新城疫分离毒HN基因的分子特性和片段同源相关性

选取国内1997-2005年分离的新城疫病毒(Newcastle disease virus,NDV)24株,经蚀斑纯化克隆其血凝素-神经氨酸酶(HN)基因,与在GenBank发表的36株国内外不同时期的NDV毒株,进行氨基酸遗传变异分析,并利用SPSS8.0软件对其不同片段的氨基酸进行同源相关比较。结果显示:国内所有NDV分离毒株氨基酸高度同源,同源性为94.4%-99.4%;与LaSota、Clone30疫苗株等的氨基酸同源性为86.9%-89%;与强毒株F48E9的氨基酸同源性为87.9%-89.9%;与国外NDV的氨基酸同源性为87.2%-96.2%。系统发育分析表明:国内NDV分离毒HN遗传距离较近,而与LaSota、Clone30和F48E9遗传距离较远。国内NDV分离毒均缺乏538-540位糖基化位点。不同片段与全长的氨基酸同源性高度相关,且与前80个氨基酸相关最密切。     

三株新城疫广西分离株全基因组序列的测定与分析

根据GenBank上所公布的新城疫病毒(NDV)的全基因组序列,设计了8对引物,运用RT-PCR方法获取了3株广西地方强毒株GX7/02、GX9/03和GX11/03的全基因组序列,并对其进行了比较分析。此3株病毒的全基因组序列均由15192个碱基组成,与GenBank公布的ZJ1、U.S/Largo/71、Italy/2736/00等7个毒株的全基因组序列长度相同,比LaSota、Clone-30和B1的全基因组序列多出6个核苷酸,此6个核苷酸位于np基因的非编码区内,相对与NDV毒株LaSota、Clone-30和B1序列的1647~1648nt位。通过序列的比较分析,发现GX7/02、GX9/03和GX11/03与ZJ1毒株的同源性较高,而与LaSota、Clone-30和B1等毒株的同源性较低。     

三株新城疫广西分离株全基因组序列的测定与分析

根据GenBank上所公布的新城疫病毒(NDV)的全基因组序列,设计了8对引物,运用RT-PCR 方法获取了3株广西地方强毒株GX7/02、GX9/03和GX11/03的全基因组序列,并对其进行了比较分析.此3株病毒的全基因组序列均由15192个碱基组成,与GenBank公布的ZJ1、U.S/Largo/71、Italy/2736/00等7个毒株的全基因组序列长度相同,比LaSota、Clone-30和B1的全基因组序列多出6个核苷酸,此6个核苷酸位于np基因的非编码区内,相对与NDV毒株LaSota、Clone-30和B1序列的1647～1648nt 位.通过序列的比较分析,发现GX7/02、GX9/03和GX11/03与ZJ1毒株的同源性较高,而与LaSota、Clone-30和B1等毒株的同源性较低.     

新城疫病毒HN基因的遗传变异与HI相关性的研究

选取国内1999~2004年分离的新城疫野毒10株,经CEF蚀斑纯化和SPF鸡胚增殖,对其血凝素-神经氨酸酶(HN)基因分别进行克隆和测序,结合在GenBank中发表的LaSota、F48E9和Clone30等参考序列,对其氨基酸序列进行遗传变异分析,绘制系统发育树。利用SPF鸡在隔离器中分别制备上述NDV毒株的单因子阳性血清,进行血凝抑制(HI)交叉试验,计算NDV不同株之间的HI相关系数(r)。利用统计学软件SPSS8.0对NDV不同株之间的HN氨基酸同源率和HI相关系数(r)进行相关比较。结果表明:NDV野毒间氨基酸高度同源,同源性为96.5%~99.8%,而与LaSota、F48E9和Clone30同源率仅为87.4%~89.9%;所有野毒均缺乏1个潜在的糖基化位点;HN基因的氨基酸同源性与HI相关系数显著相关(P<0.01,r=0.55)。     

