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。     

新城疫病毒人工感染鹅脾脏差异表达蛋白质组初步分析

脾脏是新城疫病毒(Newcastle disease virus,NDV)感染的重要靶器官,本试验旨在从分子水平上分析NDV与宿主之间的相互作用,探寻早期基因Ⅳ型强毒Herts/33和晚期基因Ⅶ型强毒JS5/05造成鹅脾脏病变差异的相关蛋白。30日龄非免疫鹅分别人工感染NDV强毒株Herts/33和JS5/05,并于感染后36、72、108h采集2个感染组和对照组的鹅脾脏,提取脾脏蛋白,以17cm、pH5～8的IPG胶条进行二维电泳,运用PDQuest 8.0.1软件对凝胶图谱进行差异蛋白分析。结果显示:与对照组相比,Herts/33感染组和JS5/05感染组脾脏组织分别有154个和148个蛋白出现了显著的差异表达,其中有86个蛋白点是不同感染组共有的差异点,包括52个感染后上调表达蛋白点,34个下调表达蛋白点;另外,有130个差异蛋白点为NDV感染鹅后不同毒株之间产生的差异表达,包括71个感染后上调表达蛋白点,59个下调表达蛋白点。基因Ⅳ型NDV强毒和基因Ⅶd亚型NDV强毒分别感染鹅后,能引起宿主脾脏组织蛋白表达谱发生不同的改变,这为进一步研究Ⅶd亚型NDV对水禽致病性增强的机制提供了重要线索。     

一株鸭源新城疫病毒毒力基因分析及其对鸭的致病性研究

为了解中国鸭源新城疫病毒(Newcastle disease virus,NDV)的毒力特点,从2009年广东地区发病鸭群中分离和鉴定出1株新城疫病毒(简称NDV-104),对其生物学特性、致病性和融合蛋白(fusion,F)、血凝素神经氨酸酶(hemagglutinin-neuraminidase,HN)基因进行了研究。结果显示,分离株的MDT、ICPI和IVPI分别为56.4 h、1.95和1.64,结合F蛋白裂解位点(112~117位)的氨基酸序列分析,确定了分离株为新城疫强毒。致病性结果表明,分离病毒对雏鸭具有感染性和致病性。F基因遗传进化结果显示,分离株属于基因Ⅶd亚型。F、HN蛋白的氨基酸同源性结果表明,分离株与2000年以来国内外分离到的基因Ⅶ型NDV同源性较高,分别为96.6%~99.3%和97.0%~99.7%,而其与常用疫苗株B1、V4、Clone30、Mukteswar和LaSota的F、HN蛋白氨基酸序列的同源性较低,且与LaSota株和V4株的同源性最低,分别仅为88.1%和87.6%,说明分离株与经典新城疫病毒毒株存在一定的差异。     

A survey of Newcastle disease in Swiss laying-hen flocks using serological testing and simulation modelling

Newcastle disease (ND) is a highly contagious viral disease of birds particularly domestic poultry. Switzerland is currently declared free from ND; since vaccination is prohibited, the detection of antibodies against ND virus (NDV) results in the destruction of the respective flock (stamping-out policy). However, in 1995 and 1996, antibody-positive flocks were detected and sporadic ND outbreaks even occurred in Switzerland. Therefore, a serosurvey was done to look for evidence of NDV infections in Swiss laying-hen flocks. The survey was designed to provide 95% confidence of detecting at least one seropositive flock if the flock prevalence were 1%. Thirty blood samples from each of 260 commercial laying-hen flocks were collected during 1996 in a central poultry slaughterhouse. Sera were screened for NDV antibodies with a commercial blocking enzyme-linked immunosorbent assay (ELISA). Samples with a questionable or positive test result were retested with the same ELISA. A stochastic computer model was applied to define a cut-off number of test-positive samples to help to differentiate between true- and false-positive flocks and to estimate the true flock prevalence of infection. Four flocks were identified as NDV-seropositive and the NDV true seroprevalence among commercial laying-hen flocks in Switzerland was most likely between 1.35 and 1.55%. This indicates that Swiss laying-hen and parental flocks with more than 150 animals have been in contact with strains of NDV that cause subclinical infection in chicken, because no clinical symptoms have been observed. In this context, computer simulation was a useful technique to interpret survey results.     

