RESUMO
Influenza and Newcastle disease are the most important poultry diseases that cause high annual damage to poultry farms worldwide. Newcastle virus fusion (F) gene and Influenza Virus Hemagglutinin (HA) gene are capable of encoding F and HA proteins that are the main factors in creating immunity, so this study aimed to clone and express these genes in Spodoptera frugiperda (Sf9) cells using baculovirus expression system. After isolating the Newcastle and Influenza virus genome, the HA gene of influenza virus and the F gene of Newcastle virus were amplified by reverse transcriptase PCR and specific primers and then cloned into pFastBacTM Dual plasmid. A recombinant sucker with these genes was produced in the DH10Bac host cell. By transfecting Sf9 cells with recombinant bacmid, expression was assessed by SDS-PAGE, western blotting, and Bradford methods. Cloning of genes into the bacmid was successful. By transfecting the recombinant bacmid into Spodoptera frugiperda cells, 218 µg/ml of the recombinant protein was obtained in the supernatant. In addition, the presence of protein was confirmed by western blotting. The PCR products of HA and F genes showed one band of 1.7 kb size using specific primers. The pFastHA1 vector was about 7 kb in size. Two bands of about 7 kb and 1.7 kb were created by ligation of the F gene and pFastHA1 vector based on enzymatic digestion, indicating the correct ligation of F gene under the P10 promoter. This is the first report on the cloning and Co-expression of two HA and F genes using baculovirus expression system and can be a candidate for dual influenza and Newcastle vaccine. Mixtures of these recombinant proteins can be used as vaccine candidates against both avian influenza and Newcastle disease.
Assuntos
Baculoviridae , Glicoproteínas de Hemaglutininação de Vírus da Influenza , Vírus da Influenza A Subtipo H9N2 , Vírus da Doença de Newcastle , Spodoptera , Animais , Baculoviridae/genética , Células Sf9 , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Vírus da Influenza A Subtipo H9N2/genética , Vírus da Doença de Newcastle/genética , Proteínas Virais de Fusão/genética , Proteínas Virais de Fusão/metabolismo , Expressão Gênica , Clonagem Molecular/métodos , Vetores Genéticos/genéticaRESUMO
The virulent form of Avian paramyxovirus-1 (APMV-1), commonly known as Newcastle Disease Virus (NDV), is a pathogen with global implications for avian health, affecting both wild and domestic bird populations. In Pakistan, recurrent Newcastle Disease (caused by NDV) outbreaks have posed significant challenges to the poultry industry. Extensive surveillance in Pakistan over 20 years has demonstrated a dynamic genetic diversity among circulating APMV-1 strains, emphasizing the potential necessity for customized vaccination strategies and continuous surveillance. In this study, 13 APMV-1-positive isolates harboring four different APMV-1 genotypes circulating throughout Pakistan were identified. These included the highly virulent genotypes VII and XIII, genotype XXI, commonly associated with Columbiformes, and genotype II, hypothesized to have been detected following vaccination. These findings underscore the intricate interplay of mutational events and host-immune interactions shaping the evolving NDV landscape. This study advances our understanding of the evolutionary dynamics of APMV-1 in Pakistan, highlighting the need for tailored vaccination strategies and continuous surveillance to enable effective APMV-1 management in avian populations, further emphasizing the importance of globally coordinated strategies to tackle APMV-1, given its profound impact on wild and domestic birds.
Assuntos
Evolução Molecular , Variação Genética , Genoma Viral , Genótipo , Doença de Newcastle , Vírus da Doença de Newcastle , Filogenia , Animais , Paquistão/epidemiologia , Vírus da Doença de Newcastle/genética , Vírus da Doença de Newcastle/classificação , Vírus da Doença de Newcastle/isolamento & purificação , Doença de Newcastle/virologia , Doença de Newcastle/epidemiologia , Aves/virologia , Aves Domésticas/virologia , Doenças das Aves Domésticas/virologia , Doenças das Aves Domésticas/epidemiologia , Galinhas/virologiaRESUMO
Newcastle disease virus (NDV) is a highly pathogenic avian infectious disease agent and also a promising oncolytic virus with broad application prospects. The Endosomal Sorting Complex Required for Transport (ESCRT) machinery has been increasingly recognized for its crucial role in the life cycles of enveloped viruses, influencing processes such as viral entry, replication, and budding. In this study, we employed an RNA interference screening approach to identify key ESCRT components that regulate NDV replication in tumor cells. qPCR, immunofluorescence, and Western blot assays demonstrated that knockdown of HRS, CHMP4A, CHMP4B, and CHMP4C significantly impaired NDV replication in HeLa cells, with HRS exhibiting the most pronounced inhibitory effect. Additionally, HRS knockout significantly inhibited viral budding and suppressed NDV-induced cell death in HeLa cells. Notably, NDV infection was shown to significantly upregulate HRS gene and protein expression in a time-dependent manner. In conclusion, this study systematically identifies critical ESCRT components involved in NDV replication within tumor cells, with a particular focus on the role of HRS in promoting NDV's replication by promoting viral budding, offering new insights for the development of NDV-based oncolytic therapies.
