新城疫病毒F48E9株病毒糖蛋白的功能分析

对抗新城疫病毒（ＮＤＶ）ＨＮ和Ｆ糖蛋白的单克隆抗体（ＭｃＡｂ）的生物学活性，应用ＥＬＩＳＡ、ＨＩ、ＨＬＩ、Ｗｅｓｔｅｒｎｂｌｏｔ等进行了分析，结果证明，ＮＤＶＦ４８Ｅ９株ＨＮ蛋白除有ＨＡ和ＮＡ功能区外，还有一个促进（启动）融合的功能区。此外，还筛选到４株ＮＤＶ强毒株特异性ＭｃＡｂ，为ＮＤＶ强弱毒株的鉴别打下了基础     

新城疫病毒致病性的研究进展

新城疫病毒（ＮＤＶ）包含一系列对鸡有不同致病性的毒株。如强毒株、中等毒力毒株和弱毒株。ＮＤＶ表面具有两种糖蛋白———血凝素神经氨酸苷酶（ＨＮ）和融合（Ｆ）蛋白。Ｆ蛋白最初是以前体多肽Ｆ０的形式被合成,然后经蛋白水解作用,裂解为两个多肽Ｆ１和Ｆ２而获得生物学活性。通常认为Ｆ蛋白的可分解性是ＮＤＶ致病性的一个重要决定因素。致病性强的毒株的Ｆ蛋白在许多细胞内可被裂解,由此,强毒可扩散到宿主全身,并引起严重的疾病。而无致病性的毒株的Ｆ蛋白只在有限的几类细胞内可裂解（如肠上皮细胞和呼吸道上皮细胞）。使用Ｕ…     

应用抗多肽抗体鉴别新城疫病毒强弱毒株

在克隆我国流行的新城疫病毒（ＮＤＶ）强毒株Ｆ４８Ｅ８株、四平株及弱毒株长春株、Ｖ４株和ＨＮ和Ｆ基因并进行测序的基础上,针对我国流行的ＮＤＶ强毒株Ｆ蛋白前体（Ｆ０）的Ｆ２片段的特异结构,人工合成特异性多肽,将其与小牛血清白蛋白（ＢＳＡ）化学偶联制备成全抗原,免疫小鼠制备出抗多肽血清。经ＥＬＩＳＡ检测,该抗体与ＮＤＶ强毒株呈强阳性反应,而与鸡痘病毒、鸡传染性法氏囊病病学、ＮＤ 弱毒株长春株和Ｖ４株呈阴     

表达ND病毒融合蛋白的重组火鸡疱疹病毒疫苗对ND和MD…

将新城疫病毒（ＮＤＶ）的融合蛋白（Ｆ）基因或血凝素－神经氨酸酶（ＨＮ）基因插入到火鸡疱疹病毒（ＦＶＴ）的一个非必需基因中，构建了重组ＨＶＴ株。ＮＤＶ抗原的表达受来自劳斯肉瘤病毒的一种强启动子的调控。１日龄腹腔内（ＩＡ）接种表达ＮＤＶ融合蛋白的重组ＨＶＴ的鸡不仅产生免疫学反应，而且在２８日龄可抵抗嗜神经ＮＤＶ强毒株Ｔｅｘａｓ ＧＢ的致死性肌肉攻毒（保护率＞９０％）。表达ＮＤＶ ＨＮ的重组ＨＶＴ仅能提     

我国部分地区NDV的分子流行病学研究

新城疫（Ｎｅｗｃａｓｔｌｅ ｄｉｓｅａｓｅ,ＮＤ）是由新城疫病毒（ＮＤＶ）引起的禽类的一种以呼吸道、消化道粘膜出血为典型症状的急性传染病,其病原——ＮＤＶ是副粘病毒科、副粘病毒亚科、腮腺炎病毒属的成员,为有囊膜的负链ＲＮＡ病毒。ＮＤＶ基因组为单股不分节ＲＮＡ,编码六种病毒结构蛋白,其中融合蛋白（Ｆ）和血凝素－神经氨酸酶蛋白（ＨＮ）构成囊膜外表面的纤突,是ＮＤＶ的功能性糖蛋白,在致病和免疫应答过程中起重要作用［１］。由于ＮＤＶ只有一种血清型,对ＮＤＶ不同毒株的差异性分析只能采用毒力、蚀斑形成能力测定等功能性试验来进行,但分型要么太细,无规律可循;要么过于粗放,无流行病学意义,使得ＮＤＶ流行病学研究一直难以突破。 近年来,随着分子生物学技术的广泛应用,使得人们在分子水平上对病毒遗传变异机制的研究取得了长足进展,发现病毒的遗传变异与疾病的流行有着密不可分的联系,并形成了病毒分子流行病学这样一门崭新学科,也为ＮＤ的流行病学研究提供了一种先进技术。已有研究表明,ＮＤＶＦ基因的遗传变异与ＮＤＶ的变异和流行密切相关,是ＮＤＶ分子流行病学研究的首选基因。 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     

