The Application of Newcastle Disease Virus (NDV): Vaccine Vectors and Tumor Therapy.

Newcastle disease virus (NDV) is an avian pathogen with an unsegmented negative-strand RNA genome that belongs to the Paramyxoviridae family. While primarily pathogenic in birds, NDV presents no threat to human health, rendering it a safe candidate for various biomedical applications. Extensive research has highlighted the potential of NDV as a vector for vaccine development and gene therapy, owing to its transcriptional modularity, low recombination rate, and lack of a DNA phase during replication. Furthermore, NDV exhibits oncolytic capabilities, efficiently eliciting antitumor immune responses, thereby positioning it as a promising therapeutic agent for cancer treatment. This article comprehensively reviews the biological characteristics of NDV, elucidates the molecular mechanisms underlying its oncolytic properties, and discusses its applications in the fields of vaccine vector development and tumor therapy.

Spillover of Newcastle disease virus to Himalayan Griffon vulture: a possible food-based transmission.

The Newcastle disease virus (NDV) affects wild and domesticated bird species, including commercial poultry. Although the diversity of NDV in domestic chickens is well documented, limited information is available about Newcastle disease (ND) outbreaks in other bird species. We report an annotated sequence of NDV/Vulture/Borjuri/01/22, an avirulent strain of NDV reported from Borjuri, Northeast India, in Himalayan Griffon vulture. The complete genome is 15,186 bases long with a fusion protein (F) cleavage site 112GRQGR↓L117. The phylogenetic analysis based on the F protein gene and the whole genome sequence revealed that the isolate from the vulture belongs to genotype II, sharing significant homology with vaccine strain LaSota. The study highlights the possible spillover of the virus from domestic to wild species through the food chain.

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.

Newcastle disease virus activates diverse signaling pathways via Src to facilitate virus entry into host macrophages.

As an intrinsic cellular mechanism responsible for the internalization of extracellular ligands and membrane components, caveolae-mediated endocytosis (CavME) is also exploited by certain pathogens for endocytic entry [e.g., Newcastle disease virus (NDV) of paramyxovirus]. However, the molecular mechanisms of NDV-induced CavME remain poorly understood. Herein, we demonstrate that sialic acid-containing gangliosides, rather than glycoproteins, were utilized by NDV as receptors to initiate the endocytic entry of NDV into HD11 cells. The binding of NDV to gangliosides induced the activation of a non-receptor tyrosine kinase, Src, leading to the phosphorylation of caveolin-1 (Cav1) and dynamin-2 (Dyn2), which contributed to the endocytic entry of NDV. Moreover, an inoculation of cells with NDV-induced actin cytoskeletal rearrangement through Src to facilitate NDV entry via endocytosis and direct fusion with the plasma membrane. Subsequently, unique members of the Rho GTPases family, RhoA and Cdc42, were activated by NDV in a Src-dependent manner. Further analyses revealed that RhoA and Cdc42 regulated the activities of specific effectors, cofilin and myosin regulatory light chain 2, responsible for actin cytoskeleton rearrangement, through diverse intracellular signaling cascades. Taken together, our results suggest that an inoculation of NDV-induced Src-mediated cellular activation by binding to ganglioside receptors. This process orchestrated NDV endocytic entry by modulating the activities of caveolae-associated Cav1 and Dyn2, as well as specific Rho GTPases and downstream effectors. IMPORTANCE: In general, it is known that the paramyxovirus gains access to host cells through direct penetration at the plasma membrane; however, emerging evidence suggests more complex entry mechanisms for paramyxoviruses. The endocytic entry of Newcastle disease virus (NDV), a representative member of the paramyxovirus family, into multiple types of cells has been recently reported. Herein, we demonstrate the binding of NDV to induce ganglioside-activated Src signaling, which is responsible for the endocytic entry of NDV through caveolae-mediated endocytosis. This process involved Src-dependent activation of the caveolae-associated Cav1 and Dyn2, as well as specific Rho GTPase and downstream effectors, thereby orchestrating the endocytic entry process of NDV. Our findings uncover a novel molecular mechanism of endocytic entry of NDV into host cells and provide novel insight into paramyxovirus mechanisms of entry.

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.

Molecular Identification and Phylogenetic Study Based on the Fusion Gene of Newcastle Disease Virus Isolated from Broiler Poultry Farms in Markazi Province, Iran.

