RESUMO
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.
Assuntos
Eritrócitos , Ácido N-Acetilneuramínico , Vírus da Doença de Newcastle , Vírus da Doença de Newcastle/fisiologia , Vírus da Doença de Newcastle/metabolismo , Eritrócitos/parasitologia , Eritrócitos/metabolismo , Animais , Ácido N-Acetilneuramínico/metabolismo , Humanos , Plasmodium yoelii/metabolismo , Camundongos , Proteína HN/metabolismo , Malária/parasitologia , Malária/metabolismoRESUMO
Sialoglycan-binding enveloped viruses often possess receptor-destroying activity to avoid being immobilized by non-functional decoy receptors. Sialic acid (Sia)-binding paramyxoviruses contain a hemagglutinin-neuraminidase (HN) protein that possesses both Sia-binding and -cleavage activities. The multivalent, dynamic receptor interactions of paramyxovirus particles provide virion motility and are a key determinant of host tropism. However, such multivalent interactions have not been exhaustively analyzed, because such studies are complicated by the low affinity of the individual interactions and the requirement of high titer virus stocks. Moreover, the dynamics of multivalent particle-receptor interactions are difficult to predict from Michaelis-Menten enzyme kinetics. Therefore, we here developed Ni-NTA nanoparticles that multivalently display recombinant soluble HN tetramers via their His tags (HN-NPs). Applying this HN-NP platform to Newcastle disease virus (NDV), we investigated using biolayer interferometry (BLI) the role of important HN residues in receptor-interactions and analyzed long-range effects between the catalytic site and the second Sia binding site (2SBS). The HN-NP system was also applicable to other paramyxoviruses. Comparative analysis of HN-NPs revealed and confirmed differences in dynamic receptor-interactions between type 1 human and murine parainfluenza viruses as well as of lab-adapted and clinical isolates of human parainfluenza virus type 3, which are likely to contribute to differences in tropism of these viruses. We propose this novel platform to be applicable to elucidate the dynamics of multivalent-receptor interactions important for host tropism and pathogenesis, particularly for difficult to grow sialoglycan-binding (paramyxo)viruses.
Assuntos
Proteína HN , Nanopartículas , Vírus da Doença de Newcastle , Receptores Virais , Proteína HN/metabolismo , Proteína HN/genética , Animais , Vírus da Doença de Newcastle/metabolismo , Vírus da Doença de Newcastle/fisiologia , Vírus da Doença de Newcastle/genética , Receptores Virais/metabolismo , Humanos , Ácido N-Acetilneuramínico/metabolismoRESUMO
Newcastle disease virus (NDV) is the pathogen of a zoonosis that is primarily transmitted by poultry and has severe infectivity and a high fatality rate. Many studies have focused on the role of the NDV fusion (F) protein in the cell-cell membrane fusion process. However, little attention has been given to the heptad repeat region, HR4, which is located in the NDV F2 subunit. Here, site-directed mutants were constructed to study the function of the NDV F protein HR4 region and identify the key amino acids in this region. Nine conserved amino acids were substituted with alanine or the corresponding amino acid of other aligned paramyxoviruses. The desired mutants were examined for changes in fusogenic activity through three kinds of membrane fusion assays and expression and proteolysis through IFA, FACS and WB. The results showed that when conserved amino acids (L81, Y84, L88, L91, L92, P94, L95 and I99) were replaced with alanine, the fusogenic activity of the F protein was abolished, possibly because of failed protein expression not only on the cell surface but also inside cells. These data indicated that the conserved amino acids above in NDV F HR4 are critical for normal protein synthesis and expression, possibly for the stability of the F protein monomer, formation of trimer and conformational changes.
