Search

Pigeon paramyxovirus-1 (PPMV-1) is a strain of Newcastle disease virus (NDV) that has adapted to infect pigeons and poses a constant threat to the commercial poultry industry. Early detection via rapid and sensitive methods, along with timely preventative and mitigating actions, is important for reducing the spread of PPMV-1. Here, we report the development of a TaqMan loop-mediated isothermal amplification assay (TaqMan-LAMP) for rapid and specific detection of PPMV-1 based on the F gene. This system makes use of six novel primers and a TaqMan probe that targets nine distinct regions of the F gene that are highly conserved among PPMV-1 isolates. The results showed that the limit of detection was 10 copies μL-1 for PPMV-1 cDNA and 0.1 ng for PPMV-1 RNA. The reaction was completed within 25 min and was thus faster than conventional RT-PCR. Moreover, no cross-reactions with similar viruses or with peste des petits ruminants virus (PPRV) or NDV LaSota vaccine strains were observed under the same conditions. To evaluate the applicability of the assay, the TaqMan-LAMP assay and a commercial RT-PCR assay were compared using 108 clinical samples, and the concordance rate between two methods was found to be 96.3%. The newly developed PPMV-1 TaqMan-LAMP assay can therefore be used for simple, efficient, rapid, specific, and sensitive diagnosis of PPMV-1 infections.

The Newcastle disease virus (NDV) matrix (M) protein is the pivotal element for viral assembly, budding, and proliferation. It traffics through the cellular nucleus but performs its primary function in the cytoplasm. To investigate the biological importance of M protein nuclear-cytoplasmic trafficking and the mechanism involved, the regulatory motif nuclear export signal (NES) and nuclear localization signal (NLS) were analyzed. Here, two types of combined NLSs and NESs were identified within the NDV-M protein. The Herts/33-type M protein was found to mediate efficient nuclear export and stable virus-like particle (VLP) release, while the LaSota-type M protein was retained mostly in the nuclei and showed retarded VLP production. Two critical residues, namely, 247 and 263, within the motif were identified and associated with nuclear export efficiency. We identified, for the first time, residue 247 as an important monoubiquitination site, of which its modification regulates the nuclear-cytoplasmic trafficking of NDV-M. Subsequently, mutant LaSota strains were rescued via reverse genetics, which contained either single or double amino acid substitutions that were similar to the M of Herts/33. The rescued LaSota (rLaSota) strains rLaSota-R247K, -S263R, and -double mutation (DM) showed about 2-fold higher hemagglutination (HA) titers and 10-fold higher 50% egg infective dose (EID(50)) titers than wild-type (wt) rLaSota. Furthermore, the mean death time (MDT) and intracerebral pathogenicity index (ICPI) values of those recombinant viruses were slightly higher than those of wt rLaSota probably due to their higher proliferation rates. Our findings contribute to a better understanding of the molecular mechanism of the replication and pathogenicity of NDV and even those of all other paramyxoviruses. This information is beneficial for the development of vaccines and therapies for paramyxoviruses. IMPORTANCE Newcastle disease virus (NDV) is a pathogen that is lethal to birds and causes heavy losses in the poultry industry worldwide. The World Organization for Animal Health (OIE) ranked Newcastle disease (ND) as the third most significant poultry disease and the eighth most important wildlife disease in the World Livestock Disease Atlas in 2011. The matrix (M) protein of NDV is very important for viral assembly and maturation. It is interesting that M proteins enter the cellular nucleus before performing their primary function in the cytoplasm. We found that NDV-M has a combined nuclear import and export signal. The ubiquitin modification of a lysine residue within this signal is critical for quick, efficient nuclear export and subsequent viral production. Our findings shed new light on viral replication and open up new possibilities for therapeutics against NDV and other paramyxoviruses; furthermore, we demonstrate a novel approach for improving paramyxovirus vaccines.

