Newcastle Disease Virus Infection Research Articles

Duration of Highly Pathogenic Avian Influenza Virus and Newcastle Disease Virus Infectivity in Dried Ornithologic Study Skins.

Ornithologic study skins are specimens of avian skins that have been preserved by drying after removing the viscera and muscle. Because of the high value of study skins for scientific studies, specimens are shared among researchers. There is concern that study skins might be contaminated with high-consequence diseases such as highly pathogenic avian influenza virus (HPAIV) or Newcastle disease virus (NDV). To mitigate risk, thermal or chemical treatment of study skins may be required before transfer; however, such treatments might damage the specimens. Therefore, a study was conducted to evaluate the duration of infectivity of HPAIV and NDV in study skins prepared from infected chickens (Gallus gallus). Study skins were prepared from 10 chickens infected with each virus. Skin and feather pulp samples were taken at the time of study skin preparation to establish starting titers. Mean starting titers in skins were 4.2 log10 and 5.1 log10 50% egg infectious doses (EID50) for HPAIV and NDV groups respectively, and were 6.7 log10 EID50 for HPAIV, and 6.4 log10 EID50 for NDV in feather pulp. Samples were collected at 2 and 4 wk of drying to quantify viable virus. At 2 wk, fewer samples had detectable virus and mean titers were 1.8 log10 (skin) and 2.1 log10 (feathers) EID50 for HPAIV, and 1.7 log10 (skin) and 3.5 log10 (feathers) EID50 for NDV. At 4 wk viable virus could not be detected in either tissue type.

The haemagglutinin–neuraminidase protein of velogenic Newcastle disease virus enhances viral infection through NF-κB-mediated programmed cell death

The haemagglutinin–neuraminidase (HN) protein, a vital membrane glycoprotein, plays a pivotal role in the pathogenesis of Newcastle disease virus (NDV). Previously, we demonstrated that a mutation in the HN protein is essential for the enhanced virulence of JS/7/05/Ch, a velogenic variant NDV strain originating from the mesogenic vaccine strain Mukteswar. Here, we explored the effects of the HN protein during viral infection in vitro using three viruses: JS/7/05/Ch, Mukteswar, and an HN-replacement chimeric NDV, JS/MukHN. Through microscopic observation, CCK-8, and LDH release assays, we demonstrated that compared with Mukteswar and JS/MukHN, JS/7/05/Ch intensified the cellular damage and mortality attributed to the mutant HN protein. Furthermore, JS/7/05/Ch induced greater levels of apoptosis, as evidenced by the activation of caspase-3/8/9. Moreover, JS/7/05/Ch promoted autophagy, leading to increased autophagosome formation and autophagic flux. Subsequent pharmacological experiments revealed that inhibition of apoptosis and autophagy significantly impacted virus replication and cell viability in the JS/7/05/Ch-infected group, whereas less significant effects were observed in the other two infected groups. Notably, the mutant HN protein enhanced JS/7/05/Ch-induced apoptosis and autophagy by suppressing NF-κB activation, while it mitigated the effects of NF-κB on NDV infection. Overall, our study offers novel insights into the mechanisms underlying the increased virulence of NDV and serves as a reference for the development of vaccines.

The Ca2+-dependent phosphatase calcineurin dephosphorylates TBK1 to suppress antiviral innate immunity.

