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Objectives The aim of this study was to explore the antimicrobial resistance, virulence genes and molecular characteristics of Staphylococcus aureus from bovine mastitis cases. Methods A total of 125 non-duplicate S. aureus isolates from bovine mastitis cases in Ningxia, China, were characterised by antimicrobial susceptibility and molecular testing to determine the antimicrobial resistance, virulence genes and molecular characteristics. Results All methicillin-resistant S. aureus (MRSA) isolates were resistant to ampicillin, oxacillin, ceftiofur, erythromycin, gentamicin and clindamycin, with resistance to nine different categories of antibiotics observed amongst the MRSA isolates. Of the methicillin-susceptible S. aureus (MSSA) isolates, 62.1% were resistant to ampicillin and sulfisoxazole. Nine clonal complexes (CCs) and 16 spa types were identified by multilocus sequence typing (MLST) and spa typing. The dominant CCs were CC97 (51.2%) and CC50 (30.4%), while t224 (30.4%), t518 (20.0%) and t359 (16.8%) were the most common spa types. A relatively high proportion (27.2%) of the S. aureus isolates belonged to ST4053, a novel sequence type identified in this study. In addition, two CC30 MSSA isolates and two CC59 MRSA isolates were positive for Panton–Valentine leukocidin, while one CC239 MRSA isolate and three CC5 MSSA isolates were positive for TSST-1. All MRSA isolates carried the immune evasion cluster (IEC) genes, including scn (100%; 9/9) and sak (100%; 9/9), which were classified into type E. Conclusion Our study indicates severe antibiotic resistance and complicated molecular characterisation of S. aureus causing bovine mastitis. Additional studies should be conducted to monitor infection and transmission of S. aureus.

Dexamethasone (DEX) is widely used because of its anti-inflammatory, anti-endotoxin, anti-shock, and stress-enhancing response activities. It can increase the risk of diabetes and hypertension if it is abused or used improperly. However, there is a lack of sensitive and rapid screening methods for DEX in food. In this study, a time-resolved fluorescent microspheres immunochromatographic assay (TRFM-ICA) integrated with a portable fluorescence reader was developed for the quantitative detection of DEX in milk and pork. The cut-off values of the TRFM-ICA were 0.25 ng/mL and 0.7 µg/kg, respectively. The limits of quantitation (LOQs) were 0.003 ng/mL and 0.062 µg/kg, respectively. The recovery rates were 80.0–106.7%, and 78.6–83.6%, respectively, with the coefficients of variation ranging 6.3–12.5%, and 7.5–10.3%, respectively. A parallel experiment for 20 milk and 10 pork samples with LC-MS/MS was carried out to confirm the performance of the on-site application of the developed TRFM-ICA. The results of the two methods are basically the same, the correlation (R2) was &gt, 0.98. The establishment of TRFM-ICA will provide a new sensitive and efficient technical support for the rapid screening of DEX in food.

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.

Elucidating virus-cell interactions is fundamental to understanding viral replication and identifying targets for therapeutic control of viral infection. The extracellular signal-regulated kinase (ERK) pathway has been shown to regulate pathogenesis during many viral infections, but its role during coronavirus infection is undetermined. Infectious bronchitis virus is the representative strain of Gammacoronavirus, which causes acute and highly contagious diseases in the poultry farm. In this study, we investigated the role of ERK1/2 signaling pathway in IBV infection. We found that IBV infection activated ERK1/2 signaling and the up-regulation of phosphatase DUSP6 formed a negative regulation loop. Pharmacological inhibition of MEK1/2-ERK1/2 signaling suppressed the expression of DUSP6, promoted cell death, and restricted virus replication. In contrast, suppression of DUSP6 by chemical inhibitor or siRNA increased the phosphorylation of ERK1/2, protected cells from apoptosis, and facilitated IBV replication. Overexpression of DUSP6 decreased the level of phospho-ERK1/2, promoted apoptosis, while dominant negative mutant DUSP6-DN lost the regulation function on ERK1/2 signaling and apoptosis. In conclusion, these data suggest that MEK-ERK1/2 signaling pathway facilitates IBV infection, probably by promoting cell survival; meanwhile, induction of DUSP6 forms a negative regulation loop to restrict ERK1/2 signaling, correlated with increased apoptosis and reduced viral load. Consequently, components of the ERK pathway, such as MEK1/2 and DUSP6, represent excellent targets for the development of antiviral drugs.

