Newcastle Disease Virus Induces Profound Lymphoid Depletion with Different Patterns of Necroptosis, Necrosis, and Oxidative DNA Damage in Bursa, Spleen, and Other Lymphoid Tissues.

This study delves into the pathogenesis of virulent genotype VII strains of the Newcastle disease virus (NDV), focusing on experimentally infected birds. Predominant and consistent lesions observed include bursal atrophy and extensive depletion of all lymphoid tissues. Immunohistochemistry (IHC) analysis, targeting apoptosis (Caspase-3), necroptosis (MLKL), and NDV markers, indicates that bursal atrophy is linked to a non-apoptotic programmed cell death pathway known as "necroptosis". Repair assisted damage detection (RADD) of the bursa reveal oxidative DNA damage patterns consistent with programmed cell death, aligning with MLKL expression. Contrastingly, in the spleen, our findings suggest that necrosis (non-programmed cell death) predominantly contributes to lymphoid depletion. This conclusion is supported by evidence of karyorrhexis, fibrinous inflammation, RADD analyses, and IHC. Moreover, in addition to being pathogenic in its own right, NDV caused extensive and rapid lymphoid depletion that should be expected to contribute to profound immunosuppression. The elucidation of necroptosis in NDV-infected chickens provides a good rationale to investigate this mechanism in other paramyxoviral diseases such as human measles.

NDV targets the invasion pathway in malaria parasite through cell surface sialic acid interaction.

Merozoites utilize sialic acids on the red blood cell (RBC) cell surface to rapidly adhere to and invade the RBCs. Newcastle disease virus (NDV) displays a strong affinity toward membrane-bound sialic acids. Incubation of NDV with the malaria parasites dose-dependently reduces its cellular viability. The antiplasmodial activity of NDV is specific, as incubation with Japanese encephalitis virus, duck enteritis virus, infectious bronchitis virus, and influenza virus did not affect the parasite propagation. Interestingly, NDV is reducing more than 80% invasion when RBCs are pretreated with the virus. Removal of the RBC surface proteins or the NDV coat proteins results in disruption of the virus binding to RBC. It suggests the involvement of specific protein: ligand interaction in virus binding. We established that the virus engages with the parasitized RBCs (PRBCs) through its hemagglutinin neuraminidase (HN) protein by recognizing sialic acid-containing glycoproteins on the cell surface. Blocking of the HN protein with free sialic acid or anti-HN antibodies abolished the virus binding as well as its ability to reduce parasite growth. Interestingly, the purified HN from the virus alone could inhibit the parasite's growth in a dose-dependent manner. NDV binds strongly to knobless murine parasite strain Plasmodium yoelii and restricted the parasite growth in mice. Furthermore, the virus was found to preferentially target the PRBCs compared to normal erythrocytes. Immunolocalization studies reveal that NDV is localized on the plasma membrane as well as weakly inside the PRBC. NDV causes neither any infection nor aggregation of the human RBCs. Our findings suggest that NDV is a potential candidate for developing targeted drug delivery platforms for the Plasmodium-infected RBCs.

Oncolytic Newcastle disease virus carrying the IL24 gene exerts antitumor effects by inhibiting tumor growth and vascular sprouting.

The second-leading cause of death, cancer, poses a significant threat to human life. Innovations in cancer therapies are crucial due to limitations in traditional approaches. Newcastle disease virus (NDV), a nonpathogenic oncolytic virus, exhibits multifunctional anticancer properties by selectively infecting, replicating, and eliminating tumor cells. To enhance NDV's antitumor activity, four oncolytic NDV viruses were developed, incorporating IL24 and/or GM-CSF genes at different gene loci using reverse genetics. In vitro experiments revealed that oncolytic NDV virus augmented the antitumor efficacy of the parental virus rClone30, inhibiting tumor cell proliferation, inducing tumor cell fusion, and promoting apoptosis. Moreover, NDV carrying the IL24 gene inhibited microvessel formation in CAM experiments. Evaluation in a mouse model of liver cancer confirmed the therapeutic efficacy of oncolytic NDV viral therapy. Tumors in mice treated with oncolytic NDV virus significantly decreased in size, accompanied by tumor cell detachment and apoptosis evident in pathological sections. Furthermore, oncolytic NDV virus enhanced T cell and dendritic cell production and substantially improved the survival rate of mice with hepatocellular carcinoma, with rClone30-IL24(P/M) demonstrating significant therapeutic effects. This study establishes a basis for utilizing oncolytic NDV virus as an antitumor agent in clinical practice.

