| Newcastle disease (ND) is one of the most serious avian diseases, which can cause substantial economic losses and remains a major threat to the poultry industry worldwide. The causative agent of the disease is virulent Newcastle disease virus (NDV). Phylogenetic analysis has revealed that NDV strains consist of two distinct classes (I and II) within a single serotype. Class I NDVs are frequently isolated from aquatic birds and most of them are avirulent. Class II NDVs are divided into nine genotypes (I-IX) with genotypes I-IV belonging to earlier genotypes and V-VIII to later genotypes. Since 1926, ND has caused four pandemics worldwide and class II isolates were responsible for all these four outbreaks. The NDVs isolated in the last decade include both earlier and later genotypes, while the dominant virulent NDV strains circulating in poultry flocks belong to subgenotype Ⅶd. Previous studies have demonstrated that NDVs of subgenotype Ⅶd cause more severe pathological lesions in immune organs when compared to the earlier genotypes, but so far the underlining mechanism remains unknown. In this study, to elucidate the different pathogenesis caused by the earlier and later genotypes of NDVs in the immune organs, we compared the pathologic changes in spleens from the chickens infected with genotype Ⅶd NDV (JS-3-05-Ch) and genotype IV NDV (Herts/33), respectively. Meanwhile, the comparative proteomics of spleens from chickens infected with these two NDVs were analyzed using two-dimensional gel electrophoresis (2-DE) together with matrix associated laser dissociation/ionization time of flight mass spectrometry (MALDI-TOF-MS). Additionally, to investigate why subgenotype Ⅶd NDV is dominant in vaccinated poultry flocks, the pathogenic differences in vaccinated chickens challenged by the above two virulent NDVs were also compared in terms of the clinical signs and virus shedding.1. Different pathological changes in vivo induced by genotype Ⅶd and genotype IV NDVsWe used genotype Ⅶd NDV (JS-3-05-Ch) and genotype IV NDV (Herts/33) to infect chickens and compared the pathology and virus replication in the spleens of the infected chickens. The two strains were both highly lethal to chickens and the chickens infected with the two NDVs showed typical ND clinical symptoms. However, the pathology observation showed that the pathological changes in the spleen from JS-3-05-Ch-infected chickens were more severe when compared with those from the Herts/33-infected chickens. Briefly, JS-3-05-Ch manifested very pathognomonic features in chicken spleen:swelling with severe necrotic lesions was observed throughout the spleens of almost all infected chickens. However, no obvious lesions were observed in the Herts/33-infected groups, except for atrophic spleens in the diseased chickens. Moreover, genotype Ⅶd NDV JS-3-05-Ch replicated at significantly higher levels in lymphoid organs than genotype IV NDV Herts/33.The histopathology of infected chickens induced by the two different genotypes NDVs in chicken spleen was consistent with the gross lesions mentioned above. Microscopically, the spleen from the JS-3-05-Ch-infected chickens exhibited extensive necrosis with marked lymphocyte depletion, while Herts/33-infected chickens presented lymphocyte depletion only in the periarterial lymphatic sheath without any remarkable lesions. Using transmission electron microscope (TEM), we found that both JS-3-05-Ch and Herts/33 induced apoptosis in the infected spleens, while more serious pathological change in the mitochondria was observed in the JS-3-05-Ch infected spleens than that of Herts/33. In addition, QRT-PCR result based on the M gene showed that the virus load in the spleen with NDV strain JS-3-05-Ch was higher than that with Herts/33.2. Comparative proteomics analysis of spleen from chickens infected with genotype Ⅶd NDV (JS-3-05-Ch) and genotype IV NDV (Herts/33)Utilizing two-dimensional gel electrophoresis (2-DE) and matrix associated laser dissociation/ionization time of flight mass spectrometry (MALDI-TOF-MS), the proteome profiles of spleens from chickens infected with genotype Ⅶd NDV (JS-3-05-Ch) and genotype IV NDV (Herts/33) were compared at different intervals. The results showed that a total of 108 different proteins were identified among the total 132 differently expressed protein spots and that the majority of different protein expression revealed at 72 and 96h after NDV infection by multiple comparisons of 2-DE gels. The identified proteins are associated with cytoskeletal change, biosynthesis, cellular metabolism, ubiquitin-mediated protein degradiation (UPP), cell deaths, translation regulation, signal transduction, gene regulation and endocytosis. Western blot