Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 783
Filtrar
1.
Onderstepoort J Vet Res ; 91(1): e1-e7, 2024 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-38708767

RESUMO

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.


Assuntos
Galinhas , Doença de Newcastle , Vírus da Doença de Newcastle , Doenças das Aves Domésticas , Vírus da Doença de Newcastle/patogenicidade , Vírus da Doença de Newcastle/genética , Vírus da Doença de Newcastle/classificação , Animais , Doença de Newcastle/virologia , Doença de Newcastle/epidemiologia , Angola/epidemiologia , Virulência , Doenças das Aves Domésticas/virologia , Doenças das Aves Domésticas/epidemiologia , Temperatura Alta
2.
Vet Res ; 55(1): 58, 2024 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-38715081

RESUMO

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.


Assuntos
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 Galinha
4.
PLoS One ; 17(2): e0263707, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35139115

RESUMO

Newcastle Disease Virus (NDV) is an avian RNA virus, which was shown to be effective and safe for use in oncolytic viral therapy for several tumour malignancies. The presence of a multi basic cleavage site (MBCS) in the fusion protein improved its oncolytic efficacy in vitro and in vivo. However, NDV with a MBCS can be virulent in poultry. We aimed to develop an NDV with a MBCS but with reduced virulence for poultry while remaining effective in killing human tumour cells. To this end, the open reading frame of the V protein, an avian specific type I interferon antagonist, was disrupted by introducing multiple mutations. NDV with a mutated V gene was attenuated in avian cells and chicken and duck eggs. Although this virus still killed tumour cells, the efficacy was reduced compared to the virulent NDV. Introduction of various mutations in the fusion (F) and hemagglutinin-neuraminidase (HN) genes slightly improved this efficacy. Taken together, these data demonstrated that NDV with a MBCS but with abrogation of the V protein ORF and mutations in the F and HN genes can be safe for evaluation in oncolytic viral therapy.


Assuntos
Neoplasias/terapia , Vírus da Doença de Newcastle/genética , Terapia Viral Oncolítica , Vírus Oncolíticos , Proteínas Estruturais Virais/genética , Células A549 , Animais , Apoptose/genética , Calibragem , Proteínas do Capsídeo/genética , Células Cultivadas , Embrião de Galinha , Chlorocebus aethiops , Patos/embriologia , Proteína HN/genética , Humanos , Mutagênese Sítio-Dirigida/métodos , Neoplasias/patologia , Vírus da Doença de Newcastle/patogenicidade , Vírus da Doença de Newcastle/fisiologia , Terapia Viral Oncolítica/efeitos adversos , Terapia Viral Oncolítica/métodos , Terapia Viral Oncolítica/normas , Vírus Oncolíticos/genética , Vírus Oncolíticos/patogenicidade , Vírus Oncolíticos/fisiologia , Fases de Leitura Aberta/genética , Segurança do Paciente , Microambiente Tumoral/genética , Células Vero , Proteínas Virais de Fusão/efeitos adversos , Proteínas Virais de Fusão/genética , Virulência/genética , Replicação Viral/genética
5.
J Virol ; 96(2): e0162921, 2022 01 26.
Artigo em Inglês | MEDLINE | ID: mdl-34705566

RESUMO

The Newcastle disease virus (NDV) matrix (M) protein is the pivotal element for viral assembly, budding, and proliferation. It traffics through the cellular nucleus but performs its primary function in the cytoplasm. To investigate the biological importance of M protein nuclear-cytoplasmic trafficking and the mechanism involved, the regulatory motif nuclear export signal (NES) and nuclear localization signal (NLS) were analyzed. Here, two types of combined NLSs and NESs were identified within the NDV-M protein. The Herts/33-type M protein was found to mediate efficient nuclear export and stable virus-like particle (VLP) release, while the LaSota-type M protein was retained mostly in the nuclei and showed retarded VLP production. Two critical residues, namely, 247 and 263, within the motif were identified and associated with nuclear export efficiency. We identified, for the first time, residue 247 as an important monoubiquitination site, of which its modification regulates the nuclear-cytoplasmic trafficking of NDV-M. Subsequently, mutant LaSota strains were rescued via reverse genetics, which contained either single or double amino acid substitutions that were similar to the M of Herts/33. The rescued LaSota (rLaSota) strains rLaSota-R247K, -S263R, and -double mutation (DM) showed about 2-fold higher hemagglutination (HA) titers and 10-fold higher 50% egg infective dose (EID50) titers than wild-type (wt) rLaSota. Furthermore, the mean death time (MDT) and intracerebral pathogenicity index (ICPI) values of those recombinant viruses were slightly higher than those of wt rLaSota probably due to their higher proliferation rates. Our findings contribute to a better understanding of the molecular mechanism of the replication and pathogenicity of NDV and even those of all other paramyxoviruses. This information is beneficial for the development of vaccines and therapies for paramyxoviruses. IMPORTANCE Newcastle disease virus (NDV) is a pathogen that is lethal to birds and causes heavy losses in the poultry industry worldwide. The World Organization for Animal Health (OIE) ranked Newcastle disease (ND) as the third most significant poultry disease and the eighth most important wildlife disease in the World Livestock Disease Atlas in 2011. The matrix (M) protein of NDV is very important for viral assembly and maturation. It is interesting that M proteins enter the cellular nucleus before performing their primary function in the cytoplasm. We found that NDV-M has a combined nuclear import and export signal. The ubiquitin modification of a lysine residue within this signal is critical for quick, efficient nuclear export and subsequent viral production. Our findings shed new light on viral replication and open up new possibilities for therapeutics against NDV and other paramyxoviruses; furthermore, we demonstrate a novel approach for improving paramyxovirus vaccines.


