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1.
Stability of canine distemper virus (CDV) after 20 passages in Vero-DST cells expressing the receptor protein for CDV 总被引：1，自引：0，他引：1
Lan NT Yamaguchi R Kawabata A Uchida K Kai K Sugano S Tateyama S 《Veterinary microbiology》2006,118(3-4):177-188
Isolates 007Lm, S124C and Ac96I and a Vero cell-adapted Onderstepoort strain of canine distemper viruses (CDV) were examined for stability after passages in Vero cells expressing the canine signaling lymphocyte activation molecule (dogSLAM, the intrinsic receptor to CDV). These viruses passage once in Vero cells expressing dogSLAM (Vero-DST) cells (original) and after 20 passages (20p) were compared by using sequence analyses and growth characteristics. All four strains of 20p grew well and were slightly better than their originals. The 20p viruses developed a cytopathic effect slightly lower than the original strains. A few changes in amino acids in the H gene were between the 20p and the original viruses, but the sites of changes were not specific. Fragments of P, M and L genes of all strains showed no nucleotide changes after the passages. These results showed that: (1) passages of CDVs in Vero-DST cells induced amino acid changes only in the H gene, not in the P, M and L genes, unlike in a previous study with Vero cells; (2) passages did not markedly affect the growth characteristics of every viral strain. These results indicate that Vero cells expressing canine SLAM allow the isolation and passaging of CDV without major changes in viral genes. 相似文献
2.
为了对1例貉源犬瘟热(CD)进行病毒检测并分析其血凝素H基因变异情况,本研究从1只疑似犬瘟热病死的貉采病料进行研磨,利用RT-PCR方法扩增犬瘟热病毒H基因,对扩增出的H基因片段进行克隆测序,并对得到的H基因序列进行分析。结果表明,该貉感染犬瘟热病毒,得到的H基因核苷酸和氨基酸序列与CDV野毒株同源性较高,分别为89.1%~98.0%和85.4%~97.5%。遗传进化分析表明其属于Asia-1型野毒株,N连接糖基化位点分析结果表明,该毒株在542aa处比参考野毒株多了1个潜在的N糖基化位点,在525aa-550aa间多出了1个抗原表位。 相似文献
3.
为了解山东地区水貂犬瘟热病毒(CDV)遗传变异特征,采集水貂养殖场的发病水貂病料,通过RT-PCR鉴定为CDV阳性,将阳性病料接种Vero/Dog SLAM细胞进行病毒分离,通过间接免疫荧光、电镜负染、测序等方法鉴定,得到4株犬瘟热病毒,分别命名为WD1株、WD2株、WX1株和WX2株。分离株H基因测序结果显示,WD1株、WD2株、WX1株均为Asia-Ⅰ型,其中WD1和WD2与近几年仅在水貂和狐狸养殖场流行的新犬瘟热毒株核苷酸和氨基酸序列同源性分别为97.5%~99.2%和97%~99%;WX-1型与国内犬源HL001株的同源性最高,核苷酸和氨基酸序列同源性分别为99.6%和99.5%;WX2与疫苗株同属于一个分支,与疫苗毒Lederle株核苷酸和氨基酸序列同源性高达99.5%和98.8%。结果表明,水貂养殖场存在多株犬瘟热病毒混合感染的情况,提醒养殖场应注意防控,该结果也为犬瘟热病毒分子流行病学积累了资料。 相似文献
4.
