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Newcastle disease virus (NDV) is an avian paramyxovirus that causes major economic losses to the poultry industry around the world, with NDV pathogenicity varying due to strain virulence differences. However, the impacts of intracellular viral replication and the heterogeneity of host responses among cell types are unknown. Here, we investigated the heterogeneity of lung tissue cells in response to NDV infection in vivo and that of the chicken embryo fibroblast cell line DF-1 in response to NDV infection in vitro using single-cell RNA sequencing. We characterized the NDV target cell types in the chicken lung at the single-cell transcriptome level and classified cells into five known and two unknown cell types. The five known cell types are the targets of NDV in the lungs with virus RNA detected. Different paths of infection in the putative trajectories of NDV infection were distinguished between in vivo and in vitro , or between virulent Herts/33 strain and nonvirulent LaSota strain. Gene expression patterns and the interferon (IFN) response in different putative trajectories were demonstrated. IFN responses were elevated in vivo , especially in myeloid and endothelial cells. We distinguished the virus-infected and non-infected cells, and the Toll-like receptor signaling pathway was the main pathway after virus infection. Cell-cell communication analysis revealed the potential cell surface receptor-ligand of NDV. Our data provide a rich resource for understanding NDV pathogenesis and open the way to interventions specifically targeting infected cells. IMPORTANCE Newcastle disease virus (NDV) is an avian paramyxovirus that causes major economic losses to the poultry industry around the world, with NDV pathogenicity varying due to strain virulence differences. However, the impacts of intracellular viral replication and the heterogeneity of host responses among cell types are unknown. Here, we investigated the heterogeneity of lung tissue cells in response to NDV infection in vivo and that of the chicken embryo fibroblast cell line DF-1 in response to NDV infection in vitro using single-cell RNA sequencing. Our results open the way to interventions specifically targeting infected cells, suggest principles of virus-host interactions applicable to NDV and other similar pathogens, and highlight the potential for simultaneous single-cell measurements of both host and viral transcriptomes for delineating a comprehensive map of infection in vitro and in vivo . Therefore, this study can be a useful resource for the further investigation and understanding of NDV., Competing Interests: The authors declare no conflict of interest.

Viruses require host cell metabolic reprogramming to satisfy their replication demands; however, the mechanism by which the Newcastle disease virus (NDV) remodels nucleotide metabolism to support self-replication remains unknown. In this study, we demonstrate that NDV relies on the oxidative pentose phosphate pathway (oxPPP) and the folate-mediated one-carbon metabolic pathway to support replication. In concert with [1,2- 13 C 2 ] glucose metabolic flow, NDV used oxPPP to promote pentose phosphate synthesis and to increase antioxidant NADPH production. Metabolic flux experiments using [2,3,3- 2 H] serine revealed that NDV increased one-carbon (1C) unit synthesis flux through the mitochondrial 1C pathway. Interestingly, methylenetetrahydrofolate dehydrogenase (MTHFD2) was upregulated as a compensatory mechanism for insufficient serine availability. Unexpectedly, direct knockdown of enzymes in the one-carbon metabolic pathway, except for cytosolic MTHFD1, significantly inhibited NDV replication. Specific complementation rescue experiments on small interfering RNA (siRNA)-mediated knockdown further revealed that only a knockdown of MTHFD2 strongly restrained NDV replication and was rescued by formate and extracellular nucleotides. These findings indicated that NDV replication relies on MTHFD2 to maintain nucleotide availability. Notably, nuclear MTHFD2 expression was increased during NDV infection and could represent a pathway by which NDV steals nucleotides from the nucleus. Collectively, these data reveal that NDV replication is regulated by the c-Myc-mediated 1C metabolic pathway and that the mechanism of nucleotide synthesis for viral replication is regulated by MTHFD2. IMPORTANCE Newcastle disease virus (NDV) is a dominant vector for vaccine and gene therapy that accommodates foreign genes well but can only infect mammalian cells that have undergone cancerous transformation. Understanding the remodeling of nucleotide metabolic pathways in host cells by NDV proliferation provides a new perspective for the precise use of NDV as a vector or in antiviral research. In this study, we demonstrated that NDV replication is strictly dependent on pathways involved in redox homeostasis in the nucleotide synthesis pathway, including the oxPPP and the mitochondrial one-carbon pathway. Further investigation revealed the potential involvement of NDV replication-dependent nucleotide availability in promoting MTHFD2 nuclear localization. Our findings highlight the differential dependence of NDV on enzymes for one-carbon metabolism, and the unique mechanism of action of MTHFD2 in viral replication, thereby providing a novel target for antiviral or oncolytic virus therapy.