Assuntos
Núcleo Celular/metabolismo , Vírus da Doença de Newcastle/fisiologia , Vírus da Doença de Newcastle/patogenicidade , Ubiquitinação , Proteínas da Matriz Viral/metabolismo , Replicação Viral , Animais , Galinhas , Citoplasma/metabolismo , Lisina , Modelos Moleculares , Mutação , Doença de Newcastle/metabolismo , Doença de Newcastle/virologia , Vírus da Doença de Newcastle/metabolismo , Sinais de Exportação Nuclear , Sinais de Localização Nuclear , Proteínas da Matriz Viral/química , Proteínas da Matriz Viral/genética , Virulência , Liberação de Vírus
6.
Microbiol Spectr ; 9(3): e0098921, 2021 12 22.
Artigo em Inglês | MEDLINE | ID: mdl-34878298

RESUMO

We provide a novel single restriction enzyme (RE; BsaHI) digestion approach for detecting distinct pathotypes of Newcastle disease virus (NDV). After scanning 4,000 F gene nucleotide sequences in the NCBI database, we discovered a single RE (BsaHI) digestion site in the cleavage site. APMV-I "F gene" class II-specific primer-based reverse transcriptase PCR was utilized to amplify a 535-bp fragment, which was then digested with the RE (BsaHI) for pathotyping avian NDV field isolates and pigeon paramyxovirus-1 isolates. The avirulent (lentogenic and mesogenic strains) produced 189- and 346-bp fragments, respectively, but the result in velogenic strains remained undigested with 535-bp fragments. In addition, 45 field NDV isolates and 8 vaccine strains were used to confirm the approach. The sequence-based analysis also agrees with the data obtained utilizing the single RE (BsaHI) digestion approach. The proposed technique has the potential to distinguish between avirulent and virulent strains in a short time span, making it valuable in NDV surveillance and monitoring research. IMPORTANCE The extensive use of the NDV vaccine strain and the existence of avirulent NDV strains in wild birds makes it difficult to diagnose Newcastle Disease virus (NDV). The intracerebral pathogenicity index (ICPI) and/or sequencing-based identification, which are required to determine virulent NDV, are time-consuming, costly, difficult, and cruel techniques. We evaluated 4,000 F gene nucleotide sequences and discovered a restriction enzyme (RE; BsaHI) digestion technique for detecting NDV and vaccine pathotypes in a short time span, which is cost-effective and useful for field cases as well as for large-scale NDV monitoring and surveillance. The data acquired using the single RE BsaHI digestion technique agree with the sequence-based analysis.


Assuntos
Enzimas de Restrição do DNA/metabolismo , Doença de Newcastle/diagnóstico , Vírus da Doença de Newcastle/genética , Proteínas Virais de Fusão/genética , Virulência/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Galinhas/virologia , Doença de Newcastle/patologia , Vírus da Doença de Newcastle/classificação , Vírus da Doença de Newcastle/patogenicidade , Técnicas de Amplificação de Ácido Nucleico , Doenças das Aves Domésticas/diagnóstico , Doenças das Aves Domésticas/virologia , RNA Viral/metabolismo , Análise de Sequência de RNA , Vacinas Virais/genética
7.
Vet Res ; 52(1): 147, 2021 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-34930432

