一株Class Ⅰ新城疫病毒中国分离株分子特性的研究

对从健康家鸭中分离到的一株Class I新城疫病毒分离株Duck/China/08-004/2008进行了遗传进化特性研究。利用RT-PCR扩增了该分离株F基因的主要功能区片段,并进行了克隆与序列分析。序列测定结果已经登录到GenBank,登录号为EU589149。该分离株F蛋白裂解位点的组成为112E-Q-Q-E-R-L117,具有典型新城疫弱毒株的分子特征。同源性分析表明其与国内普遍使用的弱毒疫苗株LaSota和V4核苷酸的同源性较低(分别为55.4%和57.7%)。通过构建F基因的遗传进化树,结果表明该分离株在分类地位上属于ClassI,与我国目前普遍使用的弱毒疫苗株LaSota(Class II中的基因II型)和V4(Class II中的基因I型)处在不同的进化分支。通过构建57株ClassI新城疫病毒的遗传进化树,表明本分离株与近年来香港活禽市场分离株较为类似,同属于基因3型。     

Characterization of pigeon-origin Newcastle disease virus isolated in China

Fourteen pigeon-origin Newcastle disease virus (NDV) isolates were obtained from sick pigeons in China between 1996 and 2005. The mean death time (MDT) of embryonated eggs and the intracerebral pathogenicity indices (ICPI) were tested to determine the virulence of the field isolates. The result indicated that most isolates were proved to be mesogenic (MDT 60-90 hr and ICPI > 1.2). The main function regions of F protein gene of the isolates were amplified and sequenced for phylogenetic and residue substitutive analysis. The fusion protein cleavage site sequences of most isolates had multiple basic amino acids R/KRQKRF at positions 112-116 and a phenyl alanine at position 117, characteristic of velogenic isolates. In the phylogenetic tree, the majority of the isolates were clustered into a single genetic lineage, termed genotype VIb, and were typical pigeon paramyxovirus type 1, whereas a small number of recent isolates (three strains) were grouped into genotype VIId, a predominant genotype responsible for most Newcastle disease outbreaks in chickens and geese since the end of last century. One isolate, PK9901, was proved to be a lentogenic strain, of genotype II NDV, to which the vaccine strain La Sota belongs.     

青海湖野生迁徙水鸟新城疫病毒的分离与鉴定

从青海湖地区野生迁徙水鸟粪便中分离到1株新城疫病毒QH0601,其脑内接种指数(ICPI)为1.88.以RT-PCR扩增该分离株F基因片段,并进行序列分析,结果显示QH0601分离株F蛋白裂解位点具有强毒株的特征性氨基酸序列112R-R-Q-K-R-F117,其101位和121位的氨基酸残基具有新城疫病毒基因Ⅶ型特征.聚类分析表明,QH0601毒株与我国普遍流行的基因Ⅶ型NDV具有很高的同源性,提示野生迁徙水鸟与家禽新城疫的发生在流行病学和生态学上有着非常密切的关系.     

Characterization of a lentogenic Newcastle disease virus isolated from broiler chickens in Japan

Newcastle disease virus (NDV), named MET95, was isolated from a non-vaccinated broiler flock in Japan in 1995. The MET95 strain was determined to be a lentogenic NDV. The strain has the properties of eluting rapidly at 4 C and has low thermostability in hemagglutinating activity with chicken erythrocytes. In these studies, no difference could be found between the MET95 strain and the Hitcher B1 vaccine strain. However, the chickens inoculated with the MET95 strain, as well as chickens that they were in contact with, had a much higher hemagglutination-inhibition antibody response than those inoculated with the B1 strain. Accordingly, the MET95 strain is thought to be a promising candidate as a live ND vaccine strain. In Japan, this is the first report on the isolation of lentogenic NDV from chickens since the paper on the Ishii strain isolated in 1966.     

