摘要
昆虫媒介病毒(虫媒病毒)是一大类主要由吸血昆虫叮咬人、家畜及野生动物而传播的病毒。其特点是病毒在节肢动物媒介体内繁殖而不发病,具有自然疫源性。目前已经证实的媒介昆虫达586种,主要为蚊(300种)和蜱(116种)。虫媒病毒引起人畜共患疾病,近年来备受关注。目前国际上发现有500多种虫媒病毒,其中130余种可引起人畜共患疾病。同时,虫媒病毒可随人群流动、宿主和媒介昆虫的迁移而传播到异地。由于大部分虫媒病毒感染没有特效药物,也没有可用的疫苗,因此虫媒病毒的研究对病毒的预防与控制具有积极意义。登革病毒隶属于黄病毒属,有四个血清型(DENV-1、-2、-3和-4),经由节肢动物白纹伊蚊(Aedesalbopictus)与埃及伊蚊(Ae.aegypti)传播,是热带、亚热带地区重要的虫媒病毒。目前登革热(Dengue Fever,DF)在超过100个国家流行,威胁着大约25亿人口,全球每年大约有5000万到1亿人受到感染,25000人死亡。由于没有有效的疫苗,病毒早期诊断与及时医疗护理显得尤为重要。我国建国后第一次登革热流行发生在1978年的广州佛山,至今登革热已经成为我国南方重要的公共卫生问题,现已是法定的传染病。系统的了解我国登革病毒三十年来分子流行病学可以阐明病毒的发生、发展,对病毒防护有指导意义。基于此,本研究以我国建国以来收集到的登革病毒为研究对象,从分子水平研究其流行与进化规律,同时开发了登革病毒的实时荧光PCR探针法诊断技术。现将研究结果总结如下:
一、虫媒登革病毒的基因组进化
1、基因组测定与比较
实验测得收集剑的14株登革病毒的DENV-1、-2和-4的基因组全长分别为10735bp、10723bp和10649bp。DENV-1的5'和3'非编码区分别为94nt和462nt,编码区从95位开始到10272位结束,全长10179bp,编码3392个氨基酸。DENV-2的5'和3'非编码区分别为96nt和454nt,编码区从97位开始到10272位结束,全长10176bp,编码3391个氨基酸。DENV-4的5'和3'非编码区分别为101nt和384nt,编码区从102位开始到10265位结束,全长10164bp,编码3387个氨基酸。
8株DENV-1(1991～2006年)全基因组核酸序列相似性在91.8%-99.7%之间,编码的氨基酸序列相似性在97.0%-99.7%之间;4株(1993～2001年)DENV-2全基因组核酸序列相似性在92.3%-98.8%之间,氨基酸序列相似性在96.5%-98.8%之间;2株(1978和1990年)DENV-4病毒株的全基因组核苷酸相似性为93.6%,编码的氨基酸序列相似性为96.0%。
同一血清型间5'UTR和3'UTR序列相似性非常高,仅有几个核苷酸差异。我们比较所测的分离株单基因水平氨基酸相似性,发现虽然氨基酸相似性在不同年代、不同病毒株、不同蛋白区域没有明显规律,但总体来讲,在NS3、NS4A、NS4B、NS5蛋白的氨基酸的相似性较高,而在结构蛋白区域氨基酸相似性较低。同时发现,2002-2003、2004、2006和2007四个时间点分离株的E461位点氨基酸分别为异亮氨酸(I)、缬氨酸(V)、亮氨酸(L)、丙氨酸(A),其疏水性参数分别为4.5、4.2、3.8和1.8,疏水性逐渐降低。病毒E蛋白C末端为E蛋白的转膜区,参与病毒与宿主的识别附着,这种疏水性变化趋势是否与病毒与人类宿主细胞相互作用的适应有关值得进一步研究。
2、虫媒登革病毒基因组进化
本文对1978至2006年登革热爆发时收集的细胞分离株基因组测序,结合GenBank上已经公布的病毒序列,总共获得145个外膜蛋白E基因序列和74个全基因组序列。基于外膜蛋白E基因与全长编码区序列所构建的系统发生树具有很好的一致性。结果显示:一、我国的大部分登革病毒与东南亚邻近国家的分离株系在进化树位置上更接近,暗示我国登革病毒大部分可能起源于登革病毒多发的东南亚国家,如泰国、菲律宾、印度尼西亚等:二、三种血清型(DENV-1、-2、-4)的虫媒登革病毒在基因型和分支水平具有丰富的遗传多样性,同时,血清型内大量基因重组现象发生也增加了遗传多样性的复杂程度;三、纯化选择是登革病毒进化的主要驱动力,但在病毒的非结构蛋白NS1的94氨基酸位点检测到强烈阳性选择。有趣的是,NS1蛋白94位氨基酸以亲水性及疏水性可以区分特定的进化血系。
二、虫媒登革病毒的实时荧光PCR诊断
1、实时荧光PCR诊断技术的建立
通过生物信息学比对分析GenBank登陆的以及本文测序的登革病毒株系序列,利用PrimerPress3软件设计实时荧光PCR特异性引物及TaqMan探针,14个登革病毒培养上清提取RNA评估引物及探针的特异性、灵敏度。研究表明:本试验设计的四种型特异性引物及TaqMan探针具有特异性好(血清型间、与相近属种、人类基因组、蚊虫细胞间无交叉反应)、敏感性高(最低检测限为15-80拷贝RNA/反应)等特点,可用于诊断登革病毒感染。
2、实时荧光PCR诊断技术的初步应用
近年来我国南方发生的登革病毒大部分是血清1型。本文收集到2006年广州1型血清型病毒流行时64份不同病程的病人临床血清样品,评估上述建立的诊断方法的应用性。结果表明:一、本研究开发的实时荧光PCR诊断方法检测阳性率为71.9%(46/64),而传统的免疫荧光分析法(IFA)检测的阳性率IgM为54.7%(35/64),IgG为34.4%(22/64),显示实时荧光PCR诊断方法具有较好的应用性;二、本文开发的实时荧光PCR诊断方法对登革热症状起始1-3天病人血清检测阳性率高达86.4%(19/22),而IFA检测中,IgM与IgG的阳性率仅为18.2%(4/22)和9.1%(2/22),表明此方法能在登革热症状起始后1-3天内较准确的检测病毒感染,克服了IFA检测速度慢及假阳性问题,因此在临床上可应用此方法进行登革病毒的早期快速诊断。