The chicken is a model animal for the study of evolution, immunity and development. In addition to their use as a model organism, chickens also represent an important agricultural product. Pathogen invasion has already been shown to modulate the expression of hundreds of genes, but the role of alternative splicing in avian virus infection remains unclear. We used RNA-seq data to analyze virus-induced changes in the alternative splicing of Gallus gallus, and found that a large number of alternative splicing events were induced by virus infection both in vivo and in vitro. Virus-responsive alternative splicing events preferentially occurred in genes involved in metabolism and transport. Many of the alternatively spliced transcripts were also expressed from genes with a function relating to splicing or immune response, suggesting a potential impact of virus infection on pre-mRNA splicing and immune gene regulation. Moreover, exon skipping was the most frequent AS event in chickens during virus infection. This is the first report describing a genome-wide analysis of alternative splicing in chicken and contributes to the genomic resources available for studying host–virus interaction in this species. Our analysis fills an important knowledge gap in understanding the extent of genome-wide alternative splicing dynamics occurring during avian virus infection and provides the impetus for the further exploration of AS in chicken defense signaling and homeostasis.

Newcastle disease virus (NDV) causes severe infectious disease in poultry and selectively kills tumor cells, by inducing apoptosis and cytokines secretion. In this report, we study the mechanisms underlying NDV-induced apoptosis by investigating the unfolded protein response (UPR). We found that NDV infection activated all three branches of the UPR signaling (PERK-eIF2α, ATF6, and IRE1α) and triggered apoptosis, in avian cells (DF-1 and CEF) and in various human cancer cell types (HeLa, Cal27, HN13, A549, H1299, Huh7, and HepG2). Interestingly, the suppression of either apoptosis or UPR led to impaired NDV proliferation. Meanwhile, the inhibition of UPR by 4-PBA protected cells from NDV-induced apoptosis. Further study revealed that activation of PERK-eIF2α induced the expression of transcription factor CHOP, which subsequently promoted apoptosis by downregulating BCL-2/MCL-1, promoting JNK signaling and suppressing AKT signaling. In parallel, IRE1α mediated the splicing of XBP1 mRNA and resulted in the translation and nuclear translocation of XBP1s, thereby promoting the transcription of ER chaperones and components of ER-associated degradation (ERAD). Furthermore, IRE1α promoted apoptosis and cytokines secretion via the activation of JNK signaling. Knock down and overexpression studies showed that CHOP, IRE1α, XBP1, and JNK supported efficient virus proliferation. Our study demonstrates that the induction of eIF2α-CHOP-BCL-2/JNK and IRE1α-XBP1/JNK signaling cascades promote apoptosis and cytokines secretion, and these signaling cascades support NDV proliferation.

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.

Lacking a self-contained metabolism network, viruses have evolved multiple mechanisms for rewiring the metabolic system of their host to hijack the host���s metabolic resources for replication. Newcastle disease virus (NDV) is a paramyxovirus, as an oncolytic virus currently being developed for cancer treatment. However, how NDV alters cellular metabolism is still far from fully understood. In this study, we show that NDV infection reprograms cell metabolism by increasing glucose utilization in the glycolytic pathway. Mechanistically, NDV induces mitochondrial damage, elevated mitochondrial reactive oxygen species (mROS) and ETC dysfunction. Infection of cells depletes nucleotide triphosphate levels, resulting in elevated AMP:ATP ratios, AMP-activated protein kinase (AMPK) phosphorylation, and MTOR crosstalk mediated autophagy. In a time-dependent manner, NDV shifts the balance of mitochondrial dynamics from fusion to fission. Subsequently, PINK1-PRKN-dependent mitophagy was activated, forming a ubiquitin chain with MFN2 (mitofusin 2), and molecular receptor SQSTM1/p62 recognized damaged mitochondria. We also found that NDV infection induces NAD+-dependent deacetylase SIRT3 loss via mitophagy to engender HIF1A stabilization, leading to the switch from oxidative phosphorylation (OXPHOS) to aerobic glycolysis. Overall, these studies support a model that NDV modulates host cell metabolism through PINK1-PRKN-dependent mitophagy for degrading SIRT3. Abbreviations: AMPK: AMP-activated protein kinase; CCCP: carbonyl cyanide 3-chlorophenylhydrazone; ECAR: extracellular acidification rate; hpi: hours post infection LC-MS: liquid chromatography-mass spectrometry; mito-QC: mCherry-GFP-FIS1[mt101���152]; MFN2: mitofusin 2; MMP: mitochondrial membrane potential; mROS: mitochondrial reactive oxygen species; MOI: multiplicity of infection; 2-NBDG: 2-(N-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl) amino)-2-deoxyglucose; NDV: newcastle disease virus; OCR: oxygen consumption rate; siRNA: small interfering RNA; SIRT3: sirtuin 3; TCA: tricarboxylic acid; TCID50: tissue culture infective doses.

