RESUMEN
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.
Asunto(s)
Proteína HN , Virus de la Enfermedad de Newcastle , Animales , Virus de la Enfermedad de Newcastle/metabolismo , Proteína HN/metabolismo , Catepsina B , Apoptosis , Lisosomas/metabolismoRESUMEN
Newcastle disease virus (NDV) has been extensively studied as a promising oncolytic virus for killing tumor cells in vitro and in vivo in clinical trials. However, the viral components that regulate the oncolytic activity of NDV remain incompletely understood. In this study, we systematically compared the replication ability of different NDV genotypes in various tumor cells and identified NP protein determines the oncolytic activity of NDV. On the one hand, NDV strains with phenylalanine (F) at the 450th amino acid position of the NP protein (450th-F-NP) exhibit a loss of oncolytic activity. This phenotype is predominantly associated with genotype VII NDVs. In contrast, the NP protein with a leucine amino acid at this site in other genotypes (450th-L-NP) can facilitate the loading of viral mRNA onto ribosomes more effectively than 450th-F-NP. On the other hand, the NP protein from NDV strains that exhibit strong oncogenicity interacts with eIF4A1 within its 366-489 amino acid region, leading to the inhibition of cellular mRNA translation with a complex 5' UTR structure. Our study provide mechanistic insights into how highly oncolytic NDV strains selectively promote the translation of viral mRNA and will also facilitate the screening of oncolytic strains for oncolytic therapy.
Asunto(s)
Virus de la Enfermedad de Newcastle , Virus Oncolíticos , Animales , Virus de la Enfermedad de Newcastle/genética , Aminoácidos , Leucina , Virus Oncolíticos/genética , ARN Mensajero/genética , Biosíntesis de ProteínasRESUMEN
Ferroptosis, a form of programmed cell death characterized by iron-dependent lipid peroxidation, has recently gained considerable attention in the field of cancer therapy. There is significant crosstalk between ferroptosis and several classical signaling pathways, such as the Hippo pathway, which suppresses abnormal growth and is frequently aberrant in tumor tissues. Yes-associated protein 1 (YAP), the core effector molecule of the Hippo pathway, is abnormally expressed and activated in a variety of malignant tumor tissues. We previously proved that the oncolytic Newcastle disease virus (NDV) activated ferroptosis to kill tumor cells. NDV has been used in tumor therapy; however, its oncolytic mechanism is not completely understood. In this study, we demonstrated that NDV exacerbated ferroptosis in tumor cells by inducing ubiquitin-mediated degradation of YAP at Lys90 through E3 ubiquitin ligase parkin (PRKN). Blocking YAP degradation suppressed NDV-induced ferroptosis by suppressing the expression of Zrt/Irt-like protein 14 (ZIP14), a metal ion transporter that regulates iron uptake. These findings demonstrate that NDV exacerbated ferroptosis in tumor cells by inducing YAP degradation. Our study provides new insights into the mechanism of NDV-induced ferroptosis and highlights the critical role that oncolytic viruses play in the treatment of drug-resistant cancers.IMPORTANCEThe oncolytic Newcastle disease virus (NDV) is being developed for use in cancer treatment; however, its oncolytic mechanism is still not completely understood. The Hippo pathway, which is a tumor suppressor pathway, is frequently dysregulated in tumor tissues due to aberrant yes-associated protein 1 (YAP) activation. In this study, we have demonstrated that NDV degrades YAP to induce ferroptosis and promote virus replication in tumor cells. Notably, NDV was found to induce ubiquitin-mediated degradation of YAP at Lys90 through E3 ubiquitin ligase parkin (PRKN). Our study reveals a new mechanism by which NDV induces ferroptosis and provides new insights into NDV as an oncolytic agent for cancer treatment.