Assuntos
Complexos Endossomais de Distribuição Requeridos para Transporte , Vírus da Doença de Newcastle , Liberação de Vírus , Replicação Viral , Vírus da Doença de Newcastle/fisiologia , Vírus da Doença de Newcastle/genética , Humanos , Células HeLa , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Vírus Oncolíticos/fisiologia , Vírus Oncolíticos/genética , AnimaisRESUMO
Introduction: Newcastle disease is one of the significant issues in the poultry industry, having catastrophic effects worldwide. The lung is one of the essential organs which harbours Bronchus-associated lymphoid tissue and plays a vital role in the immune response. Leghorn and Fayoumi breeds are known to have differences in resistance to Newcastle disease. Along with genes and long non-coding RNAs (lncRNAs) are also known to regulate various biological pathways through gene regulation. Methods: This study analysed the lung transcriptome data and identified the role of genes and long non-coding RNAs in differential immune resistance. The computational pipeline, FHSpipe, as used in our previous studies on analysis of harderian gland and trachea transcriptome was used to identify genes and lncRNAs. This was followed by differential expression analysis, functional annotation of genes and lncRNAs, identification of transcription factors, microRNAs and finally validation using qRT-PCR. Results and discussion: A total of 8219 novel lncRNAs were identified. Of them, 1263 lncRNAs and 281 genes were differentially expressed. About 66 genes were annotated with either an immune-related GO term or pathway, and 12 were annotated with both. In challenge and breed-based analysis, most of these genes were upregulated in Fayoumi compared to Leghorn, and in timepoint-based analysis, Leghorn challenge chicken showed downregulation between time points. A similar trend was observed in the expression of lncRNAs. Co-expression analysis has revealed several lncRNAs co-expressing with immune genes with a positive correlation. Several genes annotated with non-immune pathways, including metabolism, signal transduction, transport of small molecules, extracellular matrix organization, developmental biology and cellular processes, were also impacted. With this, we can understand that Fayoumi chicken showed upregulated immune genes and positive cis-lncRNAs during both the non-challenged and NDV-challenge conditions, even without viral transcripts in the tissue. This finding shows that these immune-annotated genes and coexpressing cis-lncRNAs play a significant role in Fayoumi being comparatively resistant to NDV compared to Leghorn. Our study affirms and expands upon the outcomes of previous studies and highlights the crucial role of lncRNAs during the immune response to NDV. Conclusion: This analysis clearly shows the differences in the gene expression patterns and lncRNA co-expression with the genes between Leghorn and Fayoumi, indicating that the lncRNAs and co-expressing genes might potentially have a role in differentiating these breeds. We hypothesise that these genes and lncRNAs play a vital role in the higher resistance of Fayoumi to NDV than Leghorn. This study can pave the way for future studies to unravel the biological mechanism behind the regulation of immune-related genes.