高抗体水平种鸡NDV的分离鉴定和分子特性

从抗体水平较高、产蛋下降的种鸡群分离到一株新城疫病毒(Newcastle Disease virus,NDV)SGM01,经动物回归可产生NDV典型病变,其MDT、ICPI和IVPI等分别为50．5h、1．76和2．41,表明为强毒。对其F和HN基因进行克隆测序,F基因氨基酸多肽裂解位点的基序为~(111)GRRQKRF~(117),与强毒基序一致。基因分型表明SGMO1属Ⅶ型。氨基酸同源比较显示:SGM01与LaSota、SQZ04等基因Ⅱ型的同源性为:87．7%～88．3%;(?) Taiwan95、Yunnan03等基因Ⅶ型的同源性为:95．7%～98．2%;与F_(48)E_9等基因Ⅸ型的同源率为:90．8%～91．7%。HN基因与F基因不同,具有明显的时空性。SGM01、Taiwan95、SQZ04等新近分离毒氨基酸高度同源,同源性为:95．3%～97．2%,显著高于与LaSota(经典弱毒疫苗株)和F_(48)E_9(经典强毒株)的同源性87．4%～89．0%。     

新城疫分离毒HN蛋白的抗原性初步分析及分子特性研究

运用针对NDV囊膜糖蛋白(HN)的单克隆抗体(MAbs),对2005～2006年间自我国江苏和广西部分地区的20株NDV分离株进行排谱试验,初步分析了不同毒株之间HN蛋白的抗原表位差异;并应用RT-PCR技术成功扩增了其HN基因整个编码区,经克隆、测序最终获得13株鸡源NDV与7株鹅源NDV HN基因的编码区序列,分析测定核苷酸序列及推导的氨基酸序列,并将鹅源NDV与鸡源NDV相应序列进行了比较.结果单抗排谱试验表明,20株NDV分离株之间HN蛋白的抗原表位存在差异;测序结果表明,测定的HN基因的编码区长度皆为1716nt编码571个氨基酸;分离株中18株基因Ⅶ型NDV分离株之间HN基因编码区核苷酸序列具有较高的同源性,达94.8%～100%;与近几年国内流行的其它基因Ⅶ型NDV之间的核苷酸序列同源性为92.1%～99.6%.对其推导的HN蛋白一级结构中潜在的糖基化位点及HN蛋白细胞受体结合相关区域的氨基酸序列等进行了比较分析.结果显示,单抗排谱差异显著株在部分氨基酸位点发生了突变;同时揭示我国部分地区同期流行的鹅源NDV与鸡源NDV HN基因之间具有较近的亲缘关系.     

一株基因Ⅱ型新城疫强毒株的分子特性

从患病肉鸡群分离到一株新城疫病毒(NewcastleDiseasevirus,NDV)SQZ04。经蚀斑纯化后接种40日龄SPF鸡可诱发典型病变。经蚀斑纯化前和后的MDT为50·5h和51·2h,ICPI为2·0和1·92,IVPI为2·8和2·68,表明属强毒株。但F基因分型表明SQZ04属基因Ⅱ型,而且其与已知基因Ⅱ型的疫苗株LaSota、B1和Texas48的同源性分别为99·3%、98·7%和96·9%,显著高于与基因Ⅶ或Ⅸ型强毒株的同源性88·3%~88·6%或91·3%~92·1%。这是国内第一株属于基因Ⅱ型的NDV强毒株。SQZ04F多肽氨基酸裂解位点的序列为111GGRQGRL117,与弱毒株序列完全相同,这也是国内外首次报道具有这一氨基酸序列的强毒野毒株。然而,SQZ04株与其他已知强毒株的HN氨基酸同源性高达95·3%~97·3%,显著高于与弱毒株LaSota等的同源性87·8%~89·5%。     

新城疫分离毒HN蛋白的抗原性初步分析及分子特性研究

运用针对NDV囊膜糖蛋白(HN)的单克隆抗体(MAbs), 对2005~2006年间自我国江苏和广西部分地区的20株NDV分离株进行排谱试验, 初步分析了不同毒株之间HN蛋白的抗原表位差异; 并应用RT-PCR技术成功扩增了其HN基因整个编码区, 经克隆、测序最终获得13株鸡源NDV与7株鹅源NDV HN基因的编码区序列, 分析测定核苷酸序列及推导的氨基酸序列, 并将 鹅源NDV与鸡源NDV相应序列进行了比较。结果单抗排谱试验表明, 20株NDV分离株之间 HN蛋白的抗原表位存在差异; 测序结果表明, 测定的HN基因的编码区长度皆为1716nt编码571个氨基酸; 分离株中18株基因Ⅶ型NDV分离株之间HN基因编码区核苷酸序列具有较高的同 源性,达94.8%~100%; 与近几年国内流行的其它基因Ⅶ型NDV之间的核苷酸序列同源性 为92.1%~99.6%。对其推导的HN蛋白一级结构中潜在的糖基化位点及HN蛋白细胞受体结合相关区域的氨基酸序列等进行了比较分析。结果显示, 单抗排谱差异显著株在部分氨基酸位点发生了突变; 同时揭示我国部分地区同期流行的鹅源NDV与鸡源NDV HN基因之间具有较近的亲缘关系。     

