山羊痘病毒粒子的电子显微镜观察

本研究室2003年以来对山羊痘病毒感染多种细胞进行了透射电镜观察。在感染的皮肤和黏膜上皮细胞浆中观察到大量不同成熟阶段的典型山羊痘病毒粒子，病毒粒子也见于巨噬细胞、成纤维细胞、血管内皮细胞胞浆内，甚至少量散在血管腔中。用山羊痘病毒分离物感染鸡胚绒毛尿囊膜形成痘斑，其上皮细胞和成纤维细胞中也可见成熟和不成熟的病毒粒子，同时感染BHK-21细胞也观察到典型的山羊痘病毒粒子。     

山羊痘的病理形态学观察

对发生在贵州省的山羊痘进行了病理形态学观察.山羊痘以全身皮肤、呼吸道及消化道粘膜出现痘疹为特征.皮肤疹最初为红斑,红斑肿胀隆起形成结节状丘疹,然后丘疹发生凝固性坏死,脱落而形成痂皮.组织学检查早期皮肤病变可见充血,水肿和多量的巨噬细胞和中性白细胞浸润,随着病变的发展可见更多的巨噬细胞,淋巴细胞,浆细胞和一些体积较大的所谓"山羊痘细胞"出现.在真皮的病变细胞和痘细胞中可见嗜酸性胞浆内包涵体.引起水肿和坏死的血管炎常伴有血栓和梗死.上皮的变化包括棘细胞增生,空泡变性,不全角化,过度角化和坏死等.其他器官的病变也有相似的细胞浸润和血管炎变化.但发生在上呼吸道及上消化道时常出现溃疡.透射电镜观察在感染的皮肤和肺上皮细胞的胞浆内发现有大量不同成熟阶段的典型的山羊痘病毒颗粒.受感染的细胞除可见胞浆内散在的病毒颗粒或形成病毒包涵体外,通常可见细胞水肿、线粒体肿胀、内质网和高尔基复合体扩张.     

羊痘病毒致宿主细胞病变研究进展

羊痘病毒是一种在细胞质中进行复制的DNA病毒,可侵染上皮细胞、成纤维细胞、血管内皮细胞、羔羊睾丸细胞和肾细胞等多种细胞并致细胞病变,形成胞浆包涵体。目前,已知的羊痘病毒宿主细胞不少,因此,对羊痘病毒所致宿主细胞病变做一综述,对认识羊痘病毒对体内外宿主细胞的不同作用及影响有一定帮助。同时,结合近年来对羊痘病毒宿主细胞的进一步认识,讨论了研究相关体外试验的必要性及可能性。     

有髓神经纤维被山羊痘病毒波及的证实

[目的和方法]对发生在贵州省的山羊痘皮肤病变进行了透射电镜观察.[结果]发现有髓神经纤维被山羊痘病毒和被病毒感染的巨噬细胞所波及.可见节段性脱髓鞘、轴索和雪旺氏细胞变性.[结论]有髓神经纤维被病毒侵犯可能是山羊痘病羊虽然出现全身痘疹,但临床上却表现为迟钝、沉郁、痛觉减弱甚至痛感缺失的原因.     

兔出血症病毒在细胞培养和组织中的形态发生

在电镜下系统地观察了感染后的细胞培养和组织中兔出血症病毒（ Ｒ Ｈ Ｄ Ｖ）的形态发生。感染早期，在细胞核内可见电子致密颗粒（１５ ｎｍ ）和未成熟的病毒颗粒 （２５ ｎｍ ）。中期，在细胞核和胞浆内出现大量成熟的病毒颗粒（３４ ｎｍ ），并发现部分核内病毒通过扩大的核膜孔、核膜溶解扩大的核孔和乳头状突起的核膜向胞浆释放。感染末期，核染色质消失，核内大量感染病毒清淅可见。最终细胞溶解，病毒颗粒释放至细胞间隙。提示 Ｒ Ｈ Ｄ Ｖ 是在核内复制和装配的，应归属于细小病毒科。本试验结果不排除同时存在另一种小 Ｒ Ｎ Ａ 病毒。     

