虫媒黄病毒嵌合疫苗研究进展

黄病毒科黄病毒属包括70多种病毒,分为几种抗原复合组,大多数黄病毒为节肢动物传播的,其中40％以上的病毒对人有致病性,最重要的疾病有登革热／登革出血热(DEN／DHF),日本脑炎(乙型脑炎,JE),黄热(YF),西尼罗热(WNF)和璧虱传播脑炎(森林脑炎,TBE)等。登革热每年约有数千万至1亿病人,数十万人发生严重的登革出血热。乙脑每年约发生4～5万病例,病死率25％,45％     

黄病毒科病毒编码的非结构蛋白及其功能

黄病毒科病毒是一类具有囊膜的ＲＮＡ病毒,现包括黄病毒、瘟病毒及类丙型肝炎病毒三个属。该科的许多病毒均是引起人和动物一些严重疾病的病原,其基因组为一单股正链的ＲＮＡ分子,分别编码病毒的结构蛋白和非结构蛋白,其中非结构蛋白对于病毒的复制及病毒与其宿主细胞的相互作用起着极为重要的作用。本文综述了该科病毒编码的非结构蛋白及其主要功能,旨在深入了解和研究该科病毒的复制规律,以有助于由该科病毒引起的许多严重病毒病的防治     

黄病毒属病毒复制子载体研究进展

RNA复制子是一种能自主复制的RNA载体,保留了病毒非结构蛋白(复制/转录酶)基因,而结构蛋白基因缺失或由外源抗原基因替代,复制/转录酶可控制载体RNA在细胞质中高水平复制以及外源基因的高水平表达。在黄病毒属病毒感染性克隆基础上,其复制子载体得到了成功的构建。黄病毒属病毒复制子为病毒基因组结构功能研究、表达载体构建、假病毒包装及新型疫苗制备等提供了新的技术平台。本文综述黄病毒属病毒复制子的构建原理、方法及应用。     

虫媒黄病毒嵌合疫苗研究进展

黄病毒科黄病毒属包括70多种病毒,分为几种抗原复合组,大多数黄病毒为节肢动物传播的,其中40%以上的病毒对人有致病性,最重要的疾病有登革热/登革出血热(DEN/DHF),日本脑炎(乙型脑炎,JE),黄热(YF),西尼罗热(WNF)和璧虱传播脑炎(森林脑炎,TBE)等.登革热每年约有数千万至1亿病人,数十万人发生严重的登革出血热.乙脑每年约发生4～5万病例,病死率25%,45%的患者发生神经学或生理学上的后遗症.当前对黄热病已有减毒活疫苗,对登革热还没有疫苗,对乙脑已有鼠脑和地鼠肾细胞灭活疫苗和减毒活疫苗,对璧虱脑炎已有灭活疫苗,但各种疫苗都还存在一定的不足,有必要进一步改进和提高.     

NS3蛋白在黄病毒科病毒生命活动中的作用

黄病毒科病毒包括三个属,即黄病毒属(Flavivirus),瘟病毒属(Pestivirus)和丙型肝炎病毒属(Hepacivirus)。这些病毒均能引起人和动物患严重疾病。黄病毒属黄热病毒(Yellow fever virus,YFV)、登革病毒(Dengue virus,DEN)能引起发烧、出血,患者死亡率极高,瘟病毒属牛腹泻病     

广东省三株蚊媒黄病毒的鉴定和系统进化分析

为了解广东省蚊媒病毒的种类和分子特征,本研究对从蚊子中分离的三株黄病毒提取病毒核酸并进行二代测序,经序列拼接和比对,基因组序列中缺失的gap通过RT-PCR进行扩增填补。用MEGA软件登录GenBank下载黄病毒代表株序列并绘制进化树,同时用ClustalW2软件与新毒株进行氨基酸同源性比对和编码框序列的分析。结果显示利用二代测序技术获得了三株病毒基因组序列的96.35%～98.26%,并用RT-PCR成功补齐所有gap,序列比对显示其中一株与黄斑库蚊病毒核苷酸和氨基酸同源性98.51%,两株病毒与广平病毒的核苷酸同源性分别为97.30%和89.78%。三株病毒都属于黄病毒家族中的未分类黄病毒或昆虫特异性黄病毒,编码区序列中C蛋白的同源性最低,NS5的同源性最高。本研究发现的三株蚊媒黄病毒中,库蚊黄斑病毒在国内为首次鉴定;两株广平病毒核苷酸同源性存在较大差异,可能存在不同基因亚型。     

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。     

Permethrin Induction of Multiple Cytochrome P450 Genes in Insecticide Resistant Mosquitoes,Culex quinquefasciatus