Assuntos
Mutagênese Sítio-Dirigida , Vírus da Doença de Newcastle , Proteínas Virais de Fusão , Internalização do Vírus , Vírus da Doença de Newcastle/genética , Vírus da Doença de Newcastle/metabolismo , Proteínas Virais de Fusão/genética , Proteínas Virais de Fusão/metabolismo , Animais , Substituição de Aminoácidos , Linhagem Celular , Mutação , Proteólise , Fusão de MembranaRESUMO
The transcription and replication of the Newcastle disease virus (NDV) strictly rely on the viral ribonucleoprotein (RNP) complex, which is composed of viral NP, P, L and RNA. However, it is not known whether other viral non-RNP proteins participate in this process for viral self-regulation. In this study, we used a minigenome (MG) system to identify the regulatory role of the viral non-RNP proteins V, M, W, F and HN. Among them, V significantly reduced MG-encoded reporter activity compared with the other proteins and inhibited the synthesis of viral mRNA and cRNA. Further, V interacted with NP. A mutation in residue W195 of V diminished V-NP interaction and inhibited inclusion body (IB) formation in NP-P-L-cotransfected cells. Furthermore, a reverse-genetics system for the highly virulent strain F48E9 was established. The mutant rF48E9-VW195R increased viral replication and apparently enhanced IB formation. In vivo experiments demonstrated that rF48E9-VW195R decreased virulence and retarded time of death. Overall, the results indicate that the V-NP interaction of the W195 mutant V decreased, which regulated viral RNA synthesis, IB formation, viral replication and pathogenicity. This study provides insight into the self-regulation of non-RNP proteins in paramyxoviruses.
Assuntos
Vírus da Doença de Newcastle , Proteínas Virais , Replicação Viral , Vírus da Doença de Newcastle/genética , Vírus da Doença de Newcastle/fisiologia , Vírus da Doença de Newcastle/metabolismo , Animais , Proteínas Virais/metabolismo , Proteínas Virais/genética , Nucleoproteínas/metabolismo , Nucleoproteínas/genética , Doença de Newcastle/virologia , Doença de Newcastle/metabolismo , Linhagem Celular , Regulação Viral da Expressão Gênica , RNA Viral/genética , RNA Viral/metabolismo , Galinhas , Virulência , Ligação Proteica , MutaçãoRESUMO
Lysosomes are acidic organelles that mediate the degradation and recycling of cellular waste materials. Damage to lysosomes can cause lysosomal membrane permeabilization (LMP) and trigger different types of cell death, including apoptosis. Newcastle disease virus (NDV) can naturally infect most birds. Additionally, it serves as a promising oncolytic virus known for its effective infection of tumor cells and induction of intensive apoptotic responses. However, the involvement of lysosomes in NDV-induced apoptosis remains poorly understood. Here, we demonstrate that NDV infection profoundly triggers LMP, leading to the translocation of cathepsin B and D and subsequent mitochondria-dependent apoptosis in various tumor and avian cells. Notably, the released cathepsin B and D exacerbate NDV-induced LMP by inducing the generation of reactive oxygen species. Additionally, we uncover that the viral Hemagglutinin neuraminidase (HN) protein induces the deglycosylation and degradation of lysosome-associated membrane protein 1 (LAMP1) and LAMP2 dependent on its sialidase activity, which finally contributes to NDV-induced LMP and cellular apoptosis. Overall, our findings elucidate the role of LMP in NDV-induced cell apoptosis and provide novel insights into the function of HN during NDV-induced LMP, which provide innovative approaches for the development of NDV-based oncolytic agents.
Assuntos
Proteína HN , Vírus da Doença de Newcastle , Animais , Vírus da Doença de Newcastle/metabolismo , Proteína HN/metabolismo , Catepsina B , Apoptose , Lisossomos/metabolismoRESUMO
Newcastle disease virus (NDV) typically induces severe illness in poultry and results in significant economic losses for the worldwide poultry sector. NDV, an RNA virus with a single-stranded negative-sense genome, is susceptible to mutation and immune evasion during viral transmission, thus imposing enormous challenges to avian health and poultry production. NDV is composed of six structural proteins and two nonstructural proteins that exert pivotal roles in viral infection and antiviral responses by interacting with host proteins. Nowadays, there is a particular focus on the mechanisms of virus-host protein interactions in NDV research, yet a comprehensive overview of such research is still lacking. Herein, we briefly summarize the mechanisms regarding the effects of virus-host protein interaction on viral infection, pathogenesis, and host immune responses. This review can not only enhance the present comprehension of the mechanism underlying NDV and host interplay, but also furnish a point of reference for the advancement of antiviral measures.
