犬冠状病毒的分离鉴定及其纤突蛋白基因的克隆与序列比较

以犬胎原代细胞，采用同步接种、带毒传代和添加新细胞的方法，从１例腹泻犬脾脏和１例临床健康犬肠内容物中分离出２株犬冠状病毒（ＣＣＶＹＳ１，ＣＣＶ ＣＩ１）。该２株病毒可使ＣＲＦＫ细胞出现典型的细胞融合病变，与从美国引进的ＣＣＶＮＬ－１８参考株形成的细胞病变相似；电镜负染和超薄切片观察，分离毒株细胞培养物中均含有典型的冠状病毒粒子，并形成胞浆内包涵体；经理化学、生物学鉴定，濉有ＣＣＶ的特性；间接免疫荧     

犬实验感染CDV的病理学研究

对实验感染犬瘟热病毒的病犬进行了系统的病理学观察，并用酶标ＳＰＡ法对病犬脏器组织中ＣＤＶ抗原进行了定位检查。结果表明，淋巴系统各器官组织是ＣＤＶ急性感染早期首先侵犯的靶器官。脏器组织的病理改变与ＣＤＶ抗原检出呈正相关。脏器组织中包涵体的检出与形态结构具有一定的特征性和示病意义，但采用免疫组化方法检查ＣＤＶ抗原，更具优越性。作者认为，ＣＤＶ９３０３９株和ＣＤＶ９３０４１株是致病力很强的泛嗜性ＣＤＶ。     

犬实验感染CDV的病理学研究

对实验感染犬瘟热病毒的病犬进行了系统的病理学观察，并用酶标ＳＰＡ法对病犬脏器组织中ＣＤＶ抗原进行了定位检查，结果表明淋巴系统各器官组织是ＣＤＶ急性感染早期首先侵犯的靶器官，脏器组织的病理改变与ＣＤＶ抗原出呈正相关，脏器组织中包涵体的检出与形态结构具有一定的特征性和示病意义，但采用免疫组化方法检查ＣＤＶ抗原，更具优越性，作者认为：ＣＤＶ９３０３９株和ＣＤＶ９３０４１株是致病力很强的泛嗜性ＣＤＶ。     

犬瘟热病毒小熊猫株(LP)的动物感染试验

将从死亡小熊猫肝脏病料中分离到ＣＤＶ ＬＰ株第９代ＭＤＣＫ细胞培养毒,按不同剂量各接种７只２～３月龄的健康幼犬,接种犬全部发病。于接种后７天时各捕杀２只犬进行检测,电镜检查、ＣＤＶ ＲＴ－ＰＣＲ、ＣＤＶ的分离及中和试验结果均为阳性,说明接种犬发生了ＣＤＶ感染。结合接种犬出现的临床症状及病理解剖学变化结果进行的分析表明,ＣＤＶ ＬＰ株是一株强毒,并且具有很强的免疫原性。人工感染犬试验结果表明某动物园     

犬瘟热诊疗初探

犬瘟热诊疗初探周昌贵，李真银，刘友平，何锡荣（四川省江津市农牧渔业局６３２２６０）犬瘟热是由犬瘟热病毒（ＣＤＶ）引起的主要危害犬、特别是２～４月龄幼犬的一种高度接触性传染病，病死率高，流行面广。据资料报道ＣＤＶ感染病犬自然死亡率可达８０％以上，对犬危...     