以鸡痘病毒为载体的新城疫病毒重组疫苗的免疫效力

构建了表达速发型新城疫病毒（ＮＤＶ）融合蛋白（Ｆ）和血凝素－神经氨酸酶（ＨＮ）糖蛋白的鸡痘重组病毒ＴＲＯＶＡＣ－ＮＤＶ。ＳＰＦ鸡免疫试验表明，一次注射得组病毒有使鸡产生大量血凝抑制（ＨＩ）抗体。并能维持８周以上，而且重凤苗能诱导对经肌肉和呼吸道途径攻击ＮＤＶ的免疫力。带母滞病毒（ＦＰＶ）的免疫力，但ＮＤＶ的特异性体液应签水平降低。     

新城疫病毒F48E9株病毒结构蛋白的分析

采用ＳＤＳ－ＰＡＧＥ、激光扫描技术、ＷｅｓｔｅｒｎＢｌｏｔ和糖蛋白染色方法对新城疫病毒（ＮＤＶ）Ｆ４８Ｅ９株的结构蛋白进行了分析，结果表明，它具有ＮＤＶ的典型结构特征，与ＬａＳｏｔａ毒株相比无显著差异     

应用生物素标记的NDV—cDNA探针检测感染尿囊液中DNV—RNA的初…

本文应用聚合酶链反应（ＰＣＲ）技术从构建的新城疫病毒（ＤＮＶ）ｃＤＮＡ文库中扩增含编码Ｆ糖蛋白前体－Ｆｏ酶切位点序列的３５９ｂｐ的Ｆ蛋白基因ｃＤＮＡ片段。将此３５９ｂｐ ｃＤＮＡ片段经光敏生物素标记后，即成ＤＮＶ－ｃＤＮＡ探针。该探针能特异性地从感染的尿囊液中检测出ＤＮＶ强毒株和疫苗毒株的基因组ＲＮＡ，而不与ＩＢＤＶ－ｄｓＲＮＡ，ＡＩＢＶ－ｓｓＲＮＡ，ＥＤＳ７６－ｄｓＤＮＡ、ＭＤＶ０ｄｓＤＮＡ、Ｆ     

间接夹心ELISA检测新城疫病毒抗原

以ＳＰＦ鸡抗新城疫病毒ＩｇＧ为包被抗体，兔抗ＮＤＶ ｖｅｒｏ细胞培养纯化毒ＩｇＧ为第二抗体，酶标记羊抗兔ＩｇＧ为指示抗体，建立了检测ＮＤＶ的间接夹心ＥＬＩＳＡ，并分别以兔抗ＮＤＶ鸡胚毒纯化ＨＮ糖蛋白，Ｆ糖蛋白ＩｇＧ为第二抗体进行了比较试验，并对攻毒鸡外分泌物样品进行了病原检测。     