Assuntos
Interações entre Hospedeiro e Microrganismos , Vírus da Doença de Newcastle , Viroses , Animais , Antivirais , Evasão da Resposta Imune , Vírus da Doença de Newcastle/metabolismo , Viroses/metabolismoRESUMO
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.
Assuntos
Neoplasias Colorretais , Vírus Oncolíticos , Humanos , Animais , Camundongos , Vírus da Doença de Newcastle/genética , Vírus da Doença de Newcastle/metabolismo , Proteína HN/genética , Proteína HN/metabolismo , Neuraminidase/genética , Neuraminidase/metabolismo , Hemaglutininas , Ácido N-Acetilneuramínico/metabolismo , Células HCT116 , Vírus Oncolíticos/genética , Modelos Animais de Doenças , Proteínas de Membrana , Neoplasias Colorretais/terapiaRESUMO
Newcastle disease is a highly contagious viral infection primarily affecting poultry, leading to significant economic losses worldwide due to its high morbidity and mortality rates. Given the severity of the disease and its impact on the poultry industry, there is an urgent need for a preventative approach to tackle this issue. Developing an efficient and effective vaccine is a valuable step toward reducing the burden of this virus. Consequently, investing in preventive measures, such as vaccination programs, is a top priority to mitigate the economic losses associated with Newcastle disease and protect the livelihoods of those relying on the poultry industry. Despite many vaccines against this viral disease, it still infects many wild and domestic birds worldwide. In this work, chimeric proteins, composed of the recombinant B subunit of Enterotoxigenic E. coli with one or two HN (Hemagglutinin-neuraminidase) subunits of NDV (LHN and LHN2, respectively), expressed using E.coli host. In-silico, in-vitro, and In-vivo procedures were performed to evaluate the immunogenicity of these proteins. The sera from immunized mice were analyzed using Western Blotting and ELISA. The LHN2 protein with an extra HN subunit elicited a higher antibody titer than the LHN protein (P<0.05). Both products could effectively elicit an immune response against NDV and can be considered a component of Newcastle disease vaccine candidates.
Assuntos
Doença de Newcastle , Vacinas , Vacinas Virais , Animais , Camundongos , Vírus da Doença de Newcastle/genética , Vírus da Doença de Newcastle/metabolismo , Doença de Newcastle/prevenção & controle , Hemaglutininas/metabolismo , Neuraminidase/metabolismo , Imunidade Humoral , Galinhas , Escherichia coli/genética , Temperatura Alta , Vacinas/metabolismo , Modelos Animais , Vacinas Virais/metabolismo , Anticorpos Antivirais/metabolismoRESUMO
BACKGROUND: TRAIL is an anticancer drug that induces cancer cell apoptosis by interacting with death receptors (DRs). However, owing to low cell-surface expression of DRs, certain colorectal cancer (CRC) cells resist TRAIL-induced apoptosis. Newcastle disease virus (NDV) infection can elevate DR protein expression in cancer cells, potentially influencing their TRAIL sensitivity. However, the precise mechanism by which NDV infection modulates DR expression and impacts TRAIL sensitivity in cancer cells remains unknown. METHODS: Herein, we developed nonpathogenic NDV VG/GA strain-based recombinant NDV (rNDV) and TRAIL gene-containing rNDV (rNDV-TRAIL). We observed that viral infections lead to increased DR and TRAIL expressions and activate signaling proteins involved in intrinsic and extrinsic apoptosis pathways. Experiments were conducted in vitro using TRAIL-resistant CRC cells (HT-29) and nonresistant CRC cells (HCT116) and in vivo using relevant mouse models. RESULTS: rNDV-TRAIL was found to exhibit better apoptotic efficacy than rNDV in CRC cells. Notably, rNDV-TRAIL had the stronger cancer cell-killing effect in TRAIL-resistant CRC cells. Western blot analyses showed that both rNDV and rNDV-TRAIL infections activate signaling proteins involved in the intrinsic and extrinsic apoptotic pathways. Notably, rNDV-TRAIL promotes concurrent intrinsic and extrinsic signal transduction in both HCT-116 and HT-29 cells. CONCLUSIONS: Therefore, rNDV-TRAIL infection effectively enhances DR expression in DR-depressed HT-29 cells. Moreover, the TRAIL protein expressed by rNDV-TRAIL effectively interacts with DR, leading to enhanced apoptosis in TRAIL-resistant HT-29 cells. Therefore, rNDV-TRAIL has potential as a promising therapeutic approach for treating TRAIL-resistant cancers.
