【作者】 陈锴；

【导师】 杨光友； 王琴；

【作者基本信息】 四川农业大学 ， 预防兽医学， 2012， 博士

【摘要】 猪瘟(Classical Swine Fever, CSF)是由猪瘟病毒(Classical Swine Fever Virus, CSFV)引起的猪的高度接触性、传染性疾病,给养猪业造成巨大威胁和经济损失,被世界动物卫生组织(OIE)列为必须报告的法定传染病之一。本研究旨在通过CSFV中低致病力毒株感染后,从基因、细胞、机体三个水平上对CSFV慢性感染实验猪的临床症候、病理损伤、炎症反应、免疫应答、免疫调节功能的影响进行系统的研究,阐明CSFV慢性感染的免疫机理,为CSF的防控与净化措施提供理论依据。1.中等致病力毒株HeBHHl/95全基因组测序及分析设计CSFV全基因组分段PCR扩增引物,对PCR产物进行克隆并测序,利用DNAStar和ClustalX等生物信息学软件进行序列编辑和拼接,分析全基因组的结构和组成,并从Genebank上下载参考序列进行多重序列比对及同源性分析。测序结果显示本研究获得CSFV中等致病力毒株HeBHH1/95的全基因组序列,基因组由12,297个核苷酸构成,共包括1个CDS和2个非编码区。CDS中各基因排序及长度与NCBI已登录CSFV序列均相同。与HeBHH1/95碱基差异最大的是基因3型毒株csfv94.4IL94TWN,为台湾分离株。碱基差异最小为2.2亚群毒株csfv_GXWZ02,为中国大陆分离株。遗传压力分析显示,HeBHH1/95株异意替换率(Ka)/同意替换(Ks)比值低于1,显示HeBHH1/95的ORF处于负选择压力下。使用不同致病力毒株进行归类分析,并无与致病性相关的序列特征。2.猪瘟慢性感染动物模型的构建、组织病理学及病毒在体内的动态分布研究以肌肉注射的方式对11头30日龄健康断奶仔猪人工感染CSFV中等致病力毒株HeBHH1/95株,构建了病程为45d的猪瘟慢性感染动物模型,同时设2头阴性对照猪,感染后每天测量实验猪体温并对临床症状进行打分记录。在1dpi,3dpi,6dpi,10dpi,15dpi,25dpi,35dpi,45dpi剖杀感染猪并采集组织器官和体外分泌物共30种。根据临床症状和体温变化,将整个慢性感染病程分成三个阶段,即潜伏期(1dpi～7dpi)、发病期(8～24dpi)、转归期(25dpi～45dpi)。潜伏期内感染猪体温正常,无明显临床症状；进入发病期后体温逐渐升高至41.5℃,表现猪瘟典型临床症状。19dpi后体温开始降低,临床症状逐渐消失；转归期体温正常,食欲正常,偶尔拉稀,尤其是复制了感染猪在临床上常见的耳尖尾尖发绀、坏死和断耳断尾的“僵猪”典型特征。对上述采集的不同感染时间采集的24个组织样本切片后进行HE染色,结果显示,第3dpi,腹股沟淋巴结和颌下淋巴结开始出现炎性细胞浸润,微血栓形成；脾脏小梁周围可见红细胞、炎性细胞浸润；扁桃体黏膜表面可见炎性细胞浸润,淋巴细胞变性,此时其他组织未见病变。随着病程的发展,其他组织逐渐出现病变,第15dpi,所有组织均表现出不同程度的病变。在转归期,大部分器官组织未发现明显组织病理变化,但免疫器官、肾脏、肝脏以及十二指肠仍可见组织病变。免疫组化结果显示,在3dpi,脾脏、扁桃体、腹股沟淋巴结、肠系膜淋巴结、回盲瓣、回肠等组织中开始检出CSFV阳性信号；6dpi,肝脏、胃、十二指肠、胰腺、结肠、直肠、肾脏中开始检出CSFV阳性细胞；10dpi～45dpi,所有样本组织中均可检出CSFV阳性细胞,但在转归期数量有所下降。采用猪瘟病毒荧光定量RT-PCR对24个组织器官检测,CSFV慢性感染病程中各个组织器官核酸载量都呈现从低到高,再降低的变化趋势；通过2-ddCt方法统计分析,CSFV对各组织嗜性从高到低排序为：脾脏、颌下淋巴结、回肠、腹股沟淋巴结、扁桃体、肠系膜淋巴结、胰腺、肾脏、皮肤、肺脏、回盲瓣、空肠、肝脏、食道、胃、结肠、睾丸、十二指肠、膀胱、直肠、心肌、脊髓、肌肉、大脑。病毒载量的变化与临床症候、病例剖解变化的规律是一致的。研究结果表明：采用中等致病力毒株成功复制了猪瘟病毒慢性感染动物模型,慢性感染中CSFV的主要复制场所为淋巴器官,且对淋巴器官具有持续性的、不可恢复性的损伤,对其他组织的损伤具有可恢复性；相较于CSF急性感染,扁桃体依然是病毒载量较高的组织,是进行CSFV活体检测最敏感的组织之一。组织病理学和病毒载量的研究结果显示在病程的三个阶段大部分组织器官病变规律与临床症候的发病规律相一致。3.猪瘟慢性感染及疫苗免疫对猪外周血免疫细胞及细胞因子转录水平的影响将23头实验用猪(60日龄)随机分为攻毒组、疫苗组和对照组,定期采血,采用qRT-PCR技术、流式细胞仪和ELISA技术,检测猪外周血中11种细胞因子转录水平、淋巴细胞亚群分析和猪瘟抗体水平。结果显示：慢性感染组中实验猪存活43dpi以上,在感染过程中T淋巴细胞减少和免疫抑制是猪瘟慢性感染早期的临床特征,具有直接杀伤能力的γδT细胞、杀伤性T细胞和辅助型T细胞的数量降低均开始于2dpi,而不具备直接杀伤能力的活化记忆T细胞数量的降低开始于9dpi。在HeBHH1/95感染后,所有检测抗病毒因子均显著上升。HCLV免疫组在5-7dpi时淋巴细胞绝对数量略有上升,在12dpi到达高峰,对照组差异显著(P<0.05),而后逐渐下调并趋于稳定。进一步的流式细胞仪检测结果显示淋巴细胞数量的提升主要是由于辅助性T细胞数量的增多。在HCLV免疫组中,CSFV抗体开始呈现阳性(阻断率>40%)出现在9-12dpi之间,在此期间IL-8转录水平大幅上调,这两者增长与抗体形成的相关性揭示了Th细胞通过增殖诱导B淋巴细胞成熟,诱导细胞因子表达,产生特异性CSFV抗体,并形成免疫保护的机制。综上所述,通过检测中等致病力毒株建立的猪瘟慢性感染中实验动物的临床表现、病理变化和病原动态分布,分析了CSFV慢性感染和免疫后的各免疫相关因子动态变化,比较感染前后和免疫前后各指标变化,分析了慢性感染的病理特征及病毒在逃避宿主免疫过程和形成特异性抗体中起关键作用的细胞及细胞因子的动态变化,为阐述CSFV慢性感染和免疫的机制提供理论科学依据。更多还原

