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Abstract The purpose of this study was to investigate the prevalence and relevant factors of nocturia and its impact on sleep quality in university students in Mainland China. A large-scale survey was conducted on 14,000 university students from 3 universities in Henan province, China by using an anonymous questionnaire. The questionnaire collected the information from the past six months. The relationships between the prevalence of nocturia and its relevant factors were evaluated. A total of 13,874 questionnaires were collected and 13,104 qualified for statistical analysis. A total of 659 students suffered from clinically relevant nocturia (CRN) (4.56% in male and 5.34% in female). Both univariate analysis and the logistic stepwise regression model showed that the prevalence of nocturia was significantly related to female, history of enuresis, ease of waking up, urgency, frequency and RUTI (P

Newcastle Disease (ND) and Infectious Bronchitis (IB) are two significant diseases that pose threats to the poultry industry, caused by Newcastle disease virus (NDV) and Infectious bronchitis virus (IBV), respectively. Currently, the control and prevention of these diseases primarily rely on vaccination. However, commercial ND and IB vaccines face challenges such as poor cross-protection of inactivated IBV strains and interference from live vaccines when used together, leading to immunization failures. Previously, we reported the successful rescue of a recombinant NDV expressing multiple epitopes of IBV, named rNDV-IBV-T/B, which showed promising immunoprotective efficacy against both NDV and IBV. This study focuses on the biosafety of the genetically modified recombinant vaccine candidate rNDV-IBV-T/B. Immunization was performed on day-old chicks, ducklings, goslings, and ICR mice. Observations were recorded on clinical symptoms, body weight changes, and post-mortem examination of organs, as well as histopathological preparations of tissue samples. The results indicated that the rNDV-IBV-T/B vaccine candidate had no adverse effects on the growth of targeted animals (chickens) and non-target species (ducks, geese) as well as in mammals (mice). Additionally, histopathological slides confirmed that the vaccine is safe for all tested species. Further studies evaluated the potential of rNDV-IBV-T/B to spread horizontally and vertically post-immunization, and its environmental safety. The findings revealed that the vaccine candidate lacks the capability for both horizontal and vertical transmission and does not survive in the environment. In conclusion, the rNDV-IBV-T/B strain is safe and holds potential as a new chimeric live vaccine for ND and IB.

Abstract Variations in the pathogenicity of Newcastle disease virus (NDV), the agent causing Newcastle disease, are associated with variants of different virulence. A few studies have characterized the expression of microRNAs (miRNAs) in NDV-infected avian cells. Here, the expression of miRNAs in chicken embryo fibroblasts (CEFs) infected with Herts/33 and LaSota NDV strains (highly virulent and nonvirulent, respectively) was determined using RNA sequencing. miRNAs involved in NDV infection included 562 previously documented and 184 novel miRNAs. miRNA target genes involved transcription factors, cell apoptosis, ubiquitin-mediated proteolysis, and protein processing in the endoplasmic reticulum. Potential target genes associated with autophagy were verified by qRT-PCR. No studies have documented the miRNA profiles of CEFs infected with NDVs variants. This study adds to our knowledge of the cellular miRNAs involved in NDV infection and the complex molecular mechanisms mediating virus-host interactions. The results of this study will aid the development of strategies against the chicken virus.

Summary: Newcastle disease is a global problem that causes huge economic losses and threatens the health and welfare of poultry. Despite the knowledge gained on the metabolic impact of NDV on cells, the extent to which infection modifies the plasma metabolic network in chickens remains unknown. Herein, we performed targeted metabolomic and lipidomic to create a plasma metabolic network map during NDV infection. Meanwhile, we used single-cell RNA sequencing to explore the heterogeneity of lung tissue cells in response to NDV infection in vivo. The results showed that NDV remodeled the plasma phospholipid metabolism network. NDV preferentially targets infected blood endothelial cells, antigen-presenting cells, fibroblasts, and neutrophils in lung tissue. Importantly, NDV may directly regulate ribosome protein transcription to facilitate efficient viral protein translation. In conclusion, NDV infection remodels the plasma phospholipid metabolism network in chickens. This work provides valuable insights to further understand the pathogenesis of NDV.