Assuntos
Antineoplásicos , Neoplasias Colorretais , Humanos , Animais , Camundongos , Vírus da Doença de Newcastle/genética , Vírus da Doença de Newcastle/metabolismo , Células HT29 , Células HCT116 , Antineoplásicos/metabolismo , Apoptose , Neoplasias Colorretais/genética , Neoplasias Colorretais/terapia , Ligante Indutor de Apoptose Relacionado a TNF/genéticaRESUMO
Spontaneous canine mammary carcinomas (CMCs) have been widely considered a good research model for human breast cancers, which brings much attention to CMCs. In recent years, the oncolytic effect of Newcastle disease virus (NDV) on cancer cells has been widely studied, but its effect on CMCs is still unclear. This study aims to investigate the oncolytic effect of NDV LaSota strain on canine mammary carcinoma cell line (CMT-U27) in vivo and in vitro. The in vitro cytotoxicity and immunocytochemistry experiments showed that NDV selectively replicated in CMT-U27 cells, and inhibited cell proliferation and migration but not in MDCK cells. KEGG analysis of transcriptome sequencing indicated the importance of the TNFα and NF-κB signalling pathways in the anti-tumour effect of NDV. Subsequently, the significantly increased expression of TNFα, p65, phospho-p65, caspase-8, caspase-3 and cleaved-PARP proteins in the NDV group suggested that NDV induced CMT-U27 cells apoptosis by activating the caspase-8/caspase-3 pathway and the TNFα/NF-κB signalling pathway. Nude mice tumour-bearing experiments showed that NDV could significantly decrease the growth rate of CMC in vivo. In conclusion, our study demonstrates the effective oncolytic effects of NDV on CMT-U27 cells in vivo and in vitro, and suggests NDV as a promising candidate for oncolytic therapy.
Assuntos
Carcinoma , Doenças do Cão , Terapia Viral Oncolítica , Vírus Oncolíticos , Animais , Cães , Camundongos , Apoptose , Carcinoma/veterinária , Caspase 3/metabolismo , Caspase 8/metabolismo , Linhagem Celular Tumoral , Doenças do Cão/terapia , Camundongos Nus , Vírus da Doença de Newcastle/metabolismo , NF-kappa B/metabolismo , Terapia Viral Oncolítica/veterinária , Fator de Necrose Tumoral alfa/metabolismoRESUMO
BACKGROUND: Newcastle disease virus (NDV) is an oncolytic virus that can inhibit cancer cell proliferation and kill cancer cells. The NDV nonstructural V protein can regulate viral replication; however, whether the V protein contributes to NDV oncolysis is unclear. RESULTS: This study revealed that NDV inhibited tumor cell proliferation and that V protein expression promoted the proliferation of HepG2 cells, as determined at the single-cell level. In addition, to identify the regulatory mechanism of the V protein in HepG2 cells, transcriptome sequencing was performed and indicated that the expression/activation of multiple cell proliferation-related genes/signaling pathways were changed in cells overexpressing the V protein. Hence, the MAPK and WNT signaling pathways were selected for verification, and after blocking these two signaling pathways with inhibitors, the V protein promotion of cell proliferation was found to be attenuated. CONCLUSIONS: The results showed that the V protein regulated the proliferation of cancer cells through multiple signaling pathways, providing valuable references for future studies on the mechanism by which the V protein regulates cancer cell proliferation.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Terapia Viral Oncolítica , Vírus Oncolíticos , Animais , Humanos , Vírus Oncolíticos/genética , Vírus da Doença de Newcastle/metabolismo , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/terapia , Células Hep G2 , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/terapia , Proliferação de Células , Replicação Viral , Terapia Viral Oncolítica/métodosRESUMO