应用生物素标记的NDV－cDNA探针检测感染尿囊液中NDV－RNA的初步研究

本文应用聚合酶链反应（ＰＣＲ）技术从构建的新城疫病毒（ＮＤＶ）ｃＤＮＡ文库中扩增含编码Ｆ糖蛋白前体──Ｆｏ酶切位点序列的３５９ｂｐ的Ｆ蛋白基因ｃＤＮＡ片段。将此３５９ｂｐｃＤＮＡ片段经光敏生物素标记后,即成ＮＤＶ－ｃＤＮＡ探针。该探针能特异性地从感染的尿囊液中检测出ＮＤＶ强毒株和疫苗毒株的基因组ＲＮＡ,而不与ＩＢＤｖ－ｄｓＲＮＡ、ＡＩＢｖ－ｓｓＲＮＡ、ＥＤＳ７６－ｄｓＤＮＡ、ＭＤＶ－ｄｓＤＮＡ,ＦＰＶ－ｄｓＲＮＡ及ＡＩＬＶ－ｄｓＤＮＡ发生交叉杂交反应。试验结果表明：尽管该探钎含有编码Ｆｏ蛋白酶切位点序列的碱基顺序,但它还是不能把ＮＤＶ的强、弱毒株区分开。这说明ＮＤＶ强、弱毒株比区域内的碱基存在着相当大的同源性。不过,此探针对ＮＤＶ来说具有特异性,这就为ＮＤＶ的诊断技术开创了基因水平检测的新途径。     

新城疫病毒囊膜糖蛋白生物学特性的研究进展

新城疫是当今全球范围内最严重的禽类传染病之一。其病原新城疫病毒是单股负链RNA病毒,编码NP、P、M、F、HN和L 6种结构蛋白,其中最主要的是位于囊膜上的F和HN两种糖蛋白。F蛋白具有使病毒囊膜与宿主细胞膜融合,致使病毒穿入宿主细胞膜的作用,是决定病毒毒力的关键因子;HN蛋白具有血凝素和神经氨酸酶两种活性,这两种活性对于NDV侵染细胞具有重要的作用。这两种糖蛋白不仅对NDV的毒力及致病性方面起着决定性作用,而且也是诱导产生保护性抗体的蛋白。作者主要对这两种蛋白的结构与功能作一概述。     

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。     

Enhancement of pathogenicity of Newcastle disease virus by alteration of specific amino acid residues in the surface glycoproteins F and HN

Recombinant viruses were rescued after site-specific mutagenesis of a full-length clone of the lentogenic Newcastle disease virus (NDV) strain Clone 30. To assess the contribution of different amino acids to virulence, specific alterations were introduced into the fusion (F) protein and in the hemagglutinin-neuraminidase (HN) protein based on sequence comparison between NDV strains of different virulence. Modification of the proteolytic cleavage site in the F protein to a polybasic motif increased the intracerebral pathogenicity index (ICPI) from 0.0 to 1.28. Moreover, the additional exchange of amino acid 123 of the HN protein from tryptophan to cysteine in combination with alteration of amino acid 27 of the F protein from cysteine to arginine increased the ICPI to 1.5. The HN mutation visibly altered conformation of the protein, resulting in the formation of disulfide-linked HN dimers that may indicate that this HN conformation is beneficial for the virulent phenotype.     

Protection of chickens from Newcastle disease with a recombinant baculovirus subunit vaccine expressing the fusion and hemagglutinin-neuraminidase proteins

Recombinant baculoviruses containing the fusion (F) and hemagglutinin-neuraminidase (HN) glycoprotein gene of the viscerotropic velogenic (vv) Newcastle disease virus (NDV) isolate, Kr-005/00, and a lentogenic La Sota strain of the NDV were constructed in an attempt to develop an effective subunit vaccine to the recent epizootic vvNDV. The level of protection was determined by evaluating the clinical signs, mortality, and virus shedding from the oropharynx and cloaca of chickens after a challenge with vvNDV Kr-005/00. The recombinant ND F (rND F) and recombinant HN (rND HN) glycoproteins derived from the velogenic strain provided good protection against the clinical signs and mortality, showing a 0.00 PI value and 100% protection after a booster immunization. On the other hand, the combined rND F + HN glycoprotein derived from the velogenic strain induced complete protection (0.00 PI value and 100% protection) and significantly reduced the amount of virus shedding even after a single immunization. The rND F and rND HN glycoproteins derived from the velogenic strain had a slightly, but not significantly, greater protective effect than the lentogenic strain. These results suggest that the combined rND F + HN glycoprotein derived from vvNDV can be an ideal subunit marker vaccine candidate in chickens in a future ND eradication program.     

