Newcastle Disease Virus Replication Research Articles

Effect of co-infection with Newcastle disease virus on Mycoplasma gallisepticum pathogenesis in vivo and in vitro

The co-infection of Newcastle disease virus (NDV) and Mycoplasma gallisepticum (MG) has a detrimental effect on chicken production performance, exerts a deleterious impact on poultry production performance, resulting in substantial economic losses. However, the exact impact and underlying mechanisms remain ambiguous. In this study, co-infection models were established both in vivo and in vitro. Through these models, it was found that the co-infection facilitated the replication of MG and NDV, as well as MG induced pathogenesis. The administration of lentogenic NDV resulted in the suppression of the innate immune response in vivo. At cellular level, co-infection promoted MG induced apoptosis through caspase-dependent mitochondrial endogenous pathway and suppressed the inflammatory secretion. This research contributes novel insights in co-infection.

Identification of pigeon mitochondrial antiviral signaling protein (MAVS) and its role in antiviral innate immunity.

Pigeons can be infected with various RNA viruses, and their innate immune system responds to viral infection to establish an antiviral response. Mitochondrial antiviral signaling protein (MAVS), an important adaptor protein in signal transduction, plays a pivotal role in amplifying the innate immune response. In this study, we successfully cloned pigeon MAVS (piMAVS) and performed a bioinformatics analysis. The results showed that the caspase recruitment domain (CARD) and transmembrane (TM) domain are highly conserved in poultry and mammals but poorly conserved in other species. Furthermore, we observed that MAVS expression is upregulated both in pigeons and pigeon embryonic fibroblasts (PEFs) upon RNA virus infection. Overexpression of MAVS resulted in increased levels of β-interferon (IFN-β), IFN-stimulated genes (ISGs), and interleukin (ILs) mRNA and inhibited Newcastle disease virus (NDV) replication. We also found that piMAVS and human MAVS (huMAVS) induced stronger expression of IFN-β and ISGs when compared to chicken MAVS (chMAVS), and this phenomenon was also reflected in the degree of inhibition of NDV replication. Our findings demonstrate that piMAVS plays an important role in repressing viral replication by regulating the activation of the IFN signal pathway in pigeons. This study not only sheds light on the function of piMAVS in innate immunity but also contributes to a more comprehensive understanding of the innate immunity system in poultry. Our data also provide unique insights into the differences in innate immunity between poultry and mammal.

Mitochondrial protein CHCHD10 inhibits NDV replication and reduces pathological changes

Newcastle disease (ND) is a disease that threatens the world's poultry industry, which is caused by virulent Newcastle disease virus (NDV). As its pathogenic mechanism remains not fully clear, the proteomics of NDV-infected cells were analyzed. The results revealed that coiled-coil-helix-coiled-coil-helix domain containing 10 (CHCHD10) protein displayed a significant decrease at the late stage of NDV infection. To investigate the function of CHCHD10 in NDV infection, its expression after NDV infection was detected both in vivo and in vitro. Besides, the tissue viral loads and pathological damage of C57BL/6 mice with CHCHD10 differently expressed were also investigated. The results showed that the CHCHD10 expression was significantly decreased both in vivo and in vitro at the late stage of NDV infection. The viral loads were significantly higher in CHCHD10 silenced C57BL/6 mice, along with more severe pathological damage and vice versa.

