RESUMEN
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
Cytoplasmic stress granules (SGs) are generally triggered by stress-induced translation arrest for storing mRNAs. Recently, it has been shown that SGs exert anti-viral functions due to their involvement in protein synthesis shut off and recruitment of innate immune signaling intermediates. The largest RNA viruses, coronaviruses, impose great threat to public safety and animal health; however, the significance of SGs in coronavirus infection is largely unknown. Infectious Bronchitis Virus (IBV) is the first identified coronavirus in 1930s and has been prevalent in poultry farm for many years. In this study, we provided evidence that IBV overcomes the host antiviral response by inhibiting SGs formation via the virus-encoded endoribonuclease nsp15. By immunofluorescence analysis, we observed that IBV infection not only did not trigger SGs formation in approximately 80% of the infected cells, but also impaired the formation of SGs triggered by heat shock, sodium arsenite, or NaCl stimuli. We further demonstrated that the intrinsic endoribonuclease activity of nsp15 was responsible for the interference of SGs formation. In fact, nsp15-defective recombinant IBV (rIBV-nsp15-H238A) greatly induced the formation of SGs, along with accumulation of dsRNA and activation of PKR, whereas wild type IBV failed to do so. Consequently, infection with rIBV-nsp15-H238A strongly triggered transcription of IFN-ß which in turn greatly affected rIBV-nsp15-H238A replication. Further analysis showed that SGs function as an antiviral hub, as demonstrated by the attenuated IRF3-IFN response and increased production of IBV in SG-defective cells. Additional evidence includes the aggregation of pattern recognition receptors (PRRs) and signaling intermediates to the IBV-induced SGs. Collectively, our data demonstrate that the endoribonuclease nsp15 of IBV interferes with the formation of antiviral hub SGs by regulating the accumulation of viral dsRNA and by antagonizing the activation of PKR, eventually ensuring productive virus replication. We further demonstrated that nsp15s from PEDV, TGEV, SARS-CoV, and SARS-CoV-2 harbor the conserved function to interfere with the formation of chemically-induced SGs. Thus, we speculate that coronaviruses employ similar nsp15-mediated mechanisms to antagonize the host anti-viral SGs formation to ensure efficient virus replication.
Asunto(s)
COVID-19/virología , Gránulos Citoplasmáticos/metabolismo , Endorribonucleasas/inmunología , Endorribonucleasas/metabolismo , SARS-CoV-2/fisiología , Proteínas no Estructurales Virales/inmunología , Proteínas no Estructurales Virales/metabolismo , COVID-19/metabolismo , Línea Celular , Coronavirus/inmunología , Gránulos Citoplasmáticos/inmunología , Gránulos Citoplasmáticos/virología , Humanos , Interferón beta/inmunología , Interferón beta/metabolismo , SARS-CoV-2/metabolismo , Transducción de Señal , Replicación Viral/fisiologíaRESUMEN
BACKGROUND: Felid herpesvirus 1 (FHV-1) is a major pathogenic agent of upper respiratory tract infections and eye damage in felines worldwide. Current FHV-1 vaccines offer limited protection of short duration, and therefore, do not reduce the development of clinical signs or the latency of FHV-1. METHODS: To address these shortcomings, we constructed FHV ∆gIgE-eGFP, FHV ∆TK mCherry, and FHV ∆gIgE/TK eGFP-mCherry deletion mutants (ΔgI/gE, ΔTK, and ΔgIgE/TK, respectively) using the clustered regularly interspaced palindromic repeats (CRISPR)/CRISP-associated protein 9 (Cas9) system (CRISPR/Cas9), which showed safety and immunogenicity in vitro. We evaluated the safety and efficacy of the deletion mutants administered with intranasal (IN) and IN + subcutaneous (SC) vaccination protocols. Cats in the vaccination group were vaccinated twice at a 4-week interval, and all cats were challenged with infection 3 weeks after the last vaccination. The cats were assessed for clinical signs, nasal shedding, and virus-neutralizing antibodies (VN), and with postmortem histological testing. RESULTS: Vaccination with the gI/gE-deleted and gI/gE/TK-deleted mutants was safe and resulted in significantly lower clinical disease scores, fewer pathological changes, and less nasal virus shedding after infection. All three mutants induced virus-neutralizing antibodies after immunization. CONCLUSIONS: In conclusion, this study demonstrates the advantages of FHV-1 deletion mutants in preventing FHV-1 infection in cats.
