Identification And Phylogenic Analyses Of A Pigeon-origin Class Ⅰ Newcastle Disease Virus

Newcastle disease(ND), Asian Fowl pest, is one of the most highly pathogenic viral diseases of avian species because it causes high mortality and morbidity rates. ND is caused by NDV, which is also known as avian paramyxovirus type 1(APMV-1); this virus belongs to the genus Avulavirus and subfamily Paramyxovirnae(family Paramyxivirinae, order Mononegavirales). The NDV genome is non-segmented and contains single-stranded negative-sense RNA, with six transcriptional units in the order 3′-NP-P-M-F-HN-L-5′and followed the ′rule of six′ and encode six structural proteins and two additional non-structural proteins. NDV is generally categorised based on pathogenicity in chickens into three pathotypes, namely, velogenic, mesogenic or lentogenic. NDV strains can be separated into Class Ⅰ and Ⅱ based on genomic size and sequence analysis. Class Ⅱ viruses comprise most virulent NDVs, and at least 18 genotypes have been identified in this group; Class Ⅰviruses, with a genome size of 15,198 nucleotides(nt), are mostly avirulent and frequently isolated from wild waterfowl and live bird markets(LBM). NDV has a wide host range and can infect at least 250 species in most avian orders. Wild birds as a natural reservoir play an important role in the genetic and viral virulence evolution.The main results of this research study as follows:1. In November 2014, 34 tracheal and cloacal swabs were collected from non-clinically healthy free-ranging pigeons in Huangyuan Country, Qing-hai Province, Northwest of China.After a series of passages in 10-day old SPF chicken embryos, there are two(5.88%) samples positive identified by HA, HI, and RT-PCR.2. Pigeon/QH-01/Ch/14 isolate shown an intracerebral pathogenicity index of 0.00 and a mean time of death more than 148 h, and shared a motif of-112E-R-Q-E-R-L117- at the F protein cleavage site. The results of pathogenicity assessment assay shown that this NDV isolate was a lentogenic strains according to OIE criterion.3. Ten overlapping fragments covering the full-length genome of Pigeon/QH-01/Ch/14 isolate was amplified by RT-PCR. The genome was assembled with the software suit DNASTAR. The NDV isolate genome, with 15,198 nt in length, comprised six ORFs in the order 3′-NP-P-M-F-HN-L-5′. A phylogenetic tree was constructed based on the completesequences of the F gene by using MEGA 6.02. This NDV isolate were classified into genotype 1b in Class Ⅰ. Pigeon/QH-01/14 in this study showed 97.6% identities and the smallest evolutionary distance 0.010 with the Duck/LH/LHuB/085/2013 strain(Accession number: KM885161), a lentogenic NDV strain belonging to Class Ⅰ.This study show that pigeon as a susceptible host not merely for classical genotype VI but also for 1b in Class ⅠNDV.4. The results of deduced amino acids and nucleotide acids of F and HN of pigeon/QH-01/14. 7 and 13 amino acid substitutions were identified in the functional domains of F and HN proteins, respectively, which were compared with mostly other Class Ⅰ and Class Ⅱ NDV isolates. Analyses of the amino acids of neutralising epitopes of F and HN proteins showed 3 and 10 amino acid substitutions, respectively, in the isolate, which were compared with other Class Ⅰ NDV strains. Those results show that the continuous evolution and variation in sub-genotype 1 in Class Ⅰ.