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 &deg, 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 &deg, 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 &lt, 0.05). The rLS-T-HN-T/B vaccine stored at 25 &deg, 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.

Newcastle disease virus (NDV), a member of the Paramyxoviridae family, can activate PKR/eIF2α signaling cascade to shutoff host and facilitate viral mRNA translation during infection, however, the mechanism remains unclear. In this study, we revealed that NDV infection up-regulated host cap-dependent translation machinery by activating PI3K/Akt/mTOR and p38 MAPK/Mnk1 pathways. In addition, NDV infection induced p38 MAPK/Mnk1 signaling participated 4E-BP1 hyperphosphorylation for efficient viral protein synthesis when mTOR signaling is inhibited. Furthermore, NDV NP protein was found to be important for selective cap-dependent translation of viral mRNAs through binding to eIF4E during NDV infection. Taken together, NDV infection activated multiple signaling pathways for selective viral protein synthesis in infected cells, via interaction between viral NP protein and host translation machinery. Our results may help to design novel targets for therapeutic intervention against NDV infection and to understand the NDV anti-oncolytic mechanism., Author summary Viruses are obligate intracellular parasites and have no protein translation machinry of their own. Therefore, viruses remain exclusively dependent on host translation machinery to ensure viral protein synthesis and progeny virion production during infection. We previous reported that Newcastle disease virus (NDV) shutoff host and facilitate viral mRNA translation by activating PKR/eIF2α signaling cascade. Here, we demonstrated that NDV infection up-regulated host cap-dependent translation machinery by activating PI3K/Akt/mTOR and p38 MAPK/Mnk1 pathways. Furthermore, NDV NP protein was found to be important for selective cap-dependent translation of viral mRNAs. Our findings highlight a new strategy how virus used host translation machinery for selective viral protein synthesis.

The cell cycle, as a basic cellular process, is conservatively regulated. Consequently, subversion of the host cell replication cycle is a common strategy employed by many viruses to create a cellular environment favorable for viral replication. Newcastle disease virus (NDV) causes disease in poultry and is also an effective oncolytic agent. However, the effects of NDV infection on cell cycle progression are unknown. In this study, we showed that NDV replication in asynchronized cells resulted in the accumulation of infected cells in the G0/G1 phase of the cell cycle, which benefitted the proliferation of NDV. Examination of various cell cycle-regulatory proteins showed that expression of cyclin D1, was significantly reduced following NDV infection. Importantly, the decreased expression of cyclin D1 was reversed by inhibition of CHOP expression, indicating that induction of the PERK-eIF-2a-ATF4-CHOP signaling pathway was involved in the G0/G1 phase cell cycle arrest observed following NDV infection.

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.

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)2-6C) 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.

Paramyxovirus establishes an intimate and complex interaction with the host cell to counteract the antiviral responses elicited by the cell. Of the various pattern recognition receptors in the host, the cytosolic RNA helicases interact with viral RNA to activate the mitochondrial antiviral signaling protein (MAVS) and subsequent cellular interferon (IFN) response. On the other hand, viruses explore multiple strategies to resist host immunity. In this study, we found that Newcastle disease virus (NDV) infection induced MAVS degradation. Further analysis showed that NDV V protein degraded MAVS through the ubiquitin-proteasome pathway to inhibit IFN-β production. Moreover, NDV V protein led to proteasomal degradation of MAVS through Lys362 and Lys461 ubiquitin to prevent IFN production. Further studies showed that NDV V protein recruited E3 ubiquitin ligase RNF5 to polyubiquitinate and degrade MAVS. Compared with levels for wild-type NDV infection, V-deficient NDV induced attenuated MAVS degradation and enhanced IFN-β production at the late stage of infection. Several other paramyxovirus V proteins showed activities of degrading MAVS and blocking IFN production similar to those of NDV V protein. The present study revealed a novel role of NDV V protein in targeting MAVS to inhibit cellular IFN production, which reinforces the fact that the virus orchestrates the cellular antiviral response to its own benefit. IMPORTANCE Host anti-RNA virus innate immunity relies mainly on the recognition by retinoic acid-inducible gene I and melanoma differentiation-associated protein 5 and subsequently initiates downstream signaling through interaction with MAVS. On the other hand, viruses have developed various strategies to counteract MAVS-mediated signaling. The mechanism for paramyxoviruses regulating MAVS to benefit their infection remains unknown. In this article, we demonstrate that the V proteins of NDV and several other paramyxoviruses target MAVS for ubiquitin-mediated degradation through E3 ubiquitin ligase RING-finger protein 5 (RNF5). MAVS degradation leads to the inhibition of the downstream IFN-β pathway and therefore benefits virus proliferation. Our study reveals a novel mechanism of NDV evading host innate immunity and provides insight into the therapeutic strategies for the control of paramyxovirus infection.