Asunto(s)
Enfermedades de los Gatos , Infecciones por Herpesviridae , Varicellovirus , Gatos , Animales , Virulencia , Varicellovirus/genética , Vacunación , Anticuerpos Neutralizantes , Infecciones por Herpesviridae/prevención & control , Infecciones por Herpesviridae/veterinaria , Enfermedades de los Gatos/prevención & controlRESUMEN
Virulent systemic feline calicivirus (VS-FCV) is a newly emerging FCV variant that is associated with a severe acute multisystem disease in cats that is characterized by jaundice, oedema, and high mortality (approximately 70%). VS-FCV has spread throughout the world, but there are no effective vaccines or therapeutic options to combat infection. VS-FCV may therefore pose a serious threat to the health of felines. The genomic characteristics and functions of VS-FCV are still poorly understood, and the reason for its increased pathogenicity is unknown. Reverse genetics systems are powerful tools for studying the molecular biology of RNA viruses, but a reverse genetics system for VS-FCV has not yet been reported. In this study, we developed a plasmid-based reverse genetics system for VS-FCV in which infectious progeny virus is produced in plasmid-transfected CRFK cells. Using this system, we found that the 3' untranslated region (UTR) and poly(A) tail are important for maintaining the infection and replication capacity of VS-FCV and that shortening of the poly(A) tail to less than 28 bases eliminated the ability to rescue infectious progeny virus. Whether these observations are unique to VS-FCV or represent more-general features of FCV remains to be determined. In conclusion, we successfully established a rapid and efficient VS-FCV reverse genetics system, which provides a good platform for future research on the gene functions and pathogenesis of VS-FCV. The effects of the deletion of 3' UTR and poly(A) tail on VS-FCV infectivity and replication also provided new information about the pathogenesis of VS-FCV.
Asunto(s)
Infecciones por Caliciviridae , Calicivirus Felino , Enfermedades de los Gatos , Gatos , Animales , Regiones no Traducidas 3'/genética , Calicivirus Felino/genética , ADN Complementario , Genética Inversa , Replicación Viral/genéticaRESUMEN
BACKGROUND: Peste des petits ruminants (PPR) disease is a cross-species infectious disease that severely affects small ruminants and causes great losses to livestock industries in various countries. Distinguishing vaccine-immunized animals from naturally infected animals is an important prerequisite for the eradication of PPR. At present PPRV are classified into lineages I through IV, and only one vaccination strain, Nigeria/75/1, belongs to lineage II, but all of the epidemic strains in China at present are from lineage IV. RESULTS: To achieve this goal, we developed an SYBR Green I real-time qRT-PCR method for rapid detection and identification of PPRV lineages II and IV by analyzing different melting curve analyses. The negative amplification of other commonly circulating viruses such as orf virus, goat poxvirus, and foot-and-mouth disease virus demonstrated that primers targeting the L gene of PPRV were extremely specific. The sensitivity of the assay was assessed based on plasmid DNA and the detection limit achieved was 100 copies of PPRV lineages II and IV. CONCLUSION: Since the method has high sensitivity, specificity, and reproducibility, it will be effectively differentiated PPRV lineages II from PPRV lineages IV in PPRV infected animals.
Asunto(s)
Enfermedades de las Cabras , Peste de los Pequeños Rumiantes , Virus de la Peste de los Pequeños Rumiantes , Animales , Virus de la Peste de los Pequeños Rumiantes/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/veterinaria , Reproducibilidad de los Resultados , Peste de los Pequeños Rumiantes/epidemiología , Rumiantes , Cabras , Enfermedades de las Cabras/epidemiologíaRESUMEN
Newcastle disease virus (NDV), a member of the Paramyxoviridae family, can activate PKR/eIF2α signaling cascade to shutoff host and facilitate viral mRNA translation during infection, however, the mechanism remains unclear. In this study, we revealed that NDV infection up-regulated host cap-dependent translation machinery by activating PI3K/Akt/mTOR and p38 MAPK/Mnk1 pathways. In addition, NDV infection induced p38 MAPK/Mnk1 signaling participated 4E-BP1 hyperphosphorylation for efficient viral protein synthesis when mTOR signaling is inhibited. Furthermore, NDV NP protein was found to be important for selective cap-dependent translation of viral mRNAs through binding to eIF4E during NDV infection. Taken together, NDV infection activated multiple signaling pathways for selective viral protein synthesis in infected cells, via interaction between viral NP protein and host translation machinery. Our results may help to design novel targets for therapeutic intervention against NDV infection and to understand the NDV anti-oncolytic mechanism.