鸭病毒性肠炎病毒强毒在人工感染鸭体内形态结构和发生学的电镜观察

为研究鸭病毒性肠炎病毒(DEV)CH强毒株在感染鸭体内的分布和形态学发生规律，应用透射电镜和超薄切片技术对人工感染DEV的成年鸭各组织器官进行观察。结果表明：感染后12h在脾脏和法氏囊首先观察到少量的DEV出现，24h后在脾、胸腺和法氏囊以及死亡鸭的肝、肠和胰中均观察到具有典型的疱疹病毒粒子及其核衣壳形态的DEV。DEV病毒核衣壳有空心型、致密核心型、双环型和内壁附有颗粒型4种形态，存在胞核和胞浆两种装配方式。病毒成熟有两种方式：一为细胞核内核衣壳在核内获得皮层，通过核内膜获得囊膜成为成熟病毒；二为核内核衣壳通过内外核膜进入胞浆，核内和胞浆内的核衣壳在细胞浆中获得皮层，然后在各种质膜上获得囊膜，最后成熟病毒通过细胞破裂或其他方式释放到细胞外。伴随着病毒的复制、装配和成熟，细胞中出现多种核内和胞浆包涵体、核内致密颗粒、核内微管和中空短管、胞浆电子致密小体等结构。     

山羊痘病毒的分离鉴定及生物学特性的研究

本研究对2003年广西部分地区山羊群发生的疑似山羊痘进行了病毒分离鉴定及生物特性的研究。取疑似山羊痘病羊的皮肤丘疹、水泡或脓泡组织的病毒悬液，接种初生羔羊睾丸细胞观察到明显的细胞病变，免疫荧光试验结果显示，病毒能与山羊痘标准阳性血清反应，在感染的细胞浆内发出特异性的黄绿色荧光。病毒悬液接种乳鼠、小鼠、豚鼠、兔子都未发病，而接种3月龄山羊则出现典型的山羊痘症状和病理变化，接种9～10日龄鸡胚绒毛尿囊膜，未见出现痘斑，连传3代，均无异常变化。通过病理组织学观察可以看到在细胞浆内有大小不一圆形或椭圆形的包涵体，在电子显微镜下可以观察到150nm～300nm大小，卵圆形、砖形，有囊膜的病毒颗粒。利用一对山羊痘病毒P32基因引物进行了PCR扩增，将所得序列与GenBank收录的5株山羊痘病毒P32基因的核苷酸及氨基酸序列比较分析。结果与疫苗株的同源性分别为99．8％和99．4％。与国外其它毒株的同源性为99．6％和98、8％～99．4％。研究结果表明，所分离的病毒为山羊痘病毒，在生物学特性上与资料记载存在一定的差异，P32基因与疫苗毒和国外毒株之间同源性非常高。将该毒株命名为山羊痘病毒LiuJiang／2003株。     

斑点叉尾(鱼回)疑似疱疹病毒感染的病理形态学观察

对四川省一些养殖场斑点又尾鮰大规模死亡的病鱼组织进行了光镜和电镜观察．在其肾和肝组织的细胞中发现一种圆形或椭圆形病毒颗粒，有囊膜的病毒颗粒存在于胞浆中，直径150～200nm．无囊膜的病毒颗粒既存在于胞浆中，也存在于胞棱中，直径为80～110nm，同时在细胞核内可见无病毒核心的空衣壳，根据其形态特征初步确定为一种疱疹病毒。病鱼主要表现为鳍条基部和皮肤（特别是腹部和尾柄）充血、出血，腹部膨大，眼球突出．鳃发白．腹腔内充有淡黄色或淡红色的腹水，胃肠道扩张，其内充满大量淡黄色的粘液，肝、脾和肾肿大。病理组织学变化主要为全身组织器官广泛性水肿、出血、变性、坏死和炎症细胞浸润，特别是肾、肝、胃肠道、脾和脑的损伤较为严重。肾间质水肿，造血组织坏死，巨噬细胞和中性拉细胞浸润．肾小管上皮细胞空泡变性和坏死；肝水肿，狄氏间隙增宽．肝细胞空泡变性及坏死；胃肠道粘膜上皮变性，坏死．脱落，固有膜，粘膜下层水肿，淋巴细胞、巨噬细胞和中性枉细胞浸润；脾淤血．出血．淋巴细胞减少，大量巨噬细胞和中性粒细胞浸润；脑水肿，神经细胞肿胀．甚至坏死固缩。超微结构上．被病毒感染的细胞发生明显的痛变，线粒体肿胀，嵴断裂．溶解，粗面内质网扩张，核糖体颗粒脱落．细胞核体积增大，染色质浓缩．边集。     

