Virion functions of RNA+ temperature-sensitive mutants of Newcastle disease virus.

Virions from Newcastle disease virus mutants in four temperature-sensitive RNA+ groups were grown in embryonated hen eggs at the permissive temperature, purified, and then analyzed for biological properties at both the permissive and nonpermissive temperatures. At the permissive temperature, virions of mutants in groups B, C, and BC (11 mutants) were all lower in specific (per milligram of protein) hemagglutination, neuraminidase, and hemolysis activities compared with the wild type. These deficiencies were related to decreased amounts of hemagglutinin-neuraminidase glycoprotein in the virions. Activities of these mutant virions at both the permissive and nonpermissive temperatures were similar, indicating that hemagglutinin-neuraminidase synthesized at the permissive temperature was not temperature sensitive in function. The three group D mutants displayed a different pattern. At the permissive temperature, they had wild-type hemagglutination and neuraminidase activities but were deficient compared with the wild type in hemolysis. Again, functions were similar at both temperatures. Most of the B, C, and BC mutants had specific infectivities similar to that of the wild type despite lower hemagglutination, neuraminidase, and hemolysis functions. However, the D mutants were all less infectious. This evidence is consistent with a shared hemagglutinin-neuraminidase defect in the B, C, and BC mutants and a defect in either the F glycoprotein or the M protein in the D mutants.     

Revertant analysis of a temperature-sensitive mutant of Newcastle disease virus with defective glycoproteins: implication of the fusion glycoprotein in cell killing and isolation of a neuraminidase-deficient hemagglutinating virus

Biological and molecular properties of a temperature-sensitive mutant (C1) of Newcastle disease virus and its revertants were analyzed. C1 exhibited three temperature-sensitive alterations (plaque formation, virion assembly, and cytopathogenicity) and several defects which were also present at the permissive temperature. C1 virions contained low amounts of hemagglutinin-neuraminidase glycopeptides and consequently were deficient in hemagglutinating and neuraminidase activities. These virions also contained defective fusion glycoproteins which rendered them poorly hemolytic and slow to penetrate cultured chicken embryo cells. The biological activities of the membrane glycoproteins were recovered sequentially in a series of plaque-forming revertants. The coreversion of hemolysis, membrane-penetrating activities, and cytopathogenicity in the first-step revertant (S1) suggested that fusion glycoproteins were major contributors to cellular destruction. This revertant also provided evidence of a role for fusion glycoproteins in virion assembly. From S1 we isolated a large-plaque-forming revertant (L1) that assembled wild-type amounts of biologically active hemagglutinin-neuraminidase glycoproteins into virions. Although it was normal for hemagglutination, L1 had less than 3% of the neuraminidase activity of the wild type, demonstrating that these two activities can be uncoupled genetically. The neuraminidase deficiency of L1 did not impair its virulence in ovo or its reproduction in cultured cells.     

Hemolytic Interaction of Newcastle Disease Virus and Chicken Erythrocytes. II. Determining Factors

Standard procedures have been described for measuring paramyxovirus-induced hemolysis. The choice of these procedures is based on the analysis of the behavior of eight different strains of Newcastle disease virus. Significant strain-specific differences in hemolytic activity have been found. The presence of at least two kinds of inhibitors of hemolysis in virus preparations necessitates the use of purified virus when comparisons of hemolytic activities are to be made. In addition, it has been stressed that both hemagglutination titers and hemolysis determinations provide only relative values. Thus, quantitative comparisons can be made only with results obtained on the same day and with the same erythrocyte preparation.     

Infective and noninfective hemagglutinating particles of Newcastle disease virus: biological and chemical characterization.