Asunto(s)
Proteínas Aviares , Factor 4E Eucariótico de Iniciación , Sistema de Señalización de MAP Quinasas , Virus de la Enfermedad de Newcastle , Nucleoproteínas , ARN Mensajero , ARN Viral , Proteínas Virales , Animales , Proteínas Aviares/genética , Proteínas Aviares/metabolismo , Embrión de Pollo , Pollos , Factor 4E Eucariótico de Iniciación/genética , Factor 4E Eucariótico de Iniciación/metabolismo , Células HEK293 , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Virus de la Enfermedad de Newcastle/genética , Virus de la Enfermedad de Newcastle/metabolismo , Proteínas de la Nucleocápside , Nucleoproteínas/biosíntesis , Nucleoproteínas/genética , Nucleoproteínas/metabolismo , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Biosíntesis de Proteínas , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Viral/genética , ARN Viral/metabolismo , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismo , Proteínas Virales/biosíntesis , Proteínas Virales/genética , Proteínas Virales/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/genética , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Deoxyribonucleic acid (DNA) damage response (DDR) is the fundamental cellular response for maintaining genomic integrity and suppressing tumorigenesis. The activation of ataxia telangiectasia-mutated (ATM) kinase is central to DNA double-strand break (DSB) for maintaining host-genome integrity in mammalian cells. Oncolytic Newcastle disease virus (NDV) can selectively replicate in tumor cells; however, its influence on the genome integrity of tumor cells is not well-elucidated. Here, we found that membrane fusion and NDV infection triggered DSBs in tumor cells. The late replication and membrane fusion of NDV mechanistically activated the ATM-mediated DSB pathway via the ATM-Chk2 axis, as evidenced by the hallmarks of DSBs, i.e., auto-phosphorylated ATM and phosphorylated H2AX and Chk2. Immunofluorescence data showed that multifaceted ATM-controlled phosphorylation markedly induced the formation of pan-nuclear punctum foci in response to NDV infection and F-HN co-expression. Specific drug-inhibitory experiments on ATM kinase activity further suggested that ATM-mediated DSBs facilitated NDV replication and membrane fusion. We confirmed that the Mre11-RAD50-NBS1 (MRN) complex sensed the DSB signal activation triggered by NDV infection and membrane fusion. The pharmacological inhibition of MRN activity also significantly inhibited intracellular and extracellular NDV replication and syncytia formation. Collectively, these data identified for the first time a direct link between the membrane fusion induced by virus infection and DDR pathways, thereby providing new insights into the efficient replication of oncolytic NDV in tumor cells.
Asunto(s)
Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Roturas del ADN de Doble Cadena , Células Gigantes , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Virus de la Enfermedad de Newcastle/fisiología , Virus Oncolíticos/fisiología , Replicación Viral , Células A549 , Ácido Anhídrido Hidrolasas/genética , Ácido Anhídrido Hidrolasas/metabolismo , Animales , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Embrión de Pollo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Células Gigantes/metabolismo , Células Gigantes/virología , Células HEK293 , Humanos , Proteína Homóloga de MRE11/genética , Proteína Homóloga de MRE11/metabolismo , Proteínas de Neoplasias/genética , Neoplasias/genética , Neoplasias/virología , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Transducción de Señal/genéticaRESUMEN
Elucidating virus-cell interactions is fundamental to understanding viral replication and identifying targets for therapeutic control of viral infection. The extracellular signal-regulated kinase (ERK) pathway has been shown to regulate pathogenesis during many viral infections, but its role during coronavirus infection is undetermined. Infectious bronchitis virus is the representative strain of Gammacoronavirus, which causes acute and highly contagious diseases in the poultry farm. In this study, we investigated the role of ERK1/2 signaling pathway in IBV infection. We found that IBV infection activated ERK1/2 signaling and the up-regulation of phosphatase DUSP6 formed a negative regulation loop. Pharmacological inhibition of MEK1/2-ERK1/2 signaling suppressed the expression of DUSP6, promoted cell death, and restricted virus replication. In contrast, suppression of DUSP6 by chemical inhibitor or siRNA increased the phosphorylation of ERK1/2, protected cells from apoptosis, and facilitated IBV replication. Overexpression of DUSP6 decreased the level of phospho-ERK1/2, promoted apoptosis, while dominant negative mutant DUSP6-DN lost the regulation function on ERK1/2 signaling and apoptosis. In conclusion, these data suggest that MEK-ERK1/2 signaling pathway facilitates IBV infection, probably by promoting cell survival; meanwhile, induction of DUSP6 forms a negative regulation loop to restrict ERK1/2 signaling, correlated with increased apoptosis and reduced viral load. Consequently, components of the ERK pathway, such as MEK1/2 and DUSP6, represent excellent targets for the development of antiviral drugs.