Insect-vectored or arthropod-borne viruses(arboviruses) are a large group of viruses that are spread mainly by blood-sucking insects such as mosquito,sandfly and midge.Those viruses are just carried and transmitted by insects,rather than causing their disease,therefore as natural sources for human disease emergence.Currently,more than 586 specieses of insect have been identified for transmission of 500 viruses,of which 130 viruses could cause serious zoonotic disease.Arboviruses could also be transmitted to other yet-epidemic area by host(human,insect) movement.Despite the serious threat of arbovirus to human health,few specific medicine or vaccine are available for their control,highlighting the necessity of continuing research.Dengue viruses(DENVs),which are transmitted by Aedes albopictus and Ae.Aegypti,belong to the genus of flavivirus with more than one of the four serotypes(DENV-1,-2,and -3) circulating in tropical and subtropical regions. Dengue virus is epidemic in over one hundred countries,threatening 2.5 billion people all over the world.Due to the unavailability of effective vaccine,early diagnosis and timely medical care are of significance for dengue patient.In China,the first epidemic of dengue happens in 1978 in Foshan city,Guangzhou.But now dengue has caused serious public health impact in southern China,and has been documented as an official infectious disease.A systematic understanding of the molecular aspects of dengue epidemiology over thirty-year period is of meaningful for dengue control.As such, this work is focusing on 1) molecular epidemiology of dengue viruses in China from its first emergence in 1978 to the latest outbreak in 2006,and 2) development of real-time PCR TaqMan probe for rapid and early diagnosis of dengue virus.The main results were as follows:
1.Genomic evolution of dengue virus
1.1 Genomic sequencing and comparison
Based on published dengue sequences from GenBank,different specific primers for DENV-1, -2 and -4 full genome amplification were designed,followed by RT-PCR amplification,cloning into pGEM-T vector,and sequencing.By assembling of sequenced fragments,totally,six E gene and fourteen full-genome sequences were obtained.The full-length of genome of DENV-1,-2 and -4 are 10735bp,10723bp,and 10649bp,encoding 3392,3391,3387 amino acids,respectively.The 5' and 3' non-coding region of DENV-1 were 94 and 462 nts,respectively,while DENV-2 were 94 and 452 nts,and DENV-1 were 101 and 485 nts,respectively.