Evaluation of the Immunity Responses in Mice to Recombinant Bacillus subtilis Displaying Newcastle Disease Virus HN Protein Truncations.

Bacillus subtilis, a probiotic bacterium with engineering potential, is widely used for the expression of exogenous proteins. In this study, we utilized the integrative plasmid pDG364 to integrate the hemagglutinin-neuraminidase (HN) gene from Newcastle disease virus (NDV) into the genome of the B. subtilis 168 model strain. We successfully constructed a recombinant B. subtilis strain (designated B. subtilis RH) that displays a truncated HN antigen fragment on the surface of its spores and further evaluated its immunogenic effects in mice. Using ELISA, we quantified the levels of IgG in serum and secretory IgA (sIgA) in intestinal contents. The results revealed that the recombinant B. subtilis RH elicited robust specific mucosal and humoral immune responses in mice. Furthermore, B. subtilis RH demonstrated potential mucosal immune adjuvant properties by fostering the development of immune organs and augmenting the number of lymphocytes in the small intestinal villi. Additionally, the strain significantly upregulated the relative expression of inflammatory cytokines such as IL-1ß, IL-6, IL-10, TNF-α, and IFN-γ in the small intestinal mucosa. In conclusion, the B. subtilis RH strain developed in this study exhibits promising mucosal immunogenic effects. It holds potential as a candidate for an anti-NDV mucosal subunit vaccine and offers a novel preventive strategy for the poultry industry against this disease.

Recombinant Newcastle disease viruses expressing immunological checkpoint inhibitors induce a pro-inflammatory state and enhance tumor-specific immune responses in two murine models of cancer.

Introduction: Tumor microenvironments are immunosuppressive due to progressive accumulation of mutations in cancer cells that can drive expression of a range of inhibitory ligands and cytokines, and recruitment of immunomodulatory cells, including myeloid-derived suppressor cells (MDSC), tumor-associated macrophages, and regulatory T cells (Tregs). Methods: To reverse this immunosuppression, we engineered mesogenic Newcastle disease virus (NDV) to express immunological checkpoint inhibitors anti-cytotoxic T lymphocyte antigen-4 and soluble programmed death protein-1. Results: Intratumoral administration of recombinant NDV (rNDV) to mice bearing intradermal B16-F10 melanomas or subcutaneous CT26LacZ colon carcinomas led to significant changes in the tumor-infiltrating lymphocyte profiles. Vectorizing immunological checkpoint inhibitors in NDV increased activation of intratumoral natural killer cells and cytotoxic T cells and decreased Tregs and MDSCs, suggesting induction of a pro-inflammatory state with greater infiltration of activated CD8+ T cells. These notable changes translated to higher ratios of activated effector/suppressor tumor-infiltrating lymphocytes in both cancer models, which is a promising prognostic marker. Whereas all rNDV-treated groups showed evidence of tumor regression and increased survival in the CT26LacZ and B16-F10, only treatment with NDV expressing immunological checkpoint blockades led to complete responses compared to tumors treated with NDV only. Discussion: These data demonstrated that NDV expressing immunological checkpoint inhibitors could reverse the immunosuppressive state of tumor microenvironments and enhance tumor-specific T cell responses.

The artificial amino acid change in the sialic acid-binding domain of the hemagglutinin neuraminidase of newcastle disease virus increases its specificity to HCT 116 colorectal cancer cells and tumor suppression effect.

