Newcastle disease virus isolated from recent outbreaks in Taiwan phylogenetically related to viruses (genotype VII) from recent outbreaks in western Europe

Three major outbreaks of Newcastle disease (ND) occurred in Taiwan in the last three decades (in 1969, 1984, and 1995). Newcastle disease viruses (NDVs) isolated in the three outbreaks, together with those isolated in 1998, were sequenced between nucleotides 47 and 435 of the fusion gene. A phylogenetic tree based on sequences obtained showed that the NDV isolated in 1969 was similar to the genotype III viruses. In contrast, all isolates in 1984 and seven of the eight isolates in 1995, together with all isolates in 1998, fell into the genotype VII. These results suggest that the 1969 outbreak of ND in Taiwan was caused by the genotype III virus, whereas the 1984 and 1995 outbreaks were caused by the genotype VII viruses. To date, the genotype VII viruses have caused many outbreaks in east Asia and western Europe. We suspect that these outbreaks have constituted the fourth panzootic of ND, which is distinct from the third panzootic caused by the "pigeon PMV-1 viruses." NDV isolated in Taiwan in 1984 was the earliest isolation of the genotype VII virus.     

Molecular epidemiological investigation of Newcastle disease virus from domestic ducks in Korea

To expand the epidemiological understanding of Newcastle disease virus (NDV) found in domestic ducks in Korea, 14 NDV isolates from apparently healthy domestic ducks were biologically and genetically characterized. Thirteen and 1 isolates of NDV were categorized into lentogenic and velogenic viruses, respectively, based on in vivo pathogenicity tests. Twelve lentogenic viruses showed HA activity to horse RBCs, while 1 lentogenic virus and the velogenic virus were negative. Lentogenic viruses (n=13) had sequence motifs of (112)ERQERL(117) (n=1) or (112)GRQGRL(117) (n=12) at the F0 cleavage site, while the velogenic virus (n=1) had a sequence motif of (112)RRQKRF(117) at the same site. Phylogenetic analysis revealed that at least three distinct genotypes may exist in domestic ducks in Korea; one class I genotype (genotype 2), and two class II (genotypes I and VII) genotypes. The class I virus was most closely related to strains of genotype 2 which were isolated in birds from the USA, Germany and Denmark. Twelve lentogenic class II viruses were grouped together in genotype I, and were then divided into at least three clusters, namely Aomori-like, Ulster2C-like, and V4-like. The velogenic class II virus was assigned to genotype VII which represents viruses responsible for recent epidemics in many Asian countries including Korea. The epidemiological importance of domestic duck isolates of NDV in Korea is discussed.     

疫苗免疫压力下新城疫病毒的动态演化

国内大量新城疫病毒(NDV)血凝素基因F和HN的测序表明,NDV主要免疫原HN和F基因与生产中广泛应用的经典疫苗La Sota株的核苷酸同源性不足80%,而NDV流行株之间则高达94.4%～100%,从分子遗传学角度证实了VIId型NDV是导致新城疫免疫失败的重要原因。HI交叉抑制和鸡胚中和试验等则从抗原性的角度证实了NDV在免疫压力下抗原性的变化,尽管NDV不同毒株间抗原性变异的程度已出现明显差异,但仍局限在同一个血清型中。     

Characterisation of genotype VII Newcastle disease virus (NDV) isolated from NDV vaccinated chickens,and the efficacy of LaSota and recombinant genotype VII vaccines against challenge with velogenic NDV

A Newcastle disease virus (NDV) isolate designated IBS002 was isolated from a commercial broiler farm in Malaysia. The virus was characterised as a virulent strain based on the multiple basic amino acid motif of the fusion (F) cleavage site ¹¹²RRRKGF¹¹⁷ and length of the C-terminus extension of the hemagglutinin-neuraminidase (HN) gene. Furthermore, IBS002 was classified as a velogenic NDV with mean death time (MDT) of 51.2 h and intracerebral pathogenicity index (ICPI) of 1.76. A genetic distance analysis based on the full-length F and HN genes showed that both velogenic viruses used in this study, genotype VII NDV isolate IBS002 and genotype VIII NDV isolate AF2240-I, had high genetic variations with genotype II LaSota vaccine. In this study, the protection efficacy of the recombinant genotype VII NDV inactivated vaccine was also evaluated when added to an existing commercial vaccination program against challenge with velogenic NDV IBS002 and NDV AF2240-I in commercial broilers. The results indicated that both LaSota and recombinant genotype VII vaccines offered full protection against challenge with AF2240-I. However, the LaSota vaccine only conferred partial protection against IBS002. In addition, significantly reduced viral shedding was observed in the recombinant genotype VII-vaccinated chickens compared to LaSota-vaccinated chickens.     