Type III interferons (IFNLs) have critical roles in the host's innate immune system, also serving as the first line against pathogenic infections of mucosal surfaces. In mammals, several IFNLs have been reported; however, only limited data on the repertoire of IFNLs in avian species is available. Previous studies showed only one member in chicken (chIFNL3). Herein, we identified a novel chicken IFNL for the first time, termed chIFNL3a, which contains 354 bp, and encodes 118 amino acids. The predicted protein is 57.1% amino acid identity with chIFNL. Genetic, evolutionary, and sequence analyses indicated that the new open reading frame (ORF) groups with type III chicken IFNs represent a novel splice variant. Compared to IFNs from different species, the new ORF is clustered within the type III IFNs group. Further study showed that chIFNL3a could activate a panel of IFN-regulated genes and function mediated by the IFNL receptor, and chIFNL3a markedly inhibited the replication of Newcastle disease virus (NDV) and influenza virus in vitro. These data collectively shed light on the repertoire of IFNs in avian species and provide useful information that further elucidate the interaction of the chIFNLs and viral infection of poultry. IMPORTANCE Interferons (IFNs) are critical soluble factors in the immune system, and are composed of 3 types (I, II, and III) that utilize different receptor complexes (IFN-αR1/IFN-αR2, IFN-γR1/IFN-γR2, and IFN-λR1/IL-10R2, respectively). Herein, we identified IFNL from the genomic sequences of chicken and termed it chIFNL3a, located on chromosome 7 of chicken. Phylogenetically clustered with all known types of chicken IFNs, the finding of this IFN is considered a type III IFN. To further evaluate the biological properties of chIFNL3a, the target protein was prepared by the baculovirus expression system (BES), which could markedly inhibit the replication of NDV and influenza viruses. In this study, we uncovered a new interferon lambda splice variant of chicken, termed chIFNL3a, which could inhibit viral replication in cells. Importantly, these novel findings may extend to other viruses, offering a new direction for therapeutic interventions.
Assuntos
Galinhas , Orthomyxoviridae , Animais , Interferon lambda , Antivirais/farmacologia , Interferons/metabolismo , Orthomyxoviridae/metabolismo , Vírus da Doença de Newcastle/metabolismo , MamíferosRESUMO
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.
Assuntos
Metilenotetra-Hidrofolato Desidrogenase (NADP) , Doença de Newcastle , Vírus da Doença de Newcastle , Replicação Viral , Animais , Metilenotetra-Hidrofolato Desidrogenase (NADP)/genética , Metilenotetra-Hidrofolato Desidrogenase (NADP)/metabolismo , Doença de Newcastle/enzimologia , Doença de Newcastle/fisiopatologia , Doença de Newcastle/virologia , Vírus da Doença de Newcastle/genética , Vírus da Doença de Newcastle/metabolismo , Nucleotídeos/metabolismo , Serina/metabolismo , Replicação Viral/genética , Linhagem Celular , Células A549 , Humanos , Mesocricetus , Técnicas de Silenciamento de Genes , Transporte Proteico/genética , Mitocôndrias/enzimologia , Regulação para Cima/fisiologiaRESUMO
NDV as an attractive candidate for oncolytic immunotherapy selectively lyses tumor cells but shows limited anti-tumor immunity. Immune co-stimulator OX40 ligand (OX40L) boosts anti-tumor immunity response by delivering a potent costimulatory signal to CD4+ and CD8+ T cells. To improve the anti-tumor immunity of NDV, the recombinant NDV expressing the murine OX40L (rNDV-mOX40L) was engineered. The viral growth kinetics was examined in CT26 cell lines. The ability of rNDV-mOX40L to express mOX40L was detected in the infected tumor cells and tumor tissues. The anti-tumor activity of rNDV-mOX40L was studied in the CT26 animal model. Tumor-specific CD4+, CD8+ and OX40+ T cells were examined by immunohistochemistry staining. The virus growth curve showed that the insertion of the mOX40L gene did not affect the growth kinetics of NDV. rNDV-mOX40L expresses mOX40L and effectively inhibits the growth of CT26 colorectal cancer in vivo. The tumor inhibition rate of the rNDV-mOX40L-treated group was increased by 15.8% compared to that of NDV-treated group in the CT26 model. Furthermore, immunohistochemistry staining of tumor tissues removed from the CT26 model revealed that intense infiltration of tumor-specific CD4+, CD8+ T cells, especially OX40+ T cells were found in the rNDV-mOX40L-treated group. FACS showed that rNDV-mOX40L significantly enhanced the number of CD4+ and CD8+ T cells in spleen. Moreover, compared to the NDV-treated group, the level of mouse IFN-γ protein in the tumor site increased significantly in the rNDV-mOX40L-treated group. Taken together, rNDV-mOX40L exhibited superior anti-tumor immunity by stimulating tumor-specific T cells and may be a promising agent for cancer immunotherapy.
