Newcastle Disease Virus Infection of L Cells

Newcastle disease virus (NDV) California strain reportedly grows poorly in L cells but replicates very well in chicken embryo cells. NDV-infected L cell cultures show a characteristic virus growth curve with respect to uridine incorporation, but plaque assays of the virus produced 24 h postinfection (PI) show no infectious particles when assayed on L cell monolayers and only a very low titer on chick cell monolayers. Plasma membranes isolated and purified from infected L cells 8 h PI contain all of the major virion proteins. In addition, NDV-infected L cells show a 50% loss of H-2 antigenic activity, a phenomenon previously observed in cells productively infected with vesicular stomatitis virus. These results suggest that at least part of the normal process of NDV maturation occurs in NDV-infected L cells. Sodium dodecyl sulfate-polyacrylamide gel patterns of supernatant virus purified from cells radiolabeled with amino acids from 3 to 24 h PI in the presence of actinomycin D show that all the major NDV structural proteins are present. Electron micrographs of NDV-infected L cells show extensive virus maturation at cell membranes. It can be concluded that infection of L cells with NDV results in a normal production of virus-specific RNA, synthesis of all the major structural proteins, association of the viral envelope proteins with the L cell plasma membrane, and the loss of cell surface H-2 antigenic activity. However, most of the virus particles produced are noninfectious.     

Recombinant Newcastle Disease Virus as a Vaccine Vector

A complete cDNA clone of the Newcastle disease virus (NDV) vaccine strain Hitchner B1 was constructed, and infectious recombinant virus expressing an influenza virus hemagglutinin was generated by reverse genetics. The rescued virus induces a strong humoral antibody response against influenza virus and provides complete protection against a lethal dose of influenza virus challenge in mice, demonstrating the potential of recombinant NDV as a vaccine vector.     

Use-Dilution Test and Newcastle Disease Virus

The use-dilution test for evaluating the effectiveness of disinfectants against bacteria was modified to determine the effectiveness of disinfectants against a group of viruses. Modifications were kept to a minimum to retain the general principles of the test and thereby retain the test's familiarity among testing laboratory personnel. Modifications included the use of a standard allantoic fluid suspension of Newcastle disease virus instead of a standard bacterial culture. The only other modification was the inoculation of six embryonated chicken eggs (10 to 12 days old) with 0.1 ml of nutrient broth into which a carrier ring was transferred after a standard period in diluted disinfectant. The death or survival of 60 embryos, then, is the criterion by which a disinfectant can be judged effective at use-dilution. Experiments are described which establish the validity of the modified test procedure. The effectiveness of nine common disinfectants against Newcastle disease virus as judged by this test procedure is reported.     

Influence of Viral Envelope on Newcastle Disease Virus Infection

The adsorption characteristics of Newcastle disease virus (NDV) propagated in chicken cells (NDV-C) and in human cells (NDV-H) were examined. Adsorption experiments performed at different temperatures indicated that virus propagated in a particular cell infected that cell type more readily than did virus propagated in a different host. For example, NDV-C was more efficient in initiating infection of chicken cells at 22 C than was NDV-H; the reverse was true when human cells were employed. The results indicate that infection of susceptible cells by NDV is influenced by the host cell in which the virus was propagated. The data also suggest that NDV may be useful in studies on homologous and heterologous membrane-membrane interactions.     

几株益生菌的体外抗新城疫病毒作用

【目的】探讨益生菌的抗新城疫病毒(NDV)作用并分析其可能的机制。【方法】采用NDV血凝试验和MTT比色法,分别在体外和鸡胚成纤维细胞(CEF)上评价益生菌对NDV血凝价和抑制率的影响。【结果】所选择的5株益生菌及其代谢产物都极显著地降低了NDV的血凝价,而2株致病菌及其代谢产物对NDV的血凝价均没有影响,这一结果说明益生菌可能对NDV具有直接破坏的作用,并且具有菌株特异性。益生菌可以显著地提高CEF对NDV的抑制率,并且这种作用具有量效关系(P0.01)。益生菌与细胞作用后再感染病毒,对NDV抑制率升高的结果反映了益生菌对NDV吸附细胞的阻断作用;从益生菌与病毒同时接入细胞后降低病毒对细胞侵害的现象,可以看出益生菌可能对病毒具有直接破坏作用;在细胞感染病毒后再接入益生菌对NDV抑制率极低的现象说明,病毒感染后益生菌再很难起作用。【结论】益生菌对NDV既具有直接破坏的作用,又可以阻断NDV对细胞的感染、抑制其在细胞内的增殖。     