The receptor tyrosine kinases TYRO3, AXL, and MERTK (TAM) are transmembrane proteins associated with the regulation of the innate immune response. In this study, the role of the chicken-derived MERTK protein (chMertk) in the regulation of the type I interferon (IFN) signaling pathway and its antiviral effect were investigated in vitro. Newcastle disease (ND) caused by the Newcastle disease virus (NDV) is able to widely spread in chickens and give rise to massive losses in the poultry industry around the world. We found that the overexpression of the exogenous chMertk upregulated the STAT1 phosphorylation and the expression of IFN-stimulated gene IFITM3 and significantly reduced the NDV titer (p < 0.05). A mutation assay showed that three tyrosine residues (Y739, Y743, and Y744) in chMertk promoted STAT1 phosphorylation and inhibited NDV replication. However, the chicken-derived E3 ubiquitin ligase CBL significantly negatively regulated chMertk expression, thus attenuating STAT1 phosphorylation. chMertk function was restored by the ubiquitin-proteasome inhibitor MG132, demonstrating that chMertk was controlled by Casitas B-lineage proto-oncogene (CBL) ubiquitination and degradation. Together, these results suggested that chMertk participated in regulating the immune responses to NDV infection, and that CBL significantly downregulated the expression of chMertk through its ubiquitination and degradation, to maintain cellular homeostasis. Overall, our study provided new insights into the role of chMertk in regulating the innate immune response and its anti-NDV activity., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

Cold stress in poultry is a global problem that causes huge economic losses and threatens the health and welfare of poultry. However, knowledge of chicken responses to virus infection under cold stress is limited. The purpose of this research was to investigate the effects of cold stress on gene expression and viral replication in chicken DF-1 cells in hypothermia. In addition, the characterization of circulating steroid hormone profiles in the plasma of chickens under cold stress was analyzed by liquid chromatography-tandem mass spectrometry. Herein, we performed RNA sequencing to obtain DF-1 cell transcriptional profiles under cold stress. A total of 9499 differentially expressed genes (DEGs) were identified in DF-1 cells. Overexpressed DEGs were related to the proteasome, cell cycle, spliceosome, ribosome biogenesis, and mammalian target of rapamycin (mTOR). Down-regulated DEGs were related to ribosomes, oxidative phosphorylation, apoptosis, and the p53 signaling pathway. Gene set enrichment analysis showed that the DEGs mainly affect host ribosome translation and mitochondrial respiratory electron transport. The principal steroid hormone alterations in chickens subjected to cold stress included dihydrotestosterone, testosterone, β-sitosterol, androstenedione, 7a,27-dihydroxycholesterol,7-ketocholesterol, and desmosterol, which are associated with endocrine resistance, ovarian steroidogenesis, and steroid hormone biosynthesis. In addition, Infectious bronchitis virus (IBV), Newcastle disease virus (NDV), and Influenza A (H9N2) Virus replication in DF-1 cells is significantly inhibited by cold stress. Moreover, the plasma concentrations of corticosterone, an important stress hormone in poultry, were significantly elevated in chickens subjected to cold stress, and we found that IBV and vesicular stomatitis virus (VSV) replication were strongly inhibited in DF-1 cells pretreated with CORT, but NDV and H9N2 replication were unaffected. In conclusion, in response to cold stress, the translation efficiency and mitochondrial respiratory chain are temporarily weakened in DF-1 cells, which affects virus replication. Chickens may regulate aromatase deficiency, androstenedione metabolism, androgen and estrogen metabolism, and 17-beta hydroxysteroid dehydrogenase III deficiency through steroid hormones in response to cold stress. This study provides valuable insights into the molecular regulatory mechanisms of poultry under cold stress and may support further research on the intrinsic link between steroid hormones and virus replication under stress., Competing Interests: Conflict of interest This is the first submission of this manuscript and no parts of this manuscript are being considered for publication elsewhere. All authors have approved this manuscript. No author has financial or other contractual agreements that might cause conflicts of interest., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The genotype VII Newcastle disease virus (NDV) vaccine has begun to replace the traditional genotype II NDV vaccine and is widely used in the commercial poultry of China. However, the effect of homologous and heterogeneous anti-NDV serum on the evolution of prevalent NDV is unknown. To understand the effect of genotype II and VII anti-NDV serum on the evolution of genotype VII NDV strains, ZJ1 (waterfowl origin) and CH/SD/2008/128 (ND128; chicken origin) were used for serial passage of 30 generations in DF-1 cells without anti-NDV serum or with genotype II and VII anti-NDV serum independently. The F and HN genes of the 2 viruses were amplified for the 10th, 20th, and 30th generations of each serial passage group and compared with their respective original viruses. We found that there was only one mutation at position 248 in the F gene of ZJ1 due to the serum pressure of genotype VII anti-NDV. Similarly, mutations at residue 527 of the F gene, and position 9 and 319 of the HN gene of ND128 were noted in both anti-NDV serum groups. The results show that the nonsynonymous (NS)-to-synonymous (S) ratio of the F gene of ZJ1 virus was 1.6, and for the HN gene, it was 2.5 in the anti-II serum group. In the anti-VII serum group, the NS/S ratio for the F gene was 2.1, and for the HN gene, it was 2.5. The NS/S ratio of the F gene of the ND128 virus was 0.8, and for the HN gene, it was 3 in the anti-II serum group. Furthermore, the NS/S ratio of the F gene was 0.8, and the HN gene was 2.3 in the anti-VII group. Taken together, our findings highlight that there was no significant difference in the variation of protective antigens in genotype VII NDV under the selection pressure of homologous and heterogeneous genotype NDV inactivated vaccines., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Newcastle disease virus (NDV) causes a highly contagious and devastating disease in poultry. ND causes heavy economic losses to the global poultry industry by decreasing the growth rate, decrease in egg production high morbidity and mortality. Although significant advances have been made in the vaccine development, outbreaks are reported in vaccinated birds. In this study, we report the damage caused by NDV infection in the pancreatic tissues of vaccinated and specific-pathogen-free chickens. The histopathological examination of the pancreas showed severe damage in the form of partial depletion of zymogen granules, acinar cell vacuolization, necrosis, apoptosis, congestion in the large and small vessels, sloughing of epithelial cells of the pancreatic duct, and mild perivascular edema. Increased plasma levels of corticosterone and somatostatin were observed in NDV-infected chicken at three- and five- days post infection (DPI). A slight decrease in the plasma concentrations of insulin was noticed at 5 DPI. Significant changes were not observed in the plasma levels of glucagon. Furthermore, NDV infection decreased the activity and mRNA expression of amylase, lipase, and trypsin from the pancreas. Taken together, our findings highlight that NDV induces extensive tissue damage in the pancreas, decreases the activity and expression of pancreatic enzymes, and increases plasma corticosterone and somatostatin. These findings provide new insights that a defective pancreas may be one of the reasons for decreased growth performance after NDV infection in chickens.