Characterization of a velogenic Newcastle disease virus isolated from broilers in Saudi Arabia

A highly virulent Newcastle disease virus (SA84) was isolated from a large broiler operation in Saudi Arabia. The mean death time of chicken embryos given the minimum lethal dose, the pathogenicity of the isolate for 8-week-old chickens, the plaque characteristics, and the intracerebral pathogenicity index indicated that the isolate is of the viscerotropic velogenic pathotype.     

Characterization of Newcastle disease virus (avian paramyxovirus-1) isolated from pigeons

Newcastle disease virus (avian paramyxovirus-1) was isolated from pigeons in 12 states between May 1984 and December 1985. One of the isolates was from a feral pigeon; the remainder were from privately owned pigeon lofts. Use of monoclonal antibodies showed seven of the eight isolates tested to be indistinguishable from the 1982 and 1983 Great Britain and European isolates. Clinical signs were paralysis, torticollis, tremors, incoordination, and death. Pigeons inoculated with the paramyxovirus-1 isolates intravenously or intramuscularly developed clinical disease identical to that described for natural infection; however, only one pigeon inoculated intranasally developed clinical signs. The mean death time for inoculated pigeons was 9.5 days, with a range of 4 to 25. Virus was shed for up to 20 days. Primary lesions observed on necropsy were gastroenterocolitis and pancreatic necrosis. Chickens experimentally infected by the cloacal, intranasal, or caudal thoracic air-sac route remained healthy. However, the intracerebral pathogenicity index (ICPI) in day-old chickens was similar to that observed with velogenic Newcastle disease virus isolates. Four of six isolates inoculated intravenously into 6-week-old chickens induced neurotropic disease.     

Characterization of Newcastle disease virus isolated from northern pintail (Anas acuta) in Japan

A field isolate of Newcastle disease virus (NDV) isolated from northern pintail (Anas acuta) in Tohoku district, northeast Japan, was characterized. Phylogenetic analysis of the fusion protein indicated that the isolate belonged to genotype I and was closely related to isolates from the Far East corresponded to the migration route for this bird species. The isolate had the typical avirulent cleavage site of the fusion protein (112)GKQGR*L(117). In addition, pathogenicity tests indicated the isolate to have avirulent characteristics. However, the isolate has been shown to cause fusion cytopathic effects and form plaques on chicken embryo fibroblasts (CEF) in the absence of trypsin. The present results suggest that the CEF-adapted NDV, which is avirulent, is circulating among waterfowl populations.     

Epizootiology of Newcastle disease in waterfowl.

Antibodies to Newcastle disease virus (NDV) as measured by hemagglutination-inhibition and virus-neutralization tests were detected in 40/236 Canada geese captured while in their southward migration or in their wintering grounds. Antibodies were also found in 37/267 wild ducks and in 20/31 domestic geese. Adult geese were readily infected by several routes. Inapparent disease usually resulted, and only 1/13 cases were fatal. Goose embryos responded differently to inoculation with selected NDV strains than did chicken embryos of comparative developmental stages. Some goslings that hatched from inoculated embryos died and were found to have virus, whereas others survived and developed active antibodies. Four strains of virus isolated from migratory ducks of the Pacific flyway were characterized. All 4 strains were lentogenic but differed from lentogenic strains prevalent in chickens by being thermostable. It is proposed that wild waterfowl neither receive their ND infection from domestic poultry nor pass their disease to poultry. The virus reservoir probably exists in nature.     

Characterization of Newcastle disease viruses isolated from field cases in Japan

Seven Newcastle disease viruses isolated in Japan from 1930 to 1984 were cloned on chicken embryo fibroblasts (CEFs) and characterized biologically. All seven produced two or more types of plaques on CEFs. The plaques were classified into four types. Plaque cloning was carried out five times, and 22 cloned viruses were established. The biological characters of the cloned viruses suggested that the strains contain different clones and that their clones are different even among close cases, such as G strain and H strain.     

