Research On The Molecular Epidemiology Of Newcastle Disease Virus Isolates And Inactivated Vaccine Immunogenicity

13 strains of Newcastle disease virus (NDV), 11 from vaccinated chickens and 2 from pigeons, were isolated from Heilongjiang Province during 2004～2005. After plaque purification, all isolates'the mean death time (MDT) of embryonated eggs, interacerebral pathogenicity index (ICPI)hemagglutination spectrum of erythrocytes, and hemagglutination-separation reactions were performed. Then partial F gene of all NDV isolates were cloned and sequenced, along with the reference strains, phylogenetic analysis was conducted on the basis of these sequences. Biological characterization results showed that all NDV isolates could form round and clear plaques on the monolayer cell of chicken embryonate fibrocytes; MDT are between 36.8 hours and 73.6 hours; ICPI are between 1.51and 2.00, belong to velogenic NDV. All 13 strains can agglutinate the red blood cell of chicken and human O type. However, their ability of agglutinating the erythrocytes of swine, horse, cow, rabbit and guinea pig were variable. The hemagglutinin of lentogenic strains were stable to heat and belonged to slow type of agglutination-separation, virulent strains were not stable to heat and belonged to fast type of agglutination-separation. To further understand the relationship of isolates, main functional fragment of F gene (540bp) of 13 NDV isolates were amplified by reverse transcription-polymerase chain reaction (RT-PCR) and sequenced after being cloned into pMD-18-T plasmids. The nucleotide sequence from the coding region of the F gene between nucleotides 1 and 374 was compared by phylogenetic analysis. Total 76 NDV strains from ND outbreaks in China between 1979 and 2005 were analyzed. Based on nucleotide sequence, the virulence and genotype of 13 NDV strains were confirmed. Among the isolates, 10 from chicken were placed into genotype Ⅶe; 2 from pigeon formed genotype Ⅵ; and 1 from chick belonged to genotype Ⅸ. It also indicated that genotype Ⅶe of the NDV was main epidemic strains, genotypes I,Ⅱ,Ⅵ,Ⅷand Ⅸwere still present in China. In conclusion, genotype Ⅶe of the NDV prevails in Heilongjiang Province. Monovalent and polyvalent oil-adjuvant inactivated vaccine was prepared successfully with two isolated strains of genotype VI, Ⅶ(HLJ-8-04 and HLJ-4-04) and La Sota of NDV. The results demonstrated that the HI titer of all immunization groups come to a peak in 21 days post-vaccination, and immunization protective rate of all vaccine groups was 100%. After being challenged with two different genotypes VI and Ⅶ, the HI titer of all immunization groups respectively reached two peaks in 7 days (11.0log2～12.8log2) and 16 days (10.0log2～11.4log2)post-challenge. It was worth to note that the HI titer of monovalent vaccine immunized group challenged with the same type reached the peak earlier than that challenged with different type virus, whereas there was no such difference among multivalent vaccine immunized groups. It suggested that NDV immunogenicity among epizootic isolates and vaccine strains were also variable in some extent.