| Pigeon Newcastle Disease (ND) which caused by virulent Newcastle Disease Virus (NDV) also known as pigeon Paramyxovirus 1 (PPMV-1), is an acute and highly contagious disease and resembles the clinical symptoms to chicken ND. Pigeon ND was spread to China in the 1980s and has been a serious threat to the pigeon industry since then. To explore the genetic variation of NDVs in pigeon flocks, we characterized 11 pigeon-origin NDVs isolated in China during 2011 to 2015 biologically and phylogentically. To benefit the study in the pathogenesis and enrich the molecular biology information database of PPMV-1, the whole genome sequences of two PPMV-1 strains, from different isolation sites and with significantly different biological properties, were analyzed. Based on the result of molecular epidemiological investigation and the lack of suitable vaccines for pigeon ND, the immune efficacy of inactivated vaccine VIb-14 specific for pigeon ND was also conducted in this study.To elucidate the circulation of the NDVs among the pigeon flocks, eleven pigeon-origin NDVs isolated from the pigeons during 2011-2015 were characterized biologically and phylogenetically. All strains were grouped into sub-genotype VIb according to the phylogenetic analysis based on F and HN genes. Therefore, the pigeon ND was mainly caused by sub-genotype VIb NDVs which exhibited the host--specific infection tropism. The nucleotide sequence identities of F and HN genes among the 11 PPMV-1 strains were 94.3-99.3% and 93.9-99.9% respectively. All the isolated strains were located in the same branch with the EU/re virus, but exhibited long distance with the vaccine strain La Sota. The nucleotide sequence identities of F and HN genes between the isolated strains and La Sota were 83.9-84.9% and 82.7-83.6%, respectively. The MDT of the isolates ranged from 50h to 114h and their ICPI ranged from 0.98 to 1.51, although they shared the same cleavage site motif 112R-R-Q-K-R-F117 in the F protein, suggesting that the amino acid sequence at the F cleavage site was not the sole determinant of the virulence. Compared with La Sota there were 12 amino acid substitutions in/around the five antigenic epitopes on the HN protein, and eight substitutions could induce the alteration in polarity, charge and structure. Compared with La Sota, substitution E347G was present at the linear epitope on the HN and the HI titer of monoclonal antibody 6B1 against the isolates was 61og2 lower than that against La Sota. Meanwhile, the antigen similarity coefficient between NT-23-11-Pi and La Sota was 0.42 indicating the significant antigenic difference between them.Both the isolates NT-23-11-Pi and ZJ-08-13-Pi possessed the full-length genome of 15192 nts and distributed in the EU/re branch in the phylogenic tree. The amino acid sequence identities of NP, P, M, F, HN, L between them were 99%,97%,99.5%,98.6%,99.5% and 99.6% respectively. Between NT-23-11-Pi and ZJ-08-13-Pi there were 25 amino acid nonconservative substitutions among the six proteins, and these substitutions provided useful messages to elucidate the molecular mechanisms of the virulence difference for sub-genotype VIb NDVs.Vaccination is the main method to prevent and control the ND, however, there is no efficient vaccine suitable for pigeon ND in China currently. Previously, the candidate vaccine strain VIb-I4 specific for pigeon ND has been developed by reverse genetics in our laboratory. In order to accelerate this vaccine industrialization, the efficacy tests of the VIb-I4 based killed vaccine were also conducted in this study. The vaccine strain VIb-I4 was serially passaged in SPF chicken embryo for 15 generations and the 1st,5,10th and 15th passaged viruses were subjected to genetic stability assay. The results showed that no change was detected in the amino acid sequence at the F cleavage site and each generation was detected as lentogenic strains. The reproductive performance of the vaccine strain was excellent in terms of the high HA titer (≧ 9log2) and EID50(≧1090/0.1ml). According to the biological characteristics and pathogenicity in pigeons, the virus NT-23-11-Pi was selected as a standard virulent challenge strain. Based on the vaccination and challenge experiment, the minimum immune dose for the inactivated vaccine VIb-I4 was determined as 200μl. When the pigeons were vaccinated with this dose, the mean HI titer of the serum from the vaccinated pigeons could reach 7.2 log2 five weeks post-immunization and the vaccinated pigeons could be efficiently protected against the NT-23-11-Pi infection. More importantly, the vaccine strain VIb-I4 could induce higher HI titers and better immune efficacy than the traditional vaccine strain La Sota. |
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