2008年江苏地区基因Ⅶd亚型新城疫病毒遗传变异分析

为了研究基因Ⅶ d亚型新城疫病毒(Newcastle disease virus,NDV)的分子流行病学及遗传变异规律,作者对2008年分离自江苏地区16株具有一定代表性NDV分离株的F和HN基因片段进行了扩增.根据F基因和HN基因序列绘制的2个遗传进化树基本一致,表明分离株中不存在囊膜糖蛋白基因发生重组的NDV;同时,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.基因Ⅶd亚型变异株的出现应引起相关领域从业者们的高度重视.     

近年来典型ND分离株的致病性和抗原性变异研究

8株ND分离株经过毒力测定(MDT小于47h；ICPI大于1．65)均属于NDV强毒，交互凝集抑制试验表明：分离株与C30和48株在HI效价上差1～3个滴度；攻毒试验表明不同毒株致病性明显不同；鸡胚中和试验则表明，La Sota单因子血清对分离株有中和作用，但中和滴度有明显差异。     

Coexpression of avian influenza virus H5 and N1 by recombinant Newcastle disease virus and the impact on immune response in chickens

To analyze the contribution of neuraminidase (NA) toward protection against avian influenza virus (AIV) infection, three different recombinant Newcastle disease viruses (NDVs) expressing hemagglutinin (HA) or NA, or both, of highly pathogenic avian influenza virus (HPAIV) were generated. The lentogenic NDV Clone 30 was used as backbone for the insertion of HA of HPAIV strain A/chicken/Vietnam/P41/05 (H5N1) and NA of HPAIV strain A/duck/Vietnam/TG24-01/05 (H5N1). The HA was inserted between the genes encoding NDV phosphoprotein (P) and matrixprotein (M), and the NA was inserted between the fusion (F) and hemagglutinin-neuraminidase protein (HN) genes, resulting in NDVH5VmPMN1FHN. Two additional recombinants were constructed carrying the HA gene between the NDV P and M genes (NDVH5VmPM) or the NA between F and HN (NDVN1FHN). All recombinants replicated well and stably expressed the HA gene, the NA gene, or both. Chickens immunized with NDVH5VmPMN1FHN or NDVH5VmPM were protected against two different HPAIV H5N1 and also against HPAIV H5N2. In contrast, immunization of chickens with NDVN1FHN induced NDV- and AIV N1-specific antibodies but did not protect the animals against a lethal dose of HPAIV H5N1. Furthermore, expression of AIV N1, in addition to AIV H5 by NDV, did not increase protection against HPAIV H5N1.     

人工感染鸭源新城疫病毒对绍鸭β-防御素和细胞因子基因表达的影响

试验旨在探讨人工感染鸭源新城疫病毒（Newcastle disease virus,NDV）对绍鸭β-防御素（avian β-defensins,AvBDs）和细胞因子基因表达的影响。选用45只20日龄SPF绍鸭,随机分为攻毒组（27只）和对照组（18只）,攻毒组采用滴鼻点眼的途径（10^8.38 EID₅₀）接种鸭源NDV强毒株（Md/CH/LGD/1/2005）,对照组接种磷酸盐缓冲液。在攻毒后24和48 h,从对照组及攻毒组各随机取6只鸭屠宰,分别采集肾脏、肺脏、气管、腺胃、骨髓、脾脏、盲肠扁桃体、哈德氏腺、法氏囊9个组织,运用实时荧光定量PCR法检测组织样品中9种AvBDs和细胞因子的表达量;剩余鸭每天持续观察,每隔4 d采血1次,直至24 d,检测血清抗体水平;于攻毒后24、48、72和120 h采集咽喉及泄殖腔拭子,以确定排毒周期。结果表明,感染该NDV毒株后,鸭没有临床发病症状和死亡情况发生;感染后鸭排毒开始于攻毒后第1天,到第5天停止,在攻毒组咽喉及泄殖腔拭子中均检测到NDV。抗体水平检测结果显示,该NDV能够诱导鸭体内产生抗NDV特异抗体。实时荧光定量PCR结果发现,与对照组相比,感染后24 h,部分鸭组织中AvBD1、AvBD2、AvBD5、AvBD6、AvBD9和AvBD16表达量均显著升高（P<0.05）;感染后48 h,AvBD1和AvBD6在部分鸭组织中表达量均显著上调（P<0.05）;感染后48 h法氏囊中白细胞介素6（IL-6）和脾脏中干扰素γ（IFN-γ）的表达量明显低于感染后24 h的表达量。综上所述,该鸭源NDV毒株感染可诱导绍鸭体内在早期产生天然免疫反应,这些反应可能与病毒在机体内复制有关。     