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)-&beta, 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.

Oncolytic Newcastle disease virus (NDV) reportedly employs direct fusion of the viral envelope with the plasma membrane and caveolae-dependent endocytosis to enter cells. Here, we show that macropinocytosis and clathrin-mediated endocytosis are involved in NDV entry into a galline embryonic fibroblast cell line. Upon specific inhibition of clathrin assembly, GTPase dynamin, Na+/H+ exchangers, Ras-related C3 botulinum toxin substrate 1, p21 activated kinase 1 or protein kinase C, entry of NDV and its propagation were suppressed. NDV entry into cells triggers Rac1-Pak1 signaling and elicits actin rearrangement and plasma membrane ruffling. Moreover, NDV internalization within macropinosomes and trafficking involve Rab5a-positive vesicles. This is the first report demonstrating that NDV utilizes clathrin-mediated endocytosis and macropinocytosis as alternative endocytic pathways to enter cells. These findings shed new light on the molecular mechanisms underlying NDV entry into cells, and provide potential targets for NDV-mediated therapy in cancer.

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.

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.

Viral infections result in cellular stress responses, which can trigger protein translation shutoff via phosphorylation of eukaryotic initiation factor 2 alpha (eIF2α). Newcastle disease virus (NDV) causes severe disease in poultry and selectively kills human tumour cells. In this report, we determined that infection of HeLa human cervical cancer cells and DF-1 chicken fibroblast cells with NDV maintained protein at early infection times, 0-12 h post-infection (p.i.), and gradually inhibited global protein translation at late infection times, 12-24 h p.i. Mechanistic studies showed that translation inhibition at late infection times was accompanied by phosphorylation of eIF2α, a checkpoint of translation initiation. Meanwhile, the eIF2α kinase, PKR, was upregulated and activated by phosphorylation and another eIF2α kinase, PERK, was phosphorylated and cleaved into two fragments. Pharmacological inhibition experiments revealed that only PKR activity was required for eIF2α phosphorylation, suggesting that recognition of viral dsRNA by PKR was responsible for translation shutoff. High levels of phospho-eIF2α led to preferential translation of the transcription factor ATF4 and an increase in GADD34 expression. Functionally, GADD34, in conjunction with PP1, dephosphorylated eIF2a and restored protein translation, benefiting virus protein synthesis. However, PP1 was degraded at late infection times, functionally counteracting the upregulation of GADD34. Taken together, our data support that NDV-induced translation shutoff at late infection times was attributed to sustaining phosphorylation of eIF2α, which is mediated by continual activation of PKR and degradation of PP1.

Deoxyribonucleic acid (DNA) damage response (DDR) is the fundamental cellular response for maintaining genomic integrity and suppressing tumorigenesis. The activation of ataxia telangiectasia-mutated (ATM) kinase is central to DNA double-strand break (DSB) for maintaining host-genome integrity in mammalian cells. Oncolytic Newcastle disease virus (NDV) can selectively replicate in tumor cells; however, its influence on the genome integrity of tumor cells is not well-elucidated. Here, we found that membrane fusion and NDV infection triggered DSBs in tumor cells. The late replication and membrane fusion of NDV mechanistically activated the ATM-mediated DSB pathway via the ATM-Chk2 axis, as evidenced by the hallmarks of DSBs, i.e., auto-phosphorylated ATM and phosphorylated H2AX and Chk2. Immunofluorescence data showed that multifaceted ATM-controlled phosphorylation markedly induced the formation of pan-nuclear punctum foci in response to NDV infection and F-HN co-expression. Specific drug-inhibitory experiments on ATM kinase activity further suggested that ATM-mediated DSBs facilitated NDV replication and membrane fusion. We confirmed that the Mre11-RAD50-NBS1 (MRN) complex sensed the DSB signal activation triggered by NDV infection and membrane fusion. The pharmacological inhibition of MRN activity also significantly inhibited intracellular and extracellular NDV replication and syncytia formation. Collectively, these data identified for the first time a direct link between the membrane fusion induced by virus infection and DDR pathways, thereby providing new insights into the efficient replication of oncolytic NDV in tumor cells., Author summary Various viruses have evolved a plethora of strategies to manipulate DNA damage machinery and commandeer cells to maximize their own replication. On the contrary, host cells have also evolved to maintain cellular genomic stability and suppress carcinogenesis through multifaceted cellular DNA damage response (DDR). Most studies have focused on the relationship between DNA virus replication and the host DDR pathway, but few have investigated the interface between the DDR pathway and ribonucleic acid (RNA) viruses, such as oncolytic Newcastle disease virus (NDV). In the present study, we report for the first time that F-HN co-expression and NDV infection trigger ataxia telangiectasia-mutated (ATM)-dependent DSB lesions in tumor cells to promote viral replication and syncytium formation, respectively. We found that late NDV infection and membrane fusion activated the ATM-Chk2 axis in tumor cells, which expands prior understanding of the membrane fusion process and NDV infection. Our findings reveal new links among oncolytic NDV replication, membrane fusion, and DDR and deepen insights into NDV replication.