Asunto(s)
Ferroptosis , Neoplasias , Virus de la Enfermedad de Newcastle , Viroterapia Oncolítica , Proteínas Señalizadoras YAP , Animales , Humanos , Proteínas Adaptadoras Transductoras de Señales , Línea Celular Tumoral , Hierro , Neoplasias/terapia , Virus Oncolíticos/fisiología , Factores de Transcripción/genética , Ubiquitina-Proteína Ligasas , UbiquitinasRESUMEN
The avian influenza virus (AIV) PA protein contributes to viral replication and pathogenicity; however, its interaction with innate immunity is not well understood. Here, we report that the H5 subtype AIV PA protein strongly suppresses host antiviral defense by interacting with and degrading a key protein in interferon (IFN) signaling, Janus kinase 1 (JAK1). Specifically, the AIV PA protein catalyzes the K48-linked polyubiquitination and degradation of JAK1 at lysine residue 249. Importantly, the AIV PA protein harboring 32T/550L degrades both avian and mammalian JAK1, while the AIV PA protein with residues 32M/550I degrades avian JAK1 only. Furthermore, the residues 32T/550L in PA protein confer optimum polymerase activity and AIV growth in mammalian cells. Notably, the replication and virulence of the AIV PA T32M/L550I mutant are attenuated in infected mice. Collectively, these data reveal an interference role for H5 subtype AIV PA protein in host innate immunity, which can be targeted for the development of specific and effective anti-influenza therapeutics.
Asunto(s)
Virus de la Influenza A , Gripe Aviar , Animales , Ratones , Virulencia , Pollos/metabolismo , Virus de la Influenza A/metabolismo , Proteínas no Estructurales Virales/metabolismo , MamíferosRESUMEN
Caspase-8, an aspartate-specific cysteine protease that primarily functions as an initiator caspase to induce apoptosis, can downregulate innate immunity in part by cleaving RIPK1 and IRF3. However, patients with caspase-8 mutations or deficiency develop immunodeficiency and are prone to viral infections. The molecular mechanism underlying this controversy remains unknown. Whether caspase-8 enhances or suppresses antiviral responses against influenza A virus (IAV) infection remains to be determined. Here, we report that caspase-8 is readily activated in A549 and NL20 cells infected with the H5N1, H5N6, and H1N1 subtypes of IAV. Surprisingly, caspase-8 deficiency and two caspase-8 inhibitors, Z-VAD and Z-IETD, do not enhance but rather downregulate antiviral innate immunity, as evidenced by decreased TBK1, IRF3, IκBα, and p65 phosphorylation, decreased IL-6, IFN-ß, MX1, and ISG15 gene expression; and decreased IFN-ß production but increased virus replication. Mechanistically, caspase-8 cleaves and inactivates CYLD, a tumor suppressor that functions as a deubiquitinase. Caspase-8 inhibition suppresses CYLD cleavage, RIG-I and TAK1 ubiquitination, and innate immune signaling. In contrast, CYLD deficiency enhances IAV-induced RIG-I and TAK1 ubiquitination and innate antiviral immunity. Neither caspase-3 deficiency nor treatment with its inhibitor Z-DEVD affects CYLD cleavage or antiviral innate immunity. Our study provides evidence that caspase-8 activation in two human airway epithelial cell lines does not silence but rather enhances innate immunity by inactivating CYLD.
Asunto(s)
Caspasa 8 , Proteína 58 DEAD Box , Enzima Desubiquitinante CYLD , Inmunidad Innata , Virus de la Influenza A , Gripe Humana , Quinasas Quinasa Quinasa PAM , Ubiquitinación , Humanos , Enzima Desubiquitinante CYLD/metabolismo , Enzima Desubiquitinante CYLD/genética , Caspasa 8/metabolismo , Caspasa 8/genética , Quinasas Quinasa Quinasa PAM/metabolismo , Quinasas Quinasa Quinasa PAM/genética , Quinasas Quinasa Quinasa PAM/inmunología , Virus de la Influenza A/inmunología , Proteína 58 DEAD Box/metabolismo , Proteína 58 DEAD Box/genética , Proteína 58 DEAD Box/inmunología , Gripe Humana/inmunología , Gripe Humana/virología , Células A549 , Animales , Transducción de Señal/inmunología , Receptores InmunológicosRESUMEN
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.
