Antigenic Comparisons And Analysis Of F And HN Gene Of Newcastle Disease Virus Isolated From Hatching Eggs

Newcastle disease (ND) is regarded worldwide one of urgent and highly contagious disease which had done great harm to poultry enterprise. Its causative agent, Newcastle disease virus (NDV), is classified as a member of the Rubulavirus genus sub-family Paramyxovirinae in the family Paramyxoviridae, belonging to Mononegavirales and is designated avian paramyxovirus-1 (APMV-1). It caused high morbidity and mortality in non-immunized chickens. Although an intensive vaccination program against ND is carried out, the prevalence of ND is still very serious as the poultry-breeding is more and more florescent in china.Especially since 1996, non-typical ND appears, which are characterized by the performance of similar clinical symptoms and pathological changes in chickens and a common drop in egg production in breeders.We suspect that it is probably congenital infection about similar clinical symptoms and pathological changes in chickens. At the same time, when the isolation of virus are practiced from different organs of infectious breeders, NDV is usually first found in the oviduct not in the brian and spleen. Therefore, we suspected the existence of such a strain, which has a certain tropism for the sexual organ such as the oviduct. Three NDV are isolated from from the eggs of breeders that only showed egg dropping in Shandong and another NDV is isolated from the the oviduct of infectious breeders in Henan. The difference between these four isolates and other NDV are explained through the comparision of antigenicity and the analysis of F and HN gene in our research.Part 1 The isolation and identification of NDVThree NDV are isolated from from the eggs of chickens that only showed egg dropping in Shandong and another NDV is isolated from the oviduct of infectious breeders in Henan. The four isolates are identification by HA and HI, and purified by cloning plague technology.Part 2 Comparasion of the antigenicity among the isolates through across HI testPositive serums of 3 isolates from eggs associated with La sota and F48E8 are produced. Across HI tests are practioned according 5 antigen of NDV and corresponding serums, then HI correlation coefficients are accessed according to the average HI data. The results showed the HI titers of La sota positive serum against 3 field strains are 7.5~8Log2, lower then those of the field stains serum against themselves antigens which is 8~11Log2. It may partly explain the reason why the tranditional protection threshold 8Log2 can not resist the attack of the field virus. The HI correlation coefficients display that the HI antigenicity between the filed virus and the vaccine strain La sota had occurred changes.3 The amplification and analysis of F and HN genesTwo pairs of primers were designed specially and synthetized. This test amplified Fusion F and HN gene of isolates by reverse transcription-polymerase chain reaction (RT-PCR) technique, then sequenced them after the products of amplification being cloned. The results showed that the F genes of the four isolates all have the 1662bp opening reading fragment (ORF) length and encoding 553 amino acids. The amino acids sequences of cleavage site region (112-117) of F gene were similar to that of all other virulent strains. The results also showed that the four isolate strains were virulent strains of NDV. The HN genes of the four isolates all have the 1716bp opening reading fragment (ORF) length and encoding 571 amino acids.By means of sequence comparisions, the amino homologies among the F gene range from 99.5% to 99.8% for the four isolates, those of HN are between 99.6% to 100.0%. The isolated strains share the amino homologies among the F and HN gene of 95.3% to 98.7% and 90.7% to 99.1% respectively with NDV of genetype VII; and 87.9% to 88.1% and 88.1% to 88.4% respectively with La sota; and 91.1% to 91.3% and 89.7% to 90.0% respectively with F48E8. It illustrate the mutation in both F and HN gene had occurred between the isolates and La sota. The constructed phylogenetic tree showed that the 4 isolates belonged to NDV genotypeâ…¦in both F and HN gene.