Newcastle disease virus (Herts strain), grown in embryonated eggs or in a line of bovine kidney cells, was purified and then separated by sucrose density gradient centrifugation into infectious (IH) and noninfectious hemagglutinating (NIH) particles. These particles were morphologically similar, although the average size of IH was twice that of NIH particles. The activity of hemagglutinin per milligram of virus protein was two- to threefold higher in NIH particles than in IH particles, whereas the specific activity of neuraminidase did not differ in the two particle types. This was consistent with the observed particle size difference. The distribution of the major proteins in IH and NIH particles from egg-grown virus, determined by polyacrylamide gel electrophoresis (PAGE), was significantly different. In IH particles the molar ratio of protein 1 (74,000 daltons) to proteins 2 and 3 (56,000 daltons): protein 6 (41,000 daltons) was 1.0:2.5:2.5; in NIH particles the ratio was 1.0:0.6:1.0. When Newcastle disease virus was grown in bovine kidney cells, the molar ratio of proteins in IH particles resembled that of of egg-grown virus. However, in NIH particles from bovine kidney cells, only protein bands corresponding to protein 1 and proteins 2-3 were present and their molar ratio was 1.0:0.6. Protein 6 was marginally detectable in these particles. Analysis of the proteins in [3H]isoleucine- and [14C]glucosamine-labeled virus showed proteins 1 and 2 (glycoproteins) present in the ratio of 1.0:0.5; protein 3, the nucleoprotein, was not detected. These results are compatible with previous findings by others that NIH particles are deficient in RNA and nucleoprotein antigen, and suggest that formation of discrete particles of Newcastle disease virus by budding requires at most minimal amounts of proteins 3 or 6. The fatty acid composition of egg-grown IH and NIH particles was not significantly different and resembled that of normal allantoic fluid.     

Action of ortho- and paramyxovirus neuraminidase on gangliosides. Hydrolysis of ganglioside GM1 by Sendai virus neuraminidase

The action of neuraminidase of influenza A virus, Sendai virus and Newcastle disease virus particles on bovine brain ganglioside GM1 and the properties of Sendai virus neuraminidase for GM1 were studied. With Sendai virus, GM1 was hydrolyzed to asialo-GM1 (GA1) and N-acetylneuraminic acid even in the absence of surfactant or other additives, while the hydrolysis of GM1 by Newcastle disease virus or influenza A virus was very low or undetectable under the same conditions. The formation of GA1 by Sendai virus neuraminidase was confirmed by thin-layer chromatography and immunodiffusion test using anti-GA1 antiserum. The apparent Km of Sendai virus neuraminidase for GM1 hydrolysis was found to be 2.67 x 10(-4) M and the optimum pH was 5.6. GM3, GM2 and oligosaccharide of GM1 were hydrolyzed more effectively than GM1 in the absence of surfactant (GM3 greater than GM2 greater than oligosaccharide of GM1 greater than GM1). The hydrolysis of GM1 by the Sendai virus enzyme was stimulated by the addition of sodium cholate or sodium taurocholate, but was inhibited by divalent cations (10 mM), Ca2+, Mg2+, ZN2+, Fe2+ and CU2+. In the absence of the surfactant, Sendai virus neuraminidase hydrolyzed GM1 more efficiently than Arthobacter ureafaciens neuraminidase which has been reported recently as being an adequate enzyme to hydrolyze ganglioside GM1 as a substrate.     

Hemagglutination-Inhibition: Rapid Assay for Neuraminic Acid-Containing Viruses

Influenza virus particles bind rapidly to vesicular stomatitis, Sindbis, or Rauscher murine leukemia virus particles, forming mixed aggregates demonstrable by electron microscopy. The normal hemagglutinating property of influenza virus is inhibited by these viruses, providing a rapid quantitative assay. Prior treatment with neuraminidase blocks the ability of other viruses to inhibit influenza virus hemagglutination.     

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.     

Hemolytic Interaction of Newcastle Disease Virus and Chicken Erythrocytes. I. Quantitative Comparison Procedure

The extent to which erythrocytes are hemolyzed by Newcastle disease virus is a function of the relative concentrations of both virus and erythrocytes. Under proper conditions, the interaction of a single virus particle with an erythrocyte is sufficient to cause lysis. The extent of hemolysis is directly proportional to virus concentration only when the virus-erythrocyte ratio is very low. At the higher virus-erythrocyte ratios usually employed in hemolysis experiments, the extent of hemolysis is proportional to the logarithm of the virus concentration. Thus, quantitative comparisons of hemolytic activities of different virus preparations cannot be made by directly comparing the extent of hemolysis. Relative hemolytic activities must be determined by comparing virus concentrations which yield equivalent amounts of hemolysis (the quantitative comparison procedure).     

