本文選題：新城疫病毒 + 唾液酸�。� 參考：《西北農(nóng)林科技大學》2017年碩士論文

【摘要】：新城疫是由禽副黏病毒1型(Avian paramyxovirus serotype 1,APMV-1)引起的禽類傳染病,與禽流感并稱危害養(yǎng)禽業(yè)最嚴重的兩大疫病。依據(jù)新城疫病毒對雞的致病性或毒力的不同而分為三種類型:速發(fā)型(Velogenic,強毒)、中發(fā)型(Mesogenic,中強毒)和緩發(fā)型(Lentogenic,弱毒)。根據(jù)強毒株對雞的臨床病理特征及其對機體組織的嗜性進一步將其分為兩種類型:嗜神經(jīng)型(Neurotropic velogenic)和嗜內(nèi)臟型(Viscerotropic velogenic)。其中,嗜神經(jīng)型強毒株引起的神經(jīng)系統(tǒng)病變會出現(xiàn)非化膿性腦炎而導(dǎo)致顯著的中樞神經(jīng)紊亂,如扭頸、頭頸震顫、轉(zhuǎn)圈、腿或翅膀麻痹和癱瘓等癥狀,其雛雞死亡率可達90%以上。生產(chǎn)上多見的神經(jīng)型新城疫往往是雞群發(fā)病后的后遺癥,出現(xiàn)神經(jīng)癥狀的家禽很難康復(fù),長期處于疾病狀態(tài)。唾液酸((Sialic acid,SA)作為NDV的受體被認為是決定NDV感染的宿主范圍和組織嗜性的重要因素,但是SA受體對NDV神經(jīng)感染的作用并不清楚。本研究通過運用凝集素染色等技術(shù)探究唾液酸受體對NDV神經(jīng)嗜性的作用,力求為新城疫病毒的神經(jīng)嗜性和致病機理研究提供有價值的線索。本論文的主要研究結(jié)果如下:1.強弱毒株感染引起雞腦組織的臨床癥狀和病理變化將4周齡的SPF雞分別感染NDV強毒株F48E9和弱毒疫苗株LaSota,并設(shè)立正常(未感染)對照組。感染強毒株F48E9的SPF雞出現(xiàn)咳嗽,呼吸困難,哮喘,食欲不振,腹瀉,嗉囊積液,以及嚴重的神經(jīng)系統(tǒng)癥狀,如頸部扭轉(zhuǎn),無外界刺激反應(yīng),單側(cè)腿麻痹且全部于3~5dpi之間死亡。HE染色顯示,強毒株F48E9引起雞大腦神經(jīng)元變性壞死,膠質(zhì)細胞增生,神經(jīng)纖維空泡化,呈現(xiàn)套管現(xiàn)象等典型的腦炎病理變化。感染弱毒株LaSota組和對照組在臨床癥狀和病理變化上均未發(fā)現(xiàn)任何異常。2.強弱毒株在雞中樞神經(jīng)系統(tǒng)的復(fù)制差異通過TCID_(50)和EID_(50)檢測1,3和5 dpi的NDV在雞中樞神經(jīng)系統(tǒng)(CNS)中的滴度,結(jié)果表明強毒株F48E9可在大腦,小腦和脊髓有效地復(fù)制,且大腦內(nèi)病毒復(fù)制更為敏感。但在感染LaSota毒株的雞CNS中未檢測到病毒。數(shù)據(jù)表明強弱毒株在CNS的復(fù)制具有顯著差異,且弱毒株不能有效復(fù)制。然而在感染的體外培養(yǎng)雞原代神經(jīng)元細胞中,弱毒株LaSota能夠復(fù)制,但是復(fù)制能力相對較弱。這些結(jié)果表明,NDV強毒株在神經(jīng)系統(tǒng)和神經(jīng)元細胞中能有效復(fù)制,但是弱毒株可能喪失了復(fù)制能力或不能有效復(fù)制。3.唾液酸受體在CNS和雞原代神經(jīng)細胞上的表達分別取各組雞的腦組織進行免疫組化和免疫熒光染色分析。雞腦組織中的SAα2,3Gal和SAα2,6Gal可分別被凝集素MAA和SNA染色。結(jié)果表明,在感染組和對照組腦組織中均檢測到兩種SA受體,雞腦中不同區(qū)域的SA分布略有不同,但不同組間分布區(qū)域與豐度無顯著差異。4.SA受體是NDV感染神經(jīng)細胞所必需的首先,將感染后的腦組織和原代神經(jīng)細胞進行NDV與唾液酸雙熒光染色,結(jié)果顯示NDV與唾液酸能夠共定位。其次,為了研究NDV與神經(jīng)細胞的結(jié)合能力,將雞原代神經(jīng)元細胞與NDV強弱毒株在4℃下孵育。PBS洗滌細胞后收集細胞,并用EID_(50)法測定結(jié)合的病毒。結(jié)果顯示F48E9株和LaSota株都能夠與神經(jīng)元細胞結(jié)合,且神經(jīng)細胞結(jié)合的兩種病毒滴度沒有顯著差異,說明強弱毒株具有相似的細胞結(jié)合能力。用神經(jīng)氨酸酶處理法將雞原代神經(jīng)元細胞表面的SA去除后測定NDV的結(jié)合和復(fù)制能力,結(jié)果顯示與對照組相比,所有處理組中細胞結(jié)合病毒數(shù)量均顯著減少(P0.05),且用霍亂弧菌神經(jīng)氨酸酶(VCNA)處理的比用α2-3,6,8,9 neuraminidase A(NeuA)處理組更有效。病毒復(fù)制的結(jié)果與用酶處理后的結(jié)合反應(yīng)基本一致,但酶處理后結(jié)合和復(fù)制能力均抑制不完全,猜測其他宿主分子可能參與該過程。綜上表明,SAα2,6Gal和SAα2,3Gal受體對NDV的神經(jīng)細胞結(jié)合和復(fù)制起著至關(guān)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.

【學位授予單位】：西北農(nóng)林科技大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S852.65

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