BACKGROUND: Oncolytic viruses are being studied and developed as novel cancer treatments. Using directed evolution technology, structural modification of the viral surface protein increases the specificity of the oncolytic virus for a particular cancer cell. Newcastle disease virus (NDV) does not show specificity for certain types of cancer cells during infection; therefore, it has low cancer cell specificity. Hemagglutinin is an NDV receptor-binding protein on the cell surface that determines host cell tropism. NDV selectivity for specific cancer cells can be increased by artificial amino acid changes in hemagglutinin neuraminidase HN proteins via directed evolution, leading to improved therapeutic effects. METHODS: Sialic acid-binding sites (H domains) of the HN protein mutant library were generated using error-prone PCR. Variants of the H domain protein were screened by enzyme-linked immunosorbent assay using HCT 116 cancer cell surface molecules. The mutant S519G H domain protein showed the highest affinity for the surface protein of HCT 116 cells compared to that of different types of cancer cells. This showed that the S519G mutant H domain protein gene replaced the same part of the original HN protein gene, and S519G mutant recombinant NDV (rNDV) was constructed and recovered. S519G rNDV cancer cell killing effects were tested using the MTT assay with various cancer cell types, and the tumor suppression effect of the S519G mutant rNDV was tested in a xenograft mouse model implanted with cancer cells, including HCT 116 cells. RESULTS: S519G rNDV showed increased specificity and enhanced killing ability of HCT 116 cells among various cancer cells and a stronger suppressive effect on tumor growth than the original recombinant NDV. Directed evolution using an artificial amino acid change in the NDV HN (S519G mutant) protein increased its specificity and oncolytic effect in colorectal cancer without changing its virulence. CONCLUSION: These results provide a new methodology for the use of directed evolution technology for more effective oncolytic virus development.

Clinico-pathological assessment of virulent Newcastle Disease Virus in ducks

Abstract Newcastle disease (ND) is an infectious, highly contagious and lethal disease of avian species. It is considered that ducks are natural reservoir or carrier for Newcastle disease virus (NDV) and are resistant against different strains of NDV. Current study was designed to evaluate the pathogenesis of Newcastle disease in domestic ducks through histopathology, immunohistochemistry (IHC) and serum biochemical changes. For this purpose, eighty ducks were reared for 42 days and divided in two groups A and B. Ducks in group A were challenged with (NDV) at rate of 0.1 ml of ELD50 (virus titer 107.32/100µl) on second week of age, whereas Group B was control negative. Splenomegaly, atrophy of thymus and necrotic lesion in kidney were observed on 9th day of post infection. Hepatic degeneration and mononuclear cell infiltration were noticed in proventriculus and intestine in challenged ducks. Viral antigen detected in lungs, intestine, proventriculus and lymphoid organs of infected ducks through IHC. Albumin and total protein values were significantly low in infected groups A as compared to control group B. ALT, AST, and ALP values were significantly high in infected group A. On 5th and 7th day of post infection oropharyngeal swabs were negative for NDV and cloacal swabs were positive for NDV through Reverse transcriptase polymerase chain reaction. It is concluded that ducks are susceptible to NDV and virulent strain of NDV caused disease in ducks.

Resumo A doença de Newcastle (DN) é uma doença infecciosa, altamente contagiosa e letal de espécies aviárias. Considera-se que os patos são reservatórios ou portadores naturais do vírus da doença de Newcastle (VDN) e são resistentes a diferentes cepas de VDN. O presente estudo foi desenvolvido para avaliar a patogênese da DN em patos domésticos por meio de histopatologia, imuno-histoquímica (IHQ) e alterações bioquímicas séricas. Para este propósito, 80 patos foram criados por 42 dias e divididos em dois grupos A e B. Os patos do grupo A foram submetidos ao VDN a uma taxa de 0,1 ml de ELD50 (título viral de 107,32 / 100 µl) na segunda semana de idade, enquanto o Grupo B foi controle negativo. Esplenomegalia, atrofia do timo e lesão necrótica no rim foram observadas no 9º dia pós-infecção. Degeneração hepática e infiltração de células mononucleares foram observadas no proventrículo e intestino em patos infectados. Antígeno viral foi detectado em pulmões, intestino, proventrículo e órgãos linfoides de patos infectados por IHQ. Os valores de albumina e proteína total foram significativamente baixos no grupo A infectado em comparação com o grupo B. Os valores de ALT, AST e ALP foram significativamente altos no grupo A. No 5º e no 7º dia após a infecção, os esfregaços orofaríngeos foram negativos para VDN, enquanto os esfregaços cloacais foram positivos para VDN por meio da reação em cadeia da polimerase via transcriptase reversa. Conclui-se que os patos são suscetíveis ao VDN e à cepa virulenta de VDN que causou doenças em patos.