The expression of some insect P450 genes can be induced by both exogenous and endogenous compounds and there is evidence to suggest that multiple constitutively overexpressed P450 genes are co-responsible for the development of resistance to permethrin in resistant mosquitoes. This study characterized the permethrin induction profiles of P450 genes known to be constitutively overexpressed in resistant mosquitoes, Culex quinquefasciatus. The gene expression in 7 of the 19 P450 genes CYP325K3v1, CYP4D42v2, CYP9J45, (CYP) CPIJ000926, CYP325G4, CYP4C38, CYP4H40 in the HAmCqG8 strain, increased more than 2-fold after exposure to permethrin at an LC50 concentration (10 ppm) compared to their acetone treated counterpart; no significant differences in the expression of these P450 genes in susceptible S-Lab mosquitoes were observed after permethrin treatment. Eleven of the fourteen P450 genes overexpressed in the MAmCqG6 strain, CYP9M10, CYP6Z12, CYP9J33, CYP9J43, CYP9J34, CYP306A1, CYP6Z15, CYP9J45, CYPPAL1, CYP4C52v1, CYP9J39, were also induced more than doubled after exposure to an LC50 (0.7 ppm) dose of permethrin. No significant induction in P450 gene expression was observed in the susceptible S-Lab mosquitoes after permethrin treatment except for CYP6Z15 and CYP9J39, suggesting that permethrin induction of these two P450 genes are common to both susceptible and resistant mosquitoes while the induction of the others are specific to insecticide resistant mosquitoes. These results demonstrate that multiple P450 genes are co-up-regulated in insecticide resistant mosquitoes through both constitutive overexpression and induction mechanisms, providing additional support for their involvement in the detoxification of insecticides and the development of insecticide resistance.     

A New Esterase Gene Amplification Involved in OP Resistance in Culex pipiens Mosquitoes from China

Two overproduced esterases (A8 and B8) not previously described were found in southern China. They provide a low resistance level to organophosphate (OP) insecticides, and correspond to a coamplification of both esterase loci (Est-2 and Est-3) classically involved in OP resistance for this mosquito species. This coamplification is distinct from all other similar events thus far reported. The peculiar situation in southern China, where numerous OP resistance alleles at these two loci were found, is discussed in comparison with the Mediterranean situation, the only one with a similar diversity of overproduced esterases.     

The Earliest Normal Flower from Liaoning Province,China

The early evolution of angiosperms has been a focus of intensive research for more than a century. The Yixian Formation in western Liaoning yields one of the earliest angiosperm macrofioras. Despite multitudes of angiosperm fossils uncovered, including Archaefructus and Sinocarpus, no bona fide normal flower has been dated to 125 Ma （mega-annum） or older. Here we report Callianthus dilae gen. et sp. nov. from the Yixian Formation （Early Cretaceous） in western Liaoning, China as the earliest normal flower known to date. The flower demonstrates a typical floral organization, including tepals, androecium, and gynoecium. The tepals are spatulate with parallel veins. The stamens have a slender filament, a globular anther, bristles at the anther apex, and in situ round-triangular pollen grains. The gynoecium is composed of two stylate carpels enclosed in a fleshy envelope, and develops into a ＂hip＂ when mature. Since the well-accepted history of angiosperms is not much longer than 125 Ma, Callianthus together with Chaoyangia, Archaefructus and Sinocarpus from the Yixian Formation demonstrate a surprisingly high diversity of angiosperms, implying a history of angiosperms much longer than currently accepted.     

The vector competence of Culex annulirostris, Aedes sagax and Aedes alboannulatus for Murray Valley encephalitis virus at different temperatures

Culex annulirostris Skuse, colonized from Brisbane, Queensland, and Mildura, Victoria, Australia, were effective vectors of Murray Valley encephalitis virus at 20, 27 and 32-35 degrees C with full extrinsic incubation periods of 15, 10 and 4 days respectively. At 20 degrees C, 7-11 days post-infection, transmission by the Mildura colony (0-20%) was less efficient than the Brisbane colony (30-70%) but both were capable of 75-100% transmission after longer extrinsic incubation periods. Discriminant analysis of body and salivary gland titres showed that these were not satisfactory indicators of transmission. Wild-caught Aedes sagax (Skuse) and Cx annulirostris from the Murray Valley showed equal competence, but Aedes alboannulatus (Macquart) was a poor vector. The results provide data on rural amplification of Murray Valley encephalitis virus during spring and suggest that further work on the potential of Ae. sagax as a natural vector is warranted.     