Tumor necrosis factor receptor-associated factor family member-associated NF-κB activator-binding kinase 1 (TBK1) plays a key role in the induction of the type 1 interferon (IFN-I) response, which is an important component of innate antiviral defense. Viruses target calcium (Ca2+) signaling networks, which participate in the regulation of the viral life cycle, as well as mediate the host antiviral response. Although many studies have focused on the role of Ca2+ signaling in the regulation of IFN-I, the relationship between Ca2+ and TBK1 in different infection models requires further elucidation. Here, we examined the effects of the Newcastle disease virus (NDV)-induced increase in intracellular Ca2+ levels on the suppression of host antiviral responses. We demonstrated that intracellular Ca2+ increased significantly during NDV infection, leading to impaired IFN-I production and antiviral immunity through the activation of calcineurin (CaN). Depletion of Ca²+ was found to lead to a significant increase in virus-induced IFN-I production resulting in the inhibition of viral replication. Mechanistically, the accumulation of Ca2+ in response to viral infection increases the phosphatase activity of CaN, which in turn dephosphorylates and inactivates TBK1 in a Ca2+-dependent manner. Furthermore, the inhibition of CaN on viral replication was counteracted in TBK1 knockout cells. Together, our data demonstrate that NDV hijacks Ca2+ signaling networks to negatively regulate innate immunity via the CaN-TBK1 signaling axis. Thus, our findings not only identify the mechanism by which viruses exploit Ca2+ signaling to evade the host antiviral response but also, more importantly, highlight the potential role of Ca2+ homeostasis in the viral innate immune response.IMPORTANCEViral infections disrupt intracellular Ca2+ homeostasis, which affects the regulation of various host processes to create conditions that are conducive for their own proliferation, including the host immune response. The mechanism by which viruses trigger TBK1 activation and IFN-I induction through viral pathogen-associated molecular patterns has been well defined. However, the effects of virus-mediated Ca2+ imbalance on the IFN-I pathway requires further elucidation, especially with respect to TBK1 activation. Herein, we report that NDV infection causes an increase in intracellular free Ca2+ that leads to activation of the serine/threonine phosphatase CaN, which subsequently dephosphorylates TBK1 and negatively regulates IFN-I production. Furthermore, depletion of Ca2+ or inhibition of CaN activity exerts antiviral effects by promoting the production of IFN-I and inhibiting viral replication. Thus, our results reveal the potential role of Ca2+ in the innate immune response to viruses and provide a theoretical reference for the treatment of viral infectious diseases.

Abstract 6656: Revolutionizing solid tumor treatment: Viral protein-targeted CAR-T with oncolytic virus

Abstract Major challenges for CAR-T cell therapy in solid tumors include various limitations due to the issue of appropriate antigen selection, tumor heterogeneity, and the immune-suppressive tumor microenvironment (TME). To overcome these hurdles, we’ve developed a novel therapy, CLM-202 which is a combination therapy of oncolytic Newcastle disease virus (NDV) and NDV F protein-targeted CAR-T cell therapy (NDV F CAR-T). After NDV infects tumor cells, NDV envelope protein F is expressed on the tumor cell surface. NDV F CAR-T recognizes NDV F protein and kills the tumor cells. This approach can be applied to patients with various indications, regardless of the presence of the target protein. Using NDV to deliver antigens minimizes the risk of tumor recurrence due to tumor heterogeneity. Furthermore, it mitigates off-target toxicity by targeting oncolytic virus-derived proteins, not human proteins. Oncolytic NDV also triggers a potent immune response within the tumor microenvironment, thereby altering the immunosuppressive conditions of the tumor and improving CAR-T efficacy. Therefore, CLM-202 can be a multipurpose solution for solid tumors. We have developed a second-generation CAR-T cell therapy, NDV F CAR-T, which can be used in combination with an oncolytic virus known as NDV. NDV F CAR-T is designed to specifically target the F protein expressed when cells are infected by NDV. It incorporates a single-chain variable fragment (scFv) derived from an antibody that can bind specifically to the F protein of NDV-infected cells. To assess the activity of NDV F CAR-T in tumor cells against a variety of solid tumors as indications, we conducted several experiments. We initially confirmed the expression of the F protein in NDV-infected tumor cells. We also conducted cytotoxicity tests to confirm NDV F CAR-T's ability to induce death in NDV-infected tumor cell lines. Furthermore, to assess the efficacy of NDV F CAR-T in an in vivo model, we established an orthotopic mouse model. In this model, we administered NDV via intratumoral injection, followed by the single-dose administration of NDV F CAR-T to perform the efficacy test. We have confirmed that NDV infects all solid tumor cell lines representing different indications and induces the expression of the F protein in infected tumor cells. Furthermore, we have observed that NDV infection in these diverse tumor cells results in robust tumor cell-killing activity by NDV F CAR-T and the subsequent activation of CAR-T. Moreover, in an orthotopic mouse model, we have demonstrated that the administration of NDV followed by NDV F CAR-T induces potent tumor growth inhibition. In summary, our research proposes a novel therapeutic strategy, CLM-202 that utilizes NDV F CAR-T and NDV to overcome the limitations of CAR-T cell therapy in solid tumors, including tumor heterogeneity and immune-suppressive tumor microenvironments. This approach aims to effectively treat a wide range of indications. Citation Format: Soyoung Choi, Kiwan Kim, Sangeun Lee, Jeongeun Choi, Song-Jae Lee, Yong Gu Lee, Jeonghun Kim, Hoyoung Lee, Kisoon Kim, Man-Seong Park, Seong-Won Song. Revolutionizing solid tumor treatment: Viral protein-targeted CAR-T with oncolytic virus [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6656.

