【作者】 王君玮；

【导师】 姜平；

【作者基本信息】 南京农业大学 ， 预防兽医学， 2008， 博士

【摘要】 犬瘟热病毒（CDV）属于副粘病毒科（family Paramyxoviridae）麻疹病毒属（genusMorbillivirus）成员,常引起貂、狐、貉等毛皮动物、犬以及熊猫、虎等珍稀动物大批发病,由于犬瘟热（CD）感染性强、发病急、死亡率高,以及与人类亚急性硬化性全脑炎（subaeute selerosing panencephalitis,SSPE）和多发性硬化症（multiple sclerosing,MS）可能存在联系,一直成为国内外学者研究的热点,但毛皮动物CDV病原学、病理学、诊断、免疫学等研究目前尚不深入。2006年6月至10月间,山东、河北、辽宁等地毛皮动物群大面积暴发犬瘟热。本研究在该病流行病学、临床表现、剖检病变及病理组织学研究基础上,从临床病料中分离获得5个CDV分离株,并进一步从该病毒基因特征、Real-time RT-PCR快速诊断方法和DNA疫苗免疫效果等方面进行了分子生物学特性研究,为毛皮动物群CD防控奠定基础.1.貂、狐、貉群犬瘟热病理学研究:对2006年6月至10月间山东、河北等地区的部分毛皮动物养殖场发生疑似犬瘟热的疫情进行了流行病学、临床表现以及剖检变化观察和分析,并对采集的病料进行了病理学研究。结果表明,这起疫情主要是由携带CDV的貉、貂调运而促进病毒传播感染引起。发病动物剖检病变以全身脏器出血性变化为特征,病情及病变严重程度依次为貉＞狐狸＞水貂。通过对发病狐狸、貉、水貂的肺脏、肝脏、脑、肾脏、膀胱等脏器病理学比较,在病变程度上水貂与狐狸、貉有差异。同一种动物,依病程、病型及有无并发症等因素,病理学变化也存在差异。2.貂、狐、貉源犬瘟热病毒的分离鉴定与生物学特性研究:对流行病学调查、临床症状检查、病理剖检以及病理组织学检查诊断为CDV阳性的发病貉、狐狸和水貂,取其肝、脾、肺、淋巴结及脑混合研磨处理后,分别接种能稳定表达犬信号淋巴细胞活性分子（SLAM）的Vero-DST细胞进行病毒分离,并通过电镜形态观察、RT-PCR检测、理化特性测定和人工感染试验进行鉴定。结果为:5份不同来源的病料接种Vero-DST细胞,36-96h后均产生明显CPE;病毒TCID₅₀分别为10^-5.2～^-7.3/ml;电镜观察可见多形态病毒粒子,,大小为110-500nm;CDV特异性引物RT-PCR检测5个分离毒株均为阳性;除鸡、鹅红细胞呈微弱阳性外,其余均未见血凝作用;对乙醚、氯仿和甲醛敏感,对酸、碱和热的抵抗力比较弱;5个病毒分离株对乳鼠的LD₅₀分别为2×10^-3.8～^-4.8/ml,对实验兔无致病性,对貉有明显致病性作用,从而均鉴定为犬瘟热病毒（CDV）,分别命名为CDV/SD/RD06/HT-P（貉源）、CDV/SD/RD06/THD1（貉源）、CDV/SD/M06/HD（貂源）、CDV/SD/M06/LN（貂源）和CDV/SD/F06/HB（狐源）。3.招、狐、貉源犬瘟热病毒不同分离株H和N基因序列分析:用RT-PCR方法对分离的5个CDV分离株的H基因和N基因进行了扩增,并分别将其克隆到PGEMT-Vector,进行序列测定。然后与GenBank中己报道的CDV毒槲目比较。结果为:（1）H基因5个分离株H基因nt序列同源性均达到97.5%以上,与国内疫苗株chn同源性为90.5～91.8%。5个CDV分离株H基因推导的aa列同源性比较表明:HT-P与THD1的aa同源性为100%,二者与其他分离毒株间aa序列同源性差别大,分别为87.9%～99.1%:与chn同源性普遍较低,为89.7%～91.8%。而chn与Onderstepoort、Convac有着很高的同源性关系（nt同源性分别为96.8%和97.2%;aa同源性分别为97.1%和95.6%）.（2）N基因5个分离株N基因nt序列同源性均达到94.5%以上,HT-P与THD1的同源性最高（99.8%）。国内疫苗株chn与分离株的nt序列同源性普遍较低（90.9～93.5%）,而与疫苗株Onderstepoort的nt同源性高,达到97.1%。从N蛋白aa序列看,分离株之间除HT-P、THD1与HD aa序列同源性较高（99.0%以上）外,其他分离株之间aa同源性低。而Clan与Onderstepoort有着很高的同源性关系,同源性比例达97.3%.本研究结果提示,HD和LN发病区域相距较远,但具有较高的同源性,而与HB具有相对较低的nt同源性,提示野毒株在易感动物上可能存在种属差异。此外,分离毒株H和N蛋白免疫原性可能与疫苗株有较大差别,可能与CD免疫失败有关。4.犬瘟热病毒实时荧光定量RT-PCR检测方法的研究:按照CDV N基因序列,设计合成了特异性引物和探针,经各反应条件的优化,建立了CDV的Real-time荧光定量RT-PCR检测技术。用20pmol/mL的引物浓度各1uL和20pmol/mL的探针浓度0.3uL,获得的荧光信号最强,曲线平滑。敏感度为1.24×10^-3ng/mL的病毒RNA;与NDV、AIV、NiPV等RNA病毒不发生交叉反应。试验重复性的变异系数（CV）分别为2.3%、2.5%和4.2%。对采集的57份临床样品进行检测,荧光RT-PCR检测阳性检出率为93%（53/57）,而BIOINDIST BIT RAPID CDV试剂盒、常规RT-PCR阳性检出率分别为70.2%（40/57）和71.9%（41/57）,与BIOINDIST试剂盒和常规RT-PCR的符合率分别为77.2%和78.9%,而BIOINDIST试剂盒与常规RT-PCR符合率达98.2%。结果表明,本研究建立的检测CDV的荧光RT-PCR方法具有快速、特异、敏感、可定量,并可同时检测大量样品等优点,能用于CDV感染的早期诊断和海关检疫。5.貉源犬瘟热病毒H、F和N基因真核表达质粒的构建与鉴定:将貉源CDV分离株的H、F和N（N1,N2）基因经RT-PCR扩增后,连接到pMD18-T载体,对比测序结果表明各基因序列正确,并且符合阅读框规则。然后将各基因亚克隆到真核表达载体pIRES1neo中,经PCR方法和限制酶酶切鉴定成功构建了四种重组真核表达质粒,分别命名为pIRES T-N1、pIREs T-N2、pIREs T-H、pIRES T-F。用构建的真核重组质粒转染VERO-DST细胞,用抗CDV阳性血清进行Western blot分析,结果表明,各重组质粒可以表达相应的目的蛋白。6.貉源犬瘟热病毒H、F和N基因表达质粒DNA免疫保护效率研究本研究将pIRES T-H、pIRES T-F、pIRES T-N1和pIRES T-N2四个真核表达质粒作为基因疫苗,单独或混合分组对42只小鼠和21只断奶貉进行了动物免疫试验,对免疫的实验貉进行了攻毒保护实验,并与chn弱毒疫苗、进口CDV-CPV二联疫苗进行了比较。结果为:（1）小鼠免疫试验采用构建的基因疫苗免疫小鼠2次后开始有抗体反应,免疫第3次后小鼠血清中抗CDV ELISA抗体和中和抗体水平与免疫前和空载体阴性对照相比明显增高（p＜0.05）。