Lan NT Yamaguchi R Kai K Uchida K Kato A Tateyama S 《The Journal of veterinary medical science / the Japanese Society of Veterinary Science》2005,67(5):491-495
To know growth profiles of canine distemper virus (CDV) on Vero cells stably expressing canine signaling lymphocyte activation molecule (Vero-DogSLAMtag; Vero-DST cells), the propagation of three strains of CDV was tested in Vero-DST cells in comparison with parental Vero cells. Strain MD77 could grow well in both cell lines, but demonstrated no syncytium formation or indistinguishable rounding cytopathic effects (CPE) in Vero cells. Strains Onderstepoort and KDK-1 also grew well in Vero-DST cells with apparent syncytium CPE, while they grew less or no efficiently, respectively, in Vero cells. All three CDV strains demonstrated the peak titers, in Vero-DST cells before reaching to an extensive CPE and drastic decrease of titers at/after full CPE. Immunohistochemistry revealed that viral antigens of all CDV strains were found exclusively in the syncytia in Vero-DST cells, while in Vero cells, viral antigen was identified in their single cells for strain MD77 but none for other strains. Thus, every strain of CDV could grow well in Vero-DST cells and behaved differently against Vero cells. These results would be of practical value for workers of CDV because 1) In Vero-DST cells, by observation of distinct syncytium CPE, the highest titer or the best growth of virus could be identified; 2) In Vero cells, various CDV strains could be readily classified after propagation in Vero-DST cells. 相似文献
5.
Dog lymphocyte cultures facilitate the isolation and growth of virulent canine distemper virus. 总被引：5，自引：0，他引：5
M J Appel S Pearce-Kelling B A Summers 《Journal of veterinary diagnostic investigation》1992,4(3):258-263
Optimal conditions for the isolation and growth of virulent canine distemper virus (CDV) in canine thymic and peripheral blood lymphocyte cultures were determined. Peak virus titers were seen from 3 to 6 days postinoculation of lymphocytes and depended on the multiplicity of infection. Dog lymphocytes were at least as susceptible as canine macrophages to infection with virulent CDV. Virus replication in lymphocytes resulted in higher virus titers than in dog lung macrophages. Peripheral blood lymphocytes (PBL) from CDV-immune dogs were as susceptible to CDV as were PBL from susceptible dogs. 相似文献
6.
为了解上海地区犬瘟热病毒（Canine distemper virus，CDV）遗传变异情况，本研究采用首尾重叠的11对特异性引物，对CDV上海株SH202003进行RT-PCR扩增，将扩增片段进行反复测序，序列拼接后最终获得了SH202003株全基因组序列，应用Lasergene 7.0和Mega 6.0软件对全基因及H基因进行序列分析，并构建系统进化树。结果显示，SH202003株基因组全长为15 690 bp，编码6种结构蛋白（N、P、M、F、H和L），H和L基因间隔序列为CUA，L和5'端尾随序列为CAA，与Hebei株核苷酸和氨基酸相似性最高，达到98.6%和96.6%，与疫苗株核苷酸相似性在92.2%~94.3%，氨基酸相似性只有82.7%~87.0%；全基因进化树中，SH202003株与流行野毒株在同一分支，与疫苗株在不同的分支；H基因同样与Hebei株亲缘关系最近，核苷酸和氨基酸相似性分别为98.7%和99.5%，与疫苗株Snyder Hill、CDV3、Convac及Onderstepoort亲缘关系较远；SH202003株处于Asia-1型分支，属于Asia-1型强毒株；SH202003株具有9个潜在N-糖基化位点，与强毒株Hebei株一致。研究表明，上海株SH202003属于CDV强毒株，为Asia-1型，其H基因序列相对保守，具有9个潜在N-糖基化位点，但是全基因序列存在较多突变，与疫苗株的匹配度较差，可能是免疫犬依然发生犬瘟热的主要原因。 相似文献
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RT-PCR检测贵州犬瘟热病毒 总被引：5，自引：3，他引：5
根据犬瘟热病毒H基因核苷酸序列 ,设计合成一对引物对贵州临床诊断为犬瘟热 (CD)病死犬的心、肝、脾、肾、粪便进行RT PCR检测。结果表明 :从心、脾、肾病料上清液中可扩增出 760bp的特异性带 ,与预期扩增片段长度相同 ;粪便和肝脏上清液RT PCR结果为阴性 ,但粪便上清液接种Vero细胞后连续传代 3代 ,每代细胞培养液RT PCR结果均为阳性 ;在检测的 2 2条病死犬中 ,有 1 9条病死犬检测到犬瘟热病毒H基因的特异性核酸片段 ,阳性率为 86 4% (1 9/ 2 2 )。 相似文献
9.
