Temperature-Sensitive Defect of Mutants Isolated from L Cells Persistently Infected with Newcastle Disease Virus

The temperature-sensitive defects of virus mutants isolated from L cells persistently infected with Newcastle disease virus (NDV) were analyzed. Genetic grouping of the mutants by complementation tests was attempted by using several different methods, including yield analysis, RNA synthesis, and heterozygote formation at 42 to 43 C, the nonpermissive temperature. In each case, specific interference prevented detection of complementation. This interference was shown to occur prior to or at the level of virus RNA synthesis. Temperature-shift experiments with five different NDV(pi) clones showed that virus replication begun at 37 C could not be completed at the nonpermissive temperature. The activity of the NDV-specific RNA-dependent RNA polymerase in the cytoplasm of infected chicken embryo cells was not stable and could not be demonstrated directly. However, indirect measurement of RNA polymerase activity at the nonpermissive temperature was accomplished by studying the kinetics of virus-specific RNA synthesis in infected cells after temperature shift. Two types of response were obtained: with three NDV(pi) clones, virus-specific RNA synthesis ceased immediately upon transfer of infected cells to 42 to 43 C, whereas in cells infected with two other NDV(pi) clones, RNA synthesis continued for several hours at this temperature. These results suggested that there may be two types of ts defects in NDV(pi), both associated with virus-specific RNA polymerase activity.     

The housefly, Musca domestica, as a possible mechanical vector of Newcastle disease virus in the laboratory and field

Newcastle disease (Paramyxoviridae) is a highly infectious virus shed in the faeces of infected birds. Non-biting Muscid flies characteristically visit manure and decaying organic material to feed and oviposit, and may contribute to disease transmission. The housefly, Musca domestica (Linnaeus, 1758) (Diptera: Muscidae), has been implicated as a mechanical vector of numerous pathogens. In this study 2000 aerial net-captured houseflies were examined for their ability to harbour Newcastle disease virus (NDV). In an adjacent study, laboratory-reared flies were experimentally exposed to NDV La Sota strain. The virus was detected in the dissected gastrointestinal tract of laboratory-exposed flies for up to 72 h post-exposure, whereas the untreated control flies were negative.     

新城疫病毒抗肿瘤作用的研究进展

新城疫病毒(newcastle disease virus,NDV)属副黏病毒,由于其安全性,自从被发现以来,即受到广大研究者们的关注。经过多年的研究,新城疫病毒在抑制人肝癌、恶性胸膜间皮瘤、纤维肉瘤以及头颈癌细胞方面都取得了可喜的成果。目前,新城疫病毒抑瘤作用的机制尚未完全阐明,研究表明主要涉及诱导肿瘤细胞凋亡,发挥抑瘤佐剂作用,增强免疫细胞活性及抑制肿瘤化疗耐药。NDV即使在缺氧环境下,也可以稳定的发挥抗肿瘤作用,且其强毒株具有高效的抗肿瘤作用。本文主要就近年来新城疫病毒抗肿瘤作用的研究进展进行了综述。     

Nuclear entry and nucleolar localization of the Newcastle disease virus (NDV) matrix protein occur early in infection and do not require other NDV proteins.

A large proportion of the Newcastle disease virus (NDV) matrix (M) protein is found in the nuclei of infected chicken embryo cells. Kinetic analysis indicated that much of the M protein enters the nucleus early in infection, concentrating in discrete regions of the nucleus and remaining there throughout infection. The M protein was found in localized regions of the nuclei of a variety of cell lines infected with NDV. Immunostaining for both M protein and nucleolar antigens indicated that most of these regions represent nucleoli. Moreover, this nucleolar localization of the M protein was observed in chicken embryo cells infected with 11 different strains of NDV. Only the M protein of strain HP displayed a modified pattern, concentrating in the nucleolus early in infection but in the cytoplasm late in infection. M protein transiently expressed in COS-1 cells also localized to the nucleus and nucleolus, indicating that the M protein does not require other NDV proteins for this localization.     

Spectrum of Newcastle disease virus stability in gradients of temperature and pH

Newcastle disease (ND) is one of the highly pathogenic viral diseases of avian species. The disease is endemic in many developing countries where agriculture serves as the primary source of national income. Newcastle disease virus (NDV) belongs to the family Paramyxoviridae and is well characterized member among the avian paramyxovirus serotypes. The failure of vaccination is one of the major causes of NDV outbreaks in field condition. The present study gives a brief picture about the biology of NDV genome and its proteins under different conditions of temperature and pH. Our results indicate that the NDV is non-infective above 42 °C and unstable above 72 °C. The study will be useful in defining an optimum storage condition for NDV without causing any deterioration in its viability.     

