Studies Of Cleavage Fusion Protein And Foreign Proteins Recombine Virus On The Histotropism And Pathogenicity Of RLaSota

Newcastle disease virus(NDV)is a member of the family Paramyxoviridae.It has been assigned to the genus Avulavirus in the subfamily Paramyxoviridae. The establishment of reverse genetic for non-segment negative single strain RNA viruses enabled Newcastle disease virus to be the extremely attractive recombinant live viral vaccine candidates. The immunication mechanism and application of recombinant NDV were investigated in this study.To study the impact of foreign gene to the impact of viral antigen histotropism and pathogenicity.Foreign gene includes HA gene and the VP2 protein of the very virulent Infectious Bursal Disease (vvIBDV) Gx strain. HA gene of H5N1 highly pathogenic avian influenza virus the virulence associated multiple basic amino acids of the HA gene were removed .We inserted foreign gene between the M gene and P gene based on the rLaSota backbone.We named rLaSota-HA(mut),rLaSota-vp2. Vaccination was given by eyedrop and intranasally instillation with rLaSota-HA(mut),rLaSota-vp2,rLaSota (100μL/chick) to 2-day-old,10-day-old and 20-day-old SPF chickens, after 1,3 ,5 ,10 ,15,18 days,we collected the trachea. Furthermore,to 2-day-old we collected lungs , livers,kidneys , brains, spleens , proventriculus , duodenum.To rLaSota-HA(mut),rLaSota-vp2,we generated a genetically tagged derivatise of LaSota strain in which the amino acid sequence of the protease cleavage site(GGRQGR↓L) of fusion protein F0 was changed to the consensus cleavage site of virulent NDV strains (GRRQRR↓F) and (GRRQRR↓L).We named them rL-F(M)-FHA,rL-F(M)-FVP2,rL-F(M)-LHA. Vaccination was given by eyedrop and intranasally instillation with them (100μL/chick) to 2-day-old SPF chickens.After 1 , 3 , 5 , 10 , 15 , 18 days,we collected trachea , lungs , livers , kidneys , brains, spleens , proventriculus , duodenum. To chickens immunication with rLaSota-HAmut(GD),rLaSota-vp2,rLaSota ,ultrastructural changes in the tracheal epithelium were examined by light microscopy and scanning electron microscopy. Light microscopy showed small vacuoles containing lymphocytes,heterophils,plasma cells in the epithelial layer.The submucosa is slightly edematous.Immature epithelium proliferated in some areas.The trachea is lined with several layers of immuture cells.There is hyperplasia of mucous gland cells causing narrowing or obliteration of glandular lumina.To the extend of lesion of tracheal epithelium.Scanning electron microscopy revealed foreign gene inserted rLaSota can resulted in the change of tracheal epithelium.Hypertrophy of goblet cell and small patches of the deciliated epithelium around the disoriented and deformed cilla .Hyperplasia of goblet cells accompanied by an increase in mucus .Globular particles found in areas of the trachea epithelium with tips of cilia adhering to them,goblet cells and basal cell regenerated.But they gradually decreased in size. This response is probably an attempt by the mucocillary defense mechanism. To chickens immunication with rL-F(M)-FHA,rL-F(M)-FVP2,rL-F(M)-LHA the entire structure of the trachea epithelium was destroyed.The histotropism of viral antigen in tissues were detected by using immunohistochemical(IHC) staining. As a resault, to chickens immunication with rLaSota,rLaSota-VP2 ,specilfic staining was localized in the trachea submucosa cells,hepatocytes;To chickens immunication with rLaSota-HAmut(GD),specilfic staining was localized in the tracheal submucosa cells,hepatocytes,bronchus epithelial cells,alveolar nacrophages; To chickens immunication with rL-F(M)-FHA , rL-F(M)-LHA , rL-F(M)-Fvp2, the viral angen could be detected in multiple organs and tissues.We found specific staining was localized in lymphocytes, epithelial cells,macrophages, hepatocytes ,neuroglia cells ,gland cells .These results showed that HAmut(GD)being inserted have great influence in virulence, rLaSota-HAmut(GD)have little lesion of tracheal epithelium. Vp2 being inserted have little influence in virulence.It was similar to rLaSota. rLaSota- Vp2 have great lesion of tracheal epithelium.Cleavage of the F protein can induce great lesion to tracheal epithelium. Foreign gene being inserted rLaSota have little influence to histotropism of viral antigen.Cleavage of the F protein had great influence to histotropism of viral antigen, rL-F(M)-FHA, rL-F(M)-FVP2, rL-F(M)-LHA can be used to reinforce immunication in immunication program.This was the first study to evaluate the NDV virulence and immunication mechanism by observing lesion of tracheal epithelium.Furthermore,it has very important academic and practical significance for the immunication rLaSota-HA(mut),rLaSota-vp2.It was a new way to probe the reinforce immunication strain.