Comparison Of Pathogenicity Between Two Virulent Newcastle Disease Virus Strains

Newcastle disease (ND) is one of the highly contagious and infectious diseases, which is caused by Newcastle disease virus (NDV). Phybgenetic analysis have revealed that Newcastle disease virus strains consist of two distinct classes (I and II) within a single serotype, and class I isolates are avirulent in chickens. Lentogenic NDV, Duck/JS/10 (HQ008337), was isolated by our lab, which was passaged in chicken air sacs for ten times (JS10-A10). During passage of the Duck/JS/10 via air sacs, its pathogenicity was increased, which MDT, ICPI,and IVPI values were 46,1.94, and 2.08, respectively. The amino acid sequence of cleavage site in F protein was changed into 112KRQKRF117. In animal experiment, we found JS10-A10 caused 100% mortality in chicken by intramuscular or intravenous injection, however nomortality can be founded in intranasal injiected chicken. In order to elucidate the different pathogenic mechanism caused by JS10-A10 and classical velogenic NDV, two NDV strains JS10-A10 and Herts/33 (classical velogenic NDV) was comprehensive compared in vivo and viro infection experiment.1. Chicken splenocytes and DF-1 cells infected of two virulent NDVIn this study, we compared the differences among virus replication ability, the interferon and interfeukin expression level and cell death response by two virulent NDV infected cells. Herts/33 replicated at slightly higher efficiency in chicken splenocytes and DF-1 cells than JS10-A10, but there is no significant difference after statistics analysis. In addition, indrect immunofluorescence and neutral red-stained plague assay also proved that the two virulent NDV strains have the similar replication ability. The gene expression levels of IFNα, IFNβ, IFNγ, IL-1β, IL-6 and IL-18 in chicken splenocytes infected with JS10-A10 and Herts/33 were detected at 6h,12h,18h and 24 h postinfection The results showed that there is no significant difference in the levels of cytokines induced by the two NDV virulent strains. The number of free nucleosomal DNA in splenocytes infected with JS10-A10 is as same as Herts/33. Annexin-V and PI double staining of infected DF-1 cells showed that there is no difference between two NDV strains. In conclusion, the replication ability, interferon and interleukin gene expression level and cell death response between JS10-A10 and Herts/33 in chicken splenocytes and DF-1 cells are similarity.2. Use SPF chicken as a model to comparison and analysis of molecular pathogenicity mechanism between two virulent NDVIn this study, the differences in the replication ability between JS10-A10 and Herts/33 in chicken spleen, lung, intestine, liver, and bursa, together with the gene expression of cytokines and lesions induced by the two strains were evaluated. In addition, virus shedding in oropharyngeal and cloacal swabs and antibody neutralization titer were detected. Herts/33 induced more severe lesions in chicken spleen, lung, intestine, liver, and bursa, which is characterized by the remarkable necrosis. Herts/33 replicated at significantly higher levels in chicken spleen, lung, intestine, liver, and bursa than JS10-A10. The expression of cytokines revealed that JS10-A10 induced stronger innate immunity response in chicken than Herts/33. Virus shedding in oropharyngeal and cloacal swabs from chicken infected with Herts/33 was higher than JS10-A10. In contrast, neutralization antibody titer in chicken infected with JS10-A10 was higher than Herts/33. Take together, JS10-A10can induce higher and earlier levels of cytokines and neutralization antibody titer than Herts/33, which can effectively inhibit the proliferation and replication of the virus in chicken tissues, causing less damage to chicken.3. Bioinformatics analysis of two virulent NDV strains and preliminary verificationThrough vivo experiment we hypothesized that the difference in pathogenicity of two NDV strains may due to F protein, HN protein and V protein, so sequence analysis was performed on F, HN, and V protein of JS10-A10 and Herts/33. Sequences were edited using DNAStar software and compared to published strains by BLAST. Reference strains were picked based on BLAST search results as well as from representatives for different lineages. The alignments and phylogenetic analyses were conducted using MEGA software version 6.0. Three-dimensional structures of F, HN, and V protein were compared using the online tools of SWISS-MODEL. The results show that amino acid sequence was differences among F, HN, and V protein. The amino acid at position 25 in F protein of JS10-A10 is alanine (A), not cysteine (C). Fourteen amino acids insertion of HN protein of JS10-A10 was presented at positions 572-585 and the amino acid at position 520 in HN protein of JS10-A10 and Herts/33 was S and R, respectively. Amino acid sequence in V protein was significant differences between JS10-A10 and Herts/33, which may lead to the different ability to antagonize IFN. Through the analyses of genetic information, binding efficiency of two NDV strains was compared using HN monoclonal antibody against NDV. The results showed that the binding ability of Herts/33 was much stronger than JS10-A10. Through the molecular biology analyses, we initially identified that the bingding ability of two strains to trachea and lung tissue sections was different.