Newcastle disease (ND), which is caused by Newcastle disease virus (NDV), can cause heavy economic losses to the poultry industry worldwide. It is characterised by extensive pathologies of the digestive, respiratory, and nervous systems and can cause severe damage to the reproductive system of egg-laying hens. However, it is unknown whether NDV replicates in the male reproductive system of chickens and induces any pathologies. In this study, we selected a representative strain (i.e. ZJ1) of the most common genotype (i.e. VII) of NDV to investigate whether NDV can induce histological, hormonal, and inflammatory responses in the testes of specific pathogen free (SPF) roosters. NDV infection increased the expression of toll like receptor TLR3, TLR7, MDA5, IFN-α, IFN-β, IFN-γ, IL-8, and CXCLi1 in the testes of NDV-infected roosters at 5 days post-infection (dpi). Severe histological changes, including decrease in the number of Sertoli cells and individualized, shrunken spermatogonia with pyknotic nuclei, were observed at 3 dpi. At 5 dpi, the spermatogenic columns were disorganized, and there were fewer cells, which were replaced by necrotic cells, lipid vacuoles, and proteinaceous homogenous material. A significant decrease in the plasma concentrations of testosterone and luteinizing hormone (LH) and the mRNA expression of their receptors in the testes, steroidogenic acute regulatory protein, cytochrome P450 side-chain cleavage enzyme, and 3β-hydroxysteroid dehydrogenase in the NDV-infected group was observed relative to those in the control group (P < 0.05). Collectively, these results indicate that NDV infection induces a severe inflammatory response and histological changes, which decrease the steroidogenesis.

The APOBEC proteins play significant roles in the innate and adaptive immune system, probably due to their deaminase activities. Because APOBEC1 (A1) and APOBEC3 (A3) are absent in the chicken genome, we were interested in determining whether chicken APOBEC4 (A4) possessed more complex functions than its mammalian homologs. In this study, chicken A4 (chA4) mRNA was identified and cloned for the first time. Based on bioinformatics analyses, the conserved zinc-coordinating motif (HXE … PC(X) 2-6 C) was identified on the surface of chA4 and contained highly conserved His97, Glu99, Pro130, Cys131 and Cys138 active sites. The highest expression levels of constitutive chA4 were detected in primary lymphocytes and bursa of Fabricius. Newcastle Disease (ND) is one of the most serious infectious diseases in birds, causing major economic losses to the poultry industry. In vitro, Newcastle Disease Virus (NDV) early infection induced significant increases in chA4 expression in the chicken B cell line, DT40, the macrophage cell line, HD11 and the CD4 + T cell line, MSB-1, but not the fibroblast cell line, DF-1. In vivo, the expression levels of chA4 were up-regulated in several tissues from NDV-infected chickens, especially the thymus, testicles, duodenum and kidney. The high level expression of exogenous chA4 displayed inhibitory effects on NDV and reduced viral RNA in infected cells. Taken together, these data demonstrate that chA4 is involved in the chicken immune system and may play important roles in host anti-viral responses., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Newcastle disease (ND) is an acute, febrile, highly contagious disease caused by the virulent Newcastle disease virus (vNDV). The disease causes serious economic losses to the poultry industry. However, the metabolic changes caused by vNDV infection remain unclear. The objective of this study was to determine the metabolomic profiling after infection with vNDV. DF-1 cells infected with the vNDV strain Herts/33 and the lungs from Herts/33-infected specific pathogen-free (SPF) chickens were analyzed via ultra-high-performance liquid chromatography/quadrupole time-of-flight tandem mass spectrometry (UHPLC-QTOF-MS) in combination with multivariate statistical analysis. A total of 305 metabolites were found to have changed significantly after Herts/33 infection, and most of them belong to the amino acid and nucleotide metabolic pathway. It is suggested that the increased pools of amino acids and nucleotides may benefit viral protein synthesis and genome amplification to promote NDV infection. Similar results were also confirmed in vivo. Identification of these metabolites will provide information to further understand the mechanism of vNDV replication and pathogenesis.

