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.     

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。     

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.     

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.     

ClassⅠ新城疫病毒概述

新城疫病毒是影响养禽业健康发展的主要病原之一。新城疫病毒只有一个血清型,但根据亲源关系可以划分为ClassⅠ和ClassⅡ两大类。ClassⅠ新城疫病毒于2006年被证实存在,当前可被细分为9个基因型(1～9)。ClassⅠ所有自然分离株均为无毒株或弱毒株,但有经人工传代返强的报道,由于其分布广泛,数量庞大,对养禽业有巨大的潜在威胁。为了更好的了解ClassⅠ新城疫毒株的生物学特性,为研究ClassⅠ新城疫病毒提供参考,现将其基因组结构、分子流行病学及检测方法等综述如下。     

半番鸭源禽1型副粘病毒FM01株的分离鉴定与F蛋白基因分析

从表现类似新城疫症状的病死半番鸭中分离到1株病毒FM01株，经血凝及血凝抑制试验证实为禽1型副粘病毒、以SPF鸡胚测定其平均致死时间为113，4h，对1d雏鸡脑内接种致病指数为0．23，表明为温和型毒株。利用RT-PCR技术一次性扩增其F蛋白全基因，克隆到pMD 18-T质粒载体，测序后获得F基因全序列，并推导出其相应的氨基酸序列。FM01株的F蛋白基因完整的编码区全长1662bp，编码553个氨基酸，其裂解位点的氨基酸序列为112G-R-Q-G-R-L117，具有温和型毒株特有的氨基酸序列结构．与常见新城疫毒株的核苷酸同源性在88．4％～99.6％之间，氨基酸同源性在89．2％～99.1％之间。     

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.     

1株鸭源Ⅰ类新城疫病毒分离株F和HN基因的分子特性分析

采用RT-PCR技术对Ⅰ类新城疫病毒(NDV)09-014分离株完整的融合蛋白(F)基因和血凝素-神经氨酸酶(HN)基因进行了扩增和遗传进化分析。F基因的序列测定结果表明:该分离株F基因全长为1 792 bp,可编码553个氨基酸,裂解位点的氨基酸组成为112E-R-Q-E-R-L117,具有典型的新城疫弱毒株特征。同源性分析表明本分离株的F基因与Ⅰ类新城疫病毒代表毒株之间核苷酸的同源性为93%～95.2%,而与Ⅱ类新城疫病毒代表毒株的同源性较低,介于70.6%～72.4%。HN基因的序列测定结果表明:HN基因全长2 001 bp,可编码616个氨基酸,同源性分析表明本分离株的HN基因与Ⅰ类新城疫病毒代表毒株之间核苷酸的同源性在92.7%～94.7%之间,而与Ⅱ类新城疫病毒同源性较低,为70.7%～71.5%。根据完整的F基因和HN基因构建的遗传进化树均表明:本分离株在分类地位上属于Ⅰ类新城疫病毒基因3型,因此Ⅰ类新城疫病毒的F基因和HN基因具有相似的进化速率。     

华东地区健康水禽携带新城疫病毒情况调查

近年来,新城疫逐渐成为了一种严重的家养水禽疾病。尽管如此,目前对于我国水禽新城疫的流行和分布情况依然知之甚少。本研究采集了2011-2012年中国华东6省1市的部分活禽市场和养殖场表观健康家鸭和家鹅泄殖腔棉拭样品,进行新城疫病毒的分离和毒力检测工作。通过测定分离株F基因高变区nt47-420,对其进行遗传进化分析。结果显示,在1200份样品中,检测到阳性样品23份,分离率为1．92％,表明眼观健康家鸭和家鹅中携带新城疫病毒。对比寒冷的春冬两季和炎热的夏季,新城疫病毒的分离率存在明显的差别。在中国家鸭和鹅中流行的新城疫毒株依然对温度敏感,高温天气不利于新城疫疫情蔓延。对分离株的鸡胚平均死亡时间和脑内接种指数进行了测定。参考76株GenBank中已发表并鉴定基因型毒株的F基因高变区nt47～420,对23株家养水禽分离株进行了遗传进化分析,结果发现分离株与流行的强毒株和疫苗株遗传距离较远。其中,13株与ClassII基因Ib亚型遗传距离最近;3株属于ClassI基因2型;7株与ClassI基因3b亚型遗传距离最近。     

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%）。     

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.     

Genetic relationship of H3 subtype avian influenza viruses isolated from domestic ducks and wild birds in Korea and their pathogenic potential in chickens and ducks

The H3 subtype avian influenza virus (AIV) is one of the most frequently isolated subtypes in domestic ducks, live poultry markets, and wild birds in Korea. In 2002-2009, a total of 45 H3 subtype AIVs were isolated from the feces of clinically normal domestic ducks (n=28) and wild birds (n=17). The most prevalent subtypes in domestic ducks were H3N2 (35.7%), H3N6 (35.7%), H3N8 (25.0%), and H3N1 (3.6%, novel subtype in domestic duck in Korea). In contrast, H3N8 (70.6%) is the most prevalent subtype in wild birds in Korea. In the phylogenetic analysis, HA genes of the Korean H3 AIVs were divided into 3 groups (Korean duck, wild bird 1, and wild bird 2) and all viruses of duck origin except one were clustered in a single group. However, other genes showed extensive diversity and at least 17 genotypes were circulating in domestic ducks in Korea. When the analysis expanded to viruses of wild bird origin, the genetic diversity of Korean H3 AIVs became more complicated. Extensive reassortments may have occurred in H3 subtype influenza viruses in Korea. When we inoculated chickens and ducks with six selected viruses, some of the viruses replicated efficiently without pre-adaptation and shed a significant amount of viruses through oropharyngeal and cloacal routes. This raised concerns that H3 subtype AIV could be a new subtype in chickens in Korea. Continuous surveillance is needed to prepare the advent of a novel subtype AIV in Korea.     