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.     

Interactions Between Sendai Virus and Human Erythrocytes

Concentrated Sendai virus, when adsorbed to erythrocytes at 4 C, caused invaginations in the plasma membrane. Following elevation of the temperature to 37 C, the plasma membrane became fused with the viral envelope before dissolution of the virions and rupture of the cells. Cell lysis was accompanied by rapid and total loss of hemoglobin to the extracellular space. Following aqueous pyridine extraction, the hemoglobin-free ghosts remaining were found to be devoid of N-acetylneuraminic acid and to have solubility properties different from those of normal erythrocyte ghosts. By the action of viral neuraminidase, bound N-acetylneuraminic acid was also liberated from purified virus receptor substance whose electrophoretic mobility was thereby substantially reduced. Cu⁺⁺ selectively inhibited hemolysis and neuraminidase without interfering with hemagglutination and attachment. Neuraminidase appeared to be essential for Sendai virus hemolysis; viral particle size may also be a critical factor in this process.     

Characterization of visna virus envelope neuraminic acid.

Visna virus particles inhibit influenza virus hemagglutination in an assay for neuraminic acid-containing viruses. Pretreatment of visna virus with neuraminidase abolished hemagglutination inhibition activity but did not significantly affect attachment, infectivity, or virus-induced cell fusion in sheep choroid plexus cell monolayers.     

重组新城疫病毒Anhinga株与TRAIL蛋白协同杀伤肿瘤细胞

将新城疫病毒（Newscastle disease virus, NDV）Anhinga株的全 长基因组cDNA克隆质粒、pTM1-L、pTM1-P、pTM1-NP表达质粒共转染稳定表 达T7 RNA聚合酶的BSRT7/5细胞,得到拯救NDV病毒．通过PCR、酶切法、测 序证明拯救病毒中存在引入的分子标签.通过血凝实验、蚀斑测定证明成功 拯救病毒.并研究了该重组病毒对4种不同人肿瘤细胞的体外杀伤效果．首 次证明重组Anhinga株对SMMC-7721细胞、A549细胞、HepG2细胞和SH-SY5Y 细胞均有杀伤作用．该重组病毒主要诱导SMMC-7721细胞和A549细胞凋亡, 诱导HepG2细胞和SH-SY5Y细胞坏死．TNF家族成员TRAIL蛋白可以显著增强 NDV杀伤肿瘤细胞的效果.本实验为进一步研究重组NDV用于肿瘤治疗奠定基 础．     

Thermal Inactivation of Newcastle Disease Virus I. Coupled Inactivation Rates of Hemagglutinating and Neuraminidase Activities

The thermal stability of Newcastle disease virus has been characterized in terms of the rate constants for inactivation of hemagglutinating activity (HA), neuraminidase activity (NA), and infectivity. Inactivation of HA results in the concomitant loss of NA. Infectivity, however, is much more thermolabile. Disintegration of the virus particle is not responsible for the identical rate constants for inactivation of HA and NA, nor is their parallel inactivation uncoupled in envelope fragments produced by pretreating the virus with phospholipase-C. The data indicate that a common envelope factor(s) can influence the thermal stability of both activities.     

Effect of β-Propiolactone on Sendai Virus

The biological properties (infectivity, hemagglutination, hemolysis, cell fusion, neuraminidase) of Sendai virus were dissociated on the basis of sensitivity to beta-propiolactone, by freeze-thawing, by heating at different temperatures, and by adsorption-elution with formalinized chicken erythrocytes. Possible mechanisms whereby beta-propiolactone selectively destroys viral infectivity are discussed.     