RESUMO

The fusion (F) and haemagglutinin-neuraminidase (HN) proteins of Newcastle disease virus (NDV) are viral entry proteins and are recognized as the major virulence determinants. Previously, a lentogenic NDV virus (CE16) was derived from a mesogenic strain (CI10) through sequential passages in chick embryos. Whole-genome sequence analysis revealed that the two homologous strains shared the same F protein but differed in HN with two amino acid (aa) substitutions (A215G and T430A). To elucidate the molecular reasons for virulence attenuation, two original plasmids (HN-CI10 and HN-CE16) and two single-point mutants (G215A and A430T) reverse-mutated from HN-CE16 were constructed to analyse the known biological functions of HN. The results showed that the A430T substitution significantly weakened the haemadsorption (HAd) activity, increased the neuraminidase (NA) activity, improved the fusion-promoting activity, and enhanced the cleavage-promoting activity of HN-CE16. However, G215A failed to induce obvious functional changes. Therefore, the aa residue HN430 may play a key role in determining virulence. To test this hypothesis, further studies on A430T were conducted through reverse genetics using an infectious cDNA clone. At the viral level, the A430T-mutated virus showed dramatic promotion of viral plaque formation, propagation, and pathogenicity in vitro and in vivo. This study demonstrates a new virulence site associated with HN protein functions, viral propagation, and pathogenicity. All these findings could lay a foundation for illuminating the molecular mechanism of NDV virulence.


Assuntos
Aminoácidos , Proteína HN , Doença de Newcastle , Vírus da Doença de Newcastle , Virulência , Aminoácidos/genética , Animais , Embrião de Galinha , Galinhas , Proteína HN/genética , Mutação , Doença de Newcastle/virologia , Vírus da Doença de Newcastle/genética , Vírus da Doença de Newcastle/patogenicidade , Virulência/genética
8.
Viruses ; 13(12)2021 12 06.
Artigo em Inglês | MEDLINE | ID: mdl-34960715

RESUMO

Newcastle disease virus (NDV) can infect over 250 bird species with variable pathogenicity; it can also infect humans in rare cases. The present study investigated an outbreak in feral pigeons in São Paulo city, Brazil, in 2019. Affected birds displayed neurological signs, and hemorrhages were observed in different tissues. Histopathology changes with infiltration of mononuclear inflammatory cells were also found in the brain, kidney, proventriculus, heart, and spleen. NDV staining was detected by immunohistochemistry. Twenty-seven out of thirty-four tested samples (swabs and tissues) were positive for Newcastle disease virus by RT-qPCR test, targeting the M gene. One isolate, obtained from a pool of positive swab samples, was characterized by the intracerebral pathogenicity index (ICPI) and the hemagglutination inhibition (HI) tests. This isolate had an ICPI of 0.99, confirming a virulent NDV strain. The monoclonal antibody 617/161, which recognizes a distinct epitope in pigeon NDV strains, inhibited the isolate with an HI titer of 512. A complete genome of NDV was obtained using next-generation sequencing. Phylogenetic analysis based on the complete CDS F gene grouped the detected isolate with other viruses from subgenotype VI.2.1.2, class II, including one previously reported in Southern Brazil in 2014. This study reports a comprehensive characterization of the subgenotype VI.2.1.2, which seems to have been circulating in Brazilian urban areas since 2014. Due to the zoonotic risk of NDV, virus surveillance in feral pigeons should also be systematically performed in urban areas.


Assuntos
Columbidae , Surtos de Doenças/veterinária , Doença de Newcastle/epidemiologia , Vírus da Doença de Newcastle/genética , Animais , Brasil/epidemiologia , Genoma Viral , Genótipo , Sequenciamento de Nucleotídeos em Larga Escala , Doença de Newcastle/patologia , Doença de Newcastle/virologia , Vírus da Doença de Newcastle/classificação , Vírus da Doença de Newcastle/isolamento & purificação , Vírus da Doença de Newcastle/patogenicidade , Filogenia , Virulência , Sequenciamento Completo do Genoma
9.
Microbiol Spectr ; 9(3): e0217321, 2021 12 22.
Artigo em Inglês | MEDLINE | ID: mdl-34937182

RESUMO

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


Assuntos
Doença de Newcastle/virologia , Vírus da Doença de Newcastle/fisiologia , Vírus da Doença de Newcastle/patogenicidade , Doenças das Aves Domésticas/virologia , Proteínas Virais de Fusão/química , Proteínas Virais de Fusão/metabolismo , Liberação de Vírus , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Linhagem Celular , Galinhas , Regulação Viral da Expressão Gênica , Vírus da Doença de Newcastle/química , Vírus da Doença de Newcastle/genética , Alinhamento de Sequência , Tirosina/genética , Tirosina/metabolismo , Proteínas Virais de Fusão/genética , Virulência , Replicação Viral
10.
Viruses ; 13(10)2021 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-34696415