The Role of Newcastle Disease Virus in Cancer Therapy: A Systematic Review

Recently, immunotherapy has become a hopeful option for cancer therapy. Taking advantage of pathogens is a well-established method of cancer immunotherapy. It has been shown that the Newcastle disease virus (NDV), an avian virus with oncolytic properties, can be used to treat cancer. This study was designed to offer a comprehensive overview of the role of NDV in cancer treatment, focusing on its attributes, mechanisms of action, preclinical and clinical trials, and future perspectives. A systematic literature review was performed to gather relevant information about NDV in cancer therapy. The inclusion criteria of this study included studies conducted in vitro and in vivo as well as clinical trials to investigate the anti-cancer effects and mechanisms behind the action of NDV. A total of 34 out of 176 academic articles, preclinical studies, clinical trials, and review articles were analyzed to collect key findings. In addition to replicating selectively through invading cancerous cells, NDV has been shown to induce apoptosis in in vivo studies. There is evidence that it can induce apoptosis, induce oncolysis, and modulate immune function in preclinical research. Studies have demonstrated that combining this therapy with chemotherapy, immunotherapies, and targeted therapies provides encouraging results regarding effectiveness and safety in animal models. As a result of NDV’s ability to induce immunogenic cell death, the immune system is activated when it reacts to cancer cells. In addition, NDV infection promotes the recruitment and activation of immune cells, especially cytotoxic T cells, by releasing cytokines and chemokines. This dual mechanism triggers anti-cancer immune responses. An interesting new approach to cancer treatment is based on the selective replication of NDV, which can induce immunogenic cell death in tumor tissues and interfere with oncogenic signaling pathways. Research in preclinical models has yielded valuable information, as well as evidence of the effectiveness and safety of clinical trials. A synergistic effect has been demonstrated when chemotherapy, immunotherapies, and targeted therapies are used in conjunction.

Newcastle disease virus activates the PI3K/AKT signaling pathway by targeting PHLPP2 degradation to delay cell apoptosis and promote viral replication

Newcastle disease (ND) is a highly pathogenic, contagious, and fatal infectious disease in poultry caused by the Newcastle disease virus (NDV). The PI3K/AKT signaling pathway is a phosphorylation cascade that participates in regulating several cellular functions. Viruses reportedly regulate the course of infection through the PI3K/AKT axis. Here, we aimed to analyze the pathogenesis of NDV infection mediated by the PI3K/AKT signaling pathway activation. We found that NDV infection can phosphorylate AKT to activate the PI3K/AKT axis both in vitro and in vivo. Flow cytometry and Caspase-3 activity assay showed that NDV infection could inhibit cell apoptosis. The activation or inhibition of the PI3K/AKT signaling pathway activity significantly inhibited or promoted NDV-mediated apoptosis. Furthermore, inhibition of cell apoptosis significantly promoted NDV replication. Overall, our results showed that NDV infection activates the PI3K/AKT signaling pathway and inhibits cell apoptosis, thus promoting viral replication. In this context, the reduced expression of PHLPP2 protein mediated by NDV infection could be inhibited by MG132. PHLPP2 expression reversely and positively regulated NDV replication and cell apoptosis, respectively. These results indicated that NDV infection-mediated activation of the PI3K/AKT signaling pathway and the inhibition of apoptosis depend on the ubiquitin-proteasome degradation of the PHLPP2 protein. Co-IP and indirect immunofluorescence results showed that NDV V protein could interact with PHLPP2 protein, indicating that NDV targeted PHLPP2 protein degradation through V protein to activate the PI3K/AKT signaling pathway. This study deepens our understanding of the molecular mechanisms of NDV infection, providing a theoretical basis for ND prevention and control.

Molecular cloning and functional characterization of pigeon IKKε

Inhibitor of nuclear factor kappa-B kinase ε (IKKε), a member of the non-canonical IκB kinase family, plays a critical role in connecting various signaling pathways associated with the initiation of type I interferon (IFN) production. Although the importance of IKKε in innate immunity has been well established in mammals and fish, its characterization and function in pigeons have remained largely unexplored. In this study, we successfully cloned pigeon IKKε (piIKKε) from pigeon embryo fibroblasts (PEFs) for the first time. This gene encodes 722 amino acids and shares high amino acid similarity with its duck and goose counterparts. piIKKε showed a diffuse cytoplasmic distribution and broad expression in all tissues examined. Overexpression of piIKKε in PEFs significantly activated the IFN-β promoter, with both the kinase and CC domains of piIKKε playing key roles in initiating IFN-β expression. Knockdown of piIKKε using small interfering RNA significantly reduced the levels of IFN-β induced by NDV, AIV, poly (I:C), or SeV. Furthermore, the presence of piIKKε resulted in a remarkable reduction in the replication of both avian influenza virus (AIV) H9N2 and Newcastle disease virus (NDV) in PEFs. Our results demonstrate that piIKKε plays a critical role in mediating antiviral innate immunity in pigeons.