Newcastle disease (ND) is an economically significant and extremely spreadable viral illness affecting a wide variety of avian species. ND can rapidly spread within poultry farms and result in considerable economic losses for the global poultry industry. This disease is endemic in Iran, and despite intensive vaccination efforts in the poultry industry, outbreaks of ND occur unexpectedly. This study aimed to isolate the Newcastle disease virus (NDV) from poultry farms with breathing problems in Markazi province, Iran, and investigate the evolutionary relationship and molecular characteristics of the isolates during 2017-2019. To this end, tissue samples (lung, brain, and trachea) were taken from 42 broiler farms exhibiting respiratory symptoms. The samples were inoculated into 9-11-day-old embryonated eggs, and the virus was isolated from 20 (47.6%) of the 42 farms. Subsequently, RT-PCR was used to amplify partial fusion gene sequences from the new isolates. The amplified products were sequenced and compared phylogenetically to the standard pilot dataset (125 selected sequences) generated by the NDV consortium. As determined by phylogenetic analysis, all nine isolates belonged to subgenotype VII.1.1 of genotype VII and were highly similar to isolates from other parts of Iran and China. Moreover, all isolates possessed a polybasic cleavage site motif (112RRQKRF117), characteristic of virulent strains. Furthermore, the present isolates shared a high nucleotide identity (96%) with viruses previously isolated from other provinces of Iran, as determined by BLAST searches and multiple alignments. In addition, they shared a high degree of sequence similarity but were distinct from the existing NDV vaccines. Therefore, the genetic dissimilarity between current vaccine strains and circulating NDVs must be considered in vaccination programs.

YLMY Tyrosine Residue within the Cytoplasmic Tail of Newcastle Disease Virus Fusion Protein Regulates Its Surface Expression to Modulate Viral Budding and Pathogenicity.

Newcastle disease virus (NDV) fusion protein mediates the virus's fusion activity, which is a determinant of NDV pathogenicity. The ectodomain of the F protein is known to have a major impact on fusion, and several reports have also indicated the role of the cytoplasmic tail (CT) in viral entry, F protein cleavage, and fusion, which are regulated by specific motifs. We found a highly conserved tyrosine residue located in the YLMY motif. The tyrosine residues at positions 524 and 527 have different roles in viral replication and pathogenicity and are associated with F protein intracellular processing. Tyrosine residues mutants affect the transportation of the F protein from the endoplasmic reticulum to the Golgi apparatus, resulting in different cleavage efficiencies. F protein is subsequently transported to the cell surface where it participates in viral budding, a process closely related to the distinctions in pathogenicity caused by the tyrosine residues. In addition, the different mutations all led to a hypofusogenic phenotype. We believe that the highly conserved tyrosine residue of the YLMY motif uses a similar mechanism to the tyrosine-based motif (YXXΦ) to regulate F protein transport and thus affect viral replication and pathogenicity. IMPORTANCE The amino-terminal cytoplasmic domains of paramyxovirus fusion glycoproteins include trafficking signals that influence protein processing and cell surface expression. This study clarified that tyrosine residues at different positions in the YLMY motif in the cytoplasmic region of the F protein regulate F protein transportation, thereby affecting viral replication and pathogenicity. This study has increased our understanding of how NDV virulence is mediated by the F protein and provides a fresh perspective on the role of CT in the virus's life cycle. This information may be useful in the development of NDV as an effective vaccine vector and oncolytic agent.

Different kinetics of chicken interferon-alpha signalling transduction responses following immunization of broiler chickens with different Newcastle disease virus vaccines and infection with virulent genotype VIId strain.

Newcastle disease virus (NDV) is a highly contagious and notifiable avian disease leading to grave economic losses in the poultry industry. Although the immune responses against NDV have been widely investigated, little is known regarding the virus interaction with the host innate immune responses. In this study, we tested the effect of different commercially applied Newcastle disease vaccines as well as virulent NDV genotype VIId on the expression pattern of the upstream regulator and downstream effector genes related to chicken interferon-alpha (chIFNα) signalling transduction pathway. Using quantitative real-time PCR analysis, mild transient induction of chIFNα-inducible genes was detected in bird spleen 72 h post-vaccination (hpv) with either live LaSota (respiratory) or VG/GA (enteric) strains. Vaccination with the enteric VG/GA strain led to stimulation of the investigated pathway as early as 24 hpv which continued up to 7 days in bird caecal tonsils. Subcutaneous injection with inactivated LaSota oil adjuvant-based vaccine led to continual stimulation of the investigated pathway up to 7 days post-vaccination (dpv). The recombinant herpesvirus of turkey (rHVT) - NDV vaccine led to remarkable stimulation of all the tested cytokines up to 17 dpv in comparison with LaSota and VG/GA NDV vaccines. Stronger but transient activation of all the tested cytokines was detected in spleens during the first 24 h post-challenge with virulent NDV (vNDV) which reduced gradually and diminished later due to the virus-induced lymphocytic depletion. This study will aid in the discovery of new approaches to control NDV.

