scholarly journals Rescue of Newcastle Disease Virus from Cloned cDNA: Evidence that Cleavability of the Fusion Protein Is a Major Determinant for Virulence

1999 ◽  
Vol 73 (6) ◽  
pp. 5001-5009 ◽  
Author(s):  
Ben P. H. Peeters ◽  
Olav S. de Leeuw ◽  
Guus Koch ◽  
Arno L. J. Gielkens
Keyword(s):  
Amino Acid ◽  
Fusion Protein ◽  
Rna Polymerase ◽  
Newcastle Disease Virus ◽  
Disease Virus ◽  
Newcastle Disease ◽  
Cleavage Site ◽  
T7 Rna Polymerase ◽  
Protease Cleavage ◽  
Cloned Cdna

ABSTRACT A full-length cDNA clone of Newcastle disease virus (NDV) vaccine strain LaSota was assembled from subgenomic overlapping cDNA fragments and cloned in a transcription plasmid between the T7 RNA polymerase promoter and the autocatalytic hepatitis delta virus ribozyme. Transfection of this plasmid into cells that were infected with a recombinant fowlpoxvirus that expressed T7 RNA polymerase, resulted in the synthesis of antigenomic NDV RNA. This RNA was replicated and transcribed by the viral NP, P, and L proteins, which were expressed from cotransfected plasmids. After inoculation of the transfection supernatant into embryonated specific-pathogen-free eggs, infectious virus derived from the cloned cDNA was recovered. By introducing three nucleotide changes in the cDNA, we generated a genetically tagged derivative of the LaSota strain in which the amino acid sequence of the protease cleavage site (GGRQGR↓L) of the fusion protein F0 was changed to the consensus cleavage site of virulent NDV strains (GRRQRR↓F). Pathogenicity tests in day-old chickens showed that the strain derived from the unmodified cDNA was completely nonvirulent (intracerebral pathogenicity index [ICPI] = 0.00). However, the strain derived from the cDNA in which the protease cleavage site was modified showed a dramatic increase in virulence (ICPI = 1.28 out of a possible maximum of 2.0). Pulse-chase labeling of cells infected with the different strains followed by radioimmunoprecipitation of the F protein showed that the efficiency of cleavage of the F0 protein was greatly enhanced by the amino acid replacements. These results demonstrate that genetically modified NDV can be recovered from cloned cDNA and confirm the supposition that cleavage of the F0 protein is a key determinant in virulence of NDV.

Expression at the cell surface of native fusion protein of the Newcastle disease virus (NDV) strain Italien from cloned cDNA

1987 ◽  
Vol 95 (1-2) ◽  
pp. 79-95 ◽  
Author(s):  
D. Espion ◽  
S. de Henau ◽  
C. Letellier ◽  
C. D. Wemers ◽  
R. Brasseur ◽  
...  
Keyword(s):  
Cell Surface ◽  
Fusion Protein ◽  
Newcastle Disease Virus ◽  
Disease Virus ◽  
Newcastle Disease ◽  
Cloned Cdna

scholarly journals A single amino acid change, Q114R, in the cleavage-site sequence of Newcastle disease virus fusion protein attenuates viral replication and pathogenicity

2011 ◽  
Vol 92 (10) ◽  
pp. 2333-2338 ◽  
Author(s):  
Sweety Samal ◽  
Sachin Kumar ◽  
Sunil K. Khattar ◽  
Siba K. Samal
Keyword(s):  
Amino Acid ◽  
Viral Replication ◽  
Newcastle Disease Virus ◽  
Disease Virus ◽  
Newcastle Disease ◽  
Cleavage Site ◽  
Single Amino Acid ◽  
Protein Cleavage ◽  
F Protein ◽  
Glutamine Residue

A key determinant of Newcastle disease virus (NDV) virulence is the amino acid sequence at the fusion (F) protein cleavage site. The NDV F protein is synthesized as an inactive precursor, F0, and is activated by proteolytic cleavage between amino acids 116 and 117 to produce two disulfide-linked subunits, F1 and F2. The consensus sequence of the F protein cleavage site of virulent [112(R/K)-R-Q-(R/K)-R↓F-I118] and avirulent [112(G/E)-(K/R)-Q-(G/E)-R↓L-I118] strains contains a conserved glutamine residue at position 114. Recently, some NDV strains from Africa and Madagascar were isolated from healthy birds and have been reported to contain five basic residues (R-R-R-K-R↓F-I/V or R-R-R-R-R↓F-I/V) at the F protein cleavage site. In this study, we have evaluated the role of this conserved glutamine residue in the replication and pathogenicity of NDV by using the moderately pathogenic Beaudette C strain and by making Q114R, K115R and I118V mutants of the F protein in this strain. Our results showed that changing the glutamine to a basic arginine residue reduced viral replication and attenuated the pathogenicity of the virus in chickens. The pathogenicity was further reduced when the isoleucine at position 118 was substituted for valine.

scholarly journals Evaluation of fusion protein cleavage site sequences of Newcastle disease virus in genotype matched vaccines

PLoS ONE ◽  
2017 ◽  
Vol 12 (3) ◽  
pp. e0173965 ◽  
Author(s):  
Shin-Hee Kim ◽  
Zongyan Chen ◽  
Asuka Yoshida ◽  
Anandan Paldurai ◽  
Sa Xiao ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Newcastle Disease Virus ◽  
Disease Virus ◽  
Newcastle Disease ◽  
Cleavage Site ◽  
Protein Cleavage ◽  
Fusion Protein Cleavage Site ◽  
Fusion Protein Cleavage

scholarly journals Generation of recombinant lentogenic Newcastle disease virus from cDNA

1999 ◽  
Vol 80 (11) ◽  
pp. 2987-2995 ◽  
Author(s):  
Angela Römer-Oberdörfer ◽  
Egbert Mundt ◽  
Teshome Mebatsion ◽  
Ursula J. Buchholz ◽  
Thomas C. Mettenleiter
Keyword(s):  
Rna Polymerase ◽  
Newcastle Disease Virus ◽  
Disease Virus ◽  
Vaccine Strain ◽  
Newcastle Disease ◽  
T7 Rna Polymerase ◽  
Wild Type Virus ◽  
Progeny Virus ◽  
Restriction Sites ◽  
Specific Pathogen

Recombinant lentogenic Newcastle disease virus (NDV) of the vaccine strain Clone-30 was reproducibly generated after simultaneous expression of antigenome-sense NDV RNA and NDV nucleoprotein, phosphoprotein and RNA-dependent RNA polymerase from plasmids transfected into cells stably expressing T7 RNA polymerase. For this purpose, the genome of Clone-30, comprising 15186 nt, was cloned and sequenced prior to assembly into a full-length cDNA clone under control of a T7 RNA polymerase promoter. Recombinant virus was amplified by inoculation of transfection supernatant into the allantoic cavity of embryonated specific-pathogen-free (SPF) chicken eggs. Two marker restriction sites comprising a total of five nucleotide changes artificially introduced into noncoding regions were present in the progeny virus. The recombinant NDV was indistinguishable from the parental wild-type virus with respect to its growth characteristics in cell culture and in embryonated eggs. Moreover, an intracerebral pathogenicity index of 0·29 was obtained for both viruses as determined by intracerebral inoculation of day-old SPF chickens, proving that the recombinant NDV is a faithful copy of the parental vaccine strain of NDV.

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