西尼罗病毒易感性研究现状

西尼罗病毒可引起鼠、人类及其他动物严重脑炎,近年来再度流行。基因研究分析表明近年来西尼罗病毒突变的速度逐渐加快,突变主要发生在基因编码区,这些突变可导致病毒蛋白E区、NS1区、NS2区及NS5区氨基酸序列的改变。西尼罗病毒感染主要发生于老龄及免疫缺陷动物或人群。西尼罗病毒的易感染性除与突变有重要关系外,与CCR5、OAS及CXCL10等受体及因子也有一定的关系。     

西尼罗病毒研究进展

西尼罗病毒（West Nile virus,WNV）属黄病毒科,为正单链RNA病毒。它在人类中的感染导致以发热为主要症状的传染性疾病,主要由蚊虫叮咬传播。自20世纪50年代首例报告西尼罗病毒自然感染所致脑炎后的几十年内,西尼罗病毒脑炎在欧洲及中亚地区散在、小规模流行。西尼罗病毒脑炎于1999年在美国的爆发及随后几年在北美的流行引起了极大的关注。这次爆发流行中新出现的种种迹象,如其中间宿主——野生鸟类的大量死亡,人类感染者中中枢神经系统受损比例的增高等,提示近期的遗传变异已使西尼罗病毒感染的病理学与流行病学发生了较显著的变化。另外,随着感染的流行,蚊虫叮咬以外的传播途径,如输血、器官移植、母婴传播等日益受到人们重视。同时,人们对阻止疫情所急需的疫苗的研制也在进行之中。本文就近几年来对西尼罗病毒的感染、免疫与流行病学方面的研究进展进行了综述。     

西尼罗病毒研究进展

西尼罗病毒(West Nile virus,WNV)首先在1937年乌干达的西尼罗地区的Omogo镇的一位发热病人血液中分离到,从而得名。西尼罗病毒属于黄病毒科黄病毒属。黄病毒科病毒共有70余种成员组成,其中黄病毒属大多属于经蚊虫、蜱等媒介传播的虫媒病毒(Arbovirus,Arthropod-borne-     

西尼罗病毒的RT-PCR检测与鉴定

建立西尼罗病毒敏感、特异、快速的RT-PCR检测方法用于实验室诊断和流行病学监测。采用一步RT-PCR和套式PCR法对西尼罗病毒感染的乳鼠脑和细胞培养上清进行扩增,并对扩增产物进行序列测定。两种方法均可分别从两种组织中扩增出与预期大小相一致的片段,套式PCR法比一步RT-PCR法更为敏感,该扩增片段与西尼罗病毒埃及Eg101株相应序列的同源性为99％。     

西尼罗病毒研究进展

西尼罗病毒 (West Nile virus,WNV) 首先在1937年乌干达的西尼罗地区的Omogo镇的一位发热病人血液中分离到,从而得名[1].西尼罗病毒属于黄病毒科黄病毒属.     

我国西尼罗病毒和Tahyna病毒的发现与流行

已发现100余种蚊传虫媒病毒在世界各地流行,其引发的人兽共患病是全世界关注的公共卫生问题。长期以来我国仅发现乙型脑炎和登革热两种蚊传虫媒病毒病,但近年来新发现西尼罗病毒和Tahyna病毒及其感染疾病流行。从我国新疆维吾尔自治区采集的蚊虫标本中分离到西尼罗病毒,大量血清学研究证明当地不仅存在西尼罗病毒感染所致疾病,还发生过西尼罗病毒感染引发的病毒性脑炎流行。目前已从新疆维吾尔自治区、青海省和内蒙古自治区采集的蚊虫标本中分离到Tahyna病毒,并发现其在自然界动物中的循环和导致的人类感染流行。西尼罗病毒和Tahyna病毒及其相关感染性疾病的发现为我国虫媒病毒及虫媒病毒病的预防与控制提出了新的挑战。     

实验感染的三带喙库蚊和来亨鸡体内西尼罗病毒的分离与鉴定

采用C6/36细胞培养分离活病毒、间接免疫荧光染色检测病毒抗原、RT-PCR扩增病毒基因片段和PCR产物测序等方法,对实验感染的三带喙库蚊Culex tritaeniorhynchus和来亨鸡血液样本中的西尼罗病毒进行分离和鉴定。结果表明,接种实验感染蚊虫研磨液和来亨鸡血液样本的C6/36细胞出现细胞融合、空泡形成的病变效应; 用西尼罗病毒抗血清进行间接免疫荧光染色,感染病毒的细胞呈现黄绿色荧光,为阳性反应; 采用3对不同引物的RT- PCR体系扩增分别出现预期的408 bp、498 bp和559 bp的基因片段,序列测定证实扩增序列与实验所用毒株相应的基因序列基本相同。从而证实实验感染三带喙库蚊和来亨鸡体血液内的西尼罗病毒与实验感染所用的西尼罗病毒Chin-01株一致。     