不同宿主源NDV毒株对SPF鸡致病性研究

为阐明不同宿主及不同基因型新城瘦病毒(NOV)对SPF鸡致病性,选择分离自鸡、番鸭、鹅、健康野鸟的基因VIId亚型NDV 6株,鸡源基因Ⅲ型和鸽源基因VIb型毒株各1株,以及基因Ⅸ型强毒F48E9,共9株NDV毒株进行致病性试验.在对各毒株的EID50及主要致病指数MDT、ICPI测定基础之上,以相同剂量感染15日龄SPF鸡,观察临床症状及剖检病变,计算发病率和死亡率,并在攻毒后不同时间采集主要组织样品.以SYBR Green I Real-time PCR检测病毒最早出现时间及病毒载量.结果表明,6株基因VIId亚型NDV毒株导致SPF鸡100%发病,死亡率90%以上,属于高致病性毒株;基因Ⅲ、VIb型毒株导致SPF鸡发病但不死亡,属于中等毒力毒株.VIId亚型毒株与F48E9株攻毒后SPF鸡在病理变化、组织嗜性及病毒载量上没有显著差异.根据毒株的MDT、ICPI指数及攻毒鸡病程综合判断,VIId亚型毒株在致病性上与F48E9株差异不显著.健康野鸟携带基因VIId亚型高致病性NDV,在NDV的自然生态传播过程中起重要作用,提示应该加强对野鸟的流行病学监测及相关研究.     

Newcastle disease and avian influenza A virus in wild waterfowl in South Africa

In an intensive ostrich farming area in South Africa with a history of ostrich influenza outbreaks, we conducted a survey of avian influenza virus (AIV) and Newcastle disease virus (NDV) in wild aquatic birds. During late autumn and winter 1998, the time of year when outbreaks in ostriches typically start to occur, 262 aquatic birds comprising 14 species were sampled and tested for both virus infections. From eight samples, AIV, serotype H10N9, could be isolated. All isolates were apathogenic as determined by the intravenous pathogenicity index (0.00). Conversely, none of 33 sera of these wild birds showed antibodies against H10. However, one bird was found serologically positive for H6 AIV. This AIV serotype was later isolated from ostriches during an avian influenza outbreak in this area. No NDV was isolated although 34 of 46 serum samples contained NDV-specific antibodies. This is the first H10N9 isolate to be reported from Africa. In addition, our data support the notion that wild aquatic birds may function as a reservoir for AIV and NDV in South Africa.     

Characterization of pigeon paramyxoviruses (Newcastle disease virus) isolated in South Africa from 2001 to 2006

Pigeon paramyxovirus type 1 (PPMV-1), a variant of Newcastle disease virus that primarily affects doves and pigeons has been isolated in South Africa since the mid-1980s. Phylogenetic evidence indicates that pigeon paramyxovirus type 1 viruses were introduced into South Africa on multiple occasions, based on the presence of two separate lineages, 4bi and 4bii, that have been circulating in Europe and the Far East since the early 1990s. During 2006, a PPMV-1 virus was isolated from an African ground hornbill (Bucorvus leadbeateri) which became acutely infected with PPMV-1 and died, probably after scavenging off infected dove carcasses in the region, since a closely-related PPMV-1 strain was also isolated from doves collected nearby. The hornbill isolate had ICPI and MDT values characteristic of PPMV-1 strains. The threat of PPMV-1 to poultry production and biodiversity in southern Africa highlights the importance of monitoring the spread of this strain.     

一株新城疫病毒广东分离株的遗传关系分析

根据新城疫病毒已报道的基因序列设计了一对引物,扩增F基因3 ′端374 bp片段,包括F基因信号肽序列和裂解位点.经过RT-PCR、PCR产物测序和序列分析,表明该毒株为基因Ⅶ型强毒株,和中国动物卫生与流行病学中心2009年分离到的毒株NDV09-038相似性最高,可达98％.基因进化树分析表明该毒株与常用疫苗株亲源关系较远,与I系疫苗株Mukteswar相似性为81.4％,与Herts' 33的相似性为84.5％.氨基酸序列分析表明该毒株为典型的强毒株.     