Newcastle disease virus nucleocapsid protein mediates the degradation of 14–3–3ε to antagonize the interferon response and promote viral replication

Newcastle disease virus (NDV) is responsible for outbreaks that pose a threat to the global poultry industry. NDV triggers an interferon (IFN) response in the host upon infection. However, it also employs mechanisms that counteract this response. One important component in IFN-related signaling pathways is 14–3–3ε, which is known to interact with retinoic acid-inducible gene I (RIG-I) and mitochondrial antiviral signaling protein (MAVS). The relationship between 14 and 3–3ε and NDV infection has not been previously explored; therefore, this study aimed to investigate this relationship in vivo and in vitro using overexpressed and knockdown 14–3–3ε experiments, along with co-immunoprecipitation analysis. We found that NDV infection led to the degradation of 14–3–3ε. Furthermore, 14–3–3ε inhibited the replication of NDV, suggesting that NDV may enhance its own replication by promoting the degradation of 14–3–3ε during infection. The study revealed that 14–3–3ε is degraded by lysosomes and the viral protein nucleocapsid protein (NP) of NDV induces this degradation. It was also observed that 14–3–3ε is involved in activating the IFN pathway during NDV infection and mediates the binding of MDA5 to MAVS. Our study reveals that NDV NP mediates the entry of 14–3–3ε into lysosomes and facilitates its degradation. These findings contribute to the existing knowledge on the molecular mechanisms employed by NDV to counteract the IFN response and enhance its own replication.

In Vitro Characterization of chIFITMs of Aseel and Kadaknath Chicken Breeds against Newcastle Disease Virus Infection.

Newcastle disease (ND) is highly contagious and usually causes severe illness that affects Aves all over the world, including domestic poultry. Depending on the virus's virulence, it can impact the nervous, respiratory, and digestive systems and cause up to 100% mortality. The chIFITM genes are activated in response to viral infection. The current study was conducted to quantify the mRNA of chIFITM genes in vitro in response to ND viral infection. It also examined its ability to inhibit ND virus replication in chicken embryo fibroblast (CEF) cells of the Aseel and Kadaknath breeds. Results from the study showed that the expression of all chIFITM genes was significantly upregulated throughout the period in the infected CEF cells of both breeds compared to uninfected CEF cells. In CEF cells of the Kadaknath breed, elevated levels of expression of the chIFITM3 gene dramatically reduced ND viral growth, and the viral load was 60% lower than in CEF cells of the Aseel breed. The expression level of the chIFITMs in Kadaknath ranged from 2.39 to 11.68 log2 folds higher than that of control CEFs and was consistently (p < 0.01) higher than Aseel CEFs. Similar to this, theIFN-γ gene expresses strongly quickly and peaks at 13.9 log2 fold at 48 hpi. Based on these cellular experiments, the Kadaknath breed exhibits the potential for greater disease tolerance than Aseel. However, to gain a comprehensive understanding of disease resistance mechanisms in chickens, further research involving in vivo investigations is crucial.

Niclosamide inhibits Newcastle disease virus replication in chickens by perturbing the cellular glycolysis

Newcastle disease virus (NDV), a member of Paramyxoviridae family, is one of the most important pathogens in poultry. To ensure optimal environments for their replication and spread, viruses rely largely on host cellular metabolism. In the present study, we evaluated the small drug molecule niclosamide for its anti-NDV activity. Our study has shown that a sublethal dose of 1 μM niclosamide could drastically reduce NDV replication. The results showed that niclosamide has antiviral activity against NDV infection during in vitro, in ovo and in vivo assays. Pharmacologically inhibiting the glycolytic pathway remarkably reduced NDV RNA synthesis and infectious virion production. Our results suggest that the effect of niclosamide on cellular glycolysis could be the possible reason for the specific anti-NDV effect. This study could help us understand antiviral strategies against similar pathogens and may lead to novel therapeutic approaches through targeted inhibition of specific cellular metabolic pathways.

Identification of a Novel Interferon Lambda Splice Variant in Chickens.

