Mutation of the F-Protein Cleavage Site of Avian Paramyxovirus Type 7 Results in Furin Cleavage, Fusion Promotion, and Increased Replication In Vitro but Not Increased Replication, Tissue Tropism, or Virulence in Chickens

Abstract

bWe constructed a reverse genetics system for avian paramyxovirus serotype 7 (APMV-7) to investigate the role of the fusion F glycoprotein in tissue tropism and virulence. The AMPV-7 F protein has a single basic residue arginine (R) at position ؊1 in the F cleavage site sequence and also is unusual in having alanine at position ؉2 (LPSSR2FA) (underlining indicates the basic amino acids at the F protein cleavage site, and the arrow indicates the site of cleavage.). APMV-7 does not form syncytia or plaques in cell culture, but its replication in vitro does not depend on, and is not increased by, added protease. Two mutants were successfully recovered in which the cleavage site was modified to mimic sites that are found in virulent Newcastle disease virus isolates and to contain 4 or 5 basic residues as well as isoleucine in the ؉2 position: (RRQKR2FI) or (RRKKR2FI), named Fcs-4B or Fcs-5B, respectively. In cell culture, one of the mutants, Fcs-5B, formed protease-independent syncytia and grew to 10-fold-higher titers compared to the parent and Fcs-4B viruses. This indicated the importance of the single additional basic residue (K) at position ؊3. Syncytium formation and virus yield of the Fcs-5B virus was impaired by the furin inhibitor decanoyl-RVKR-CMK, whereas parental APMV-7 was not affected. APMV-7 is avirulent in chickens and is limited in tropism to the upper respiratory tract of 1-day-old and 2-week-old chickens, and these characteristics were unchanged for the two mutant viruses. Thus, the acquisition of furin cleavability by APMV-7 resulted in syncytium formation and increased virus yield in vitro but did not alter virus yield, tropism, or virulence in chickens.

Generation by Reverse Genetics of an Effective, Stable, Live-Attenuated Newcastle Disease Virus Vaccine Based on a Currently Circulating, Highly Virulent Indonesian Strain

“…The cells was fixed with methanol and stained with 1% crystal violet. NDV titers in 50%-tissue-culture-infectious-dose (TCID50) units were determined as previously described [19]. Briefly, confluent DF1 cell monolayers were infected with 10-fold dilutions of virus as described for the plaque assay and incubated in medium without methylcellulose.…”

Evaluation of the Replication, Pathogenicity, and Immunogenicity of Avian Paramyxovirus (APMV) Serotypes 2, 3, 4, 5, 7, and 9 in Rhesus Macaques

“…The viruses included biologically-and recombinantly-derived wt viruses as well as several recombinant viruses in which the F protein cleavage site had been modified to be multi-basic and to contain the optimal furin protease cleavage site motif RX(R/K)RQ (signature R and K residues underlined). This was done because the presence of a furin motif in the F protein cleavage site typically facilitates cleavage and is a major determinant of virulence for NDV strains [45,46], although this paradigm is uncertain for the other APMV serotypes [16,18,19]. The present study showed that, except for APMV-5, all of the APMVs under evaluation replicated at varying levels in rhesus macaques without inducing any apparent clinical disease signs.…”

“…(ii) a recombinant derivative of wt APMV-2, called rAPMV-2 (type 1 Africa), in which the natural cleavage site was replaced by the cleavage site RRRRRQF that is present in a APMV type 1 strain isolated in Africa [16]; (iii) biologically-derived wt APMV-3 strain parakeet/Netherland/449/75 [9]; (iv) recombinant wt APMV-4 strain duck/Hong Kong/D3/75 [10]; (v) a derivative of rAPMV-4, called rAPMV-4/Fc-BC, in which its natural cleavage site was replaced by the cleavage site RRQKRQF that is present in mesogenic NDV strain Baudette C [19]; (vi) biologically-derived wt APMV-5 strain budgerigar/Kunitachi/74 [11]; (vii) biologically-derived wt APMV-7 strain dove/Tennessee/4/75 [13]; (viii) a recombinant derivative of wt APMV-7, called rAPMV-7/Fcs-5B in which its natural cleavage site was replaced by the cleavage site RRKKRQFI present in a velogenic NDV strain Nigeria/95, and which includes an amino acid substitution in the +2 position relative to the cleavage site [18]; and (ix) biologically-derived wt APMV-9 strain duck/New York/22/78 [15]. All the APMV serotypes except serotype 5 were grown in the allantoic cavity of 9day-old specific-pathogen-free (SPF) embryonated chicken eggs.…”

“…Some members of the family are responsible for major human and animal diseases, while others cause inapparent infections. Paramyxoviruses are pleomorphic and enveloped and contain a non-segmented, negative-sense, single-stranded RNA genome of [13][14][15][16][17][18][19] kb. On the basis of virus structure, genome organization and sequence relatedness, the family Paramyxoviridae is divided in to two subfamilies: Paramyxovirinae and Pneumovirinae [6].…”

“…Subfamily Pneumovirinae contains two genera, Pneumovirus (including human and bovine respiratory syncytial viruses) and Metapneumovirus (comprising human metapneumovirus and the avian metapneumoviruses) [6,7] Although a lot of information is available for APMV-1, much less is known about the molecular biology, pathogenicity and host range of the other APMV serotypes. As an initial step towards their characterization, we have recently determined the complete genome sequences of APMV-2 to -9 [8][9][10][11][12][13][14][15] and we have developed reverse genetics systems for APMV-2, -3, -4 and -7 [16][17][18][19]. However, the biological characteristics and pathogenicity of APMV-2 to -9 remain poorly understood.…”