Characterization of Nigerian strains of Newcastle disease virus

Newcastle disease virus was isolated from outbreaks of the disease in vaccinated and unvaccinated poultry flocks representing commercial and backyard farms in different parts of Nigeria. On characterization, all 12 isolates were found to be velogenic.     

鸭新城疫病毒分离株抗原表位和遗传演化分析

从规模化养殖场鸭群气管和泄殖腔试子分离到新城疫病毒(NDV)27株,用2株针对NDV HN单抗进行抗原表位分析,并选择4个分离株进行F基因高变区(374bp)和HN基因全长序列分析。抗原表位分析结果显示,27个鸭分离株均能与其中一株单抗C3-B7反应,而与另外一株单抗1E5反应为阴性。F基因(374bp)序列分析结果显示,4个鸭分离株均属于NDV ClassⅠ分支,分离株之间核苷酸同源性为99.2%～100%;分离株与NDV ClassⅡ毒株遗传距离为0.9%～9.9%,与NDV ClassⅠ毒株遗传距离为38.5%～41.7%。根据核苷酸序列推导的氨基酸序列表明,4个鸭NDV分离株F蛋白裂解位点氨基酸模式为:112-EROERL-117。HN基因序列分析结果显示,4个鸭NDV分离株HN基因全长1851bp,编码585个氨基酸;同源性比较发现4个鸭NDV分离株之间核苷酸同源性为99.7%～99.8%,与NDV ClassⅡ毒株核苷酸同源性为68.4%～70.5%,与NDV ClassⅠ毒株核苷酸同源性为95.8%～98.0%。本研究结果显示,鸭分离毒均属于NDV ClassⅠ弱毒,在抗原表位和基因序列上与广泛应用的NDV弱毒疫苗株(LaSota)不同,这些毒株的来源有待进一步深入研究。     

Differences in receptor specificity between Newcastle disease viruses originating from chickens and waterfowl.

We compared the receptor specificity of Newcastle disease viruses from a variety of avian species, including chickens and wild waterfowl, using hemagglutination tests with erythrocytes from different animal species. All isolates from wild waterfowl agglutinated horse erythrocytes, while the chicken isolates did not. The results showed that the receptor specificity of Newcastle disease viruses is different, depending on the avian species from which the viruses are isolated.     

新城疫病毒山东流行株F基因的序列测定和分析

用 SPF鸡胚从发生新城疫鸡群分离到一株病毒 ,进行了生物学特性的研究 ,结果表明 ,最小致死量鸡胚平均死亡时间 (MDT)为 5 0 ,1日龄鸡脑内接种致病指数 (ICPI)为 1.82 ,6周龄鸡静脉接种致病指数 (IVPI)为 2 .6 2 ,属强毒力毒株。以异硫氰酸胍法提取病毒基因组 RNA,RT- PCR扩增其融合蛋白 F基因重要功能区片段 ,经回收后 ,克隆到 p GEM- T载体上进行酶切鉴定与核苷酸序列测定 ,并与多株已报道的 NDV国内外参考株相应片段进行序列比较 ,F基因的同源性在 84% - 91%之间 ,氨基酸同源性为 82 % - 93% ,经基因进化树分析 ,证实流行株为基因 型毒株     

两种禽源新城疫病毒分离株F基因遗传变异分析

以鸡胚分离结合血清学鉴定,从近几年江苏省及河南省分离到来自鸡和鸽子的6株新城疫病毒(NDV).病毒蚀斑纯化后,选取3个克隆利用RT-PCR技术扩增F基因重要功能区片段,在对PCR产物直接序列测定分析基础上,选取一致克隆株进行全长F基因克隆、序列测定分析.根据分离株序列与GenBank中NDV相关毒株序列比较分析,结果表明,所分离的6个分离株归属为3个NDV基因型,其中有基因VIId亚型(3株)、基因VI型(2株)和基因Ⅱ型(1株).这6株NDV的F蛋白裂解位点的氨基酸序列均为112R-R-Q-K-R-F117,属于NDV强毒株典型序列.