猪源新城疫病毒JL01株分离鉴定及F基因遗传进化分析

2000年,在吉林省某猪场发生具有较高发病率和死亡率的急性传染病,发病率达到40%～50%,病死率15%,取其脾、肺、肾等组织进行电镜观察,可见副粘病毒样颗粒,表明其病原可能为某种猪源副粘病毒,命名为JL01株。在对该病毒的的血凝、血凝抑制等生物学特性进行鉴定后,初步确定该种猪副粘病毒为猪源新城疫病毒。病毒回归试验表明,纯化的病毒对猪仍有较强的致死性,并可从死亡猪体内分离到新城疫病毒。该病毒对鸡胚平均致死时间(MDT)、脑内致病指数(ICPI)和半数致死量(EID50)分别为55.2h、1.60和10-7.5/0.1 mL,表明该毒株属于新城疫病毒强毒株。在此基础上,采用RT-PCR方法克隆了猪源新城疫病毒F基因,与其他10株NDV毒株进行序列比较分析,结果表明,JL01株与B1、LaSota、Clone30等经典的新城疫病毒弱毒株同源性较高(91.5%～98.5%),与ZJ1、Mukteswar等强毒株的同源性较低。F基因氨基酸裂解位点的序列为112G-K-Q-G-R-L117,与弱毒株序列完全相同。基因分型结果表明JL01株属于基因Ⅰ型。因此,本研究所分离到感染猪的新城疫病毒属于基因变异的新城疫病毒弱毒株...     

4株鸽新城疫病毒的基因组序列与致病性分析

本研究对从北京、天津以及山东地区发病鸽群中分离到的4株鸽新城疫病毒（Newcastle disease virus,NDV）进行基因组测序和遗传进化分析,并进一步比较这些毒株对鸽子的致病性。研究通过特异性引物扩增4株鸽NDV的全长基因组序列后与GenBank上已登陆的所有鸽NDV毒株的序列进行遗传进化分析;通过病毒生物学特性的测定比较分离株的毒力;通过病毒感染1月龄肉鸽,检测分离株对鸽子的致病性。结果显示4株鸽NDV毒株均属于ClassⅡ类基因Ⅵb亚型病毒,其中Pigeon/China/BJ2018株、Pigeon/China/TJ2017株和Pigeon/China/BJ2013株属于VIb/4bii f亚型,Pigeon/China/SD2012株属于VIb/4bii d亚型。4株病毒与目前国内鸽NDV流行株属于同一进化分支,与鸡源经典疫苗株La Sota属于不同进化分支。对这四个毒株进行生物学特性测定,均为中等毒力毒株。4株病毒中,Pigeon/China/BJ2018株对1月龄肉鸽的致病性最强,通过肌肉注射途径攻毒后3d肉鸽开始表现临床症状,攻毒后第5d开始出现死亡,累计死亡率...     

Characterization of velogenic Newcastle disease viruses isolated from dead wild birds in Serbia during 2007

Avian paramyxoviruses type 1 or Newcastle disease viruses (NDV) are frequently recovered from wild birds and such isolates are most frequently of low virulence. Velogenic NDV are usually recovered from poultry and only occasionally from wild birds. Five NDV isolates were obtained from carcasses of four wild bird species during 2007 in Serbia: Mallard (Anas platyrhynchos), Eurasian Sparrowhawk (Accipiter nisus), feral Rock Pigeon (Columba livia), and Eurasian Collared Dove (Streptopelia decaocto). All the isolates have a typical fusion protein cleavage site motif of velogenic viruses ((112)R-R-Q-K-R-F(117)). The highest homology (99%) for the nucleotide sequences spanning the M and F gene of the studied isolates was with the genotype VII NDV isolate Muscovy duck/China(Fujian)/FP1/02. Phylogenetic analysis based on a partial F gene sequence showed that the isolates from wild birds cluster together with concurrent isolates from poultry in Serbia within the subgenotype VIId, which is the predominant pathogen involved currently in Newcastle disease outbreaks in poultry worldwide. It is unlikely that the wild birds played an important role in primary introduction or consequent spread of the velogenic NDV to domestic poultry in Serbia, and they probably contracted the virus from locally infected poultry.     