【Abstract】 Classical Swine Fever （CSF） caused by Classical Swine Fever Virus （CSFV） is a highly contagious and hemorrhagic disease of pigs and is classified as a notifiable animal disease by Office International des Epizooties （OIE）. By studying the histopathological changes and transcription level of cytokines gene after experimental infected with moderate virulent strains and the vaccine strain （HCLV） in vivo, the effects on immune system have been anlysised systematically. The results of this study will supply scientific basis for explaining the mechanism of CSFV chronic infection.1The complete genome sequencing and comparative analysis of the csfv moderate virulent strain HeBHHl/95Primers used in PCR for each fragment which cover all the genome of the moderate virulent strain HeBHHl/95.The products of PCR were sequenced, and the segmants were assembled and edited by Clustal X and DNAStar for bioinformatics analysis. The genome contains12,297nucleotides, encoding3,898amino acids flanked by a373-nt region at the5’untrans-lated region （UTR） and a227-nt region at the3’UTR. The subgroup2viruses SXDT2011, GXWZ02,0406_CH₀1_TWN, SXYL2006,96TD, Paderborn isolated from1994to2010in China,Taiwan,Europe show high similarites from97.9%to96.75%.According to the ratio of dN to dS,the CSFV under pure solections.2Histopathology and Dynamic Distribution of Classical Swine Fever Virus in Chronically Infected Pigs11piglets were challenged with CSFV moderate virulence strains HeBHHl/95strain by intramuscular injection, CSF chronic infection model animal was establi shed, while2piglets as negative control. Body temperature and clinical symptoms were recorded daily. Infected pigs were killed on different time interval,30kind s of samples including organs and vitro secretions were collected for HE staining, immunohistochemistry and FQ-PCR. The course of chronic infection could be di vided into three periods due to the symptoms,clinical score and body temperature record, as the incubation period （1dpi～7dpi）, diseased period （8-24dpi）, latent peri od （25-45dpi）. In the incubation period, temperature of infected pigs was nomal and no clinical symptom. In diseased period, body temperature rise to41.5℃and showed CSF typical symptom. In latent period, the temperature recover to normal level and clinical symptoms disappeared slowly. Results of HE staining sh ow that:3DPI, infiltration of inflammatory cells,micro-thrombosis had been detecte d on lymph nodes of inguen and submaxillary lymph nodes; infiltration of erythro cyte and inflammatory cells could be found around the trabecula of spleen; infiltr ation of inflammatory cells in the mucous membrane of tonsil, lymphocyte degen erstion in the lymphoid nodules;15dpi, all kinds of tissues had been detected histopat hological changes, such as microvascular bleeding, thrombus forming and infiltration of inflammatory cell and histiocytenecrosis.In the latent period（35dpi and45dpi）,mos t of tissues had recovered,but