Newcastle disease virus (NDV), an avian paramyxovirus, was shown to prefer to replicate in tumor cells instead of normal cells; however, this mechanism has not been fully elucidated. Exosomes play a crucial role in intercellular communication due to the bioactive substances they carry. Several studies have shown that exosomes are involved in virus infections. However, the effect that exosomes have on NDV-infected tumor cells is not known. In this study, we focus on the role of exosomes secreted by NDV-infected HeLa cells in promoting NDV replication. Three miRNA candidates (miR-1273f, miR-1184, and miR-198) embraced by exosomes were associated with enhancing NDV-induced cytopathic effects on HeLa cells. Furthermore, luciferase assays, RT-qPCR, and enzyme-linked immunosorbent assay (ELISA) all demonstrated that these miRNAs could suppress interferon (IFN)-β gene expression. Enhanced NDV replication in HeLa cells was identified by Western blot and plaque assays. Based on these results, we speculate that NDV employed exosomes entry into neighboring cells, which carry miRNAs, resulting in inhibition of the IFN pathway and promotion of viral infection. To our knowledge, this is the first report on the involvement of NDV-employed exosomes in tumor cells, and as such, it provides new insights into the development of anti-tumor therapies., Competing Interests: The authors declare that they do not have any conflicts of interest.

The fusogenically activated F and HN proteins of virulent NDV induce complete autophagic flux in DF-1 and A549 cells. However, the effect of both glycoproteins on mitochondria remains elusive. Here, we found that F and HN cooperation increases mitochondrial biogenesis but does not cause the mitochondria damage. We observed that both glycoproteins change the morphological characteristics and spatial distribution of intracellular mitochondria. F and HN cooperate cooperatively to induce ER stress and UPR mt . Our preliminary data suggested that F and HN cooperatively disturb mitochondrial fusion-fission homeostasis to enhance mitochondrial biogenesis, and eventually meet the energy demand of syncytium formation.

Autophagy triggered by glycoprotein-mediated membrane fusion has been reported for several paramyxoviruses. However, the function of HN and F glycoproteins of NDV and their role in autophagy induction have not been studied. Here, we found that co-transfection of HN and F of virulent NDV rapidly induced syncytium formation and triggered a steady state autophagy flux in adenocarcinomic human alveolar basal epithelial (A549) cells and chicken embryo fibroblast (DF-1) cells. Furthermore, we clearly identified that F and HN synergistically induced autophagosome fusion with lysosomes for subsequent degradation. The seven cleavage site mutations of F significantly decreased the autophagy induction, compared with those of wildtype virulent F. RNAi and pharmacological experiments suggested that autophagy benefitted membrane fusion and syncytium formation induced by F and HN of NDV. Activated F 1 co-operated with HN to stimulate AMPK kinase and downstream ULK1 activation to suppress mTORC1 signaling. Our data described the synergistic role of HN and F in the induction of completed autophagic flux through the activation of AMPK- mTORC1- ULK1 pathway., (Copyright © 2019 Elsevier B.V. All rights reserved.)

High mobility group box 1 (HMGB1) is a key member of the "danger associated molecular patterns" (DAMPs) which plays important roles in systemic inflammation and has a pathogenic role in infectious diseases like viral or bacterial infections. Newcastle disease virus (NDV) infection is proved to cause intense inflammatory responses that result in excessive cellular apoptosis and tissue damage, but the function of HMGB1 in NDV-induced cytokine storm has not been elucidated. Here, we report that HMGB1 is a significant inflammation factor in NDV infection. HMGB1 is widely distributed in chicken tissues, and the secretion of it was induced by NDV infection both in DF-1 and A549 cells. Moreover, inhibiting the secretion of HMGB1, NDV replication was not significantly reduced, but it is involved in NDV-induced NF-κB activation and the inflammatory response. Further investigation showed that HMGB1 promotes inflammatory cytokine production through the RAGR, TLR2, and TLR4 receptors. HMGB1-RAGE interaction also takes parts in activation of ERK1/2 and JNK induced by NDV infection. Neutralizing HMGB1 in vivo, chicken viability was increased. Pathological changes and inflammatory cytokines expression were reduced under NDV infection, which further confirmed the pathogenic roles of HMGB1 in inflammatory responses. Thus, our findings show that HMGB1 contributes to the inflammatory cytokine storm induced by NDV infection, which played a critical role in viral pathogenesis., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Newcastle disease (ND), caused by ND virus (NDV), is one of the most infectious and economically important diseases of the poultry industry worldwide. While infections are reported in a wide range of avian species, the pathogenicity of chicken-origin virulent NDV isolates in ducks remains elusive. In this study, two NDV strains were isolated and biologically and genetically characterized from an outbreak in chickens and apparently healthy ducks. Pathogenicity assessment indices, including the mean death time (MDT), intracerebral pathogenicity index (ICPI) and cleavage motifs in the fusion (F) protein, indicated that both isolates were velogenic in nature. While these isolates carried pathogenic characteristics, interestingly they showed differential pathogenicity in ducks. The chicken-origin isolate caused high (70%) mortality, whereas the duck-origin virus resulted in low (20%) mortality in 4-week-old ducks. Intriguingly, both isolates showed comparable disease pathologies in chickens. Full-genome sequence analysis showed that the virus genome contains 15 192 nucleotides and carried features that are characteristic of velogenic strains of NDV. A phylogenetic analysis revealed that both isolates clustered in class II and genotype VII. However, there were several mutations in the functionally important regions of the fusion (F) and haemagglutinin-neuraminidase (HN) proteins, which may be responsible for the differential pathogenicity of these viruses in ducks. In summary, these results suggest that NDV strains with the same genotype show differential pathogenicity in chickens and ducks. Furthermore, chicken-origin virulent NDVs are more pathogenic for ducks than duck-origin viruses. These findings propose a role for chickens in the evolution of viral pathogenicity and the potential genetic resistance of ducks to poultry viruses., (© 2018 Blackwell Verlag GmbH.)