Cytoplasmic stress granules (SGs) are generally triggered by stress-induced translation arrest for storing mRNAs. Recently, it has been shown that SGs exert anti-viral functions due to their involvement in protein synthesis shut off and recruitment of innate immune signaling intermediates. The largest RNA virus, coronavirus, mutates frequently and circulates among animals, imposing great threat to public safety and animal health; however, the significance of SGs in coronavirus infections is largely unknown. Infectious bronchitis virus (IBV) is the first identified coronavirus in 1930s and has been prevalent in poultry farm for many years. In this study, we provide evidence that IBV overcomes the host antiviral response by inhibiting SGs formation via the virus-encoded endoribonuclease nsp15. By immunofluorescence analysis, we observed that IBV infection not only did not trigger SGs formation in approximately 80% of the infected cells, but also impaired the formation of SGs triggered by heat shock, sodium arsenite, or NaCl stimuli. We show that the intrinsic endoribonuclease activity of nsp15 is responsible for the inhibition of SGs formation. In fact, nsp15-defective recombinant IBV (rIBV-nsp15-H238A) greatly induced the formation of SGs, along with accumulation of dsRNA and activation of PKR, whereas wild type IBV failed to do so. Consequently, infection with rIBV-nsp15-H238A triggered transcription of IFN-β which in turn greatly affected recombinant virus replication. Further analysis showed that SGs function as antiviral hub, as demonstrated by the attenuated IRF3-IFN response and increased production of IBV in SG-defective cells. Additional evidence includes the aggregation of PRRs and signaling intermediates to the IBV-induced SGs. Collectively, our data demonstrate that the endoribonuclease nsp15 of IBV suppresses the formation of antiviral hub SGs by regulating the accumulation of viral dsRNA and by antagonizing the activation of PKR, eventually ensuring productive virus replication. We speculate that coronaviruses employ similar mechanisms to antagonize the host anti-viral SGs formation for efficient virus replication, as the endoribonuclease function of nsp15 is conserved in all coronaviruses.Author summaryIt has been reported that stress granules (SGs) are part of the host cell antiviral response. Not surprisingly, viruses in turn produce an array of antagonists to counteract such host response. Here, we show that IBV inhibits the formation of SGs through its endoribonuclease nsp15, by reducing the accumulation of viral dsRNA, evading the activation of PKR, and by subsequently inhibiting eIF2α phosphorylation and SGs formation. Nsp15 also inhibits SG formation independent of the eIF2α pathway, probably by targeting host mRNA. Depletion of SG scaffold proteins decreases IRF3-IFN response and increases the production of IBV. All coronaviruses encode a conserved endoribonuclease nsp15, and it will be important to determine whether also other (non-avian) coronaviruses limit the formation of anti-viral SGs in a similar manner.

Cytoplasmic stress granules (SGs) are generally triggered by stress-induced translation arrest for storing mRNAs. Recently, it has been shown that SGs exert anti-viral functions due to their involvement in protein synthesis shut off and recruitment of innate immune signaling intermediates. The largest RNA viruses, coronaviruses, impose great threat to public safety and animal health; however, the significance of SGs in coronavirus infection is largely unknown. Infectious Bronchitis Virus (IBV) is the first identified coronavirus in 1930s and has been prevalent in poultry farm for many years. In this study, we provided evidence that IBV overcomes the host antiviral response by inhibiting SGs formation via the virus-encoded endoribonuclease nsp15. By immunofluorescence analysis, we observed that IBV infection not only did not trigger SGs formation in approximately 80% of the infected cells, but also impaired the formation of SGs triggered by heat shock, sodium arsenite, or NaCl stimuli. We further demonstrated that the intrinsic endoribonuclease activity of nsp15 was responsible for the interference of SGs formation. In fact, nsp15-defective recombinant IBV (rIBV-nsp15-H238A) greatly induced the formation of SGs, along with accumulation of dsRNA and activation of PKR, whereas wild type IBV failed to do so. Consequently, infection with rIBV-nsp15-H238A strongly triggered transcription of IFN-β which in turn greatly affected rIBV-nsp15-H238A replication. Further analysis showed that SGs function as an antiviral hub, as demonstrated by the attenuated IRF3-IFN response and increased production of IBV in SG-defective cells. Additional evidence includes the aggregation of pattern recognition receptors (PRRs) and signaling intermediates to the IBV-induced SGs. Collectively, our data demonstrate that the endoribonuclease nsp15 of IBV interferes with the formation of antiviral hub SGs by regulating the accumulation of viral dsRNA and by antagonizing the activation of PKR, eventually ensuring productive virus replication. We further demonstrated that nsp15s from PEDV, TGEV, SARS-CoV, and SARS-CoV-2 harbor the conserved function to interfere with the formation of chemically-induced SGs. Thus, we speculate that coronaviruses employ similar nsp15-mediated mechanisms to antagonize the host anti-viral SGs formation to ensure efficient virus replication., Author summary Coronavirus encodes the conserved endoribonuclease nsp15, which has been reported to antagonize IFN responses by mediating evasion of recognition by dsRNA sensors. SGs are part of the host cell anti-viral response; not surprisingly, viruses in turn produce an array of antagonists to counteract such host response. Here, we show that IBV prevents the formation of SGs via nsp15, by reducing the accumulation of viral dsRNA, thereby evading the activation of PKR, phosphorylation of eIF2α, and formation of SGs. Depletion of SG scaffold proteins G3BP1/2 decreases IRF3-IFN response and increases the production of IBV. When overexpressed alone, nsp15s from different coronaviruses (IBV, PEDV, TGEV, SARS-CoV, and SARS-CoV-2) interferes with chemically- and physically-induced SGs, probably by targeting essential SGs assembly factors. In this way, coronaviruses antagonize the formation of SGs by nsp15, via reducing the viral dsRNA accumulation and sequestering/depleting critical component of SGs. To our knowledge, this is the first report describing the role of coronavirus nsp15 in the suppression of integral stress response, in crosstalk with anti-innate immune response.