Precursor Protein for Newcastle Disease Virus

The course of viral protein synthesis during infection of chicken embryo fibroblasts with Newcastle disease virus (NDV) L. Kansas has been followed by using sodium dodecyl sulfate polyacrylamide gel electrophoresis. Of the three major virion polypeptide molecular weight classes, I (78,400 daltons), II (53,500 daltons), and III (37,600 daltons), only II, having the same electrophoretic mobility as nucleocapsid polypeptide, appears to be the cleavage product of a precursor polypeptide PII (64,800 daltons) detected in NDV-infected cells after brief labeling with radioactive amino acids. Nucleocapsids were isolated from NDV-infected cells which had been pulse-labeled with radioactive amino acids or pulse-labeled and further incubated with unlabeled amino acids. Gel electrophoretic analysis of proteins derived from nucleocapsids showed that an increase in the period of incubation with unlabeled amino acids resulted in an increase in the amount of radioactivity in nucleocapsid protein. Polypeptide PII was not detected as a transient component of the isolated nucleocapsid fraction. These results are consistent with two interpretations. The product of PII cleavage is (i) nucleocapsid polypeptide, or (ii) a nonvirion or minor envelope polypeptide having the same electrophoretic mobility as nucleocapsid polypeptide.     

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。     

Thermal Inactivation of Newcastle Disease Virus

The rate of destruction of hemagglutinins and infectivity of Newcastle disease virus was determined over a temperature range of 37.8 to 60 C. From the calculated values of deltaH and deltaS, it was concluded that inactivation of the hemagglutinating activity and viral infectivity was due to protein denaturation.     

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.     

Molecular Weight Determination of Sendai and Newcastle Disease Virus RNA

The molecular weights of Sendai and Newcastle disease virus RNA were estimated by sedimentation in sucrose gradients and by length measurements in the electron microscope under both denaturing and nondenaturing conditions. Sedimentation analyses under denaturing conditions yielded molecular weight estimates of 2.3 x 10(6) to 2.6 x 10(6), whereas length measurements yielded estimates of 5.2 x 10(6) to 5.6 x 10(6) for both denatured and nondenatured viral RNA. It would appear that the conditions of denaturation used (99% dimethyl sulfoxide at 26 C, and reaction with 1.1 M formaldehyde for 10 min at 60 C) do not equally denature parainfluenza virus RNA and other RNAs, such as cellular rRNA, 45S rRNA precursor, and R17 RNA.     

Microculture System for Detection of Newcastle Disease Virus Antibodies

A microculture system utilizing cytopathic effect (CPE) and hemadsorption (HAd) end points was effective in determining the level of Newcastle disease virus (NDV) antibodies. The microculture system was of comparable sensitivity to the plaque reduction test for the detection of NDV antibodies. The standards by which the CPE and HAd microculture tests would be considered reproducible were defined. The results indicate that the CPE and HAd microculture tests are reproducible within one twofold dilution.     

Nucleocapsid Protein Subunits of Simian Virus 5, Newcastle Disease Virus, and Sendai Virus

Helical nucleocapsids of each of the paramyxoviruses simian virus 5 (SV5), Newcastle disease virus (NDV), and Sendai virus have been isolated in two different forms. One form contains larger protein subunits and is obtained from mature virions or infected cells dispersed by ethylenediaminetetraacetic acid. The other form possesses smaller subunits and is obtained from infected cells dispersed by trypsin. The estimated molecular weights of the larger subunits in the three viruses are similar: SV5, 61,000; Sendai virus, 60,000; NDV, 56,000. The smaller nucleocapsid subunits are also very similar: SV5, 43,000; Sendai virus, 46,000; NDV, 47,000. The helical nucleocapsid composed of the smaller subunit appears to be less flexible and more stable than that formed by the larger subunit. There is suggestive evidence that conversion of the larger subunit to the smaller by proteolytic cleavage may occur intracellularly. The possibility that such a mechanism could be involved in the accumulation of nucleocapsid in cells persistently infected with paramyxoviruses is discussed.     

Different Regions of the Newcastle Disease Virus Fusion Protein Modulate Pathogenicity

Newcastle disease virus (NDV), also designated as Avian paramyxovirus type 1 (APMV-1), is the causative agent of a notifiable disease of poultry but it exhibits different pathogenicity dependent on the virus strain. The molecular basis for this variability is not fully understood. The efficiency of activation of the fusion protein (F) is determined by presence or absence of a polybasic amino acid sequence at an internal proteolytic cleavage site which is a major determinant of NDV virulence. However, other determinants of pathogenicity must exist since APMV-1 of high (velogenic), intermediate (mesogenic) and low (lentogenic) virulence specify a polybasic F cleavage site. We aimed at elucidation of additional virulence determinants by constructing a recombinant virus that consists of a lentogenic NDV Clone 30 backbone and the F protein gene from a mesogenic pigeon paramyxovirus-1 (PPMV-1) isolate with an intracerebral pathogenicity index (ICPI) of 1.1 specifying the polybasic sequence R-R-K-K-R*F motif at the cleavage site. The resulting virus was characterized by an ICPI of 0.6, indicating a lentogenic pathotype. In contrast, alteration of the cleavage site G-R-Q-G-R*L of the lentogenic Clone 30 to R-R-K-K-R*F resulted in a recombinant virus with an ICPI of 1.36 which was higher than that of parental PPMV-1. Substitution of different regions of the F protein of Clone 30 by those of PPMV-1, while maintaining the polybasic amino acid sequence at the F cleavage site, resulted in recombinant viruses with ICPIs ranging from 0.59 to 1.36 suggesting that virulence is modulated by regions of the F protein other than the polybasic cleavage site.     