RESUMO

Newcastle disease virus (NDV) strain R2B, with an altered fusion protein cleavage site, was used as a viral vector to deliver the immunogenic genes VP2 and VP1 of chicken infectious anaemia virus (CIAV) to generate a bivalent vaccine candidate against these diseases in chickens. The immunogenic genes of CIAV were expressed as a single transcriptional unit from the NDV backbone and the two CIA viral proteins were obtained as separate entities using a self-cleaving foot-and-mouth disease virus 2A protease sequence between them. The recombinant virus (rR2B-FPCS-CAV) had similar growth kinetics as that of the parent recombinant virus (rR2B-FPCS) in vitro with similar pathogenicity characteristics. The bivalent vaccine candidate when given in specific pathogen-free chickens as primary and booster doses was able to elicit robust humoral and cell-mediated immune (CMI) responses obtained in a vaccination study that was conducted over a period of 15 weeks. In an NDV and CIAV ELISA trial, there was a significant difference in the titres of antibody between vaccinated and control groups which showed slight reduction in antibody titre by 56 days of age. Hence, a second booster was administered and the antibody titres were maintained until 84 days of age. Similar trends were noticed in CMI response carried out by lymphocyte transformation test, CD4+ and CD8+ response by flow cytometry analysis and response of real time PCR analysis of cytokine genes. Birds were challenged with virulent NDV and CIAV at 84 days and there was significant reduction in the NDV shed on the 2nd and 4th days post challenge in vaccinated birds as compared to unvaccinated controls. Haematological parameters comprising PCV, TLC, PLC and PHC were estimated in birds that were challenged with CIAV that indicated a significant reduction in the blood parameters of controls. Our findings support the development and assessment of a bivalent vaccine candidate against NDV and CIAV in chickens.


Assuntos
Vírus da Anemia da Galinha/imunologia , Galinhas/imunologia , Vírus da Doença de Newcastle/genética , Animais , Anticorpos Antivirais/sangue , Vírus da Anemia da Galinha/patogenicidade , Galinhas/virologia , Vetores Genéticos , Imunidade/imunologia , Imunidade Celular , Doença de Newcastle/virologia , Vírus da Doença de Newcastle/imunologia , Vírus da Doença de Newcastle/patogenicidade , Doenças das Aves Domésticas/virologia , Vacinação/métodos , Vacinas Virais/imunologia
11.
Sci Rep ; 11(1): 17570, 2021 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-34475461

RESUMO

Newcastle disease virus (NDV) has caused significant outbreaks in South-East Asia, particularly in Indonesia in recent years. Recently emerged genotype VII NDVs (NDV-GVII) have shifted their tropism from gastrointestinal/respiratory tropism to a lymphotropic virus, invading lymphoid organs including spleen and bursa of Fabricius to cause profound lymphoid depletion. In this study, we aimed to identify candidate genes and biological pathways that contribute to the disease caused by this velogenic NDV-GVII. A transcriptomic analysis based on RNA-Seq of spleen was performed in chickens challenged with NDV-GVII and a control group. In total, 6361 genes were differentially expressed that included 3506 up-regulated genes and 2855 down-regulated genes. Real-Time PCR of ten selected genes validated the RNA-Seq results as the correlation between them is 0.98. Functional and network analysis of Differentially Expressed Genes (DEGs) showed altered regulation of ElF2 signalling, mTOR signalling, proliferation of cells of the lymphoid system, signalling by Rho family GTPases and synaptogenesis signalling in spleen. We have also identified modified expression of IFIT5, PI3K, AGT and PLP1 genes in NDV-GVII infected chickens. Our findings in activation of autophagy-mediated cell death, lymphotropic and synaptogenesis signalling pathways provide new insights into the molecular pathogenesis of this newly emerged NDV-GVII.


Assuntos
Proteínas Aviárias/metabolismo , Doença de Newcastle/patologia , Vírus da Doença de Newcastle/patogenicidade , Doenças das Aves Domésticas/patologia , Baço/patologia , Animais , Proteínas Aviárias/genética , Galinhas , Indonésia , Doença de Newcastle/genética , Doença de Newcastle/virologia , Doenças das Aves Domésticas/genética , Doenças das Aves Domésticas/virologia , Baço/metabolismo , Baço/virologia , Transcriptoma
12.
Microbiol Spectr ; 9(2): e0131221, 2021 10 31.
Artigo em Inglês | MEDLINE | ID: mdl-34585949

