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 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 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.     

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.     

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。     

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.     

2003年～2006年东北地区新城疫病毒部分分离株分子遗传学特征

本研究从2003年-2006年我国东北地区收集的疑似新城疫病毒感染的鸡源病料中,分离鉴定出12株可在鸡胚成纤维细胞上产生病变的新城疫病毒,并对其进行了蚀斑纯化。应用RT-PCR方法扩增含病毒融合蛋白基因47nt～420nt片段,并进行序列测定及分析。结果表明,12株NDV分离株F蛋白裂解位点序列均为^112R-R-Q-K-R-F^117。重要中和位点、糖基化位点及半胱氨酸残基高度保守。与GenBank中30株NDV参考毒株F基因序列比较和分析后,发现12株NDV分离株均为基因VIId亚型毒株,为我国目前优势流行毒株;与不同地区及宿主来源的参考毒株比较的结果表明本次分离于东北地区的毒株没有明显宿主及地域特征。     

Characterization of Newcastle disease viruses isolated from chicken,gamefowl, pigeon and quail in Mexico

Velogenic Newcastle disease has threatened the Mexican poultry industry since 1946. Seven strains of velogenic Newcastle disease virus were isolated from poultry and other avian species in central and northern Mexico from 1998 to 2006 and subjected to phylogenetic analysis and biological characterization using standard pathogenicity tests and challenge studies. Phylogenetic analysis showed that all velogenic strains belonged to genetic group V and are clearly divided in two lineages, since phylogenetic similarities between groups are of only 93–94%. Isolates from 1998 to 2001 are closely related to the strain responsible for the 2000 year outbreak raised in La Laguna region (Torreon strain), and are phylogenetically distinct from viruses isolated between 2004 and 2006 that are genetically related to the Chimalhuacan strain isolated in 1973. All the viruses of both, the Chimalhuacan and the Torreon groups, contained a virulent fusion protein cleavage site represented by the motif “GGRRQKRF”, revealing that evolutionary changes occurred at a different site. Chicken embryo mean death time value was shorter for the Chimalhuacan-like viruses (43.9 hours), when compared with the 1998–2001 average (54.3 hours). ICPI average value was higher (1.92) for viruses isolated during 2004–2006 than that for viruses isolated before 2001 (1.74). Microscopic evaluation of bursa of Fabricius and thymus of 5w-o broiler chickens challenged with 10⁶ LD₅₀/0.2 ml showed that Chimalhuacan-like isolate caused more severe lesions at 48 hpi in bursa and 72 and 96 hpi in thymus than Torreon-like isolate. Along with the MDT, ICPI and microscopic results, our findings suggest that some distinct selective pressure on the very virulent Chimalhuacan strain isolated in early 1970’s may have led to the appearance of the still velogenic but less virulent new group (Torreon-like) in the middle of 1990’s.     

一株鸽源新城疫病毒主要毒力基因分析

对2008年从广州市白云区某发病信鸽养殖场分离鉴定的一株鸽源新城疫病毒(简称CP-GD-2008),运用RT-PCR方法扩增出该病毒的F和HN基因的ORF序列,并进行序列测定。经序列对比、进化分析发现,CP-GD-2008属于基因Ⅵb亚型毒株,F蛋白具有强毒株裂解位点序列112R-R-Q-K-R-F117。该毒株与欧洲流行的鸽源基因Ⅵb亚型毒株进化关系密切,表明该毒株与欧洲分离株可能存在共同来源。对F和HN蛋白功能研究发现,除HN蛋白第347位抗原位点由E突变为G外,F和HN蛋白的中和位点、糖基化位点及半胱氨酸残基高度保守。     

蛋鸡新城疫病毒的分离与初步鉴定

采用鸡胚接种法从河北一些蛋鸡场分离到3株病毒,分别标号为WJ1、WJF2和WJF3.经血凝、血凝抑制试验及血清学中和试验鉴定3株分离毒株均为新城疫病毒;鸡胚半数致死量测定,WJ1株毒价为107.3ELD50/0.1 mL,WJ2株和WJ3株毒价分别为107.9ELD50/0.1 mL.动物回归试验鸡的临床表现与自然病例基本一致.再用分离毒攻ND Ⅳ系疫苗免疫鸡,显示ND Ⅳ系疫苗可以保护WJ1和WJ3分离毒株,而不能保护WJ2分离毒株.     

我国部分地区新城疫病毒分离株生物学特性的研究

从北京、昌黎、广州、长春、四平、青岛等地区采集疑似新城疫病鸡的病料,通过鸡胚接种分离出6株病毒,经血凝、血凝抑制试验和形态学观察,鉴定为新城疫病毒（NDV）,分别命名为北京株、昌黎株、广州株、长春株、四平株、青岛株。测定了这6株NDV的致死鸡胚的平均时间（MDT）和脑内致病指数（ICPI）,MDT分别为58．2,64．5,55．2,96。0．61．2和57．9h,ICPI分别为1．66,1．45,1．60,0．69,1．45和1．65。由这两项指标判定北京株、广州株、青岛株为强毒株,昌黎株和四平株为中强毒株,长春株为弱毒株。此外,还测定了6株NDV的血凝谱,各毒株均能凝集鸡和人（O型血）的红细胞,对猪、山羊、绵羊、牛和马的红细胞凝集作用有差异。     