Assuntos
Galinhas , Perfilação da Expressão Gênica , Pulmão , MicroRNAs , Doença de Newcastle , Vírus da Doença de Newcastle , Doenças das Aves Domésticas , RNA Longo não Codificante , Transcriptoma , Animais , Galinhas/genética , Galinhas/imunologia , Doença de Newcastle/imunologia , Doença de Newcastle/virologia , Doença de Newcastle/genética , Pulmão/imunologia , Pulmão/virologia , Vírus da Doença de Newcastle/imunologia , Vírus da Doença de Newcastle/genética , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Doenças das Aves Domésticas/virologia , Doenças das Aves Domésticas/imunologia , Doenças das Aves Domésticas/genética , MicroRNAs/genética , MicroRNAs/metabolismo , Regulação da Expressão Gênica , Resistência à Doença/genética , Biologia Computacional/métodos , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologiaRESUMO
Newcastle disease was suspected in 37 commercial poultry farms, including 12 layer and 25 broiler farms in four districts of Gujarat, India. Vaccination had been done in 32 (20 broilers and 12 layers) farms. Tissue samples from each farm were pooled as one sample. In egg embryo inoculation, HA-HI and PCR, respectively, 32/37, 29/37, and 24/37 samples were found positive. Pathotyping by mean death time calculation and primer combination PCR revealed velogenic NDV, which was later confirmed with the presence of the 112-RRQKR*F-117 sequence at the F protein cleavage site. Phylogenetic analysis of full F gene sequences (N=10) confirmed the presence of sub-genotype VII.2 in 9/10 sequences, and genotype II in one sample. These 9 sequences were only 0.7 to 2.6 % divergent with two VII.2 (=VIIi) sequences (HQ697254.1 chicken/Banjarmas/Indonesia and KU862293.1 Parakeet/Karachi/Pakistan) but had 2.2 to 3.6 % diversion from two VII.2 sequences (OR185447 and MZ546197) from India. Then branching was found from sequences of VIIh, VIIk (VII.2), and VIIa (VII.1.2), and then from sub-genotypes VII.1.1 and VII.1.2. Due to less than 5 % diversion, these sequences could not be qualified as new sub-genotype in evolutionary distance analysis. At the amino acid level, our sequences had aa N-T-I-A-L-T at 24-79-125-385-445-482. Whereas at the same positions, in most of the retrieved VII.2 sequences and vaccines, the sequence was S-A-V-T-Q/I- E/A. Two sequences revealed additional six and four amino acid differences,respectively.This indicates rapid continuous genetic evolution of sub-genotype VII.2 and partially explains vaccinal immunity escape.
Assuntos
Galinhas , Evolução Molecular , Genótipo , Doença de Newcastle , Vírus da Doença de Newcastle , Filogenia , Doenças das Aves Domésticas , Animais , Vírus da Doença de Newcastle/genética , Índia/epidemiologia , Doença de Newcastle/virologia , Galinhas/virologia , Doenças das Aves Domésticas/virologia , Vacinas Virais/genética , Vacinas Virais/imunologia , Vacinação/veterinária , FazendasRESUMO
As a founding member of the Src family of kinases, Src has been confirmed to participate in the regulation of immune responses, integrin signaling, and motility. Ducks are usually asymptomatic carriers of RNA viruses such as Newcastle disease virus and avian influenza virus, which can be deadly to chickens. The beneficial role of Src in modulating the immune response remains largely unknown in ducks. Here, we characterized the duck Src and found that it contains a 192-base-pair 5' untranslated region, a 1602-base-pair coding region, and a 2541-base-pair 3' untranslated region, encoding 533 amino acid residues. Additionally, duSrc transcripts were significantly activated in duck tissues infected by Newcastle disease virus compared to controls. The duSrc transcripts were notably widespread in all tissues examined, and the expression level was higher in liver, blood, lung, pancreas, and thymus. Moreover, we found the expression levels of IFN-ß, NF-κB, IRF3, and Src were significantly increased in DEFs after infection with 5'ppp dsRNA, but there was no significant difference before and after treatment in DF1 cells. Furthermore, overexpression of duSrc followed by stimulation with 5'ppp dsRNA led to an elevation of IFN-ß levels. The SH3 and PTKc domains of duSrc contributed to promoting the activity of IFN-ß and NF-κB in DEFs stimulated by 5'ppp dsRNA.
Assuntos
Clonagem Molecular , Patos , Animais , Patos/genética , Patos/imunologia , Patos/virologia , Quinases da Família src/genética , Quinases da Família src/metabolismo , Vírus da Doença de Newcastle/imunologia , Vírus da Doença de Newcastle/genética , Proteínas Aviárias/genética , Proteínas Aviárias/imunologia , Proteínas Aviárias/metabolismo , Doença de Newcastle/imunologia , Doença de Newcastle/virologia , Doença de Newcastle/genética , Interferon beta/genética , Interferon beta/imunologia , Interferon beta/metabolismo , Distribuição Tecidual , Doenças das Aves Domésticas/imunologia , Doenças das Aves Domésticas/virologia , Doenças das Aves Domésticas/genéticaRESUMO
Infectious bursal disease (IBD) is a widespread problem in the poultry industry, and vaccination is the primary preventive method. However, moderately virulent vaccines may damage the bursa, necessitating the development of a safe and effective vaccine. The Newcastle disease virus (NDV) has been explored as a vector for vaccine development. In this study, reverse genetic technology was used to obtain three recombinant viruses, namely, rClone30-VP2L (P/M)-chGM-CSF (NP), rClone30-chGM-CSF (P/M)-VP2L (NP), and rClone30-VP2L-chGM-CSF (P/M). Animal experiments showed that the three biological adjuvant bivalent vaccines effectively increased anti-NDV and anti-infectious bursal disease virus (IBDV) titres, enhancing both humoral and cellular immune responses in chickens without leading to any harm. Amongst the three biological adjuvant bivalent vaccines, the rClone30-chGM-CSF (P/M)-VP2L (NP) group had higher levels of anti-NDV antibodies at 14 days after the first immunization and stimulated a greater humoral immune response in 7-10 days. While, the rClone30-VP2L (P/M)-chGM-CSF (NP) group was the most effective in producing a higher level of IBDV antibody response. In conclusion, these three vaccines can induce immune responses more rapidly and effectively, streamline production processes, be cost-effective, and provide a new avenue for the development of Newcastle disease (ND) and IBD bivalent vaccines.