Molecular characterization of Newcastle disease viruses in Ostriches (Struthio camelus L.): further evidences of recombination within avian paramyxovirus type 1

Newcastle disease virus (NDV) strains isolated from ostriches have been genotyped for the first time by partial sequencing of the F gene to determine the epidemiologic role that this species can play within ND outbreaks. Fifteen additional NDV strains, mostly isolated from chickens but also from pigeons and penguins, were also included in the study to determine genetic relationships with ostriches NDV isolates. High genetic diversity was demonstrated in ostrich NDV isolates, as the 10 isolates were grouped in four distinct NDV genotypes. In agreement with the results obtained when chicken isolates have been molecularly characterized, the predominant genotype in ostriches was the genotype VII. More interestingly, evidences of recombination between genotype II and VII were observed in one ostrich isolate and in two further chicken isolates. Therefore, it seems that ostriches may play a relevant role in the ecology and epidemiology of ND particularly in those regions where they have an increasing farming importance as minor poultry species.     

2006～2008年华东地区新城疫病毒HN基因序列分析

为研究新城疫病毒（Newcastle disease virus,NDV）的HN（血凝素-神经氨酸酶,hemagglutinin—neuraminidase）基因分子流行病学规律,利用RT—PCR扩增了2006～2008年间分离自我国华东地区的43株NDV的HN基因片段。基因序列分析显示,分离株中基因Ⅰ型2株,基因Ⅱ型3株,基因Ⅵ型1株,其它37株都属于基因Ⅶd亚型。根据遗传发生进化树可将基因Ⅶd亚型进一步分为Ⅶd1和Ⅶd2两个亚型,基因Ⅶd1亚型HN蛋白的第102、118、443位氨基酸分别为T,A和T,而基因Ⅶd2亚型则分别为I,E和M。另外,研究表明HN蛋白线性表位发生E347K突变的变异株的分离率在我国呈上升趋势。     

新城疫病毒贵州不同鸡源分离株F基因的克隆与序列分析

应用PCR技术扩增NDV贵州不同鸡源分离株,包括肉鸡源P1株与BY株、蛋鸡源H2株与FW株、七彩山鸡源N98株和越南斗鸡源DQ株的F蛋白基因,将该基因片段分别进行克隆和测序,并与国内外NDV参考株的对应序列进行比较分析。结果表明:6株NDV贵州不同鸡源分离株的F基因长度均为1 662 bp,编码553个氨基酸;分离株间核苷酸同源性为86.9%~99.7%,氨基酸同源性为91.0%~99.3%;与国内外NDV代表株(LaSota株、B1株、F48E9株、CH2000株和TW 2000株)的核苷酸同源性为84.0%~98.9%,氨基酸同源性为87.2%~98.6%;经系统发育进化树分析,DQ株、N98株、P1株和H2株为基因VIId型,而BY株和FW株为基因IX型。这些结果提示贵州省不同鸡群间存在相同NDV毒株感染的可能性,不同年份间NDV毒株发生基因型改变,而近年来贵州省流行的ND疫情主要是由NDV基因VII型引起。     

Molecular characterization of Newcastle disease viruses isolated from rural chicken in northwest Ethiopia reveals the circulation of three distinct genotypes in the country