Asunto(s)
Apoptosis/fisiología , Fosfatasas de Especificidad Dual/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Virus de la Bronquitis Infecciosa/fisiología , Fosfatasas de la Proteína Quinasa Activada por Mitógenos/metabolismo , Animales , Butadienos/farmacología , Línea Celular , Pollos , Chlorocebus aethiops , Fosfatasas de Especificidad Dual/antagonistas & inhibidores , Fosfatasas de Especificidad Dual/genética , Quinasas MAP Reguladas por Señal Extracelular/genética , Fosfatasas de la Proteína Quinasa Activada por Mitógenos/antagonistas & inhibidores , Fosfatasas de la Proteína Quinasa Activada por Mitógenos/genética , Nitrilos/farmacología , Regulación hacia Arriba , Replicación ViralRESUMEN
Paramyxovirus establishes an intimate and complex interaction with the host cell to counteract the antiviral responses elicited by the cell. Of the various pattern recognition receptors in the host, the cytosolic RNA helicases interact with viral RNA to activate the mitochondrial antiviral signaling protein (MAVS) and subsequent cellular interferon (IFN) response. On the other hand, viruses explore multiple strategies to resist host immunity. In this study, we found that Newcastle disease virus (NDV) infection induced MAVS degradation. Further analysis showed that NDV V protein degraded MAVS through the ubiquitin-proteasome pathway to inhibit IFN-ß production. Moreover, NDV V protein led to proteasomal degradation of MAVS through Lys362 and Lys461 ubiquitin to prevent IFN production. Further studies showed that NDV V protein recruited E3 ubiquitin ligase RNF5 to polyubiquitinate and degrade MAVS. Compared with levels for wild-type NDV infection, V-deficient NDV induced attenuated MAVS degradation and enhanced IFN-ß production at the late stage of infection. Several other paramyxovirus V proteins showed activities of degrading MAVS and blocking IFN production similar to those of NDV V protein. The present study revealed a novel role of NDV V protein in targeting MAVS to inhibit cellular IFN production, which reinforces the fact that the virus orchestrates the cellular antiviral response to its own benefit.IMPORTANCE Host anti-RNA virus innate immunity relies mainly on the recognition by retinoic acid-inducible gene I and melanoma differentiation-associated protein 5 and subsequently initiates downstream signaling through interaction with MAVS. On the other hand, viruses have developed various strategies to counteract MAVS-mediated signaling. The mechanism for paramyxoviruses regulating MAVS to benefit their infection remains unknown. In this article, we demonstrate that the V proteins of NDV and several other paramyxoviruses target MAVS for ubiquitin-mediated degradation through E3 ubiquitin ligase RING-finger protein 5 (RNF5). MAVS degradation leads to the inhibition of the downstream IFN-ß pathway and therefore benefits virus proliferation. Our study reveals a novel mechanism of NDV evading host innate immunity and provides insight into the therapeutic strategies for the control of paramyxovirus infection.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas de Unión al ADN/metabolismo , Interferón Tipo I/antagonistas & inhibidores , Virus de la Enfermedad de Newcastle/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas Virales/metabolismo , Células A549 , Antivirales , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/inmunología , Células HEK293 , Células HeLa , Interacciones Huésped-Patógeno , Humanos , Inmunidad Innata , Interferón Tipo I/metabolismo , Interferón beta/inmunología , Interferón beta/metabolismo , Virus de la Enfermedad de Newcastle/inmunología , ARN Helicasas/metabolismo , Transducción de Señal , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/biosíntesis , Ubiquitina-Proteína Ligasas/inmunología , UbiquitinaciónRESUMEN
Newcastle disease (ND), which is caused by Newcastle disease virus (NDV), can cause heavy economic losses to the poultry industry worldwide. It is characterised by extensive pathologies of the digestive, respiratory, and nervous systems and can cause severe damage to the reproductive system of egg-laying hens. However, it is unknown whether NDV replicates in the male reproductive system of chickens and induces any pathologies. In this study, we selected a representative strain (i.e. ZJ1) of the most common genotype (i.e. VII) of NDV to investigate whether NDV can induce histological, hormonal, and inflammatory responses in the testes of specific pathogen free (SPF) roosters. NDV infection increased the expression of toll like receptor TLR3, TLR7, MDA5, IFN-α, IFN-ß, IFN-γ, IL-8, and CXCLi1 in the testes of NDV-infected roosters at 5 days post-infection (dpi). Severe histological changes, including decrease in the number of Sertoli cells and individualized, shrunken spermatogonia with pyknotic nuclei, were observed at 3 dpi. At 5 dpi, the spermatogenic columns were disorganized, and there were fewer cells, which were replaced by necrotic cells, lipid vacuoles, and proteinaceous homogenous material. A significant decrease in the plasma concentrations of testosterone and luteinizing hormone (LH) and the mRNA expression of their receptors in the testes, steroidogenic acute regulatory protein, cytochrome P450 side-chain cleavage enzyme, and 3ß-hydroxysteroid dehydrogenase in the NDV-infected group was observed relative to those in the control group (P < 0.05). Collectively, these results indicate that NDV infection induces a severe inflammatory response and histological changes, which decrease the steroidogenesis.