Lack of detection of host associated differences in Newcastle disease viruses of genotype VIId isolated from chickens and geese

ABSTRACT: BACKGROUND: The goose is usually considered to be resistant even to strains of Newcastle disease virus (NDV) that are markedly virulent for chickens. However, ND outbreaks have been frequently reported in goose flocks in China since the late 1990s with the concurrent emergence of genotype VIId NDV in chickens. Although the NDVs isolated from both chickens and geese in the past 15 years have been predominantly VIId viruses, published data comparing goose- and chicken-originated ND viruses are scarce and controversial. RESULTS: In this paper, we compared genotype VIId NDVs originated from geese and chickens genetically and pathologically. Ten entire genomic sequences and 329 complete coding sequences of individual genes from genotype VIId NDVs of both goose- and chicken-origin were analyzed. We then randomly selected two goose-originated and two chicken-originated VIId NDVs and compared their pathobiology in both geese and chickens in vivo and in vitro with genotype IV virus Herts/33 as a reference. The results showed that all the VIId NDVs either from geese or from chickens shared high sequence homology and characteristic amino acid substitutions and clustered together in phylogenetic trees. In addition, geese and chickens infected by goose or chicken VIId viruses manifested very similar pathological features distinct from those of birds infected with Herts/33. CONCLUSIONS: There is no genetic or phenotypic difference between genotype VIId NDVs originated from geese and chickens. Therefore, no species-preference exists for either goose or chicken viruses and more attention should be paid to the trans-species transmission of VIId NDVs between geese and chickens for the control and eradication of ND.     

Newcastle Disease Virus Fusion Protein Is the Major Contributor to Protective Immunity of Genotype-Matched Vaccine

Virulent strains of Newcastle disease virus (NDV) can cause devastating disease in chickens worldwide. Although the current vaccines are substantially effective, they do not completely prevent infection, virus shedding and disease. To produce genotype-matched vaccines, a full-genome reverse genetics system has been used to generate a recombinant virus in which the F protein cleavage site has been changed to that of avirulent vaccine virus. In the other strategy, the vaccines have been generated by replacing the F and HN genes of a commercial vaccine strain with those from a genotype-matched virus. However, the protective efficacy of a chimeric virus vaccine has not been directly compared with that of a full-genome virus vaccine developed by reverse genetics. Therefore, in this study, we evaluated the protective efficacy of genotype VII matched chimeric vaccines by generating three recombinant viruses based on avirulent LaSota (genotype II) strain in which the open reading frames (ORFs) encoding the F and HN proteins were replaced, individually or together, with those of the circulating and highly virulent Indonesian NDV strain Ban/010. The cleavage site of the Ban/010 F protein was mutated to the avirulent motif found in strain LaSota. In vitro growth characteristics and a pathogenicity test indicated that all three chimeric viruses retained the highly attenuated phenotype of the parental viruses. Immunization of chickens with chimeric and full-length genome VII vaccines followed by challenge with virulent Ban/010 or Texas GB (genotype II) virus demonstrated protection against clinical disease and death. However, only those chickens immunized with chimeric rLaSota expressing the F or F plus HN proteins of the Indonesian strain were efficiently protected against shedding of Ban/010 virus. Our findings showed that genotype-matched vaccines can provide protection to chickens by efficiently preventing spread of virus, primarily due to the F protein.     

