上清病毒滴度增加。结论：登革病毒感染可以通过内、外源性途径诱导RAW264.7细胞发生凋亡;凋亡发生抑制了病毒的产生。     

Role of intracellular events in the pathogenesis of dengue; An overview

Dengue is one of the most important mosquito-borne viral diseases that are relentlessly spreading in newer areas in the tropical and subtropical regions of the World. In last fifty years, in spite of intensive and extensive investigations, pathogenesis of dengue is still not clearly understood. Recently, the research focus is on studying the role of intracellular events in pathogenesis of viral infections. Entry of virion in the host cell is followed by quick succession of events, unfolded protein response, lipid bodies and lipophagy, endoplasmic reticulum stress and recent demonstration of autophagy. The turbulence caused by these events may result in clearance of the virus/enhanced replication and survival of the host cell/apoptosis. Both, increased virus load and apoptosis of host cell may have pathological effects on the host. In the present review, we have summed up the role of various intracellular events in viral infections with special emphasis on Dengue virus infection.     

Enhancement of varicella-zoster virus infection in cell lines expressing ORF4- or ORF62-encoded proteins

Varicella-Zoster virus (VZV) open reading frames 4 (ORF4) and 62 (ORF62) encode putative immediate-early proteins (ORF4p and ORF62p, respectively) which are strong transactivators of other VZV genes and are involved in the very early stages of viral infection. ORF4p and ORF62p transactivate immediate-early and early gene promoters but have little or no effect on late gene promoters. To investigate the effect of ORF4p or ORF62p overexpression on the viral replication cycle, we constructed Vero cell lines expressing those genes under the control of the human cytomegalovirus major immediate-early promoter. VZV OKA infection of these stably transformed cell lines was followed-up using VZV glycoprotein E (gE) antigen quantification and virus titration. Upon serial passaging of infection in these cell lines expressing functionally active ORF4p or ORF62p, a 5- to 10-fold increase in viral gE antigen production was observed. Viral titers also demonstrated a 2- to 5-fold increase in viral production in these transformed cell lines. These results emphasize the role that both ORF4p and ORF62p play in enhancing the VZV replicative cycle. © 1996 Wiley-Liss, Inc.     

Dengue virus infection induces upregulation of hn RNP-H and PDIA3 for its multiplication in the host cell

The pathogenic mechanism of Dengue virus (DENV) infection is related to the host responses within target cells and therefore, we assessed intracellular changes in host cell proteins following DENV infection. This study provides evidence that heterogeneous nuclear ribonucleoprotein H (hnRNP-H) and protein disulfide isomerase A3 (PDIA3) helps in DENV multiplication by suppressing TNF-α production in human monocytic THP1 cells. Proteomic analysis of infected cells, identified upregulation of the host cell proteins PDIA3 and hnRNP-H in comparison to mock infected cells. The functional role of hnRNP-H and PDIA3 in DENV infection was identified by down regulating hnRNP-H and PDIA3 genes with their specific siRNA duplexes which lead to decreased intracellular viral load. It also resulted in increased TNF-α level which mediates antiviral effect. This is the first study, which reports the role of PDIA3 and hnRNP-H in TNF-α production in DENV infected cells. Collectively, these results suggest that increased level of hnRNP-H and PDIA3 expression in DENV infected THP1 cells assist in the viral replication by suppressing the TNF-α production.     

Early activation of natural killer and B cells in response to primary dengue virus infection in A/J mice

Dengue virus (DEN) causes the most prevalent arthropod-borne viral illness in humans worldwide. Immune mechanisms that are involved in protection and pathogenesis of DEN infection have not been fully elucidated due largely to the lack of an adequate animal model. Therefore, as a first step, we characterized the primary immune response in immunocompetent inbred A/J mice that were infected intravenously with a non-mouse-adapted DEN type 2 (DEN2) strain. A subset (55%) of infected mice developed paralysis by 14 days post-infection (p.i.), harbored infectious DEN in the central nervous system (CNS), and had an elevated hematocrit and a decreased white blood cell (WBC) count. Immunologic studies detected (i). increased numbers of CD69(+) splenic natural killer (NK) and B cells at day 3 p.i., (ii). DEN-specific IgM and IgG responses by days 3 and 7 p.i., respectively, and (iii). splenocyte production of IFNgamma at day 14 p.i. We conclude that the early activities of NK cells, B cells and IgM, and later actions of IFNgamma and IgG likely play a role in the defense against DEN infection.     

