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

Virus mutants (NDV(pi)) isolated from L cells persistently infected with the Herts strain of Newcastle disease virus have been previously reported by this laboratory to differ from the wild-type virus (NDV(o)) in several physical and biological properties. It has now been determined that, in addition to these differences, the NDV(pi) mutants are also spontaneously selected temperature-sensitive mutants. The temperature sensitivity of 10 NDV(pi) clones was confirmed by temperature inhibition, plaquing efficiency, and single-cycle yield experiments. The cut-off temperature, at which more than 90% of virus replication is inhibited was between 41 and 42 C. All 10 NDV(pi) clones were also found to be defective in virus-specific ribonucleic acid (RNA) synthesis in infected chick embryo cells at 42 C and are tentatively classified as RNA(-). The possible relationships of the temperature sensitivity, the other NDV(pi) properties, and the maintenance of the persistently infected state are discussed.     

Cells Persistently Infected with Newcastle Disease Virus: II. Ribonucleic Acid and Protein Synthesis in Cells Infected with Mutants Isolated from Persistently Infected L Cells

A comparison of the replication patterns in L cells and in chick embryo (CE) cell cultures was carried out with the Herts strain of Newcastle disease virus (NDV(o)) and with a mutant (NDV(pi)) isolated from persistently infected L cells. A significant amount of virus progeny, 11 plaque-forming units (PFU)/cell, was synthesized in L cells infected with NDV(o), but the infectivity remained cell-associated and disappeared without being detectable in the medium. In contrast, in L cells infected with NDV(pi), progeny virus (30 PFU/cell) was released efficiently upon maturation. It is suggested that the term "covert" rather than "abortive" be used to describe the infection of L cells with NDV(o). In both L and CE cells, the latent period of NDV(pi) was 2 to 4 hr longer than for NDV(o). The delay in synthesis of viral ribonucleic acid (RNA) in the case of NDV(pi) coincided with the delay in the inhibition of host RNA and protein synthesis. Although both NDV(o) and NDV(pi) produced more progeny and more severe cell damage in CE cells than in L cells, the shut-off of host functions was significantly less efficient in CE cells than in L cells. Paradoxically, no detectable interferon was produced in CE cells by either of the viruses, whereas in L cells most of the interferon appeared in the medium after more than 90% of host protein synthesis was inhibited. These results suggest that the absence of induction of interferon synthesis in CE cells infected with NDV is not related to the general shut-off of host cell synthetic mechanisms but rather to the failure of some more specific event to occur. In spite of the fact that NDV(pi) RNA synthesis commenced 2 to 4 hr later than that of NDV(o), interferon was first detected in the medium 8 hr after infection with both viruses. This finding suggests that there is no relation between viral RNA synthesis and the induction of interferon synthesis.     

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.     

Viral Ribonucleic Acid Synthesis by Newcastle Disease Virus Mutants Isolated from Persistently Infected L Cells: Effect of Interferon

The synthesis of different viral ribonucleic acid (RNA) species was studied in chick embryo (CE) and mouse L-cell cultures infected with the Herts strain of Newcastle disease virus (NDV(o)) and a mutant isolated from persistently infected L cells (NDV(pi)). In CE cell cultures, both viruses synthesized significant amounts of 54, 36, and 18S RNA. However, in L cells, synthesis of 54S virion RNA was markedly reduced. From these results, it seems likely that the low yield of infective virus in L cells is due to a deficient synthesis of 54S RNA in this host. On this basis, however, it is apparent that the "covert" replication of NDV(o) in L cells is due to factors other than viral RNA synthesis. When low concentrations of interferon were used to pretreat CE cells, a differential effect on the synthesis of various RNA species was observed. The 18S RNA of NDV(o) was more sensitive to interferon action than the 36 and the 54S RNA species. In contrast, the 18S RNA of NDV(pi) was less sensitive than the 36S and the 54S RNA. The inhibition of 54S RNA synthesis correlated with the reduction of viral yield and explained the greater sensitivity of NDV(pi) to interferon.     