Phylogenetic and pathotypical analysis of two virulent Newcastle disease viruses isolated from domestic ducks in China

Two velogenic Newcastle disease viruses (NDV) obtained from outbreaks in domestic ducks in China were characterized in this study. Phylogenetic analysis revealed that both strains clustered with the class II viruses, with one phylogenetically close to the genotype VII NDVs and the other closer to genotype IX. The deduced amino acid sequence of the cleavage site of the fusion (F) protein confirmed that both isolates contained the virulent motif (112)RRQK/RRF(117) at the cleavage site. The two NDVs had severe pathogenicity in fully susceptible chickens, resulting in 100% mortality. One of the isolates also demonstrated some pathogenicity in domestic ducks. The present study suggests that more than one genotype of NDV circulates in domestic ducks in China and viral transmission may occur among chickens and domestic ducks.     

Newcastle disease virus-based live attenuated vaccine completely protects chickens and mice from lethal challenge of homologous and heterologous H5N1 avian influenza viruses

H5N1 highly pathogenic avian influenza virus (HPAIV) has continued to spread and poses a significant threat to both animal and human health. Current influenza vaccine strategies have limitations that prevent their effective use for widespread inoculation of animals in the field. Vaccine strains of Newcastle disease virus (NDV), however, have been used successfully to easily vaccinate large numbers of animals. In this study, we used reverse genetics to construct a NDV that expressed an H5 subtype avian influenza virus (AIV) hemagglutinin (HA). Both a wild-type and a mutated HA open reading frame (ORF) from the HPAIV wild bird isolate, A/Bar-headed goose/Qinghai/3/2005 (H5N1), were inserted into the intergenic region between the P and M genes of the LaSota NDV vaccine strain. The recombinant viruses stably expressing the wild-type and mutant HA genes were found to be innocuous after intracerebral inoculation of 1-day-old chickens. A single dose of the recombinant viruses in chickens induced both NDV- and AIV H5-specific antibodies and completely protected chickens from challenge with a lethal dose of both velogenic NDV and homologous and heterologous H5N1 HPAIV. In addition, BALB/c mice immunized with the recombinant NDV-based vaccine produced H5 AIV-specific antibodies and were completely protected from homologous and heterologous lethal virus challenge. Our results indicate that recombinant NDV is suitable as a bivalent live attenuated vaccine against both NDV and AIV infection in poultry. The recombinant NDV vaccine may also have potential use in high-risk human individuals to control the pandemic spread of lethal avian influenza.     

Generation by Reverse Genetics of an Effective,Stable, Live-Attenuated Newcastle Disease Virus Vaccine Based on a Currently Circulating,Highly Virulent Indonesian Strain

Newcastle disease virus (NDV) can cause severe disease in chickens. Although NDV vaccines exist, there are frequent reports of outbreaks in vaccinated chickens. During 2009–2010, despite intense vaccination, NDV caused major outbreaks among commercial poultry farms in Indonesia. These outbreaks raised concern regarding the protective immunity of current vaccines against circulating virulent strains in Indonesia. In this study, we investigated whether a recombinant attenuated Indonesian NDV strain could provide better protection against prevalent Indonesian viruses. A reverse genetics system for the highly virulent NDV strain Banjarmasin/010/10 (Ban/010) isolated in Indonesia in 2010 was constructed. The Ban/010 virus is classified in genotype VII of class II NDV, which is genetically distinct from the commercial vaccine strains B1 and LaSota, which belong to genotype II, and shares only 89 and 87% amino acid identity for the protective antigens F and HN, respectively. A mutant virus, named Ban/AF, was developed in which the virulent F protein cleavage site motif “RRQKR↓F” was modified to an avirulent motif “GRQGR↓L” by three amino acid substitutions (underlined). The Ban/AF vaccine virus did not produce syncytia or plaques in cell culture, even in the presence of added protease. Pathogenicity tests showed that Ban/AF was completely avirulent. Ban/AF replicated efficiently during 10 consecutive passages in chickens and remained genetically stable. Serological analysis showed that Ban/AF induced higher neutralization and hemagglutination inhibition antibody titers against the prevalent viruses than the commercial vaccines B1 or LaSota. Both Ban/AF and commercial vaccines provided protection against clinical disease and mortality after challenge with virulent NDV strain Ban/010 (genotype VII) or GB Texas (genotype II). However, Ban/AF significantly reduced challenge virus shedding from the vaccinated birds compared to B1 vaccine. These results suggest that Ban/AF can provide better protection than commercial vaccines and is a promising vaccine candidate against NDV strains circulating in Indonesia.     