从ELISA抗体滴度比较,四种基因疫苗联合免疫组（简称“四基因组”）与pIRES T-H组、pIRES T-F组和pIRES T-N1+pIRES T-N2组差异不显著,但其中和抗体水平明显高于单一基因疫苗组（p＜0.05）。chn组中和效价与四基因组差异不显著,但ELISA滴度明显高于四基因组。CDV-CPV组ELISA抗体滴度和中和抗体滴度均明显高于各基因疫苗组（p＜0.05）。（2）貉免疫保护试验基因疫苗免疫貉后,血清中CDV ELISA抗体滴度(OD₆₅₀值)组间差异不显著,但是四基因组比单独免疫组有较高抗体滴度。4种基因疫苗经3次免疫貉后,四基因组与其他3组相比中和滴度较高。四基因组与chn组、CDV-CPV组免疫后都有抗体升高,但是chn组上升较低,CDV-CPV组上升较高。CDV-CPV组中和抗体水平明显高于四基因组2个滴度。攻毒实验表明,除四基因组外,pIRES T-H、pIRES T-F、pIRES T-N1+pIRES T-N2组各有不同程度死亡。Chn组死亡1只,而CDV-PPV组均存活。比较存活貉的临床表现,基因疫苗免疫的四个组在攻毒后反应程度不一,四基因组均存活,分值分别为10、9、2;pIRES T-N1+pIRES T-N2组反应严重,存活貉分值:12,另2只死亡;pIRES T-H组存活的2只貉中1只垂危（分值:13）,另1只表现较严重的症状（分值:8）;pIRES T-F组存活的唯一貉也表现神经症状（分值:12）。与基因疫苗免疫组相比,CDV-CPV组、chn组在攻毒后反应相对轻微,但是chn组仍有死亡现象发生。与CDV-CPV组存活的3只貉相比,四基因组虽然都存活,但是有2只出现相对比较严重的临床症状（分值:10,9）,而CDV-CPV组只有1只有上述表现（分值:8）,另2只除短暂体温升高外,没有异常表现。剖检结果可以看出,免疫组自然死亡貉中,pIRES T-H组、pIRES T-F组和pIREST-N1+pIRES T-N2组表现中度至严重病理变化,而chn组死亡1只貉病变程度相对较低。试验结束实施安乐死的貉均呈轻微至中度变化。排毒检测结果表明,试验貉中除了CDV-PPV组的2只和四基因组中的1只一直没有检测到CDV特异核酸外,其他样品都在不同采样时段或采集的不同样品中检测到。其中血液中检出时间最早,其次为眼结膜、直肠拭子。眼结膜拭子的检出率和检出时间较直肠拭子的高,检出时间较早。自然死亡貉组织都扩增出了CDV特异性目的片段。从症状和病变看,空白对照组明显比免疫组严重,四基因联合免疫组比单基因免疫组轻微,与CDV-CPV组和chn组差别不大。四基因组的保护效果明显优于单基因免疫组,虽然仍能通过眼睛、肛门等天然孔途径向外界排毒,但能抵抗CDV强毒株的感染。本研究结果表明,采用混合H、F和N基因疫苗免疫貉可以诱导机体产生较好的体液免疫反应和较好的免疫保护作用,为毛皮动物用CDV新型疫苗研究奠定了重要基础。更多还原

【Abstract】 Canine distemper virus（CDV） belongs to genus Morbillivirus of Paramyxoviridae which can cause animal diseases such as marten,fox,raccoon dog,dog,panda and tiger. This disease has always been focussed on researching by researchers all over the world because its high infectivity,high morbidity and high mortality,and also its relationship with subacute sclerosing panencephalitis（SSPE） and multiple sclerosing（MS） of human. However,there is not a lot of systemic research on the etiology,immunology,pathology, diagnostic techniques and new technique of vaccine.During the period of June to October in 2006,this disease broadly broke out in fur-bearing animals in Shandong,Hebei,Jiangsu and Liaoning provinces.Here,we isolated and identified 5 CDV isolates from raccoon dogs, foxes and minks after observation of the pathology of the clinical ill animals.Then we analysed the molecular biological properties of the 5 isolates and studied on the immune efficacy of four recombinant plasmid DNAs expressing the H,F and N gene of CDV.1.Study on the clinical pathologySuspicious CD epidemic raised in part fur-bearing animal farms in Shandong,Hebei, Liaoning and Jiangsu provinces from June to October in 2006 was observed and analysed from epidemiology,clinical situation and pathology.Then,we studied on the pathology of selected samples.The results showed that this CDV epidemic was mainly caused by allocate and transportation of raccoon dogs and minks carrying CDVs.Pathological lesions were characterized as haemorrage of whole body tissues,and its severity level from high to low was raccoon dog,fox and mink,respectively.Pathology comparison of lung,liver, brain,kidney and bladder indicated that there was no obviously difference apart from the pathological changes between minks and fox/raccoon dogs.However,pathological lesion difference existed in the same species animals according to disease of course,type of disease and complications. 