自发性急性犬瘟热的原发性脱髓性脑病

为了进一步观察犬瘟热病毒引起的原发性脑损伤和包涵体形成的特点，调查脑组织的损伤与神经症状的关系，对10只急性犬瘟热病犬的脑组织进行了详细的病理学研究。为了仔细地观察病变，本试验按照解剖学关系将脑组织分成3个大部分和11个切面，即大脑(4个切面)，脑干(5个切面)和小脑(2个切面)。组织切片经HE、LFB和免疫组织化学染色后进行检查，结果表明：在大脑，脱髓呈弥漫性发生，程度较轻；脑干的周围或靠近第三脑室的白质脱髓较重；小脑在轻度或中度脱髓的基础上常出现严重的多发性脱髓灶。脱髓部呈空泡或海绵状，有少量胶质细胞存在，但无炎性反应。脱髓性病损是非时称性发生，对神经束没有特殊的亲和力。在脑室的室管膜细胞内发现有较多的嗜酸性胞浆或核内包涵体。用抗犬瘟热病毒抗体染色，带有包涵体的室管膜细胞呈现强阳性反应。部分锥体细胞，神经核细胞和漓氏细胞变性、溶解或胞浆深染。胞核浓缩。这种变化以小锥体神经细胞表现得最为明显。根据此研究结果，作者认为由犬瘟热病毒引起的原发性脑组织损伤是一种脱髓性脑病，而不是脑炎变化；位于室管膜细胞内的包涵体对于脑组织犬瘟热的确诊具有重要的作用；由于犬瘟热病毒引起神经细胞的损伤是非特异性的，对脑组织的侵害是非对称性的。对神经束的作用无特殊的亲和力，所以患犬瘟热的犬在临床上可出现不同的神经症状。     

犬瘟热病毒的分离鉴定、致病特点和单克隆抗体的研究

犬瘟热是由犬瘟热病毒（ Ｃａｎｉｎｅ Ｄｉｓｔｅｍ ｐｅｒ Ｖｉｒｕｓ, Ｃ Ｄ Ｖ）引起的犬的一种高度接触传染性疾病。近年来,随着我国军、警犬、实验用犬和宠物犬饲养量的大幅度增加,以及异地交流的增多,犬瘟热在我国犬群中的发病率和致死率均有升高的趋势,而且临床表现也与以往有所不同。分析原因,除母源抗体干扰、疫苗效价低及使用不当外,犬群中是否出现了 Ｃ Ｄ Ｖ 新毒株亟待研究证实。本研究在从国内病犬分离到 Ｃ Ｄ Ｖ 的基础上,进行了 Ｃ Ｄ Ｖ 理化特性、致病特点、回归动物试验以及研制成功分泌抗 Ｃ Ｄ Ｖ 单克隆抗体,并进行了初步应用,取得了较为满意的研究结果。在研究中应用免疫组化方法进行病原检查,证实可很好地解决以往病理观察中无法进行病毒性疾病的特异性诊断的问题。１）采用免疫组化间接 Ｂ Ａ 法,对临诊疑似犬瘟热病犬脏器组织进行了 Ｃ Ｄ Ｖ 抗原定位检查及系统病理学观察。结果表明：发病犬大脑、小脑、心、肺、肝、脾、肠、肾、肾上腺、淋巴结、膀胱等组织细胞及血管内皮、支气管上皮细胞胞浆内均可见抗原阳性反应。同时,抗原阳性反应组织、器官均可见不同程度的病理变化。２）从病死犬肺、肝和淋巴结分离到 ２株 Ｃ Ｄ Ｖ,即 Ｃ Ｄ Ｖ９３０３９ 和      

用RT—PCR检测犬外周血液单核细胞中犬瘟热病毒核衣壳蛋…

为了迅捷地诊断犬瘟热病毒（ＣＤＶ）感染，本研究采用及转录－聚合酶链反应（ＲＴ－ＰＣＲ）技术，检测犬外周血液核细胞（ＰＢＭＣ）中ＣＤＶ核衣壳蛋白（ＮＰ）基因。以两套引物针对ＣＤＶＯｎｄｅｒｓｔｅｐｏｏｒｔ毒株ＮＰ基因的两个片段。应用ＲＴ－ＰＣＲ〉以６株ＣＤＶ毒株感染Ｖｅｒｏ细胞，用ＣＤＶ人工感犬染的的ＰＢＭＣ，在这两处细胞的提的ＲＮＡ中，手增ＮＰ基因片段，取得了较好的结果。在３２例临床疑为ＣＤＶ感染     