Asunto(s)
Metilenotetrahidrofolato Deshidrogenasa (NADP) , Enfermedad de Newcastle , Virus de la Enfermedad de Newcastle , Replicación Viral , Animales , Metilenotetrahidrofolato Deshidrogenasa (NADP)/genética , Metilenotetrahidrofolato Deshidrogenasa (NADP)/metabolismo , Enfermedad de Newcastle/enzimología , Enfermedad de Newcastle/fisiopatología , Enfermedad de Newcastle/virología , Virus de la Enfermedad de Newcastle/genética , Virus de la Enfermedad de Newcastle/metabolismo , Nucleótidos/metabolismo , Serina/metabolismo , Replicación Viral/genética , Línea Celular , Células A549 , Humanos , Mesocricetus , Técnicas de Silenciamiento del Gen , Transporte de Proteínas/genética , Mitocondrias/enzimología , Regulación hacia Arriba/fisiologíaRESUMEN
Many DNA viruses develop various strategies to inhibit cell death to facilitate their replication. However, whether influenza A virus (IAV), a fast-replicating RNA virus, attenuates cell death remains unknown. Here, we report that IAV infection induces TAK1 phosphorylation in a murine alveolar epithelial cell line (LET1) and a murine fibroblastoma cell line (L929). The TAK1-specific inhibitor 5Z-7-Oxzeneonal (5Z) and TAK1 knockout significantly enhance IAV-induced apoptosis, as evidenced by increased PARP, caspase-8, and caspase-3 cleavage. TAK1 inhibition also increases necroptosis as evidenced by increased RIPK1S166, RIPK3T231/S232, and MLKLS345 phosphorylation. Mechanistically, TAK1 activates IKK, which phosphorylates RIPK1S25 and inhibits its activation. TAK1 also activates p38 and its downstream kinase MK2, which phosphorylates RIPK1S321 but does not affect RIPK1 activation. Further investigation revealed that the RIPK1 inhibitor Nec-1 and RIPK1 knockout abrogate IAV-induced apoptosis and necroptosis; re-expression of wild-type but not kinase-dead (KD)-RIPK1 restores IAV-induced cell death. ZBP1 knockout abrogates IAV-induced cell death, whereas RIPK3 knockout inhibits IAV-induced necroptosis but not apoptosis. 5Z treatment enhances IAV-induced cell death and slightly reduces the inflammatory response in the lungs of H1N1 virus-infected mice and prolongs the survival of IAV-infected mice. Our study provides evidence that IAV activates TAK1 to suppress RIPK1-dependent apoptosis and necroptosis, and that RIPK3 is required for IAV-induced necroptosis but not apoptosis in epithelial cells.
Asunto(s)
Apoptosis , Quinasas Quinasa Quinasa PAM , Necroptosis , Proteína Serina-Treonina Quinasas de Interacción con Receptores , Animales , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Quinasas Quinasa Quinasa PAM/metabolismo , Quinasas Quinasa Quinasa PAM/genética , Ratones , Fosforilación , Infecciones por Orthomyxoviridae/virología , Infecciones por Orthomyxoviridae/patología , Línea Celular , Virus de la Influenza A/fisiología , Ratones Endogámicos C57BL , HumanosRESUMEN
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.
Asunto(s)
Apoptosis , Proteína HN , FN-kappa B , Enfermedad de Newcastle , Virus de la Enfermedad de Newcastle , Virus de la Enfermedad de Newcastle/fisiología , Virus de la Enfermedad de Newcastle/genética , Virus de la Enfermedad de Newcastle/patogenicidad , Animales , Proteína HN/genética , Proteína HN/metabolismo , Enfermedad de Newcastle/virología , FN-kappa B/metabolismo , Enfermedades de las Aves de Corral/virología , Pollos , Embrión de PolloRESUMEN
H7N9 subtype avian influenza viruses (AIVs) cause 1567 human infections and have high mortality, posing a significant threat to public health. Previously, we reported that two avian-derived H7N9 isolates (A/chicken/Eastern China/JTC4/2013 and A/chicken/Eastern China/JTC11/2013) exhibit different pathogenicities in mice. To understand the genetic basis for the differences in virulence, we constructed a series of mutant viruses based on reverse genetics. We found that the PB2-E627K mutation alone was not sufficient to increase the virulence of H7N9 in mice, despite its ability to enhance polymerase activity in mammalian cells. However, combinations with PB1-V719M and/or PA-N444D mutations significantly enhanced H7N9 virulence. Additionally, these combined mutations augmented polymerase activity, thereby intensifying virus replication, inflammatory cytokine expression, and lung injury, ultimately increasing pathogenicity in mice. Overall, this study revealed that virulence in H7N9 is a polygenic trait and identified novel virulence-related residues (PB2-627K combined with PB1-719M and/or PA-444D) in viral ribonucleoprotein (vRNP) complexes. These findings provide new insights into the molecular mechanisms underlying AIV pathogenesis in mammals, with implications for pandemic preparedness and intervention strategies.