2.Isolation and identification of CDV isolates,and research on their biological propertiesBased on the observation of epidemiology,clinical signs,pathological changes and histology,several viruses were isolated from samples of liver,spleen,lung,lymph node and brain of ill raccoon dogs,foxes and minks,respectively,by using Vero-DST cells expressing dog’s SLAM,Then the isolates were identified by by electron microscope with phosphotungstic acid（PTA） negative staining or ultrathin section of cell cultures,specific nuclear acid detected from infected vero-DST cells by RT-PCR,detection of physical and chemical characters,and artificial infecting animal tests.The result showed that clearly CPEs were observed on Vero-DST cells at 36-96h after inoculated with treated samples from 5 different origins.TCID₅₀ of the 5 isolates was 10^-5.2～-7.3/ml.Multi-morphous virion about 110～500nm large was observed by electron microscope.All the 5 isolates were positive detected by specific RT-PCR for CDV.All the isolates were not observed hemagglutination with red blood cell（RBC） except weakly hemagglutinin of chicken and goose RBC.The isolates were sensitive to diethyl ether,chloroform,formaldehyde,and weak power of resistance to acid,alkali and high temperature.LD₅₀ of 5 isolates to suckling mouse was 2×10^-3.8～-4.8/ml.They all had powerful pathogenicity to raccoon dogs,but no pathogenicity to experiment rabbits.According to the above results,all the isolates were identified as CDV and named as CDV/SD/RD06/HT-P（raccoon dog）, CDV/SD/RD06/THDI（raccoon dog）,CDV/SD/M06/HD（mink）,CDV/SD/M06/LN（mink） and CDV/SD/F06/HB（fox）,respectivey.3.Sequence analysis of H and N protein gene of isolates from mink,fox and raccoon dogThe H and N genes of 5 CDV isolates were amplified and cloned into PGEM T-Vector.The sequences of all these H and N genes were compared with other sequences published in GenBank.Results revealed that:（1） the homology of H gene nucleic acid sequences among the 5 isolates reached more than 97.5%,however,those among the isolates and vaccine strain chn was 90.5-91.8%.Amino acid homology between HT-P and THD1 was 100%,but had great distinction with the other strains,87.9-99.1%.The deduced aa sequences of the 5 H genes were generally low compared with chn,89.7%-91.8%. However,there was high homology between chn and Onderstepoort or Convac with nucleic acid homology of 96.8%and 97.2%respectively,and amino acid homology of 97.1%and 95.6%respectively.