稳定表达貉犬瘟热病毒细胞受体SLAM的Vero细胞系的建立及应用

[目的]信号淋巴激活分子(SLAM又称CD150)为犬瘟热病毒(Canine distemper virus,CDV)的细胞受体.本研究目的在于建立稳定表达犬瘟热易感动物貉SLAM基因的Vero细胞系,用于犬瘟热诊断及CDV强毒株的快速分离.[方法]从重组质粒pMD-18-T-rSLAM扩增rSLAM基因编码区,通过分子克隆技术构建真核表达质粒pIRES2-EGFP-rSLAMhis.将该质粒转染Vero细胞后,经EGFP荧光观察、G418抗性压力和单细胞克隆化及RT-PCR筛选表达rSLAM阳性细胞系.应用获得细胞系对临床犬瘟热病例进行病毒分离,对分离得到CDV进行RT-PCR鉴定及动物回归试验.[结果]经过RT-PCR和免疫组化试验证实,本实验筛选获得一株能稳定表达rSLAM的Vero细胞系——Vero-rSLAM.该细胞系接种CDV阳性样品36-48h即可产生典型CDV致细胞融合病变,而其亲本Vero细胞接种至6d均无可见细胞病变出现.应用Vero-rSLAM细胞系从狐狸、貉犬瘟热阳性病料中分离获得了3株犬瘟热病毒.其中犬瘟热病毒LN(10) f1株对易感狐狸、貉均可产生致死性感染.[结论]成功建立了稳定表达rSLAM的Vero细胞系,用它分离的CDV对本动物保持较强的毒力.     

犬瘟热病毒临床流行野毒株F蛋白信号肽区分析

根据GenBank上发表的犬瘟热病毒(CDV)融合蛋白基因(F)序列, 设计引物扩增F蛋白部分信号肽区, 片段长369 bp。对2005年~2007年收集的犬瘟热阳性的水貂、狐、貉实质脏器、血液、尿液等样品进行扩增, 获得了13个CDV F蛋白部分信号肽区基因片段。序列分析发现, CDV野毒F蛋白该区段核苷酸与氨基酸序列与目前我国使用疫苗株CDV3及其他国内外疫苗株比较存在较大差异, 与CDV3对应区段的核苷酸同源性在80.7%~83.2%之间, 而推导的对应氨基酸序列同源性只有64.8%~71.3%。部分信号肽区的氨基酸疏水性分析, 推测其调控功能也发生变化, 本研究为CDV遗传变异和分子流行病学提供理论数据。     

犬瘟热病毒临床流行野毒株F蛋白信号肽区分析

根据GenBank上发表的犬瘟热病毒(CDV)融合蛋白基因(F)序列,设计引物扩增F蛋白部分信号肽区,片段长369 bp.对2005年～2007年收集的犬瘟热阳性的水貂、狐、貉实质脏器、血液、尿液等样品进行扩增,获得了13个CDVF蛋白部分信号肽区基因片段.序列分析发现,CDV 野毒F蛋白该区段核苷酸与氨基酸序列与目前我国使用疫苗株CDV3及其他国内外疫苗株比较存在较大差异,与CDV3对应区段的核苷酸同源性在80.7%～83.2%之间,而推导的对应氨基酸序列同源性只有64.8%～71.3%.部分信号肽区的氨基酸疏水性分析,推测其调控功能也发生变化,本研究为CDV遗传变异和分子流行病学提供理论数据.     

犬瘟热病毒受体及其跨宿主感染

犬瘟热是由犬瘟热病毒(Canine distemper virus,CDV)感染引起的一种高度接触性传染病,其可跨宿主感染不同科属的多种动物并造成较高的死亡率,因此该病对养犬业、经济动物养殖业和野生动物保护业造成了很大危害。近年来,随着生态环境变化及病毒变异,CDV的宿主范围也不断扩大。目前已鉴定的CDV两种受体—SLAM和nectin-4,分别在宿主免疫细胞和上皮细胞上表达,二者与CDV血凝素(H)蛋白的相互作用是决定CDV跨宿主感染和病毒诱导宿主免疫抑制等致病性的基础。本文就近年来国内外对CDV受体及其跨宿主感染研究进展进行了综述。     