Failure of multinucleated giant cell formation in k562 cells infected with newcastle disease virus and human parainfluenza type 2 virus

When K562 cells were infected with Newcastle disease virus (NDV) or human parainfluenza type 2 virus (hPIV-2), polykaryocyte formation could not be detected. Failure of multinucleated giant cell formation in K562 cells infected with either NDV or hPIV-2 is due to disturbance of the viral envelope-cell fusion step or to defect in the cell-cell fusion step, respectively. Especially, NDV completely replicated in K562 cells, and the hemagglutinin-neuraminidase and fusion proteins expressed on the cell surface of NDV-infected K562 cell were fully functional for fusion inducing activity. Therefore, the cell membranes of K562 cells are considered to be resistant to virus-induced cell fusion. Membrane fusion is regulated by many host factors including membrane fluidity, cytoskeletal systems, and fusion regulatory proteins system. An unknown regulatory mechanism of virus-induced cell fusion may function on the cell surface of K562 cells.     

新城疫病毒抗肿瘤效应机制的研究现状

溶瘤病毒疗法是一种重要的抗癌手段。经研究,新城疫病毒(Newcastlediseasevirus,NDV)是一种非常有效的溶瘤病毒(oncolyticvirus,OV),它能选择性杀伤肿瘤细胞,对正常细胞几乎无影响。本文从NDV诱导肿瘤细胞发生凋亡、自噬、抑制细胞代谢、刺激机体免疫反应和诱导肿瘤细胞发生核糖体应激反应等方面综述了新城疫病毒的抗肿瘤效应机制,并着重探讨了NDV通过诱导核糖体压力应激反应调控肿瘤细胞翻译系统并诱导细胞发生凋亡的具体机制,旨在为今后NDV抗肿瘤作用的深入研究及靶向治疗癌症提供更加扎实丰富的理论基础。     

Newcastle disease virus fusion protein expressed in a fowlpox virus recombinant confers protection in chickens.

A cDNA copy of the RNA encoding the fusion (F) protein of Newcastle disease virus (NDV) strain Texas, a velogenic strain of NDV, was obtained and the sequence was determined. The 1,792-base-pair sequence encodes a protein of 553 amino acids which has essential features previously established for the F protein of virulent NDV strains. These include the presence of three strongly hydrophobic regions and pairs of dibasic amino acids in the pentapeptide Arg-Arg-Gln-Arg-Arg preceding the putative cleavage site. When inserted into a fowlpox virus vector, a glycosylated protein was expressed and presented on the surface of infected chicken embryo fibroblast cells. The F protein expressed by the recombinant fowlpox virus was cleaved into two polypeptides. When inoculated into susceptible birds by a variety of routes, an immunological response was induced. Ocular or oral administration of the recombinant fowlpox virus gave partial protection, whereas both intramuscular and wing-web routes of inoculation gave complete protection after a single inoculation.     

鹅源新城疫病毒ZJ1株微型基因组的构建及其初步应用

在获得鹅源新城疫病毒ZJ1株全基因组序列的基础上,用增强型绿色荧光蛋白（eGFP）报告基因取代鹅源新城疫病毒ZJ1株整个编码区,只保留与病毒复制、转录和病毒粒子包装相关的调控序列,将其反向克隆入转录载体TVT7R(0.0)中,构建了该毒株的微型基因组。当转染用辅助病毒ZJ1株感染的Hep_2细胞时报告基因得到表达,表明此微型 基因组RNA可被辅助病毒提供的NP、P和L蛋白翻译。同时将该病毒NP、P和L蛋白基因分别克隆入真核表达载体pCI_neo中,构建了表达该病毒NP、P与L蛋白的辅助质粒,用此微型基因组对辅助质粒的表达产物进行了功能鉴定并对该病毒拯救过程中痘苗病毒的最适感染剂量进行了摸索。以上研究为该病毒的成功拯救及开展其它相关研究奠定了基础。     

A 38,000-dalton antigen found in Namalwa cells induced by Newcastle disease virus

An antigen has been isolated from Namalwa cells, a Burkitt lymphoma line, that was induced by Newcastle disease virus (NDV) for interferon production. The antigen was extracted by 3 M NaCl from ribonucleoprotein particles (RNP), obtained from the nuclear 0.01 M Tris extract, and was purified by hydroxylapatite chromatography, phosphocellulose chromatography, and preparative sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Its molecular weight was 38 kilodalton (kDa) as determined by SDS-PAGE. The tryptic peptide map of 125I-labeled antigen contained seven major peptides. The antigen was not found in HeLa cells, normal human liver or in Namalwa cells that had not been induced by the virus. This result suggests that this antigen was produced in Namalwa cells as a result of induction by the NDV virus.     

