Velogenic Newcastle Disease Virus Research Articles

Isolation and molecular characterization of velogenic Newcastle disease virus genotype VII.1.1 from commercial broilers and wild birds in Matrouh governorate, Egypt

Isolation and molecular characterization of velogenic Newcastle disease virus genotype VII.1.1 from commercial broilers and wild birds in Matrouh governorate, Egypt

Ultrastructure Microscopic Characterization of Apoptosis in Myelomoncytic Leukemia Cell Line Treated with Velogenic Viscerotropic Newcastle Disease Virus Strain AF 2240.

Leukemia is a group of hematologic malignancies in the bonemarrow that arise from the dysfunctional proliferation of developing leukocytes. It is classified as either acute or chronic based on the rapidity of proliferation and as myelocytic or lymphocytic based on the cell of origin. Newcastle Disease Virus (NDV) is an avian paramyxovirus, which has been demonstrated to possess significant oncolytic activity against mammalian cancers because its ability to kill tumor cells with limited toxicity to normal cells. In this study, the morphophical changes and apoptosis induction of WEHI 3B leukemia cell line treated with NDV strain AF2240 were studied by scanning electron microscopes and transmission electron microscopes techniques. Electron microscopy indicated that NDV strain AF 2240 significantly altered cell morphology and reduced cell viability. Furthermore, early apoptosis was observed 6 h post-inoculation by fluorescence microscope. Our results suggest that NDV has ability to induce significant apoptoic structural changes in WEHI 3B leukemia cell line. These findings provide new insights into the mechanism of action of NDV virotherapy and could lead to the development of more effective treatments for leukemia.

Experimental Velogenic Viscerotropic Newcastle Disease Virus Infection in Chickens Immunologically Impaired by Treatment with Cyclophosphamide

This study investigated whether lymphocytic depletion following chemical bursectomy influenced the severity of infection and development of lesions in chickens challenged with velogenic viscerotropic Newcastle disease virus (vvNDV). Cockerel chickens treated with cyclophosphamide on days 2, 3 and 4 post-hatch showed loss of weight, atrophy and lymphocytic depletion in the bursa of Fabricius and spleen. At 6 weeks of age, the chickens were assigned to four groups- Bursectomized intramuscularly vvNDV inoculated (BI), bursectomized uninfected (BU), non-bursectomized infected (NBI) and non-bursectomized uninfected (NBU) chickens. The BI and NBI chickens showed significant (P < .05) loss of weight than their uninfected controls. Depression, anorexia, greenish diarrhea, listlessness, tremor, and oculo-nasal discharges were observed in both infected groups, but were more severe and frequent in the NBI than in the BI chickens. Total mortalities were 100% and 95.5% for the NBI and BI chickens, respectively (P > .05). Lesions in both infected groups included atrophy of the bursa, spleen and thymus. Hemorrhages in the proventricular mucosa, intestines and cecal tonsils, as well as congestion and enlargement of the kidneys were significantly (P < .05) more severe and frequent in NBI than BI chickens. Histopathology showed necrosis and depletion of lymphocytes in the three lymphoid organs in both infected groups with more severity in the NBI than BI chickens. These results show that depletion of lymphocytes by treatment with cyclophosphamide may influence the severity of infection and development of lesions in vvNDV infection in cockerel chickens.

RT-PCR based detection and pathotypical characterization of Newcastle disease virus isolated from layer chickens in Mymensingh, Bangladesh

The Newcastle disease virus (NDV) poses a significant threat to commercial chicken operations and is prevalent in Bangladesh. This rapidly spreading viral illness has severe economic implications for the poultry industry globally. In Bangladesh, the velogenic strain of NDV is particularly widespread. This study utilized primers from the Fusion (F) and Matrix (M) genes along with haemagglutination (HA) testing and reverse transcription-polymerase chain reaction (RT-PCR) to isolate and identify NDV. Post-mortem examinations provided 12 samples (from brain, trachea, and proventriculus) taken from four birds suspected of having NDV from Trishal and Mymensingh Sadar. The samples were injected into 10-day-old embryonated chicken eggs using the chorioallantoic membrane technique. Embryos and allantoic fluid (AF) were collected at 48 and 96 hours post-infection. NDV-infected embryos showed edema and hemorrhage. Significant hemagglutination was observed in the allantoic fluid tested with HA. Through the HA test and lesion observation, four NDV isolates were identified. Pathotypic characterization of the field isolates using the Intracerebral Pathogenicity Index (ICPI) and Mean Death Time (MDT) indicated velogenic strains (ICPI: 1.5–2.0, MDT: <60). RT-PCR confirmed the NDV status of all four isolates using primers for partial amplification of the F gene (839 bp) and the common sequence of the ‘F-M’ genes (970 bp). This study demonstrates that RT-PCR is an effective molecular technique for the rapid and confirmatory identification of velogenic NDV strains, particularly prevalent in Mymensingh at the field level.