Nidovirales is one order of RNA virus, with the largest single-stranded positive sense RNA genome enwrapped with membrane envelope. It comprises four families (Arterividae, Mesoniviridae, Roniviridae, and Coronaviridae) and has been circulating in humans and animals for almost one century, posing great threat to livestock and poultry，as well as to public health. Nidovirales shares similar life cycle: attachment to cell surface, entry, primary translation of replicases, viral RNA replication in cytoplasm, translation of viral proteins, virion assembly, budding, and release. The viral RNA synthesis is the critical step during infection, including genomic RNA (gRNA) replication and subgenomic mRNAs (sg mRNAs) transcription. gRNA replication requires the synthesis of a negative sense full-length RNA intermediate, while the sg mRNAs transcription involves the synthesis of a nested set of negative sense subgenomic intermediates by a discontinuous strategy. This RNA synthesis process is mediated by the viral replication/transcription complex (RTC), which consists of several enzymatic replicases derived from the polyprotein 1a and polyprotein 1ab and several cellular proteins. These replicases and host factors represent the optimal potential therapeutic targets. Hereby, we summarize the Nidovirales classification, associated diseases, “replication organelle,” replication and transcription mechanisms, as well as related regulatory factors.

IntroductionThis mixed-methods study evaluates the impact of AI-assisted language learning on Chinese English as a Foreign Language (EFL) students’ writing skills and writing motivation. As artificial intelligence (AI) becomes more prevalent in educational settings, understanding its effects on language learning outcomes is crucial.MethodsThe study employs a comprehensive approach, combining quantitative and qualitative methods. The quantitative phase utilizes a pre-test and post-test design to assess writing skills. Fifty EFL students, matched for proficiency, are randomly assigned to experimental (AI-assisted instruction via ChatGPT) or control (traditional instruction) groups. Writing samples are evaluated using established scoring rubrics. Concurrently, semi-structured interviews are conducted with a subset of participants to explore writing motivation and experiences with AI-assisted learning.ResultsQuantitative analysis reveals significant improvements in both writing skills and motivation among students who received AI-assisted instruction compared to the control group. The experimental group demonstrates enhanced proficiency in various aspects of writing, including organization, coherence, grammar, and vocabulary. Qualitative findings showcase diverse perspectives, ranging from recognition of AI’s innovative instructional role and its positive influence on writing skills and motivation to concerns about contextual accuracy and over-reliance. Participants also reflect on the long-term impact and sustainability of AI-assisted instruction, emphasizing the need for ongoing development and adaptation of AI tools.DiscussionThe nuanced findings offer a comprehensive understanding of AI’s transformative potential in education. These insights have practical implications for practitioners and researchers, emphasizing the benefits, challenges, and the evolving nature of AI’s role in language instruction.

As obligate intracellular parasites, viruses rely completely on host metabolic machinery and hijack host nutrients for viral replication. Newcastle disease virus (NDV) causes acute, highly contagious avian disease and functions as an oncolytic agent. NDV efficiently replicates in both chicken and tumour cells. However, how NDV reprograms host cellular metabolism for its efficient replication is still ill-defined. We previously identified a significantly upregulated glutamate transporter gene, solute carrier family 1 member 3 (SLC1A3), during NDV infection via transcriptome analysis. To investigate the potential role of SLC1A3 during NDV infection, we first confirmed the marked upregulation of SLC1A3 in NDV-infected DF-1 or A549 cells through p53 and NF-κB pathways. Knockdown of SLC1A3 inhibited NDV infection. Western blot analysis further confirmed that glutamine, but not glutamate, asparagine, or aspartate, was required for NDV replication. Metabolic flux data showed that NDV promotes the decomposition of glutamine into the tricarboxylic acid cycle. Importantly, the level of glutamate and glutaminolysis were reduced by SLC1A3 knockdown, indicating that SLC1A3 propelled glutaminolysis for glutamate utilization and NDV replication in host cells. Taken together, our data identify that SLC1A3 serves as an important regulator for glutamine metabolism and is hijacked by NDV for its efficient replication during NDV infection. These results improve our understanding of the interaction between NDV and host cellular metabolism and lay the foundation for further investigation of efficient vaccines.