Clinico-pathological assessment of virulent Newcastle Disease Virus in ducks / Avaliação clínico-patológica do Vírus da Doença de Newcastle em patos

Abstract Newcastle disease (ND) is an infectious, highly contagious and lethal disease of avian species. It is considered that ducks are natural reservoir or carrier for Newcastle disease virus (NDV) and are resistant against different strains of NDV. Current study was designed to evaluate the pathogenesis of Newcastle disease in domestic ducks through histopathology, immunohistochemistry (IHC) and serum biochemical changes. For this purpose, eighty ducks were reared for 42 days and divided in two groups A and B. Ducks in group A were challenged with (NDV) at rate of 0.1 ml of ELD50 (virus titer 107.32/100µl) on second week of age, whereas Group B was control negative. Splenomegaly, atrophy of thymus and necrotic lesion in kidney were observed on 9th day of post infection. Hepatic degeneration and mononuclear cell infiltration were noticed in proventriculus and intestine in challenged ducks. Viral antigen detected in lungs, intestine, proventriculus and lymphoid organs of infected ducks through IHC. Albumin and total protein values were significantly low in infected groups A as compared to control group B. ALT, AST, and ALP values were significantly high in infected group A. On 5th and 7th day of post infection oropharyngeal swabs were negative for NDV and cloacal swabs were positive for NDV through Reverse transcriptase polymerase chain reaction. It is concluded that ducks are susceptible to NDV and virulent strain of NDV caused disease in ducks.

Resumo A doença de Newcastle (DN) é uma doença infecciosa, altamente contagiosa e letal de espécies aviárias. Considera-se que os patos são reservatórios ou portadores naturais do vírus da doença de Newcastle (VDN) e são resistentes a diferentes cepas de VDN. O presente estudo foi desenvolvido para avaliar a patogênese da DN em patos domésticos por meio de histopatologia, imuno-histoquímica (IHQ) e alterações bioquímicas séricas. Para este propósito, 80 patos foram criados por 42 dias e divididos em dois grupos A e B. Os patos do grupo A foram submetidos ao VDN a uma taxa de 0,1 ml de ELD50 (título viral de 107,32 / 100 µl) na segunda semana de idade, enquanto o Grupo B foi controle negativo. Esplenomegalia, atrofia do timo e lesão necrótica no rim foram observadas no 9º dia pós-infecção. Degeneração hepática e infiltração de células mononucleares foram observadas no proventrículo e intestino em patos infectados. Antígeno viral foi detectado em pulmões, intestino, proventrículo e órgãos linfoides de patos infectados por IHQ. Os valores de albumina e proteína total foram significativamente baixos no grupo A infectado em comparação com o grupo B. Os valores de ALT, AST e ALP foram significativamente altos no grupo A. No 5º e no 7º dia após a infecção, os esfregaços orofaríngeos foram negativos para VDN, enquanto os esfregaços cloacais foram positivos para VDN por meio da reação em cadeia da polimerase via transcriptase reversa. Conclui-se que os patos são suscetíveis ao VDN e à cepa virulenta de VDN que causou doenças em patos.

Genetic Diversity of Newcastle Disease Virus Involved in the 2021 Outbreaks in Backyard Poultry Farms in Tanzania.