Oviposition bioassay responses of Culex tarsalis and Culex quinquefasciatus to neem products containing azadirachtin

Neem (Azadirachta indica A. Juss) products have been shown to exert pesticidal properties against a variety of insect species. In mosquito control programs, such products may have the potential to be used successfully as larvicides. In exploring other advantages of neem products, we studied the oviposition responses of Culex tarsalis Coquillett and Cx. quinquefasciatus Say to two experimental azadirachtin (AZ) formulations, wettable powder Azad ^– WP10 (WP) and emulsifiable concentrate Azad ^– EC4.5 (EC). Gravid Cx. tarsalis exhibited a distinct preference for the neem suspension of the WP, where significantly more egg rafts were collected from the treatment than from the control. The minimum effective AZ concentration for this activity was 0.5 ppm. The aged suspensions from 1–7 days at 0.5 and 1 ppm AZ were more active in eliciting oviposition responses in Cx. tarsalis than the fresh preparations. This activity of the aged suspensions lasted up to 14 and 21 days at 0.5 and 1 ppm AZ, respectively. Negative ovipositional responses were indicated in the tests of the EC vs. Cx. tarsalis, as well as both neem formulations vs. Cx. quinquefasciatus. In the tests of the EC formulation, significantly less gravid females were trapped by oviposition cups in the treatment than in the control, and in the tests of the WP significantly less egg rafts were collected from the treatment than from the control. The minimum effective concentrations for oviposition avoidance activity were 5 ppm AZ for Cx. tarsalis and 10 ppm AZ for Cx. quinquefasciatus, which lasted up to 1 and 4 days for these two species respectively. Neem products potentially used as mosquito larvicides may have many additional benefits in mosquito control programs, the oviposition modification noted in the current studies is one such example.     

一株牛源阿卡斑病毒的分离鉴定

阿卡斑病毒(Akabane virus,AKV)是能引起牛、绵羊、山羊流产、早产、新生胎儿畸形的虫媒性RNA病毒。为了解家畜虫媒病毒在我国西南边境地区的分布和流行情况,本研究对中缅边境西盟县的52份牛抗凝血和140份血清(牛70份、羊70份)中的蓝舌病病毒(Bluetongue virus,BTV)、鹿流行性出血热病毒(Epizootic hemorrhagic disease virus,EHDV)、AKV等虫媒病毒进行检测与分离,通过ELISA及qRT-PCR方法检测病毒,通过核酸阳性抗凝血接种BHK细胞传代以分离病毒,通过设计特异性引物,扩增分离毒株S基因721bp片段及M基因816bp片段,通过克隆测序及中和试验以鉴定病毒,最终从38号牛的抗凝血中分离到一株AKV,TCID50为10-3.5/0.1mL,经比对,分离株S片段与日本KS-2/Mo/06毒株亲缘关系最近,核苷酸同源性为97.66%,M片段与中国DHL10M110毒株亲缘关系最近,核苷酸同源性为96.56%。本研究首次报告了从云南边境地区牛群中分离到AKV,证实了西南边境存在AKV的流行,为AKV在我国的流行病学和边境地区疫病风险防控提供了重要参考及有力依据。     

四个不同地理区域的库蚊复合组居群的遗传多样性研究

库蚊种群在农药强选择压下的生存及抗性进化是与其种群的遗传多样性密切相关的。本文主要从居群的遗传多样性角度出发 ,研究其抗性遗传行为。分别对采自 4个不同地理区域的库蚊复合组样群 ,采用水平切片淀粉凝胶电泳的方法 ,分析了 3个酶系统 (苹果酸酶Me、苹果酸脱氢酶Mdh、甘油醛-3-磷酸脱氢酶Gpd) ,获得了 5个位点共 2 7个等位基因的资料。并计算了 4个库蚊居群的遗传多样性指标为 (A ,P ,He,Ho) ,同时分析了 5种居群间的遗传距离。分析结果表明 :遗传多样性主要存在于各居群之内 (FIS=0 .5 4 37) ,但居群之间的遗传分化程度也相对较大 (Gst =0 .4 498)。     

Fossil coniferous wood from the Middle Jurassic of Liaoning Province, China

Silicified coniferous wood was collected from the Lanqi Formation (late Middle Jurassic in age) at Shebudaigou Village, Liaoning Province, China. Three taxa are identified, namely Pinoxylon dacotense Knowlton, Xenoxylon phyllocladoides Gothan, and Araucariopitys sp. Based on these new data, and those of other fossil plants reported previously from the same formation, we consider the climate during the deposition of the Lanqi Formation was subtropical, humid and seasonal. In this respect the Lanqi flora differs from the coeval Shimengou and Longmen floras from North China. The Longmen flora was deposited during more humid, subtropical conditions, while the Shimengou Formation indicates that the climate was warm temperate and dry. Our data would suggest that the Late Jurassic climatic pattern was initiated as early as the late Middle Jurassic.