ABSTRACT Newcastle disease virus (NDV) is an avian paramyxovirus that causes major economic losses to the poultry industry around the world, with NDV pathogenicity varying due to strain virulence differences. However, the impacts of intracellular viral replication and the heterogeneity of host responses among cell types are unknown. Here, we investigated the heterogeneity of lung tissue cells in response to NDV infection in vivo and that of the chicken embryo fibroblast cell line DF-1 in response to NDV infection in vitro using single-cell RNA sequencing. We characterized the NDV target cell types in the chicken lung at the single-cell transcriptome level and classified cells into five known and two unknown cell types. The five known cell types are the targets of NDV in the lungs with virus RNA detected. Different paths of infection in the putative trajectories of NDV infection were distinguished between in vivo and in vitro, or between virulent Herts/33 strain and nonvirulent LaSota strain. Gene expression patterns and the interferon (IFN) response in different putative trajectories were demonstrated. IFN responses were elevated in vivo, especially in myeloid and endothelial cells. We distinguished the virus-infected and non-infected cells, and the Toll-like receptor signaling pathway was the main pathway after virus infection. Cell-cell communication analysis revealed the potential cell surface receptor-ligand of NDV. Our data provide a rich resource for understanding NDV pathogenesis and open the way to interventions specifically targeting infected cells. IMPORTANCE Newcastle disease virus (NDV) is an avian paramyxovirus that causes major economic losses to the poultry industry around the world, with NDV pathogenicity varying due to strain virulence differences. However, the impacts of intracellular viral replication and the heterogeneity of host responses among cell types are unknown. Here, we investigated the heterogeneity of lung tissue cells in response to NDV infection in vivo and that of the chicken embryo fibroblast cell line DF-1 in response to NDV infection in vitro using single-cell RNA sequencing. Our results open the way to interventions specifically targeting infected cells, suggest principles of virus-host interactions applicable to NDV and other similar pathogens, and highlight the potential for simultaneous single-cell measurements of both host and viral transcriptomes for delineating a comprehensive map of infection in vitro and in vivo. Therefore, this study can be a useful resource for the further investigation and understanding of NDV.

Although the involvement of the ubiquitin-proteasome system (UPS) in several coronavirus-productive infections has been reported, whether the UPS is required for infectious bronchitis virus (IBV) and porcine epidemic diarrhea virus (PEDV) infections is unclear. In this study, the role of UPS in the IBV and PEDV life cycles was investigated. When the UPS was suppressed by pharmacological inhibition at the early infection stage, IBV and PEDV infectivity were severely impaired. Further study showed that inhibition of UPS did not change the internalization of virus particles; however, by using R18 and DiOC-labeled virus particles, we found that inhibition of UPS prevented the IBV and PEDV membrane fusion with late endosomes or lysosomes. In addition, proteasome inhibitors blocked the degradation of the incoming viral protein N, suggesting the uncoating process and genomic RNA release were suppressed. Subsequently, the initial translation of genomic RNA was blocked. Thus, UPS may target the virus-cellular membrane fusion to facilitate the release of incoming viruses from late endosomes or lysosomes, subsequently blocking the following virus uncoating, initial translation, and replication events. Similar to the observation of proteasome inhibitors, ubiquitin-activating enzyme E1 inhibitor PYR-41 also impaired the entry of IBV, enhanced the accumulation of ubiquitinated proteins, and depleted mono-ubiquitin. In all, this study reveals an important role of UPS in coronavirus entry by preventing membrane fusion and identifies UPS as a potential target for developing antiviral therapies for coronavirus.

ABSTRACT: 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.