RESUMO

The large (L) polymerase proteins of most nonsegmented, negative-stranded (NNS) RNA viruses have conserved methyltransferase motifs, (G)-G-G-D and K-D-K-E, which are important for the stabilization and translation of mRNA. However, the function of the (G)-G-G-D and K-D-K-E motifs in the NNS RNA virus Newcastle disease virus (NDV) remains unclear. We observed G-G-D and K-D-K-E motifs in all NDV genotypes. By using the infection cloning system of NDV rSG10 strain, recombinant NDVs with a single amino acid mutated to alanine in one motif (G-G-D or K-D-K-E) were rescued. The intracerebral pathogenicity index and mean death time assay results revealed that the G-G-D motif and K-D-K-E motif attenuate the virulence of NDV to various degrees. The replication, transcription, and translation levels of the K-D-K-E motif-mutant strains were significantly higher than those of wild-type virus owing to their altered regulation of the affinity between nucleocapsid protein and eukaryotic translation initiation factor 4E. When the infection dose was changed from a multiplicity of infection (MOI) of 10 to an MOI of 0.01, the cell-to-cell spread abilities of G-G-D- and K-D-K-E-mutant strains were reduced, according to plaque assay and dynamic indirect immunofluorescence assay results. Finally, we found that NDV strains with G-G-D or K-D-K-E motif mutations had less pathogenicity in 3-week-old specific-pathogen-free chickens than wild-type NDV. Therefore, these methyltransferase motifs can affect virulence by regulating the translation and cell-to-cell spread abilities of NDV. This work provides a feasible approach for generating vaccine candidates for viruses with methyltransferase motifs. IMPORTANCE Newcastle disease virus (NDV) is an important pathogen that is widespread globally. Research on its pathogenic mechanism is an important means of improving prevention and control efforts. Our study found that a deficiency in its methyltransferase motifs (G-G-D and K-D-K-E motifs) can attenuate NDV and revealed the molecular mechanism by which these motifs affect pathogenicity, which provides a new direction for the development of NDV vaccines. In addition to the (G)-G-G-D and K-D-K-E motifs of many nonsegmented, negative-stranded RNA viruses, similar motifs have been found in dengue virus, Zika virus, Japanese encephalitis virus (JEV), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This suggests that such motifs may be present in more viruses. Our finding also provides a molecular basis for the discovery and functional study of (G)-G-G-D and K-D-K-E motifs of other viruses.


Assuntos
Motivos de Aminoácidos/genética , Metiltransferases/genética , Doença de Newcastle/transmissão , Vírus da Doença de Newcastle/crescimento & desenvolvimento , Vírus da Doença de Newcastle/genética , Proteínas Virais/genética , Animais , Linhagem Celular , Galinhas , Chlorocebus aethiops , Cricetinae , Genoma Viral/genética , Vírus da Doença de Newcastle/patogenicidade , Doenças das Aves Domésticas/transmissão , Doenças das Aves Domésticas/virologia , RNA Viral/biossíntese , RNA Viral/genética , Células Vero , Virulência/genética , Replicação Viral/genética
13.
Viruses ; 13(8)2021 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-34452385

RESUMO

Newcastle disease virus (NDV) is a significant pathogen of poultry; however, variants also affect other species, including pigeons. While NDV is endemic in Bangladesh, and poultry isolates have been recently characterized, information about viruses infecting pigeons is limited. Worldwide, pigeon-derived isolates are commonly of low to moderate virulence for chickens. Here, we studied a pigeon-derived NDV isolated in Bangladesh in 2010. To molecularly characterize the isolate, we sequenced its complete fusion gene and performed a comprehensive phylogenetic analysis. We further studied the biological properties of the virus by estimating mean death time (MDT) and by experimentally infecting 5-week-old naïve Sonali chickens. The studied virus clustered in sub-genotype XXI.1.2 with NDV from pigeons from Pakistan isolated during 2014-2018. Deduced amino acid sequence analysis showed a polybasic fusion protein cleavage site motif, typical for virulent NDV. The performed in vivo pathogenicity testing showed a MDT of 40.8 h, and along with previously established intracerebral pathogenicity index of 1.51, these indicated a velogenic pathotype for chickens, which is not typical for pigeon-derived viruses. The experimental infection of chickens resulted in marked neurological signs and high mortality starting at 7 days post infection (dpi). Mild congestion in the thymus and necrosis in the spleen were observed at an advanced stage of infection. Microscopically, lymphoid depletion in the thymus, spleen, and bursa of Fabricius were found at 5 dpi, which progressed to severe in the following days. Mild to moderate proliferation of glial cells was noticed in the brain starting at 2 dpi, which gradually progressed with time, leading to focal nodular aggregation. This study reports the velogenic nature for domestic chickens of a pigeon-derived NDV isolate of sub-genotype XXI.1.2. Our findings show that not all pigeon-derived viruses are of low virulence for chickens and highlight the importance of biologically evaluating the pathogenicity of NDV isolated from pigeons.