Asunto(s)
Subtipo H7N9 del Virus de la Influenza A , Mutación , Infecciones por Orthomyxoviridae , Proteínas Virales , Animales , Ratones , Subtipo H7N9 del Virus de la Influenza A/genética , Subtipo H7N9 del Virus de la Influenza A/patogenicidad , Subtipo H7N9 del Virus de la Influenza A/fisiología , Infecciones por Orthomyxoviridae/virología , Infecciones por Orthomyxoviridae/veterinaria , Virulencia , Femenino , Proteínas Virales/genética , Proteínas Virales/metabolismo , Ratones Endogámicos BALB C , Replicación ViralRESUMEN
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.
Asunto(s)
Núcleo Celular/metabolismo , Virus de la Enfermedad de Newcastle/fisiología , Virus de la Enfermedad de Newcastle/patogenicidad , Ubiquitinación , Proteínas de la Matriz Viral/metabolismo , Replicación Viral , Animales , Pollos , Citoplasma/metabolismo , Lisina , Modelos Moleculares , Mutación , Enfermedad de Newcastle/metabolismo , Enfermedad de Newcastle/virología , Virus de la Enfermedad de Newcastle/metabolismo , Señales de Exportación Nuclear , Señales de Localización Nuclear , Proteínas de la Matriz Viral/química , Proteínas de la Matriz Viral/genética , Virulencia , Liberación del VirusRESUMEN
The haemagglutinin-neuraminidase (HN) protein plays a crucial role in the infectivity and virulence of Newcastle disease virus (NDV). In a previous study, the mutant HN protein was identified as a crucial virulence factor for the velogenic variant NDV strain JS/7/05/Ch, which evolved from the prototypic vaccine strain Mukteswar. Furthermore, macrophages are the main susceptible target cells of NDV. However, the possible involvement of cellular molecules in viral infectivity remains unclear. Herein, we elucidate the crucial role of vimentin, an intermediate filament protein, in regulating NDV infectivity through targeting of the HN protein. Using LCâMS/MS mass spectrometry and coimmunoprecipitation assays, we identified vimentin as a host protein that differentially interacted with prototypic and mutant HN proteins. Further analysis revealed that the variant NDV strain induced more significant rearrangement of vimentin fibres compared to the prototypic NDV strain and showed an interdependence between vimentin rearrangement and virus replication. Notably, these mutual influences were pronounced in HD11 chicken macrophages. Moreover, vimentin was required for multiple infection processes of the variant NDV strain in HD11 cells, including viral internalization, fusion, and release, while it was not necessary for those of the prototypic NDV strain. Collectively, these findings underscore the pivotal role of vimentin in NDV infection through targeting of the HN protein, providing novel targets for antiviral treatment strategies for NDV.
Asunto(s)
Enfermedad de Newcastle , Virus de la Enfermedad de Newcastle , Animales , Virus de la Enfermedad de Newcastle/fisiología , Proteína HN/genética , Vimentina/genética , Cromatografía Liquida/veterinaria , Espectrometría de Masas en Tándem/veterinaria , PollosRESUMEN
Highly pathogenic (HP) avian influenza A H7N9 virus has emerged in China since 2016. In recent years, it has been most prevalent in northern China. However, several strains of HP H7N9 reappeared in southwestern China (Yunnan Province) in 2021. As a result, we are wondering if these viruses have re-emerged in situ or been reintroduced. Here, we present phylogenetic evidence that the HP H7N9 viruses isolated in Yunnan emigrated from northern to southwestern China in 2020. The northern subregion of China has become a novel epicenter in HP H7N9 dissemination. Meanwhile, a cleavage motif re-emerged due to the T341I mutation, implying a parallel evolution. This cross-region transmission, which originated in non-adjacent provinces and traveled a great geographic distance in an unknown way, indicates that HP H7N9 dissemination did not halt in 2020, even under the shadow of the COVID-19 pandemic. Additional surveillance studies in poultry are required to determine the HP H7N9 virus's geographic distribution and spread.