Newcastle disease virus is a significant avian pathogen with the potential to decimate poultry populations all over the world and cause enormous economic losses. Distinct NDV genotypes are currently causing outbreaks worldwide. Due to the high genetic diversity of NDV, virulent strains that may result in a lack of vaccine protection are more likely to emerge and ultimately cause larger epidemics with massive economic losses. Thus, a more comprehensive understanding of the circulating NDV genotypes is critical to reduce Newcastle disease (ND) burden. In this study, NDV strains were isolated and characterized from backyard poultry farms from Tanzania, East Africa in 2021. Reverse-transcription polymerase chain reaction (RT-PCR) based on fusion (F) gene amplification was conducted on 79 cloacal or tracheal swabs collected from chickens during a suspected ND outbreak. Our results revealed that 50 samples out 79 (50/79; 63.3%) were NDV-positive. Sequencing and phylogenetic analyses of the selected NDV isolates showed that 39 isolates belonged to subgenotype VII.2 and only one isolate belonged to subgenotype XIII.1.1. Nucleotide sequences of the NDV F genes from Tanzania were closely related to recent NDV isolates circulating in southern Africa, suggesting that subgenotype VII.2 is the predominant subgenotype throughout Tanzania and southern Africa. Our data confirm the circulation of two NDV subgenotypes in Tanzania, providing important information to design genotype-matched vaccines and to aid ND surveillance. Furthermore, these results highlight the possibility of the spread and emergence of new NDV subgenotypes with the potential of causing future ND epizootics.

The production of Newcastle disease virus-like particles in Nicotiana benthamiana as potential vaccines.

Newcastle disease (ND) is a highly contagious viral respiratory and neurological disease that has a severe impact on poultry production worldwide. In the present study, an expression platform was established for the transient production in N.bethamiana of ND virus-like particles (VLPs) for use as vaccines against ND. The expression of the ND Fusion (F) and/or Hemagglutinin-neuraminidase (HN) proteins of a genotype VII.2 strain formed ND VLPs in planta as visualized under the transmission electron microscope, and HN-containing VLPs agglutinated chicken erythrocytes with hemagglutination (HA) titres of up to 13 log2.The immunogenicity of the partially-purified ND VLPs was confirmed in specific-pathogen-free White leghorn chickens. Birds receiving a single intramuscular immunization with 1024 HA units (10 log2) of the F/HN ND VLPs administered with 20% [v/v] Emulsigen®-P adjuvant, seroconverted after 14 days with F- and HN-specific antibodies at ELISA titres of 5705.17 and HI geometric mean titres (GMTs) of 6.2 log2, respectively. Furthermore, these ND-specific antibodies successfully inhibited viral replication in vitro of two antigenically closely-related ND virus isolates, with virus-neutralization test GMTs of 3.47 and 3.4, respectively. Plant-produced ND VLPs have great potential as antigen-matched vaccines for poultry and other avian species that are highly immunogenic, cost-effective, and facilitate prompt updating to ensure improved protection against emerging ND field viruses.

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

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

Adaptor complex-mediated trafficking of Newcastle disease virus fusion protein is regulated by the YLMY motif of its cytoplasmic tail.

Previously, we reported that the mediation of Newcastle disease virus (NDV) pathogenicity by the 524YLMY527 motif depends mainly on the regulation of F protein transport to the cell surface. The virus and host determinants that govern this intracellular trafficking remain unknown. Here, we confirmed that host adaptor protein (AP) complexes are involved in NDV infection using small interfering RNA. The transport of viral F protein to the cell surface depends on host transport proteins. We observed that the trends for host expression of AP complexes AP1M1 and AP2M1 were similar to those of mutated F proteins, especially in the membrane protein. NDV F protein interacted with AP1M1 and AP2M1, and the YLMY motif influenced this interaction. Knockdown of AP1M1 or AP2M1 suppressed the intracellular and extracellular virus titre of mutated-YLMY-motif NDVs, especially rSG10*-F/Y527A and rSG10*-F/Y524AY527A, to varying degrees. Therefore, the YLMY motif regulates AP-mediated viral F protein transportation from the cytoplasm to the cell surface and subsequently affects viral titer. We further found that the YLMY-motif mutants were differently associated with the process of AAK1 and GAK kinase-mediated AP - viral F protein interaction. These data demonstrate that the essential YLMY motif located in the NDV F protein cytoplasmic tail recruits AP to direct the F protein to the cell surface, which is necessary for its ability to affect virus budding. This study provides support for a deeper understanding of virus and host determinants that facilitate virus trafficking, which can be exploited in the design of novel antiviral therapies.