Type III interferons (IFNLs) have critical roles in the host's innate immune system, also serving as the first line against pathogenic infections of mucosal surfaces. In mammals, several IFNLs have been reported; however, only limited data on the repertoire of IFNLs in avian species is available. Previous studies showed only one member in chicken (chIFNL3). Herein, we identified a novel chicken IFNL for the first time, termed chIFNL3a, which contains 354 bp, and encodes 118 amino acids. The predicted protein is 57.1% amino acid identity with chIFNL. Genetic, evolutionary, and sequence analyses indicated that the new open reading frame (ORF) groups with type III chicken IFNs represent a novel splice variant. Compared to IFNs from different species, the new ORF is clustered within the type III IFNs group. Further study showed that chIFNL3a could activate a panel of IFN-regulated genes and function mediated by the IFNL receptor, and chIFNL3a markedly inhibited the replication of Newcastle disease virus (NDV) and influenza virus in vitro. These data collectively shed light on the repertoire of IFNs in avian species and provide useful information that further elucidate the interaction of the chIFNLs and viral infection of poultry. IMPORTANCE Interferons (IFNs) are critical soluble factors in the immune system, and are composed of 3 types (I, II, and III) that utilize different receptor complexes (IFN-αR1/IFN-αR2, IFN-γR1/IFN-γR2, and IFN-λR1/IL-10R2, respectively). Herein, we identified IFNL from the genomic sequences of chicken and termed it chIFNL3a, located on chromosome 7 of chicken. Phylogenetically clustered with all known types of chicken IFNs, the finding of this IFN is considered a type III IFN. To further evaluate the biological properties of chIFNL3a, the target protein was prepared by the baculovirus expression system (BES), which could markedly inhibit the replication of NDV and influenza viruses. In this study, we uncovered a new interferon lambda splice variant of chicken, termed chIFNL3a, which could inhibit viral replication in cells. Importantly, these novel findings may extend to other viruses, offering a new direction for therapeutic interventions.

Newcastle Disease Virus Manipulates Mitochondrial MTHFD2-Mediated Nucleotide Metabolism for Virus Replication.

Viruses require host cell metabolic reprogramming to satisfy their replication demands; however, the mechanism by which the Newcastle disease virus (NDV) remodels nucleotide metabolism to support self-replication remains unknown. In this study, we demonstrate that NDV relies on the oxidative pentose phosphate pathway (oxPPP) and the folate-mediated one-carbon metabolic pathway to support replication. In concert with [1,2-13C2] glucose metabolic flow, NDV used oxPPP to promote pentose phosphate synthesis and to increase antioxidant NADPH production. Metabolic flux experiments using [2,3,3-2H] serine revealed that NDV increased one-carbon (1C) unit synthesis flux through the mitochondrial 1C pathway. Interestingly, methylenetetrahydrofolate dehydrogenase (MTHFD2) was upregulated as a compensatory mechanism for insufficient serine availability. Unexpectedly, direct knockdown of enzymes in the one-carbon metabolic pathway, except for cytosolic MTHFD1, significantly inhibited NDV replication. Specific complementation rescue experiments on small interfering RNA (siRNA)-mediated knockdown further revealed that only a knockdown of MTHFD2 strongly restrained NDV replication and was rescued by formate and extracellular nucleotides. These findings indicated that NDV replication relies on MTHFD2 to maintain nucleotide availability. Notably, nuclear MTHFD2 expression was increased during NDV infection and could represent a pathway by which NDV steals nucleotides from the nucleus. Collectively, these data reveal that NDV replication is regulated by the c-Myc-mediated 1C metabolic pathway and that the mechanism of nucleotide synthesis for viral replication is regulated by MTHFD2. IMPORTANCE Newcastle disease virus (NDV) is a dominant vector for vaccine and gene therapy that accommodates foreign genes well but can only infect mammalian cells that have undergone cancerous transformation. Understanding the remodeling of nucleotide metabolic pathways in host cells by NDV proliferation provides a new perspective for the precise use of NDV as a vector or in antiviral research. In this study, we demonstrated that NDV replication is strictly dependent on pathways involved in redox homeostasis in the nucleotide synthesis pathway, including the oxPPP and the mitochondrial one-carbon pathway. Further investigation revealed the potential involvement of NDV replication-dependent nucleotide availability in promoting MTHFD2 nuclear localization. Our findings highlight the differential dependence of NDV on enzymes for one-carbon metabolism, and the unique mechanism of action of MTHFD2 in viral replication, thereby providing a novel target for antiviral or oncolytic virus therapy.