Newcastle disease virus (NDV) V protein is crucial for viral interferon (IFN) antagonism and virulence, determining its host range restriction. However, little information is available on the B cell epitopes of V protein and the subcellular movement of V protein in the process of NDV infection. In this study, the monoclonal antibody (mAb) clone 3D7 against genotype VII NDV V protein was generated by immunizing mice with a purified recombinant His-tagged carboxyl-terminal domain (CTD) region of V protein. Fine epitope mapping analysis and B-cell epitope prediction indicated that mAb 3D7 recognized a linear epitope 152 RGPAELWK 159 , which is located in the V protein CTD region. Sequence alignment showed that the mAb clone 3D7-recognized epitope is highly conserved among Class II genotype VII NDV strains, but not among other genotypes, suggesting it could serve as a genetic marker to differentiate NDV genotypes. Furthermore, the movement of V protein during NDV replication in infected cells were determined by using this mAb. It was found that V protein localized around the nucleus during virus replication. The establishment of V protein-specific mAb and identification of its epitope extend our understanding of the antigenic characteristics of V protein and provide a basis for the development of epitope-based diagnostic assays., (Copyright © 2018. Published by Elsevier B.V.)

Avian avulaviruses serotype 1 (abbreviated as APMV-1 for the historical name avian paramyxovirus 1) are capable of infecting a wide spectrum of avian species with variable clinical symptoms and outcomes. Ease of transmission has allowed the virus to spread worldwide with varying degrees of virulence depending upon the virus strain and host species. The emergence of new virulent genotypes from global epizootics, and the year-to-year genomic changes in low and high virulence APMV-1 imply that distinct genotypes of APMV-1 are simultaneously evolving at different geographic locations across the globe. This vast genomic diversity may be favoured by large variety of avian species susceptibility to APMV-1 infection, and by the availability of highly mobile wild birds. It has long been considered that waterfowls are not sensitive to APMV-1 and are unable to show any clinical signs, however, outbreaks from the 90's contradict these concepts. The APMV-1 isolates are increasingly reported from the waterfowl. Waterfowl have strong innate immune responses, which minimize the impact of virus infection, however, are unable to prevent the viral shedding. Numerous APMV-1 are carried by domestic waterfowl intermingling with terrestrial poultry. Therefore, commercial ducks and geese should be vaccinated against APMV-1 to minimize the virus shedding and for the prevention the transmission. Genetic diversity within APMV-1 demonstrates the need for continual monitoring of viral evolution and periodic updates of vaccine seed-strains to achieve efficient control and eradication of APMV-1 in waterfowls.

Newcastle disease (ND), characterized by visceral, respiratory, and neurological pathologies, causes heavy economic loss in the poultry industry around the globe. While significant advances have been made in effective diagnosis and vaccine development, molecular mechanisms of ND virus (NDV)-induced neuropathologies remain elusive. In this study, we report the magnitude of oxidative stress and histopathological changes induced by the virulent NDV (ZJ1 strain) and assess the impact of vitamin E in alleviating these pathologies. Comparative profiling of plasma and brains from mock and NDV-infected chicken demonstrated alterations in several oxidative stress makers such as nitric oxide, glutathione, malondialdehyde, total antioxidant capacity, glutathione S-transferase, superoxide dismutase, and catalases. While decreased levels of glutathione and total antioxidant capacity and increased concentrations of malondialdehyde and nitric oxide were observed in NDV-challenged birds at all time points, these alterations were eminent at latter time points (5 days post infection). Additionally, significant decreases in the activities of glutathione S-transferase, superoxide dismutase, and catalase were observed in the plasma and brains collected from NDV-infected chickens. Intriguingly, we observed that supplementation of vitamin E can significantly reduce the alteration of oxidative stress parameters. Under NDV infection, extensive histopathological alterations were observed in chicken brain including neural inflammation, capillary hyperemia, necrosis, and loss of prominent axons, which were reduced with the treatment of vitamin E. Taken together, our findings highlight that neurotropic NDV induces extensive tissue damage in the brain and alters plasma oxidative stress profiles. These findings also demonstrate that supplementing vitamin E ameliorates these pathologies in chickens and proposes its supplementation for NDV-induced stresses., Competing Interests: The authors declare no conflicts of interest.