基因免疫制备西尼罗病毒NS5蛋白特异性单抗

研究了NS5蛋白在西尼罗病毒的特异性检测方面的应用及NS5在黄病毒复制中的作用机理。采用RT．PCR方法扩增了西尼罗病毒株的NS5基因片段,将其克隆至真核表达载体pVAX1,构建真核表达质粒。以重组质粒免疫BALB／c小鼠后取脾脏进行杂交瘤细胞融合,建立能稳定分泌西尼罗NS5单克隆抗体的杂交瘤细胞株。构建了真核表达质粒pVAX1-WNV—NS5,免疫动物后获得了28289等4株稳定分泌特异性抗体的杂交瘤细胞株,均为IgM型。真核表达质粒免疫后成功地诱导了针对NS5蛋白的体液免疫应答,单抗特异性分析显示4株单抗与其他黄病毒存在一定交叉反应。     

一种致命性西尼罗病毒

这种病毒首次在乌干达西尼罗河省被发现 ,故称西尼罗病毒 (westnilevirus) ,属于黄热病毒属成员。同属这一科的还有登革热病病毒、日本脑炎病毒以及丙型肝炎病毒等。由于此病毒的宿主为各种鸟类。因此 ,该病毒也称之为“鸟病毒” (1 937年 )。它通过蚊子叮咬或输血传播给人类或其它牲畜 ,大有蔓延之势。当蚊子叮咬宿主后立即受到感染 ,病毒存在于唾腺内 ,经 1 0～ 1 4d的潜伏期 ,它能攻击人的神经系统和脑组织 ,一般有 3～ 1 2d的发病潜伏期 ,病发后出现肌肉无力 ,不辨方向 ,头痛 ,类似感冒等症状 ;重者则出现致命性脑炎和脑膜炎。 1 997年…     

Application of nile blue and nile red, two fluorescent probes, for detection of lipid droplets in human skeletal muscle

Using frozen sections from human muscle biopsies, we assessed the value of Nile blue and Nile red, two fluorescent probes, as stains for lipid droplets in normal and pathological skeletal muscle fibers. In normal muscle, lipid storage disorders, and mitochondrial myopathies, Nile blue stained the lipid droplets as yellow-gold fluorescent structures. The lipid droplets were also seen as yellow-gold fluorescent structures in Nile red-stained sections, but the outstanding feature in these preparations was the staining of the membrane network of the muscle fibers and membrane proliferations in pathological muscle as red-orange fluorescent structures. These results suggest that both Nile blue and Nile red stains are useful for visualization of lipid droplets and membrane proliferations in pathological muscle biopsies.     

Antiviral peptides targeting the west nile virus envelope protein

West Nile virus (WNV) can cause fatal murine and human encephalitis. The viral envelope protein interacts with host cells. A murine brain cDNA phage display library was therefore probed with WNV envelope protein, resulting in the identification of several adherent peptides. Of these, peptide 1 prevented WNV infection in vitro with a 50% inhibition concentration of 67 muM and also inhibited infection of a related flavivirus, dengue virus. Peptide 9, a derivative of peptide 1, was a particularly potent inhibitor of WNV in vitro, with a 50% inhibition concentration of 2.6 muM. Moreover, mice challenged with WNV that had been incubated with peptide 9 had reduced viremia and fatality compared with control animals. Peptide 9 penetrated the murine blood-brain barrier and was found in the brain parenchyma, implying that it may have antiviral activity in the central nervous system. These short peptides serve as the basis for developing new therapeutics for West Nile encephalitis and, potentially, other flaviviruses.     

Spectrofluorometric studies of the lipid probe, nile red

We found that the dye nile red, 9-diethylamino-5H-benzo[alpha]phenoxazine-5-one, can be applied as a fluorescent vital stain for the detection of intracellular lipid droplets by fluorescence microscopy and flow cytofluorometry (J. Cell. Biol. 1985. 100: 965-973). To understand the selectivity of the staining, we examined the fluorescence properties of nile red in the presence of organic solvents and model lipid systems. Nile red was found to be both very soluble and strongly fluorescent in organic solvents. The excitation and emission spectra of nile red shifted to shorter wavelengths with decreasing solvent polarity. However, the fluorescence of nile red was quenched in aqueous medium. Nile red was observed to fluoresce intensely in the presence of aqueous suspensions of phosphatidylcholine vesicles (excitation maximum: 549 nm; emission maximum: 628 nm). When neutral lipids such as triacylglycerols or cholesteryl esters were incorporated with phosphatidylcholine to form microemulsions, nile red fluorescence emission maxima shifted to shorter wavelengths. Serum lipoproteins also induced nile red fluorescence and produced spectral blue shifts. Nile red fluorescence was not observed in the presence of either immunoglobulin G or gelatin. These results demonstrate that nile red fluorescence accompanied by a spectral blue shift reflects the presence of nile red in a hydrophobic lipid environment and account for the selective detection of neutral lipid by the dye. Nile red thus serves as an excellent fluorescent lipid probe.     