Chicken-derived MERTK protein inhibits Newcastle disease virus replication by increasing STAT1 phosphorylation in DF-1 cells

The receptor tyrosine kinases TYRO3, AXL, and MERTK (TAM) are transmembrane proteins associated with the regulation of the innate immune response. In this study, the role of the chicken-derived MERTK protein (chMertk) in the regulation of the type I interferon (IFN) signaling pathway and its antiviral effect were investigated in vitro. Newcastle disease (ND) caused by the Newcastle disease virus (NDV) is able to widely spread in chickens and give rise to massive losses in the poultry industry around the world. We found that the overexpression of the exogenous chMertk upregulated the STAT1 phosphorylation and the expression of IFN-stimulated gene IFITM3 and significantly reduced the NDV titer (p < 0.05). A mutation assay showed that three tyrosine residues (Y739, Y743, and Y744) in chMertk promoted STAT1 phosphorylation and inhibited NDV replication. However, the chicken-derived E3 ubiquitin ligase CBL significantly negatively regulated chMertk expression, thus attenuating STAT1 phosphorylation. chMertk function was restored by the ubiquitin-proteasome inhibitor MG132, demonstrating that chMertk was controlled by Casitas B-lineage proto-oncogene (CBL) ubiquitination and degradation. Together, these results suggested that chMertk participated in regulating the immune responses to NDV infection, and that CBL significantly downregulated the expression of chMertk through its ubiquitination and degradation, to maintain cellular homeostasis. Overall, our study provided new insights into the role of chMertk in regulating the innate immune response and its anti-NDV activity.

Mutation of Basic Residues R283, R286, and K288 in the Matrix Protein of Newcastle Disease Virus Attenuates Viral Replication and Pathogenicity.

The matrix (M) protein of Newcastle disease virus (NDV) contains large numbers of unevenly distributed basic residues, but the precise function of most basic residues in the M protein remains enigmatic. We previously demonstrated that the C-terminus (aa 264-313) of M protein interacted with the extra-terminal (ET) domain of chicken bromodomain-containing protein 2 (chBRD2), which promoted NDV replication by downregulating chBRD2 expression and facilitating viral RNA synthesis and transcription. However, the key amino acid sites determining M's interaction with chBRD2/ET and their roles in the replication and pathogenicity of NDV are not known. In this study, three basic residues-R283, R286, and K288-in the NDV M protein were verified to be responsible for its interaction with chBRD2/ET. In addition, mutation of these basic residues (R283A/R286A/K288A) in the M protein changed its electrostatic pattern and abrogated the decreased expression of endogenic chBRD2. Moreover, a recombinant virus harboring these mutations resulted in a pathotype change of NDV and attenuated viral replication and pathogenicity in chickens due to the decreased viral RNA synthesis and transcription. Our findings therefore provide a better understanding of the crucial biological functions of M's basic residues and also aid in understanding the poorly understood pathogenesis of NDV.

A novel short transcript isoform of chicken IRF7 negatively regulates interferon-β production

Type I interferon (IFN-I) provides an important first line to protect avian species against pathogens invasion. IFN regulatory factor 7 (IRF7) has been identified as the most important regulator for both DNA and RNA virus-induced IFN-I production in chickens. Although four splicing variants of IRF7 have been identified in mammals, it is still unclear whether alternative splicing patterns and the function of IRF7 isoform(s) exist in chickens. In this study, we reported a novel short transcript isoform of chicken IRF7 (chIRF7), termed chIRF7-iso, which contained an intact N-terminal DNA-binding domain (DBD) and 14 amino acids different from chIRF7 in the C-terminal. Overexpression of chIRF7 in chicken leghorn male hepatocellular (LMH) cells activated the IFN-β promoter and significantly inhibited Newcastle disease virus (NDV) and fowl adenovirus serotype 4 (FAdV-4) replication. Conversely, overexpression of chIRF7-iso blocked the IFN-β promoter activity and was favorable for NDV and FAdV-4 replication in vitro. Collectively, our results confirm that a novel chIRF7 isoform-mediated negative regulates IFN-β production, which will contribute to understanding the role of chIRF7 in innate antiviral response in chicken.