Asunto(s)
COVID-19 , Subtipo H7N9 del Virus de la Influenza A , Gripe Aviar , Gripe Humana , Animales , Humanos , Subtipo H7N9 del Virus de la Influenza A/genética , Filogenia , Pandemias , China/epidemiología , COVID-19/epidemiologíaRESUMEN
Mineral dust serves as a significant source of sulfate aerosols by mediating heterogeneous sulfur dioxide (SO2) oxidation in the atmosphere. Given that a considerable proportion of small organic acids are deposited onto mineral dust via long-range transportation, understanding their impact on atmospheric SO2 transformation and sulfate formation is of great importance. This study investigates the effect of oxalate on heterogeneous SO2 uptake and oxidation phenomenon by in situ FTIR, theoretical calculation, and continuous stream experiments, exploiting hematite (Fe2O3) as an environmental indicator. The results highlight the critical role of naturally deposited oxalate in mononuclear monodentate coordinating surface Fe atoms of Fe2O3 that enhances the activation of O2 for oxidizing SO2 into sulfate. Meanwhile, oxalate increases the hygroscopicity of Fe2O3, facilitating H2O dissociation into reactive hydroxyl groups and further augmenting the SO2 uptake capacity of Fe2O3. More importantly, other conventional iron minerals, such as goethite and magnetite, as well as authentic iron-containing mineral dust, exhibit similar oxalate-promoted sulfate accumulation behaviors. Our findings suggest that oxalate-assisted SO2 oxidation on iron minerals is one of the important contributors to secondary sulfate aerosols, especially during the nighttime with high relative humidity.
Asunto(s)
Hierro , Oxalatos , Sulfatos , Minerales , Oxidación-Reducción , Óxidos de Azufre , AerosolesRESUMEN
Nucleoprotein (NP) functions crucially in the replicative cycle of influenza A virus (IAV) via forming the ribonucleoprotein complex together with PB2, PB1, and PA proteins. As its high conservation, NP ranks one of the hot targets for design of universal diagnostic reagents and antiviral drugs for IAV. Here, we report an anti-NP murine monoclonal antibody (mAb) 5F10 prepared from traditional lymphocyte hybridoma technique with the immunogen of a clade 2.3.4.4 H5N1 subtype avian influenza virus. The specificity of mAb 5F10 to NP protein was confirmed by immunofluorescence assay and western blotting, and the mAb 5F10 could be used in immunoprecipitation and immunohistochemistry assays. Importantly, mAb 5F10 possessed broad-spectrum reactivity against H1~H11 subtypes of avian influenza viruses, including various HA clades of H5Nx subtype. In addition, mAb 5F10 also showed good affinity with H1N1 and H3N2 subtype influenza viruses of swine and human origin. Furthermore, the recognized antigenic epitope of mAb 5F10 was identified to consist of the conserved amino acid motif 81EHPSA85 in the second flexible loop region of NP protein through screening the phage display peptide library. Collectively, the mAb 5F10 which recognizes the novel universal NP linear B-cell epitope of IAV with diverse origins and subtypes will be a powerful tool for NP protein-based structural, functional, and mechanistic studies, as well as the development of detection methods and universal vaccines for IAV. KEY POINTS: ⢠A broad-spectrum mAb against various subtypes and sources of IAV was developed ⢠The mAb possessed good reactivity in IFA, western blot, IP, and IHC assays ⢠The mAb targeted a novel conserved linear B-cell epitope involving 81EHPSA85 on NP protein.
Asunto(s)
Subtipo H1N1 del Virus de la Influenza A , Subtipo H5N1 del Virus de la Influenza A , Virus de la Influenza A , Gripe Humana , Animales , Humanos , Ratones , Porcinos , Anticuerpos Monoclonales , Nucleoproteínas , Epítopos de Linfocito B , Subtipo H3N2 del Virus de la Influenza A , Anticuerpos AntiviralesRESUMEN
We investigated genetic and biologic characteristics of 2 Eurasian avian-like H1N1 swine influenza viruses from pigs in China that belong to the predominant G4 genotype. One swine isolate exhibited strikingly great homology to contemporaneous human Eurasian avian-like H1N1 isolates, preferential binding to the human-type receptor, and vigorous replication in mice without adaptation.