Isolation and Genetic Characterization of Genotype VII Velogenic Pathotype Newcastle Disease Virus from Commercial Chicken Farms in Central Ethiopia, Distinct from the Local Vaccine Strains.

Newcastle disease (ND) is caused by virulent strains of avian paramyxovirus type 1, also known as Newcastle disease virus (NDV). Despite vaccination, the frequency of reported outbreaks in Ethiopia has increased. From January to June 2022, an active outbreak investigation was conducted in six commercial chicken farms across areas of central Ethiopia to identify the circulating NDV strains. Thirty pooled tissue specimens were collected from chickens suspected of being infected with NDV. A questionnaire survey of farm owners and veterinarians was also carried out to collect information on the farms and the outbreak status. NDV was isolated using specific-pathogen-free (SPF)-embryonated chicken eggs and detected using haemagglutination and the reverse transcriptase-polymerase chain reaction (RT-PCR). The genotype and virulence of field NDV isolates were determined using phylogenetic analysis of fusion (F) protein gene sequences and the mean death time (MDT) test in SPF-embryonated chicken eggs. The questionnaire results revealed that ND caused morbidity (23.1%), mortality (16.3%), case fatality (70.8%), and significant economic losses. Eleven of thirty tissue specimens tested positive for NDV using haemagglutination and RT-PCR. The MDT testing and sequence analysis revealed the presence of virulent NDV classified as genotype VII of class II velogenic pathotype and distinct from locally used vaccine strains (genotype II). The amino acid sequences of the current virulent NDV fusion protein cleavage site motif revealed 112RRQKR↓F117, unlike the locally used avirulent vaccine strains (112GRQGR↓L117). The epidemiological data, MDT results, cleavage site sequence, and phylogenetic analysis all indicated that the present NDV isolates were virulent. The four NDV sequences were deposited in GenBank with accession numbers F gene (PP726912-15) and M gene (PP726916-19). The genetic difference between avirulent vaccine strains and circulating virulent NDV could explain the low level of protection provided by locally used vaccines. Further studies are needed to better understand the circulating NDV genotypes in different production systems.

Velogenic Newcastle disease virus invades chicken brain by infecting brain microvascular endothelial cells to increase blood-brain barrier permeability1

The blood-brain barrier (BBB) keeps poisons and infections out of the brain. Some viruses can pass through this barrier and replicate in the central nervous system (CNS). Velogenic Newcastle disease virus (VNDV) is a neurotropic virus that causes avian nonsuppurative encephalitis. VNDV often develops into a chronic infection that seriously affects poultry health in partially immune birds. The routes by which the virus enters the chicken brain are poorly understood. In this study, we discovered that VNDV increased BBB permeability in vivo and in vitro by breaking the tight junction protein zona occludens-1 (ZO-1) continuity of chicken brain microvascular endothelial cells (chBMECs). By investigating the susceptibility of chBMECs to NDV infection, we found that VNDV but not lentogenic NDV was detected in the basolateral compartment in transwell assays after apical infection, suggesting efficient replication and transcellular transport of the virus across the BBB in vitro. Furthermore, viral replication and BBB permeability were reduced during the early stage of infection by using the dynamin inhibitor dynasore. Our data demonstrate that VNDV invades the chicken brain by infecting and damaging the tight junction of chBMECs directly to increase BBB permeability. VNDV could infect chBMECs via endocytosis. As a result, our findings provide compelling evidence for VNDV entrance into the brain via the BBB, paving the way for the development of medications for NDV prevention and therapy.