绵羊痘病毒吉林株的分离鉴定及其RPO30、P32和Kl基因的克隆与进化分析

从病理学和病原学角度对临床一起疑似羊痘病例进行了系统的检测分析,确诊该起病例为绵羊痘病毒(sheep poxvirus,SPPV)感染所致。对病死羊进行大体病理剖检,病羊口腔黏膜、尾根及肺脏等内脏器官表面可见有大量痘疹样结节;病理组织学观察可见,肺脏支气管黏膜上皮细胞、肺泡上皮细胞发生变性、坏死、脱落,并伴有上皮化生;此外,在肺泡上皮细胞胞浆中可见有嗜酸性病毒包涵体,该结果提示这起病例可能是羊痘病毒感染所致。之后从病原学角度进行了检测分析,电镜负染观察可见典型的痘病毒粒子;RPO30、P32、Kl基因的PCR检测结果显示,均扩增出与预期目的片段大小相一致的条带;P32基因的系统发育进化分析结果显示,该分离株与SPPV-AV40的亲缘关系较近,证实了该起病例的病因为SPPV感染所致,命名为SPPV-Jilin分离株(KF991006)。该分离株RPO30和Kl基因的测序分析结果发现,与山羊痘病毒相应序列相比,其RPO30基因缺少21bp的核酸序列,而Kl基因在902～925bp位置处存在缺失,该结果表明RPO30和Kl基因将可能用于绵羊痘病毒与山羊痘病毒的鉴别诊断。     

山羊痘病例的病理学诊断及病毒鉴定

菏泽市某养羊场从散户购买青山羊50余只,购进后1周左右陆续发病,发病羊体温41~42℃,2周内死亡率超过60%,部分羊体表有痘疹样病变,疑似羊痘。对该病例进行了现场调查、尸体剖检、病理组织学检查及病毒分离鉴定。结果显示:病羊眼睑、口、鼻部有痘疹结节,皮肤有大量红色丘疹。剖检病死羊可见肺、胃肠黏膜、肾及肝表面有数量不等的灰白色丘疹。皮肤及黏膜痘疹部的上皮细胞增生、水泡变性,并见胞浆红染包涵体;肺、肝、肾丘疹完全符合痘疹的病变特征,通过Macehiavello包涵体染色在皮肤、黏膜增生的上皮细胞及肝、肾痘细胞的胞浆内有紫红染包涵体。将获得p32基因序列与GenBank上登录的相应序列进行同源性分析表明该分离株与山羊痘病毒分离株的同源性为99.4%~99.8%,但与绵羊痘病毒的同源性偏低(97.2%~98.1%)。系统进化树分析表明该分离株与山羊痘病毒处于同一分支。将纯化后的p32基因扩增产物用Hinf I酶切位点分析表明,该病毒仅在688bp处存在1个酶切位点,酶切后产生2条目的片段(688和311bp)。对p32基因序列进一步分析表明,在169~171位点上存在3个碱基(GAT)的插入,而山羊痘病毒和其他痘病毒在该位点上有3个碱基缺失,证明此次疫情是由山羊痘病毒引起的。据病理学及病原学诊断结果判定该病例为山羊痘重症感染。按《中华人民共和国动物防疫法》规定,扑杀并无害化处理发病羊群,羊舍、场地、用具严格消毒,粪便堆积发酵处理,受威胁区的羊群实施疫苗紧急接种羊痘疫苗,疫情未发生进一步扩散。     

Experimental peste des petits ruminants (goat plague) in goats and sheep.

In order to study the pathomorphology and immunohistochemistry of peste des petits ruminants, four goats and two sheep were inoculated intranasally with the Malig-Yemen strain of peste des petits ruminants virus. The animals developed fever, nasal discharge, oral erosions, cough and diarrhea. One goat and one sheep died and one moribund goat was killed. Three animals survived the infection. At necropsy, erosive stomatitis, pneumonia and gastroenteritis were found. Histopathologically the pneumonocytes and epithelial cells of the ileum had eosinophilic cytoplasmic and nuclear inclusions. By an indirect immunoperoxidase method, the nuclei and cytoplasm of the ileal epithelial cells of one goat contained positively (brown) stained antigen, which corresponded to viral nucleocapsids by electron microscopy. Virus appeared to be released through the microvilli of the epithelial cells. We also confirmed the formation of giant cells due to peste des petits ruminants virus.     