Spatiotemporal Patterns and Risk Factors for Newcastle Disease Virus among Chickens in a Tanzania Live Bird Market

Village poultry plays a vital role in providing essential nutrition and income for rural communities in Africa. In this context, poultry are often traded through live bird markets (LBMs), which serve as central trading hubs where producers connect with traders and consumers, facilitating the flow of poultry products along the value chain. While they serve as important trading hubs, these markets create an environment where avian pathogens, like Newcastle disease virus (NDV) and avian influenza virus, can easily emerge and spread. Improving our understanding of the epidemiology of NDV in LBMs is important for assessing disease risks and identifying factors that contribute to its persistence. Local chickens at the Mawenzi LBM in Morogoro municipality were surveyed for NDV presence, its temporal and spatial distribution, and risk factors for NDV infection. Twenty-three percent of 659 local chickens sampled over a 1-year period were positive for NDV based on PCR. Increased odds of NDV infection were identified in chickens that had been in the market for 2 or more days prior to sampling and during the period extending from August through October. Four significant spatiotemporal clusters of NDV-positive chickens encompassing 13 villages were detected between August and October 2020, illustrating geographic hotspots of infection when NDV was most prevalent. Similar to the other LBMs, this market had enclosures with high densities of birds of mixed species, limited biosecurity, and the presence of birds with observable illness. Bird traders who source the chickens from the villages, described long transit times in mixed enclosures with limited sanitation practices and without consideration of sick birds or vaccination status prior to arriving at the LBM. This study highlights the need to invest in improvements to infrastructure and biosecurity for LBMs as well as training opportunities for increasing traders’ knowledge on hygiene and sanitation practices, animal welfare, and poultry biosecurity measures.

Newcastle disease virus suppresses antigen presentation via inhibiting IL-12 expression in dendritic cells.

As a potential vectored vaccine, Newcastle disease virus (NDV) has been subject to various studies for vaccine development, while relatively little research has outlined the immunomodulatory effect of the virus in antigen presentation. To elucidate the key inhibitory factor in regulating the interaction of infected dendritic cells (DCs) and T cells, DCs were pretreated with the NDV vaccine strain LaSota as an inhibitor and stimulated with lipopolysaccharide (LPS) for further detection by enzyme-linked immunosorbent assay (ELISA), flow cytometry, immunoblotting, and quantitative real-time polymerase chain reaction (qRT-PCR). The results revealed that NDV infection resulted in the inhibition of interleukin (IL)-12p40 in DCs through a p38 mitogen-activated protein kinase (MAPK)‍-dependent manner, thus inhibiting the synthesis of IL-12p70, leading to the reduction in T cell proliferation and the secretion of interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), and IL-6 induced by DCs. Consequently, downregulated cytokines accelerated the infection and viral transmission from DCs to T cells. Furthermore, several other strains of NDV also exhibited inhibitory activity. The current study reveals that NDV can modulate the intensity of the innate‍‒‍adaptive immune cell crosstalk critically toward viral invasion improvement, highlighting a novel mechanism of virus-induced immunosuppression and providing new perspectives on the improvement of NDV-vectored vaccine.