Assuntos
Anticorpos Antivirais , Infecções por Birnaviridae , Galinhas , Vírus da Doença Infecciosa da Bursa , Doença de Newcastle , Vírus da Doença de Newcastle , Doenças das Aves Domésticas , Vacinas Virais , Animais , Vacinas Virais/imunologia , Doenças das Aves Domésticas/prevenção & controle , Doenças das Aves Domésticas/virologia , Doenças das Aves Domésticas/imunologia , Infecções por Birnaviridae/prevenção & controle , Infecções por Birnaviridae/imunologia , Infecções por Birnaviridae/veterinária , Vírus da Doença de Newcastle/imunologia , Vírus da Doença de Newcastle/genética , Vírus da Doença Infecciosa da Bursa/imunologia , Vírus da Doença Infecciosa da Bursa/genética , Doença de Newcastle/prevenção & controle , Doença de Newcastle/imunologia , Anticorpos Antivirais/sangue , Imunidade Humoral , Adjuvantes Imunológicos/administração & dosagem , Adjuvantes de Vacinas , Imunidade Celular , VacinaçãoRESUMO
The extensive protein production in virus-infected cells can disrupt protein homeostasis and activate various proteolytic pathways. These pathways utilize post-translational modifications (PTMs) to drive the ubiquitin-mediated proteasomal degradation of surplus proteins. Protein arginylation is the least explored PTM facilitated by arginyltransferase 1 (ATE1) enzyme. Several studies have provided evidence supporting its importance in multiple physiological processes, including ageing, stress, nerve regeneration, actin formation and embryo development. However, its function in viral pathogenesis is still unexplored. The present work utilizes Newcastle disease virus (NDV) as a model to establish the role of the ATE1 enzyme and its activity in pathogenesis. Our data indicate a rise in levels of N-arginylated cellular proteins in the infected cells. Here, we also explore the haemagglutinin-neuraminidase (HN) protein of NDV as a presumable target for arginylation. The data indicate that the administration of Arg amplifies the arginylation process, resulting in reduced stability of the HN protein. ATE1 enzyme activity inhibition and gene expression knockdown studies were also conducted to analyse modulation in HN protein levels, which further substantiated the findings. Moreover, we also observed Arg addition and probable ubiquitin modification to the HN protein, indicating engagement of the proteasomal degradation machinery. Lastly, we concluded that the enhanced levels of the ATE1 enzyme could transfer the Arg residue to the N-terminus of the HN protein, ultimately driving its proteasomal degradation.
Assuntos
Aminoaciltransferases , Vírus da Doença de Newcastle , Complexo de Endopeptidases do Proteassoma , Processamento de Proteína Pós-Traducional , Proteólise , Animais , Embrião de Galinha , Cricetinae , Humanos , Aminoaciltransferases/metabolismo , Aminoaciltransferases/genética , Arginina/metabolismo , Linhagem Celular , Proteína HN/metabolismo , Proteína HN/genética , Interações Hospedeiro-Patógeno , Doença de Newcastle/virologia , Doença de Newcastle/metabolismo , Vírus da Doença de Newcastle/genética , Vírus da Doença de Newcastle/metabolismo , Vírus da Doença de Newcastle/fisiologia , Complexo de Endopeptidases do Proteassoma/metabolismoRESUMO
Newcastle disease virus (NDV), an avian paramyxovirus, causes major economic losses in the poultry industry worldwide. NDV strains are classified as avirulent, moderately virulent, or virulent according to the severity of the disease they cause. In order to gain a deeper understanding of the molecular mechanisms of virus-host interactions, we conducted Illumina HiSeq-based RNA-Seq analysis on chicken embryo fibroblast (DF1) cells during the first 24 hours of infection with NDV strain Komarov. Comparative analysis of uninfected DF1 cells versus NDV-infected DF1 cells at 6, 12, and 24 h postinfection identified 462, 459, and 410 differentially expressed genes, respectively. The findings revealed an increase in the expression of genes linked to the MAPK signalling pathway in the initial stages of NDV infection. This overexpression potentially aids viral multiplication while hindering pathogen detection and subsequent immune responses from the host. Our findings provide initial insights into the early responses of DF1 cells to NDV infection.
