Research On The Biological Characteristics And Molecular Epidemiology Of Newcastle Disease Virus Isolates

Newcastle disease virus (NDV) is classified as a member of the newly defined genus Avulavirus in the family of Paramyxoviridae. It is the causative agent of Newcastle disease (ND), which is widely distributed and has inflicted substantial economic costs to poultry industry worldwide. During 2005 and 2006, some sporadic ND outbreaks occurred in chicken and goose flocks in some regions of Jiangsu and Guangxi, with substantial economic losses. The clinical signs shown by the affected chickens and geese included anorexia, listlessness, greenish-white diarrhea, and paralysis of the legs and wings. Twenty NDV isolates were obtained from chickens or geese showing typical signs of ND in these outbreaks.In the present study, we determined the mean death time (MDT) in embryonated chicken eggs and the intracerebral pathogenicity index (ICPI) in day-old chickens, investigated the stability of haemagglutinin at 56℃and haemagglutination elution pattern, and analyed the antigenic variation with a panel of monoclonal antibodies for these isolates. We genetically characterized them on the basis of partial sequence analysis of the fusion (F) and hemagglutinin-neuraminidase (HN) glycoprotein genes. The results demonstrated that all the 20 NDV isolates were velogenic, and 18 strains belonged to subgenotypeⅦd and the others belonged to an old genotypeⅢ, indicating the continuous predominance of subgenotypeⅦd viruses in poultry flocks in China. In addition, the close genetic similarity between the NDV isolates of chicken origin and those of goose origin derived in the same period suggested that transspecies transmission might occur.1 Isolation and identification of high virulent strains of Newcastle disease virus and their biological characteristicsTwenty NDV strains were isolated from diseased chickens and geese in field outbreaks during 2005 and 2006 in some regions of Jiangsu and Guangxi and their biological characteristics were identified and studied. MDT and ICPI of the isolates were 45.3h~58.2h and 1.61~2.00 respectively, showing that they were velogenic viruses. And their hemagglutinin was not resistant to heat and belonged to fast elution pattern. Based on the reaction spectrum with a panel of monoclonal antibodies to the F or HN glycoprotein, the antigenic variation of the isolates was detected.2 Sequence analysis of fusion(F) protein genes of Newcastle disease virus isolatesIn order to determine the epidemiological relationships and phylogenetic relationships among these NDV isolates, the F glycoprotein gene variable regions were amplified by RT-PCR, cloned and sequenced. The results of nucleotide sequencing and phylogentic analysis of F glycoprotein gene showed that the 20 strains shared homology from 79.7% to100% among themselves, from 78.1% to 83.4% and from 80.2% to 90.1% with NDV LaSota, F48E8, respectively. Comparison of amino acid sequences in variable region of NDV F glycoprotein revealed that all NDV isolates, except tow NDV isolates (JS-7-05-Ch, JS-9-05-Go), possessed K101 and V121 characteristic of the genotypeⅦviruses and were most similar to subgenotypeⅦd. However, a unique amino acid substitution (L23 to M23) was found in all four NDV strains isolated in Guangxi. And the deduced amino acid sequences of F glycoprotein at the cleavage sites of all the isolates were 112R-R-Q-R/K-R-F117, with the motif characteristics of the virulent NDV strain, which was accordant with the results of assessment of the pathogenicity. The phylogentic tree based on sequences of F glycoprotein gene variable regions (47-420nt) revealed that the 18 strains belonged to subgenotypeⅦd and the others belonged to an old genotypeⅢof NDV. The results showed that subgenotypeⅦd virus was responsible for the NDV outbreaks in some regions of Jiangsu and Guangxi recently.3 Sequence analysis of hemagglutinin-neuraminidase(HN) gene of NDV isolatesIn order to further identify the genetic characteristics of these NDV isolates, the entire ORFs encoding HN protein of them were amplified by RT-PCR, cloned and sequenced. The resultant sequences of HN genes of 13 isolates of chicken origin and 7 isolates of goose origin were gained and analyzed. The results of reaction spectrum showed that there were some distinct differences in the antigenic epitopes among the 20 NDV isolates. And the sequences revealed that the coding regions of the HN genes of these isolates all consisted of 1716 nt characteristic of virulent strains of NDV, coding for 571 amino acids. Neucleotides sequence homology were found to be from 94.8% to 100% among 18 NDV isolates of genotypeⅦ, and the neucleotides sequence homology between all the isolates and the other genotypeⅦstrains of recent years in China ranged from 92.1% to 99.6%. The deduced amino acid sequences and the receptor-binding regions of HN proteins between the NDV isolates of chicken origin and of goose origin were compared and analyzed. The results showed that thirteen cysteine residues of the HN protein of these isolates were highly conserved throughout the deduced amino acid sequences, whereas the number of potential glycosylation sites varied from 4 to 6. No distinct differences were found in the receptor-binding regions of HN proteins between the NDV isolates of chicken origin and of goose origin in the same period.In conclusion, this study suggested that some unique amino acid substitutions were found in the genome of the NDV isolates, and the close genetic similarity provided evidence for epidemiological linkage between the NDV isolates of chicken origin and of goose origin in the same period.