Antigenic heterogeneity amongst the field isolates of Newcastle Disease Virus (NDV) in relation to the vaccine strain. Part II: studies on viruses isolated from domestic birds in Israel

Forty three Newcastle disease virus (NDV) strains isolated before and during 1997 in Israel from domestic birds were studied by means of the three panels of monoclonal antibodies prepared against all the viral envelope proteins in order to reveal the possible antigenic differences between them and the VH strain used in Israel for poultry vaccination. Three isolates were found to have significant antigenic differences in the hemagglutinin-neuraminidase (HN) and fusion (F) glycoproteins as compared to the vaccine strain. As to the matrix protein, almost all the viruses isolated during the year 1997 were found to have considerable differences from the vaccine strain in two of four antigenic sites.     

副黏病毒糖蛋白的结构与功能研究进展

副黏病毒是一类包含多种能引起人和动物严重疾病的负链RNA病毒。其病毒颗粒囊膜上排列有两种糖蛋白，分别是识别并结合宿主受体的附着蛋白(hemagglutinin neuraminidase/hemagglutinin/glycoprotein, HN/H/G)和介导病毒颗粒囊膜与宿主细胞膜融合的融合蛋白(fusion protein, F)。这两种糖蛋白通过与病毒受体及细胞膜的互相作用介导了病毒与宿主细胞的特异性识别、结合及融合过程，最终使病毒基因组进入宿主细胞质开启复制过程。由于糖蛋白是主要的病毒抗原及中和抗体靶点，因此近年来对副黏病毒糖蛋白的研究在不断的深入探索。就副黏病毒糖蛋白在其结构、功能及其相互作用方面的研究进展进行了综述，以期为治疗性抗体和疫苗的合理开发提供理论基础。     

Protective immunity against Newcastle disease: the role of antibodies specific to Newcastle disease virus polypeptides

Studies were performed to determine if passive immunization with hyperimmune sera generated to specific Newcastle disease virus (NDV) proteins conferred protection against virus challenge. Six groups of 3-wk-old chickens were passively immunized with antiserum against either hemagglutinin-neuraminidase/fusion, (HN/F) protein, nucleoprotein/phosphoprotein (NP/P), Matrix (M) protein, a mixture of all NDV proteins (ALL), intact ultraviolet-inactivated NDV (UVNDV), or negative sera. Blood samples were collected 2 days postimmunization, and the birds were challenged with Texas GB strain of NDV. Antibody titers were detected from those recipient birds that had received the antisera against the HN/F, ALL, or UVNDV by a hemagglutination inhibition test, an enzyme-linked immunosorbent assay (ELISA), and a virus neutralization test. Antibodies were detected only by the ELISA from the birds that had received antisera against NP/P and M protein. Antibody titers in the recipient birds dropped by two dilutions (log2) after 2 days postinjection. Birds passively immunized with antisera against HN/F, ALL, and UVNDV were protected from challenge, whereas chickens passively immunized with antisera against NP/P and M protein and specific-pathogen-free sera developed clinical signs of Newcastle disease. The challenge virus was recovered from the tracheas of all passively immunized groups. The presence of neutralizing antibodies to NDV provided protection from clinical disease but was unable to prevent virus shedding from the trachea.     

Improved protection from velogenic Newcastle disease virus challenge following multiple immunizations with plasmid DNA encoding for F and HN genes

Specific-pathogen free (SPF) chickens were inoculated with the plasmid constructs encoding the fusion (F) and haemagglutinin-neuraminidase (HN) glycoproteins of Newcastle disease virus (NDV), either individually or in combination and challenged with velogenic NDV. The antibody level against NDV was measured using commercial enzyme linked immunosorbent assay (ELISA). In the first immunization regimen, SPF chickens inoculated twice with NDV-F or NDV-HN constructs elicited antibody responses 1 week after the second injection. However, the levels of the antibody were low and did not confer significant protection from the lethal challenge. In addition, administration of the plasmid constructs with Freund's adjuvant did not improve the level of protection. In the second immunization regimen, chickens inoculated twice with the plasmid constructs emulsified with Freund's adjuvant induced significant antibody titers after the third injection. Three out of nine (33.3%) chickens vaccinated with pEGFP-HN, five of ten (50.0%) chickens vaccinated with pEGFP-F and nine of ten (90.0%) chickens vaccinated with combined pEGFP-F and pEGFP-HN were protected from the challenge. No significant differences in the levels of protection were observed when the chickens were vaccinated with linearized pEGFP-F. The results suggested that more than two injections with both F and HN encoding plasmid DNA were required to induce higher level of antibodies for protection against velogenic NDV in chickens.