Stress in poultry can lead to changes in body metabolism and immunity, which can increase susceptibility to infectious diseases. However, knowledge regarding chicken responses to viral infection under stress is limited. Dexamethasone (Dex) is a synthetic glucocorticoid similar to that secreted by animals under stress conditions, and has been widely used to induce stress in chickens. Herein, we established a stress model in 7-day-old chickens injected with Dex to elucidate the effects of stress on IBV replication in the kidneys. The metabolic changes, immune status and growth of the chickens under stress conditions were comprehensively evaluated. Furthermore, the metabolic profile, weight gain, viral load, serum cholesterol levels, cytokines and peripheral blood lymphocyte ratio were compared in chickens treated with Dex and infected with IBV. An LC-MS/MS-based metabolomics method was used to examine differentially enriched metabolites in the kidneys. A total of 113 metabolites whose abundance was altered after Dex treatment were identified, most of which were lipids and lipid-like molecules. The principal metabolic alterations in chicken kidneys caused by IBV infection included fatty acid, valine, leucine and isoleucine metabolism. Dex treatment before and after IBV infection mainly affected the host’s tryptophan, phenylalanine, amino sugar and nucleotide sugar metabolism. In addition, Dex led to up-regulation of serum cholesterol levels and renal viral load in chickens, and to the inhibition of weight gain, peripheral blood lymphocytes and IL-6 production. We also confirmed that the exogenous cholesterol in DF-1 cells promoted the replication of IBV. However, whether the increase in viral load in kidney tissue is associated with the up-regulation of cholesterol levels induced by Dex must be demonstrated in future experiments. In conclusion, chick growth and immune function were significantly inhibited by Dex. Host cholesterol metabolism and the response to IBV infection are regulated by Dex. This study provides valuable insights into the molecular regulatory mechanisms in poultry stress, and should support further research on the intrinsic link between cholesterol metabolism and IBV replication under stress conditions.

Infectious bronchitis virus (IBV) has been prevalent in chicken farms for many years, and its control relies on extensive vaccine administration. The continuous emergence of new variants and the low cross-protection efficiency prompt the development of new vaccines. In this study, we develop a reverse genetics technique based on the classical vaccine strain H120 genome, via in vitro ligation method. Using the H120 genome as the backbone, we constructed the recombinant virus rH120-QX(S) by replacing the H120 S gene with the QX S gene, a prevalent strain in China. Biological characteristics of the rH120-QX(S) virus, such as 50% egg lethal dose (ELD50), 50% egg infectious dose (EID50), dwarf embryo, growth curve, and genetic stability, are measured, which are comparable to the parental virus H120. There are no clinical symptoms and tissue lesions in the trachea and kidney in the rH120-QX(S)-infected specific-pathogen-free (SPF) chickens, demonstrating that this recombinant virus does not confer pathogenicity. Furthermore, protection studies show that there is 100% homologous protection of rH120-QX(S) to the virulent QX strain, as shown by the absence of clinical signs and no lethality. Taken together, our results demonstrate that swapping the S gene onto the H120 genetic backbone is a precise and effective way to produce genetically defined IBV vaccine candidates.

Abstract 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.

Virus infection can lead to the production of interferon, which activates the JAK/STAT pathway and induces the expression of multiple downstream interferon-stimulated genes (ISGs) to achieve their antiviral function. Cytidine/uridine monophosphate kinase 2 (CMPK2) gene has been identified as an ISG in human and fish, and is also known as a rate-limiting enzyme in mitochondria to maintain intracellular UTP/CTP levels, which is necessary for de novo mitochondrial DNA synthesis. By mining previous microarray data, it was found that both Avian Influenza Virus (AIV) and Newcastle Disease Virus (NDV) infection can lead to the significant upregulation of chicken CMPK2 gene. However, little is known about the function of CMPK2 gene in chickens. In the present study, the open reading frame (ORF) of chicken CMPK2 (chCMPK2) was cloned from DF-1, a chicken embryo fibroblasts cell line, and subjected to further analysis. Sequence analysis showed that chCMPK2 shared high similarity in amino acid with CMPK2 sequences from all the other species, especially reptiles. A thymidylate kinase (TMK) domain was identified in the C-terminus of chCMPK2, which is highly conserved among all species. In vitro, AIV infection induced significant increases in chCMPK2 expression in DF-1, HD11, and the chicken embryonic fibroblasts (CEF), while obvious increase only detected in DF-1 cells and CEF cells after NDV infection. In vivo, the expression levels of chCMPK2 were up-regulated in several tissues from AIV infected chickens, especially the brain, spleen, bursa, kidney, intestine, heart and thymus, and notable increase of chCMPK2 was detected in the bursa, kidney, duodenum, lung, heart, and thymus during NDV infection. Here, using MDA5 and IFN-β knockdown cells, we demonstrated that as a novel ISG, chCMPK2 could be regulated by the MDA5/IFN-β pathway. The high expression level of exogenous chCMPK2 displayed inhibitory effects on AIV and NDV as well as reduced viral RNA in infected cells. We further demonstrated that Asp135, a key site on the TMK catalytic domain, was identified as critical for the antiviral activities of chCMPK2. Taken together, these data demonstrated that chCMPK2 is involved in the chicken immune system and may play important roles in host anti-viral responses.