Background/aims: Newcastle disease virus (NDV) causes a highly devastating and contagious disease in poultry, which is mainly attributed to extensive tissue damages in the digestive, respiratory and nervous systems. However, nature and dynamics of NDV-induced oxidative stresses in the intestine of chickens remain elusive., Methods: In this study, we examined the magnitude of intestinal oxidative stress and histopathological changes caused by the virulent NDV infection, and explored the protective roles of vitamin E (vit. E) in ameliorating these pathological changes. For these purposes, chickens were divided into four groups namely i) non supplemented and non-challenged (negative control, CON); ii) no supplementation of vit. E but challenged with ZJ1 (positive control, NS+CHA); iii) vit. E supplementation at the dose of 50 IU/day/Kg body weight and ZJ1 challenge (VE50+CHA); and 4) vit. E supplementation at the dose of 100 IU/day/Kg body weight and ZJ1 challenge (VE100+CHA). In all groups, we analyzed concentrations of glutathione (GSH), malondialdehyde (MDA), nitric oxide (NO), total antioxidant capacity (T-AOC), and activity of glutathione S-transferase (GST), superoxide dismutase (SOD), catalase (CAT) using biochemical methods. The virus loads were determined by quantitative RT-PCR and antibody titers by hemagglutination inhibition assays. We also examined the histopathological changes in the duodenal and jejunal mucosa at 3 and 5-day post infection (dpi) with NDV., Results: A significant elevation in the NO level was observed in NDV challenged chickens compared to the CON chickens at 2 dpi. The MDA contents were significantly increased whereas GSH was significantly decreased in NDV-challenged chickens compared to control. Furthermore, activities of GST, CAT, SOD, as well as the TOAC were markedly decreased in challenged chickens in comparison with control. Virus copy numbers were higher in NDV infected NS+CHA group compared to other groups. Severe histopathological changes including inflammation, degeneration and broken villi were observed in the intestine of NDV challenged chickens. However, all these malfunctions of antioxidant system and pathological changes in the intestine were partially or completely reversed by the vit. E supplementation., Conclusions: Our results suggest that NDV infection causes oxidative stress and histopathological changes in the duodenum and jejunum of chickens, which can be partially or fully ameliorated by supplementation of vit. E. Additionally, these findings suggest that oxidative stress contributes to the intestinal damages in NDV infected chickens. These findings will help to understand the pathogenesis of NDV and further investigation of therapeutic agents for control of Newcastle disease., (© 2018 The Author(s). Published by S. Karger AG, Basel.)

Background: For many years, ND has been one of the most important infectious pigeon diseases in China. In recent years, a high mortality has been observed in ND-infected pigeons in China. Mortality is from 40% to 80% or 100% in some cases., Methods: The full-length genomes of four pigeon paramyxovirus type 1 (PPMV-1) strains, which were isolated from infected pigeons in China in 2012 and 2013, were sequenced and analyzed to determine the phylogenetic characteristics of PPMV-1 circulating in pigeons of China in recent years. Furthermore, cross hemagglutination inhibition and cross virus neutralization assays, as well as animal experiments were conducted to determine the antigenicity and pathogenicity of those viruses. Proteolytic cleavage sites (residues 112-117) of the F proteins were identified as the typical virulence motif, 112 RRQKR↓F 117 for all four PPMV-1 strains investigated., Results: Phylogenetic analysis based on sequences of complete genomes and F gene revealed that the four PPMV-1 isolates and most of recent isolates in China were highly homologous to European isolates from 1998 to 2011. All those isolates were clustered in one clade of genotype VI NDV, termed as subgroup 4bii f. The R value was calculated based on cross hemagglutination inhibition and cross virus neutralization results, and confirmed antigenic difference of the PPMV-1 strains isolated in 2013 from the LaSota vaccine strain. Several mutations were identified in the surface glycoproteins F and HN, which probably gave rise to those antigenic differences., Conclusion: Our result suggested that the PPMV-1 strain prevailing in China in the last decade diverged from a common ancestor and was presumably transmitted from Europe. PPMV-1 isolates displayed obvious antigenic differences from vaccine strain LaSota. Even though PPMV-1 did not cause high mortality in experimental pigeons, the infected pigeons were exhibiting viral shedding for 3 weeks after infection, suggesting PPMV-1 is a potential threat to NDV control worldwide.

Newcastle disease virus (NDV) is an oncolytic virus which selectively replicates in tumor cells and exerts anti-tumor cytotoxic activity by promoting cell death. In this study, we focus on characterization of the underlying mechanisms of NDV-induced cell death in HeLa cells. We find that NDV Herts/33 strain triggers both extrinsic and intrinsic apoptosis at late infection times. The activation of NF-кB pathway and subsequent up-regulation of TNF-α/TRAIL initiates extrinsic apoptosis, leading to activation of caspase 8 and cleavage of Bid into tBid. tBid transmits the extrinsic apoptotic signals to mitochondria and mediates intrinsic apoptosis, which is hallmarked by cleavage of caspase 9. Moreover, RIP1 is cleaved into RIP1-N and RIP1-C at D324 by caspase 8, and this cleavage promotes apoptosis. Surprisingly, over expression of RIP1 reduces apoptosis and depletion of RIP1 promotes apoptosis, suggesting full length RIP1 is anti-apoptotic. Moreover, necroptosis hallmark protein MLKL is activated by phosphorylation at 12-24 h.p.i., and RIP1 regulates the level of phosphor-MLKL. Immunostaining shows that RIP1 aggregates to stress granules (SGs) at 8-24 h.p.i., and phosphor-MLKL is also recruited to SGs, instead of migrating to plasma membrane to exert its necrotic function. Immunoprecipitation study demonstrates that RIP1 bind to phosphor-MLKL, and depletion of RIP1 reduces the aggregation of MLKL to SGs, suggesting that RIP1 recruits MLKL to SGs. Altogether, NDV infection initiates extrinsic apoptosis via activation of NF-кB and secretion of TNF-α/TRAIL. Activation of caspase 8 by TNF-α/TRAIL and subsequent cleavage of Bid and RIP1 transmit the death signals to mitochondria. Meanwhile, virus subverts the host defensive necroptosis via recruiting phosphor-MLKL by RIP1 to SGs. Thus, RIP1 is a central signaling protein in regulation of apoptosis and necroptosis during NDV infection.