犬瘟热病毒基因变异及其细胞受体研究进展

由犬瘟热病毒(Canine distemper virus,CDV)引起的犬瘟热(CD)一直是对世界养犬业、毛皮动物养殖业以及野生动物保护事业危害最为严重的传染病之一,甚至在广泛应用疫苗防制CD的近年,世界各地仍有犬或野生动物感染CDV并造成CD流行的报道.通过对CDV主要抗原基因--血凝基因(H基因)氨基酸序列分析,CDV可分为6个基因型.研究证实,CDV野毒株在抗原性上与疫苗株存在较大差异,因此推测H蛋白抗原变异造成了弱毒疫苗免疫效力的降低进而导致了某些地区CD的爆发.CDV作为一种宿主范围广泛的病毒,其细胞受体--信号淋巴细胞激活因子(SLAM)和硫酸乙酰肝素(HS)在哺乳动物体内的广泛存在是CDV跨物种间感染的重要因素.本文就国内外最新研究进展并结合作者的工作,对上述问题进行了综述和探讨.     

TaqMan 荧光定量RT-PCR 检测犬瘟热病毒方法的建立与初步应用

犬瘟热（Canine distemper,CD）是由犬瘟热病毒（Canine distemper virus,CDV）感染引起的一种急性、高度接触性、致死性传染病.CDV属副粘病毒科麻疹病毒属成员,宿主谱很广,可感染犬（Canis familiaris）、狐狸（Vulpes vulpes）、貉（Ussuriensis）、狼（Canis lupus）、雪貂（Lepus zibellina）、虎（Panthera tigris）、狮（Panthera leo）、大熊猫（Ailuropoda melanoleuca）、小熊猫（Ailures fulgens）、黑熊（Selenarctas thibetanus）等动物,严重危害养犬业、经济动物养殖业的健康发展以及野生动物的生存.     

犬瘟热病毒体外感染人源细胞的分子研究进展北大核心CSCD

犬瘟热病毒(Canine distemper virus,CDV)是一种主要通过呼吸道传播对犬科动物造成严重危害的烈性传染病。近年随着CDV在灵长类动物中暴发,研究者开始关注CDV是否具有潜在感染人的风险。目前虽然无直接证据证明CDV可在人体内定殖,但CDV在体外能感染人源细胞,且人体内CDV关键受体SLAM和Nectin-4与灵长类动物具有极高的同源性,因此我们必须高度重视CDV对人类的潜在威胁。本文对CDV分子结构特征、CDV感染宿主靶细胞过程、CDV入侵宿主所需关键受体以及CDV野毒株体外感染人源细胞等方面进行综述,分析CDV对人是否具有潜在感染的风险。     

犬瘟热病毒体外感染人源细胞的分子研究进展

犬瘟热病毒(Canine distemper virus,CDV)是一种主要通过呼吸道传播对犬科动物造成严重危害的烈性传染病。近年随着CDV在灵长类动物中暴发,研究者开始关注CDV是否具有潜在感染人的风险。目前虽然无直接证据证明CDV可在人体内定殖,但CDV在体外能感染人源细胞,且人体内CDV关键受体SLAM和Nectin-4与灵长类动物具有极高的同源性,因此我们必须高度重视CDV对人类的潜在威胁。本文对CDV分子结构特征、CDV感染宿主靶细胞过程、CDV入侵宿主所需关键受体以及CDV野毒株体外感染人源细胞等方面进行综述,分析CDV对人是否具有潜在感染的风险。     

貂、狐、貉源犬瘟热病毒的分离鉴定与序列分析（英文）

采用能稳定表达犬信号淋巴细胞活性分子（SLAM）的Vero-DST细胞从发病貉、狐狸和水貂病料,分离获得5株病毒,经电镜形态观察、RT-PCR检测、理化特性测定和人工感染发病试验鉴定,均为犬瘟热病毒（CDV）,分别命名为HT-P（貉源）、THD1（貉源）、HD（貂源）、LN（貂源）和HB（狐源）。5个分离毒株TCID50为10-5.2～-7.3/ml;除鸡、鹅红细胞呈微弱阳性外,其余均未见血凝作用;对乳鼠的LD50分别为2×10-3.8～-4.8/ml,对实验兔无致病性,对貉有明显致病性作用。5株CDV H和N基因序列分析结果为5个分离株之间H基因nt序列同源性均达到97.5%以上,HT-P（貉源,山东青岛）与THD1（貉源,山东潍坊）的aa同源性为100%,二者与其他分离株间aa序列同源性为87.9%～99.1%;与chn等疫苗株基因nt序列和推导aa序列同源性普遍较低,分别为90.5～91.8和89.%～91.8%。5个分离株之间N基因nt序列同源性均≥94.5%,HT-P与THD1同源性最高（99.8%）,分离株之间aa序列除HT-P、THD1与HD（貂源,山东青岛）同源性较高（99.0%以上）外,其他．分离株之间同源性低。与chn等疫苗株nt序列同源性较低（90．9～93．5％）。此外,HD和LN（貂源,辽宁大连）发病区域相距较远,但具有较高的同源性,但与HB（狐源,河北沦州）具有相对较低的nt同源性,提示野毒株在易感动物上可能存在种属差异。分离毒株与疫苗株之间H和N蛋白基因差异,可能与CD免疫失败有关。     