奶山羊羔羊山羊痘的病理学、血清学及分子生物学诊断

目的 确定内蒙古某规模化奶山羊养殖场部分引进羔羊发病死亡的原因,为该场加强重点疫病免疫防控提供科学依据。方法 对出现典型临床症状的发病羊只进行病理解剖学观察,无菌采集肺脏病变样本,制备病理组织切片;无菌采集发病羊只的血液制备血清,采用高敏荧光技术检测血清中的山羊痘病毒抗体;无菌采集发病羊只的肺组织、鼻拭子、眼拭子,利用PCR技术检测山羊痘病毒P32基因,对测序获得的序列进行BLAST比对并进行同源性分析。结果 剖检可见病羊口腔、鼻腔、喉头、气管黏膜有圆形疹痘;肺部明显水肿,表面存在红色疹痘,切开后呈不透明、灰白色胶冻样;瘤胃、网胃、瓣胃、皱胃黏膜均出现疹痘。病理组织学检查可见肺泡红色深染,间质变宽,腔内存在脱落的上皮细胞,Ⅱ型肺泡上皮细胞增生,并化生为腺泡样状;支气管腔内存在大量的炎性细胞。发病山羊血清经高敏荧光技术检测,判定为山羊痘病毒抗体阳性。采集肺组织（GTPV-YP1）、鼻拭子（GTPV-YP2）、眼拭子（GTPV-YP3）样品各1份,3份样品经P32基因PCR检测均获得983 bp的扩增片段,与预期大小一致;GTPV-YP1、GTPV-YP2样品P32基因序列与山羊痘病毒中国分离株KC951854.1、Oman分离株 MN072621.1 P32基因的同源性为100%,GTPV-YP3与山羊痘病毒中国分离株EF514892.1、HM572329.1、JN596275.1、MG817382.1的同源性为99.0%。结论 经病理解剖学观察、病理组织学检查、血清学检测以及病原PCR鉴定,确定引起该场引进奶山羊羔羊发病的原因为山羊痘病毒感染。     

Immunohistochemical evaluation of inflammatory infiltrate in the skin and lung of lambs naturally infected with sheeppox virus

The present study describes immunophenotypic characteristics of inflammatory infiltrate in the skin and lung of lambs naturally infected with sheeppox virus (SPV). Three lambs revealed typical cutaneous and pulmonary lesions of sheeppox. Histologically, cutaneous and pulmonary lesions consisted of hyperplastic and/or degenerative changes in the epithelium with mononuclear cells, neutrophils, and typical sheeppox cells (SPCs), which had a vacuolated nucleus and marginated chromatin with occasional granular intracytoplasmic inclusions. The inflammatory infiltrate in pox lesions in both skin and lung was characterized by the presence of MHC II+ dendritic cells, CD4+, CD8+, gammadelta+ T cells, IgM+ cells, and CD21+ cells. Loss of expression of MHC I and MHC II antigens was observed in the affected areas of skin and lung. SPCs, stained with anti-SPV antibody, were also positive for CD14 and CD172A, antigens expressed on monocytes and macrophages. CD14 and CD172A negative SPCs were considered to be SPV infected degenerated epithelial cells or fibroblasts.     

An immunohistochemical study of the pneumonia caused by peste des petits ruminants virus

Ten goats were inoculated with peste des petits ruminants virus, a paramyxovirus closely related to rinderpest virus. All goats developed severe clinical disease, 8/10 having coughing or dyspnea as prominent clinical signs. In addition, all of the goats had stomatitis and diarrhea. Histopathologic and immunohistochemical studies were done only on the respiratory tracts. Pathologic changes ranged from mild multifocal bronchiolitis and bronchitis to severe bronchointerstitial pneumonia. Lesions were more severe in anteroventral than caudal lobes. The histologic nature of the viral process in the goat lungs had many features in common with the processes of pneumonia in dogs, due to canine distemper, or pneumonia in human beings, due to measles virus. Immunohistochemical staining of formalin-fixed, paraffin-embedded respiratory tract tissue was performed using an indirect system with rabbit anti-rinderpest virus serum, biotinylated anti-rabbit antibody, streptavidin-alkaline phosphatase, and nitroblue tetrazolium chromogen. Staining was sensitive, highlighting the presence of viral antigen in both lung and trachea of all goats. Viral antigen was found in both cytoplasm and nucleus of tracheal, bronchial, and bronchiolar epithelial cells, type II pneumocytes, syncytial cells, and alveolar macrophages. In general, the amount of staining correlated directly with the severity of the inflammatory process.     