Antiviral activity of a novel mixture of natural antimicrobials, in vitro, and in a chicken infection model in vivo.

The aim of this study was to test in vitro the ability of a mixture of citrus extract, maltodextrin, sodium chloride, lactic acid and citric acid (AuraShield L) to inhibit the virulence of infectious bronchitis, Newcastle disease, avian influenza, porcine reproductive and respiratory syndrome (PRRS) and bovine coronavirus viruses. Secondly, in vivo, we have investigated its efficacy against infectious bronchitis using a broiler infection model. In vitro, these antimicrobials had expressed antiviral activity against all five viruses through all phases of the infection process of the host cells. In vivo, the antimicrobial mixture reduced the virus load in the tracheal and lung tissue and significantly reduced the clinical signs of infection and the mortality rate in the experimental group E2 receiving AuraShield L. All these effects were accompanied by a significant reduction in the levels of pro-inflammatory cytokines and an increase in IgA levels and short chain fatty acids (SCFAs) in both trachea and lungs. Our study demonstrated that mixtures of natural antimicrobials, such AuraShield L, can prevent in vitro viral infection of cell cultures. Secondly, in vivo, the efficiency of vaccination was improved by preventing secondary viral infections through a mechanism involving significant increases in SCFA production and increased IgA levels. As a consequence the clinical signs of secondary infections were significantly reduced resulting in recovered production performance and lower mortality rates in the experimental group E2.

Protective efficacy of inactivated Newcastle disease virus vaccines prepared in two different oil-based adjuvants.

Despite the availability of Newcastle disease (ND) vaccines for more than six decades, disease outbreaks continue to occur with huge economic consequences to the global poultry industry. The aim of this study is to develop a safe and effective inactivated vaccine based on a recently isolated Newcastle disease virus (NDV) strain IBS025/13 and evaluate its protective efficacy in chicken following challenge with a highly virulent genotype VII isolate. Firstly, high titre of IBS025/13 was exposed to various concentrations of binary ethylenimine (BEI) to determine the optimal conditions for complete inactivation of the virus. The inactivated virus was then prepared in form of a stable water-in-oil emulsion of black seed oil (BSO) or Freund's incomplete adjuvant (FIA) and used as vaccines in specific pathogen-free chicken. Efficacy of various vaccine preparations was also evaluated based on the ability of the vaccine to protect against clinical disease, mortality and virus shedding following challenge with highly virulent genotype\VII NDV isolate. The results indicate that exposure of NDV IBS025/13 to 10 mM of BEI for 21 h at 37 °C could completely inactivate the virus without tempering with the structural integrity of the viral hemagglutin-neuraminidase protein. More so, the inactivated vaccines adjuvanted with either BSO- or FIA-induced high hemagglutination inhibition antibody titre that protected the vaccinated birds against clinical disease and in some cases virus shedding, especially when used together with live attenuated vaccines. Thus, genotype VII-based NDV-inactivated vaccines formulated in BSO could substantially improve poultry disease control particularly when combined with live attenuated vaccines.

Ionic Strength-Dependent, Reversible Pleomorphism of Recombinant Newcastle Disease Virus.