Newcastle disease (ND) is a highly contagious disease that affects many species of birds and causes significant economic losses to the poultry industry worldwide. Fifteen Newcastle disease virus (NDV) isolates obtained from rural chickens in northwest Ethiopia in 2011 and 2012 were characterized genotypically. The main functional region of the F gene was amplified and sequenced (260 nucleotides). Among the Ethiopian NDV isolates, 2 isolates had the virulent motif ¹¹²R-R-Q-K-R-F¹¹⁷ at the cleavage site of the fusion protein while 13 isolates contained the lentogenic motif ¹¹²G-G/R-Q-G-R-L¹¹⁷. Phylogenetic analysis based on the variable region of the F gene indicated that the two isolates exhibiting the virulent motif belonged to lineage 5 (genotype VII) subgenotype d and the remaining 13 isolates were grouped into lineage 2 (genotype II). The nucleotide sequences of lineage 5 isolates were genetically related to the Sudanese NDV isolates, suggesting potential epidemiological link of ND outbreaks between neighbouring countries. The lentogenic strains shared similarities with La Sota vaccine strain and probably originated from the vaccine strain either through direct exposure of birds to the live vaccine or to infectious La Sota-like strains circulating in rural poultry. This study provides genetic evidence on the existence of different NDV genotypes circulating in the rural poultry in Ethiopia. The virulent NDV continues to be a problem in poultry sector in Ethiopia, and their continuous circulation in rural and commercial poultry calls for improved surveillance and intensified vaccination and other control measures.     

鸡新城疫病毒河北分离株F基因的分子特性分析

通过毒力测定、RT-PCR及F基因的序列测定与遗传进化分析,对2005-2008年从河北省部分地区的发病鸡群中分离到的10株新城疫病毒（NDV）进行了研究。各分离株经典毒力测定结果显示：MDT在37.6～54.4h之间,ICPI在1.71～2.0之间,IVPI值在2.16～2.8之间,均为新城疫病毒强毒株特征。F基因的序列测定表明,分离株之间的核苷酸序列具有77.4%～98.0%的同源性,与疫苗株Lasota的同源性为87.0%～98.9%,与国内标准强毒株F48E9同源性为89.7%～98.9%。推导其氨基酸序列分析表明,8个分离株的F蛋白的裂解位点氨基酸组成为112 R-R-Q-K-R-F117,具有NDV强毒株特征,与毒力测定结果相符,2个分离株的F蛋白的裂解位点氨基酸组成为112 G-R-Q-G-R-L117,与弱毒株特征相符。F基因分型和同源性比较显示：目前河北新城疫的流行以基因Ⅶ型为主（占70%）,同时兼有基因Ⅱ型（占20%）和基因Ⅸ型（占10%）。     

Hemagglutinin-neuraminidase gene of genotype VII Newcastle disease virus strains isolated in Japan

Recently, genotype VII of Newcastle disease virus (NDV) has become the most prevalent NDV genotype in Asia. Here the hemagglutinin-neuraminidase (HN) gene of genotype VII NDV strains isolated in Japan was analyzed. Notably, point amino acid substitutions in the HN protein at position 347, which is located on the major linear epitope of the HN protein, were found in two strains. However, by a hemagglutination inhibition assay, major antigenic differences did not exist between the studied strains. Additionally, chickens vaccinated with the B1 strain did not exhibit clinical effects after challenge with variants possessing the substitution at position 347 (E to K), whereas all unvaccinated chickens subjected to this challenge died within 5 days.     

Phylogenetic Analysis of HN Gene of Eight Pigeon NDV Isolates in Guangxi

[Objective] The paper was to provide a basis for scientific prevention and control of pigeon Newcastle disease(ND).[Method] The HN gene of eight pigeon NDV strains isolated from different pigeon farms in Guangxi were amplified by RT-PCR,sequenced and analyzed.The molecular evolution characteristics of HN gene of pigeon NDV isolates in Guangxi was discussed.[Result] The nucleotide sequence length of HN gene of the eight NDV isolates was 1 716 bp,encoding 571 amino acids.They belonged to virulent group C,and the gene length characteristic of HN gene accorded with virulent strain.Analysis of nucleotide homologies indicated that the eight NDV isolates shared higher homology with genotype VIb,ranging from 90.4% to 99.5%.Phylogenetic tree analysis demonstrated that the genetic relationship between the eight NDV strains in Gangxi and the NDV isolates from Guangxi,Guangdong,Jilin,Liaoning,Yunnan and Heilongjiang during 2011 and 2013 was close.They were located in the same cladogram branch.[Conclusion] We assume that the eight pigeon NDV isolates in Guangxi all belong to the gene class II genotype VI b NDV.     