Genomic Regions and Candidate Genes Affecting Response to Heat Stress with Newcastle Virus Infection in Commercial Layer Chicks Using Chicken 600K Single Nucleotide Polymorphism Array.

Heat stress results in significant economic losses to the poultry industry. Genetics plays an important role in chickens adapting to the warm environment. Physiological parameters such as hematochemical parameters change in response to heat stress in chickens. To explore the genetics of heat stress resilience in chickens, a genome-wide association study (GWAS) was conducted using Hy-Line Brown layer chicks subjected to either high ambient temperature or combined high temperature and Newcastle disease virus infection. Hematochemical parameters were measured during three treatment phases: acute heat stress, chronic heat stress, and chronic heat stress combined with NDV infection. Significant changes in blood parameters were recorded for 11 parameters (sodium (Na+, potassium (K+), ionized calcium (iCa2+), glucose (Glu), pH, carbon dioxide partial pressure (PCO2), oxygen partial pressure (PO2), total carbon dioxide (TCO2), bicarbonate (HCO3), base excess (BE), and oxygen saturation (sO2)) across the three treatments. The GWAS revealed 39 significant SNPs (p < 0.05) for seven parameters, located on Gallus gallus chromosomes (GGA) 1, 3, 4, 6, 11, and 12. The significant genomic regions were further investigated to examine if the genes within the regions were associated with the corresponding traits under heat stress. A candidate gene list including genes in the identified genomic regions that were also differentially expressed in chicken tissues under heat stress was generated. Understanding the correlation between genetic variants and resilience to heat stress is an important step towards improving heat tolerance in poultry.

Newcastle disease virus regulates its replication by instigating oxidative stress-driven Sirtuin 7 production.

Reactive oxygen species (ROS) accumulation inside the cells instigates oxidative stress, activating stress-responsive genes. The viral strategies for promoting stressful conditions and utilizing the induced host proteins to enhance their replication remain elusive. The present work investigates the impact of oxidative stress responses on Newcastle disease virus (NDV) pathogenesis. Here, we show that the progression of NDV infection varies with intracellular ROS levels. Additionally, the results demonstrate that NDV infection modulates the expression of oxidative stress-responsive genes, majorly sirtuin 7 (SIRT7), a NAD+-dependent deacetylase. The modulation of SIRT7 protein, both through overexpression and knockdown, significantly impacts the replication dynamics of NDV in DF-1 cells. The activation of SIRT7 is found to be associated with the positive regulation of cellular protein deacetylation. Lastly, the results suggested that NDV-driven SIRT7 alters NAD+ metabolism in vitro and in ovo. We concluded that the elevated expression of NDV-mediated SIRT7 protein with enhanced activity metabolizes the NAD+ to deacetylase the host proteins, thus contributing to high virus replication.

The HN protein of Newcastle disease virus induces cell apoptosis through the induction of lysosomal membrane permeabilization.

Lysosomes are acidic organelles that mediate the degradation and recycling of cellular waste materials. Damage to lysosomes can cause lysosomal membrane permeabilization (LMP) and trigger different types of cell death, including apoptosis. Newcastle disease virus (NDV) can naturally infect most birds. Additionally, it serves as a promising oncolytic virus known for its effective infection of tumor cells and induction of intensive apoptotic responses. However, the involvement of lysosomes in NDV-induced apoptosis remains poorly understood. Here, we demonstrate that NDV infection profoundly triggers LMP, leading to the translocation of cathepsin B and D and subsequent mitochondria-dependent apoptosis in various tumor and avian cells. Notably, the released cathepsin B and D exacerbate NDV-induced LMP by inducing the generation of reactive oxygen species. Additionally, we uncover that the viral Hemagglutinin neuraminidase (HN) protein induces the deglycosylation and degradation of lysosome-associated membrane protein 1 (LAMP1) and LAMP2 dependent on its sialidase activity, which finally contributes to NDV-induced LMP and cellular apoptosis. Overall, our findings elucidate the role of LMP in NDV-induced cell apoptosis and provide novel insights into the function of HN during NDV-induced LMP, which provide innovative approaches for the development of NDV-based oncolytic agents.