Assuntos
Neoplasias Colorretais , Vírus Oncolíticos , Animais , Camundongos , Vírus da Doença de Newcastle/genética , Vírus da Doença de Newcastle/metabolismo , Linfócitos T CD8-Positivos , Adjuvantes Imunológicos/metabolismo , Ligante OX40/genética , Ligante OX40/metabolismo , Vírus Oncolíticos/genética , Interleucina-2 , Neoplasias Colorretais/terapiaRESUMO
The development of thermostable vaccines can relieve the bottleneck of existing vaccines caused by thermal instability and subsequent poor efficacy, which is one of the predominant reasons for the millions of deaths caused by vaccine-preventable diseases. Research into the mechanism of viral thermostability may provide strategies for developing thermostable vaccines. Using Newcastle disease virus (NDV) as model, we identified the negative surface charge of attachment glycoprotein as a novel determinant of viral thermostability. It prevented the temperature-induced aggregation of glycoprotein and subsequent detachment from virion surface. Then structural stability of virion surface was improved and virus could bind to and infect cells efficiently after heat-treatment. Employing the approach of surface charge engineering, thermal stability of NDV and influenza A virus (IAV) vaccines was successfully improved. The increase in the level of vaccine thermal stability was determined by the value-added in the negative surface charge of the attachment glycoprotein. The engineered live and inactivated vaccines could be used efficiently after storage at 37°C for at least 10 and 60 days, respectively. Thus, our results revealed a novel surface-charge-mediated link between HN protein and NDV thermostability, which could be used to design thermal stable NDV and IAV vaccines rationally.
Assuntos
Doença de Newcastle , Vacinas Virais , Animais , Galinhas/metabolismo , Glicoproteínas , Proteína HN/metabolismo , Doença de Newcastle/prevenção & controle , Vírus da Doença de Newcastle/metabolismoRESUMO
The aim of this study was to develop a semi-quantitative immunochromatographic method for rapid detection of Newcastle disease virus (NDV) antibodies by expressing HN protein in rice endosperm bioreactor. The recombinant plasmid pUC57-HN was digested by Mlyâ and Xhoâ to retrieve the HN gene, while the intermediate vector pMP3 containing promoter, signal peptide and terminator was digested by Naeâ and Xhoâ . The HN gene and the linearized pMP3 were purified and ligated to form a recombinant plasmid pMP3-HN1. Subsequently, pMP3-HN1 and plant vector pCAMBIA1300 were digested by EcoRâ and Hind â ¢, and the HN1 gene was cloned into pCAMBIA1300. The recombinant plasmid pCAMBIA1300-HN1 was introduced into Agrobacterium tumefaciens EHA105 by electrotransformation, and the pCAMBIA1300-HN1 was transferred into rice callus by agrobacterium-mediated method. After dark culture, callus screening, differentiation, rooting and transplanting, transgenic rice seeds were obtained 4 months later. PCR identified that the HN gene has been inserted into the rice genome. SDS-PAGE and Western blotting indicated that the HN protein was successfully expressed in the positive rice endosperm. The purity of the HN protein was more than 90% by SP cation exchange chromatography and gel filtration chromatography. According to the national standards for the diagnostic techniques of Newcastle disease HI test (HI≥4log2, positive antibody reaction), a colloidal gold labeled purified HN protein was used to prepare a semi-quantitative test strip by double-antibody sandwich method for rapid detection of NDV antibody. The results showed that the test strip did not cross-react with positive sera against other viruses, and the sensitivity of the test strip reached 1:102 400 for standard positive sera of Newcastle disease. Testing of a total of 308 clinical sera showed that the compliance rate of the test strip with HI test was 97.08%, and the Kappa value was 0.942. In conclusion, high purity recombinant HN protein was obtained from rice endosperm, and a simple, rapid, highly sensitive and highly specific semi-quantitative immunochromatographic strip was developed. The test strip could be used for immune evaluation of the Newcastle disease vaccine.