NDV CN株对几种人肿瘤细胞的杀伤作用及其机理的分析

本文研究了NDV-CN株对5株不同的人肿瘤细胞的体外杀伤作用。结果表明5株肿瘤细胞对NDV-CN敏感,于感染早期出现细胞收缩变圆,失去贴壁性,感染第5天时细胞存活率低于10％。其中尤以HEP3B细胞最敏感。但NDV-CN株对人二倍体细胞2BS有弱杀伤性。病毒感染早期可检测到感染细胞中有病毒核酸的复制、感染细胞表面有病毒蛋白的表达,脑浆内有病毒粒子存在。NDV-CN株主要诱导HEP3B及T24细胞产生凋亡,主要诱导Hep2、Hela及A549细胞产生坏死。     

Factors Affecting the Sensitivity of Different Viruses to Interferon

When the sensitivities to interferon of Newcastle disease virus (NDV) and vesicular stomatitis virus (VSV) were compared by the plaque reduction method in chick embryo cell cultures, NDV was found to be 45-fold more resistant than VSV. This difference was exaggerated when a multiple-cycle yield inhibition method was employed. In marked contrast, when the same viruses were tested by a single-cycle yield inhibition method, the difference in sensitivity to interferon of the two viruses was virtually eliminated. Further investigation showed that, in chick embryo cells exposed to interferon, the resistance to NDV decayed more rapidly than resistance to VSV. This finding explained the divergent results obtained with the two viruses when single- or multiple-cycle replication techniques were employed. Experiments carried out with L cells showed that cellular antiviral resistance decayed much more slowly in these cells than in chick embryo cells. Consequently, when measured by the plaque reduction method in L cells, no difference was observed in the sensitivity to interferon of VSV and NDV(pi), a mutant of NDV which replicates efficiently in L cells. A procedure is suggested for determining the relative sensitivities to interferon of different viruses under conditions which minimize the role of decay of antiviral resistance in the host cells.     

Virus-induced alterations of lymphoid tissues. II. Lymphocyte receptors for Newcastle disease virus

Newcastle disease virus (NDV) agglutinates rat, mouse and human lymphocytes. Viral agglutination of rat thoracic duct lymphocytes was specifically inhibited by N-acetylneuraminic acid implying that the receptors terminate in sialic acid. While the attachment of virus to lymphocytes was rapid the reaction was unstable and NDV was shown to elute at 37 °C. Evidence was obtained that the eluting virus cleaved sialic acid from the surface of lymphocytes and concomitantly destroyed this lymphocyte receptor.     

Detection of Newcastle disease virus using nucleic acid sequence-based amplification.

Newcastle disease (ND) is a contagious and widespread avian disease affecting most species of birds. ND virus (NDV) is the only member of the avian paramyxovirus serotype 1 (APMV1) causing ND outbreak in bird flocks. The technique of nucleic acid sequence-based amplification (NASBA) is a potential method to rapidly and reliably detect NDV isolates. Here, we describe an effective and unprecedented method for detecting NDV strains of all pathotypes. A conserved region of the fusion protein gene was used for designing oligonucleotides specific to all NDV pathotypes. The dynamic range of this NDV NASBA detection method is comparable to virus culture and therefore the NDV NASBA method is a potential alternative for NDV screening and surveillance.     

Construction and characterization of recombinant fowlpox virus co-expressing F and HN genes of newcastle disease virus and gB gene of infectious larygnotracheitis virus

The Fusion (F) and Haemagglutinin-Neuraminidase (HN) genes of Newcastle disease virus (NDV) and the glycoprotein B (gB) gene of infectious laryngothracheitis virus (ILTV) as well as a LacZ reporter gene were all inserted into a nonessential gene of fowlpox virus (FPV) 017 strain by homologous recombination. The NDV and ILTV genes were each under the control of a fowlpox virus immediate early/late promoter (LP2EP2), whereas the LacZ reporter gene expression cassette was regulated by a P11 late promoter. A recombinant FPV harboring the F, HN and gB genes as well as the LacZ gene, designated as rFPV-F/HN/gB/LacZ, was obtained after ten cycles of blue plaque purification. The presence of the NDV and ILTV genes was confirmed by PCR. The expression of the recombinant proteins in rFPV-F/HN/gB/LacZ was characterized by Western blot (F and gB proteins) and indirect immunofluorescence tests (F, HN and gB proteins). The results demonstrated that all four foreign proteins, which were encoded within a 10-kb gene fragment, could be expressed authentically and efficiently. Compared with the parental virus, rFPV-F/HN/gB/LacZ showed no obvious difference with respect to virus replication and cytopathogenic effects in the cell culture of chicken embryo fibroblasts (CEF). Overall, this study suggests that FPV can be a useful live virus vector for the expression of multiforeign genes against multiple avian pathogens.     