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.

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.

Summary A number of new cell death processes have been discovered in recent years, including ferroptosis, which is characterized by the accumulation of lipid peroxidation products derived from iron metabolism. The evidence suggests that ferroptosis has a tumor-suppressor function. However, the mechanism by which ferroptosis mediates the response of tumor cells to oncolytic viruses remains poorly understood. The Newcastle disease virus (NDV) can selectively replicate in tumor cells. We show that NDV-induced ferroptosis acts through p53-SLC7A11-GPX4 pathway. Meanwhile, the levels of intracellular reactive oxygen species and lipid peroxides increased in tumor cells. Ferritinophagy was induced by NDV promotion of ferroptosis through the release of ferrous iron and an enhanced Fenton reaction. Collectively, these observations demonstrated that the NDV can kill tumor cells through ferroptosis. Our study provides novel insights into the mechanisms of NDV-induced ferroptosis and highlights the critical role of viruses in treating therapy-resistant cancers., Graphical abstract, Highlights • Oncolytic viruses NDV caused tumor cells death through ferroptosis • NDV-induced ferroptosis acts through nutrient deprivation by suppression of System Xc− • P53 activation is required for NDV-induced ferroptosis initiation • Ferritinophagy induced by NDV promotes ferroptosis through release of ferrous iron, Biological sciences; Virology; Cell biology; Cancer

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.

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.

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.

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.

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.

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.

Although several reports suggest that the entry of infectious bronchitis virus (IBV) depends on lipid rafts and low pH, the endocytic route and intracellular trafficking are unclear. In this study, we aimed to shed greater light on early steps in IBV infection. By using chemical inhibitors, RNA interference, and dominant negative mutants, we observed that lipid rafts and low pH was indeed required for virus entry; IBV mainly utilized the clathrin mediated endocytosis (CME) for entry; GTPase dynamin 1 was involved in virus containing vesicle scission; and the penetration of IBV into cells led to active cytoskeleton rearrangement. By using R18 labeled virus, we found that virus particles moved along with the classical endosome/lysosome track. Functional inactivation of Rab5 and Rab7 significantly inhibited IBV infection. Finally, by using dual R18/DiOC labeled IBV, we observed that membrane fusion was induced after 1 h.p.i. in late endosome/lysosome., Highlights • Intact lipid rafts is involved in IBV entry. • Low pH in intracyplasmic vesicles is required for IBV entry. • IBV penetrates cells via clathrin mediated endocytosis. • IBV moves along with the classical endosome/lysosome track, finally fuses with late endosome/lysosome.

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), 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.

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.

