The Molecular Mechanism For The Increased Virulence Of A Genotype â…¢ NDV Strain Mukteswar

Newcastle disease (ND), which is caused by Newcastle disease virus (NDV), is one of the highly contagious and infectious diseases with worldwide distribution that can cause substantial economical losses to the poultry industry. Up to now, ND vaccines are still being widely used to protect chickens from NDV infection in China. Currently, the genogtype â…¢ NDV Mukteswar has been used widely as a moderate virulent vaccine strain in China. In recent years, genotype â…¢ NDVs, which have the high similarity with Mukteswar in genome, are continually isolated from clinical samples, but the virulence of these NDV isolates is much higher than that of Mukteswar. Most of the isolated genotype â…¢ NDVs are thought to be generated from the long-term use of Mukteswar-based vaccine in poultry, which caused the virulence increase of the isolates in turn. One virulent NDV strain JS/7/05/Ch sharing more than99%identities in nucleotide sequence with Mukteswar was isolated in2005in our laboratory, but it possessed significantly higher virulence when compared with Mukteswar. In order to elucidate the molecular mechanism of the increased virulence of Mukteswar, the difference in the pathogenicity of chickens infected with the two NDV strains JS/7/05/Ch and Mukteswar was compared. Moreover, reverse genetic approach was applied to further determine the key sites in the HN protein responsible for the increased virulence of Mukteswar.1. Pathogenicity analysis of two genotype â…¢ NDVs with different virulenceIn this study, the differences in the replication ability of two NDV strains JS/7/05/Ch and Mukteswar in chicken spleen, bursa of Fabricius, thymus and lung, together with the expression of cytokines induced by the two strains were evaluated. We found that the virus titers in the spleen and bursa of chickens infected with JS/7/05/Ch were much higher than Mukteswar at5days post-infection (dpi). In addition, the expression level of interferon induced by JS/7/05/Ch was also higher than Mukteswar at24hour post-infection (hpi). Moreover, the related genes of inflammatory reaction caused by JS/7/05/Ch were more intense when compared to that of Mukteswar, especially the expression level of interleukin6at12hpi, indicating that JS/7/05/Ch induced stronger inflammatory response in chickens. Together, our results suggest that the increased replication and the early overexpression of inflammatory cytokines caused by JS/7/05/Ch are the main reason for the severe pathogenicity of the newly isolated genotype III NDVs.2. The molecular mechanism of the increased virulence of genotype â…¢ NDV strain MukteswarIn our previous study, we have demonstrated that the HN gene is the key factor for the virulence increase of ND vaccine strain Mukteswar. To further determine the functional domain and key sites of HN protein responsible for the increased virulence of Mukteswar, the receptor binding sites494and495in the HN protein of Mukteswar were mutated to that of JS/7/05/Ch HN protein in the cDNA clone of Mukteswar. The mutant virus was successfully rescued and named as MukHN494+495JS. We found that the pathogenicity, the replication ability and the expression level of cytokines induced by the mutant virus MukHN494+495JS were similar to those of JS/7/05/Ch, but were much higher than those of the parent virus Mukteswar. Together, our results indicate that the amino acids494and495in the HN protein play an important role in the viral virulence, efficient replication and inflammation response of genotype III NDV strain Mukteswar, which may be the main sites for the increased virulence of Mukteswar.