【作者】 陈红莲；

【导师】 江育林；

【作者基本信息】 华中农业大学 ， 水产养殖， 2009， 博士

【摘要】 狗鱼幼鱼弹状病毒(Pike fry rhabdovirus, PFRV)是一种与鲤春病毒血症病毒(Spring viraemia of carp virus, SVCV)同源性很高的病毒,目前还未引起人们高度重视。为了对PFRV有一个较全面的了解,对该病毒今后的监测奠定基础,本论文测定了PFRV的生物学特性,克隆并测序了其全基因组序列,并尝试建立RT-PCR (Reverse transcription polymerase chain reaction, RT-PCR)、实时荧光RT-PCR (Real time RT-PCR)及RT-LAMP (Reverse transcription loop-mediated isothermal amplification, RT-LAMP)3种检测方法。1、PFRV的生物学特征。PFRV能在铜吻磷鳃太阳鱼成纤维细胞(Blue gill fry, BF-2)、鲑鱼胚胎细胞(Chinook salmon embryo, CHSE)、鲤鱼上皮瘤细胞(Epithelioma papulosum cyprinid, EPC)、肥头鲤细胞(Fathead minnow, FHM)和草鱼性腺细胞(Overy of grass carp, CO)上复制,产生明显的细胞病变效应(Cytopathogenic effect, CPE); PFRV在BF-2细胞中的适宜复制温度为15℃-25℃；理化性质测定表明,氯仿处理、pH 3处理60min、50℃水浴30min,病毒彻底灭活,但病毒对DNA抑制剂(5’-碘-2’-去氧尿嘧啶,5-iodo-2’-deoxyuridine, IUdR)处理不敏感；细胞超薄切片电镜观察显示病毒粒子呈典型的弹状,大小为105nm-130nm×60nm-70nm；SDS-PAGE电泳显示病毒含有5个结构蛋白,其分子量分别为165 kDa,65 kDa,46 kDa,38 kDa和29 kDa。2、PFRV全基因组序列的克隆、测序及分子进化分析。本研究共设计10对简并引物,4对特异性引物及2套cDNA末端快速扩增(Rapid-amplification of cDNA ends, RACE)引物扩增PFRV全基因组序列,获得了16个相互重叠的克隆,测得序列拼接后确定PFRV的全基因组序列为11097个核苷酸。序列分析表明PFRV全基因组含有5个基因,基因间有间隔序列,在基因组的3’和5’端分别有前导序列和拖尾序列。参考其他弹状病毒,这5个开放阅读框(Open reading frame, ORF)编码的5个蛋白分别为核蛋白(Nucleoprotein, N)、磷酸化蛋白(Phosphoprotein, P)、基质蛋白(Matrix protein, M)、糖蛋白(Glycoprotein, G)和RNA依赖的RNA聚合酶蛋白(RNA-dependent RNA polymerase protein, L),在基因组中的排列顺序为3’N-P-M-G-L5’。PFRV全基因组序列已经被Genebank接受,收录号为FJ872827。PFRV5个基因的转录起始信号和转录终止信号分别与其他水疱性口膜炎病毒的相应信号相同。PFRV3’端和5’端序列反向互补,且分别与水疱性口膜炎病毒的相应序列有很高的同源性。氨基酸序列两两比对显示：在4个动物弹状病毒属中,PFRV编码的5个蛋白均与水疱性口膜炎病毒同源性最高,特别是SVCV,同源性分别为91.4%(N)、55.3%(P)、76.6%(M)、70.7%(G)和87.8%(L)。依据动物弹状病毒N、P、M和G蛋白的全长氨基酸序列以及L蛋白的blockⅢ氨基酸序列构建5个蛋白的系统进化树,5个进化树均显示PFRV与SVCV聚为一支,然后再与水疱性口膜炎病毒正式种类聚为一支,最后与弹状病毒903/87(Rhabdovirus 903/87,903/87)、海龟弹状病毒28/97(Sea trout rhabdovirus 28/97, STRV)和胭脂鱼弹状病毒(Chinese sucker rhabdovirus, SCRV)聚集。3、RT-PCR检测方法。依据PFRV G基因保守序列,设计2对特异性引物。对RT-PCR的反应组分Mg2+、dNTP、引物浓度及反应的退火温度、循环数进行优化。按照优化好的RT-PCR条件进行特异性试验和灵敏度试验,结果显示：检测传染性造血器官坏死病毒(Infectious haematopoietic necrosis virus, IHNV)、SVCV、病毒性出血性败血症病毒(Viral haemorrhagic septicaemia virus, VHSV)、病毒性神经坏死病毒(Virus nervous necrosis virus, VNNV)、传染性胰脏坏死病毒(Infectious pancreatic necrosis virus, IPNV)、牙鲆弹状病毒(Hirame rhabdovirus, HRV)和PFRV时,只有PFRV扩增产物电泳出现特征条带；检测PFRV的灵敏度为102.5TCID50。4、实时荧光RT-PCR。根据PFRV G基因保守序列,设计1条特异性Taqman荧光探针和1对特异性引物。对实时荧光RT-PCR的引物和探针浓度及反应的退火温度进行优化。按照优化好的实时荧光RT-PCR条件进行特异性试验和灵敏度试验,结果显示：检测IHNV、SVCV、VHSV、VNNV、IPNV、HRV和PFRV时,只有PFRV样品检测结果为阳性；检测PFRV的灵敏度为101.5TCID50。5、RT-LAMP。依据PFRV G基因6个区域,设计1套特异性LAMP引物(2条内引物,2条外引物)；对反应温度及时间进行优化,确定最优反应条件为63℃恒温反应60 min。按照优化好的RT-LAMP条件进行特异性试验和灵敏度试验,结果显示：检测IHNV、SVCV、VHSV、VNNV、IPNV、HRV和PFRV时,只有PFRV的检测结果为阳性；RT-LAMP检测PFRV的灵敏度为100.5TCID50,比用RT-PCR检测的灵敏度高100倍,比用实时荧光RT-PCR检测的灵敏度高10倍。然而,这3种PFRV分子生物学检测方法能否成为监测的标准,还需要大量的临床数据支持。更多还原