Recent advances in the molecular biology of west nile virus

Since the mid-1990s, West Nile virus (WNV) has emerged as a significant agent of arboviral encephalitis in several regions of the world. In 1999, WNV was introduced into the northeastern United States and was associated with an outbreak of encephalitis affecting humans, birds and horses. Subsequently, the virus has spread across the country, and across southern Canada, and in 2002 and 2003 was associated with the largest outbreaks of arboviral encephalitis recorded in the Western hemisphere. Interestingly, the more recent spread of WNV into Mexico, Central America and the Caribbean has not been associated with the high levels of clinical disease observed in North America. This review addresses the most recent results from studies investigating the molecular biology and evolution of WNV, as well as progress in the development of diagnostic and therapeutic reagents.     

Crystal structure of west nile virus envelope glycoprotein reveals viral surface epitopes

West Nile virus, a member of the Flavivirus genus, causes fever that can progress to life-threatening encephalitis. The major envelope glycoprotein, E, of these viruses mediates viral attachment and entry by membrane fusion. We have determined the crystal structure of a soluble fragment of West Nile virus E. The structure adopts the same overall fold as that of the E proteins from dengue and tick-borne encephalitis viruses. The conformation of domain II is different from that in other prefusion E structures, however, and resembles the conformation of domain II in postfusion E structures. The epitopes of neutralizing West Nile virus-specific antibodies map to a region of domain III that is exposed on the viral surface and has been implicated in receptor binding. In contrast, we show that certain recombinant therapeutic antibodies, which cross-neutralize West Nile and dengue viruses, bind a peptide from domain I that is exposed only during the membrane fusion transition. By revealing the details of the molecular landscape of the West Nile virus surface, our structure will assist the design of antiviral vaccines and therapeutics.     

Respiratory adaptations to diving in the nile monitor lizard,Varanus niloticus

Summary The objectives of this study included directin vivo measurements of circulating blood gases, pH, heart rate, and blood pressure during voluntary dives of unrestrained Nile monitor lizards. A Radiometer flow-through cuvette was employed for continuous recording of arterial PO₂, PCO₂ and pH. Hematological properties revealed no particular adaptations for diving. Mean values were: hematocrit = 24%; hemoglobin concentration = 7.1 g %; oxygen capacity = 9.3 vol %; red cell dimensions = 22×12 ; red cell count = 0.67 million/l. The respiratory properties of the blood, studiedin vitro andin vivo, show distinct adaptations to habitual diving. Oxygen affinity of blood is low (P₅₀ = 42.4 mm Hg at pH 7.45, 25 °) and the dissociation curve is markedly sigmoid (n = 3.1). These features, coupled with a Bohr factor ( logP ₅₀/pH) of –0.48, ensure increased utilization of oxygen while maintaining relatively high tissue PO₂. Arterial pH decreases during diving from about 7.5 to 7.1 due to combined respiratory and metabolic acidosis. High plasma bicarbonate (30 mM/l at PCO₂ = 25 mm Hg) and a buffering capacity of H C₃ ^–O/ pH = 18.9 mM/l increase the tolerance to this acidosis and prolong diving time. Thein vivo oxygen dissociation curve shows a 90 % depletion of arterial oxygen content during typical dives. Diving elicited a rapidly developing bradycardia with maximum of 85 % reduction in heart rate. The temperature sensitivity of HbO₂ binding was very low (H = –3kcal). This would minimize the HbO₂ affinity increase accompanying the decrease in body temperature likely to occur in lizards going from sun basking to submergence in water.Supported by a grant from the Danish Natural Science Research Council.     

West nile virus antibodies in bats from New Jersey and New York

Eighty-three serum samples were obtained from big brown (Eptesicus fuscus), little brown (Myotis lucifugus), and northern long-eared (Myotis septentriotalis) bats (Chiroptera: Vespertilionidae), from New Jersey and New York (USA) between July and October 2002. Samples were analyzed for neutralizing antibodies to West Nile virus (WNV) and St. Louis encephalitis (SLE) virus. One little brown bat and one northern long-eared bat tested positive for WNV neutralizing antibodies. No bats had antibodies to SLE virus. This was the first large-scale investigation of WNV infection in bats in New Jersey. Additional work is needed to determine the effects of WNV on bat populations.