Asunto(s)
Pollos , Enfermedad de Newcastle/complicaciones , Virus de la Enfermedad de Newcastle/fisiología , Enfermedades de las Aves de Corral/virología , Enfermedades Testiculares/veterinaria , Animales , Masculino , Enfermedades de las Aves de Corral/metabolismo , Organismos Libres de Patógenos Específicos , Enfermedades Testiculares/metabolismo , Enfermedades Testiculares/virología , Testículo/patologíaRESUMEN
The fusogenically activated F and HN proteins of virulent NDV induce complete autophagic flux in DF-1 and A549 cells. However, the effect of both glycoproteins on mitochondria remains elusive. Here, we found that F and HN cooperation increases mitochondrial biogenesis but does not cause the mitochondria damage. We observed that both glycoproteins change the morphological characteristics and spatial distribution of intracellular mitochondria. F and HN cooperate cooperatively to induce ER stress and UPRmt. Our preliminary data suggested that F and HN cooperatively disturb mitochondrial fusion-fission homeostasis to enhance mitochondrial biogenesis, and eventually meet the energy demand of syncytium formation.
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Retículo Endoplásmico/virología , Hemaglutininas/metabolismo , Mitocondrias/metabolismo , Neuraminidasa/metabolismo , Enfermedad de Newcastle/metabolismo , Virus de la Enfermedad de Newcastle/metabolismo , Respuesta de Proteína Desplegada , Células A549/metabolismo , Células A549/virología , Animales , Western Blotting , Retículo Endoplásmico/metabolismo , Homeostasis , Humanos , Mitocondrias/virologíaRESUMEN
BACKGROUND/AIMS: Newcastle disease virus (NDV) causes a highly devastating and contagious disease in poultry, which is mainly attributed to extensive tissue damages in the digestive, respiratory and nervous systems. However, nature and dynamics of NDV-induced oxidative stresses in the intestine of chickens remain elusive. METHODS: In this study, we examined the magnitude of intestinal oxidative stress and histopathological changes caused by the virulent NDV infection, and explored the protective roles of vitamin E (vit. E) in ameliorating these pathological changes. For these purposes, chickens were divided into four groups namely i) non supplemented and non-challenged (negative control, CON); ii) no supplementation of vit. E but challenged with ZJ1 (positive control, NS+CHA); iii) vit. E supplementation at the dose of 50 IU/day/Kg body weight and ZJ1 challenge (VE50+CHA); and 4) vit. E supplementation at the dose of 100 IU/day/Kg body weight and ZJ1 challenge (VE100+CHA). In all groups, we analyzed concentrations of glutathione (GSH), malondialdehyde (MDA), nitric oxide (NO), total antioxidant capacity (T-AOC), and activity of glutathione S-transferase (GST), superoxide dismutase (SOD), catalase (CAT) using biochemical methods. The virus loads were determined by quantitative RT-PCR and antibody titers by hemagglutination inhibition assays. We also examined the histopathological changes in the duodenal and jejunal mucosa at 3 and 5-day post infection (dpi) with NDV. RESULTS: A significant elevation in the NO level was observed in NDV challenged chickens compared to the CON chickens at 2 dpi. The MDA contents were significantly increased whereas GSH was significantly decreased in NDV-challenged chickens compared to control. Furthermore, activities of GST, CAT, SOD, as well as the TOAC were markedly decreased in challenged chickens in comparison with control. Virus copy numbers were higher in NDV infected NS+CHA group compared to other groups. Severe histopathological changes including inflammation, degeneration and broken villi were observed in the intestine of NDV challenged chickens. However, all these malfunctions of antioxidant system and pathological changes in the intestine were partially or completely reversed by the vit. E supplementation. CONCLUSIONS: Our results suggest that NDV infection causes oxidative stress and histopathological changes in the duodenum and jejunum of chickens, which can be partially or fully ameliorated by supplementation of vit. E. Additionally, these findings suggest that oxidative stress contributes to the intestinal damages in NDV infected chickens. These findings will help to understand the pathogenesis of NDV and further investigation of therapeutic agents for control of Newcastle disease.