【Abstract】 Pike fry rhabdovirus (PFRV) shares high homology to Spring viraemia of carp virus (SVCV), but has not attached great importance to people. In order to make a more comprehensive understanding of PFRV, and lay the foundation for the future inspection and control of PFRV, its biological characteristics were studied, the complete genome sequence was cloned and sequenced, and three detection methods were developed, including reverse transcription polymerase chain reaction (RT-PCR), real time RT-PCR and reverse transcription loop-mediated isothermal amplification (RT-LAMP).1. The biological characteristics of PFRV. PFRV was inoculated onto the monolayers of Bluegill fry-2 (BF-2), Fathead minnow (FHM), Epithelioma papulosum cyprini (EPC), Grass carp ovary (CO), and Chinook salmon embryo-214 (CHSE-214), CPE were observed. The range of the feasible replication temperature of PFRV was 15℃to 25℃. The results of study on the physico-chemical characterization of PFRV showed that the virus was completely inactivated by treatment with acid (pH 3), chloroform and heat (50℃for 30 min); however, the virus was stable to 5-iodo-2’deoxyuridine (IUdR). Ultrathin sections of cell infected with PFRV revealed the size of the typical bullet-shaped viral particle was approximately 60 nm to 70 nm in width and 105 nm to 130 nm in length. SDS-PAGE analysis of the purified PFRV indicated that PFRV genome contains 5 structural proteins, and the estimated molecular weights were 165 kDa,65 kDa, 46 kDa,38 kDa and 29 kDa, respectively.2. Cloning, sequencing and phylogenetic analysis of PFRV genome. In this study,10 pairs of degenerate primers,4 pairs of specific primers and 2 sets of rapid-amplification of cDNA ends (RACE) primers were designed.16 overlapped clones were obtained and sequenced, and the complete genome of PFRV was determined to be 11097 nucleotides in length. The PFRV genome contains five genes which are separated by the conserved sequences called’junction sequences’and are flanked with the additional sequences called’leader region’and’trailer region’, respectively. In analogy to other rhabdoviruses, five open reading frames (ORFs) encoding in antigenome are named the nucleocapsid protein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G) and RNA-dependent RNA polymerase protein (L), respectively, and are organized in the order 3’N-P-M-G-L 5’. The complete nucleotide sequence has been deposited in GenBank with the accession no. FJ872827. Five genes share the identical transcription initiation and transcription stop/polyadenylation signals with those of other vesiculoviruses, respectively.3’leader-and 5’trailer-sequence in PFRV show inverse complementarity, and each exhibits high homology to the respective sequences of other vesiculoviruses. Comparation of five proteins of PFRV to those of other rhabdoviruses indicates that PFRV shares higher homologies with vesiculoviruses than the rhabdoviruses belonged to other 3 animal genera, specially sharing the highest identities of 91.4%(N),55.3%(P), 76.6%(M),70.7%(G) and 87.8%(L) with SVCV. The phylogenetic trees were calculated from the deduced amino acid sequences of N, P, M, G proteins and block III within the L proteins of rhabdoviruses, the results exhibit that PFRV is mostly close to SVCV and groups with the official vesiculoviruses, and then groups with 903/87, STRV and SCRV.3. RT-PCR.2 pairs of specific primers were designed according to the conserved sequence of PFRV G gene. The concentration of Mg2+, dNTP and primers, and reaction condition including the anneal temperature and the optimal cycles were optimized. The specificity assay and the sensitivity assay were carried out according to the optimized RT-PCR condition, the results showed that only the column with PFRV served as the amplification template in gel electrophoresis exhibited the characteristic strip in detection of Infectious haematopoietic necrosis virus (IHNV), SVCV, Viral haemorrhagic septicaemia virus (VHSV), Virus nervous necrosis virus (VNNV), Infectious pancreatic necrosis virus (IPNV), Hirame rhabdovirus (HRV) and PFRV. The sensitivity in detection of PFRV was 102.5TCID50.4. Real time RT-PCR. One pair of specific primers and one probe were designed according to the conserved sequence of PFRV G gene. The concentration of primers and the probe, and the anneal temperature were optimized. The specificity assay and the sensitivity assay were carried out according to the optimized real time RT-PCR condition, the result of PFRV was positive in detection of IHNV, PFRV, SVCV, VHSV, VNNV, IPNV and HRV. The sensitivity in detection of PFRV was 101.5 TCID50.5. RT-LAMP. A set of specific primers, two outer and two inner primers, were designed based on 6 regions of PFRV G gene for RT-LAMP assay. The reaction temperature and the reaction time were optimized, and the final optimum reaction condition is keeping at 63℃for 60 min. The specificity assay and the sensitivity assay were carried out according to the optimized RT-LAMP condition, the result of PFRV was positive in detection of IHNV, PFRV, SVCV, VHSV, VNNV, IPNV and HRV. The sensitivity in detection of PFRV was 100.5TCID50,100 times more sensitive than RT-PCR,10 times more sensitive than real time RT-PCR.更多还原