新城疫病毒西藏分离株的生物学特性鉴定及遗传进化分析

从西藏病死藏鸡中分离到具有血凝活性的病毒、经血凝抑制试验、电镜观察、PCR扩增和测序鉴定为新城疫病病毒(NDV),通过动物致病性试验证明该病毒对鸡具有致病性;分离毒株毒力测定结果显示,MDT为120h,EID50为10-8.44、IVPI为0.5、ICPI为0.6,均符合NDV弱毒株特征。血凝解脱及血凝素热稳定性试验显示:各分离株的血凝解脱时间短,血凝素热稳定性较差,符合NDV弱毒株的特征。F基因的序列测定遗传进化分析表明,西藏分离毒株之间的核苷酸序列具有99%的同源性,与疫苗株LaSota的同源性为90%;与国内标准强毒株F48E8同源性为81%。推导其氨基酸序列分析表明,各分离株的F蛋白的裂解位点氨基酸112 G-K-Q-G-R-L117,具有NDV弱毒株特征,与毒力测定结果相符。本研究首次报道了NDV西藏分离毒株遗传进化情况和生物学特性情况,为进一步研究高海拔、缺氧环境下NDV生物学特性变化研究奠定了基础。     

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。     

Isolation and Identification,F Gene Cloning and Evolutionary Analysis of Newcastle Disease Virus Isolated from Wild Birds in Xinjiang

In order to understand inflection of Newcastle disease virus (NDV) of Xinjiang wild birds, through analysis of molecular characteristics and genetic variation, to prevent the outbreak and spread of Newcastle disease, isolation of the virus was performed in 9 days old specific pathogen free (SPF) chicken embryos, detected the wild birds in Fuhai county which was located in the migration line of East Africa-West Asia by HA, HI test and RT-PCR method. And then 1 strain of NDV from wild birds was isolated, named as NDV/Pintail/CH(XJ)/01/2016. As the result, ORF of F gene from the NDV isolate was 1 662 bp, encoding 553 amino acids. The motif of the cleavage site was ¹¹²G-R-Q-G-R-L¹¹⁷, which was consistent with the characteristics of avirulent NDV strains. The phylogenetic analysis showed that NDV/Pintail/CH(XJ)/01/2016 nucleotide sequence homologies of F gene between reference strain Doneck/3/968 and mule Simeonovgrad strain homology were high and reached 99.7%, it belonged to genotype Ⅱ of Class Ⅱ NDV. All of the three viruses had high homology to the vaccine strain La Sota,which were 99.5% to 99.8%. According to this research conclusion we provided evidence that poultry NDV outflowed into the natural environment.     

Pathogenicity and immunogenicity of different recombinant Newcastle disease virus clone 30 variants after in ovo vaccination

Even though Newcastle disease virus (NDV) live vaccine strains can be applied to 1-day-old chickens, they are pathogenic to chicken embryos when given in ovo 3 days before hatch. Based on the reverse genetics system, we modified recombinant NDV (rNDV) established from lentogenic vaccine strain Clone 30 by introducing specific mutations within the fusion (F) and hemagglutinin-neuraminidase (HN) proteins, which have recently been suggested as being responsible for attenuation of selected vaccine variants (Mast et al. Vaccine 24:1756-1765, 2006) resulting in rNDV49. Another recombinant (rNDVGu) was generated to correct sequence differences between rNDV and vaccine strain NDV Clone 30. Recombinant viruses rNDV, rNDV49, and rNDVGu have reduced virulence compared with NDV Clone 30, represented by lower intracerebral pathogenicity indices and elevated mean death time. After in ovo inoculation, hatchability was comparable for all infected groups. However, only one chicken from the NDV Clone 30 group survived a 21-day observation period; whereas, the survival rate of hatched chicks from groups receiving recombinant NDV was between 40% and 80%, with rNDVGu being the most pathogenic virus. Furthermore, recombinant viruses induced protection against challenge infection with virulent NDV 21 days post hatch. Differences in antibody response of recombinant viruses indicate that immunogenicity is correlated to virulence. In summary, our data show that point mutations can reduce virulence of NDV. However, alteration of specific amino acids in F and HN proteins of rNDV did not lead to further attenuation as indicated by their pathogenicity for chicken after in ovo inoculation.     

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。     

2005-2006年流行的致病性鹅新城疫病毒的分子流行病学特征

为了对能导致鹅临诊疾病的新城疫病毒(Newcastle disease virus,NDV)进行监测,2005-2006年从江苏、安徽2省不同地区的发病鹅群分离、鉴定了10株NDV,并对其中8株病毒的部分生物学特性及分子生物学特征进行了研究.鸡胚平均死亡时间(MDT)、1日龄雏鸡脑内接种致病指数(ICPI)测定结果表明8株病毒皆为NDV强毒株.对融合蛋白(F)和血凝素-神经氨酸酶(HN)基因序列的分析发现,8株病毒中有7株病毒在基因型分类上属于基因Ⅶd亚型,并与1997-2001年间流行的对鹅具有高度致病性的NDV高度同源(95.5%～99.8%),而且这7株病毒的F、HN蛋白具有出现于2001年以前流行的致病性鹅NDV的绝大部分特征性氨基酸,进一步说明这些特征性氨基酸非常保守,可以作为对鹅具有高度致病性的NDV的分子标志.