A genetically modified, recombinant form of Newcastle disease virus (rNDV) undergoes ionic strength-dependent changes in morphology, as observed by cryo-electron microscopy (cEM). In hypotonic solutions with ionic strengths ranging from < 0.01 to 0.02 M, rNDV virions are spherical or predominantly spherical. In isotonic and hypertonic solutions, rNDV displays pleomorphism and contains a mixed population of spherical and elongated particles, indicating that a change from spherical to elongated shape is induced with increasing salt concentration. This ionic strength-dependent transition is largely reversible, as determined by cEM. Concomitantly, we measured infectious titers of these same rNDV samples at different ionic strengths using a fluorescent focus assay (FFA). The infectivity of oncolytic rNDV was found to be independent of ionic strength, ranging from 0.01 M to approximately 0.5 M. These structural and functional observations, in combination, suggest that infectivity (and, by inference, oncolytic activity) of rNDV virions is fully maintained in their pleomorphic forms.IMPORTANCE Oncolytic viruses are being developed for cancer therapy, as they selectively target, infect, and kill cancer cells. NDV is particularly attractive because while it is pathogenic to avians (e.g., chickens), it does not cause significant viremia in humans. We have developed a genetically modified recombinant NDV (rNDV) that has much reduced pathogenicity in chickens but is highly oncolytic. The morphology of rNDV transitions from spherical at very low salt concentrations to a heterogeneous population of spherical and elongated virions in isotonic (physiologic salt concentration) and hypertonic solutions. The infectivity (cell-killing activity by infecting cells) of rNDV is unaltered by changes in salt concentration despite morphological changes. These observations are significant for purification and formulation of rNDV, as exposure to different salt concentrations may be needed. Importantly, at physiological salt concentration, relevant to clinical testing, infectivity and, therefore, oncolytic activity will not be compromised despite morphological heterogeneity.

Molecular Characterization of Newcastle Disease Viruses Isolated from Chickens in Tanzania and Ghana.

Newcastle disease (ND) is one of the most challenging infectious diseases affecting poultry production in Africa, causing major economic losses. To date, Newcastle disease virus isolates from several African countries have been grouped into class II NDV genotypes I, IV, V, VI, VII, XI, XIII, XIV, XVII, XVIII and XXI. Although ND is endemic in many African countries, information on circulating genotypes is still scarce. In Tanzania, outbreaks with genotypes V and XIII have been reported. In West and Central Africa, genotypes XIV, XVII, and XVIII are the most predominant. To investigate other genotypes circulating in Tanzania and Ghana, we performed molecular genotyping on isolates from Tanzania and Ghana using the MinION, a third-generation portable sequencing device from Oxford Nanopore Technologies. Using the MinION, we successfully sequenced the NDV F gene hypervariable region of 24 isolates from Tanzania and four samples from Ghana. In Tanzania, genotypes V, VII and XIII were detected. All isolates from Ghana belonged to genotype XVIII. The data obtained in this study reflect the genetic diversity of NDV in Africa and highlight the importance of surveillance for monitoring the distribution of NDV genotypes and viral evolution.

Immune effect of a Newcastle disease virus DNA vaccine with IL-12 as a molecular adjuvant delivered by electroporation.

Newcastle disease (ND), caused by virulent Newcastle disease virus (NDV) strains, has been one of the most problematic diseases affecting the poultry industry worldwide. Conventional vaccines provide effective protection for birds to survive ND outbreaks, but they may not completely suppress NDV shedding. NDV strains circulate on farms for a long time after the initial infection and cause potential risks. A new vaccine with fast clearance ability and low viral shedding is needed. In this study, we used interleukin-12 (IL-12) as an adjuvant and electroporation (EP) as an advanced delivery system to improve a DNA vaccine candidate. The fusion (F) protein gene from an NDV strain of the prevalent genotype VII.1.1 was cloned to prepare the vaccine. Chickens immunized with the F gene DNA vaccine co-delivered with an IL-12-expressing plasmid DNA showed higher neutralizing antibody levels and stronger concanavalin-A-induced lymphocyte proliferation than those treated with the F gene DNA vaccine alone. The co-delivered vaccine provided 100% protection, and less viral shedding and a shorter release time were observed in challenged chickens than when the F gene DNA vaccine was administered alone. The use of F gene DNA combined with IL-12 delivered by electroporation is a promising approach for vaccination against ND.

Interference chromatography: a novel approach to optimizing chromatographic selectivity and separation performance for virus purification.

BACKGROUND: Oncolytic viruses are playing an increasingly important role in cancer immunotherapy applications. Given the preclinical and clinical efficacy of these virus-based therapeutics, there is a need for fast, simple, and inexpensive downstream processing methodologies to purify biologically active viral agents that meet the increasingly higher safety standards stipulated by regulatory authorities like the Food and Drug Administration and the European Agency for the Evaluation of Medicinal Products. However, the production of virus materials for clinical dosing of oncolytic virotherapies is currently limited-in quantity, quality, and timeliness-by current purification technologies. Adsorption of virus particles to solid phases provides a convenient and practical choice for large-scale fractionation and recovery of viruses from cell and media contaminants. Indeed, chromatography has been deemed the most promising technology for large-scale purification of viruses for biomedical applications. The implementation of new chromatography media has improved process performance, but low yields and long processing times required to reach the desired purity are still limiting. RESULTS: Here we report the development of an interference chromatography-based process for purifying high titer, clinical grade oncolytic Newcastle disease virus using NatriFlo® HD-Q membrane technology. This novel approach to optimizing chromatographic performance utilizes differences in molecular bonding interactions to achieve high purity in a single ion exchange step. CONCLUSIONS: When used in conjunction with membrane chromatography, this high yield method based on interference chromatography has the potential to deliver efficient, scalable processes to enable viable production of oncolytic virotherapies.