Molecular characteristics and phylogenetic analysis of pigeon paramyxovirus type 1 isolates from pigeon meat farms in Shanghai (2009–2012)

The majority of pigeon paramyxovirus type 1 (PPMV-1) strains are generally non-pathogenic to chickens; however, they can induce severe illness and high mortality rates in pigeons, leading to substantial economic repercussions. The genomes of 11 PPMV-1 isolates from deceased pigeons on meat pigeon farms during passive monitoring from 2009 to 2012 were sequenced and analyzed using polymerase chain reaction and phylogenetic analysis. The complete genome lengths of 11 isolates were approximately 15,192 nucleotides, displaying a consistent gene order of 3′-NP-P-M-F-HN-L-5′. ALL isolates exhibited the characteristic motif of 112RRQKRF117 at the fusion protein cleavage site, which is characteristic of velogenic Newcastle disease virus. Moreover, multiple mutations have been identified within the functional domains of the F and HN proteins, encompassing the fusion peptide, heptad repeat region, transmembrane domains, and neutralizing epitopes. Phylogenetic analysis based on sequences of the F gene unveiled that all isolates clustered within genotype VI in class II. Further classification identified at least two distinct sub-genotypes, with seven isolates classified as sub-genotype VI.2.1.1.2.2, whereas the others were classified as sub-genotype VI.2.1.1.2.1. This study suggests that both sub-genotypes were implicated in severe disease manifestation among meat pigeons, with sub-genotype VI.2.1.1.2.2 displaying an increasing prevalence among Shanghai’s meat pigeon population since 2011. These results emphasize the value of developing pigeon-specific vaccines and molecular diagnostic tools for monitoring and proactively managing potential PPMV-1 outbreaks.

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.

A study of the immunomodulatory effects of coconut oil extract in broilers experimentally infected with velogenic Newcastle disease virus.

This study aims to evaluate the immunomodulatory effects of coconut oil extract (COE) in broilers experimentally infected with velogenic Newcastle disease virus (vNDV). A total of 150 broiler birds (day-old) were equally divided into five study groups i.e., negative control, positive control, COE-1, COE-2, and COE-3. On day 10, broilers of groups COE-1, COE-2, and COE-3 were supplemented with 1, 2, and 3 mL of COE respectively per liter of drinking water for 15 days. On day 13, 0.1 mL/bird (10-5.25 ELD50) of vNDV was inoculated in broilers of positive control, COE-1, COE-2, and COE-3 groups intramuscularly. During this study, growth performance, morbidity, and mortality rates of each study group were recorded. The antibody titer against NDV was determined on days 7, 14, 21, 28, and 35. The levels of immunoglobulins (IgY and IgM) were also determined on the 7th, 14th, and 21st days post-sheep red blood cells (SRBCs) inoculation. On day 33, avian tuberculin was injected between the 1st and 2nd toes of the left side (intradermally) to measure lymphoproliferative responses. On day 35, the phagocytic activity in the blood was assessed through a carbon clearance assay by injecting carbon black ink into the right-wing vein. The visceral organs having gross lesions were also collected for histopathology. The COE significantly improved the growth performance, and lowered the morbidity and mortality rates of broilers. There was a significant rise in antibody titers against NDV and levels of IgY and IgM antibodies against SRBC in COE-supplemented broilers. The lymphoproliferative response and phagocytic activity were also enhanced. Among COEsupplemented groups, the broilers of the COE-3 group showed a significant increase in growth performance and boosted immune defense. Coconut oil extract has the potential to boost the growth performance and immune status of broilers. It can be used effectively as a feed additive and alternative to antibiotics to prevent the spread of infectious poultry pathogens.

Effects of Hydromethanol Extract of Stem Bark of Anacardium occidentale on Gross and Histopathological changes Associated with Experimental Velogenic Newcastle Disease Virus Infection in Chickens

Effects of Hydromethanol Extract of Stem Bark of Anacardium occidentale on Gross and Histopathological changes Associated with Experimental Velogenic Newcastle Disease Virus Infection in Chickens

Isolation and molecular characterization of velogenic Newcastle disease virus genotype VII.1.1 from commercial broilers and wild birds in Matrouh governorate, Egypt

Isolation and molecular characterization of velogenic Newcastle disease virus genotype VII.1.1 from commercial broilers and wild birds in Matrouh governorate, Egypt

Sequential histopathological changes of the lower respiratory and gastrointestinal tracts in chickens infected with velogenic Newcastle disease virus