Severe persistent orf in young goats.

Orf (contagious ecthyma) is a viral disease of small and wild ruminants, humans, and less frequently other species. In sheep and goats, the disease is characterized by the formation of vesiculo-proliferative lesions in the skin of lips and nostril. Here, a form of generalized orf in 16 goat kids from 2 different locations in west Texas is described. The disease was characterized by multifocal, severe, proliferative dermatitis that persisted from about 2 months of age until the goat kids were euthanized 3 months later. All affected goats were Boer or Boer crosses under 1 year of age. The mean immunoglobulin concentration in sera of affected goats was elevated compared with healthy control goats. Severe to moderate lymphadenomegaly of the nodes draining the areas of the skin affected with orf lesions was present in all 16 goat kids. Suppurative arthritis, chronic fibrinous pneumonia, and premature thymic involution were found in 3, 5, and 7 of the goat kids, respectively. The skin lesions of 3 goat kids were infested with larvae of the opportunistic black garbage fly (Ophira sp.). The orf virus was identified in skin lesions by isolation in Marbin-Darby ovine kidney cells, electron microscopy, and amplification of viral DNA by polymerase chain reaction. The orf virus was not detected in peripheral blood or lymph node mononuclear cells of any of the goats. Cross-neutralization experiments showed that an ovine orf virus antiserum raised in sheep was more effective in neutralizing a sheep orf virus isolate than a caprine orf virus isolate. The clinical and epidemiological characteristics of these orf cases may be the result of susceptibility factors within some individuals of the Boer breed of goats.     

Lesions in lambs experimentally infected with bovine respiratory syncytial virus

Respiratory syncytial virus was inoculated intratracheally into five 1-week-old lambs. Three of the lambs responded clinically with fever, hyperpnea, and listlessness. Pulmonary lesions consisted of multifocal areas of consolidation, with necrosis of individual epithelial cells of the airways and accumulation of necrotic debris, macrophages, and few neutrophils in terminal airways and alveoli. Pulmonary septa in affected areas were infiltrated with numerous macrophages and lymphocytes. Viral particles were seen as buds on epithelial cells and free in bronchioles and alveoli.     

Immunohistochemical detection of parainfluenza type 3 virus antigens in paraffin sections of pneumonic caprine lungs

Pneumonia is a leading cause of loss to ruminants throughout the world. Parainfluenza type-3 virus (PI-3) is one of the most important respiratory pathogens of bovine and ovine. In this study, prevalence of PI-3 virus infection as causative agent of pneumonia in goats was investigated. For this purpose, a total of 1505 goat lungs slaughtered in Bitlis and Van slaughterhouses were grossly examined and pneumonia was detected in 74 cases (4.91%). Lesions were more frequently encountered in anteroventral lobes than caudal lobes. With the exception of verminous pneumonia observed in 32 cases, immunohistochemical examinations were performed on 42 pneumonic lungs. Formalin-fixed and paraffin-embedded lung tissue samples were immunohistochemically stained by the avidin-biotin-peroxidase complex procedure using polyclonal antibodies to detect PI-3 viral antigens. The presence of PI-3 viral antigens was detected in 28 (66.6%) of 42 pneumonic lungs. Viral antigens were found most frequently in the cytoplasm of bronchiolar epithelial cells, type II pneumocytes, and less frequently in the epithelial cells of bronchial glands, syncytial cells, alveolar macrophages, and lymphocytes and plasma cells. In conclusion, it was found that there was a close relationship between the pneumonia in goats and the presence of PI-3 viral antigens. Incidence of PI-3 virus in pneumonic lungs of goats was detected to be very high in the present study performed in the region of Bitlis and Van, Turkey.     

人工感染IBDV鸡法氏囊的电镜研究

通过透射电镜系统观察了人工感染传染性法氏囊病病毒（ＩＢＤＶ）后鸡法氏囊各类细胞的病理变化。感染后１２￣２４ｈ,病毒粒子主要见于髓质淋巴细胞中,细胞中可见到大量纤维样病毒发生基质及无囊膜包围的大型病毒晶格,细胞核染色质浓缩,核中出现纤维样结构。感染后３６ｈ,淋巴细胞开始大量裂解死亡。无囊膜包围的病毒晶格也出现于髓质网状细胞中,被感染的网状细胞并不裂解,而表现出细胞凋亡的特征：染色质固缩呈颗粒块状,胞