唾液酸受体在新城疫病毒神经嗜性中的作用

发布时间：2018-04-25 20:42

本文选题：新城疫病毒 + 唾液酸　；参考：《西北农林科技大学》2017年硕士论文

【摘要】：新城疫是由禽副黏病毒1型(Avian paramyxovirus serotype 1,APMV-1)引起的禽类传染病,与禽流感并称危害养禽业最严重的两大疫病。依据新城疫病毒对鸡的致病性或毒力的不同而分为三种类型:速发型(Velogenic,强毒)、中发型(Mesogenic,中强毒)和缓发型(Lentogenic,弱毒)。根据强毒株对鸡的临床病理特征及其对机体组织的嗜性进一步将其分为两种类型:嗜神经型(Neurotropic velogenic)和嗜内脏型(Viscerotropic velogenic)。其中,嗜神经型强毒株引起的神经系统病变会出现非化脓性脑炎而导致显著的中枢神经紊乱,如扭颈、头颈震颤、转圈、腿或翅膀麻痹和瘫痪等症状,其雏鸡死亡率可达90%以上。生产上多见的神经型新城疫往往是鸡群发病后的后遗症,出现神经症状的家禽很难康复,长期处于疾病状态。唾液酸((Sialic acid,SA)作为NDV的受体被认为是决定NDV感染的宿主范围和组织嗜性的重要因素,但是SA受体对NDV神经感染的作用并不清楚。本研究通过运用凝集素染色等技术探究唾液酸受体对NDV神经嗜性的作用,力求为新城疫病毒的神经嗜性和致病机理研究提供有价值的线索。本论文的主要研究结果如下:1.强弱毒株感染引起鸡脑组织的临床症状和病理变化将4周龄的SPF鸡分别感染NDV强毒株F48E9和弱毒疫苗株LaSota,并设立正常(未感染)对照组。感染强毒株F48E9的SPF鸡出现咳嗽,呼吸困难,哮喘,食欲不振,腹泻,嗉囊积液,以及严重的神经系统症状,如颈部扭转,无外界刺激反应,单侧腿麻痹且全部于3~5dpi之间死亡。HE染色显示,强毒株F48E9引起鸡大脑神经元变性坏死,胶质细胞增生,神经纤维空泡化,呈现套管现象等典型的脑炎病理变化。感染弱毒株LaSota组和对照组在临床症状和病理变化上均未发现任何异常。2.强弱毒株在鸡中枢神经系统的复制差异通过TCID_(50)和EID_(50)检测1,3和5 dpi的NDV在鸡中枢神经系统(CNS)中的滴度,结果表明强毒株F48E9可在大脑,小脑和脊髓有效地复制,且大脑内病毒复制更为敏感。但在感染LaSota毒株的鸡CNS中未检测到病毒。数据表明强弱毒株在CNS的复制具有显著差异,且弱毒株不能有效复制。然而在感染的体外培养鸡原代神经元细胞中,弱毒株LaSota能够复制,但是复制能力相对较弱。这些结果表明,NDV强毒株在神经系统和神经元细胞中能有效复制,但是弱毒株可能丧失了复制能力或不能有效复制。3.唾液酸受体在CNS和鸡原代神经细胞上的表达分别取各组鸡的脑组织进行免疫组化和免疫荧光染色分析。鸡脑组织中的SAα2,3Gal和SAα2,6Gal可分别被凝集素MAA和SNA染色。结果表明,在感染组和对照组脑组织中均检测到两种SA受体,鸡脑中不同区域的SA分布略有不同,但不同组间分布区域与丰度无显著差异。4.SA受体是NDV感染神经细胞所必需的首先,将感染后的脑组织和原代神经细胞进行NDV与唾液酸双荧光染色,结果显示NDV与唾液酸能够共定位。其次,为了研究NDV与神经细胞的结合能力,将鸡原代神经元细胞与NDV强弱毒株在4℃下孵育。PBS洗涤细胞后收集细胞,并用EID_(50)法测定结合的病毒。结果显示F48E9株和LaSota株都能够与神经元细胞结合,且神经细胞结合的两种病毒滴度没有显著差异,说明强弱毒株具有相似的细胞结合能力。用神经氨酸酶处理法将鸡原代神经元细胞表面的SA去除后测定NDV的结合和复制能力,结果显示与对照组相比,所有处理组中细胞结合病毒数量均显著减少(P0.05),且用霍乱弧菌神经氨酸酶(VCNA)处理的比用α2-3,6,8,9 neuraminidase A(NeuA)处理组更有效。病毒复制的结果与用酶处理后的结合反应基本一致,但酶处理后结合和复制能力均抑制不完全,猜测其他宿主分子可能参与该过程。综上表明,SAα2,6Gal和SAα2,3Gal受体对NDV的神经细胞结合和复制起着至关重要的作用。
[Abstract]:Newcastle disease is a avian infectious disease caused by avian paramyxovirus type 1 (Avian paramyxovirus serotype 1, APMV-1), and avian influenza, which is called the two most serious disease in poultry industry. It is divided into three types according to different pathogenicity or virulence of Newcastle disease virus to chickens: fast onset (Velogenic, strong poison), middle hair (Mesogenic, medium strong poison) and slow Hairstyle (Lentogenic, weak poison). According to the clinicopathological features of the virulent strain and the eosinophilia to the body tissue, it can be further divided into two types: the Neurotropic velogenic and the visceral (Viscerotropic velogenic). Among them, the neuropathy caused by the neurovirulent strain can cause non suppurative encephalitis. Significant central nervous disorders, such as torsional neck, head and neck tremor, circle, leg or wing paralysis and paralysis, and other symptoms such as paralysis and paralysis of the chicks can reach more than 90%. The production of neurotype Newcastle disease is often a sequelae after the onset of the chicken group, and it is difficult to recover and be in a state of disease for a long time. (Sialic acid, SA). The receptor of NDV is considered to be an important factor in determining the host range and tissue eosinophilia of NDV infection, but the role of SA receptor on NDV nerve infection is not clear. The purpose of this study is to explore the role of sialic acid receptor on the NDV neurotropism by using lectin staining and so on to provide a study of the neurotropism and pathogenesis of Newcastle disease virus. The main results of this paper are as follows: 1. the clinical symptoms and pathological changes of the chicken brain tissue caused by the infection of the strong and weak strains of the virus, the SPF chickens of 4 weeks of age were infected with the NDV strong strain F48E9 and the weakly toxic