Velogenic Newcastle disease is a highly contagious and fatal disease that affects domestic poultry, and is clinically manifested by haemorrhagic gastroenteritis and respiratory distress. This study investigated the sequential histopathogic changes in the lungs, proventriculus, and intestines of broiler chickens infected with velogenic Newcastle disease virus (vNDV). Fifty broiler chickens (6-weeks-old) were randomly assigned into two groups of 25 each: UC – unvaccinated with Newcastle disease (ND) vaccines and intranasallly inoculated with vNDV, UU – unvaccinated uninfected. At 4, 5 and 6 days post-infection (d.p.i) tissue samples were collected from birds in each group for histopathology. The lungs showed hyperaemia, haemorrhages, inflammatory oedema and cellular infiltrations in the secondary bronchi, parabronchi (atria, infundibular, air and blood capillaries of the exchange tissue), depletion of bronchial-asociated lymphoid tissues, loss of cilia and hyperplasia of surface epithelium of bronchi and parabronchi at 4 d.p.i. The lesions progressed to collapsed air capillaries with fibroplasia and fibrosis of the area at 6 d.p.i. Similar inflammatory changes were observed in the proventriculus, but accompanied by degeneration and attenuation of surface epithelium of proventiculus and proventricular glands at 4 d.p.i., and severe necrosis and disintegration of the proventricular glands at 6 d.p.i. The intestine showed severe inflammatory changes in the mucosa, submucosa and serosa and depletion of gut-associated lymphoid tissues at 4 d.p.i., and ulcerative enteritis at 5 d.p.i. The lumen was filled with necrotic debris at 6 d.p.i. This study reinforces the concept making pathologic animal experiments important tools for a complete characterization of strains in susceptible animals, as this information will be helpful in the accurate diagnosis of velogenic Newcastle disease in chickens.

Protective effectiveness of two vaccination schemes against the prevalent Egyptian strain of Newcastle disease virus genotype VII.

Despite the strict preventive immunization used in Egypt, Newcastle disease remained a prospective risk to the commercial and backyard chicken industries. The severe economic losses caused by the Newcastle disease virus (NDV) highlight the importance of the trials for the improvement and development of vaccines and vaccination programs. In the present study, we evaluated the effectiveness of two vaccination schemes for protection against the velogenic NDV (vNDV) challenge. Four groups (A-D) of commercial broiler chickens were used. Two groups (G-A and G-B) were vaccinated with priming live HB1 GII simultaneously with inactivated GVII vaccines at 5 days of age, then boosted with live LaSota GII vaccine in group A and live recombinant NDV GVII vaccine in group B on day 16. Groups A to C were challenged with NDV/Chicken/Egypt/ALEX/ZU-NM99/2019 strain (106 Embryo infective dose 50/0.1 ml) at 28 days of age. Two vaccination schemes achieved 93.3% clinical protection against NDV with body gain enhancement; whereas, 80% of the unvaccinated-challenged birds died. On day 28, the mean HI antibody titers were 4.3 ± 0.33 and 5.3 ± 0.33 log2 in groups A and B, respectively. As well as both programs remarkably reduced virus shedding. The two vaccination schemes displayed close protection efficacy against the vNDV challenge. Therefore, using the combination of a live attenuated vaccine with an inactivated genetically matched strain vaccine and then boosting it with one of the available live vaccines could be considered one of the most effective programs against current field vNDV infection in Egypt.

The evaluation and importance of Newcastle disease’s economic loss in commercial layer poultry

Velogenic Newcastle disease virus (VNDV) causes a highly contagious and devastating disease in poultry and causes up to 100 percent mortality in susceptible populations during devastating outbreaks. Newcastle disease causes heavy economic losses to the global poultry industry by decreasing the growth rate, decreasing egg production, and increasing morbidity and mortality. The households face substantial economic losses throughout the year due to Newcastle disease. It is ranked first among other poultry diseases in village chickens. The economic losses caused by this disease in the poultry industry have given awareness about the importance of Newcastle disease and made it easier to justify the operations against this disease. So, estimating the economic losses caused by Newcastle disease is very important. Regarding the Newcastle disease's huge impact on production and causing a lack of eggs, we will have to compensate for this reduction by importing eggs from other countries. Hence, a tremendous amount of money will be needed to compensate for the shortage of eggs, which will result in a huge economic loss. Finally, these will directly affect the poultry industry and its financial resources, which clarifies the importance of correct and on-time vaccination and caring for Newcastle disease in poultry flocks to prevent enormous economic losses. In this study, the economic impact of Newcastle Disease on egg-laying farms was quantitatively assessed. Data were collected on the total number of egg-laying poultry monthly and annually. Subsequently, the economic loss for each period was calculated based on the total production period. The minimal damage caused by this disease is about 116,591 Tons. Summing all these periods' production decreases, the total loss will be 10538 Tons. To estimate the losses of the disease, it is necessary to estimate the losses in breeding herds and broiler herds so that the loss resulting from the endemic Newcastle disease of the country can be measured.