Isolation and Properties of Newcastle Disease Virus Nucleocapsid

Deoxycholate (DOC) disrupted virions of Newcastle disease virus (NDV), releasing viral nucleocapsids. The nucleocapsids sedimented at about 200S in sucrose gradients and measured from 1.3 to 1.4 mu long by electron microscopy. NDV nucleo-capsids were resistant to pancreatic ribonuclease. These nucleocapsids contained all the 50S ribonucleic acid (RNA) in NDV virions, while virus-associated RNA sedimenting at less than 50S was external to the virions.     

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。     

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.     

Inhibition of Host-Cell Protein and Ribonucleic Acid Synthesis by Newcastle Disease Virus

The mechanisms of Newcastle disease virus-(NDV) induced inhibition of cell protein and ribonucleic acid (RNA) synthesis were investigated. It was observed that the ability of NDV to inhibit cell RNA synthesis is dependent on the virus strain. The inhibitors, azauridine and cycloheximide, were added to cell cultures at different times after infection to study the roles of protein and RNA synthesis in the viral inhibition process. Viral inhibition of cell RNA synthesis and viral inhibition of cell protein synthesis become resistant to cycloheximide at a different time after infection than that in which they become resistant to azauridine. The results indicate that the inhibition of cell RNA synthesis by the Texas strain involves the synthesis of inhibitory proteins which are coded by the viral genome. The Texas and Beaudette strains of NDV appear to employ different mechanisms for the inhibition of host-cell protein synthesis. Viral inhibition of cell protein synthesis does not appear to cause, or be the result of, viral inhibition of cell RNA synthesis.     

用反向遗传技术致弱基因VIId型鹅源新城疫病毒ZJI株

将新城疫病毒ZJI株基因组cDNA全长分成7个片段,依次连接并克隆至TVT7R转录载体中,构建了含ZJI株全基因组cDNA的转录载体(pNDV/ZJI),pNDV/ZJI与3个辅助表达质粒pCI-NP、pCI-P和pCI-L共转染BSR-T7/5细胞,成功拯救出了具有感染性的新城疫病毒粒子。设计两对引物,经overlapPCR方法将该毒株F蛋白裂解位点的112、115和117位碱性氨基酸突变成弱毒株特征的非碱性氨基酸后,替换pNDV/ZJI上的对应序列,构建了转录载体pNDV/ZJIFM,将pNDV/ZJIFM与3个辅助表达质粒共转染BSR-T7/5细胞,成功拯救出了致弱的基因VIId型鹅源新城疫病毒NDV/ZJIFM,获救病毒的鸡胚最小致死剂量平均死亡时间(MDT)大于120h,同时该病毒的脑内接种致病指数(ICPI)为0.16,上述结果表明,获救病毒的毒力已被致弱,是一个较为理想的疫苗候选株。     

Differentially regulated interferon response determines the outcome of Newcastle disease virus infection in normal and tumor cell lines

Newcastle disease virus (NDV) is a negative-strand RNA virus with oncolytic activity against human tumors. Its effectiveness against tumors and safety in normal tissue have been demonstrated in several clinical studies. Here we show that the spread of NDV infection is drastically different in normal cell lines than in tumor cell lines and that the two cell types respond differently to beta interferon (IFN-beta) treatment. NDV rapidly replicated and killed HT-1080 human fibrosarcoma cells but spread poorly in CCD-1122Sk human skin fibroblast cells. Pretreatment with endogenous or exogenous IFN-beta completely inhibited NDV replication in normal cells but had little or no effect in tumor cells. Thus, the outcome of NDV infection appeared to depend on the response of uninfected cells to IFN-beta. To investigate their differences in IFN responsiveness, we analyzed and compared the expression and activation of components of the IFN signal transduction pathway in these two types of cells. The levels of phosphorylated STAT1 and STAT2 and that of the ISGF3 complex were markedly reduced in IFN-beta-treated tumor cells. Moreover, cDNA microarray analysis revealed significantly fewer IFN-regulated genes in the HT-1080 cells than in the CDD-1122Sk cells. This finding suggests that tumor cells demonstrate a less-than-optimum antiviral response because of a lesion in their IFN signal transduction pathway. The rapid spread of NDV in HT-1080 cells appears to be caused by their deficient expression of anti-NDV proteins upon exposure to IFN-beta.     