2018年广西活禽市场新城疫病毒分子流行病学分析

为跟踪调查广西活禽市场新城疫病毒(Newcastle disease virus,NDV)的流行分布和分子演化特征,2018年在广西不同城市10个活禽市场随机采集鸡、鸭、鹅和鸽子泄殖腔和咽喉拭子样品2820份,通过病毒分离与F基因扩增和测序,分析其遗传进化特征。结果显示:从4种宿主中共分离得到54份NDV,包含CLASSⅠ基因1b亚型、CLASSⅡ基因Ⅰ型和CLASSⅡ基因Ⅻ型三种基因型,且发现同一宿主存在不同基因型混合感染的情况。CLASSⅠ基因1b亚型NDV占比最大(50/54,92.5%)。遗传进化分析显示,广西分离株与同基因型参考株处于不同的小分支,存在地域差异。     

Surveillance and characterization of Newcastle disease viruses isolated from northern pintail (Anas acuta) in Japan during 2006-09

A total of 38 Newcastle disease virus (NDV) isolates were obtained from 6060 fecal samples from northern pintail (Anas acuta) ducks collected in the Tohoku district in Japan during 2006-09. One isolate from each sampling location and date was selected for a total of 38 isolates, then 15 of these were characterized for their pathogenicity by mean death time of minimum lethal dose (MDT/MLD) using chicken embryos and by plaque formation on chicken embryo fibroblasts. Furthermore, nine isolates were randomly selected from these 15 isolates, and the fusion protein genes were sequenced to characterize amino acid sequences around the cleavage site. All 15 were confirmed to be nonvirulent by MDT/MLD test, and nine isolates were also confirmed as nonvirulent by the cleavage site of the fusion protein 112G/E-K/R-Q-G/E-R*L117 that was specific for nonvirulent NDVs. The characteristics of nine isolates identified by phylogenic analysis of the fusion protein gene indicated that the isolates belong to genotype I or II. In addition, we also isolated 68 avian influenza viruses and 28 other hemagglutinating viruses. Our data indicate that northern pintails are subclinically infected by, perpetuate, and distribute NDV along with different subtypes of avian influenza viruses and other hemagglutinating viruses during their migrations across vast areas over the Northern Hemisphere to Japan.     

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.     

Identification of a variable epitope on the Newcastle disease virus hemagglutinin–neuraminidase protein

Fifteen virulent Newcastle disease viruses (NDVs) were isolated from diseased birds in Eastern China in 2005. To investigate the antigenic variation in the epitopes on NDV hemagglutinin–neuraminidase (HN) protein, these isolates, together with six reference strains, were subjected to the hemagglutination inhibition (HI) tests using five HI-positive monoclonal antibodies (MAbs) against velogenic NDV strain ZJ1. The MAbs 2G5, 3A4, 3B5 and 6B1 recognized 12 of the 15 NDV isolates, and exhibited HI activity towards the six reference strains. However, these MAbs did not react with three local isolates, JS-02/05, JS-06/05 and JS-10/05. HN gene sequence analysis of all NDV strains revealed that these MAb-resistant NDV isolates possessed residue K at position 347 of the HN protein, whereas all remaining strains possessed E or G at the same site. To determine the contribution of the residue at position 347 to antigenic epitope formation, we generated by reverse genetics two recombinant viruses, ZJ1HNK with an E347K mutation on ZJ1 HN, and JSHNE with a K347E mutation on JS-06/05 HN. The HI test demonstrated that ZJ1HNK lost reactivity with MAbs 2G5, 3A4, 3B5 and 6B1, whereas JSHNE did react with these MAbs. Further verification by immunofluorescent assay demonstrated that residue 347 was a critical determinant for formation of the antigenic epitope (residues 345–353) on the HN protein.     