Inflammatory responses are important aspects of the innate immune system during virus infection. We found that Newcastle disease virus can induce inflammasome activation in the human macrophage-like cell line THP-1. Viral replication was required for inflammasome activation, and small hairpin RNA knockdown experiments indicated that IL-1β secretion was mediated by the NLRP3 inflammasome. We also verified the results in LPS-primed bone marrow-derived macrophages (BMDMs) from NLRP3-deficient and wild type mice. NDV is considered to be a promising oncolytic virus. Stimulating the immune system has been proposed as a key mechanism of oncolytic specificity, and the inflammasome appears to be an important mechanism by which NDV is controlled. Knockdown of inflammasome components or chemical inhibition of caspase-1 activity shows that cell survival was augmented and benefited NDV replication. This study shows that NLRP3 inflammasome activation is an innate cellular response to NDV infection and offers insights into the oncolytic specificity of NDV., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Newcastle disease virus (NDV) encodes a highly phosphorylated P protein; however, the phosphorylation sites have not been identified, and the relationship between phosphorylation and protein function is still unclear. In this study, we bioinformatically predicted 26 amino acid residues in the P protein as potential phosphorylation sites. Furthermore, we treated infected cells with kinase inhibitors to investigate NDV propagation and found that protein kinase C (PKC) is involved in the NDV life cycle and that PKC-activated phosphorylation functions in NDV replication. Using an NDV minigenome assay, we found that expression of a reporter protein decreased when the minigenome system contained P mutants lacking T44, S48, T271, S373 and especially T111. The phosphorylation status of S48, T111, S125 and T271 was determined by Phos-tag SDS-PAGE analysis. Coimmunoprecipitation assays showed that the binding activity of NP and the P-T111A mutant was stronger than that of NP and the wild-type P, suggesting that P-T111 is involved in NP-P interaction. This study sheds light on the mechanism by which P protein phosphorylation affects NDV replication and transcription.

Unlabelled: It has been reported that lentogenic Newcastle disease virus (NDV) isolates have the potential to become velogenic after their transmission and circulation in chickens, but the underlying mechanism is unclear. In this study, a highly velogenic NDV variant, JS10-A10, was generated from the duck-origin lentogenic isolate JS10 through 10 consecutive passages in chicken air sacs. The velogenic properties of this selected variant were determined using mean death time (MDT) assays, intracerebral pathogenicity index (ICPI), the intravenous pathogenicity index (IVPI), histopathology, and the analysis of host tissue tropism. In contrast, JS10 remained lentogenic after 20 serial passages in chicken eggs (JS10-E20). The JS10, JS10-A10, and JS10-E20 genomes were sequenced and found to be nearly identical, suggesting that both JS10-A10 and JS10-E20 were directly generated from JS10. To investigate the mechanism for virulence enhancement, the partial genome covering the F0 cleavage site of JS10 and its variants were analyzed using ultradeep pyrosequencing (UDPS) and the proportions of virulence-related genomes in the quasispecies were calculated. Velogenic NDV genomes accumulated as a function of JS10 passaging through chicken air sacs. Our data suggest that lentogenic NDV strains circulating among poultry might be a risk factor to future potential velogenic NDV outbreaks in chickens., Importance: An avirulent isolate, JS10, was passaged through chicken air sacs and embryos, and the pathogenicity of the variants was assessed. A virulent variant, JS10-A10, was generated from consecutive passage in air sacs. We developed a deep-sequencing approach to detect low-frequency viral variants across the NDV genome. We observed that virulence enhancement of JS10 was due to the selective accumulation of velogenic quasispecies and the concomitant disappearance of lentogenic quasispecies. Our results suggest that because it is difficult to avoid contact between natural waterfowl reservoirs and sensitive poultry operations, circulating lentogenic NDV strains may represent a potential reservoir for emergent velogenic NDV strains that could cause outbreaks in chickens., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Newcastle disease virus (NDV) is an avian paramyxovirus that can selectively replicate in and destroy human tumor cells. In this report, we demonstrate that NDV infection in HeLa cells leads to the activation of the pattern recognition Toll-like receptor 3 (TLR3). Overexpression of TLR3 enhanced the activity of the IFN-β promoter and the transcription factor NF-kappa B (NF-κB), thereby decreasing viral protein synthesis and the virus titer. In addition, the reduction of endogenous TLR3 by small interfering RNA (siRNA) increased NDV replication. Similar anti-NDV effects were observed in DF-1 chicken fibroblast cells with the exogenous expression of chicken TLR3 (cTLR3). Immunofluorescence staining of HeLa cells indicated that the dsRNA generated during NDV replication colocalized with TLR3 in punctate subcellular structures. Altogether, our results strongly suggest that TLR3 actively participates in the recognition of the innate pro-inflammatory response after NDV infection and leads to the consequent antiviral cytokine/interferon secretion.