Duck hepatitis virus (DHV) affects 1-week-old but not 3-week-old ducks, and it causes a more severe disease in the younger ducks. These differences may be partially due to the host response to DHV infection. In order to understand this difference, we characterized the pathobiology of and innate immune response to DHV infection in 1-day-old (1D) and 3-week-old (3 W) ducks. Viral RNA was detected in duck livers at 24, 36 and 72 h after inoculation with DHV at a dose of 10(3) LD50. Virus-induced pathology ranged from no clinical signs to severe disease and death, and it was more severe in the 1D ducks. Infection with DHV induced up-regulation of gene expression of Toll-like receptor (TLR)-7, TLR3, retinoic-acid-inducible gene I (RIG-I), melanoma differentiation-associated gene 5 (MDA-5), interleukin (IL)-6, interferon (IFN)-α, interferon-induced transmembrane protein 1 (IFITM1), interferon-stimulated gene 12 (ISG12), and 2'-5' oligoadenylate synthetase-like gene (OASL) in the livers of 3 W ducks. Of these, IL-6, OASL and ISG12 mRNA levels were more than 100-fold higher in infected 3 W ducks than in mock-infected ducks of the same age. These genes were induced much less in infected 1D ducklings. We present evidence that a lower level of viral replication in the hepatocytes of 3 W ducks, whose basal level of cytokines is higher than that in 1D ducklings, may be related to the strong innate immunity induced. From our data, we conclude that duck age plays an important role in the pathogenicity of and innate immune responses to DHV.

T-cell internal antigen-1 (TIA-1) has roles in regulating alternative pre-mRNA splicing, mRNA translation, and stress granule (SG) formation in human cells. As an evolutionarily conserved response to environmental stress, SGs have been reported in various species. However, SG formation in chicken cells and the role of chicken TIA-1 (cTIA-1) in SG assembly has not been elucidated. In the present study, we cloned cTIA-1 and showed that it facilitates the assembly of canonical SGs in both human and chicken cells. Overexpression of the chicken prion-related domain (cPRD) of cTIA-1 that bore an N-terminal green fluorescent protein (GFP) tag (pntGFP-cPRD) or Flag tag (pFlag-cPRD) induced the production of typical SGs. However, C-terminal GFP-tagged cPRD induced notably large cytoplasmic granules that were devoid of endogenous G3BP1 and remained stable when exposed to cycloheximide, indicating that these were not typical SGs, and that the pntGFP tag influences cPRD localization. Finally, endogenous cTIA-1 was recruited to SGs in chicken cells and tissues under environmental stress. Taken together, our study provide evidence that cTIA-1 has a role in canonical SG formation in chicken cells and tissues. Our results also indicate that cPRD is necessary for SG aggregation.

VP22 of Marek's disease virus serotype 1 (MDV-1) could function in protein transduction. In this study, an infectious bursal disease virus VP2 gene was fused to the carboxyl termini of VP22. It showed that the fusion protein did not spread into the bystander cells from the cells transfected with pVP22-VP2, as the VP22 alone could. The VP22 proteins were found to be translocated into all the nuclei in the neighboring COS-1 cells, as analyzed by a fluorescence assay. Although mice were immunized with the recombinant DNAs mixed with polyethylenimine (PEI) at a dose of 1:2, it failed to enhance the antibody response against IBDV VP2, as measured by the indirect ELISA assay, yet the cell mediated immune response was significantly increased. The ratio of CD8(+)/CD4(+) T cells was significantly increased in the immunized group with the fusion genes, compared with the group immunized with VP2 (P

The viral protein 22 (VP22) in the tegument of Marek's disease virus serotype 1 (MDV-1) plays an important role in cell-to-cell spread and viral propagation. Antiserum against the carboxyl terminus of VP22 was prepared by immunizing mice with recombinant VP22 expressed in E. coli, and used to investigate its expression in chicken embryo fibroblast (CEF) cells infected with different MDV-1 strains. At an infection dose of PFU=50, intercellular trafficking of the VP22 into the nuclei of the surrounding receipt cells was detected as early as 3 hours post infection. By 6 hours after infection (before viral plague formation), the protein was detected in the whole nuclei of the recipient cells with no difference among MDV-1 strains CVI988/Rispens, GA and RB1B. Intra-nuclear accumulation of the VP22 protein was further increased when the viral plagues started to form. These results indicate that, albeit the existence of the 201TKSERT206 deletion, the VP22 of the CVI988/Rispens vaccine strain has also intercellular-trafficking function, which might serve as a potential alternative delivering protein instead of virulent strains VP22.