贵州犬瘟热病毒的分离鉴定

对流行病学调查、临床症状检查和ELISA检测为犬瘟热阳性的自然发病犬,取肠内容物为病料,采用同步培养方法接种于犬肾细胞系(MDCK)进行病毒的分离,并对分离株进行了形态学特征、血凝特性、动物感染及RT-PCR鉴定。结果表明:病料接种MDCK细胞产生明显的细胞病变(CPE),电镜负染观察接毒细胞培养物见有典型的犬瘟热病毒粒子。分离株不凝集鸡及人“O”型红细胞,接种犬出现明显的临床症状和病理变化。用RT-PCR技术检测病毒细胞培养液,扩增出的片段长为760 bp,与预期设计的长度相同,由此确证分离株为犬瘟热病毒,命名为CDV-GZ2株。     

犬瘟热疫苗弱毒的复壮与免疫试验

将一株犬瘟热疫苗株病毒通过Ｖｅｒｏ细胞２０代（ＣＤＶ／Ｒ－２０）后又通过敏感犬的腹腔巨噬细胞，获得一株免疫原性更高的弱毒株（ＣＤＶ／Ｒ－２０／８）。接种同样剂量１０４ＴＣＩＤ５０的ＣＤＶ／Ｒ－２０／８和ＣＤＶ／Ｒ－２０的犬，产生出血清中和（ＳＮ）抗体价的几何平均数分别为１：５４和１：２６。前者为后者的２倍多。接种１０５ＴＣＩＤ５０的ＣＤＶ／Ｒ－２０／８能完全保护犬（１０／１０）抵抗ＣＤＶ强毒的攻击，接种１０４ＴＣＩＤ５０的保护９／１０犬，而对照犬５只全部发病，其中４只死亡。犬体内ＳＮ价可持续一年之久。冻干培养物在４和－３０℃中可分别保存９个月和１２个月而效力不减。     

Canine distemper virus causes apoptosis of Vero cells

Apoptosis of Vero cells infected with two canine distemper virus (CDV) vaccine strains was detected using TdT (terminal deoxynucleotidyl transferase)-mediated dUTP nick end-labelling (TUNEL), flow cytometric analysis, agarose gel electrophoresis and electron microscopy (EM). By TUNEL, apoptotic cells were found in CDV-Onderstepoort (CDV-Ond)-infected cells. DNA fragments isolated from infected cells were separated by agarose gel electrophoresis and a 'ladder' pattern appeared. EM observations demonstrated that the cells undergoing cytopathic effect (CPE) possessed morphological characteristics of apoptotic cells. Flow cytometric analysis indicated that CDV could induce apoptosis of Vero cells, but the percentages of the apoptotic cells were correlated with the CPE types. The strain showing the cell-rounding type of CPE produced a much higher percentage of apoptotic cells than CDV-Ond with the syncytium type of CPE (P < 0.01). It was concluded that CDV vaccine strains could induce apoptosis of Vero cells and the apoptosis was virus strain-dependent and cell-dependent. The mechanism remains to be studied.     

In vitro propagation of canine distemper virus: establishment of persistent infection in Vero cells

Primary cultures of bovine fibroblast (BF) and canine brain cells, persistently infected with virulent R252-canine distemper virus (CDV), were cocultured with African green monkey (Vero) cells. Transfer of persistent CDV from BF to Vero cells varied inversely with the in vitro passage level (age) of the CDV-infected BF cells. Successful transfer of CDV to Vero cells was signaled by the transient appearance of viral syncytia, rapid spread of viral antigen to all Vero cells in the culture, and by recovery of cell-free Vero-infectious virus in culture fluids. With time, viral cytopathic effects in Vero cells containing CDV disappeared, and the infected lines could not be distinguished from noninfected control Vero cells, except by immunoassay for viral antigen.     