Genetic characterization of pigeon- origin avian avulavirus-1 reveals unique substitutions in F and HN proteins.

Since decades, Newcastle disease (ND) has become endemic in the poultry population of the Indian subcontinent. ND is a highly contagious disease of poultry and other avian species. However, the genetic nature of ND viruses circulating in the rock pigeons is unraveled. The present investigation is a part of Newcastle disease virus (NDV) surveillance in wild birds. Two velogenic NDV strains could be isolated from apparently healthy rock pigeons, thus establishing the status of carrier/reservoir host. The fusion protein cleavage site in the fusion protein has multiple basic amino acid (RRRKRF) motifs similar to velogenic isolates. Phylogenetic analysis based on complete fusion gene sequences confirmed that the isolates belong to NDV sub genotype XIII 2.2. Further analysis revealed several amino acid substitutions in the hypervariable region, heptad repeat regions and neutralizing epitopes of the fusion protein and heptad repeat regions and antigenic sites of the hemagglutinin-neuraminidase (HN) protein that are critical for fusion. A unique D170A substitution in the neutralizing epitope is identified that is critical for structure and function of the fusion protein. Mutations within the virulence determinants including fusion (F) and HN, elucidate continuous evolution of the viruses among the rock pigeons. Accidental spillover of these mutated viruses into commercial poultry operations may result in disease outbreaks with economic breakdown.

Determination of median embryo lethal dose for a velogenic Newcastle disease virus isolated in Sulaimani/Iraq

Determination of median embryo lethal dose for a velogenic Newcastle disease virus isolated in Sulaimani/Iraq

The in vitro Potential Oncolytic Effect of Lentogenic and Velogenic Newcastle Disease Viruses on MCF-7 and Caco-2 Cell Lines Compared to Chemotherapies

The in vitro Potential Oncolytic Effect of Lentogenic and Velogenic Newcastle Disease Viruses on MCF-7 and Caco-2 Cell Lines Compared to Chemotherapies

Superior Efficacy of Apathogenic Genotype I (V4) over Lentogenic Genotype II (LaSota) Live Vaccines against Newcastle Disease Virus Genotype VII.1.1 in Pathogen-Associated Molecular Pattern-H9N2 Vaccinated Broiler Chickens.

A comparison of the efficacy of apathogenic genotype I (V4) and lentogenic genotype II (LaSota) strains of live Newcastle disease virus (NDV) vaccines was performed following vaccination with pathogen-associated molecular pattern (PAMP) H9N2 avian influenza vaccine and challenge with velogenic NDV genotype VII.1.1 (vNDV-VII.1.1). Eight groups (Gs) of day-old chicks were used (n = 25). Groups 1-4 received a single dose of PAMP-H9N2 subcutaneously, while Gs (1, 5) and (2, 6) received eye drops of V4 and LaSota, respectively, as two doses. All Gs, except for 4 and 8, were intramuscularly challenged with vNDV-VII.1.1 at 28 days of age. No signs were detected in Gs 1, 5, 4, and 8. The mortality rates were 0% in Gs 1, 4, 5, and 8; 40% in G2; 46.66% in G6; and 100% in Gs 3 and 7. Lesions were recorded as minimal in Gs 1 and 5, but mild to moderate in Gs 2 and 6. The lowest significant viral shedding was detected in Gs 1, 2, and 5. In conclusion, two successive vaccinations of broilers with a live V4 NDV vaccine provided higher protection against vNDV-VII.1.1 challenge than LaSota. PAMP-H9N2 with live NDV vaccines induced more protection than the live vaccine alone.

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.

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.