感染CDV的犬病料N、H、F基因的克隆与序列分析

目的了解病料中犬瘟热病毒（CDV）的变异情况,为犬瘟热的预防与治疗提供理论依据。方法采用RT-PCR方法对犬病料中CDV的血凝素蛋白（H）、融合蛋白（F）和核衣壳蛋白（N）基因进行扩增,将扩增产物克隆到pGEM-T-easy载体中测序,并进行序列分析。结果测定一株CDV的H、F和N基因大小分别为1863 bp、1653 bp和2235 bp。同源性分析表明,未发现碱基的插入和缺失。该病毒的H、F和N基因分别与国内强毒株BJ080127株的H基因、GN株的F基因和HL株的N基因亲缘关系最近,氨基酸同源性分别为97.3%、97.7%和99.3%。与国外标准野毒株A75-17在核苷酸水平上同源性依次为94.4%、93.8%和97.2%,与疫苗株CDV3在核苷酸水平上同源性分别为88.5%、88.8%和93.9%。系统进化树表明该病毒与国内强毒株在同一谱系。糖基化分析显示,该病毒在F基因3～5位氨基酸处多出1个糖基化位点。结论本研究从犬病料中成功克隆了犬瘟热病毒血凝素蛋白、融合蛋白和核衣壳蛋白,在F基因中新增了一个糖基化位点,为犬瘟热的遗传变异和流行学研究提供了分子生物学依据。     

Phylogenetic implications of the 38 putative ancestral chromosome segments for four canid species.

Chromosome homologies between the Japanese raccoon dog (Nectereutes procyonoides viverrinus, 2n = 39 + 2-4 B chromosomes) and domestic dog (Canis familiaris, 2n = 78) have been established by hybridizing a complete set of canine paint probes onto high-resolution G-banded chromosomes of the raccoon dog. Dog chromosomes 1, 13, and 19 each correspond to two raccoon dog chromosome segments, while the remaining 35 dog autosomes each correspond to a single segment. In total, 38 dog autosome paints revealed 41 conserved segments in the raccoon dog. The use of dog painting probes has enabled integration of the raccoon dog chromosomes into the previously established comparative map for the domestic dog, Arctic fox (Alopex lagopus), and red fox (Vulpes vulpes). Extensive chromosome arm homologies were found among chromosomes of the red fox, Arctic fox, and raccoon dog. Contradicting previous findings, our results show that the raccoon dog does not share a single biarmed autosome in common with the Arctic fox, red fox, or domestic cat. Comparative analysis of the distribution patterns of conserved chromosome segments revealed by dog paints in the genomes of the canids, cats, and human reveals 38 ancestral autosome segments. These segments could represent the ancestral chromosome arms in the karyotype of the most recent ancestor of the Canidae family, which we suggest could have had a low diploid number, based on comparisons with outgroup species.     

Association of MC3R gene polymorphisms with body weight in the red fox and comparative gene organization in four canids

There are five genes encoding melanocortin receptors. Among canids, the genes have mainly been studied in the dog (MC1R, MC2R and MC4R). The MC4R gene has also been analysed in the red fox. In this report, we present a study of chromosome localization, comparative sequence analysis and polymorphism of the MC3R gene in the dog, red fox, arctic fox and Chinese raccoon dog. The gene was localized by FISH to the following chromosome: 24q24‐25 in the dog, 14p16 in the red fox, 18q13 in the arctic fox and NPP4p15 in the Chinese raccoon dog. A high identity level of the MC3R gene sequences was observed among the species, ranging from 96.0% (red fox – Chinese raccoon dog) to 99.5% (red fox – arctic fox). Altogether, eight polymorphic sites were found in the red fox, six in the Chinese raccoon dog and two in the dog, while the arctic fox appeared to be monomorphic. In addition, association of several polymorphisms with body weight was analysed in red foxes (the number of genotyped animals ranged from 319 to 379). Two polymorphisms in the red fox, i.e. a silent substitution c.957A>C and c.*185C>T in the 3′‐flanking sequence, showed a significant association (P < 0.01) with body weight.     