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 152RGPAELWK159, 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.

Mitochondrial antiviral-signaling protein (MAVS) is an essential adaptor protein in retinoic acid-inducible gene I (RIG-I)-mediated antiviral innate immunity in mammals. In this study, the goose MAVS gene (goMAVS) was identified. The 2019 bp-long goMAVS exhibits 96.2% amino acid similarity compared to the predicted goMAVS. Quantitative real-time polymerase chain reactions showed that goMAVS mRNA was widely expressed in different tissues. The overexpression of goMAVS in goose embryo fibroblast cells up-regulated the mRNA levels of goose interferon-stimulated genes. We concluded that MAVS mediates the activation of type I interferon (IFN) pathway in a species-specific manner. We further demonstrated that a CARD-like domain, transmembrane domain and two previously unidentified domains of goMAVS were essential for the activation of type I IFN pathway. GoMAVS inhibited Newcastle disease virus replication by activating type I IFN pathways, especially at the early stages of infection. Finally, the interaction between goMAVS and goose RIG-I was confirmed. The CARD domain of goMAVS plays a vital role in the interaction. Together, we identified goMAVS as a goRIG-I interactive protein and concluded that goMAVS is involved in the activation of type I IFN pathways in goose cells.

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.

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.

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.

Newcastle disease virus (NDV) is an avian paramyxovirus that causes significant economic losses to the poultry industry worldwide, with variations in NDV pathogenicity due to the differences in virulence between strains. However, there is limited knowledge regarding the avian innate immune response to NDV infection. In this study, transcriptional profiles were obtained from chick embryo fibroblasts (CEFs) that were infected with the highly virulent NDV Herts/33 strain or the nonvirulent LaSota strain using RNA-seq. This yielded 8433 transcripts that were associated with NDV infection. This list of candidate genes was then further examined using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. It showed a high enrichment in the areas of cellular components and metabolic processes, with the cellular components possibly being associated with NDV pathogenicity. Among these 8433 transcripts, 3616 transcripts associated with interferon-stimulated genes (ISGs) were obtained; these transcripts are involved in metabolic processes, including protein phosphorylation and protein modification. These results provide further insight into the identification of genes that are involved in NDV infection. The global survey of changes in gene expression performed herein provides new insights into the complicated molecular mechanisms underlying virus and host interactions and will enable the use of new strategies to protect chickens against this virus.

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.

Newcastle disease virus (NDV) causes major economic losses in the poultry industry. Previous studies have shown that NDV utilizes different pathways to infect various cells, including dendritic cells (DCs). Here, we demonstrate that NDV gains entry into DCs mainly via macropinocytosis and clathrin-mediated endocytosis. The detection of cytokines interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), interleukin-12 (IL-12), interleukin-4 (IL-4) and interleukin-10 (IL-10) indicates that NDV significantly induces Th1 responses and lowers Th2 responses. Furthermore, NDV entry into DCs resulted in the upregulation of TNF-related apoptosis-inducing ligand (TRAIL) and cleaved caspase-3 proteins, which in turn activated the extrinsic apoptosis pathway and induced DCs apoptosis. Transwell® co-culture demonstrated that direct contact between live NDV-stimulated DCs and T cells, rather than heated-inactivated NDV, inhibited CD4+ T cell proliferation. Taken together, these findings provide new insights into the mechanism underlying NDV infections, particularly in relation to antigen presentation cells and suppression of T cell proliferation.