登革2型病毒在经口感染的白纹伊蚊不同个体体内分布的比较研究

本研究用石蜡切片和免疫组织化学技术 ,对白纹伊蚊经口感染登革 2型病毒的分布定位进行了研究。白纹伊蚊感染登革 2型病毒 1 4天后 ,切片染色可直观地定位出病毒在蚊虫组织中的分布 ,比较不同蚊虫之间的染色可分为 3种类型 :1 白纹伊蚊的中肠 ,唾液腺 ,复眼 ,神经节等较多组织呈阳性着色 ;2 白纹伊蚊的中肠呈阳性 ,其它组织无阳性着色 ;3 白纹伊蚊的中肠及其它组织均无阳性着色。以不同病毒滴度经口接种白纹伊蚊时 ,白纹伊蚊中肠 ,唾液腺的感染率和感染病毒滴度呈正相关。以上结果提示白纹伊蚊部分个体对登革 2型病毒的感染存在中肠感染和中肠释放屏障 ,并且和感染病毒剂量有关     

登革2型病毒在C6/36细胞上受体蛋白的鉴定

应用VOPBA (病毒辅覆蛋白结合分析 )技术 ,结合免疫印迹化学发光的方法在C6 36细胞上鉴定出了登革 2型病毒唯一的大小约为 35kDa的受体蛋白成分。为研究病毒对白纹伊蚊的感染机理打下了良好的基础。     

A model of laboratory surveillance for neuro-arbovirosis applied during 2012 in the Emilia-Romagna region,Italy

Arboviruses with neuroinvasive potential are gaining more attention due to the increased number of cases of autochthonous and imported infections in the human host. Diagnosis of infection caused by these viruses in patients with central nervous system (CNS) diseases is still underestimated and these infections represent an emerging threat to public health. We describe a model suitable for the laboratory surveillance of neuro-arbovirosis that was applied in the Emilia-Romagna region, north-eastern Italy, during the 2012 summer season. One hundred and twenty cases of suspected neuroinvasive infection were tested for arboviral agents on the basis of clinical and laboratory signs and epidemiological data. The most common virus detected was Toscana virus (TOSV): anti-TOSV specific antibodies or viral components were detected in 28.3% of the cases; 79.4% of the TOSV cases were in the acute phase of infection. No cases resulted in acute phase for West Nile (WNV), Usutu (USUV), Chikungunya (CHIKV) or Dengue (DENV) virus infection. Conversely, two patients with a history of staying in a tick-borne encephalitis virus (TBEV) endemic area showed a probable TBEV infection. These results emphasize the importance of a complete and →ready to act‘ laboratory diagnostic system to be implemented within the larger frame of a regional integrated surveillance system.     

Mosquito cells accommodate balanced, persistent co-infections with a densovirus and Dengue virus

To study persistent viral co-infections in arthropods, we first produced stable, persistently infected C6/36 mosquito cell cultures by serial passage of exponentially growing whole cells infected with either a densovirus (AalDNV) or Dengue virus (DEN-2). We then obtained stable, persistent co-infections by reciprocal super-challenge and similar passaging. Persistently infected cultures did not differ from naïve-cell cultures in growth rate and cell morphology. Nor did they differ in high production of both viruses with high infection rates for naïve C6/36 cells. Immunocytochemistry revealed that 99–100% of the cells were coinfected but that super-infection order had some effect on antigen distribution for the two viruses. Our results combined with existing field information and previously published experimental work suggest that the capacity to support stable, viral co-infections may be a general phenomenon for arthropod cells, and that they may be achieved easily and rapidly by serial passaging of whole cultured cells. Such persistent infections would facilitate studies on interactions between co-infecting viruses.