Assessment of implementation of biosecurity measures and its impacts on Newcastle disease in some poultry farms and live bird markets in Sokoto State, Nigeria

There is considerable global concern over the outbreaks of Newcastle disease (ND) affecting domestic poultry flocks around the world including Nigeria. There have been little investigations on the biosecurity measures taken by poultry stakeholders in relation to the rate of ND in Sokoto State, Nigeria. Thus, this study evaluated the biosecurity practices in commercial poultry farms and live bird markets in Sokoto State, Nigeria. Data were collected through questionnaire administered to commercial farmers, flock owners at live bird markets as well as other poultry value chain intermediaries. A traffic light system model was used to test for relative risks based on the biosecurity measures put in place at the farm and live bird markets. The result indicates that both commercial farms and live bird markets were at high risk (56.9% and 77.3% respectively) of ND virus infection. The high risk practices identified include unsafe disposal of poultry carcasses (50%), locating farms on high ways (75%), absence of high pressure sprayer at gate (100%), presence of water body (37.5%), raising different species of birds (25% for farms and 100% for live bird markets), presence of multi age groups (100%), feed spillage (100%), fly and rodent infestation (100%), lack of personal protective equipment (50% for farms and 100% for live bird markets), absence of changing room (87.5%), poor handling of sick birds (75% for farms and 100% for live bird markets), inadequate washing and disinfection of hands and equipment (100%). Using chi square, presence of fence, washing and disinfection of pen, washing and disinfection of equipment, always allowing visitors to enter farm, borrowing of equipment, sale of runt and sick birds, isolation of sick/new birds, culling of diseased birds, safe and clean store for feed, observing signs of ND in the last 3 months showed significant statistical difference (P&lt;0.05) between live bird markets and poultry houses. Thus, that may contribute to the risk of environmental contamination and disease transmission. Outreach and biosecurity awareness raising initiatives should be specifically developed for all the poultry value chain stakeholders with the objective of improving general poultry management and thus preventing ND and other poultry diseases.

Phylogenetic Analysis of Newcastle Disease Virus Isolated from Poultry in Live Bird Markets and Wild Waterfowl in Zambia.

Poultry production is essential to the economy and livelihood of many rural Zambian households. However, the industry is threatened by infectious diseases, particularly Newcastle disease virus (NDV) infection. Therefore, this study employed next-generation sequencing to characterise six NDV isolates from poultry in Zambia's live bird markets (LBMs) and wild waterfowl. Four NDV isolates were detected from 410 faecal samples collected from chickens in LBMs in Lusaka and two from 2851 wild birds from Lochinvar National Park. Phylogenetic analysis revealed that the four NDVs from LBM clustered in genotype VII and sub-genotype VII.2 were closely related to viruses previously isolated in Zambia and other Southern African countries, suggesting possible local and regional transboundary circulation of the virus. In contrast, the two isolates from wild birds belonged to class I viruses, genotype 1, and were closely related to isolates from Europe and Asia, suggesting the possible introduction of these viruses from Eurasia, likely through wild bird migration. The fusion gene cleavage site motif for all LBM-associated isolates was 112RRQKR|F117, indicating that the viruses are virulent, while the isolates from wild waterfowl had the typical 112ERQER|L117 avirulent motif. This study demonstrates the circulation of virulent NDV strains in LBMs and has, for the first time, characterised NDV from wild birds in Zambia. The study further provides the first whole genomes of NDV sub-genotype VII.2 and genotype 1 from Zambia and stresses the importance of surveillance and molecular analysis for monitoring the circulation of NDV genotypes and viral evolution.

Newcastle disease virus infection remodels plasma phospholipid metabolism in chickens

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 invivo. 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.

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.