Assessment of newcastle disease vaccination of houbara bustard breeders (Chlamydotis undulata undulata)

The houbara bustard (Chlamydotis undulata undulata) is endangered in North Africa. Through a captive-breeding program established in Morocco by The Emirates Center for Wildlife Propagation, wild populations are being supplemented by the releasing of captive-reared birds. Newcastle disease, which is caused by Newcastle disease virus (NDV; Avian paramyxovirus type 1), can infect houbara bustards and is a significant threat through contact with backyard poultry and possibly wild birds. Three vaccination schedules for Newcastle disease were evaluated by serologic monitoring to assess the efficiency and safety of various types of vaccines (live vs. inactivated), vaccine strains (Hitchner B1 and Clone 30), and administration routes (intranasal vs. injection). We evaluated antibody titers in 211 adult houbara bustards for 10 mo. Antibody titers to NDV in both sera and egg yolks were monitored by hemagglutination inhibition test. The inactivated vaccine provided a high, homogeneous, and durable serologic response in breeders; titers were higher than log2 11 after 4 wk and remained higher than log2 7 after 10 mo. The response to the two live vaccines was similar, and antibody titers did not exceed log2 6 at sero-conversion. Maternally derived antibodies were efficiently transmitted in vitellus, further confirming that offspring of females hyperimmunized with the inactivated vaccine received high titers of maternal antibodies.     

Homologous Interference by Incomplete Sendai Virus Particles: Changes in Virus-Specific Ribonucleic Acid Synthesis

Incomplete Sendai virus particles (I particles) interfered with the replication of several strains of infectious Sendai virions (standard virus) but not with the replication of Newcastle disease virus, mumps virus, or Sindbis virus. I particles did not induce interferon, and ultraviolet irradiation of I particles abolished their ability to interfere. Protein synthesis was not necessary to establish interference. The degree of interference depended on the interval between exposure of cells to the I particles and challenge by standard virus, and this was reflected in the degree of inhibition of virus-specific ribonucleic acid (RNA) synthesis in infected cells. The most dramatic change was decreased accumulation of 50S virus-specific RNA in infected cells. RNA species sedimenting slower than 50S were not as markedly reduced in total amount, but hybridization experiments showed that a substantial portion of these slowly sedimenting RNA species were plus strands, presumably representing replicas of the RNA species in I particles. When I particles in insufficient numbers to interfere were added to cells as late as 8 hr after standard virus, there were no obvious changes in virus-specific RNA species in the cells; however, significant amounts of 19 and 25S RNA species, representing progeny of the I particles, appeared in the culture medium. It was concluded that interference was an intracellular event affecting an early step in virus replication. Competition by I particles for cell sites or substrates needed by standard virus seemed a less likely mechanism of interference than competition for enzymes specified by standard virus.     

Isolation and characterization of receptor sialoglycoprotein for hemagglutinating virus of Japan (Sendai virus) from bovine erythrocyte membrane

Sialoglycoprotein which exhibits inhibitory activity for hemagglutination by Hemagglutinating Virus of Japan (HVJ, Sendai virus) was isolated from the membrane of bovine erythrocytes. Purification steps for this sialoglycoprotein included extraction with lithium diiodosalicylate, phenol partition, precipitation with ethanol, and chromatography on a phosphocellulose column and an SDS-Sepharose CL-4B column. Purified sialoglycoprotein (GP-2) has high specific activity for inhibiting the hemagglutination with HVJ, and a lesser activity for that with Newcastle disease virus, but it does not inhibit the hemagglutination by influenza A virus. Inhibitory activity of GP-2 on hemagglutination by HVJ is 2,500-fold higher than that of fetuin. Liposomes containing a 10,000-fold larger amount of ganglioside mixture of bovine erythrocytes and those containing a 5,000-fold larger amount of each ganglioside of bovine erythrocytes, N-glycolylneuraminosyl-lactosyl ceramide, sialosyllacto-N-neotetraosyl- and sialosyl-lacto-N-norhexaosyl ceramide, had no inhibitory activity toward hemagglutination with HVJ. GP-2 (mol. wt. 250 K daltons) behaved homogeneously in SDS-polyacrylamide gel electrophoresis. It contained 70% carbohydrate and 30% protein, by weight. N-Acetylgalactosamine, N-acetylglucosamine, galactose, sialic acid (N-glycolylneuraminic acid, 96%; N-acetylneuraminic acid, 4%) were identified as carbohydrate components, in molar ratios of 1.0:4.0:5.2:2.9. All the oligosaccharides of GP-2 appeared to be linked to polypeptide chains by alkali-labile O-glycosidic linkages. Sialidase treatment of GP-2 and conversion of sialic acid residue of the glycoprotein to C8 and C7 analogues resulted in the loss of the inhibitory activity on hemagglutination by HVJ. Oligosaccharides isolated by gel filtration after treatment of GP-2 with alkaline borohydride had also lost the ability to inhibit the hemagglutination by HVJ. The above results indicate that isolated sialoglycoprotein is the endogenous receptor in bovine erythrocyte membrane specific to HVJ, and the hydroxy group linked to the 9-carbon atom of sialic acid and probably also the hydrophobic protein moiety are important for the recognition of HVJ attachment.     