Development of a cytogenetic map for the Chinese raccoon dog (Nyctereutes procyonoides procyonoides) and the arctic fox (Alopex lagopus) genomes, using canine-derived microsatellite probes

New chromosomal assignments of canine-derived cosmid clones containing microsatellites to the Chinese raccoon dog and arctic fox genomes are presented in the study. The localizations are in agreement with data obtained from comparative chromosome painting experiments between the dog and arctic fox genomes. However, paracentric inversions have been detected by comparing the loci order in canid karyotypes. The number of physically mapped loci increased to thirty-five both in the Chinese raccoon dog and in the arctic fox. Furthermore, the present status of the cytogenetic map of the Chinese raccoon dog and arctic fox is presented in this study.     

犬科的线粒体细胞色素b DNA序列及其分子系统学研究

通过对犬科的赤狐、蓝狐、貉和狼４种的线粒体细胞色素ｂ约３７２ｂｐＤＮＡ片段序列分析,结合ＧｅｎＢａｎｋ中狗、西门豺和非洲野犬３种的该区段ＤＮＡ序列的比较,共发现１１３个核苷酸位点存在变异（约３０％）。ＮＪ法构建的分子系统树显示,非洲野犬最先从犬科动物中分化出来;犬属的我狼、狗和西门豺等３种为系统树上独立的一支,且其分歧的时间较赤狐、蓝狐和貉早;赤狐和蓝狐具有较近的亲缘关系。上述结果与形态的观点基本     

Chromosomal evolution of the Canidae. II. Divergence from the primitive carnivore karyotype

The Giemsa-banding patterns of chromosomes from the arctic fox (Alopex lagopus), the red fox (Vulpes vulpes), the kit fox (Vulpes macrotis), and the raccoon dog (Nyctereutes procyonoides) are compared. Despite their traditional placement in different genera, the arctic fox and the kit fox have an identical chromosome morphology and G-banding pattern. The red fox has extensive chromosome arm homoeology with these two species, but has only two entire chromosomes in common. All three species share some chromosomes with the raccoon dog, as does the high diploid-numbered grey wolf (Canis lupus, 2n = 78). Moreover, some chromosomes of the raccoon dog show partial or complete homoeology with metacentric feline chromosomes which suggests that these are primitive canid chromosomes. We present the history of chromosomal rearrangements within the Canidae family based on the assumption that a metacentric-dominated karyotype is primitive for the group.     

A comparative analysis of MC4R gene sequence, polymorphism, and chromosomal localization in Chinese raccoon dog and Arctic fox

Numerous mutations of the human melanocortin receptor type 4 (MC4R) gene are responsible for monogenic obesity, and some of them appear to be associated with predisposition or resistance to polygenic obesity. Thus, this gene is considered a functional candidate for fat tissue accumulation and body weight in domestic mammals. The aim of the study was comparative analysis of chromosome localization, nucleotide sequence, and polymorphism of the MC4R gene in two farmed species of the Canidae family, namely the Chinese raccoon dog (Nycterutes procyonoides procyonoides) and the arctic fox (Alopex lagopus). The whole coding sequence, including fragments of 3'UTR and 5'UTR, shows 89% similarity between the arctic fox (1276 bp) and Chinese raccoon dog (1213 bp). Altogether, 30 farmed Chinese raccoon dogs and 30 farmed arctic foxes were searched for polymorphisms. In the Chinese raccoon dog, only one silent substitution in the coding sequence was identified; whereas in the arctic fox, four InDels and two single-nucleotide polymorphisms (SNPs) in the 5'UTR and six silent SNPs in the exon were found. The studied gene was mapped by FISH to the Chinese raccoon dog chromosome 9 (NPP9q1.2) and arctic fox chromosome 24 (ALA24q1.2-1.3). The obtained results are discussed in terms of genome evolution of species belonging to the family Canidae and their potential use in animal breeding.     

