Mutation Of Newcastle Disease Virus Hemagglutinin-Neuraminidase From A Waterfowl Isolate By Passaging In Chickens

Newcastle disease (ND) is one of the most serious infectious diseases of poultry .The causative agent, Newcastle disease virus (NDV), is a member of the family Paramyxoviridae and has been placed in the genus Rubulavirus in the subfamily Paramyxovirinae. In the past records there were three times of outbreaks in the world, mostly caused by parrot and pigeon. It is possible that NDV isolated from other species of wild could acquire high virulence by direct passaging in chickens.Thus, the past study experimentally showed that outbreaks by high pathogenic NDVs could occur by the introduction of benign viruses into poultry followed by passages in these bird populations. To demonstrate experimentally the generation of velogenic NDV from an avirulent waterfowl isolate passaged an avirulent goose isolate in chickens. After nine consecutive passages by air sac inoculation (9a), followed by five passages in chick brain(5b), the virus became highly virulent in chickens, producing a 100% mortality and demonstrating typical velogenic properties in pathogenicity tests. Sequence analysis at the fusion protein cleavage site showed that the original isolate contained the typical avirulent type sequence E-R-Q-E-R/L, which progressed incrementally to a typical virulent type K-R-Q-K-R/F, during passages in chickens. The results demostrated that avirulent viruses, maintained in wild waterfowl in nature and bearing the consensus avirulent type sequence, had the potential to become velogenic after transmission to and circulation in chicken population. The result also suggested that chickens provide a mechanism for the evolution of virulent viruses from an avirulent background.The HN protein is a kind of glycoprotein, which contains both hemagglutination and neuraminidase activities of NDV and is responsible for the initial attachment of the virus particles to the receptor and receptor-destroying activity, with an additional function of fusion promotion.In this test, we had worked on the mutation of HN protein on virulence evolution of NDV. The HN genes of 15 viruses were amplified by RT-PCR. The products of RT-PCR were cloned into"pGEM-T easy"vector, then sequenced, to reserch the molecular mechanism for pathogenicity of NDV. In summary, the results showed that after passaging through the air sacs of chicks two times (2a), HN protein ,an G-to-T substitution at nucleotide 1717, resulted in the terminal of HN gene changing from 1864TAA1866 to 1717TAG1719 , and the HN protein from 616 amino acid residues to 572. This mutation was maintained in 9a5b.The results suggested that the HN protein activity changed after 2 consecutive passages through air sacs. By passaging, the original avirulent pathopoiesis nucleotide sequence had generated sensetively mutation. The mutation is happened on the transcriptional level, not on the translational level. No direct evidence suggested the single HN codn mutation is the necessary condition for the enhancing of NDV virulence. But it is possible that the change of the HN protein, which have relationship with pathopoiesis, is the major reason for the enhancing of NDV virulence before F protein cleavage site mutation, namely, the mutation of HN gene was earlier than that of F protein and followed with the change in the virulence.