Nucleotide sequence similarity of eight DENV-1(2001-2006) full genomes was ranging from 91.8%to 99.7%,while their corresponding amino acid sequence similarity was ranging from 97.0% to 98.8%.Nucleotide sequence similarity of four DENV-2(1993-2001) full genomes was ranging from 92.3%to 98.8%,while their corresponding amino acid sequence similarity was ranging from 96.5%to 98.8%.Nucleotide sequence similarity of two DENV-4(1978 and 1990) full genomes was 93.6%,while their corresponding amino acid sequence similarity was ranging from 96%.
The sequences of 5'UTR and 3'UTR of the same serotype were really conservative,with only several nucleotides difference.According to the amino acid comparison of single protein encoded by sequences determined in the study,the amino acid sequences of NS3,NS4A,NS4B,and NS5 showed superior similarity to that of structural proteins and other nonstructural proteins.We further explored the amino acid change of envelope protein genes collected in the article,and found amino acid position 461 of envelope protein(E461) was Ile(I),Val(V),L(Leu),and A(Ala) at the time point of 2002-2003,2004,2006 and 2007,respectively.And the corresponding hydrophobic parameters for each amino acid were 4.5,4.2,3.8 and 1.8,demonstrating a decreasing trend of hydrophobic parameters.E461 was on the C-terminal of envelope protein,a transmembrane region involving in the host identification and attachment.Whether the trend indicates an adaption of the dengue virus to host remains undetermined.
1.2 Genomic evolution of dengue virus
A dataset of 14 full-length DENV genome sequences from 1978 to 2006 and six complete E gene sequences from patients' sera in 2006 was obtained.These sequences were combined with 60 DENV full coding region sequences and 125 E gene sequences taken from GenBank representing a wide range in geographic localities.The longitudinal study of dengue(DENV-1,-2 and -4) molecular epidemiology in south China over the thirty-year period revealed,that 1) introduction from neighboring countries contributes most to the dengue outbreak in China,although endemic strain was inferred and China might also be a dengue source,that 2) genetic diversity of Chinese isolates in genotype and clade level was seen,but it was further complicated by diverse potential intra-serotype recombination,that 3) purifying selection was predominating evolutionary drive, while positively selected site on NS1 of DENV-1 was detected that shows to drive specific lineage evolution by changing hydrophilic amino acids to hydrophobic ones.Those highlight the necessity of concern on natural recombination and nonstructural protein gene for dengue evolution.
2.Development of real-time PCR diagnosis of dengue viruses
2.1 Establishment of real-time PCR diagnosis
All the available sequences from GenBank and the sequences determined in the study were aligned to find their serotype-specific regions for real-time PCR primers and probe exploration by Primer Press 3,followed by evaluation of their specificity and sensitivity by RNA extracted from dengue virus cell culture suspension.Results show that 1) the four sets of primers and TaqMan probes have significant specificity,with no cross reaction between different serotypes,close genus, human genome,and mosquito cell RNA,high sensitivity,with the detection limitation ranging from 15 to 80 copies/reaction.Those indicate the developed real-time PCR methods were reliable for dengue diagnosis.
2.2 Preliminary evaluation of real-time PCR for dengue diagnosis
Recently,DENV-1 is the main serotype circulating in south China.Sixty-four clinical blood samples with different time after disease onset were collected during dengue epidemic in 2006 in Guangzhou to evaluate the applicability of the established method.Results show that 1) the positive rate of real-time PCR for detecting dengue is 71.9%(46/64),while the immuno-fluorescence assay (IFA) of IgM is 54.7%(35/64),IgG is 34.4%(22/64),showing superior performance of real-time PCR over IFA,that 2) during the first 1-3 days after the symptom onset,the positive rate of real-time PCR is as high as 86.4%(19/22),while in IFA,the IgM and IgG are 18.2%(4/22) and 9.1(2/22), respectively,indicating real-time PCR detecting dengue in the study is superior,especially in early dengue diagnosis.Those above results indicate real-time PCR developed in the study can be used to dengue diagnosis,especially in early diagnosis.
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