Lipoprotein Inhibitor of Newcastle Disease Virus from Chicken Lung

A lipoprotein inhibitor of Newcastle disease virus was obtained from chicken lung tissue by means of dilute alkaline extraction procedures. The inhibitor was further purified by ammonium sulfate fractionation, isoelectric precipitation, and density gradient centrifugation. The purified lipoprotein inhibited active Newcastle disease virus hemagglutination at a concentration of 2.0 mug/ml which represented a 30-fold purification over the original extract. Infection of chicken embryo fibroblasts by Newcastle disease virus was also inhibited by the purified lipoprotein, the degree of inhibition depending upon the inhibitor-to-virus ratio. Chemical analysis of the purified inhibitor provided a composition of 72% lipid, 26% protein, and 3% carbohydrate, although some compositional variation was observed from one preparation to another. The chloroform-soluble lipids were shown to contain 40 to 50% phospholipid and 10 to 20% cholesterol; of the fatty acids recovered from the saponified lipoprotein, 39% was palmitic, 22% oleic, and 17% stearic. Careful analyses of large quantities of the inhibitor revealed a small (0.84%) but significant content of sialic acid. Removal of sialic acid from the lipoprotein by means of digestion with neuraminidase produced a sharp diminution in inhibitory properties. A delipidized form of the inhibitor was obtained by ether extraction, and this material produced a single broad band of precipitate in gel immunodiffusion tests.     

Phospholipids in Newcastle Disease Virus infected cells.

Infection of chicken cells with Newcastle Disease Virus modifies phosphatidylserine and phosphatidylcholine synthesis in the host cell. The virion contains cellular phospholipids synthesized both before and after infection. Relative concentration of various labeled phospholipids in the virus differ from those in the corresponding cells and their surface membranes. Late in infection, fragments of membranes with a distribution of labeled phospholipids similar but not identical to that of the virus can be found in the supernatant of infected cells. The significance of these findings is discussed in relation to the origin of viral phospholipids and the intervention of the host cell membrane in the assembly of the viral envelope.     

Isolation and Purification of the Envelope Proteins of Newcastle Disease Virus

A procedure has been developed for the isolation of Newcastle disease virus (NDV) envelope proteins. The two surface glycoproteins and the non-glycosylated membrane protein were solubilized with 2% Triton X-100 and 1 m KCl. Removal of the KCl by dialysis yielded by precipitation a pure preparation of the non-glycosylated membrane protein, which is insoluble in solutions of low ionic strength. The soluble fraction consisting of the two glycoproteins possessed full neuraminidase and hemagglutinating activities. The two glycoproteins could be separated by rate zonal sedimentation in a sucrose gradient containing 1% Triton X-100 and 1 m KCl. Under these conditions, the sedimentation coefficient of the larger glycoprotein, virus protein 1, was 9.3s, and that of the smaller, virus protein 2, was 6.1s. Both hemagglutinating and neuraminidase activities were associated with virus protein 1; virus protein 2 had neither activity. The results suggest that both activities reside on a single NDV glycoprotein. Similar results were obtained previously with another paramyxovirus, simian virus 5. These findings suggest that the association of hemagglutinating and neuraminidase activities with one glycoprotein is a general property of the paramyxovirus group.     

Comparison of RNA Polymerase Associated With Newcastle Disease Virus and a Temperature-Sensitive Mutant of Newcastle Disease Virus Isolated from Persistently Infected L Cells

An in vitro comparison was made of the RNA polymerase activity associated with Newcastle disease virus (NDVo) and three clones of the temperature-sensitive mutant (NDVpi) isolated from persistently infected L cells. Less polymerase activity was associated with the NDVpi clones. Also, compared to NDVo, an increase in incubation temperature from 32 to 37 or 42 C resulted in a marked decrease in polymerase activity for the temperature-sensitive mutants which coincided with their inability to replicate at 42 C.     

Effect of Temperature on Radiosensitivity of Newcastle Disease Virus

Newcastle disease virus was irradiated at temperatures ranging from 2.2 to 60 C. An interaction between the thermal and ionizing energy was observed in the temperature region of 49 to 60 C. At 2.2 C, the hemagglutinin was considerably more radioresistant than the infectivity property. It is believed that radiation inactivation of Newcastle disease virus infectivity at low temperatures was due to nucleic acid degradation and at higher temperatures was due to protein denaturation.     

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.