Infectious bronchitis virus (IBV) is a major pathogen common in the poultry industry. Broad cytotoxic T lymphocyte (CTL) response against IBV is one of the crucial factors that help to control viral replication. Spike glycoproteins on the surface of the IBV virion harbor major T cell epitopes. In this study, based on the peptide-binding motifs of chicken MHC I molecules for the BF2*4, BF2*12, BF2*15, and BF2*19 haplotypes, potential CTL epitopes were predicted using S1 proteins from different IBV strains. Twenty-one peptides were predicted to be potential CTL epitopes; they were manually synthesized and the CTL responses to them tested in vitro. Spleen lymphocytes were collected from specific-pathogen free (SPF) chicken that had been immunized with the S1 protein expression plasmid, pV-S1, and were stimulated by the synthesized peptides. IFN-γ secretion and CD8(+) T cell proliferation in chickens were tested by ELISpot array and flow cytometry, respectively. Four epitopes (P8SRIQTATDP, P9SRNATGSQP, P18GAYAVVNV, and P19SRIQTATQP) were identified to stimulate CD8(+) T cell proliferation and IFN-γ secretion, indicating their efficacy as CTL epitopes in chicken. Poly-CTL-epitope DNA vaccine (pV-S1T) was constructed by inserting nucleotide sequences encoding the P8, P9, P18, and P19 CTL epitopes into the pVAX1 vector. Chickens were vaccinated with either pV-S1, pV-S1T, or pVAX1 and the protection efficacy was analyzed, revealing that ninety percent of chickens immunized with pV-S1T were protected after challenge with 10(6) ELD50 of IBV, demonstrating that these novel CTL epitopes were effective against IBV challenge. This study provides a new method to screen virus CTL epitopes in chicken and to develop poly-CTL-epitope DNA vaccines.

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.

The α-enolase protein is reported to be an adhesin in several pathogenic bacterial species, but its role in Mycoplasma gallisepticum is unknown. In this study, the M. gallisepticum α-enolase gene was adapted to heterologous expression in Escherichia coli by performing overlapping polymerase chain reaction with site-directed mutagenesis to introduce A960G and A1158G mutations in the nucleotide sequence. The full-length mutated gene was cloned into a pGEM-T Easy vector and subcloned into the expression vector pET32a(+) to construct the pET-rMGEno plasmid. The expression of rMGEno in E. coli strain DE3 was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis with Coomassie blue staining. Purified rMGEno exhibited α-enolase catalytic activity that it could reflect the conversion of NADH to NAD+. Mouse antiserum to α-enolase was generated by immunization with rMGEno. Immunoblotting and immunofluorescence assay with the antiserum identified α-enolase on the surface of M. gallisepticum cells. Enzyme-linked immunosorbent assay characterized rMGEno as a chicken plasminogen binding protein. An adherence inhibition assay on immortalized chicken fibroblasts (DF-1) demonstrated more than 77% inhibition of adhesion in the presence of mouse antiserum, suggesting that α-enolase of M. gallisepticum participates in bacterial adhesion to DF-1 cells.

Eperythrozoonosis of swine (also designated as porcine mycoplasmosis) is a disease of swine under stress, expressed as a febrile condition with development of an acute ictero-anemia. It is caused by Eperythrozoon suis and usually causes a subclinical infection with a latent carrier state that persists for extended periods. In China, this disease has gradually developed as an important intercurrent disease and an emerging swine disease that, in recent years, has spread throughout all provinces except Tibet. Classical swine fever (hog cholera), porcine influenza, swine enzootic pneumonia, porcine reproductive and respiratory syndrome (blue ear disease), streptococci, and toxoplasmosis were detected in Eperythrozoonosis-infected pig herds, and caused serious economic losses. National epidemiology surveillance in 2002 revealed that this disease caused a total morbidity of 30% and a mortality of 10-20%. Total mortality (which includes culling sick pigs) was more than 60%. The morbidity within infected herds was near 100%, has spread throughout with a total mortality rate usually over 50%. Mortality of piglets in some districts was as high as 50%. The highest infection rate on pig farms was more than 90%. The farms with higher infection rates occurred in pig-raising areas during epidemic seasons. New diagnostic tests, such as ELISA and PCR, have been developed for the detection of porcine eperythrozoonosis, but traditionally the diagnosis of the disease is still based on clinical history and optical microscopic examination of the causative agent in blood smears. Efficient preventive and control measures include the detection of carriers in pig herds and treatment of sick pigs with drugs, such as long-acting oxytetracycline, doxycycline, or aceturate of diminazene. Oxytetracyclines as feed additives have been introduced for eperythrozoonosis prevention in uninfected pig herds, and pig producers have taken measures to reduce stress and improve sanitary conditions.