Molecular epidemiologic investigation of lentogenic Newcastle disease virus from domestic birds at live bird markets in Korea

A Newcastle disease surveillance program was conducted at live bird markets in Korea to expand our epidemiologic understanding of the disease in Korea. During the surveillance program, 10 lentogenic Newcastle disease viruses (NDVs) were isolated and identified from apparently healthy chickens and ducks at live bird markets. The lentogenic viruses had sequence motifs of either 112GKQGRL117 (n = 8) or 112GRQGRL117 (n = 2) at the F0 cleavage site. Sequencing and phylogenetic analyses of NDV isolates based on the hypervariable region of the F protein revealed two different genotypes: genotypes I (n = 8) and II (n = 2). Genotype I viruses were most closely related to the NDV V4 strain (n = 7) or the NDV Ulster 2C strain (n = 1). In contrast, genotype II viruses clustered with the NDV vaccine strains (LaSota and VG/GA) that are commonly used as live vaccines in Korea. The epidemiologic importance of NDV at live bird markets in Korea is discussed.     

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.     

Natural infection of broiler breeder chickens with endemic apathogenic Newcastle disease virus and their subsequent response to vaccination with a live V4 Newcastle disease virus vaccine

Flocks of broiler breeder chickens housed on a commercial farm were monitored from 13 w of age for natural infection with endemic lentogenic Newcastle disease virus (NDV). Seroconversion was first detected at 17 w. By 24 w, all 8 flocks had achieved peak log2 mean haemagglutination inhibiting antibody titres of up to 4.8. Antibody titres then declined and rose again over several months, suggesting cyclic reinfection with NDV. A lentogenic NDV indistinguishable from V4 was isolated from the cloaca of one bird at 18 weeks of age. At 54 weeks of age, 6 of 8 flocks were vaccinated en masse with live V4 NDV vaccine, 3 flocks by drinking water and 3 flocks by aerosol. All flocks were serologically monitored for a further 8 w. Drinking water vaccination induced an anamnestic response in 3 flocks, showing that flocks with pre-existing active immunity to NDV may be successfully vaccinated with V4. However, in all aerosol vaccinated flocks, the procedures failed to induce a response different to that observed in unvaccinated flocks. The serological response to vaccination was greater in sires than in dams.     

Rapid serological profiling by enzyme-linked immunosorbent assay. IV. Association of infectious bursal disease serology with broiler flock performance

Breeder and broiler flocks were serologically evaluated using a multiple enzyme-linked immunosorbent assay (M-ELISA). The serologic status of two commercial broiler-breeder flocks and their progeny was monitored, and 840 sera were promptly assessed for antibodies against six infectious agents using the M-ELISA. Breeder flocks were sampled at lay, and broiler chicks were hatched from fertile eggs collected on the scheduled lay date of the breeders. The broiler chicks were placed for growout as eight separate flocks (four from each breeder), and the serologic survey of broilers included sequentially sampling each flock five times between 1 day of age and market. Association of broiler vaccination schedules, mortality, and condemnation data with the temporal serologic data obtained indicated that the earlier appearance of active antibody against infectious bursal disease (IBD) in some unvaccinated flocks was associated with subsequent higher growout mortality and with the poorer overall performance that these flocks experienced. The results of this serologic survey also demonstrated that if a constant, well-timed monitoring program had not been used, major serologic differences between flocks would not have been detected. Serologic profiles of selected broiler flocks by virus-neutralization (VN) tests for infectious bronchitis virus (IBV) and reovirus or by hemagglutination-inhibition (HI) tests for Newcastle disease virus (NDV) compared favorably with the serologic profiles obtained by M-ELISA. Comparison of vaccination histories with serologic results derived from M-ELISA, VN or HI tests indicated that response to vaccination for IBV and NDV at 1 day was either blocked or significantly delayed by moderate levels of maternal antibody and/or were suppressed by an apparent field outbreak of IBD that occurred in all eight broiler flocks.     