Modification of low virulent Newcastle disease virus infection in chickens infected with reticuloendotheliosis virus

One-day-old SPF chicks were inoculated with reticuloendotheliosis virus (REV) which had been isolated from contaminated Marek's disease vaccine. Then they were subjected to super infection with the B1 or TCND strain of Newcastle disease virus (NDV) and examined for virus recovery, antibody response and the appearance of symptoms. Regardless of the time, from 0 to 8 weeks, of inoculation with the NDV-B1 strain after the REV infection, the antibody response was suppressed and the duration of the NDV recovery prolonged. Specific death preceded by severe respiratory or neural signs occurred more frequently to chicks inoculated with REV than to uninoculated controls after inoculation with the NDV-B1 strain in the neonatal stage or with the NDV-TCND strain at 5 weeks of age.     

Isolation and characterization of mouse FM3A cell mutants which are devoid of Newcastle disease virus receptors.

A method was developed to select host cell mutants which did not permit the replication of Newcastle disease virus (NDV), and 14 isolates of NDV-nonpermissive mutants of mouse FM3A cells were obtained. All these isolates were judged to be deficient in NDV receptors, since their ability to adsorb 3H-labeled NDV virions was markedly decreased. They were tested for genetic complementation in pairs by cell fusion and shown to fall into a single recessive complementation group, which was designated as Had-1. Vesicular stomatitis virus was able to replicate in this mutant to produce infectious progeny, but the glycoprotein of the released virion was abnormal in size, suggesting a defective processing of the asparagine-linked carbohydrate chains in the mutant cell. The Had-1 mutant was resistant to wheat germ agglutinin, but sensitive to a Griffonia simplicifolia lectin, GS-II, which recognizes terminal N-acetylglucosamine residues. The altered sensitivity to these plant lectins compared with that of the parental FM3A cells indicates that sialylated sugar chains on the cell surface are almost absent from the Had-1 cells, thereby rendering the cells NDV receptor deficient.     

Oncolytic specificity of Newcastle disease virus is mediated by selectivity for apoptosis-resistant cells

Newcastle disease virus (NDV) is a negative-sense RNA virus that has been shown to possess oncolytic activity. NDV's selective replication in tumor cells has been previously suggested to be due to the lack of a proper antiviral response in these cells. Here we demonstrate that NDV possesses oncolytic activity in tumor cells capable of a robust type I interferon (IFN) response, suggesting that another mechanism underlies NDV's tumor specificity. We show that the oncolytic selectivity of NDV for tumor cells is dependent upon tumor cell resistance to apoptosis. Utilizing the human non-small-cell lung cancer cell line A549 overexpressing the antiapoptotic protein Bcl-xL, we show significant enhancement of oncolytic activity and NDV replication. Interestingly, while the Bcl-xL-overexpressing cells were resistant to apoptotic stimuli induced by chemotherapeutic agents and early viral replication, during the subsequent viral cycles, we observed a paradoxical increase in apoptosis in response to NDV. The increased oncolytic activity seen was secondary to enhanced viral replication and syncytium formation. The induction of a type I IFN response was enhanced in Bcl-xL cells. Overall, these findings propose a new mechanism for cancer cell specificity for NDV, making it an attractive anticancer agent for chemoresistant tumors with enhanced antiapoptotic activity.     

鸭源新城疫病毒M蛋白核定位信号突变影响病毒的毒力和复制能力

【目的】研究鸭源新城疫病毒(Newcastle disease virus,NDV)M蛋白核定位信号(nuclear localization signal,NLS)突变对其毒力和复制能力的影响。【方法】利用鸭源NDV SS1株P基因和F基因上的AgeⅠ和Bstz17Ⅰ酶切位点,将overlapPCR方法获得的M蛋白NLS突变的片段替换到p NDV/SS1GFP中获得全长质粒pNDV/SS1GFP-M/NLSm。通过反向遗传学技术拯救M蛋白NLS突变体病毒,并对拯救的病毒进行血凝(hemagglutination,HA)试验、荧光试验和M基因测序鉴定。另外,对突变体病毒进行M蛋白的亚细胞定位观察,以及病毒的生物学特性、空斑形成能力和体外增殖能力测定。【结果】成功构建M蛋白NLS突变的全长质粒pNDV/SS1GFP-M/NLSm。细胞转染物接种鸡胚后的第1代尿囊液无HA效价,盲传3代才能检测到拯救病毒的HA效价。进一步的荧光试验和M基因测序确定拯救的病毒是突变体病毒r SS1GFP-M/NLSm。与亲本病毒rSS1GFP相比,突变体病毒M蛋白由细胞核定位变为细胞质定位。此外,突变体病毒的毒力、在鸡胚上的复制能力以及在细胞中的空斑形成能力显著降低,并且感染细胞后产生的细胞病变轻微,M蛋白和绿色荧光蛋白的表达量均降低,说明M蛋白NLS突变使病毒的体外增殖能力受到抑制。【结论】NLS突变导致的M蛋白细胞核定位功能丧失可明显降低鸭源NDV的毒力和复制能力。