Host cholesterol metabolism remodeling is significantly associated with the spread of human pathogenic coronaviruses, suggesting virus-host relationships could be affected by cholesterol-modifying drugs. Cholesterol has an important role in coronavirus entry, membrane fusion, and pathological syncytia formation, therefore cholesterol metabolic mechanisms may be promising drug targets for coronavirus infections. Moreover, cholesterol and its metabolizing enzymes or corresponding natural products exert antiviral effects which are closely associated with individual viral steps during coronavirus replication. Furthermore, the coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 infections are associated with clinically significant low cholesterol levels, suggesting cholesterol could function as a potential marker for monitoring viral infection status. Therefore, weaponizing cholesterol dysregulation against viral infection could be an effective antiviral strategy. In this review, we comprehensively review the literature to clarify how coronaviruses exploit host cholesterol metabolism to accommodate viral replication requirements and interfere with host immune responses. We also focus on targeting cholesterol homeostasis to interfere with critical steps during coronavirus infection.

Human activities beyond ecosystem capacity have resulted in serious effects on corals worldwide. Nowadays, many studies have focused on the influence of diving activities on coral communities, while the knowledge of physiological changes under corresponding environmental stresses remains largely undetermined. In the study, we aimed to investigate the physiological effects of touching, ammonia nitrogen enrichment (5 μmol⋅L–1), and sediment cover (particle size of less than 0.3 mm), which simulated improper self-contained underwater breathing apparatus (SCUBA) diving behaviors, on Acropora hyacinthus and Porites cylindrica in Wuzhizhou Island, the South China Sea. For A. hyacinthus, continuous touching caused the tentacles to shrink and secrete mucus, which consumed energy and dissolved oxygen. The skeletal growth rate was decreased by 72% compared with the control group. There was a rapid decline of Fv/Fm and alpha under the dual impacts of high ammonia nitrogen and touching, while the Chl a concentration and tissue biomass were decreased by 36 and 28% compared with touching alone, respectively. High ammonia nitrogen and touching increased the concentrations of lipid and protein. Nevertheless, zooxanthellae density was increased by 23% to relieve the effects of a lower concentration of Chl a in a high nutrient environment. Constant touching and sediment cover in diving areas with elevated ammonia content affected the photosynthesis and respiration of corals, and a significant decrease was observed for lipid, zooxanthellae density, and Chl a concentration. Coral bleaching occurred on day 7. For P. cylindrica, the decreasing magnitude of Fv/Fm and alpha under different stresses in the subsequent phase was less compared with A. hyacinthus. The contents of carbohydrate and protein under continuous touching were decreased by 7 and 15% compared with the control group, respectively, causing negative growth. Under the dual influences of high ammonia nitrogen and continuous touching, all energy reserves were significantly lower. Repeated touching and sediment cover in diving areas containing high ammonia content increased the concentrations of lipid and protein compared with the touching and high nutrient treatment group likely because that Porites associated with C15 zooxanthella increased heterotrophic feeding to compensate for restricted symbiodiniaceae photosynthesis. Additionally, P. cylindrica produced mucus to aid the removal of sediment, so that corals didn’t obviously bleach during the experiment. Collectively, P. cylindrica was more resistant to diving activities than A. hyacinthus which only relies on photoautotrophy. To ensure the sustainable development of coral reef dive tourism, it is necessary to strengthen the supervision of diving behaviors, rotate the diving areas, and conduct regular assessments on the coral status.

ABSTRACT: 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.

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.