Assuntos
Galinhas , Fibroblastos , Perfilação da Expressão Gênica , Interações Hospedeiro-Patógeno , Doença de Newcastle , Vírus da Doença de Newcastle , Vírus da Doença de Newcastle/genética , Vírus da Doença de Newcastle/patogenicidade , Vírus da Doença de Newcastle/fisiologia , Animais , Doença de Newcastle/virologia , Doença de Newcastle/imunologia , Galinhas/virologia , Fibroblastos/virologia , Interações Hospedeiro-Patógeno/genética , Embrião de Galinha , Linhagem Celular , Transcriptoma , Doenças das Aves Domésticas/virologia , Doenças das Aves Domésticas/genética , Replicação Viral/genéticaRESUMO
Newcastle disease (ND) is caused by virulent strains of avian paramyxovirus type 1, also known as Newcastle disease virus (NDV). Despite vaccination, the frequency of reported outbreaks in Ethiopia has increased. From January to June 2022, an active outbreak investigation was conducted in six commercial chicken farms across areas of central Ethiopia to identify the circulating NDV strains. Thirty pooled tissue specimens were collected from chickens suspected of being infected with NDV. A questionnaire survey of farm owners and veterinarians was also carried out to collect information on the farms and the outbreak status. NDV was isolated using specific-pathogen-free (SPF)-embryonated chicken eggs and detected using haemagglutination and the reverse transcriptase-polymerase chain reaction (RT-PCR). The genotype and virulence of field NDV isolates were determined using phylogenetic analysis of fusion (F) protein gene sequences and the mean death time (MDT) test in SPF-embryonated chicken eggs. The questionnaire results revealed that ND caused morbidity (23.1%), mortality (16.3%), case fatality (70.8%), and significant economic losses. Eleven of thirty tissue specimens tested positive for NDV using haemagglutination and RT-PCR. The MDT testing and sequence analysis revealed the presence of virulent NDV classified as genotype VII of class II velogenic pathotype and distinct from locally used vaccine strains (genotype II). The amino acid sequences of the current virulent NDV fusion protein cleavage site motif revealed 112RRQKR↓F117, unlike the locally used avirulent vaccine strains (112GRQGR↓L117). The epidemiological data, MDT results, cleavage site sequence, and phylogenetic analysis all indicated that the present NDV isolates were virulent. The four NDV sequences were deposited in GenBank with accession numbers F gene (PP726912-15) and M gene (PP726916-19). The genetic difference between avirulent vaccine strains and circulating virulent NDV could explain the low level of protection provided by locally used vaccines. Further studies are needed to better understand the circulating NDV genotypes in different production systems.
Assuntos
Galinhas , Surtos de Doenças , Genótipo , Doença de Newcastle , Vírus da Doença de Newcastle , Filogenia , Doenças das Aves Domésticas , Vacinas Virais , Animais , Vírus da Doença de Newcastle/genética , Vírus da Doença de Newcastle/isolamento & purificação , Vírus da Doença de Newcastle/classificação , Vírus da Doença de Newcastle/patogenicidade , Galinhas/virologia , Etiópia/epidemiologia , Doença de Newcastle/virologia , Doença de Newcastle/epidemiologia , Doenças das Aves Domésticas/virologia , Doenças das Aves Domésticas/epidemiologia , Vacinas Virais/imunologia , Vacinas Virais/genética , Vacinas Virais/administração & dosagem , Virulência , Fazendas , Proteínas Virais de Fusão/genéticaRESUMO
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
BACKGROUND: Haemagglutinin-neuraminidase (HN) is one of the membrane proteins of Newcastle disease virus (NDV) that plays a significant role during host viral infection. Therefore, antibodies against HN are vital for the host's ability to protect itself against NDV infection due to their critical functions in viral infection. As a result, HN has been a candidate protein in vaccine development against the Newcastle disease virus. METHODS: This report used the full-length sequence of the HN protein of NDV isolated in Iran (VIId subgenotype). We characterize and identify amino acid substitutions in comparison to other more prevalent NDV genotypes, VII subgenotypes and vaccine strains. Furthermore, bioinformatics tools were applied to determine the three-dimensional structure, molecular dynamics simulation and prediction of B-cell antigenic epitopes. RESULTS: The results showed that the antigenic regions of our isolate are quite comparable to the other VII subgenotypes of NDV isolated from different geographical places. Moreover, by employing the final 3D structure of our HN protein, the amino acid residues are proposed as a B-cell epitope by epitope prediction servers, which leads to the introduction of linear and conformational antigenic sites. CONCLUSIONS: Immunoinformatic vaccine design principles currently exhibit tremendous potential for developing a new generation of candidate vaccines quickly and economically to eradicate infectious viruses, including the NDV. In order to accomplish this, focus is directed on residues that might be considered antigenic.