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.     

Enhancement of Newcastle Disease Virus-Induced Fusion of Mouse L Cells by Sodium Vanadate

Sodium vanadate enhanced Newcastle disease virus (NDV)-induced cell fusion in L cells, and there was a direct correlation between the degree of cell fusion and the dose of vanadate added. When anti-F protein of NDV monospecific antiserum was added to the culture fluid of L cells infected with NDV, the enhancement of cell fusion was suppressed. In contrast, neither anti-HN nor anti-M protein monospecific antiserum inhibited the enhancement. Incubation at low temperature (4 C) and addition of sodium azide to the culture fluid suppressed the enhancement. The suppression by azide was seen only when the drug was added within 5 min after the beginning of incubation of NDV-infected L cells with vanadate. On the other hand, incubation at low temperature inhibited the enhancement at any time during incubation with vanadate. Cytochalasin D also inhibited the enhancement if it was added at any time during incubation with vanadate.     

Autophagy Benefits the Replication of Newcastle Disease Virus in Chicken Cells and Tissues

Newcastle disease virus (NDV) is an important avian pathogen. We previously reported that NDV triggers autophagy in U251 glioma cells, resulting in enhanced virus replication. In this study, we investigated whether NDV triggers autophagy in chicken cells and tissues to enhance virus replication. We demonstrated that NDV infection induced steady-state autophagy in chicken-derived DF-1 cells and in primary chicken embryo fibroblast (CEF) cells, evident through increased double- or single-membrane vesicles, the accumulation of green fluorescent protein (GFP)-LC3 dots, and the conversion of LC3-I to LC3-II. In addition, we measured autophagic flux by monitoring p62/SQSTM1 degradation, LC3-II turnover, and GFP-LC3 lysosomal delivery and proteolysis, to confirm that NDV infection induced the complete autophagic process. Inhibition of autophagy by pharmacological inhibitors and RNA interference reduced virus replication, indicating an important role for autophagy in NDV infection. Furthermore, we conducted in vivo experiments and observed the conversion of LC3-I to LC3-II in heart, liver, spleen, lung, and kidney of NDV-infected chickens. Regulation of the induction of autophagy with wortmannin, chloroquine, or starvation treatment affects NDV production and pathogenesis in tissues of both lung and intestine; however, treatment with rapamycin, an autophagy inducer of mammalian cells, showed no detectable changes in chicken cells and tissues. Moreover, administration of the autophagy inhibitor wortmannin increased the survival rate of NDV-infected chickens. Our studies provide strong evidence that NDV infection induces autophagy which benefits NDV replication in chicken cells and tissues.     

表达绿色荧光蛋白重组新城疫病毒 LaSota疫苗株的构建

新城疫病毒是理想的新型活病毒疫苗载体,具有巨大的优势和应用前景。采用生产实践中广泛应用、免疫效果良好的NDV LaSota弱毒疫苗株,建立了反向遗传操作系统。在此基础上,进一步构建了表达绿色荧光蛋白(GFP)的重组NDV基因组cDNA克隆,成功救获了重组病毒rLaSota-EGFP,病毒F1代尿囊病毒液按1×104EID50接种9~10日龄SPF鸡胚尿囊腔,接种后分别于24h、48h、72h及96h收获尿囊液,检测平均HA滴度分别为28、210.3、211.3和211,每mL尿囊液病毒量EID50分别为108.64、109.22、109.21和109.64,重组病毒与亲本株生长滴度在相近时间达到峰值,生长动力学特性与亲本株无明显差异。各代次重组病毒按1×106EID50病毒量接种9~10日龄SPF鸡胚,96h内完全不致死鸡胚。救获重组病毒保持了LaSota弱毒疫苗亲本毒株对鸡胚良好的高滴度生长适应和低致病特性,并且鸡胚连续传9代次仍保持GFP的稳定表达及生物学特性不变。重组病毒rLaSota-EGFP的成功救获为开展新城疫病毒活载体疫苗研制提供了可行的技术平台。