Assuntos
Doença de Newcastle , Oryza , Animais , Anticorpos Antivirais , Galinhas , Proteína HN/genética , Proteína HN/metabolismo , Doença de Newcastle/prevenção & controle , Vírus da Doença de Newcastle/genética , Vírus da Doença de Newcastle/metabolismo , Oryza/genéticaRESUMO
Interferon regulatory factors (IRFs) play a key role in many aspects of immune response, and IRF1, IRF3, and IRF7 are positive regulators of IFN induction in mammals. However, IRF3, as the most critical regulatory factor in mammals, is naturally absent in birds, which attracts us to study the functions of other members of the avian IRF family. In the present study, we cloned goose IRF1 (GoIRF1) and conducted a series of bioinformatics analyses to compare the protein homology of GoIRF1 with that of IRF1 in other species. The overexpression of GoIRF1 in DF-1 cells induced the activation of IFN-ß, and this activation is independent of the dosage of the transfected GoIRF1 plasmids. The overexpression of GoIRF1 in goose embryonic fibroblasts (GEFs) induced the expression of IFNs, proinflammatory cytokines, and IFN-stimulated genes (ISGs); it also inhibited the replication of green fluorescent protein (GFP)-tagged Newcastle disease virus (NDV) (NDV-GFP) and GFP-tagged vesicular stomatitis virus (VSV) (VSV-GFP). Our results suggest that GoIRF1 is an important regulator of IFNs, proinflammatory cytokines, and ISGs and plays a role in antiviral innate immunity in geese.
Assuntos
Gansos , Vírus da Doença de Newcastle , Animais , Imunidade Inata/genética , Interferon beta/metabolismo , Mamíferos , Vírus da Doença de Newcastle/metabolismo , Replicação Viral/genéticaRESUMO
The LaSota strain of Newcastle disease virus (NDV) is a commonly used vaccine to control Newcastle disease. However, improper immunization is a common reason for vaccine failure. Hence, it is imperative to thoroughly explore innate immunity-related molecular regulatory responses to the LaSota vaccine. In this text, 140 long non-coding RNAs (lncRNAs), 8 microRNAs (miRNAs), and 1514 mRNAs were identified to be differentially expressed by RNA sequencing analysis in the thymic tissues of Chinese Partridge Shank chickens after LaSota vaccine inoculation. Moreover, 70 dysregulated genes related to innate immunity were identified based on GO, Reactome pathway, and InnateDB annotations and differential expression analysis. Additionally, dysregulated lncRNAs and innate immunity-related mRNAs that could interact with dysregulated miRNAs were identified based on bioinformatics prediction analysis via the miRanda software and differential expression analysis. Among these transcripts, expression patterns of five lncRNAs, seven miRNAs, and six mRNAs were further examined by RT-qPCR assay. Both RNA-seq and RT-qPCR outcomes showed that 10 transcripts (MSTRG.22689.1, ENSGALT00000065826, ENSGALT00000059336, ENSGALT00000060887, gga-miR-6575-5p, gga-miR-6631-5p, gga-miR-1727, paraoxonase 2 (PON2), mitogen-activated protein kinase 10, and cystic fibrosis transmembrane conductance regulator (CFTR) were highly expressed, and 4 transcripts (MSTRG.188121.10, gga-miR-6655-5p, gga-miR-6548-3p, and matrix metallopeptidase 9 (MMP9) were low expressed after NDV infection. Additionally, two potential competing endogenous RNA networks (ENSGALT00000060887/gga-miR-6575-5p/PON2 or MSTRG.188121.10/gga-miR-6631-5p/MMP9) and some co-expression axes (ENSGALT00000065826/gga-miR-6631-5p, MSTRG.188121.10/gga-miR-6575-5p, MSTRG.188121.10/CFTR, ENSGALT00000060887/MMP9) were identified based on RT-qPCR and co-expression analyses. In conclusion, we identified multiple dysregulated lncRNAs, miRNAs, and mRNAs after LaSota infection and some potential regulatory networks for these dysregulated transcripts.
