Investigation of the molecular pathogenesis of newly emerged Newcastle disease virus in Indonesia

Abstract

Newcastle Disease Virus (NDV) has caused significant outbreaks in South-East Asia, particularly in Indonesia. Australia is currently free from virulent strains of NDV. However, the introduction of virulent NDV strains is a severe risk for the Australian poultry industry. Indonesia is the closest country to Australia and spillover might occur through migratory birds. Two viral strains from recent outbreaks have been isolated and analyses by full genome sequencing and phylogenetic assays. This study was conducted to determine the pathogenesis, candidate genes, biological pathways and tissue tropism of recently emerged genotype VII of NDV (NDV-GVII) viral strains. Chickens were experimentally infected with live NDV-GVII virus. Tissue samples were collected after euthanasia of birds. A transcriptomic analysis based on RNA sequencing (RNA-Seq) of spleen was performed in chickens challenged with NDV-GVII and a control group. Repair Assisted Damage Detection (RADD), and immunohistochemistry staining of Viral HN, caspase-3 and MLKL antigens were employed to analyse DNA damage levels, viral load, apoptosis and necroptosis markers, respectively. Phylogenetic study results revealed that these two strains were identical and belong to genotype VII.1, class II cluster of avian paramyxoviruses, and have significant differences in amino acid identities with La Sota strain. In total, 6361 genes were differentially expressed that included 3506 up-regulated genes and 2855 down-regulated genes. Real-time PCR of ten selected differentially expressed genes (DEGs) from the RNA-Seq results showed agreement between the two technologies in detecting DEGs as the correlation between them is 0.98. Functional and network analysis of DEGs showed down regulation of ElF2 signalling, mTOR signalling, the proliferation of lymphatic system cells, signalling by Rho family GTPases and synaptogenesis signalling in spleen. We have also identified increased expression of IFIT5, PI3K, AGT and PLP1 genes in NDV-GVII infected chickens. Bursal atrophy was associated with profound expression of MLKL and only patchy distribution of viral antigen, providing evidence that the mechanism of lymphoid depletion involved a non-apoptotic pathway of programmed cell death termed necroptosis. RADD and oxididative RADD analysis of bursa showed a DNA damage pattern consistent with the programmed cell death rather than necrosis, consistent with MLKL stain results. MLKL expression in the spleen was less pronounced and largely restricted to the central portion of periarteriolar lymphoid sheaths, while other regions of white pulp expressed neither MLKL nor caspase-3. A shift in tissue tropism from neurologic and gastrointestinal to the immune system compared to previously reported NDV-GVII strains was also observed in this strain. Significant differences in amino acid identities of circulation viruses and La Sota strain as the most common vaccine strain used in Indonesia shed more light on the probable reason for vaccine failure against these NDV strains and highlights the urgent need for updated vaccine development strategies in South-East Asia. Our findings in activation of autophagy-mediated cell death, lymphotropic and synaptogenesis signalling pathways provide new insights into the molecular pathogenesis of this newly emerged NDV-GVII. This study is the first report of using RADD assay for DNA damage analysis in NDV infection and revealed the persistence of oxidative lesion in the genome after viral challenge. Together with observations of karyorhexis, fibrinous inflammation, and RADD analyses, we conclude that necrosis was responsible for the majority of lymphoid depletion in the spleen. Therefore, we speculate that the progression of NDV infection may deplete various subsets of lymphocytes by different mechanisms.