Oncolytic Newcastle Disease Virus Co-Delivered with Modified PLGA Nanoparticles Encapsulating Temozolomide against Glioblastoma Cells: Developing an Effective Treatment Strategy.

Glioblastoma multiforme (GBM) is considered to be one of the most serious version of primary malignant tumors. Temozolomide (TMZ), an anti-cancer drug, is the most common chemotherapeutic agent used for patients suffering from GBM. However, due to its inherent instability, short biological half-life, and dose-limiting characteristics, alternatives to TMZ have been sought. In this study, the TMZ-loaded PLGA nanoparticles were prepared by employing the emulsion solvent evaporation technique. The prepared TMZ-PLGA-NPs were characterized using FT-IR, zeta potential analyses, XRD pattern, particle size estimation, TEM, and FE-SEM observations. The virotherapy, being safe, selective, and effective in combating cancer, was employed, and TMZ-PLGA-NPs and oncolytic Newcastle Disease Virus (NDV) were co-administered for the purpose. An AMHA1-attenuated strain of NDV was propagated in chicken embryos, and the virus was titrated in Vero-slammed cells to determine the infective dose. The in vitro cytotoxic effects of the TMZ, NDV, and the TMZ-PLGA-NPs against the human glioblastoma cancer cell line, AMGM5, and the normal cell line of rat embryo fibroblasts (REFs) were evaluated. The synergistic effects of the nano-formulation and viral strain combined therapy was observed on the cell lines in MTT viability assays, together with the Chou-Talalay tests. The outcomes of the in vitro investigation revealed that the drug combinations of NDV and TMZ, as well as NDV and TMZ-PLGA-NPs exerted the synergistic enhancements of the antitumor activity on the AMGM5 cell lines. The effectiveness of both the mono, and combined treatments on the capability of AMGM5 cells to form colonies were also examined with crystal violet dyeing tests. The morphological features, and apoptotic reactions of the treated cells were investigated by utilizing the phase-contrast inverted microscopic examinations, and acridine orange/propidium iodide double-staining tests. Based on the current findings, the potential for the use of TMZ and NDV as part of a combination treatment of GBM is significant, and may work for patients suffering from GBM.

Detection of Velogenic Avian Paramyxoviruses in Rock Doves in New York City, New York.

Avian paramyxovirus 1 (APMV-1), also known as Newcastle disease virus (NDV), causes severe and economically important disease in poultry around the globe. Although a limited amount of APMV-1 strains in urban areas have been characterized, the role of the urban wild bird population as an APMV-1 reservoir is unclear. Because urban birds may have an important role for long-term circulation of the virus, fecal and swab samples were collected by community scientists from wild birds in New York City (NYC), New York, United States. These samples were screened for APMV-1 and genotypically characterized by sequencing of the complete genome. A total of 885 samples were collected from NYC parks and from a local wildlife rehabilitation clinic from October 2020 through June 2021, and 255 samples obtained from 197 birds have been processed to date. Eight birds (4.1%) screened positive for the APMV-1 nucleoprotein gene by conventional reverse transcription PCR (RT-PCR), and two live viruses were isolated via egg culture. A multibasic F protein cleavage sequence, 112R R K K R F117, an indicator of highly pathogenic velogenic APMV-1 strains, was present in the two samples fully sequenced by next generation sequencing. Phylogenetic analysis of the F gene coding sequence classified both isolates into genotype VI, a diverse and predominant genotype responsible for APMV-1 outbreaks in pigeon and dove species worldwide. IMPORTANCE Here we describe the first large-scale effort to screen for APMV-1 in New York City's wild bird population as part of the New York City Virus Hunters program, a community science initiative. We characterized two isolates of APMV-1, with phylogenetic analyses suggesting diversity in established and circulating strains of pigeon paramyxoviruses. Our isolates are also domestic reference strains for future APMV-1 vaccine developments. Future surveillance in this region may contribute to our understanding of APMV-1's evolution and genetic diversity, as well as inform poultry husbandry and vaccination practices in New York State.