analysis further demonstrated the alteration of beta-actin during infection which was consistent with the expression changes presented by comparative proteomics analysis. Furthermore, STRING database and Ingenuity pathway analysis (IPA) softwares ware were also used to examine the differentially expressed proteins data set, and these different proteins were ascribed into specific function classes, specific function networks, subcellular annotations and relevant canonical pathways based on the published reports and databases. The results indicated that NDV infection and the host response involved a complex interaction between the host and virus. Both the two virulent NDVs induced intense innate immune response, UPP disorders and cell death of the hosts. However, the genotype Ⅶd virus strain JS-3-05-Ch induced more intense innate immune responses and apoptosis in the chicken spleen than the Herts/33 strain did. Moreover, there were more categories of proteins associated with apoptosis, UPP pathway and cell cytoskeleton alteration, which induced higher grade of cytoskeletal disruption and functional disorders of the UPP system in JS-3-05-Ch infected group than in Herts/33 infected group. The data in the present study will cast light on elucidating the host mechanism of the differential pathological in the spleen that caused by the earlier and later genotype NDVs and also give some clues to explain the potential biological mechanism for the dominance of the Ⅶd NDV in recent years.3. Pathogenic difference of NDV strains JS-3-05-Ch and Herts/33 in the vaccinated chickensOne hundred 3-week-old SPF chickens were randomly divided into three groups. The group 1 and 2 consisting of 40 chickens each were injected subcutaneously with 20ul inactivated oil emulsion-A-VII (subgenotype Ⅶd NDV strain) and oil emulsion-LaSota (routine vaccine strain) respectively. Twenty chickens in group 3 were served as control, which were sham-vaccinated with phosphate buffered saline (PBS) through the same route. Blood samples were taken on day 21 post-immunization (pi), and sera were isolated for HI assays. Subsequently, the chickens in each group were randomly divided into two subgroups which were challenged with 103 ELD50 subgenotype Ⅶd NDV strain JS-3-05-Ch and genotype IV strain Herts/33, respectively. Following the challenge, the clinical signs in experiment chickens were monitored daily for 10 days post-challenge (pc) and oropharyngeal and cloacal swabs were collected on days 3,5,7 and 9 pc for virus isolation and titration.All birds in the sham-vaccinated group displayed 100% mortality on day 5 pc. According to the clinical signs present in chickens, the immune efficacies of oil emulsion-LaSota against the virulent NDV JS-3-05-Ch and Herts/33 were 80% and 95% respectively, while the oil emulsion-A-VII induced 100% immune efficacy against the two challenge viruses. Both in group 1 and group 2, the virus isolation rate was significant higher in chickens challenged with JS-3-05-Ch than that in the chickens infected with Herts/33. The virus titration of the positive swabs from the LaSota-vaccinated chickens showed that titers of shedding virus from the JS-3-05-Ch-injected chickens were significant higher than those from the Herts/33-injected chickens both on day 5 and 7 pc.Additionally, twenty 6-week-old SPF chickens with HI titer of 6 log2 were divided into 2 groups and challenged with JS-3-05-Ch and Herts/33, respectively. On day 3 and 5 post-infection, the spleens were taken for immune-related cytokines detection. The results showed that except IL-10 and IFN-y, the expression of cytokines IL-1β, IL-6, IL-18, LITAF, IFN-a and IFN-β from the JS-3-05-Ch infected chicken spleens were lower than that from the Herts/33 infected ones, indicating that the genotype Ⅶd NDV possessed higher ability to suppress the host immune response and might be responsible for this genotype NDV infection in the vaccinated chicken flocks.Conclusions:(1) Compared with the early genotype IV strain Herts/33, genotype Ⅶd NDVs induce extensive necrotic foci in the spleen and more severe mitochondrion lesion.(2) Genotype Ⅶd NDV (JS-3-05-Ch) infection elicits stronger innate immune response, changes in the cellular cytoskeleton networks, cell death and ubiquitin-proteosome pathway than genotype IV NDV (Herts/33).(3) Significant high replication and the intense inflammatory response may contribute to the severe spleen injury caused by genotype Ⅶd NDV (JS-3-05-Ch).(4) The routine vaccine strain LaSota does not confer sufficient protection against the infection of the dominant subgenotype Ⅶd NDV which possesses high ability to suppress the host immune response. |
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