NDV inhibited IFN-β secretion through impeding CHCHD10-mediated mitochondrial fusion to promote viral proliferation

Newcastle disease virus (NDV) is an RNA virus that can promote its own replication through the inhibition of cellular mitochondrial fusion. The proteins involved in mitochondrial fusion, namely mitofusin 1 (Mfn1) and optic atrophy 1 (OPA1) are associated with interferon-beta (IFN-β) secretion during NDV infection. However, the precise mechanism by which NDV modulates the Mfn1-mediated or OPA1-mediated fusion of mitochondria, thereby impacting IFN-β, remains elusive. This study revealed that the downregulation of the mitochondrial protein known as coiled-coil-helix-coiled-coil-helix domain containing 10 (CHCHD10) exerts a negative regulatory effect on OPA1 and Mfn1 in human lung adenocarcinoma (A549) cells during the late stage of NDV infection. This reduction in CHCHD10 expression impeded cellular mitochondrial fusion, subsequently leading to a decline in the activation of interferon regulatory factor 3 (IRF3) and nuclear factor kappa B (NF-κB), ultimately resulting in diminished secretion of IFN-β. In contrast, the overexpression of CHCHD10 alleviated infection-induced detrimental effect in mitochondrial fusion, thereby impeding viral proliferation. In summary, NDV enhances its replication by inhibiting the CHCHD10 protein, which impedes mitochondrial fusion and suppresses IFN-β production through the activation of IRF3 and NF-κB.

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.

Protective effects of a novel chimeric virus–like particle vaccine against virulent NDV and IBDV challenge

Newcastle disease (ND) and infectious bursal disease (IBD) pose significant threats to the chicken industry, causing substantial economic losses. Currently, immunization through vaccination is the most effective strategy to prevent ND and IBD but currently used traditional vaccines, including inactivated or attenuated vaccines, face challenges in achieving a balance between immunogenicity and safety. To develop a green and efficient novel vaccine for ND and IBD, we developed a bivalent chimeric virus-like particle vaccine (ND-IBD cVLPs) displaying the ND virus (NDV) HN protein and the IBD virus (IBDV) VP2 protein based on the ND VLPs carrier platform and insect baculovirus expression system. This study aimed to evaluate the immunogenicity and protective efficacy of ND-IBD cVLPs in specific pathogen-free chickens. Chickens were immunized with 50 µg of purified ND-IBD cVLPs at 7 days old, boosted at 21 days old, and challenged at 42 days old. The results demonstrated that ND-IBD cVLPs stimulated highly effective hemagglutination inhibition antibody levels against NDV HN protein and enzyme-linked immunosorbent assay antibody levels against the IBDV VP2 protein. Furthermore, ND-IBD cVLPs provided complete protection against virulent NDV and IBDV challenges and mitigated pathological damage to the lung caused by NDV infection and the bursa of Fabricius caused by IBDV infection. These findings suggest that ND-IBD cVLPs hold promise as a safe and efficient novel vaccine candidate for the effective prevention of ND and IBD, extending the development of a foreign protein delivery platform of ND VLPs.

Cellular vimentin regulates the infectivity of Newcastle disease virus through targeting of the HN protein

The haemagglutinin-neuraminidase (HN) protein plays a crucial role in the infectivity and virulence of Newcastle disease virus (NDV). In a previous study, the mutant HN protein was identified as a crucial virulence factor for the velogenic variant NDV strain JS/7/05/Ch, which evolved from the prototypic vaccine strain Mukteswar. Furthermore, macrophages are the main susceptible target cells of NDV. However, the possible involvement of cellular molecules in viral infectivity remains unclear. Herein, we elucidate the crucial role of vimentin, an intermediate filament protein, in regulating NDV infectivity through targeting of the HN protein. Using LC‒MS/MS mass spectrometry and coimmunoprecipitation assays, we identified vimentin as a host protein that differentially interacted with prototypic and mutant HN proteins. Further analysis revealed that the variant NDV strain induced more significant rearrangement of vimentin fibres compared to the prototypic NDV strain and showed an interdependence between vimentin rearrangement and virus replication. Notably, these mutual influences were pronounced in HD11 chicken macrophages. Moreover, vimentin was required for multiple infection processes of the variant NDV strain in HD11 cells, including viral internalization, fusion, and release, while it was not necessary for those of the prototypic NDV strain. Collectively, these findings underscore the pivotal role of vimentin in NDV infection through targeting of the HN protein, providing novel targets for antiviral treatment strategies for NDV.