Innate immunity serves as the first barrier against virus infection. DEAD (Glu-Asp-Ala-Glu) box RNA helicases, originally considered to be involved in RNA processing and RNA unwinding, have been shown to play an important role in antiviral innate immunity.

Multi-functional DEAD-box helicase 5 (DDX5), which is important in transcriptional regulation, is hijacked by diverse viruses to facilitate viral replication. However, its regulatory effect in antiviral innate immunity remains unclear. We found that DDX5 interacts with the N6-methyladenosine (m6A) writer METTL3 to regulate methylation of mRNA through affecting the m6A writer METTL3-METTL14 heterodimer complex. Meanwhile, DDX5 promoted the m6A modification and nuclear export of transcripts DHX58, p65, and IKKγ by binding conserved UGCUGCAG element in innate response after viral infection. Stable IKKγ and p65 transcripts underwent YTHDF2-dependent mRNA decay, whereas DHX58 translation was promoted, resulting in inhibited antiviral innate response by DDX5 via blocking the p65 pathway and activating the DHX58-TBK1 pathway after infection with RNA virus. Furthermore, we found that DDX5 suppresses antiviral innate immunity in vivo. Our findings reveal that DDX5 serves as a negative regulator of innate immunity by promoting RNA methylation of antiviral transcripts and consequently facilitating viral propagation.

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.

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.

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.

Abstract Background Anaplastic thyroid cancer (ATC) is one of the most aggressive of all solid tumors for which no effective therapies are currently available. Oncolytic Newcastle disease virus (NDV) has shown the potential to induce oncolytic cell death in a variety of cancer cells of diverse origins. However, whether oncolytic NDV displays antitumor effects in ATC remains to be investigated. We have previously shown that the oncolytic NDV strain FMW (NDV/FMW) induces oncolytic cell death in several cancer types. In the present study, we investigated the oncolytic effects of NDV/FMW in ATC. Methods In this study, a recombinant NDV expressing green fluorescent protein (GFP) was generated using an NDV reverse genetics system. The resulting virus was named after rFMW/GFP and the GFP expression in infected cells was demonstrated by direct fluorescence and immunoblotting. Viral replication was evaluated by end-point dilution assay in DF-1 cell lines. Oncolytic effects were examined by biochemical and morphological experiments in cultural ATC cells and in mouse models. Results rFMW/GFP replicated robustly in ATC cells as did its parent virus (NDV/FMW) while the expression of GFP protein was detected in lungs and spleen of mice intravenously injected with rFMW/GFP. We further showed that rFMW/GFP infection substantially increased early and late apoptosis in the ATC cell lines, THJ-16 T and THJ-29 T and increased caspase-3 processing and Poly (ADP-ribose) polymerase (PARP) cleavage in ATC cells as assessed by immunoblotting. In addition, rFMW/GFP induced lyses of spheroids derived from ATC cells in three-dimensional (3D) cultures. We further demonstrated that rFMW/GFP infection resulted in the activation of p38 MAPK signaling, but not Erk1/2 or JNK, in THJ-16 T and THJ-29 T cells. Notably, inhibition of p38 MAPK activity by SB203580 decreased rFMW/GFP-induced cleavage of caspase-3 and PARP in THJ-16 T and THJ-29 T cells. Finally, both rFMW/GFP and its parent virus inhibited tumor growth in mice bearing THJ-16 T derived tumors. Conclusion Taken together, these data indicate that both the recombinant reporter virus rFMW/GFP and its parent virus NDV/FMW, display oncolytic activities in ATC cells in vitro and in vivo and suggest that oncolytic NDV may have potential as a novel therapeutic strategy for ATC.

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.

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.

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 >0.98. The establishment of TRFM-ICA will provide a new sensitive and efficient technical support for the rapid screening of DEX in food.