野禽新城疫病毒F和HN基因的遗传变异趋势

野禽携带的新城疫病毒是新城疫流行传播的重要的潜在传染源。结合本实验室克隆测序的鸽NDV(PB9601)、企鹅NDV(QE01)以及GenBank下载的44株野禽(包括野鸭、鹦鹉、鸽子、天鹅、鸳鸯、雁、火鸡等)的NDV毒株的融合蛋白(Fusion gene,F)基因和血凝素蛋白(Hemagglutinin-neuraminidase gene,HN)基因分别进行了F蛋白裂解位点、基因分型以及F和HN基因氨基酸全长的遗传距离分析。结果表明:野禽感染NDV F基因的基因型以Ⅶ和Ⅵ为主,同时Ⅰ、Ⅱ、Ⅴ和Ⅸ多种基因型并存。同一种属、同一地区的毒株间的遗传距离较近,具有明显的种属性和区域性,且绝大多数分离株与经典毒株LaSota、V4、B1、TEX-48等的遗传距离相对较远;对HN基因的遗传进化关系分析得出了相似的结论。     

Unexpected newcastle disease virus in day old commercial chicks and breeder hen

Newcastle disease virus (NDV) specific antigen in the gut contents and NDV specific antibody in blood circulation were seen in day old chicks belonging to nine different commercial hatcheries of Tamil Nadu, India. Antigen disappeared by 4th week and antibody by 6th week of age. Fourteen NDV isolates obtained from the gut contents of day old chicks of different commercial hatcheries, one NDV isolate from dead in shell eggs and one NDV isolate from breeder hen were characterized and grouped under velogenic, mesogenic and lentogenic pathotypes. Four isolates were grouped under F and another four isolates were grouped under E based on reaction with monoclonal antibodies (Mabs) but found to be velogenic based on pathogenicity tests. In one particular flock velogenie NDV was isolated from breeder hen, dead in shell embryos and day old chicks and they all belong to Mabs group E. Vertical transmission of velogenic, mesogenic and lentogenic NDVs and role of NDVs in the gut contents have been discussed.     

Biological and phylogenetic characterization of virulent Newcastle disease virus circulating in Mexico

In 2002-2003, velogenic Newcastle Disease Virus outbreaks, closely related to the Mexican isolates, were confirmed in the United States (U.S.) in southern California, Arizona, Nevada, and Texas. In this report, virulent NDVs isolated in Mexico between 1998 and 2006 were subjected to biologic characterization, using standard pathogenicity tests, and to phylogenetic analysis. Chicken embryo mean death time (MDT) test results ranged from 39.7 to 61.5 hours, and intracerebral pathogenicity index (ICPI) values were between 1.59 and 1.94, compared to a possible maximum value of 2.0. These isolates showed a dibasic amino acid motif at the fusion protein cleavage site sequence required for host systemic replication. Phylogenetic analysis indicated that the Mexican virulent NDVs belong to the class II, genotype V viruses and can be clearly divided in two groups as follows: isolates from 1998 to 2001 with close epidemiologic relationship with the latest U.S. NDV outbreaks, and phylogenetically distinct viruses, isolated from 2004 to 2006, which showed higher virulence. The assessment of the evolution of viruses from Mexico and other neighboring countries will aid in the U.S surveillance efforts for early detection of highly virulent NDV.     

Newcastle disease--seroepidemiologic study of a highly contagious epizootic in poultry and in wild birds in Switzerland

Newcastle disease (ND) is a highly contagious viral disease particularly of domestic poultry. Switzerland is currently declared free from ND. A serosurvey using an ELISA was performed to investigate infections with ND-Virus (NDV) in 260 Swiss laying hen flocks, 169 backyard poultry flocks and 1576 wild birds. For laying hen flocks, a stochastic model was applied to analyse the results from serological testing. Four laying hen flocks were identified as NDV-seropositive, and the true NDV seroprevalence in this population was most likely between 1.3 and 1.5%. NDV antibodies were also detected in five of the 169 backyard poultry-flocks. ND-antibody positive birds were found in 10% of all wild birds examined, with the highest proportions among cormorants, grebes, birds of prey, owls, and swifts. The study indicated that positive flocks must have been in contact with NDV strains causing sub-clinical infection, since no clinical signs had been observed. Moreover, trade of poultry or poultry eggs was considered to be an important factor associated with seropositivity in backyard poultry flocks. Contact to wild birds did not seem to be of major importance.     

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.     

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.