Assuntos
Genótipo , Proteína HN , Vírus da Doença de Newcastle , Vírus da Doença de Newcastle/genética , Vírus da Doença de Newcastle/imunologia , Proteína HN/genética , Proteína HN/química , Sequência de Aminoácidos , Animais , Irã (Geográfico) , Sequência de Bases , Galinhas , Doenças das Aves Domésticas/virologia , Doença de Newcastle/virologiaRESUMO
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.
Assuntos
Vetores Genéticos , Neoplasias , Vírus da Doença de Newcastle , Terapia Viral Oncolítica , Vírus Oncolíticos , Vírus da Doença de Newcastle/genética , Vírus da Doença de Newcastle/imunologia , Animais , Humanos , Vetores Genéticos/genética , Neoplasias/terapia , Neoplasias/imunologia , Terapia Viral Oncolítica/métodos , Vírus Oncolíticos/genética , Vírus Oncolíticos/imunologia , Terapia Genética/métodos , Vacinas Virais/imunologia , Vacinas Virais/genética , Doença de Newcastle/prevenção & controle , Doença de Newcastle/terapia , Doença de Newcastle/virologia , Doença de Newcastle/imunologia , Desenvolvimento de Vacinas/métodosRESUMO
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.
Assuntos
Interleucinas , Vírus da Doença de Newcastle , Terapia Viral Oncolítica , Vírus Oncolíticos , Animais , Vírus da Doença de Newcastle/genética , Vírus da Doença de Newcastle/fisiologia , Terapia Viral Oncolítica/métodos , Vírus Oncolíticos/genética , Vírus Oncolíticos/fisiologia , Humanos , Camundongos , Linhagem Celular Tumoral , Interleucinas/genética , Interleucinas/metabolismo , Neoplasias Hepáticas/terapia , Camundongos Endogâmicos BALB C , Carcinoma Hepatocelular/terapia , Apoptose , Neovascularização Patológica/terapia , Proliferação de Células , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Células Dendríticas/imunologia , Linfócitos T/imunologiaRESUMO
BACKGROUND: In order to prevent the emergence and spread of future variants of concern of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), developing vaccines capable of stopping transmission is crucial. The SARS-CoV-2 vaccine NDV-HXP-S can be administered live intranasally (IN) and thus induce protective immunity in the upper respiratory tract. The vaccine is based on Newcastle disease virus (NDV) expressing a stabilised SARS-CoV-2 spike protein. NDV-HXP-S can be produced as influenza virus vaccine at low cost in embryonated chicken eggs. METHODS: The NDV-HXP-S vaccine was genetically engineered to match the Omicron variants of concern (VOC) BA.1 and BA.5 and tested as an IN two or three dose vaccination regimen in female mice. Furthermore, female mice intramuscularly (IM) vaccinated with mRNA-lipid nanoparticles (LNPs) were IN boosted with NDV-HXP-S. Systemic humoral immunity, memory T cell responses in the lungs and spleens as well as immunoglobulin A (IgA) responses in distinct mucosal tissues were characterised. FINDINGS: NDV-HXP-S Omicron variant vaccines elicited high mucosal IgA and serum IgG titers against respective SARS-CoV-2 VOC in female mice following IN administration and protected against challenge from matched variants. Additionally, antigen-specific memory B cells and local T cell responses in the lungs were induced. Host immunity against the NDV vector did not interfere with boosting. Intramuscular vaccination with mRNA-LNPs was enhanced by IN NDV-HXP-S boosting resulting in improvement of serum neutralization titers and induction of mucosal immunity. INTERPRETATION: We demonstrate that NDV-HXP-S Omicron variant vaccines utilised for primary immunizations or boosting efficiently elicit humoral and cellular immunity. The described induction of systemic and mucosal immunity has the potential to reduce infection and transmission. FUNDING: This work was partially funded by the NIAIDCenters of Excellence for Influenza Research and Response (CEIRR) and by the NIAID Collaborative Vaccine Innovation Centers and by institutional funding from the Icahn School of Medicine at Mount Sinai. See under Acknowledgements for details.