北京地区犬瘟热病毒分离株H基因的分型 总被引：2，自引：0，他引：2
根据发表的犬瘟热病毒(CDV)参考毒株Onderstepoort的序列设计了一对引物,以感染犬瘟热病毒的犬的粪便总RNA为模板,RT-PCR扩增出17株CDV的H全基因1877bp的片段,将这个片段连接到pGEM-Teasy载体上,经酶切鉴定得到阳性克隆,将阳性质粒进行序列测定,并与国内外毒株进行同源性比较和系统发育分析,结果显示17株均为Asia-1型。北京地区流行的毒株主要是America-1型和Asia-1型,其中2007年主要流行America-1(疫苗型),而2008年主要是Asia-1型。 相似文献
10.
本试验采用Vero细胞从临床疑似患犬瘟热的犬组织病料中分离出一株病毒,通过观察细胞病变,提取细胞培养物RNA并扩增克隆分析N蛋白基因部分序列进行了鉴定。结果表明,该毒株接种于Vero细胞后,出现了典型的细胞病变;对接种病料后的Vero细胞培养物提取总RNA进行RT-PCR扩增,得到了287 bp的目的片段;序列分析表明该分离株与犬瘟热病毒弱毒(Onderstepoort和Snyder Hill)的同源性为95.5%~96.9%,与标准强毒株(A75/17和2544/Han95)及野毒株(XJ-4、ZJ7、98-2646等)的同源性较高,为97.2%~99.0%,证明该毒株为犬瘟热病毒野毒株,命名为QD10。 相似文献
11.
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。 相似文献
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Cha SY Kim EJ Kang M Jang SH Lee HB Jang HK 《Comparative immunology, microbiology and infectious diseases》2012,35(5):497-504
Raccoon dogs (Nyctereutes procyonoides) are widespread and common in South Korea. In 2011, we obtained serum samples from 102 wild raccoon dogs to survey their exposure to canine distemper virus (CDV). Forty-five of the 102 animals (44.1%) were seropositive. Field cases of canine distemper in wild raccoon dogs from 2010 to 2011 were investigated. Fourteen cases of CDV infection were identified by a commercially available CDV antigen detection kit. These cases were used for virus isolation and molecular analysis. Sequence analysis of hemagglutinin genes indicated that all viruses isolated belonged to the Asia-2 genotype. H protein residues which are related to the receptor and host specificity (residues 530 and 549) were analyzed. A glutamic acid (E) residue is present at 530 in all isolates. At 549, a histidine (H) residue was found in five isolates and tyrosine (Y) residue was found in 6 isolates. Our study demonstrated that CDV infection was widespread in wild raccoon dogs in South Korea. 相似文献
14.
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. 相似文献
15.
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。 相似文献
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Gámiz C Martella V Ulloa R Fajardo R Quijano-Hernandéz I Martínez S 《Veterinary research communications》2011,35(6):381-390
Canine Distemper is a highly contagious viral systemic disease that affects a wide variety of terrestrial carnivores. Canine
Distemper virus (CDV) appears genetically heterogeneous, markedly in the hemagglutinin protein (H), showing geographic patterns
of diversification that are useful to monitor CDV molecular epidemiology. In Mexico the activity of canine distemper remains
high in dogs, likely because vaccine prophylaxis coverage in canine population is under the levels required to control effectively
the disease. By phylogenetic analysis based on the nucleoprotein (N) and on the H genes, Mexican CDV strains collected between
2007 and 2010 were distinguished into several genovariants, all which constituted a unique group, clearly distinct from field
and vaccine strains circulating worldwide, but resembling a CDV strain, 19876, identified in Missouri, USA, 2004, that was
genetically unrelated to other North-American CDV strains. Gathering information on the genetic heterogeneity of CDV on a
global scale appears pivotal in order to investigate the origin and modalities of introduction of unusual/novel CDV strains,
as well as to understand if vaccine breakthroughs or disease epidemics may be somewhat related to genetic/antigenic or biological
differences between field and vaccine strains. 相似文献
17.