The tumor suppressive effect and apoptotic mechanism of TRAIL gene-containing recombinant NDV in TRAIL-resistant colorectal cancer HT-29 cells and TRAIL-nonresistant HCT116 cells, with each cell bearing a mouse model.

TRAIL is an anticancer drug that induces cancer cell apoptosis by interacting with death receptors (DRs). However, owing to low cell-surface expression of DRs, certain colorectal cancer (CRC) cells resist TRAIL-induced apoptosis. Newcastle disease virus (NDV) infection can elevate DR protein expression in cancer cells, potentially influencing their TRAIL sensitivity. However, the precise mechanism by which NDV infection modulates DR expression and impacts TRAIL sensitivity in cancer cells remains unknown. Herein, we developed nonpathogenic NDV VG/GA strain-based recombinant NDV (rNDV) and TRAIL gene-containing rNDV (rNDV-TRAIL). We observed that viral infections lead to increased DR and TRAIL expressions and activate signaling proteins involved in intrinsic and extrinsic apoptosis pathways. Experiments were conducted invitro using TRAIL-resistant CRC cells (HT-29) and nonresistant CRC cells (HCT116) and invivo using relevant mouse models. rNDV-TRAIL was found to exhibit better apoptotic efficacy than rNDV in CRC cells. Notably, rNDV-TRAIL had the stronger cancer cell-killing effect in TRAIL-resistant CRC cells. Western blot analyses showed that both rNDV and rNDV-TRAIL infections activate signaling proteins involved in the intrinsic and extrinsic apoptotic pathways. Notably, rNDV-TRAIL promotes concurrent intrinsic and extrinsic signal transduction in both HCT-116 and HT-29 cells. Therefore, rNDV-TRAIL infection effectively enhances DR expression in DR-depressed HT-29 cells. Moreover, the TRAIL protein expressed by rNDV-TRAIL effectively interacts with DR, leading to enhanced apoptosis in TRAIL-resistant HT-29 cells. Therefore, rNDV-TRAIL has potential as a promising therapeutic approach for treating TRAIL-resistant cancers.

Circular RNAs are associated with the resistance to Newcastle disease virus infection in duck cells.

Newcastle disease virus (NDV) is prevalent worldwide with an extensive host range. Among birds infected with velogenic NDV strains, chickens experience high pathogenicity and mortality, whereas ducks mostly experience mild symptoms or are asymptomatic. Ducks have a unique, innate immune system hypothesized to induce antiviral responses. Circular RNAs (circRNAs) are among the most abundant and conserved eukaryotic transcripts. These participate in innate immunity and host antiviral response progression. In this study, circRNA expression profile differences post-NDV infection in duck embryo fibroblast (DEF) cells were analyzed using circRNA transcriptome sequencing. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were used to reveal significant enrichment of differentially expressed (DE) circRNAs. The circRNA-miRNA-mRNA interaction networks were used to predict the related functions of circRNAs. Moreover, circ-FBXW7 was selected to determine its effect on NDV infection in DEFs. NDV infection altered circRNA expression profiles in DEF cells, and 57 significantly differentially expressed circRNAs were identified post-NDV infection. DEF responded to NDV by forming circRNAs to regulate apoptosis-, cell growth-, and protein degradation-related pathways via GO and KEGG enrichment analyses. circRNA-miRNA-mRNA interaction networks demonstrated that DEF cells combat NDV infection by regulating cellular pathways or apoptosis through circRNA-targeted mRNAs and miRNAs. circ-FBXW7 overexpression and knockdown inhibited and promoted viral replication, respectively. DEF cells mainly regulated cell cycle alterations or altered cellular sensing to combat NDV infection. These results demonstrate that DEF cells exert antiviral responses by forming circRNAs, providing novel insights into waterfowl antiviral responses.