Assuntos
Administração Intranasal , Anticorpos Antivirais , Vacinas contra COVID-19 , COVID-19 , Imunidade Humoral , Imunidade nas Mucosas , Vírus da Doença de Newcastle , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Animais , Feminino , SARS-CoV-2/imunologia , SARS-CoV-2/genética , Camundongos , Vacinas contra COVID-19/imunologia , Vacinas contra COVID-19/administração & dosagem , COVID-19/prevenção & controle , COVID-19/imunologia , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/sangue , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/genética , Vírus da Doença de Newcastle/imunologia , Vírus da Doença de Newcastle/genética , Imunidade Celular , Imunoglobulina A/imunologia , Nanopartículas/administração & dosagem , Nanopartículas/química , Anticorpos Neutralizantes/imunologia , Vacinação/métodos , Humanos , LipossomosRESUMO
The emergence of new virulent genotypes and the continued genetic drift of Newcastle disease virus (NDV) implies that distinct genotypes of NDV are simultaneously evolving in different geographic locations across the globe, including throughout Africa, where NDV is an important veterinary pathogen. Expanding the genomic diversity of NDV increases the possibility of diagnostic and vaccine failures. In this review, we systematically analyzed the genetic diversity of NDV genotypes in Africa using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. Information published between 1999 and 2022 were used to obtain the genetic background of different genotypes of NDV and their geographic distributions in Africa. The following genotypes were reported in Africa: I, II, III, IV, V, VI, VII, VIII, XI, XIII, XIV, XVII, XVIII, XX, and XXI. A new putative genotype has been detected in the Democratic Republic of the Congo. However, of 54 African countries, only 26 countries regularly report information on NDV outbreaks, suggesting that this number may be vastly underestimated. With eight different genotypes, Nigeria is the country with the greatest genotypic diversity of NDV among African countries. Genotype VII is the most prevalent group of NDV in Africa, which was reported in 15 countries. A phylogeographic analysis of NDV sequences revealed transboundary transmission of the virus in Eastern Africa, Western and Central Africa, and in Southern Africa. A regional and continental collaboration is recommended for improved NDV risk management in Africa.
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Variação Genética , Genótipo , Doença de Newcastle , Vírus da Doença de Newcastle , Filogenia , Vírus da Doença de Newcastle/genética , Vírus da Doença de Newcastle/classificação , Vírus da Doença de Newcastle/isolamento & purificação , Doença de Newcastle/virologia , Doença de Newcastle/epidemiologia , África/epidemiologia , Animais , Genoma Viral , Vacinação/veterinária , Galinhas/virologia , Vacinas Virais/genética , Vacinas Virais/imunologia , Doenças das Aves Domésticas/virologia , Doenças das Aves Domésticas/epidemiologia , FilogeografiaRESUMO
The majority of pigeon paramyxovirus type 1 (PPMV-1) strains are generally non-pathogenic to chickens; however, they can induce severe illness and high mortality rates in pigeons, leading to substantial economic repercussions. The genomes of 11 PPMV-1 isolates from deceased pigeons on meat pigeon farms during passive monitoring from 2009 to 2012 were sequenced and analyzed using polymerase chain reaction and phylogenetic analysis. The complete genome lengths of 11 isolates were approximately 15,192 nucleotides, displaying a consistent gene order of 3'-NP-P-M-F-HN-L-5'. ALL isolates exhibited the characteristic motif of 112RRQKRF117 at the fusion protein cleavage site, which is characteristic of velogenic Newcastle disease virus. Moreover, multiple mutations have been identified within the functional domains of the F and HN proteins, encompassing the fusion peptide, heptad repeat region, transmembrane domains, and neutralizing epitopes. Phylogenetic analysis based on sequences of the F gene unveiled that all isolates clustered within genotype VI in class II. Further classification identified at least two distinct sub-genotypes, with seven isolates classified as sub-genotype VI.2.1.1.2.2, whereas the others were classified as sub-genotype VI.2.1.1.2.1. This study suggests that both sub-genotypes were implicated in severe disease manifestation among meat pigeons, with sub-genotype VI.2.1.1.2.2 displaying an increasing prevalence among Shanghai's meat pigeon population since 2011. These results emphasize the value of developing pigeon-specific vaccines and molecular diagnostic tools for monitoring and proactively managing potential PPMV-1 outbreaks.
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Columbidae , Genoma Viral , Doença de Newcastle , Vírus da Doença de Newcastle , Filogenia , Animais , Columbidae/virologia , China/epidemiologia , Vírus da Doença de Newcastle/genética , Vírus da Doença de Newcastle/isolamento & purificação , Vírus da Doença de Newcastle/classificação , Doença de Newcastle/virologia , Doença de Newcastle/epidemiologia , Genótipo , Fazendas , Carne/virologiaRESUMO
Vectored RNA vaccines offer a variety of possibilities to engineer targeted vaccines. They are cost-effective and safe, but replication competent, activating the humoral as well as the cellular immune system.This chapter focuses on RNA vaccines derived from negative-strand RNA viruses from the order Mononegavirales with special attention to Newcastle disease virus-based vaccines and their generation. It shall provide an overview on the advantages and disadvantages of certain vector platforms as well as their scopes of application, including an additional section on experimental COVID-19 vaccines.
