Biological Characterization Of Newcastle Disease Virus Isolates From Domestic Ducks In China

Newcastle disease(ND) is one of the most devastating diseases to the poultry industry. The causative agents of ND are virulent strains of Newcastle disease virus(NDV), which are members of the genus Avulavirus within the family Paramyxoviridae. Waterfowl such as ducks and geese are generally considered potential reservoirs of NDV and may show few or no clinical signs when infected with viruses that are obviously virulent in chickens. However, ND outbreaks in domestic waterfowl have been frequently reported in many countries in the past decades.In this study, 19 NDV strains were isolated from domestic duck samples, 18 strains were isolated from cloacal swabs from clinically healthy ducks, and one strain was isolated from kidney samples from a disease duck. All of the samples were diluted and inoculated into the allantoic cavity of 9-day-old SPF eggs. Hemagglutination(HA) assays were used to identify NDV-positive embryos and then hemagglutination-positive samples were tested for HI using four HA units of antigen and anti-La Sota chicken serum. The virulent viruses which can induce cytopathic effect plaque-purified in primary chicken embryo fibroblasts, and for the viruses of lower virulence, they were purified by end point dilution method using embryonated chicken eggs. Each of the purified viruses was amplified in 9-day-old SPF eggs. Viral RNA was extracted from infective allantoic fluid, and complete genomes were amplified with a one-step RT-PCR kit. The PCR products were purified and cloned into the p MD18-T Vector. The complete genomes of these strains were sequenced, and they exhibited genomes sizes of 15186 nt, 15192 nt and 15198 nt, which follow the “rule of six” that is required for the replication of NDV strains. All 19 genomes consisted of six ORFs in the order 3?-NP-P-M-F-HN-L-5?, which is in accordance with all NDV strains. The complete genomes of all the isolates exhibited sequence differences of 0-28.3%. The F protein cleavage site is important to predict the pathotypes of NDV isolates. Of the 19 isolates, Md/CH/LGD/1/2005 had the sequence motifs 112RRQKRF117, which is a velogenic site that is phylogenetically similar to that of genotype VIId class II strains; Du/CH/LAH/224/2011 and Du/CH/LAH/209/2011 had the sequence motifs 112GKQGRL117 and 112GRQGRL117, which are phylogenetically similar to class II genotype I and genotype II lentogenic strains, respectively; the remaining 16 isolates had the sequence motifs 112ERQERL117 of class I lentogenic strains, which had been reported in wild birds and domestic ducks. Phylogenetic analysis based on the partial sequences of the F gene and the complete genome sequences showed that there are at least four genotypes of NDV circulating in domestic ducks: Md/CH/LGD/1/2005 was subdivided into genotype VIId of class II, a predominant genotype responsible for most ND outbreaks since the end of the last century; Du/CH/LAH/224/2011 was grouped into genotype Ib of class II, and it was similar to the CBU2374 strains; Du/CH/LAH/209/2011 belonged to genotype II of class II; 16 isolates could be categorized as genotype 1b, which is the epidemic genotype for waterfowl in China.Seven duck-origin NDV strains were selected for in vivo pathogenicity tests based on the sequence and phylogenetic analysis. In addition, eight strains from different hosts were also used as references according to the phylogenetic tree of the partial sequence of the F gene. Md/CH/LGD/1/2005 had an ICPI value of 1.88 and an MDT value of 50 h, demonstrating that it was a velogenic strain. The ICPI values of the other strains in this study ranged from 0 to 0.34, and the MDT values ranged from 125 to >168h, which is characteristic of lentogenic viruses. Meanwhile, we also used the original host(duck) as an experimental animal to conduct pathogenicity tests.Six strains were selected for the cross-reactive hemagglutination inhibition tests based on the results of the phylogenetic and pathogenic characterizations. In addition 17 NDV strains, representing different genotypes and hosts, which were isolated in our laboratory, used for the cross-reactive HI tests to determine their antigenic differences. The results showed that the antigenic relatedness between NDV strains may be associated with their genotypes, rather than their hosts.These results demonstrated that those NDV isolates were from duck were clustered with NDV strains isolated from chickens, rather than forming an independent clade like NDV strains from pigeons. The NDV strains isolated from ducks may have been transmitted from chickens; however, these strains adapted and evolved in ducks after the transmission. Therefore, the use of ducks as the hosts for duck-origin NDVs could precisely show the real virulence of these viruses and it was necessary to properly adjust the evaluation standard. However, they may play an important role in promoting the evolution of NDVs.