Assuntos
MicroRNAs , RNA Longo não Codificante , Vacinas , Animais , Galinhas/genética , Galinhas/metabolismo , China , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Metaloproteinase 9 da Matriz/genética , MicroRNAs/genética , MicroRNAs/metabolismo , Vírus da Doença de Newcastle/genética , Vírus da Doença de Newcastle/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transcriptoma/genética , Vacinas/metabolismoRESUMO
Newcastle disease virus (NDV) is a lethal virus in avian species with a disastrous effect on the poultry industry. NDV is enveloped by a host-derived membrane with two glycosylated haemagglutinin-neuraminidase (HN) and Fusion (F) proteins. NDV infection usually leads to death within 2-6 days, so the preexisting antibodies provide the most critical protection for this infection. The HN and F glycoproteins are considered the main targets of the immune system. In the present study, two constructs harboring the HN or F epitopes are sub-cloned separately under the control of a root-specific promoter NtREL1 or CaMV35S (35S Cauliflower Mosaic Virus promoter) as a constitutive promoter. The recombinant vectors were transformed into the Agrobacterium tumefaciens strain LBA4404 and then introduced to tobacco (Nicotiana tabacum L.) leaf disk explants. PCR with specific primers was performed to confirm the presence of the hn and f genes in the genome of the regenerated plants. Then, the positive lines were transformed via non-recombinant A. rhizogenes (strain ATCC15834) to develop hairy roots.HN and F were expressed at 0.37% and 0.33% of TSP using the CaMV35S promoter and at 0.75% and 0.54% of TSP using the NtREL1 promoter, respectively. Furthermore, the mice fed transgenic hairy roots showed a high level of antibody responses (IgG and IgA) against rHN and rF proteins.
Assuntos
Proteína HN , Nicotiana , Animais , Galinhas , Glicoproteínas/genética , Proteína HN/genética , Proteína HN/metabolismo , Camundongos , Vírus da Doença de Newcastle/genética , Vírus da Doença de Newcastle/metabolismo , Nicotiana/genética , Nicotiana/metabolismoRESUMO
BACKGROUND: Glioblastoma is one of the most serious brain cancer. Previous studies have demonstrated that PTEN function disorder affects the causing and exacerbation of glioblastoma. Newcastle disease virus (NDV) has been studied as a cancer virotherapeutics. In this study, PTEN gene was delivered to glioblastoma by recombinant NDV (rNDV) and translated into protein at the cytoplasm of the glioblastoma. METHODS: We did comparison tests PTEN protein expression efficiency and oncolytic effect depend on the PTEN gene insertion site at the between NP and P genes and the between P and M gene. PTEN protein mRNA transcription, translation in glioblastoma cell, and functional PTEN protein effect of the rNDV in vitro and in vivo test performed using western blotting, RT-qPCR, MTT assay, and Glioblastoma xenograft animal model test. RESULTS: The result of this study demonstrates that rNDV-PTEN kills glioblastoma cells and reduces cancer tissue better than rNDV without the PTEN gene. In molecular immunological and cytological assays, PTEN expression level was high at located in the between NP and P gene, and PTEN gene was successfully delivered to the glioblastoma cell using rNDV and PTEN gene translated to functional protein and inhibits hTERT and AKT gene. CONCLUSIONS: PTEN gene enhances the oncolytic effect of the rNDV. And our study demonstrated that NP and P gene site is better than P and M gene site which is commonly and conventionally used. PTEN gene containing rNDV is a good candidate virotherapeutics for glioblastoma.