Summer food composition and food niche overlap of the raccoon dog, red fox and badger in Finland

Diets of raccoon dog, red fox and badger were studied in southern Finland from 1990 to 1996 by collecting faeces from dens and latrines during May-July of each year. The frequency of occurrence and relative volume of each food item in the faeces were calculated. The raccoon dog was the most and the red fox the least omnivorous of these carnivores, according to the diversity index. Diet composition of all these species varied among areas, indicating that they are opportunistic feeders. Mammals and birds constituted the bulk of the fox diet, while invertebrates, frogs and plants were most frequently eaten by the badger. The frequency of earthworms in badger faeces varied between 16 and 77%, according to area. Voles and shrews were important to the raccoon dog, but it also frequently consumed other food items. The food niches of the badger and the raccoon dog overlapped more than that of the red fox and other species. The red fox was more carnivorous and fed on larger prey items than the others, but the food composition of the red fox in the present study reflects the prey carried to the den for the pups; thus, the diet of adult foxes may be more similar to that of the raccoon dog. In conclusion, these 3 carnivores share many resources, suggesting that competition may occur among them: however, their diets also differ to some extent, which helps them to avoid competition. Furthermore, the badger and the raccoon dog are dormant during winter, when food is scarcest, which may be the reason why all these species can coexist in rather unproductive boreal forests.     

Comparative chromosomal localization of the canine-derived BAC clones containing LEP and IGF1 genes in four species of the family Canidae

In the present report we show the chromosomal localization of two BAC clones, carrying the leptin (LEP) and insuline-like growth factor 1 (IGF1) genes, respectively, in four species belonging to the family Canidae: the dog, red fox, arctic fox and the Chinese raccoon dog. The assignments are in agreement with earlier data obtained from comparative chromosome painting for the dog, red fox and arctic fox.     

Use of RAPD technique in evolution studies of four species in the family Canidae

The RAPD-PCR technique was applied to identify genetic markers able to distinguish between four canid species: the arctic fox (Alopex lagopus), red fox (Vulpes vulpes), Chinese raccoon dog (Nyctereutes procyonoides procyonoides) and six breeds of the domestic dog (Canis familiaris). A total of 29 ten-nucleotide arbitrary primers were screened for their potential use in the differentiation of these species. Ten primers amplified RAPD profiles that made it possible to distinguish between the investigated taxa. A number of species-specific bands was scored within RAPD profiles produced by these primers: 35.6% of all the polymorphic bands were unique to the Chinese raccoon dog, 29.6% were unique to the domestic dog, 21.2% were diagnostic for the red fox and 13.6% for the arctic fox. No breed-specific fragments were amplified from canine DNA; however, three primers produced bands characteristic for the dog, but not present in all of the investigated breeds. A Neighbor-Joining tree constructed on the basis of the analysis of RAPD profiles amplified by six primers revealed that the phylogenetic distance between the dog and the arctic fox is larger than the distance between the dog and the red fox. The phylogenetic branch of the Chinese raccoon dog was the most distinct on the dendrogram, suggesting that this species belongs to a different phylogenetic lineage. Obtained results make it possible to conclude that RAPD analysis can be a powerful tool for developing molecular markers useful in distinguishing between species of the family Canidae and for studying their phylogenetic relations.     

The diet of feral raccoon dog (<Emphasis Type="Italic">Nyctereutes procyonoides</Emphasis>) and native badger (<Emphasis Type="Italic">Meles meles</Emphasis>) and red fox (<Emphasis Type="Italic">Vulpes vulpes</Emphasis>) in Denmark

The raccoon dog (Nyctereutes procyonoides) is an East Asian Canid that has been introduced in Europe. Introduction of alien species is an increasing conservation issue. We examined the diet of a recently established raccoon dog population in Denmark by analysing stomach content in 249 carcasses collected in 2008–2016. Raccoon dog diet was compared to the diet of native badger (Meles meles) and red fox (Vulpes vulpes) in Denmark. The most common food for raccoon dogs were invertebrates (frequency of occurrence, FO 69%), small mammals (FO 68%), birds (FO 41%), fruits (FO 38%), amphibians (FO 36%) and carrions (FO 34%). The occurrence of invertebrates was highest during spring and summer, while fruits, cereals and carrions were eaten most often during autumn and winter. As expected, raccoon dog shared the major food categories with badger and red fox, but generally, it had a wider dietary niche. Overall, dietary overlap between raccoon dog and badger was 0.74 (Pianka index, O_jk). The dietary overlap with red fox was relatively high in all seasons, peaking in summer (O_jk 0.87) and dropping in winter (O_jk 0.79). Despite the dietary overlap between the alien racoon dog and native red fox and badger, the species may coexist due to partitioning of feeding habitats and/or because the red fox is limited by other factors, e.g. diseases and anthropogenic activities. The introduced raccoon dog seems to fit a dietary niche between badger and red foxes in human-dominated landscapes in north-western Europe.