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.

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.

Mammalian cells respond to various environmental stressors to form stress granules (SGs) by arresting cytoplasmic mRNA, protein translation element, and RNA binding proteins. Virus-induced SGs function in different ways, depending on the species of virus; however, the mechanism of SG regulation of virus replication is not well understood. In this study, Newcastle disease virus (NDV) triggered stable formation of

Newcastle disease virus (NDV) V protein is considered as an effector for IFN antagonism, however, the mechanism remains unknown. In this study, the expression of STAT1 and phospho-STAT1 in cells infected with NDV or transfected with V protein-expressing plasmids were analyzed. Our results showed that NDV V protein targets phospho-STAT1 reduction in the cells depends on the stimulation of IFN-α. In addition, a V-deficient genotype VII recombinant NDV strain rZJ1-VS was constructed using reverse genetic technique to confirm the results. The rZJ1-VS lost the ability to reduce phospho-STAT1 and induced higher expression of IFN-responsive genes in infected cells. Furthermore, treatment with an ubiquitin E1 inhibitor PYR-41 demonstrated that phospho-STAT1 reduction was caused by degradation, but not de-phosphorylation. We conclude that NDV V protein targets phospho-STAT1 degradation to block IFN-α signaling, which adds novel knowledge to the strategies used by paramyxoviruses to evade IFN.

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.

Fourteen pigeon-origin Newcastle disease virus (NDV) isolates were obtained from sick pigeons in China between 1996 and 2005. The mean death time (MDT) of embryonated eggs and the intracerebral pathogenicity indices (ICPI) were tested to determine the virulence of the field isolates. The result indicated that most isolates were proved to be mesogenic (MDT 60-90 hr and ICPI1.2). The main function regions of F protein gene of the isolates were amplified and sequenced for phylogenetic and residue substitutive analysis. The fusion protein cleavage site sequences of most isolates had multiple basic amino acids R/KRQKRF at positions 112-116 and a phenyl alanine at position 117, characteristic of velogenic isolates. In the phylogenetic tree, the majority of the isolates were clustered into a single genetic lineage, termed genotype VIb, and were typical pigeon paramyxovirus type 1, whereas a small number of recent isolates (three strains) were grouped into genotype VIId, a predominant genotype responsible for most Newcastle disease outbreaks in chickens and geese since the end of last century. One isolate, PK9901, was proved to be a lentogenic strain, of genotype II NDV, to which the vaccine strain La Sota belongs.

Newcastle disease virus (NDV) causes large losses in the global fowl industry. To better understand NDV replication and transcription cycle, quantitative detection methods for distinguishing NDV genomic RNA (gRNA), antigenomic RNA (cRNA), and messenger RNA (mRNA) in NDV-infected cells are indispensible. Three reverse transcription primers were designed to specifically target the nucleoprotein (NP) region of gRNA, cRNA, and NP mRNA, and a corresponding real-time RT-PCR assay was developed to simultaneously quantify the three types of RNAs in NDV-infected cells. This method showed very good specificity, sensitivity, and reproducibility. The detection range of the assay was between5.5×102and1.1×109copies/μL of the target gene. These methods were applied to investigate the dynamics of the gRNA, cRNA, and mRNA synthesis in NDV La Sota infected DF-1 cells. The results showed that the copy numbers of viral gRNA, cRNA, and NP mRNA all exponentially increased in the beginning. The viral RNA copy number then plateaued at 10’h postinfection and gradually decreased from 16 h postinfection. No synthesis priority was observed between replication (gRNA and cRNA amounts) and transcription (mRNA amounts) during NDV infection. However, the cRNA accumulated more rapidly than gRNA, as the cRNA copy number was three- to tenfold higher than gRNA starting from 2 h postinfection.Conclusion. A real-time RT-PCR for absolute quantitation of specific viral RNA fragments in NDV-infected cells was developed for the first time. The development of this assay will be helpful for further studies on the pathogenesis and control strategies of NDV.