Asunto(s)
Subtipo H1N1 del Virus de la Influenza A , Gripe Humana , Infecciones por Orthomyxoviridae , Enfermedades de los Porcinos , Animales , Aves , China/epidemiología , Genotipo , Humanos , Subtipo H1N1 del Virus de la Influenza A/genética , Gripe Humana/epidemiología , Ratones , Infecciones por Orthomyxoviridae/veterinaria , Filogenia , Virus Reordenados/genética , Porcinos , Enfermedades de los Porcinos/epidemiologíaRESUMEN
Production of broadly-reactive antibodies is critical for universal immunodiagnosis of rapidly-evolving influenza viruses. Most monoclonal antibodies (mAbs) are generated in mice using the hybridoma technology which involves labor- and time-consuming screening and low yield issues. In this study, a recombinant antibody based on a broadly-reactive mAb against the hemagglutinin (HA) stalk of H7N9 avian influenza virus was expressed in CHO cells and its biological characteristics, cross-reactivity and epitope recognition were identified. The variable genes of the parental antibody were amplified and cloned into the antibody-expressing plasmids containing the constant genes of murine IgG1. The recombinant antibody was expressed in high yield and purity in CHO cells and showed similar features to the parental antibody, including negative hemagglutination inhibition activity against H7N9 virus and high binding activity with the H7N9 HA protein. Notably, the recombinant antibody exhibited a broad reactivity with different influenza subtypes belonging to group 1 and group 2, which was associated with its recognition of a highly-conserved epitope in the stalk, as observed for the parental antibody. Our results suggest that cell-based antibody expression system can be utilized as an important alternative to the hybridoma technology for antibody production for influenza virus diagnostics.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacología , Anticuerpos Antivirales/inmunología , Anticuerpos Antivirales/farmacología , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Orthomyxoviridae/efectos de los fármacos , Animales , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/aislamiento & purificación , Anticuerpos Antivirales/genética , Anticuerpos Antivirales/aislamiento & purificación , Células CHO , Cricetinae , Cricetulus , Reacciones Cruzadas , Expresión Génica , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Humanos , Subtipo H7N9 del Virus de la Influenza A/genética , Subtipo H7N9 del Virus de la Influenza A/inmunología , Gripe Humana/virología , Ratones , Orthomyxoviridae/clasificación , Orthomyxoviridae/inmunologíaRESUMEN
Newcastle disease (ND) is one of the most economically devastating infectious diseases affecting the poultry industry. Virulent Newcastle disease virus (NDV) can cause high mortality and severe tissue lesions in the respiratory, gastrointestinal, neurological, reproductive and immune systems of poultry. Tremendous progress has been made in preventing morbidity and mortality caused by ND based on strict biosecurity and wide vaccine application. In recent decades, the continual evolution of NDV has resulted in a total of twenty genotypes, and genetic variation may be associated with disease outbreaks in vaccinated chickens. In some countries, the administration of genotype-matched novel vaccines in poultry successfully suppresses the circulation of virulent NDV strains in the field. However, virulent NDV is still endemic in many regions of the world, especially in low- and middle-income countries, impacting the livelihood of millions of people dependent on poultry for food. In ND-endemic countries, although vaccination is implemented for disease control, the lack of genotype-matched vaccines that can reduce virus infection and transmission as well as the inadequate administration of vaccines in the field undermines the effectiveness of vaccination. Dissection of the profiles of existing ND vaccines is fundamental for establishing proper vaccination regimes and developing next-generation vaccines. Therefore, in this article, we provide a broad review of commercial and experimental ND vaccines and promising new platforms for the development of next-generation vaccines.