Assuntos
Galinhas/virologia , Columbidae/virologia , Doença de Newcastle/mortalidade , Vírus da Doença de Newcastle/genética , Vírus da Doença de Newcastle/patogenicidade , Doenças das Aves Domésticas/mortalidade , Animais , Bangladesh , Ovos/virologia , Genoma Viral , Genótipo , Vírus da Doença de Newcastle/classificação , Vírus da Doença de Newcastle/isolamento & purificação , Filogenia , Doenças das Aves Domésticas/virologia , Análise de Sequência de DNA , Virulência
14.
Vet Microbiol ; 261: 109181, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34399297

RESUMO

The V protein of Newcastle disease virus (NDV) has been shown to inhibit the secretion of interferon (IFN) during infection, which is responsible for the promotion of NDV pathogenicity. However, the ability of the V protein to suppress host innate immunity is not well understood. In this study, we explored the function of V protein and its relationship with virulence by generating V protein-inserted recombinant (r) NDVs. Using rNDVs as a model, we examined the efficiency of infection, IFN responses, and apoptosis of host cells during infection. We found that viral propagation occurred smoothly when V protein from lentogenic NDV is inserted instead of the V protein from the velogenic strain. The infection of lentogenic V protein-inserted rNDV induced less expression of IFNs and downstream antiviral proteins via efficient degradation of p-STAT1 and MDA5. Moreover, velogenic V protein triggered a higher apoptosis rate during infection thereby restricting the replication of NDV. Conversely, lentogenic V protein inhibits IFN responses efficiently and induces less apoptosis compared to the velogenic strain. Our findings provide a novel understanding of the role of V protein in NDV pathogenicity.


Assuntos
Doença de Newcastle/imunologia , Doença de Newcastle/virologia , Vírus da Doença de Newcastle/genética , Vírus da Doença de Newcastle/patogenicidade , Doenças das Aves Domésticas/imunologia , Doenças das Aves Domésticas/virologia , Proteínas Virais , Animais , Apoptose , Regulação da Expressão Gênica/imunologia , Interações entre Hospedeiro e Microrganismos/imunologia , Interferons/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo
15.
Vet Microbiol ; 260: 109167, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34274763

RESUMO

Virulent Newcastle disease virus (NDV) is a violent infection in avian species. The understanding of its pathogenic mechanism is consistently evolving along with the development of molecular biological advancement. Exosomes derived from NDV infected cells (NDV Ex) were reported to promote virus replication through transportation of viral proteins and miRNAs. However, the function of mRNAs in NDV Ex remains unknown. In this study, a novel mechanism of NDV Ex to facilitate NDV infection was explored. Through transcriptome analysis, seven immune related genes were found to up-regulate in NDV Ex. Among them, NLRX1 mRNA was notably enriched in NDV Ex, and decreased inside the cells after virulent NDV infection. Further investigation suggested that NLRX1 mRNA decrease was in accordance with the NLRX1 protein expression reduction. This process can be reversed by the inhibition of exosome release. Therefore, NDV infection could utilize NDV Ex to export NLRX1 mRNA and reduce cellular NLRX1 protein. As NLRX1 is a crucial anti-viral protein of MAVS signal pathway, and NDV Ex transported NLRX1 cannot counteract its function in recipient cells, it can be concluded that NDV could benefit its replication through exporting NLRX1 mRNA to relieve the anti-viral pressure on its survival.


Assuntos
Exossomos/metabolismo , Proteínas Mitocondriais/metabolismo , Doença de Newcastle/virologia , Vírus da Doença de Newcastle/patogenicidade , Transdução de Sinais , Transcriptoma , Proteínas Virais/metabolismo , Animais , Transporte Biológico , Embrião de Galinha , Fibroblastos/virologia , Células HeLa , Humanos , Proteínas Mitocondriais/genética , Vírus da Doença de Newcastle/genética , Vírus da Doença de Newcastle/fisiologia , RNA Mensageiro/genética , Organismos Livres de Patógenos Específicos , Proteínas Virais/genética , Replicação Viral
16.
Res Vet Sci ; 139: 159-165, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34332418