（2）the homology of N gene nucleic acid sequences among 5 isolates was more than 94.5%,with highest homology between HT-P and THD1,99.8%.HD origin site was far from LN’s,but they had higher homology,however,it had relatively lower nt homology to HB.This result might hint that species differences existed between field isolates when they infected susceptive animals.Generally lower homology of nt sequences were observed between elan and isolates,90.9%-93.5%,but there was higher homology of nt between chn and Onderspoort,97.1%.On view of aa sequence of N proteins,there was lower homology between isolates except HT-P,THD1 and HD.Chn had very high homology with Onderstepoort,with their homology reaching 97.3%.These results showed that there was powerful difference of immunocity both H and N protein with vaccine strains, which might be the key factor of CD failure immunization.4.Study on rapid test method for CDV by Real-time TaqMan RT-PCRSpecific primer&probe were designed and synthesized according to CDV N gene. After optimization,a Real-time TaqMan RT-PCR was established.Powerful fluoescence signal and smooth curve were gained with 20pmol/mL primer concentration 1uL and 20pmol/mL probe concentration 0.3uL.High sensitivity,with testing RNA concentration as low as 1.24×10^-3ng/uL;High specificity,with no cross reaction to NDV,AIV and NiPV. Coefficient variation（CV） of test repeatability was 2.3%,2.5%and 4.2%,respectively.In order to examine the correspondency level among new established menthod and routine RT-PCR,BIOINDIST BIT RAPID CDV kit,57 clinical suspicious CDV samples were detected with three above-metioned methods.Results revealed that positive rate was 93%（53/57） with established TaqMan RT-PCR method,while BIOINDIST BIT RAPID CDV kit and routine RT-PCR was 70.2%（40/57） and 71.9%（41/57）,respectively. Coinfidence of TaqMan RT-PCR to BIOINDIST BIT RAPID CDV kit was 77.2%,and to routine RT-PCR.was 78.9%.However,there was high correspondency level between BIOINDIST BIT RAPID CDV kit and routine RT-PCR with their coinfidence 98.2%. Results showed that the Real-time TaqMan RT-PCR,developed in this study,had good characters of rapid,specificity,sensitivity,and could tested large quantity of samples at the same time.It could be used in early detection of CDV infection and quarantine at customs.5.Construction and identification of recombinant plasmid expressing H,F and N gene of CDV isolated from raccoon dogsThe H,F and N（N1,N2） genes were amplified from CDV RNA by RT-PCR, respectively,and cloned into pMD18-T vector.After sequence determined and analysed, these gene were sub-cloned into pIRES1neo expression vector,respectively.The recombinant plasmids DNA were transfered into VERO-DST cell,respectively.Results of Western-blotting showed that aim protein gene could be expressed and used to study on the immunogenicity of CDV genes in animals.6.Protective efficacy of recombinant plasmids expressing CDV H,F and N gene in raccoon dogIn order to observe immune reaction of the recombinant plasmids pIRES H,pIRES F, pIRES N1 and pIRES N2,fourty-two 16-18g body weight experimental white-male-mice were randomly assigned to seven groups each with six.Group 1 was vaccinated posterior limb intramuscularly multi-site injection with pIRES H and boosted two times with 2 weeks interval.Group 