新城疫不同毒株交叉鸡胚中和指数及其与F和HN基因变异的相关性

选取13株国内2001～2004年分离的新城疫流行病毒(Newcastle disease virus,NDV),经蚀斑纯化,克隆其融合蛋白(F)和血凝素.神经氨酸酶(HN)基因,结合疫苗株La Sota、Clone30和国内标准强毒株F48E9等的基因序列,进行遗传变异分析.利用纯化的病毒制备特异阳性血清,进行鸡胚交叉中和试验,确定不同NDV毒株之间的抗原相关性,并与NDV不同毒株之间的HN和F基因核苷酸(氨基酸)同源性进行相关比较.结果表明:病毒中和指数与HN基因的核苷酸(氨基酸)同源性显著相关(P<0.01,r=-0.35),与F基因呈弱相关(P<0.05,r=0.20),而与F基因前374bp的核甘酸同源性不相关.这表明,NDV的分子变异已经对NDV的抗原性变异产生了影响,研制新型的疫苗成为必然.     

A recombinant Newcastle disease virus (NDV) expressing VP2 protein of infectious bursal disease virus (IBDV) protects against NDV and IBDV

Infectious bursal disease virus (IBDV) causes a highly immunosuppressive disease in chickens. Currently available, live IBDV vaccines can lead to generation of variant viruses. We have developed an alternative vaccine that will not create variant IBDV. By using the reverse genetics approach, we devised a recombinant Newcastle disease virus (NDV) vector from a commonly used vaccine strain LaSota to express the host-protective immunogen VP2 of a variant IBDV strain GLS-5. The gene encoding the VP2 protein of the IBDV was inserted into the most 3'-proximal locus of a full-length NDV cDNA for high-level expression. We successfully recovered the recombinant virus, rLaSota/VP2. The rLaSota/VP2 was genetically stable, at least up to 12 serial passages in chicken embryos, and was shown to express the VP2 protein. The VP2 protein was not incorporated into the virions of recombinant virus. Recombinant rLaSota/VP2 replicated to a titer similar to that of parental NDV strain LaSota in chicken embryos and cell cultures. To assess protective efficacy of the rLaSota/VP2, 2-day-old specific-pathogen-free chickens were vaccinated with the recombinant virus and challenged with a highly virulent NDV strain Texas GB or IBDV variant strain GLS-5 at 3 weeks postvaccination. Vaccination with rLaSota/VP2 generated antibody responses against both NDV and IBDV and provided 90% protection against NDV and IBDV. Booster immunization induced higher levels of antibody responses against both NDV and IBDV and conferred complete protection against both viruses. These results indicate that the recombinant NDV can be used as a vaccine vector for other avian pathogens.     

Preparation and Efficacy of a Live Newcastle Disease Virus Vaccine Encapsulated in Chitosan Nanoparticles

Background

Newcastle disease (ND) is a highly contagious viral disease of poultry caused by pathogenic strains of the Newcastle disease virus (NDV). Live NDV vaccines are administered by drinking water, eyedrops or coarse aerosol spray. To further enhance mucosal immune responses, chitosan nanoparticles were developed for the mucosal delivery of a live NDV vaccine.

Methodology/Principal Findings

A lentogenic live-virus vaccine (strain LaSota) against NDV encapsulated in chitosan nanoparticles were developed using an ionic crosslinking method. Chitosan nanoparticles containing the lentogenic live-virus vaccine against NDV (NDV-CS-NPs) were produced with good morphology, high stability, a mean diameter of 371.1 nm, an encapsulation rate of 77% and a zeta potential of +2.84 mV. The Western blotting analysis showed that NDV structural proteins were detected in NDV-CS-NPs. The virus release assay results of NDV-CS-NPs indicated that NDV was released from NDV-CS-NPs. Chickens immunized orally or intranasally with NDV-CS-NPs were fully protected whereas one out of five chickens immunized with the LaSota live NDV vaccine and three out of five chickens immunized with the inactivated NDV vaccine were dead after challenge with the highly virulent NDV strain F48E9.

Conclusions/Significance

NDV-CS-NPs induced better protection of immunized specific pathogen free chickens compared to the live NDV vaccine strain LaSota and the inactivated NDV vaccine. This study lays a foundation for the further development of mucosal vaccines and drugs encapsulated in chitosan nanoparticles.