RESUMO

Recombinant Newcastle disease virus vectors have gained a lot of interest for its oncolytic virus therapy and cancer immune therapeutic properties due to its selective replication to high titers in cancer cells. The aim of this study was to find out the oncolytic effects of mesogenic recombinant NDV strain R2B-GFP on murine mammary tumor cell line 4T1 and murine melanoma cell line B16-F10. The anti-tumor effects of R2B-GFP virus were studied via expression of virus transgene GFP in cancer cells, evaluating its cytotoxicity and cell migration efficacies by MTT and wound healing assays respectively. In addition, the underlying apoptotic mechanism of R2B-GFP virus was estimated by TUNEL assay, colorimetric estimation of Caspase-3, 8 and 9 and the estimation of Bax to Bcl-2 ratio. The results showed a significant decrease in viability of both 4T1 and B16-F10 cells infected with R2B-GFP virus at 0.1 and 1 MOI. R2B-GFP virus could significantly induce apoptosis in the 4T1 and B16-F10 cells as compared to the uninfected control. Further, a flow cytometry analysis on apoptotic cells percentage and mitochondria membrane permeability test was also studied in R2B-GFP virus treated 4T1 and B16-F10 cell lines. The R2B-GFP virus caused an increase in loss of mitochondrial membrane permeability in both 4T1 and B16-F10 cells indicating the involvement of mitochondrial regulated cell death. Thus, the recombinant virus R2B-GFP virus proved to be a valid candidate for oncolytic viral therapy in 4T1 and B16-F10 cells.


Assuntos
Neoplasias Mamárias Animais , Melanoma , Vírus da Doença de Newcastle , Terapia Viral Oncolítica , Doenças dos Roedores , Animais , Apoptose , Linhagem Celular Tumoral , Neoplasias Mamárias Animais/terapia , Melanoma/terapia , Melanoma/veterinária , Camundongos , Vírus da Doença de Newcastle/patogenicidade , Terapia Viral Oncolítica/veterinária
17.
Sci Rep ; 11(1): 10270, 2021 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-33986327

RESUMO

Newcastle disease (ND), caused by virulent Newcastle disease virus (NDV), is a contagious viral disease affecting various birds and poultry worldwide. In this project, differentially expressed (DE) circRNAs, miRNAs and mRNAs were identified by high-throughput RNA sequencing (RNA-Seq) in chicken thymus at 24, 48, 72 or 96 h post LaSota NDV vaccine injection versus pre-inoculation group. The vital terms or pathways enriched by vaccine-influenced genes were tested through KEGG and GO analysis. DE genes implicated in innate immunity were preliminarily screened out through GO, InnateDB and Reactome Pathway databases. The interaction networks of DE innate immune genes were established by STRING website. Considering the high expression of gga-miR-6631-5p across all the four time points, DE circRNAs or mRNAs with the possibility to bind to gga-miR-6631-5p were screened out. Among DE genes that had the probability to interact with gga-miR-6631-5p, 7 genes were found to be related to innate immunity. Furthermore, gga-miR-6631-5p promoted LaSota NDV replication by targeting insulin induced gene 1 (INSIG1) in DF-1 chicken fibroblast cells. Taken together, our data provided the comprehensive information about molecular responses to NDV LaSota vaccine in Chinese Partridge Shank Chickens and elucidated the vital roles of gga-miR-6631-5p/INSIG1 axis in LaSota NDV replication.


Assuntos
Doença de Newcastle/genética , Pequeno RNA não Traduzido/genética , Replicação Viral/genética , Animais , Galinhas/genética , Galinhas/virologia , Expressão Gênica/genética , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica/genética , Imunidade Inata , MicroRNAs/genética , Doença de Newcastle/virologia , Vírus da Doença de Newcastle/genética , Vírus da Doença de Newcastle/patogenicidade , Doenças das Aves Domésticas/virologia , RNA Circular/genética , RNA Mensageiro/genética , Timo/metabolismo , Timo/virologia , Transcriptoma/genética , Vacinação
18.
Molecules ; 26(9)2021 Apr 24.
Artigo em Inglês | MEDLINE | ID: mdl-33923304

RESUMO

Chitosan is a non-toxic biological material, but chitosan is insoluble in water, which hinders the development and utilization of chitosan. Chitosan derivatives N-2-Hydroxypropyl trimethyl ammonium chloride (N-2-HACC) and carboxymethyl chitosan (CMCS) with good water solubility were synthesized by our laboratory. In this study, we synthesized mesoporous SiO2 nanoparticles by the emulsion, and then the mesoporous SiO2 nanoparticles were modified with γ-aminopropyltriethoxysilane to synthesize aminated mesoporous SiO2 nanoparticles; CMCS and N-2-HACC was used to cross-link the aminated mesoporous SiO2 nanoparticles to construct SiO2@CMCS-N-2-HACC nanoparticles. Because the aminated mesoporous SiO2 nanoparticles with positively charged can react with the mucous membranes, the virus enters the body mainly through mucous membranes, so Newcastle disease virus (NDV) was selected as the model drug to evaluate the performance of the SiO2@CMCS-N-2-HACC nanoparticles. We prepared the SiO2@CMCS-N-2-HACC nanoparticles loaded with inactivated NDV (NDV/SiO2@CMCS-N-2-HACC). The SiO2@CMCS-N-2-HACC nanoparticles as delivery carrier had high loading capacity, low cytotoxicity, good acid resistance and bile resistance and enteric solubility, and the structure of NDV protein encapsulated in the nano vaccine was not destroyed. In addition, the SiO2@CMCS-N-2-HACC nanoparticles could sustain slowly released NDV. Therefore, the SiO2@CMCS-N-2-HACC nanoparticles have the potential to be served as delivery vehicle for vaccine and/or drug.