In silico, in ovo and in vitro antiviral efficacy of phosphorylated derivatives of abacavir: an experimental approach.

Outstanding increase of oral absorption, bioavailability, and antiviral efficacy of phosphorylated nucleosides and basic antiviral influence of abacavir is the central idea for the development of new series of phosphorylated abacavir (ABC) derivatives. The designed compounds were primarily screened for antiviral nature against HN protein of NDV and VP7 protein of BTV using the molecular environment approach. Out of all the designed compounds, the compounds which are having higher binding energies against these two viral strains were prompted for the synthesis of the target compounds (5A-K). Among the synthesized title compounds (5A-K), the compounds which have exhibited higher dock scores akin to the rest of the compounds were then selected and screened for the antiviral activity against NDV and BTV infected embryonated eggs and BHK 21 cell lines through the in ovo and in vitro approaches. The results revealed that all the designed compounds have formed higher binding energies against both the targets. Among all, the compounds which are selected based on their dock scores such as 5A, 5F, 5G, 5H, 5I, and 5K against NDV and 5J, 5E, 5I, 5C, 5A, and 5K against BTV have shown significant antiviral activity against HN protein of NDV, VP7 protein of Bluetongue virus in both NDV- and BTV-treated embryonated eggs and BHK 21 cell lines. Hence, it is concluded that, the best lead compounds will stand as the potential antiviral agents and prompted them as virtuous therapeutics against NDV and BTV in future.

A comparative evaluation of serum biochemistry profile and antigenic relatedness among velogenic and mesogenic Avian avulavirus 1 infection in chickens and pigeons.

Newcastle disease (ND), caused by virulent Avian avulavirus 1 (AAvV 1), affects variety of avian species around the globe. Several AAvV 1 viruses of different genotypes have recently emerged with varying clinical impacts on their susceptible hosts. Although experimental infection with velogenic and mesogenic strains in chickens and pigeons is well-studied, nevertheless, there exists a paucity of data for comparative variations in serum biochemistry profile of susceptible hosts upon challenge with isolates of varying pathogenicities. With this background, a comparative assessment of a range of serum biochemical parameters was made following challenge with duck-originated velogenic strain (sub-genotype VIIi; MF437287) and pigeon-originated mesogenic strain (sub-genotype VIm; KU885949) in chickens and pigeons. For each of the isolate, commercial broiler chickens and wild pigeons were challenged (10-6.51 EID50/0.1 mL for sub-genotype VIIi and 10-6.87 EID50/0.1 mL sub-genotype Vim) separately via intranasal and intraocular route. Sera were collected on 0, 3rd, 5th, 7th, and 9th day post-infection (dpi), and processed for quantitative analysis of different biochemical parameters. By day 3 post-infection (pi), a substantial decrease (p < 0.0001) in serum alkaline phosphatase (ALP) was observed in chickens and pigeons challenged with velogenic isolate. On the other hand, from day 5 pi and onward, a significant increase (p < 0.001) in serum ALP and total protein concentration was observed exclusively in pigeons challenged with mesogenic isolate. For serum aspartate aminotransferase (AST), a significant increase (p < 0.05) in concentration was observed on day 3 pi which decreased from day 5 pi and onward in pigeons and chickens challenged with mesogenic isolate. Also, to reveal antigenic differences among homologous and heterologous vaccine and field-prevalent strains, cross-hemagglutination inhibition assay demonstrated antigenically diverse nature (R-value < 0.5) of both strains from vaccine strain (LaSota, genotype II). The study concludes antigenic differences among prevalent genotypes than vaccine strain and, although requires further studies to ascertain study outcomes, the serum biochemical profile may facilitate presumptive diagnosis of disease in their susceptible hosts.

Role of the head-stalk linker region (aa115-122) of Newcastle disease virus haemagglutinin-neuraminidase in regulating fusion triggering.