一株新疆野鸟源新城疫病毒的分离鉴定、F基因克隆及遗传进化分析

为了解新疆野鸟新城疫病毒（Newcastle disease virus,NDV）感染情况,分析其分子特征和基因变异特点,防止新城疫的暴发和蔓延,本试验用9日龄SPF鸡胚进行病毒的分离传代,HA、HI试验和RT-PCR方法对位于"东非-西亚迁徙线"福海县的野鸟进行了NDV检测,分离到1株野鸟源NDV,并命名为NDV/Pintail/CH（XJ）/01/2016。结果表明,该NDV分离株F基因ORF长1 662 bp,编码553个氨基酸,F基因碱性裂解位点序列为¹¹²G-R-Q-G-R-L¹¹⁷,符合弱毒株特征;遗传进化分析显示,NDV/Pintail/CH（XJ）/01/2016与乌克兰鸽源分离株Doneck/3/968、比利时鸭源分离株Simeonovgrad核苷酸同源性均为99.7%,属于NDV ClassⅡ基因Ⅱ型。而上述3株病毒均与疫苗毒La Sota具有较高同源性（99.5%~99.8%）。本研究结果为家禽NDV外流进入自然环境提供了论据。     

Phylogenetic analysis of Newcastle disease viruses isolated from wild birds in the Poyang Lake region of China

Newcastle disease virus (NDV) causes a highly contagious viral disease in poultry and wild birds, and it can cause significant economic loss worldwide. Eight viral strains were isolated by inoculating embryonated chicken eggs from the Poyang Lake region of China with swab samples. All eight of the NDV isolates were identified as class I genotype 3 strains, but they diverged notablely from class II viruses. Further analysis revealed that all eight NDV isolates were lentogenic strains containing the ¹¹²ERQER↓L¹¹⁷ motif at the F protein cleavage site. The strains were highly identical and were more species specific (chicken and waterfowl) than site specific (Nanchang and Duchang regions). The close phylogenetic proximity of these isolates indicates that viral transmission may happen between poultry and wild birds. Our study demonstrates that lentogenic class I NDVs exist in clinically healthy wild waterfowl and poultry within the Poyang Lake region. Active surveillance of these viruses to determine their evolution and origin is one of the most realistic strategies for preventing and controlling NDV outbreaks.     

我国部分地区ＮＤＶ的分子流行病学研究

本研究根据新城疫病毒（ＮＤＶ）Ｆ基因编码区１－３７４位核苷酸序列计算其遗传距离并给出了ＮＤＶ的系统发育进化树，将６８株ＮＤＶ分为９个基因型（３０株为国内分离株），其中Ⅰ－Ⅵ是早已存在的老基因型，Ⅶ、Ⅷ、Ⅸ为新发现的基因型，特别是Ⅸ为我国特有的基因型（Ｆ４８ＥＯ、Ｍ３、ＨＬＪ－３、ＨeB-1P和ＮＭ－５）。１９９７－１９９９年我国云南、广西、甘肃、陕西、新疆等地分离的ＹＮ－１Ｐ、ＧＸ－３、Ｈ１、Ｈ２、Ｐ１、ＧＸ－１、ＧＸ－２、ＧＳ－３、ＳＨＸ－２、ＳＨＸ－３、ＳＨＸ－６、ＳＨＸ－７、ＸＪ－２和１９９１年分离的ＨuB-1均属于Ⅶ基因型，该基因型的病毒是９０年以来引起新城疫发生的主要病原。根据遗传距离和分离年代可将此基因型进一步划分为５个基因亚型，分别是Ⅶa、Ⅶb、Ⅶc、Ⅶd和Ⅶe。此外ＨuN-1/98、HLJ-4/95和ＨeH-1P属一个老的基因Ⅵ，１９７９－１９８５年分离自青海的ＱＨ－１、ＱＨ－２、ＱＨ－４属于一个新的基因型－Ⅷ型。可见在我国新城疫的流行是极其复杂的，既有老基因型的危害（Ⅰ－Ⅵ），又有新基因型（Ⅶ）的流行，更有我国独特Ⅷ和Ⅸ基因潜伏。