Mammalian retinoic acid-inducible gene I (RIG-I) is a chief antiviral gene sensing viral RNA molecules including Newcastle disease virus (NDV). In this study, goose RIG-I gene (gRIG-I) was identified. The 2805 bp-long gene encodes a gRIG-I protein that exhibits 93.8% amino acid identity to duck RIG-I. DF-1 chicken fibroblast cells transfected with full-length of gRIG-I or CARD domain of gRIG-I plasmids respond significantly to the agonist of 21-mer 5'ppp RNA, evident through enhancement of IFN-β promoter activity. Goose RIG-I transfected 293T/17 cells were then tested for the response to NDV infection, resulting in up-regulated activity of IFN-β promoter, and mRNA levels of IRF-3 and IFIT1, but decreased virus titer. Similar results were obtained in transfected DF-1 chicken fibroblast cells and goose embryo fibroblast cells in response to NDV infections Animal experiments further support a role of gRIG-I in goose innate immunity against NDV infections by showing increased gRIG-I mRNA levels and decreased virus titer in geese lung and air sac post-infection., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Newcastle disease (ND) and infectious bronchitis (IB) are two highly contagious diseases that severely threaten the poultry industry. The goal of this study is to prevent these two diseases and reduce the vaccine costs during storage and transportation. In this study, we design a thermostable recombinant Newcastle disease virus (NDV) candidate live vaccine strain designated as rLS-T-HN-T/B, which expresses the multiple epitope cassette of the identified infectious bronchitis virus (IBV) (S-T/B). The rLS-T-HN-T/B strain was found to possess similar growth kinetics, passage stability, morphological characteristics, and virulence to the parental LaSota strain. After incubation at 56 °C at the indicated time points, the rLS-T-HN-T/B strain was determined by the hemagglutination (HA), and 50% embryo infectious dose (EID50) assays demonstrated that it accords with the criteria for thermostability. The thermostable rLS-T-HN-T/B and parental LaSota vaccines were stored at 25 °C for 16 days prior to immunizing the one-day-old specific pathogen-free (SPF) chicks. Three weeks postimmunization, the virus challenge results suggested that the chicks vaccinated with the rLS-T-HN-T/B vaccine were protected by 100% and 90% against a lethal dose of NDV and IBV, respectively. Furthermore, the trachea ciliary activity assay indicated that the mean ciliostasis score of the chicks vaccinated with thermostable rLS-T-HN-T/B vaccine was significantly superior to that of the LaSota and PBS groups (p < 0.05). The rLS-T-HN-T/B vaccine stored at 25 °C for 16 days remained capable of eliciting the immune responses and protecting against IBV and NDV challenges. However, the same storage conditions had a great impact on the parental LaSota strain vaccinated chicks, and the NDV challenge protection ratio was only 20%. We conclude that the thermostable rLS-T-HN-T/B strain is a hopeful bivalent candidate vaccine to control both IB and ND and provides an alternative strategy for the development of cost-effective vaccines for village chickens, especially in the rural areas of developing countries. [ABSTRACT FROM AUTHOR]

Infectious bronchitis (IB) and Newcastle disease (ND) are two major infectious diseases that are a threat to the domestic poultry industry. In this study, we successfully generated a recombinant LaSota candidate vaccine strain, rNDV-IBV-T/B, which expresses a short, synthetic, previously identified IBV S1 multi-epitope cassette using the reverse genetic system. The recombinant virus was propagated in nine-day-old embryonated chicken eggs for 20 passages and genetic stability was confirmed by whole genome DNA sequencing. The recombinant virus had a hemagglutination (HA) titer of 210, mean death time (MDT) of 118 hours, and intracerebral pathogenicity index (ICPI) of 0.05. None of these were significantly different from the parental Newcastle disease virus (NDV) LaSota strain (p > 0.05). Vaccination of white leghorn chickens at one day of age with 106 EID50 rNDV-IBV-T/B provided 90% protection against virulent IBV M41 challenge at three weeks of age, which was significantly higher than the protection of the control group vaccinated with phosphate-buffered saline (PBS) (p < 0.05). The ciliostasis scores of rNDV-IBV-T/B-vaccinated and LaSota-vaccinated groups were 4.2 and 37.6, respectively, which indicated that rNDV-IBV-T/B vaccination reduced the pathogenicity of IBV toward the trachea. Furthermore, real-time RT-PCR assay showed that the rNDV-IBV-T/B vaccination resulted in low levels of viral load (647.80 ± 49.65 RNA copies) in the trachea four days post-challenge, which is significantly lower than groups vaccinated with PBS (8591.25 ± 311.10 RNA copies) or LaSota (7742.60 ± 298.50 RNA copies) (p < 0.05). Meanwhile, the same dose of rNDV-IBV-T/B vaccination provided complete protection against velogenic NDV F48E9 challenge. These results demonstrate that the rNDV-IBV-T/B strain is a promising vaccine candidate to control both IB and ND simultaneously. Furthermore, epitope-based live vector vaccines provide an alternative strategy for the development of cost-effective and, broadly, cross-protective vaccines. [ABSTRACT FROM AUTHOR]