Studies On Biological Characterization Of One Strain Of Newcastle Disease Virus Isolated From China In 2006

Newcastle disease (ND), one of the most important diseases of poultry, caused by Newcastle disease virus (NDV), also called avian paramyxovirus type 1, is classified as a List A disease by the World Animal Health Organization (Office International des Epizooties, OIE) because it is highly contagious and can causes severe disease and high mortalities in susceptible birds. NDV has been placed in the genus Avulavirus, sub-family Paramyxovirinae, family Paramyxoviridae, order Mononegavirales in the current taxonomy, which has a negative-sense, single-stranded RNA genome of 15 kb that contains 6 genes in the order of 3'-NP-P-M-F-HN-L-5', encoding six proteins (nucleoprotein, phosphorprotein, matrix protein, fusion protein, hemagglutinin-neuraminidase protein and large protein respectively). Although NDV has only one serotype, there are differences in pathogenicity and virulence among different strains. All NDV isolates are categorized as three pathotypes depending on the severity of disease caused, i.e., velogenic, mesogenic, and lentogenic. Newcastle disease has been endemic for decades in China since it was first described in village chicken flocks in 1946. Since 1980s, strict vaccination program had been practiced in both large-scale poultry operations and small poultry farming in China, so the occurrences of ND were mild and sporadic. But it still remains high mortality rate in some regions in China.An outbreak of ND in one farm of poultry was identified and one strain of Newcastle disease virus was isolated successfully from the case. Parts of serums were collected during the early state of the outbreak to evaluate the HI antibody levels of ND. The result showed the HI levels were changed from 10log2 to 41og2. The samples collected from the diseased poultry during the outbreak were used to inoculate 10-day -old SPF chicken embryo by standard procedural. The infective allantoidal fluid was used to identify the pathogen. The results proved the isolate could agglutinate chicken red blood cells, which could be inhibited and neutralized by avian Newcastle disease virus (NDV) standard antiserum, but can not be inhibited by avian influenza virus (AIV) standard antiserum (H5 and H9 subtype). So the pathogen resulted the outbreak in the farm was identified as Newcastle disease virus.The pathogenicity tests were used to identify the virulence of the isolate of Newcastle disease virus, namely NDV06-025. The results of MDT (53.2h) and ICPI (1.88) proved that the isolate was virulent strain. The main function region of the fusion gene of the isolate was amplified by RT-PCR, and further cloned into pGEM-T vector and sequencing. The sequence analysis showed that the nucleotide sequences of the fragment were 535 bp. The amino acid sequence of cleavage site region was ¹¹²R-R-Q-K-R-F¹¹⁷, matching to virulent NDV strains. The isolate possess K101 and V121, which was similar to the characteristic of genotypeâ…¦NDV. A phylogenetic tree based on the nucleotide sequences of the F gene were constructed by MEGA3.1. The phylogenetic analysis revealed all the NDV strains used in the study were divided into eight genotypes, and the isolate NDV06-025 was similar to the strain Y98 and Ch2000, and was placed into genotypeâ…¦d virus.Efficacy of two kinds of in-activate Newcastle disease virus vaccines against velogenic isolate NDV06-025 challenge were evaluated in this study. The two vaccines were made by La Sota and NDV06-025 respectively according to standard procedures. Thirty 28-day-old SPF chickens were random divided into three groups, named group A, group B and group C respectively. The SPF chickens of Group A and B were inoculated by LaSota and NDV06-025 in-activated vaccine respectively, and group C was inoculated by PBS buffer for a control. All the chickens used in this study were raised in three separate isolators according to different groups. All chickens were injected intramuscularly with 10⁵ ELD₅₀ of NDV06-025 strains 21 days after immunization. All the control chickens were dead within 6 days after challenged with velogenic virus. All the chickens belonged to group A and B were survival in the test. The result indicated that the two kinds of vaccines could protect chickens against challenge of genotypeâ…¦velogenic strain NDV06-025.