Asunto(s)
Pollos , Duodeno , Yeyuno , Enfermedad de Newcastle , Virus de la Enfermedad de Newcastle , Estrés Oxidativo/efectos de los fármacos , Enfermedades de las Aves de Corral , Vitamina E/farmacología , Animales , Embrión de Pollo , Pollos/metabolismo , Pollos/virología , Duodeno/metabolismo , Duodeno/patología , Duodeno/virología , Yeyuno/metabolismo , Yeyuno/patología , Yeyuno/virología , Enfermedad de Newcastle/metabolismo , Enfermedad de Newcastle/patología , Enfermedades de las Aves de Corral/metabolismo , Enfermedades de las Aves de Corral/patología , Enfermedades de las Aves de Corral/virologíaRESUMEN
Mammalian cells respond to various environmental stressors to form stress granules (SGs) by arresting cytoplasmic mRNA, protein translation element, and RNA binding proteins. Virus-induced SGs function in different ways, depending on the species of virus; however, the mechanism of SG regulation of virus replication is not well understood. In this study, Newcastle disease virus (NDV) triggered stable formation of bona fide SGs on HeLa cells through activating the protein kinase R (PKR)/eIF2α pathway. NDV-induced SGs contained classic SG markers T-cell internal antigen (TIA)-1, Ras GTPase-activating protein-binding protein (G3BP)-1, eukaryotic initiation factors, and small ribosomal subunit, which could be disassembled in the presence of cycloheximide. Treatment with nocodazole, a microtubule disruption drug, led to the formation of relatively small and circular granules, indicating that NDV infection induces canonical SGs. Furthermore, the role of SGs on NDV replication was investigated by knockdown of TIA-1 and TIA-1-related (TIAR) protein, the 2 critical components involved in SG formation from the HeLa cells, followed by NDV infection. Results showed that depletion of TIA-1 or TIAR inhibited viral protein synthesis, reduced extracellular virus yields, but increased global protein translation. FISH revealed that NDV-induced SGs contained predominantly cellular mRNA rather than viral mRNA. Deletion of TIA-1 or TIAR reduced NP mRNA levels in polysomes. These results demonstrate that NDV triggers stable formation of bona fide SGs, which benefit viral protein translation and virus replication by arresting cellular mRNA.-Sun, Y., Dong, L., Yu, S., Wang, X., Zheng, H., Zhang, P., Meng, C., Zhan, Y., Tan, L., Song, C., Qiu, X., Wang, G., Liao, Y., Ding, C. Newcastle disease virus induces stable formation of bona fide stress granules to facilitate viral replication through manipulating host protein translation.
Asunto(s)
Gránulos Citoplasmáticos/metabolismo , Interacciones Huésped-Patógeno , Virus de la Enfermedad de Newcastle/fisiología , Replicación Viral , Animales , Proteínas Portadoras/metabolismo , Células Cultivadas , Pollos , ADN Helicasas , Factor 2 Eucariótico de Iniciación/metabolismo , Células HeLa , Humanos , Virus de la Enfermedad de Newcastle/metabolismo , Virus de la Enfermedad de Newcastle/patogenicidad , Proteínas de Unión a Poli(A)/metabolismo , Proteínas de Unión a Poli-ADP-Ribosa , Biosíntesis de Proteínas , ARN Helicasas , Proteínas con Motivos de Reconocimiento de ARN , Proteínas de Unión al ARN , Subunidades Ribosómicas/metabolismo , Antígeno Intracelular 1 de las Células T , eIF-2 Quinasa/metabolismoRESUMEN
Avian avulaviruses serotype 1 (abbreviated as APMV-1 for the historical name avian paramyxovirus 1) are capable of infecting a wide spectrum of avian species with variable clinical symptoms and outcomes. Ease of transmission has allowed the virus to spread worldwide with varying degrees of virulence depending upon the virus strain and host species. The emergence of new virulent genotypes from global epizootics, and the year-to-year genomic changes in low and high virulence APMV-1 imply that distinct genotypes of APMV-1 are simultaneously evolving at different geographic locations across the globe. This vast genomic diversity may be favoured by large variety of avian species susceptibility to APMV-1 infection, and by the availability of highly mobile wild birds. It has long been considered that waterfowls are not sensitive to APMV-1 and are unable to show any clinical signs, however, outbreaks from the 90's contradict these concepts. The APMV-1 isolates are increasingly reported from the waterfowl. Waterfowl have strong innate immune responses, which minimize the impact of virus infection, however, are unable to prevent the viral shedding. Numerous APMV-1 are carried by domestic waterfowl intermingling with terrestrial poultry. Therefore, commercial ducks and geese should be vaccinated against APMV-1 to minimize the virus shedding and for the prevention the transmission. Genetic diversity within APMV-1 demonstrates the need for continual monitoring of viral evolution and periodic updates of vaccine seed-strains to achieve efficient control and eradication of APMV-1 in waterfowls.
Asunto(s)
Anseriformes , Enfermedad de Newcastle/inmunología , Virus de la Enfermedad de Newcastle/fisiología , Enfermedades de las Aves de Corral/inmunología , Animales , Enfermedad de Newcastle/virología , Enfermedades de las Aves de Corral/virologíaRESUMEN
A field canine parvovirus (CPV) strain, CPV-SH14, was previously isolated from an outbreak of severe gastroenteritis in Shanghai in 2014. The complete genome of CPV-SH14 was determined by using PCR with modified primers. When compared to other CPV-2 strains, several insertions, deletions, and point mutations were identified in the 5' and 3' UTR, with key amino acid (aa) mutations (K19R, E572K in NS1 and F267Y, Y324I and T440A in VP2) also being observed in the coding regions of CPV-SH14. These results indicated that significant and unique genetic variations have occurred at key sites or residues in the genome of CPV-SH14, suggesting the presence of a novel genetic variant of new CPV-2a. Phylogenetic analysis of the VP2 gene revealed that CPV-SH14 may have the potential to spread worldwide. In conclusion, CPV-SH14 may be a novel genetic variant of new CPV-2a, potentially with a selective advantage over other strains.