Infection of the footpad epidermis can occur in natural canine distemper virus (CDV) infection of dogs. Footpads from 19 dogs experimentally inoculated with virulent distemper strain A75/17 and from two nonexposed dogs were examined histopathologically and assessed for the presence of viral antigen and nucleoprotein mRNA, as well as number of inflammatory and apoptotic cells. Dogs were divided into four groups based on inoculation status and postmortem examination: inoculated dogs with severe distemper (group 1, n = 7); inoculated dogs with mild distemper (group 2, n = 4); inoculated dogs without distemper (group 3, n = 8); and noninoculated dogs (group 4, n = 2). Footpads from dogs of all groups had a comparably thick epidermis. Eosinophilic viral inclusions and syncytial cells were present in footpad epidermis of one dog of group 1. Footpads of group 1 dogs contained viral antigen and mRNA in the epidermis with strongest staining in a subcorneal location. Additionally, in these dogs footpad dermal structures including eccrine glands and vascular walls were positive for virus particles. No CDV antigen or mRNA was present in the footpad epidermis and dermis of any other dog. Group 1 dogs had more CD3-positive cells and apoptotic cells within the basal layer of the epidermis when compared to the other groups. These findings demonstrate that in experimental infection CDV antigen and mRNA were colocalized in all layers of the infected canine footpad epidermis. The scarcity of overt pathological reactions with absence of keratinocyte degeneration indicates a noncytocidal persisting infection of footpad keratinocytes by CDV. 相似文献
18.
根据GenBank上登录的犬瘟热病毒（Canine distemper virus，CDV）基因组全序列，选择CDV强、弱毒株间有区别保守区设计了一对通用引物P1和P4，并在该对引物跨越区域的内部设计了CDV强毒株特异性引物P2及弱毒株特异性引物P3，用引物P1／P4进行RT—PCR，然后用引物P2／P3／P4进行复合套式PCR，建立了一种能区分CDV强、弱毒株的复合反转录-套式聚合酶链式反应（RT—nPCR）的鉴别诊断方法。应用该方法从CDV强、弱毒株的基因组中分别扩增出了大小为247bp和177bp的特异性片段，从两种病毒基因组混合物中扩增出了大小为247bp和177bp的两条特异性片段，与犬细小病毒、犬腺病毒、犬冠状病毒、狂犬病病毒、新城疫病毒的细胞培养物以及正常细胞对照组进行复合RT—nPCR扩增时均为阴性。对从黑龙江省和吉林省采集的20份疑似CDV病料进行的检测结果表明，有15份类似CDV强毒，5份类似CDV弱毒。本研究建立的复合RT—nPCR可以有效检测CDV感染，能够将强、弱毒株区分开，可用于临床快速检测、流行病学监测以及追踪疫苗免疫效果等。 相似文献
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水貂犬瘟热CDV_3疫苗株基因组序列测定及H基因遗传稳定性分析 总被引：1，自引：0，他引：1
对水貂犬瘟热CDV3疫苗株基因组进行全序列测定与分析,以阐明该疫苗株的来源亲本毒株,基因特征及H基因遗传稳定性。将CDV3疫苗株基因组cDNA分9个重叠片段进行RT-PCR并克隆入pMD 18-T载体中,测定全长cDNA序列。并与CDV野毒参考株和疫苗株N、F和H基因氨基酸序列比对分析其基因变异情况。通过对CDV3疫苗株6个不同生产批次(Vero细胞培养代次)H基因序列测定以分析毒株的分子遗传稳定性。基因序列分析结果表明:CDV3疫苗株基因组全长15 690 bp,与CDV野毒株基因组同源性较低(93.0%~95.3%)。H基因核苷酸和氨基酸序列与Lederle疫苗株(EF418783)同源性最高,分别为99.6%和98.7%。而6个不同培养代次CDV3疫苗株H基因氨基酸序列无任何差异。由此证实CDV3疫苗株的亲本毒株与Lederle疫苗株有着最密切关系,不同培养代次的CDV3疫苗株H基因具有较高的遗传稳定性。 相似文献