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Avian paramyxoviruses 1 has the ability to edit its P gene to generate three amino-coterminal proteins (P, V and W), but its kinetic change is unclear. In this study, next-generation sequencing (NGS) was used to analyze the P-gene editing of Newcastle disease virus (NDV). Transcriptome analysis of chicken embryonic tissues and bursa of fabricius showed the P-gene editing frequencies were 45.46–52.70%. To investigate the rules of P-gene editing along time, the ratio of PVW was determined by PCR based deep sequencing at multiple time points in cells infected with velogenic and lentogenic strain respectively. The results confirmed similar editing frequencies with transcriptome data and the PVW ratios were stable along time among different NDVs, but had a greater V-gene transcript on velogenic strain infection (P

Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) is the causative agent for the coronavirus disease 2019 (COVID-19) pandemic and there is an urgent need to understand the cellular response to SARS-CoV-2 infection. Beclin 1 is an essential scaffold autophagy protein that forms two distinct subcomplexes with modulators Atg14 and UVRAG, responsible for autophagosome formation and maturation, respectively. In the present study, we found that SARS-CoV-2 infection triggers an incomplete autophagy response, elevated autophagosome formation but impaired autophagosome maturation, and declined autophagy by genetic knockout of essential autophagic genes reduces SARS-CoV-2 replication efficiency. By screening 26 viral proteins of SARS-CoV-2, we demonstrated that expression of ORF3a alone is sufficient to induce incomplete autophagy. Mechanistically, SARS-CoV-2 ORF3a interacts with autophagy regulator UVRAG to facilitate PI3KC3-C1 (Beclin-1-Vps34-Atg14) but selectively inhibit PI3KC3-C2 (Beclin-1-Vps34-UVRAG). Interestingly, although SARS-CoV ORF3a shares 72.7% amino acid identity with the SARS-CoV-2 ORF3a, the former had no effect on cellular autophagy response. Thus, our findings provide the mechanistic evidence of possible takeover of host autophagy machinery by ORF3a to facilitate SARS-CoV-2 replication and raise the possibility of targeting the autophagic pathway for the treatment of COVID-19.

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 F1 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.

Background Newcastle disease virus (NDV) is pathogenic to chickens, which is characterized by dyspnea, diarrhea, nervous disorder and hemorrhages. However, the influence of different virulent NDV infection on the host gut microbiota composition is still poorly understood. In this study, twenty 21-day-old specific pathogen free chickens were inoculated with either the velogenic Herts33 NDV strain, lentogenic La Sota NDV strain or sterile phosphate buffer solution (PBS). Through 16S rRNA sequencing, the collected fecal samples of control and NDV infected chickens were examined. Results The results showed that the gut microbiota were mainly dominated by Firmicutes, Bacteroidetes and Proteobacteria in both healthy and NDV infected chickens. NDV infection altered the structure and composition of gut microbiota. As compared to PBS group, phylum Firmicutes were remarkably reduced, whereas Proteobacteria was significantly increased in velogenic NDV infected group. While the gut community structure has no significant differences between lentogenic NDV infected group and PBS group at phylum level. At genus level, Escherichia-Shigella was significantly increased in both velogenic and lentogenic NDV infected groups, but the lactobacillus was only remarkably decreased in velogenic NDV infected group. Collectively, different virulent NDV infection resulted in a different alteration of the gut microbiota in chickens, including a loss of probiotic bacteria and a expansion of pathogenic bacteria. Conclusion These results indicated that NDV with different virulence have different impact on chicken gut microbiota and may provide new insights into the intestinal pathogenesis of NDV.

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.

SARS-CoV-2 is the causative agent for the COVID-19 pandemic and there is an urgent need to understand the cellular response to SARS-CoV-2 infection. Beclin-1 is an essential scaffold autophagy protein that forms two distinct subcomplexes with modulators Atg14 and UVRAG, responsible for autophagosome formation and maturation, respectively. In the present study, we found that SARS-CoV-2 infection triggers an incomplete autophagy response, elevated autophagosome formation but impaired autophagosome maturation, and declined autophagy by genetic knockout of essential autophagic genes reduces SARS-CoV-2 replication efficiency. By screening 28 viral proteins of SARS-CoV-2, we demonstrated that expression of ORF3a alone is sufficient to induce incomplete autophagy. Mechanistically, SARS-CoV-2 ORF3a interacts with autophagy regulator UVRAG to facilitate Beclin-1-Vps34-Atg14 complex but selectively inhibit Beclin-1-Vps34-UVRAG complex. Interestingly, although SARS-CoV ORF3a shares 72.7% amino acid identity with the SARS-CoV-2 ORF3a, the former had no effect on cellular autophagy response. Thus, our findings provide the mechanistic evidence of possible takeover of host autophagy machinery by ORF3a to facilitate SARS-CoV-2 replication and raises the possibility of targeting the autophagic pathway for the treatment of COVID-19.

Viruses have evolved different strategies to hijack subcellular organelles during their life cycle to produce robust infectious progeny. Successful viral reproduction requires the precise assembly of progeny virions from viral genomes, structural proteins, and membrane components. Such spatial and temporal separation of assembly reactions depends on accurate coordination among intracellular compartmentalization in multiple organelles. Here, we overview the rearrangement and morphology remodeling of virus-triggered intracellular organelles. Focus is given to the quality control of intracellular organelles, the hijacking of the modified organelle membranes by viruses, morphology remodeling for viral replication, and degradation of intracellular organelles by virus-triggered selective autophagy. Understanding the functional reprogram and morphological remodeling in the virus-organelle interplay can provide new insights into the development of broad-spectrum antiviral strategies.