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Vetores Genéticos , Vírus da Doença de Newcastle , Vacinas de mRNA , Animais , Humanos , COVID-19/prevenção & controle , COVID-19/imunologia , COVID-19/virologia , Vetores Genéticos/genética , Vírus da Doença de Newcastle/genética , Vírus da Doença de Newcastle/imunologia , Vírus de RNA/genética , Vírus de RNA/imunologia , SARS-CoV-2/imunologia , SARS-CoV-2/genética , Vacinas Virais/imunologia , Vacinas Virais/genética , Vacinas de mRNA/genética , Vacinas de mRNA/imunologiaRESUMO
The haemagglutinin-neuraminidase (HN) protein, a vital membrane glycoprotein, plays a pivotal role in the pathogenesis of Newcastle disease virus (NDV). Previously, we demonstrated that a mutation in the HN protein is essential for the enhanced virulence of JS/7/05/Ch, a velogenic variant NDV strain originating from the mesogenic vaccine strain Mukteswar. Here, we explored the effects of the HN protein during viral infection in vitro using three viruses: JS/7/05/Ch, Mukteswar, and an HN-replacement chimeric NDV, JS/MukHN. Through microscopic observation, CCK-8, and LDH release assays, we demonstrated that compared with Mukteswar and JS/MukHN, JS/7/05/Ch intensified the cellular damage and mortality attributed to the mutant HN protein. Furthermore, JS/7/05/Ch induced greater levels of apoptosis, as evidenced by the activation of caspase-3/8/9. Moreover, JS/7/05/Ch promoted autophagy, leading to increased autophagosome formation and autophagic flux. Subsequent pharmacological experiments revealed that inhibition of apoptosis and autophagy significantly impacted virus replication and cell viability in the JS/7/05/Ch-infected group, whereas less significant effects were observed in the other two infected groups. Notably, the mutant HN protein enhanced JS/7/05/Ch-induced apoptosis and autophagy by suppressing NF-κB activation, while it mitigated the effects of NF-κB on NDV infection. Overall, our study offers novel insights into the mechanisms underlying the increased virulence of NDV and serves as a reference for the development of vaccines.
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Apoptose , Proteína HN , NF-kappa B , Doença de Newcastle , Vírus da Doença de Newcastle , Vírus da Doença de Newcastle/fisiologia , Vírus da Doença de Newcastle/genética , Vírus da Doença de Newcastle/patogenicidade , Animais , Proteína HN/genética , Proteína HN/metabolismo , Doença de Newcastle/virologia , NF-kappa B/metabolismo , Doenças das Aves Domésticas/virologia , Galinhas , Embrião de GalinhaRESUMO
Newcastle disease (ND) is endemic in Angola. Several outbreaks of ND occurred in small backyard flocks and village chickens with high mortality in the southern provinces of the country, Cunene, Namibe and Huíla, in 2016 and 2018. In those years, 15 virulent ND virus (NDV) strains were isolated and grouped within subgenotype 2 of genotype VII (subgenotype VII.2). We now present a study on the thermostability of the isolates, aiming at the selection of the most thermostable strains that, after being genetically modified to reduce their virulence, can be adapted to the production of vaccines less dependent on cold chain and more adequate to protect native chickens against ND. Heat-inactivation kinetics of haemagglutinin (Ha) activity and infectivity (I) of the isolates were determined by incubating aliquots of virus at 56 °C for different time intervals. The two isolates from Namibe province showed a decrease in infectivity of 2 log10 in ≤ 10 min, therefore belonging to the I-phenotype, but while the NB1 isolate from 2016 maintained the Ha activity up to 30 min and was classified as thermostable virus (I-Ha+), the Ha activity of the 2018 NB2 isolate decreased by 2 log2 in 30 min, being classified as a thermolabile virus (I-Ha-). Of the 13 NDV isolates from Huíla province, 10 isolates were classified as thermostable, eight with phenotype I+Ha+ and 2 with phenotype I-Ha+. The other three isolates from this province were classified as thermolabile viruses (I-Ha-).Contribution: This study will contribute to the control and/or eradication of Newcastle disease virus in Angola. The thermostable viral strains isolated from chickens in the country can be genetically manipulated by reverse genetic technology in order to reduce their virulence and use them as a vaccine in the remote areas of Angola.