从种蛋中分离的新城疫病毒抗原性及其F和HN基因的分析

对3株首次从种蛋中分离到的NDV进行交叉HI试验表明,这3株分离毒的交叉HI同源性为98.1%～100%。对这3株病毒及另1株从输卵管分离到的NDV的F与HN完整基因进行扩增并克隆测序,序列结果登陆GenBank(FJ011441～FJ011448)。氨基酸同源性分析表明:这4个毒株的F与HN基因同源性分别为99.5%～99.8%和99.6%～100%,远高于其与Lasota、F48E8株以及目前国内流行的其他基因Ⅶ型毒株的同源性。分离株F基因裂解位点的氨基酸序列为112R-R-Q-K-R-F117,符合NDV强毒株特征,根据绘制的遗传进化树分析,属于基因Ⅶ型。研究结果显示这4个不同来源毒株其2个基因的同步高度同源性可能与它们均是与生殖道感染有关。     

Determination of minimum hemagglutinin units in an inactivated Newcastle disease virus vaccine for clinical protection of chickens from exotic Newcastle disease virus challenge

The potency of inactivated Newcastle disease virus (NDV) vaccines in the United States is currently determined using vaccination and challenge of experimental animals against a velogenic strain of NDV. Because velogenic strains of NDV are now classified as select agents in the United States, all vaccine potency testing must be performed in live animals under biosafety level 3 agriculture conditions. If the minimum amount of inactivated viral antigen required for clinical protection can be determined using other methods, vaccines meeting these criteria might be considered of adequate potency. The linearity of correlation between the hemagglutination (HA) assay measurement and the 50% embryo infectious dose titer ofNDV Hitchner B1 vaccine virus was determined. Correlation between hemagglutinin units (HAU) per vaccine dose, clinical protection, and antibody response was then determined using a vaccinate-and-challenge model similar to Chapter 9 of the U.S. code of federal regulations approved method for vaccine potency testing. The dose providing 50% protection of an in-house water-in-oil emulsion vaccine formulated with inactivated NDV B1 was determined to be between 400 and 600 HAU from two separate trials. A positive correlation (R2 = 0.97) was observed between antibody response and HAU per vaccine dose. Serum antibody responses from vaccinated birds indicate HA inhibition titers >2(5) log2 would provide 100% protection from morbidity and mortality and require a minimum protective dose of 1000 HAU per bird. These are the first studies to examine establishing both a minimum protective HAU content for inactivated ND vaccines and a minimum serologic response necessary to ensure potency.     

一株鸽新城疫病毒的分离鉴定

对广东地区疑似发生鸽新城疫感染的鸽病料进行病毒分离,通过血凝试验(HA)、中和试验、F基因扩增及序列测定.结果分离到1株血凝效价为4log2,且能被NDV阳性血清中和的病毒;用针对NDV F基因设计的特异性鉴定引物对该分离株进行PCR扩增,可扩增出相应的目的片段;测序及Blast分析表 明其与山东分离株chicken/...     

3株新城疫山东分离株HN基因全序列测定和分析

用设计的特异性引物,通过RT-PCR法对1999和2004年间分离自山东省的具有一定代表性的3株鸡新城疫病毒(ShD-2-04,ShD-3-99,ShD-5-04)进行HN基因的扩增和克隆,并对其进行核苷酸序列测定和分析。结果表明:3个毒株HN基因开放性阅读框架(ORF)均为1716bp,编码571个氨基酸,属强毒的C群;3株山东分离毒的核苷酸同源性为98.2%~98.8%,氨基酸同源性为98.4%~98.8%,其中ShD-2-04和ShD-5-04核苷酸及氨基酸同源性均为最高,达到99.8%和99.8%;将3株山东分离毒株与国内外标准强毒株及弱毒株的HN基因核苷酸及氨基酸同源性分别进行比较:3毒株与国内外标准毒株HN的核苷酸同源性为82.7%~87.2%,氨基酸同源性为87.6%~90.0%,表明已发生一定的变异。     

Molecular characterization and phylogenetic analysis of new Newcastle disease virus isolates from the mainland of China

Seventy-nine velogenic Newcastle disease virus (NDV) isolates were obtained from infected chicken flocks during the outbreaks of Newcastle disease (ND) in various regions of the mainland of China in 2006. The F gene fragment (535 bp, from nt 47 to 581 of the F gene) which codes the main functional region of the F protein was obtained by RT-PCR and sequenced. All sequences obtained in this study have been submitted to GenBank. All the isolates have the motif ¹¹²R-R-Q/R-K/R-R-F¹¹⁷ at the cleavage site of the fusion protein, which is typical of velogenic NDV isolates. For genotyping, a phylogenetic tree based on nucleotides 47–435 of the F gene was constructed, and the 79 isolates could be divided into two genotypes, namely VIId and III. Most of the isolates proved to be of genotype VIId; only two isolates were of genotype III. Genotype VIId NDV has been the predominant pathogen responsible for most Newcastle disease outbreaks in China. The proportion of isolates of genotype VIId NDV shows an increasing trend, according to studies on the molecular epidemiology of NDV in China from 2002 to 2006.