Asunto(s)
Enfermedades de los Perros/virología , Genoma Viral , Infecciones por Parvoviridae/veterinaria , Parvovirus Canino/genética , Parvovirus Canino/aislamiento & purificación , Animales , Proteínas de la Cápside/genética , China , Perros , Variación Genética , Mutación , Infecciones por Parvoviridae/virología , Parvovirus Canino/clasificación , FilogeniaRESUMEN
UNLABELLED: It has been reported that lentogenic Newcastle disease virus (NDV) isolates have the potential to become velogenic after their transmission and circulation in chickens, but the underlying mechanism is unclear. In this study, a highly velogenic NDV variant, JS10-A10, was generated from the duck-origin lentogenic isolate JS10 through 10 consecutive passages in chicken air sacs. The velogenic properties of this selected variant were determined using mean death time (MDT) assays, intracerebral pathogenicity index (ICPI), the intravenous pathogenicity index (IVPI), histopathology, and the analysis of host tissue tropism. In contrast, JS10 remained lentogenic after 20 serial passages in chicken eggs (JS10-E20). The JS10, JS10-A10, and JS10-E20 genomes were sequenced and found to be nearly identical, suggesting that both JS10-A10 and JS10-E20 were directly generated from JS10. To investigate the mechanism for virulence enhancement, the partial genome covering the F0 cleavage site of JS10 and its variants were analyzed using ultradeep pyrosequencing (UDPS) and the proportions of virulence-related genomes in the quasispecies were calculated. Velogenic NDV genomes accumulated as a function of JS10 passaging through chicken air sacs. Our data suggest that lentogenic NDV strains circulating among poultry might be a risk factor to future potential velogenic NDV outbreaks in chickens. IMPORTANCE: An avirulent isolate, JS10, was passaged through chicken air sacs and embryos, and the pathogenicity of the variants was assessed. A virulent variant, JS10-A10, was generated from consecutive passage in air sacs. We developed a deep-sequencing approach to detect low-frequency viral variants across the NDV genome. We observed that virulence enhancement of JS10 was due to the selective accumulation of velogenic quasispecies and the concomitant disappearance of lentogenic quasispecies. Our results suggest that because it is difficult to avoid contact between natural waterfowl reservoirs and sensitive poultry operations, circulating lentogenic NDV strains may represent a potential reservoir for emergent velogenic NDV strains that could cause outbreaks in chickens.
Asunto(s)
Variación Genética , Enfermedad de Newcastle/patología , Enfermedad de Newcastle/virología , Virus de la Enfermedad de Newcastle/patogenicidad , Enfermedades de las Aves de Corral/patología , Enfermedades de las Aves de Corral/virología , Pase Seriado , Adaptación Biológica , Sacos Aéreos/virología , Animales , Encéfalo/patología , Pollos , Patos , Genoma Viral , Histocitoquímica , Datos de Secuencia Molecular , Virus de la Enfermedad de Newcastle/genética , Virus de la Enfermedad de Newcastle/crecimiento & desarrollo , Análisis de Secuencia de ADN , Análisis de Supervivencia , Tropismo Viral , VirulenciaRESUMEN
BACKGROUND: For many years, ND has been one of the most important infectious pigeon diseases in China. In recent years, a high mortality has been observed in ND-infected pigeons in China. Mortality is from 40% to 80% or 100% in some cases. METHODS: The full-length genomes of four pigeon paramyxovirus type 1 (PPMV-1) strains, which were isolated from infected pigeons in China in 2012 and 2013, were sequenced and analyzed to determine the phylogenetic characteristics of PPMV-1 circulating in pigeons of China in recent years. Furthermore, cross hemagglutination inhibition and cross virus neutralization assays, as well as animal experiments were conducted to determine the antigenicity and pathogenicity of those viruses. Proteolytic cleavage sites (residues 112-117) of the F proteins were identified as the typical virulence motif, 112RRQKR↓F117 for all four PPMV-1 strains investigated. RESULTS: Phylogenetic analysis based on sequences of complete genomes and F gene revealed that the four PPMV-1 isolates and most of recent isolates in China were highly homologous to European isolates from 1998 to 2011. All those isolates were clustered in one clade of genotype VI NDV, termed as subgroup 4bii f. The R value was calculated based on cross hemagglutination inhibition and cross virus neutralization results, and confirmed antigenic difference of the PPMV-1 strains isolated in 2013 from the LaSota vaccine strain. Several mutations were identified in the surface glycoproteins F and HN, which probably gave rise to those antigenic differences. CONCLUSION: Our result suggested that the PPMV-1 strain prevailing in China in the last decade diverged from a common ancestor and was presumably transmitted from Europe. PPMV-1 isolates displayed obvious antigenic differences from vaccine strain LaSota. Even though PPMV-1 did not cause high mortality in experimental pigeons, the infected pigeons were exhibiting viral shedding for 3 weeks after infection, suggesting PPMV-1 is a potential threat to NDV control worldwide.