2,group 3,group 4,and group 7 was vaccinated pIRES F,pIRES N1 + pIRES N2,pIRES H+ pIKES F+ pIRES N1 +pIRES N2,pIRES empty vector, respectively.Immunization route and inoculate dosage were the same as group 1.Group 5 was vaccinated with live attenuated vaccine which was used broadly in fur-bearing animal farms in China.Group 6 was injected subcutaneously with CDV-CPV imported vaccine according to the instruction.Group 5 and group 6 were also boosted two times after first immunization.Each group was blooded before first immunization and each boosting immunization by cutting tail of the mice.Two weeks after the last boosted,blood was collected from mice by removal their eyeballs.When serum was isolated from the blood, ELISA and neutralization antibody titers were detected respectively.The results showed that high level of CDV-specific ELISA antibody and neutralizing antibody could be induced after two boosted vaccination compared with pre-vaccination and group 7（p＜0.05）. Compared to ELISA antibody titer,the difference between group 4 and group 1,2,3 was not significant,but its neutralization antibody titer was obviously higher than single DNA vaccine（p＜0.05）.Neutralization titer distinction between group 5 and group 4 was not significant,but its ELISA titer manifest higher than group 4.Group 6 was obviously higher than any of the gene groups both ELISA and neutralization titers.To furtherly assess the immuno-efficacy of the above recombinant plasmids against CDV,twenty-one 45～60-day-old raccoon dogs（NT≤1:2） were assigned to seven groups each with three.Each group was treated as the above mice immunization test.Two weeks after the last boosted,each group was oronasally and intraperitoneally challenged with virulent CDV isolate THD1.Results revealed that high level of CDV-specific ELISA antibody and neutralizing antibody could be induced after three times vaccination.After challenged,raccoon dogs in vaccinated group pIRES H,pIRES F,pIRES N1 + pIPES N2 had different level mortality,except Group pIRES H+ pIRES F+ pIRES N1 +pIRES N2.No raccoon dog died in Group CDV-CPV imported vaccine,and live raccoon dogs were also showing slighter signs than those of other groups.Clinical signs and pathological lesions in single gene vaccine vaccinated-challenged group was similar to each other,while slighter than that in empty control group.But it was significantly high compared to Group pIRES H+ pIRES F+ pIRES N1 +pIRES N2 and Group CDV-CPV imported vaccine（P＜0.05）. Meanwhile,virema presented in vaccinated group were milder than those in control group. It indicated that the recombinant plasmids were able to confer significant protection against clinical diseases and reduce pathogenic lesions induced by virulent CDV challenge,even though it could not provide complete virological protection and could not resist clinical signs development.The recombinant plasmids might be an attractive candidate vaccine for preventing the disease associated with CDV infection.更多还原