Immunohistochemical demonstration of canine distemper virus antigen as an aid in the diagnosis of canine distemper encephalomyelitis

Brain tissue from 33 dogs with non-suppurative encephalitis was examined for evidence of canine distemper virus (CDV) encephalitis. Sections were examined for lesions, inclusion bodies, syncytial cells and CDV antigen using a double bridge unlabelled antibody enzyme technique. Histopathological lesions considered to be typical of granulomatous meningoencephalomyelitis were found in seven dogs. They all lacked inclusion bodies, syncytial cells and CDV antigen. The remaining 26 dogs all had histopathological lesions typical of CDV encephalitis. Inclusion bodies were found in 24 dogs, four of which also had syncytial cells and CDV antigen was detected immunocytochemically in 25. One dog had no inclusion bodies or syncytial cells and was immunohistochemically negative. Syncytial cells have been found to be of limited diagnostic value for the diagnosis of CDV encephalitis. While inclusion bodies proved to be a good diagnostic criterion for the confirmation of CDV infection, the immunohistochemical demonstration of CDV antigen proved to be superior. CDV antigen was more prevalent than inclusion bodies in tissue sections and much more easily detectable.     

Partial protection and intrathecal invasion of CD8(+) T cells in acute canine distemper virus infection

Initial non-inflammatory demyelination in canine distemper virus infection (CDV) develops against a background of severe immunosuppression and is therefore, thought to be virus-induced. However, recently we found a marked invasion of T cells throughout the central nervous system (CNS) in dogs with acute distemper despite drastic damage to the immune system. In the present study, this apparent paradox was further investigated by immunophenotyping of lymphocytes, following experimental CDV challenge in vaccinated and non-vaccinated dogs. In contrast to CDV infected, unprotected dogs, vaccinated dogs did not become immunosuppressed and exhibited a strong antiviral immune response following challenge with virulent CDV. In unprotected dogs rapid and drastic lymphopenia was initially due to depletion of T cells. In peripheral blood, CD4(+) T cells were more sensitive and depleted earlier and for a longer time than CD8(+) cells which recovered soon. In the cerebrospinal fluid (CSF) we could observe an increase in the T cell to B cell and CD8(+) to CD4(+) ratios. Thus, partial protection of the CD8(+) cell population could explain why part of the immune function in acute distemper is preserved. As found earlier, T cells invaded the CNS parenchyma in these dogs but also in the protected challenged dogs, which did not develop any CNS disease at all. Since markers of T cell activation were upregulated in both groups of animals, this phenomenon could in part be related to non-specific penetration of activated T cells through the blood brain barrier. However, in diseased animals much larger numbers of T cells were found in the CNS than in the protected dogs, suggesting that massive invasion of T cells in the brain requires CDV expression in the CNS.     

Phenotypical characterization of T and B cell areas in lymphoid tissues of dogs with spontaneous distemper

CD3, CD4, CD5, and CD8 antigen expression of T cells and IgG expression of B cells and canine distemper virus (CDV) antigen distribution were immunohistochemically examined in lymphoid tissues (lymph node, spleen, thymus, and tonsil) of control dogs and animals with spontaneous canine distemper. In addition, CNS tissue of all animals was studied for neuropathological changes and CDV antigen distribution. Based on the degree of depletion distemper dogs were classified into two groups. Group I represented animals with moderate to marked lymphoid depletion, while group II dogs displayed mild or no depletion. CDV antigen was mainly found in lymphocytes and macrophages of group I dogs, whereas CDV expression was most prominent in dendritic cells of group II animals. In group I dogs, a marked loss of CD3, CD4, CD5, CD8, and IgG expression was noticed, hereby loss of CD4+ cells was more prominent than depletion of CD8+ cells. In the lymphoid tissues of group II animals, a significant increase in the number of T and B cells was observed compared to group I dogs. The number of CD3+, CD4+, and CD8+ cells in group II dogs was similar to the findings in controls, however, CD5 and IgG expression was mildly reduced in T and B cell areas, respectively. Additionally, in groups I and II dogs, CD3+ and CD5- T cells were detected in T cell areas. Whether this cell population represents a cell type with autoimmune reactive potential remains to be determined. Surprisingly in group II animals, viral antigen was found predominantly in dendritic cells indicating a change in the cell tropism of CDV during chronic infection and a possible mechanism of viral persistence. The two patterns of lymphoid depletions correlated to two different types of canine distemper encephalitis (CDE). Group I dogs displayed acute non-inflammatory CDE, whereas group II dogs suffered from chronic inflammatory demyelinating CDE, indicating a pathogenic relationship between lymphocytic depletion and inflammatory brain lesions in distemper.     