Thermostabilities of Virion Activities of Newcastle Disease Virus: Evidence that the Temperature-Sensitive Mutants in Complementation Groups B, BC, and C Have Altered HN Proteins

Four virion activities of Newcastle disease virus (hemagglutinating, neuraminidase, hemolytic, and infectious activities) were examined before and after heat stress in low-salt buffer and physiological salt buffer (phosphate-buffered saline). The hemagglutinating and neuraminidase activities of the Australia-Victoria wild-type (AV-WT) strain were thermostable at both salt concentrations tested, whereas the thermostabilities of the hemolytic and infectious activities were salt dependent (thermostable in phosphate-buffered saline but not in low-salt buffer). Virions of RNA⁺ temperature-sensitive (ts) mutants of AV-WT were tested for the stabilities of the four activities. Some mutants in groups B, BC, and C were as stable as AV-WT in all functions, but others were much less stable in all functions. The unstable mutants in groups B, BC, and C affirmed the assignment of the ts lesions of these mutants to the hemagglutinin/neuraminidase (HN) protein gene because HN function(s) are required for all four activities. The instability of these ts mutants was not related to their decreased virion HN protein content and was not due to physical loss of the HN protein from the virions. Three of four ts⁺ plaque-forming revertants of the least stable mutant, BC2, coreverted for stability, confirming that the unstable phenotype is indeed the result of the mutation responsible for the ts phenotype. Group D mutants were approximately as stable as AV-WT in hemagglutinating, neuraminidase, and hemolytic activities; this is consistent with this group representing a lesion in a gene other than the HN protein gene. However, the infectivities of two of the three group D mutants were less stable than the infectivity of AV-WT in low-salt buffer.     

HVJ (Sendai virus)-induced envelope fusion and cell fusion are blocked by monoclonal anti-HN protein antibody that does not inhibit hemagglutination activity of HVJ

Two kinds of monoclonal antibodies against HN protein of HVJ were isolated. In competitive binding assay, binding of one of these antibodies to HN protein did not inhibit binding of the other antibody to the same molecule. One of the antibodies, named HN-1 antibody, inhibited hemagglutination activity of HVJ and also blocked neuraminidase activity of the virus when fetuin and Ehrlich ascites tumor cells were used as substrates, but it did not inhibit the activity when neuramine-lactose was used as substrate. The other antibody, HN-2, did not inhibit hemagglutination activity or neuraminidase activity, but blocked HVJ-induced viral envelope-cell fusion, cell-cell fusion and hemolysis. The mechanism by which HN-2 antibody blocked the fusion process is discussed.     

Ribonucleic Acid Synthesis of Vesicular Stomatitis Virus: I. Species of Ribonucleic Acid Found in Chinese Hamster Ovary Cells Infected with Plaque-forming and Defective Particles

Plaque-forming B particles of vesicular stomatitis virus (VSV) induce the synthesis of virus-specific ribonucleic acid (RNA) in Chinese hamster ovary cells, whereas defective T particles do not. Infection with low input multiplicities of B results in the formation of four species of RNA. During infection with high multiplicities, RNA synthesis begins with mainly these four species of RNA but gradually shifts to a new pattern of RNA synthesis involving five other species of RNA. The change can also be induced by superinfection with T at 2.5 hr after infection with a low multiplicity of B. T added at the same time as B prevents virtually all RNA synthesis. Synthesis of the first group of RNA species correlates with the formation of B particles, whereas synthesis of the second group correlates with the formation of T particles. The various species of RNA formed after infection with VSV particles include single-stranded RNA, a completely double-stranded RNA, and RNA with partially double-stranded regions. These observations begin to establish a molecular basis for understanding the ability of T particles to interfere with the growth of B particles.