Abstract Background For many years, ND has been one of the most important infectious pigeon diseases in China. In recent years, a high mortality has been observed in ND-infected pigeons in China. Mortality is from 40% to 80% or 100% in some cases. Methods The full-length genomes of four pigeon paramyxovirus type 1 (PPMV-1) strains, which were isolated from infected pigeons in China in 2012 and 2013, were sequenced and analyzed to determine the phylogenetic characteristics of PPMV-1 circulating in pigeons of China in recent years. Furthermore, cross hemagglutination inhibition and cross virus neutralization assays, as well as animal experiments were conducted to determine the antigenicity and pathogenicity of those viruses. Proteolytic cleavage sites (residues 112–117) of the F proteins were identified as the typical virulence motif, 112RRQKR↓F117 for all four PPMV-1 strains investigated. Results Phylogenetic analysis based on sequences of complete genomes and F gene revealed that the four PPMV-1 isolates and most of recent isolates in China were highly homologous to European isolates from 1998 to 2011. All those isolates were clustered in one clade of genotype VI NDV, termed as subgroup 4bii f. The R value was calculated based on cross hemagglutination inhibition and cross virus neutralization results, and confirmed antigenic difference of the PPMV-1 strains isolated in 2013 from the LaSota vaccine strain. Several mutations were identified in the surface glycoproteins F and HN, which probably gave rise to those antigenic differences. Conclusion Our result suggested that the PPMV-1 strain prevailing in China in the last decade diverged from a common ancestor and was presumably transmitted from Europe. PPMV-1 isolates displayed obvious antigenic differences from vaccine strain LaSota. Even though PPMV-1 did not cause high mortality in experimental pigeons, the infected pigeons were exhibiting viral shedding for 3 weeks after infection, suggesting PPMV-1 is a potential threat to NDV control worldwide.

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

Recently, chickens vaccinated with the CVI988/Rispens vaccine showed increased tumor incidence. Moreover, many strains of Marek’s disease virus (MDV) that were naturally integrated with the long terminal repeat (LTR) of the avian reticuloendotheliosis virus (REV) have been isolated, which means it is necessary to develop a new vaccine. In this study, two LTR sequences were inserted into Rispens to construct a recombinant MDV (rMDV). Then, the safety and efficacy of rMDV were evaluated separately in chickens. The growth rate curves showed that the insertion of REV-LTR into MDV enabled a faster replication in vitro than Rispens. Chickens immunized with high or repeated dose rMDV had no MD clinical signs. Further, no tumor, tissue lesions, or evident pathological changes were observed in the chicken organs. Polymerase chain reaction (PCR) and virus isolation revealed that rMDV had the ability to spread horizontally to non-immunized chickens and had no impact on the environment. After five passages in chickens, there were no obvious lesions, and the LTR insertion was stable. There were also no deletions or mutations, which indicates that rMDV is safe in chickens. In addition, rMDV has an advantage over Rispens against vvMDV Md5 at low doses. All results demonstrate that the transgenic strain of rMDV with REV-LTR can be used as a live attenuated vaccine candidate.

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

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

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

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.

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.

Urban flooding occurs frequently in many regions of China. To reduce the losses caused by urban flooding, sponge city (SPC) and low-impact development (LID) have been carried out in many Chinese cities. However, urban flooding is influenced by various factors, such as climate, land cover characteristics and nearby river networks, so it is necessary to evaluate the effectiveness of LID measures. In this study, the Storm Water Management Model (SWMM) was adopted to simulate historical urban storm processes in the mountainous Fragrance Hills region of Beijing, China. Subsequently, numerical simulations were performed to evaluate how various LID measures (concave greenbelt, permeable pavement, bio-retention, vegetative swales, and comprehensive measures) influenced urban runoff reduction. The results showed that the LID measures are effective in controlling the surface runoff of the storm events with return periods shorter than five years, in particular, for one-year events. Furthermore, the effectiveness on traffic congestion mitigation of several LID measures (concave greenbelt, vegetative swales, and comprehensive measures) was evaluated. However, the effective return periods of storm events are shorter than two years if the effectiveness on traffic congestion relief is considered. In all evaluated aspects, comprehensive measures and concave greenbelts are the most effective, and vegetative swale is the least effective. This indicated that LID measures are less effective for removing ponding from most storm events in a mountainous, low-lying and backward pipeline infrastructure region with pressures from interval flooding and urban waterlogging. The engineering measures including water conservancy projects and pipeline infrastructure construction combined with the non-engineering measures were suggested to effectively control severe urban storms.

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 between 5.5×102 and 1.1×109 copies/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.