Contribution of mutation I142M in fusion protein and Q44R in matrix protein of Newcastle disease virus to virulence in ducks.

Although verogenic Newcastle disease viruses (NDVs) generally cause subclinical infection in waterfowls such as ducks, NDVs with high virulence in waterfowl have been sporadically reported. We previously reported that the NDV d5a20b strain, which is obtained by serial passaging of the velogenic 9a5b strain in domestic ducks, showed increased virulence in ducks (Hidaka et al., 2021). The d5a20b strain had 11 amino acid substitutions in its P/V, M, F, HN, and L proteins as compared to 9a5b. In the present study, we generated a series of recombinant (r) NDVs with these amino acid substitutions to identify the molecular basis of virulence of NDV in ducks, and evaluated their influences on virulence and in vitro viral properties. Each of the single amino acid substitutions in either the F protein I142M or the M protein Q44R contributed to the enhancement of intracerebral and intranasal pathogenicity in domestic ducks. The cell-cell fusion activity of the virus with F I142M was five times higher than that of the parental r9a5b. The virus with M Q44R rapidly replicated in duck embryo fibroblasts. Additionally, the rM+F+HN strain, which has the same amino acid sequences as d5a20b in M, F, and HN proteins, showed the highest level of virulence and replication efficiency among the generated recombinant viruses, nearly comparable to rd5a20b. These results suggest that multiple factors are involved in the high growth ability of NDV in duck cells, leading to increased virulence in vivo.

Bi/tri-specific antibodies (HN-Fc-CD16 and HN-Fc-IL-15-CD16) cross-linking natural killer (NK)-CD16 and Newcastle Disease Virus (NDV)-HN, enhanced NK activation for cancer immunotherapy.

Cancer/tumor cells infected with the "avian paramyxovirus Newcastle Disease Virus (TC-NDV)" express the viral hemagglutinin-neuraminidase (HN) on the cell surface that is used as both the danger signal and anchor for bi/tri-specific antibodies (bs/tsAbs).We constructed a bs-Ab (HN-Fc-CD16) that bindsto HN and natural killer (NK)-CD16 receptor (FcgRIII)and a ts-Ab (HN-Fc-IL15-CD16) harbouring NK-activating cytokine "IL-15" within the bs-Ab.In silicoand computational predictions indicated proper exposure of both Abs in bs/tsAbs.Properbinding of thebi/tsAbstoHN on surface of TC-NDVandCD16+-cells was demonstrated by flow cytometry.The bi/tsAbstriggeredspecificcytotoxicity of NK cells againstTC-NDVand elicited substantial IFN-γproduction by activated NK cells(higher for ts-Ab) that sound promising for cancer immunotherapy purposes.

In Vitro and In Vivo Characterization of a Pigeon Paramyxovirus Type 1 Isolated from Domestic Pigeons in Victoria, Australia 2011.

Significant mortalities of racing pigeons occurred in Australia in late 2011 associated with a pigeon paramyxovirus serotype 1 (PPMV-1) infection. The causative agent, designated APMV-1/pigeon/Australia/3/2011 (P/Aus/3/11), was isolated from diagnostic specimens in specific pathogen free (SPF) embryonated eggs and was identified by a Newcastle Disease virus (NDV)-specific RT-PCR and haemagglutination inhibition (HI) test using reference polyclonal antiserum specific for NDV. The P/Aus/3/11 strain was further classified as PPMV-1 using the HI test and monoclonal antibody 617/161 by HI and phylogenetic analysis of the fusion gene sequence. The isolate P/Aus/3/11 had a slow haemagglutin-elution rate and was inactivated within 45 min at 56 °C. Cross HI tests generated an R value of 0.25, indicating a significant antigenic difference between P/Aus/3/11 and NDV V4 isolates. The mean death time (MDT) of SPF eggs infected with the P/Aus/3/11 isolate was 89.2 hr, characteristic of a mesogenic pathotype, consistent with other PPMV-1 strains. The plaque size of the P/Aus/3/11 isolate on chicken embryo fibroblast (CEF) cells was smaller than those of mesogenic and velogenic NDV reference strains, indicating a lower virulence phenotype in vitro and challenge of six-week-old SPF chickens did not induce clinical signs. However, sequence analysis of the fusion protein cleavage site demonstrated an 112RRQKRF117 motif, which is typical of a velogenic NDV pathotype. Phylogenetic analysis indicated that the P/Aus/3/11 isolate belongs to a distinct subgenotype within class II genotype VI of avian paramyxovirus type 1. This is the first time this genotype has been detected in Australia causing disease in domestic pigeons and is the first time since 2002 that an NDV with potential for virulence has been detected in Australia.