RNA-seq and LC-MS/MS analysis of antiviral effects mediated by cold stress and stress hormone corticosterone in chicken DF-1 cells

Cold stress in poultry is a global problem that causes huge economic losses and threatens the health and welfare of poultry. However, knowledge of chicken responses to virus infection under cold stress is limited. The purpose of this research was to investigate the effects of cold stress on gene expression and viral replication in chicken DF-1 cells in hypothermia. In addition, the characterization of circulating steroid hormone profiles in the plasma of chickens under cold stress was analyzed by liquid chromatography-tandem mass spectrometry. Herein, we performed RNA sequencing to obtain DF-1 cell transcriptional profiles under cold stress. A total of 9499 differentially expressed genes (DEGs) were identified in DF-1 cells. Overexpressed DEGs were related to the proteasome, cell cycle, spliceosome, ribosome biogenesis, and mammalian target of rapamycin (mTOR). Down-regulated DEGs were related to ribosomes, oxidative phosphorylation, apoptosis, and the p53 signaling pathway. Gene set enrichment analysis showed that the DEGs mainly affect host ribosome translation and mitochondrial respiratory electron transport. The principal steroid hormone alterations in chickens subjected to cold stress included dihydrotestosterone, testosterone, β-sitosterol, androstenedione, 7a,27-dihydroxycholesterol,7-ketocholesterol, and desmosterol, which are associated with endocrine resistance, ovarian steroidogenesis, and steroid hormone biosynthesis. In addition, Infectious bronchitis virus (IBV), Newcastle disease virus (NDV), and Influenza A (H9N2) Virus replication in DF-1 cells is significantly inhibited by cold stress. Moreover, the plasma concentrations of corticosterone, an important stress hormone in poultry, were significantly elevated in chickens subjected to cold stress, and we found that IBV and vesicular stomatitis virus (VSV) replication were strongly inhibited in DF-1 cells pretreated with CORT, but NDV and H9N2 replication were unaffected. In conclusion, in response to cold stress, the translation efficiency and mitochondrial respiratory chain are temporarily weakened in DF-1 cells, which affects virus replication. Chickens may regulate aromatase deficiency, androstenedione metabolism, androgen and estrogen metabolism, and 17-beta hydroxysteroid dehydrogenase III deficiency through steroid hormones in response to cold stress. This study provides valuable insights into the molecular regulatory mechanisms of poultry under cold stress and may support further research on the intrinsic link between steroid hormones and virus replication under stress.

SLC1A3 facilitates Newcastle disease virus replication by regulating glutamine catabolism

ABSTRACT 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.

Screening and identification of nucleocapsid protein-nanobodies that inhibited Newcastle disease virus replication in DF-1 cells.

Newcastle disease (ND) is an acute and highly contagious infectious disease found in poultry. Although commercial ND virus (NDV) vaccines are universally used, some case reports persistently documented vaccination failure. Therefore, novel strategies are still required to control the occurrence of the disease in chickens. Recently, nanobodies (Nbs), which have the advantages of small molecular weight and low production costs, have been shown to be promising therapeutics against viral infection. In the present study, a total of 16 Nbs against NDV nucleocapsid protein (NP) were screened from two libraries against NDV using phage display technology. Of the 16 screened Nbs, eight were prevented from binding to NDV NP protein through administering positive chicken sera for anti-NDV antibodies, indicating that the epitopes recognized by these eight Nbs were able to induce the immune response after the chickens were infected with NDV stock. Subsequently, transfection assay, construction of recombinant DF-1 cells capable of expressing different nanobodies and viral inhibition assay were used to screen the nanobodies inhibiting NDV replication. The results demonstrated that Nb18, Nb30, and Nb88 significantly inhibited the replication of Class I and different genotypes of Class II NDV strains in DF-1 cells when they were expressed in the cytoplasm. Collectively, these nanobodies provided new tools for researching the functions of NDV NP protein and may be used as a novel strategy for designing drugs against NDV infection in chickens.