International audience; AbstractThe 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 UPRmt. 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.

Constant monitoring is performed to elucidate the role of natural hosts in the ecology of Newcastle disease virus (NDV). In this study, an NDV strain isolated from an asymptomatic pigeon was sequenced and analysed. Results showed that the full-length genomes of this isolate were 15,198 nucleotides with the gene order of 3'-NP-P-M-F-HN-L-5'. This NDV isolate was lentogenic, with an intracerebral pathogenicity index of 0.00 and a mean time of death more than 148 h. The isolate possessed a motif of -(112)E-R-Q-E-R-L(117)- at the F protein cleavage site. In addition, 7 and 13 amino acid substitutions were identified in the functional domains of fusion protein (F) and haemagglutinin-neuraminidase protein (HN) proteins, respectively. Analysis of the amino acids of neutralizing epitopes of F and HN proteins showed 3 and 10 amino acid substitutions, respectively, in the isolate. Phylogenetic analysis classified the isolate into genotype Ib in Class I. This isolate shared high homologies with the NDV strains isolated from wild birds and waterfowl in southern and eastern parts of China from 2005 to 2013. To our knowledge, this study is the first to report a NDV strain isolated from pigeon that belongs to genotype Ib in Class I, rather than to the traditional genotype VI or other sub-genotypes in Class II. This study provides information to elucidate the distribution and evolution of Class I viruses for further NDV prevention.

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.

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.

Pigeon paramyxovirus type-1 (PPMV-1) is enzootic in pigeons, causing severe economic loss in the poultry industry in many countries. However, the exact epidemic process of PPMV-1 transmission is still unclear. In this study, we analyzed the complete genome of the PPMV-1/SX-01/15 isolate. Sequence results show that the virus genome contains 15,192 nucleotides, with the gene order 3'-NP-P-M-F-HN-L-5'. Phylogenetic analysis revealed that this genome belongs to subgenotype VIc in class II. The mean death time (MDT) and intracerebral pathogenicity index (ICPI) were 62.4 h and 1.13, respectively, indicating that this isolate is a mesogenic PPMV-1 strain. To our knowledge, this is the first report of a subgenotype VIc mesogenic PPMV-1 strain circulating in commercial pigeon flocks in the northwest region of China. In a comparative infection experiment, the morbidity and mortality rates were 100% and 80%, respectively, in 4-week-old pigeons, whereas they were 50% and 30%, respectively, in 5-week-old chickens. Furthermore, this virus caused severe neurological symptoms in a 4-week-old pigeon and mild neurological symptoms in a 5-week-old chicken. A histopathological examination of the brain showed a classical nonsuppurative encephalitis lesion. The pattern of viral shedding, and viral load, and virus distribution differed between infected chickens and pigeons. Genomic characteristics suggest that there was cross-species transmission of PPMV-1 subgenotype VIc in this region at least from the years 2006 to 2015.

Pigeon paramyxovirus type-1(PPMV-1) is an enzootic in pigeon flocks and causes severe economic losses in the poultry industry in many countries. A PPMV-1 isolate, abbreviated as PPMV-1/QL-01/CH/15, was isolated from a great spotted woodpecker in the northwest region of China in 2015. The complete genome was sequenced, and the results showed that the virus genome was 15,192 nt in length, in the gene arrangement 3'-NP-P-M-F-HN-L-5'. Several amino acid mutations were identified in the functional domains of the F and HN proteins. The pathogenicity index of the isolate was evaluated, and the mean death time (MDT) was 72 h and the intracerebral pathogenicity index (ICPI) was 0.925, indicating that this isolate was mesogenic. Sequencing results showed that it had a virulent Newcastle disease virus cleavage motif 112R-R-Q-K-R-F117 at the fusion protein cleavage site. Morbidity and mortality were 70% and 50%, after inoculation of pigeons, whereas this virus was nonpathogenic in chickens. Different immune responses of pigeons and chickens were induced in vivo, which led to different HI serum antibody titers. The results of phylogenetic and evolutionary distance analysis showed that this PPMV-1 strain belonged to sub-genotype VIa in class II. To our knowledge, this is the first identification and analysis of PPMV-1 co-circulation among wild birds and domestic pigeon flocks in China. The data from this study highlight the important role of wild birds in the dissemination of PPMV-1 and provide useful references for improving our understanding of the distribution and evolution of PPMV-1 in China.

The genetic variation and molecular evolution of the V gene of the class II Newcastle disease virus (NDV) isolates with genotypes I-XVIII were determined using bioinformatics. Results indicated that low homology existed in different genotype viruses, whereas high homology often for the same genotypes, exception may be existed within genotypes I, V, VI, and XII. Sequence analysis showed that the genetic variation of V protein was consistent with virus genotype, and specific signatures on the V protein for nine genotypes were identified. Phylogenetic analysis demonstrated that the phylogenetic trees were highly consistent between the V and F genes, with slight discrepancies in the sub-genotypes. Evolutionary rate analyses based on V and F genes revealed the evolution rates varied in genotypes. These data indicate that the genetic variation of V protein is genotype-related and will help in elucidating the molecular evolution of NDV.