Combining vanadyl sulfate with Newcastle disease virus potentiates rapid innate immune-mediated regression with curative potential in murine cancer models.

The avian paramyxovirus, Newcastle disease virus (NDV), is a promising oncolytic agent that has been shown to be safe and effective in a variety of pre-clinical cancer models and human clinical trials. NDV preferentially replicates in tumor cells due to signaling defects in apoptotic and antiviral pathways acquired during the transformation process and is a potent immunostimulatory agent. However, when used as a monotherapy NDV lacks the ability to consistently generate durable remissions. Here we investigate the use of viral sensitizer-mediated combination therapy to enhance the anti-neoplastic efficacy of NDV. Intratumoral injection of vanadyl sulfate, a pan-inhibitor of protein tyrosine phosphatases, in combination with NDV significantly increased the number and activation status of natural killer (NK) cells in the tumor microenvironment, concomitant with increased expression of interferon-ß, granulocyte-macrophage colony-stimulating factor, and monocyte chemoattractant protein-1, leading to rapid tumor regression and long-term cures in mice bearing syngeneic B16-F10 melanomas. The anti-tumor efficacy of this combination therapy was abrogated when NK cells were depleted and when interferon-ß expression was transiently suppressed. Tumor-specific CD8+ T cell responses were not detected, nor were mice whose tumors regressed protected from re-challenge. This suggested efficacy of the combination therapy predominantly relied on the innate immune system. Importantly, efficacy was not limited to melanoma; it was also demonstrated in a murine prostate cancer model. Taken together, these results suggest that combining NDV with vanadyl sulfate potentiates an innate immune response that can potentiate rapid clearance of tumors, with type I interferon signaling and NK cells being important mechanisms of action.

Expression and Purification of a Bispecific Antibody against CD16 and Hemagglutinin Neuraminidase (HN) in E. Coli for Cancer Immunotherapy.

BACKGROUND: : Immunotherapy of cancer by bispecific antibodies (bsAb) is an attractive approach for retargeting immune effector cells including natural killer (NK) cells to the tumor if the proper expression and purification of the bsAb for such applications could be addressed. Herein, we describe E. coli expression of a recombinant bsAb (bsHN-CD16) recognizing NK-CD16 and hemagglutinin neuraminidase (HN) of Newcastle Disease Virus (NDV). This bsAb might be efficient for ex vivo stimulation of NK cells via coupling to HN on the surface of the NDV-infected tumor cells. METHODS: A bsAb-encoding pcDNA3.1 vector (anti-HN scFv-Fc-anti-CD16 scFv) was used as a template, and the scFv segments (after enzymatic digestion and cutting of the Fc part) were rejoined to construct the Fc-deprived bsAb (anti-HN scFv-anti-CD16 scFv; bsHN-CD16). The constructed bsHN-CD16 was inserted into the HindIII and BamHI site of the T7 promoter-based pET28a plasmid. Following restriction analyses and DNA sequencing to confirm the cloning steps, bsHN-CD16 encoding pET28a was transformed into the E. coli (Rosetta DE3 strain), induced for protein expression by IPTG, and the protein was purified under native condition by Ni/NTA column using imidazole. RESULTS: Analyses by SDS-PAGE and Western Blotting using Rabbit anti-human whole IgG-HRP conjugate, confirmed the expression and purification of the bsAb with the expected full size of 55 kDa and yields around 8% of the total protein. CONCLUSION: Results showed efficient production of the bsAb in E. coli for future large-scale purification.