Asunto(s)
Enfermedad de Newcastle , Enfermedades de las Aves de Corral , Vacunas Virales , Animales , Pollos , Virus de la Enfermedad de Newcastle , Aves de CorralRESUMEN
Based on differences in the amino acid sequence of the protein haemagglutinin (HA), the H9N2 avian influenza virus (H9N2 virus) has been clustered into multiple lineages, and its rapidly ongoing evolution increases the difficulties faced by prevention and control programs. The HA protein, a major antigenic protein, and the amino acid mutations that alter viral antigenicity in particular have always been of interest. Likewise, it has been well documented that some amino acid mutations in HA alter viral antigenicity in the H9N2 virus, but little has been reported regarding how these antibody escape mutations affect antigenic variation. In this study, we were able to identify 15 HA mutations that were potentially relevant to viral antigenic drift, and we also found that a key amino acid mutation, A180V, at position 180 in HA (the numbering for mature H9 HA), the only site of the receptor binding sites that is not conserved, was directly responsible for viral antigenic variation. Moreover, the recombinant virus with alanine to valine substitution at position 180 in HA in the SH/F/98 backbone (rF/HAA180V virus) showed poor cross-reactivity to immune sera from animals immunized with the SH/F/98 (F/98, A180), SD/SS/94 (A180), JS/Y618/12 (T180), and rF/HAA180V (V180) viruses by microneutralization (MN) assay. The A180V substitution in the parent virus caused a significant decrease in cross-MN titres by enhancing the receptor binding activity, but it did not physically prevent antibody (Ab) binding. The strong receptor binding avidity prevented viral release from cells. Moreover, the A180V substitution promoted H9N2 virus escape from an in vitro pAb-neutralizing reaction, which also slightly affected the cross-protection in vivo. Our results suggest that the A180V mutation with a strong receptor binding avidity contributed to the low reactors in MN/HI assays and slightly affected vaccine efficacy but was not directly responsible for immune escape, which suggested that the A180V mutation might play a key role in the process of the adaptive evolution of H9N2 virus.
Asunto(s)
Subtipo H9N2 del Virus de la Influenza A , Gripe Aviar , Gripe Humana , Sustitución de Aminoácidos , Aminoácidos , Animales , Variación Antigénica , Antígenos Virales/genética , Pollos , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Hemaglutininas , Humanos , Subtipo H9N2 del Virus de la Influenza A/genética , Vacunas contra la Influenza , MutaciónRESUMEN
The zoonotic H7N9 avian influenza virus emerged with the H9N2-origin internal gene cassette. Previous studies have reported that genetic reassortments with H9N2 were common in the first five human H7N9 epidemic waves. However, our latest work found that the circulating high pathogenicity H7N9 virus has established a dominant internal gene cassette and has decreased the frequency of reassortment with H9N2 since 2018. This dominant cassette of H7N9 was distinct from the cocirculating H9N2, although they shared a common ancestor. As a result, we suppose that this dominant cassette may benefit the viral population fitness and promote its continuous circulation in chickens.
Asunto(s)
Subtipo H7N9 del Virus de la Influenza A , Subtipo H9N2 del Virus de la Influenza A , Gripe Aviar , Gripe Humana , Animales , Humanos , Subtipo H7N9 del Virus de la Influenza A/genética , Subtipo H9N2 del Virus de la Influenza A/genética , Virulencia/genética , Pollos , FilogeniaRESUMEN
Newcastle disease virus (NDV) is an important pathogen for poultry and is used as a vector for developing novel poultry vaccines. Previous studies showed that foreign gene insertion in NDV vector decreases virulence determined by in vitro assays; however, the impact of foreign gene expression on the pathogenicity of NDV in susceptible chickens is not fully investigated. In this study, a recombinant NDV based on a velogenic strain carrying the orange fluorescent protein (OFP) gene between the phosphoprotein (P) and matrix (M) genes was generated using reverse genetics. Biological characteristics, including virus replication, virulence, and OFP expression, and the pathogenicity in chickens were evaluated. The recombinant NDV showed comparable replication capacity in eggs and cells as the parental virus, whereas OFP insertion resulted in a mild impairment of virulence, evidenced by longer mean death time in embryos. High OFP expression was detected in the cells inoculated with the recombinant NDV. In addition, the recombinant NDV induced delayed onset of disease, lower severity of clinical signs, and lower mortality in chickens compared to the parental virus. Moreover, high titers of the parental virus were detected in the spleen, lung, and intestinal tract, while no recombinant NDV was recovered from these tissues. Our findings suggest that in vitro characteristics related to the insertion of the OFP gene in a virulent NDV do not correlate to alteration of the pathogenicity in chickens. Our results provided new information regarding assessment of the impact of foreign gene expression on the pathogenicity of NDV.