Relationships among the polypeptides of Newcastle disease virus.

We have studied the relationships among the polypeptides of Newcastle disease virus by using both kinetic and tryptic peptide analyses. The results of our tryptic peptide analyses suggest that there are at least six unique viral polypeptides--L, HN, FO(F), NP, M, and a 47,000-dalton polypeptide. The small virion glycopolypeptide F is related to FO, a glycopolypeptide found only in infected cells. In addition, several smaller polypeptides, including a 53,000-dalton polypeptide found both in purified virions and in infected cells, are related to the nucleocaspid protein. Kinetic analysis of each viral polypeptide reveals that all of the major viral polypeptides, with the possible exception of L, are stable after an amino acid chase. A precursor-product relationship between FO and F was not demonstrable by pulse-chase experiments. Also, almost the same relative amount of F, the putative product, was present in infected cultures after either 5 or 30 min of radioisotopic labeling. These results suggest that FO is processed rapidly.     

Fatty acid modification of Newcastle disease virus glycoproteins.

The fatty acid acylation of Newcastle disease virus hemagglutininin-neuraminidase and fusion glycoproteins was assayed. [3H]palmitate label was associated with cytoplasmic fusion proteins (F0 and F1) and virion-associated F1. In contrast, there was no detectable [3H]palmitate label associated with the hemagglutin-neuraminidase protein in Newcastle disease virus-infected Chinese hamster ovary cells or chicken embryo cells or in virions released from these cells. Thus, fatty acid modification may not be important for the maturation of some glycoproteins.     

Intracellular processing of the Newcastle disease virus fusion glycoprotein.

The fusion glycoprotein (Fo) of Newcastle disease virus is cleaved at an intracellular site (Nagai et al., Virology 69:523-538, 1976) into F1 and F2. This result was confirmed by comparing the transit time of the fusion protein to the cell surface with the time course of cleavage of Fo. The time required for cleavage of half of the pulse-labeled Fo protein is ca. 40 min faster than the half time of the transit of the fusion protein to the cell surface. To determine the cell compartment in which cleavage occurs, use was made of inhibitors which block glycoprotein migration at specific points and posttranslational modifications known to occur in specific cell membranes. Cleavage of Fo is inhibited by carbonyl cyanide m-chlorophenylhydrazone; thus, cleavage does not occur in the rough endoplasmic reticulum. Monensin blocks the incorporation of Newcastle disease virus glycoproteins into virions and blocks the cleavage of the fusion glycoprotein. However, Fo cannot be radioactively labeled with [3H] fucose, whereas F1 is readily labeled. These results argue that cleavage occurs in the trans Golgi membranes or in a cell compartment occupied by glycoproteins quite soon after their transit through the trans Golgi membranes. The implications of the results presented for the transit times of the fusion protein between subcellular organelles are discussed.     

Biological consequences of neuraminidase deficiency in Newcastle disease virus.

A second-step revertant (L1) of a temperature-sensitive mutant (C1) of Newcastle disease virus agglutinated erythrocytes normally but had less than 3% of the wild-type (strain AV) levels of neuraminidase activity. Revertant L1 had seven times more virion-associated N-acetylneuraminic acid (NANA) than strain AV. NANA residues on purified virions were specifically labeled with periodate and tritiated borohydride. Analyses of radiolabeled L1 virions on sodium dodecyl sulfate-polyacrylamide gels showed that most of the virion-associated NANA was in a high-molecular-weight component with an electrophoretic mobility different from that of any known viral protein. NANA was also detected in molecules with the electrophoretic mobility of the viral glycoproteins HN and F1. Revertant L1 had a twofold lower rate constant of attachment to HeLa cells than that of the wild-type. Treatment of L1 virions with Vibrio cholerae neuraminidase removed the excess NANA and returned L1 attachment kinetics to normal. Revertant N1, which has 10-fold more neuraminidase activity than L1, penetrated host cells at the same rate as L1. L1 was impaired in elution from erythrocytes. Removal of virion-associated NANA exacerbated this defect. Despite a small disadvantage in attachment and a major defect in elution relative to strain AV, revertant L1 enjoyed a slight advantage over the wild-type during a single reproductive cycle in cultured chicken embryo cells.     

Noncytopathic mutants of Newcastle disease virus

We have isolated a novel class of mutants of Newcastle disease virus which are less cytopathic than their virulent parent but are still capable of infectious virus production. Unlike wild-type virus, the mutants did not form plaques after 2 days of incubation; they did, however, make hemadsorbing spots. The mutants range in production of infectious virus from 10 to 200% of that of the wild type. They were less cytopathic in a single cycle of infection by light microscopy, loss of protein from the plate, and inhibition of total protein accumulation. All of the mutants exhibited extended mean embryo death times, a correlate of virulence in the adult animal.     

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.     

微生物介导的新城疫病毒疫苗的研制现状

新城疫病毒引起的新城疫是一种禽类急性接触性传染病,采用疫苗防治该病是当前研究的热点领域之一。HN/F蛋白是2种有效的疫苗候选分子,乳酸乳球菌、鼠伤寒沙门菌、单增李斯特菌、根癌农杆菌、鸡痘病毒、牛痘病毒、鸽痘病毒、禽红白血病病毒、禽副粘病毒3型、传染性法氏囊病病毒和传染性气管炎病毒等微生物经过改造后可成为理想的疫苗载体。本文综述了重组乳酸乳球菌(rLL-HN)、重组鼠伤寒沙门菌(rSt-F和rSt-HN)、重组单增李斯特菌(rLm-F)、重组根癌农杆菌(rAt-F)、重组鸡痘病毒(rFPV-HN、rFPV-F-gB和rFPV-F-HN-gB)、重组牛痘病毒(rVV-F)、重组鸽痘病毒(rPPV-F)、重组禽红白血病病毒(rAEV-HN)、重组禽副粘病毒3型(rAPMV-F)、重组传染性法氏囊病病毒(rIBDV-HN)和重组传染性气管炎病毒(rIBV-HN)等疫苗的构建及其免疫机制的研制现状。