histopathological change still harmful on the immun e organs and kidney,liver. These results indicated that there are harmful lesion and histopathological chamge still present on the immune organs although the clinical symptoms were not significant on the CSF chronic infection piglets during the la tent period.In the all CSF chronic infection period, all tissues and organs were foun d typical CSF histopathological changes;In the latent period, most organs recovered but some organs responsible for the immune function still present histopathological chan ges. Histopathological lesions are found on the organs which cannot recovered first ly after infection.3The influence of CSFV chronic infection on immune cell and production of cytokines23piglets were divided into3groups randomly as HeBHHl/95infected group, HCLV vaccinated group and control group. PBMC and serum were prepared at different phases. qRT-PCR, flow cytometry and ELISA were used for detecting mRNA accumulation of10cytokines, analysis of lymphocyte subsets and levels of CSFV antibody.Results showed that the experimental pigs of HeBHHl/95infected group can survived more than43dpi. CSFV chronic infection can reduce T lymphocytes and immunosuppression. The numbers of γδT cells, killer T cells and helper T cells reduceed from2dpi, those cells have direct killing ability, while activated memory T cells without direct killing ability reduced in9dpi. All detected antiviral factor （IL-1β, IL-2, IL-4, IL-8, IFN-α, IFN-β, TNF-α, Mx1, PKR and OAS） of HeBHH1/95infected group were significant up-regulation. Lymphocyte cells of HCLV vaccinated group rose slightly on5-7dpi, reached peak on12dpi, showed significantly different from the control group （P <0.05）, then gradually decreased and became stable. Moreover, flow cytometry results showed that the increment of the lymphocyte count mainly due to the helper T cells increases. CSFV antibody of HCLV vaccinated group showed positive （blocking rate>40%） on9-12dpi, IL-8transcription levels were significantly increased in this period. The results revealed that the Th cells induced B lymphocyte maturation and expression of cytokines produced specific CSFV antibody and form the mechanisms of immune protection by proliferation.In summary, clinical symptom, pathological change,and dynamic distribution of virus after CSFV chronic infection had been systemic studied in this research. Additionally,analyze the dynamic changes of cells and cytokines that play a key role in the process of virus to evade host immune and induce specific antibodies, by comparing each index change of infection before/after and immunity before/after. This study will provide a scientific basis to in order to explain the mechanism of CSFV chronic infection and attenuated vaccine strain immunization.更多还原