vaccine strain LaSota, and the normal (uninfected) control group was set up. The SPF chicken infected with the strong virus strain F48E9 appeared cough, dyspnea, asthma, and food. Inactive, diarrhea, crop fluid, and severe nervous system symptoms such as torsion of the neck, no external stimulus, unilateral leg paralysis and all 3~5dpi death between.HE, F48E9 caused degeneration and necrosis of the brain neurons of the brain, glial cell proliferation, vacuolation of the cereal fibers, and the typical pathological pathology of the brain. Changes in the clinical symptoms and pathological changes of the LaSota and control groups were not detected by any abnormal.2. strong and weak strains in the central nervous system. The drops of 1,3 and 5 DPI NDV in the chicken central nervous system (CNS) were detected by TCID_ (50) and EID_ (50). The results showed that the strong strain F48E9 could be in the brain, the cerebellum and the spinal cord. Replication is effective, and virus replication in the brain is more sensitive. But the virus is not detected in chicken CNS infected with LaSota strain. The data indicate that the replication of strong and weak strains in CNS is significantly different, and the weak strains can not be replicated effectively. However, the weak strain LaSota can be replicated in the cultured chicken primary neuron cells infected in vitro, but the replicating energy of the virus can be replicated. These results suggest that the NDV strains can be effectively replicated in the nervous system and neuron cells, but the weak strains may lose their replicative ability or can not effectively copy the expression of.3. sialic acid receptors on CNS and chicken primary nerve cells for immunohistochemistry and immunofluorescence analysis of each group of chickens. SA alpha 2,3Gal and SA alpha 2,6Gal in chicken brain tissue were stained with lectin MAA and SNA respectively. The results showed that two SA receptors were detected in the brain tissues of the infected and control groups. The distribution of SA in different regions of the chicken brain was slightly different, but there was no significant difference in the distribution area and abundance between different groups. First, the infected brain tissue and the primary nerve cell were stained with NDV and sialic acid. The results showed that NDV and sialic acid could be Co located. Secondly, in order to study the binding ability of NDV and nerve cells, the cells were incubated with the strong and weak NDV strain of the chicken and the.PBS cells were incubated at 4 centigrade, and the EID_ (50) method was used to measure the cells. The results showed that the F48E9 and LaSota strains were able to combine with the neuron cells, and there was no significant difference in the titer of the two kinds of virus, indicating that the strong and weak strains had similar cell binding ability. The binding and replication of the NDV on the cell surface of the primary neurons of the chicken was removed by the neuraminidase treatment. The results showed that the number of cell binding viruses in all the treated groups decreased significantly (P0.05), and the treatment group treated with Vibrio cholerae neuraminidase (VCNA) was more effective than that of the alpha 2-3,6,8,9 neuraminidase A (NeuA) treatment group. The results of the virus replication were basically consistent with the binding reaction after the enzyme treatment, but the enzyme treatment was combined. It is suggested that SA alpha 2,6Gal and SA alpha 2,3Gal receptors play an important role in the binding and replication of NDV.

【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.65

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