Background Newcastle Disease virus (NDV) causes severe and economically significant disease in almost all birds. However, factors that affect NDV replication in host cells are poorly understood. NDV generates long double-stranded RNA (dsRNA) molecules during transcription of single-stranded genomic RNA. Protein kinase R (PKR) is activated by dsRNA. The aim of this study was to elucidate the role of PKR in NDV infection. Results NDV infection led to the activation of dsRNA-dependent PKR and phosphorylation of its substrate, translation initiation factor eIF2α, in a dose-dependent manner by either the lentogenic strain LaSota or a velogenic strain Herts/33. PKR activation coincided with the accumulation of dsRNA induced by NDV infection. PKR knockdown remarkably decreased eIF2α phosphorylation as well as IFN-β mRNA levels, leading to the augmentation of extracellular virus titer. Furthermore, siRNA knockdown or phosphorylation of eIF2α or okadaic acid treatment significantly impaired NDV replication, indicating the critical role of the PKR/eIF2α signaling cascade in NDV infection. Conclusion PKR is activated by dsRNA generated by NDV infection and inhibits NDV replication by eIF2α phosphorylation. This study provides insight into NDV-host interactions for the development of candidate antiviral strategies.

Newcastle disease virus (NDV) is an important avian pathogen. We previously reported that NDV triggers autophagy in U251 glioma cells, resulting in enhanced virus replication. In this study, we investigated whether NDV triggers autophagy in chicken cells and tissues to enhance virus replication. We demonstrated that NDV infection induced steady-state autophagy in chicken-derived DF-1 cells and in primary chicken embryo fibroblast (CEF) cells, evident through increased double- or single-membrane vesicles, the accumulation of green fluorescent protein (GFP)-LC3 dots, and the conversion of LC3-I to LC3-II. In addition, we measured autophagic flux by monitoring p62/SQSTM1 degradation, LC3-II turnover, and GFP-LC3 lysosomal delivery and proteolysis, to confirm that NDV infection induced the complete autophagic process. Inhibition of autophagy by pharmacological inhibitors and RNA interference reduced virus replication, indicating an important role for autophagy in NDV infection. Furthermore, we conducted in vivo experiments and observed the conversion of LC3-I to LC3-II in heart, liver, spleen, lung, and kidney of NDV-infected chickens. Regulation of the induction of autophagy with wortmannin, chloroquine, or starvation treatment affects NDV production and pathogenesis in tissues of both lung and intestine; however, treatment with rapamycin, an autophagy inducer of mammalian cells, showed no detectable changes in chicken cells and tissues. Moreover, administration of the autophagy inhibitor wortmannin increased the survival rate of NDV-infected chickens. Our studies provide strong evidence that NDV infection induces autophagy which benefits NDV replication in chicken cells and tissues.

Airborne Newcastle disease (ND) viruses in the air of five chicken houses were detected and differentiated by reverse transcriptase polymerase chain reaction (RT-PCR) using degenerate primers. Fifteen air samples were collected with All Glass Impinger-30 (AGI-30) air samplers in each house. Airborne ND viruses were also isolated and virulence identified by in vivo tests. Avirulent viruses were detected both in air samples and swab samples in four houses by degenerate primers based RT-PCR. Virulent viruses were detected only in the air samples by degenerate primers based RT-PCR in two houses. Seven strains viruses were isolated from the RT-PCR positive air samples. Of the seven strains, three strains were virulent viruses and four strains were avirulent viruses identified by in vivo tests. The results showed that it was feasible to detect and differentiate NDV in the air samples using degenerate primers based RT-PCR. This technique could decrease the time it required identify NDV infected flocks while distinguishing between virulent and avirulent viruses. It will help effectively to control Newcastle disease.

The main function region of the fusion (F) protein gene of 124 strains of Newcastle disease virus isolated from 2002 to 2004 in China was amplified and sequenced for further phylogenetic and residue substitutive analysis. Most of the isolates were classified into genotype VIIc, VIId, VIf, and VIb, while others into genotype IX, III, or II. The genotype IX, a unique genotype which includes strain F48, the first Chinese virulent NDV strain isolated in 1948, were still found inducing sporadic infections in certain areas. Subgenotype VIIc, VIId, and VIIe viruses, which were distributed in clusters in the phylogenetic tree distinct from members of subgenotypes VIIa and VIIb, were responsible for most outbreaks in China and circulated predominantly in China in recent years. Strain NDV03-026, an isolate of the genotype II which was normally lentogenic, was found carrying (112)RRQKRF(117) motif at the cleavage site of F protein as the virulent strain.