Goose IRF7 is involved in antivirus innate immunity by mediating IFN activation

Interferon regulatory factor (IRF) 3 and IRF7 are the most important nuclear transcription factors regulating type-I interferon (IFN) production in mammals and the IRF3 is missing in birds. Our previous study found that IFR7 is the most important IRF in chickens, however, its functions in geese remain unknown. We cloned goose IRF7 (GoIRF7) and conducted bioinformatics analyses to compare the chromosomal location and protein homology of IRF7 in different species. Overexpression of GoIRF7 in DF-1 cells induced the activation of IFN-β, and this activation correlated positively with the dosage of transfected plasmids. Overexpression of GoIRF7 in goose embryonic fibroblasts (GEFs) induced the expression of IFNs, proinflammatory cytokines, and IFN-stimulated genes (ISGs); it also inhibited replication of Newcastle disease virus (NDV) and vesicular stomatitis virus (VSV). Our results suggest that GoIRF7 is an important regulator of IFNs, proinflammatory cytokines, and ISGs and plays a role in antiviral innate immunity in geese.

Chicken ISG12(2) attenuates Newcastle disease virus and enhances the efficiency of Newcastle disease vaccine via activating immune pathways.

Low virulence and strong immunogenicity are quite important for Newcastle disease virus (NDV) producing Newcastle disease (ND) living-attenuated vaccine. However, immunogenicity of NDV positively correlates to its virulence. Usually, the velogenic NDV induces stronger immune responses of poultry than the lentogenic strain, but virulent NDV poses a risk for chicken. In this study, we identified the chicken interferon (IFN)-stimulated gene 12-2 (ISG12(2)) not only attenuated NDV, but also increased immunogenicity of ND vaccine strain. First, we found that NDV infection or IFNs stimulation induced expression of chicken ISG12(2) that reinforced expression of IFNs. Over-expression or knock-down proved that chicken ISG12(2) inhibited NDV replication. Then, recombinant NDV LaSota strains (rLaSota/Fmut/ISG12(2) and rLaSota/ISG12(2)), expressing ISG12(2), were rescued. Pathogenicity tests showed that ISG12(2) expression attenuated NDV. RNA-seq or RT-qPCR demonstrated that, comparing to rLaSota/Fmut and rLaSota, rLaSota/Fmut/ISG12(2) and rLaSota/ISG12(2) induced hosts to produce cytokines enriching in innate and adaptive immune pathways in vitro and in vivo. Finally, we showed that rLaSota/ISG12(2) vaccination improved immune condition of chicken to quickly respond NDV infection and then enhance protection. These results suggest that chicken ISG12(2) is a potential novel molecular adjuvant to regulate immune responses, which decrease virulence and increase immunogenicity of NDV. The chicken ISG12(2) may contribute to the development of high efficient poultry vaccine.

Lentogenic NDV V protein inhibits IFN responses and represses cell apoptosis

The V protein of Newcastle disease virus (NDV) has been shown to inhibit the secretion of interferon (IFN) during infection, which is responsible for the promotion of NDV pathogenicity. However, the ability of the V protein to suppress host innate immunity is not well understood. In this study, we explored the function of V protein and its relationship with virulence by generating V protein-inserted recombinant (r) NDVs. Using rNDVs as a model, we examined the efficiency of infection, IFN responses, and apoptosis of host cells during infection. We found that viral propagation occurred smoothly when V protein from lentogenic NDV is inserted instead of the V protein from the velogenic strain. The infection of lentogenic V protein-inserted rNDV induced less expression of IFNs and downstream antiviral proteins via efficient degradation of p-STAT1 and MDA5. Moreover, velogenic V protein triggered a higher apoptosis rate during infection thereby restricting the replication of NDV. Conversely, lentogenic V protein inhibits IFN responses efficiently and induces less apoptosis compared to the velogenic strain. Our findings provide a novel understanding of the role of V protein in NDV pathogenicity.