To gain insights into the role of the head-stalk linker region in the fusion triggering, we constructed mutants by deleting or substituting the linker region (115-NGAANNSG-122) of Newcastle disease virus (NDV) haemagglutinin-neuraminidase (HN) with the corresponding sequences of other paramyxoviruses. The results showed that these HN mutants exhibited different levels of fusion-triggering activity, but most of them maintained comparable levels with wide-type HN in both receptor recognition and neuraminidase activity. We tried to figure out reasons for fusion alteration through assessing the expression and the oligomeric state of HN mutants. Moreover, four mutants with significant fusion changes were introduced into the headless HN stem (HN1-123) to intensively investigate the role of the linker region in fusion triggering. Consequently, the stability of HN oligomers and the structural integrity of the 4 helical-bundle of stalk have complicated influences on the alteration of fusion-triggering activities for different mutants. These data suggested that the head-stalk linker could regulate the fusion triggering at both full-length and headless HN levels.

Engineering Newcastle Disease Virus as an Oncolytic Vector for Intratumoral Delivery of Immune Checkpoint Inhibitors and Immunocytokines.

Newcastle disease virus (NDV) is an attractive candidate for oncolytic immunotherapy due to its ability to replicate in tumor cells and potentially to overcome the inherently immunosuppressive nature of the tumor microenvironment. The advent of checkpoint blockade immunotherapy over the past few years represents a paradigm shift in cancer therapy. However, the prevalence of severe immune-related adverse events with CTLA4 and PD1 pathway blockade in clinical studies, especially in combination therapy groups, is a cause for concern. Immunotherapies with cytokines have also been extensively explored, but they have been associated with adverse events in clinical trials. Oncolytic vectors engineered to express checkpoint blockade antibodies and cytokines could provide an avenue for reducing the clinical toxicity associated with systemic therapy by concentrating the immunomodulatory payload at the site of disease. In this study, we engineered six different recombinant viruses: NDVs expressing checkpoint inhibitors (rNDV-anti-PD1 and rNDV-anti-PDL1); superagonists (rNDV-anti-CD28); and immunocytokines, where the antibodies are fused to an immunostimulatory cytokine, such as interleukin 12 (IL-12) (rNDV-anti-CD28-murine IL-12 [mIL-12], rNDV-anti-PD1-mIL-12, and rNDV-anti-PDL1-mIL-12). These six engineered viruses induced tumor control and survival benefits in both highly aggressive unilateral and bilateral B16-F10 murine melanoma models, indicative of an abscopal effect. The data represent a strong proof of concept on which further clinical evaluation could build.IMPORTANCE Checkpoint inhibitor therapy has shown tremendous efficacy, but also frequent and often severe side effects-especially when multiple drugs of the class are used simultaneously. Similarly, many investigational immunotherapy agents, which have shown promise in animal models, have failed in clinical trials due to dose-limiting toxicity when administered systemically. This study utilized a murine melanoma model to evaluate the efficacy of intratumoral injections of recombinant NDVs engineered to express multiple immunotherapeutic proteins with well-documented side effects in humans. Our results indicate that intratumoral administration of these recombinant NDVs, particularly when combined with systemic CTLA4 checkpoint inhibition, exerts a robust effect in treated and nontreated tumors, indicative of a systemic antitumoral response. The intratumoral delivery of rNDVs expressing immunotherapeutic proteins may be an effective method of targeting the immune cell populations most relevant for antitumoral immunity and allowing us to restrict the use of systemic immunotherapy agents.

Evaluation of the safety and protection efficacy of an attenuated genotype vii newcastle disease virus strain as a candidate vaccine.

Newcastle disease (ND) is a serious avian infectious disease, causing severe economic loss worldwide. Its prevention depends on comprehensive vaccination scheme against Newcastle disease virus (NDV). However, current vaccine strains are of different genotypes with prevalent circulating strains (genotype VII), with significant genetic distance. Our team previously generated a genotype matched attenuated NDV strain (rmNA-1). In this study, its safety and immunization efficacy were evaluated. Its lentogenic characteristic was stable for 25 generations in embryonated chicken eggs and for six generations in SPF chickens. Overdosed administration did not cause any clinical signs or pathogenic changes in chickens. As to its immunization effect, rmNA-1 stimulated a comparable serum NDV specific antibody level to a LaSota (genotype II) strain based commercial vaccine, and provided full protection against virulent genotype VII strain challenge, with significantly reduced virus shedding period.