水貂犬瘟热病毒LD-1株的分离与鉴定

取山东某貂场疑似犬瘟热病毒（canine distemper virus,CDV）感染的水貂肝脏等组织病料,通过RT-PCR检测呈CDV阳性,且无水貂细小病毒存在,将病料接种原代CEF细胞、传代系Vero细胞和DF1细胞3种细胞进行病毒分离,通过优化细胞培养条件,最终在Vero细胞上传代培养成功,出现露珠状典型细胞病变（CPE）。分离毒应用RT-PCR、PCR产物测序、抗体中和试验（SN）、间接免疫荧光试验（IFA）及病毒包涵体检查等多种方法进行了CDV的鉴定,结果显示CDV阳性,表明分离到的病毒为水貂CDV,并将其命名为CDV LD-1株。     

Apoptosis in the cerebellum of dogs with distemper

Canine distemper virus (CDV) may induce multifocal demyelination in the central nervous system of infected dogs. The pathogenesis of this process is not clear. The present work identifies the presence of apoptotic cells in white and grey matter of dogs'cerebellum, naturally infected with CDV. Fifteen dogs with clinical signs of canine distemper that tested positive for CDV nucleoprotein were used. Brain specimens were processed and embedded in paraffin. Sections 5 microm thick were stained with hematoxylin-eosin and Shorr. Other sections were submitted to TUNEL reaction and to immunohistochemistry for CDV nucleoprotein detection. Acute and chronic demyelinated plaques were observed in the white matter, while apoptosis occurred particularly in the granular layer of grey matter. Apoptosis seems to play an important role in the pathogenesis of canine distemper demyelination.     

Sandwich-dot enzyme-linked immunosorbent assay for the detection of canine distemper virus

A sandwich-dot enzyme-linked immunosorbent assay (dot ELISA) was developed for the detection of canine distemper virus (CDV). In 56 dogs suspected to have CD the rates of detection of CDV antigen in samples of blood lymphocytes and palpebral conjunctiva by dot ELISA and ELISA were, respectively, 91% (49/54) and 81% (44/54) for the lymphocyte samples and 88% (28/32) and 75% (24/32) for the conjunctival samples. The CDV detection limits were 10 ng/50 μL for dot ELISA and 40 ng/50 μL for ELISA. The reliability of dot ELISA relative to electron microscopy was 96% with 22 samples: all 21 samples in which CDV particles were observed by electron microscopy yielded positive results with dot ELISA; the single sample in which particles were not observed yielded false-positive results with dot ELISA. The results indicate that the dot ELISA developed can serve as a reliable rapid diagnostic test in suspected cases of CD and also be useful for epidemiologic surveillance of the disease.     

大熊猫犬瘟热病毒LG株的分离鉴定

为获得大熊猫犬瘟热病毒株,采集死亡大熊猫的心脏、肺、肝病料,研磨并反复冻融后收集上清液接种Vero细胞,待出现细胞病变(CPE)后,收集病毒液。用逆转录-聚合酶链反应(RT-PCR)鉴定病毒分离株,测定病毒TCID_(50),动物回归试验测定其毒力。结果显示,盲传到第5代时,Vero细胞出现圆缩、聚集、脱落等病变;PCR扩增出287bp片段,与预期相符;测序结果显示,该毒株与已发表的CDV SD(14)11毒株(亚洲-Ⅰ型)的同源性为98%;毒株的TCID_(50)为10~(-5.2)/mL;幼犬感染分离毒株后出现体温升高、鼻头发干、拉稀、眼鼻分泌出水样分泌物等症状。本研究成功分离出大熊猫源犬瘟热病毒株。