Asunto(s)
Columbidae/virología , Enfermedad de Newcastle/inmunología , Enfermedad de Newcastle/virología , Virus de la Enfermedad de Newcastle/clasificación , Virus de la Enfermedad de Newcastle/fisiología , Filogenia , Animales , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , China/epidemiología , Reacciones Cruzadas/inmunología , Genoma Viral , Pruebas de Inhibición de Hemaglutinación , Pruebas de Neutralización , Enfermedad de Newcastle/epidemiología , ARN Viral , Secuenciación Completa del GenomaRESUMEN
Newcastle disease virus (NDV) encodes a highly phosphorylated P protein; however, the phosphorylation sites have not been identified, and the relationship between phosphorylation and protein function is still unclear. In this study, we bioinformatically predicted 26 amino acid residues in the P protein as potential phosphorylation sites. Furthermore, we treated infected cells with kinase inhibitors to investigate NDV propagation and found that protein kinase C (PKC) is involved in the NDV life cycle and that PKC-activated phosphorylation functions in NDV replication. Using an NDV minigenome assay, we found that expression of a reporter protein decreased when the minigenome system contained P mutants lacking T44, S48, T271, S373 and especially T111. The phosphorylation status of S48, T111, S125 and T271 was determined by Phos-tag SDS-PAGE analysis. Coimmunoprecipitation assays showed that the binding activity of NP and the P-T111A mutant was stronger than that of NP and the wild-type P, suggesting that P-T111 is involved in NP-P interaction. This study sheds light on the mechanism by which P protein phosphorylation affects NDV replication and transcription.
Asunto(s)
Enfermedad de Newcastle/virología , Virus de la Enfermedad de Newcastle/metabolismo , Fosfoproteínas/metabolismo , Enfermedades de las Aves de Corral/virología , Proteínas Virales/metabolismo , Secuencias de Aminoácidos , Animales , Pollos , Virus de la Enfermedad de Newcastle/química , Virus de la Enfermedad de Newcastle/genética , Proteínas de la Nucleocápside , Nucleoproteínas/genética , Nucleoproteínas/metabolismo , Fosfoproteínas/química , Fosfoproteínas/genética , Fosforilación , Unión Proteica , Proteínas Virales/química , Proteínas Virales/genéticaRESUMEN
Newcastle disease virus (NDV) is an important avian pathogen. We previously reported that NDV triggers autophagy in U251 glioma cells, resulting in enhanced virus replication. In this study, we investigated whether NDV triggers autophagy in chicken cells and tissues to enhance virus replication. We demonstrated that NDV infection induced steady-state autophagy in chicken-derived DF-1 cells and in primary chicken embryo fibroblast (CEF) cells, evident through increased double- or single-membrane vesicles, the accumulation of green fluorescent protein (GFP)-LC3 dots, and the conversion of LC3-I to LC3-II. In addition, we measured autophagic flux by monitoring p62/SQSTM1 degradation, LC3-II turnover, and GFP-LC3 lysosomal delivery and proteolysis, to confirm that NDV infection induced the complete autophagic process. Inhibition of autophagy by pharmacological inhibitors and RNA interference reduced virus replication, indicating an important role for autophagy in NDV infection. Furthermore, we conducted in vivo experiments and observed the conversion of LC3-I to LC3-II in heart, liver, spleen, lung, and kidney of NDV-infected chickens. Regulation of the induction of autophagy with wortmannin, chloroquine, or starvation treatment affects NDV production and pathogenesis in tissues of both lung and intestine; however, treatment with rapamycin, an autophagy inducer of mammalian cells, showed no detectable changes in chicken cells and tissues. Moreover, administration of the autophagy inhibitor wortmannin increased the survival rate of NDV-infected chickens. Our studies provide strong evidence that NDV infection induces autophagy which benefits NDV replication in chicken cells and tissues.
Asunto(s)
Autofagia , Virus de la Enfermedad de Newcastle/fisiología , Replicación Viral , Animales , Células Cultivadas , Embrión de Pollo , Microscopía Electrónica de Transmisión , Interferencia de ARNRESUMEN
The complete genome sequence of a novel duck orthoreovirus, designated DRV strain TH11(DRV-TH11), was determined and characterized. The DRV-TH11 genome is comprised of 23,417 bp and its genome organization is more similar to that of avian orthoreoviruses (ARVs) of chicken origin than other reoviruses. The results of comparative sequence analysis and dendrograms based on the µB- and σC-encoding genes indicated that TH11 may be derived from the reassortment of ARVs and classic Muscovy duck reovirus (MDRV). A possible recombinant event was identified using the SimPlot program, and it occurred in the M2 segment. The results indicated that reassortment and mutation play a role in the evolution of duck reovirus.