Assuntos
Quitosana/farmacologia , Sistemas de Liberação de Medicamentos , Nanopartículas/química , Doença de Newcastle/tratamento farmacológico , Animais , Proliferação de Células/efeitos dos fármacos , Quitosana/análogos & derivados , Humanos , Nanopartículas/uso terapêutico , Doença de Newcastle/patologia , Doença de Newcastle/virologia , Vírus da Doença de Newcastle/efeitos dos fármacos , Vírus da Doença de Newcastle/patogenicidade , Dióxido de Silício/química , Vacinas/química , Vacinas/farmacologia , Água/química
19.
Sci Rep ; 11(1): 3491, 2021 02 10.
Artigo em Inglês | MEDLINE | ID: mdl-33568682

RESUMO

To understand the dynamics of a pathogen in an animal population, one must assess how the infection status of individuals changes over time. With wild animals, this can be very challenging because individuals can be difficult to trap and sample, even more so since they are tested with imperfect diagnostic techniques. Multi-event capture-recapture models allow analysing longitudinal capture data of individuals whose infection status is assessed using imperfect tests. In this study, we used a two-year dataset from a longitudinal field study of peridomestic wild bird populations in the United Arab Emirates during which thousands of birds from various species were captured, sampled and tested for Newcastle disease virus exposure using a serological test. We developed a multi-event capture-recapture model to estimate important demographic and epidemiological parameters of the disease. The modelling outputs provided important insights into the understanding of Newcastle disease dynamics in peridomestics birds, which varies according to ecological and epidemiological parameters, and useful information in terms of surveillance strategies. To our knowledge, this study is the first attempt to model the dynamics of Newcastle disease in wild bird populations by combining longitudinal capture data and serological test results. Overall, it showcased that multi-event capture-recapture models represent a suitable method to analyse imperfect capture data and make reliable inferences on infectious disease dynamics in wild populations.


Assuntos
Aves/virologia , Doenças Transmissíveis/virologia , Doença de Newcastle/virologia , Vírus da Doença de Newcastle/patogenicidade , Animais , Animais Selvagens , Dinâmica Populacional , Emirados Árabes Unidos
20.
Poult Sci ; 100(2): 603-614, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33518113

RESUMO

Pigeon paramyxovirus type 1 (PPMV-1) is a globally distributed, virulent member of the avian paramyxovirus type-1. The PPMV-1-associated disease poses a great threat to the pigeon industry. The innate immune response is crucial for antiviral infections and revealing the pathogenic mechanisms of PPMV-1. In this study, we evaluated the pathogenicity of a PPMV-1 strain LHLJ/110822 in one-month-old domestic pigeons, as well as the host immune responses in PPMV-1-infected pigeons. We observed typically clinical sign in infected pigeons by 3 dpi. The morbidity rate and the mortality in pigeons inoculated with the PPMV-1 strain were up to 100% and 30%, respectively. The virus could replicate in all of the examined tissues, namely trachea, lung, liver, spleen, and bursa of Fabricius. In addition, the infected pigeons had developed anti-PPMV-1 antibodies as early as 8 dpi; and the antibody level increased over the time in this study. The expression level of toll-like receptor (TLR) 2, TLR3 TLR15, IFN-γ, and IL-6 were significantly upregulated by the PPMV-1 infection in some tissues of pigeons. By contrast, PPMV-1 infection results in downregulation of IL-18 expression in most of investigated tissues except for bursa of Fabricius in this study. The current results confirmed that this virus could replicate in pigeons and induce host immune responses, then leading to produce serum antibody titers. Meanwhile, the PPMV-1 infection induces strong innate immune responses and intense inflammatory responses at early stage in pigeon which may associate with the viral pathogenesis.


Assuntos
Columbidae , Doença de Newcastle/imunologia , Vírus da Doença de Newcastle/imunologia , Animais , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/fisiologia , Embrião de Galinha , Ovos/virologia , Imunidade Inata , Vírus da Doença de Newcastle/patogenicidade , Organismos Livres de Patógenos Específicos
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...