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1.
Int J Mol Sci ; 24(2)2023 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-36675290

RESUMO

Rice dwarf virus (RDV) is transmitted by insect vectors Nephotettix virescens and Nephotettix cincticeps (Hemiptera: Cicadellidae) that threatens rice yield and results in substantial economic losses. RDV induces two volatiles ((E)-ß-caryophyllene (EBC) and 2-heptanol) to emit from RDV-infected rice plants. However, the effects of the two volatiles on the olfactory behavior of both non-viruliferous and viruliferous N. virescens are unknown, and whether the two volatiles could facilitate the spread and dispersal of RDV remains elusive. Combining the methods of insect behavior, chemical ecology, and molecular biology, we found that EBC and 2-heptanol influenced the olfactory behavior of non-viruliferous and viruliferous N. virescens, independently. EBC attracted non-viruliferous N. virescens towards RDV-infected rice plants, promoting virus acquisition by non-viruliferous vectors. The effect was confirmed by using oscas1 mutant rice plants (repressed EBC synthesis), but EBC had no effects on viruliferous N. virescens. 2-heptanol did not attract or repel non-viruliferous N. virescens. However, spraying experiments showed that 2-heptanol repelled viruliferous N. virescens to prefer RDV-free rice plants, which would be conducive to the transmission of the virus. These novel results reveal that rice plant volatiles modify the behavior of N. virescens vectors to promote RDV acquisition and transmission. They will provide new insights into virus-vector-plant interactions, and promote the development of new prevention and control strategies for disease management.


Assuntos
Hemípteros , Oryza , Vírus de Plantas , Reoviridae , Animais , Heptanol , Insetos Vetores , Doenças das Plantas
2.
Dev Comp Immunol ; 140: 104614, 2023 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-36502963

RESUMO

From mammals to fish, interferons (IFNs) play vital roles in the immune response. In this study, a newly identified type IV interferon (bcIFN-υ) from black carp (Mylopharyngodon piceus) has been cloned and characterized. The CDS of bcIFN-υ consists of 489 nucleotides, encoding 163 amino acids, with the first 20 amino acids predicted to be the signal peptide region. The immunoblot and immunofluorescence assays verified that bcIFN-υ was a secreted cytokine. qPCR analysis and reporter assay demonstrated that bcIFN-υ participated in innate immune defense and activated the transcription of fish ISRE promoter under spring viremia of carp virus (SVCV) stimulation. Additionally, compared with control group, EPC cells transfected with bcIFN-υ or incubated with the bcIFN-υ-containing conditioned media before SVCV infection showed greatly enhanced antiviral activity, and the transcription levels of MX1, PKR, ISG15 and Viperin genes were significantly increased. The subsequential co-immunoprecipitation assay identified the interaction between bcIFN-υ proteins. Collectively, our data conclude that bcIFN-υ is a kind of secretory protein with self-interaction and triggering the expression of downstream ISGs to enhance the antiviral activity of host cells.


Assuntos
Carpas , Doenças dos Peixes , Infecções por Reoviridae , Reoviridae , Infecções por Rhabdoviridae , Animais , Interferons/genética , Carpas/genética , Carpas/metabolismo , Reoviridae/fisiologia , Imunidade Inata/genética , Sequência de Aminoácidos , Clonagem Molecular , Antivirais , Proteínas de Peixes/metabolismo , Mamíferos/genética
3.
Proc Natl Acad Sci U S A ; 119(50): e2203054119, 2022 Dec 13.
Artigo em Inglês | MEDLINE | ID: mdl-36469786

RESUMO

Mammalian reovirus (reovirus) is a multilayered, turreted member of Reoviridae characterized by transcription of dsRNA genome within the innermost capsid shell. Here, we present high-resolution in situ structures of reovirus transcriptase complex in an intact double-layered virion, and in the uncoated single-layered core particles in the unloaded, reloaded, pre-elongation, and elongation states, respectively, obtained by cryo-electron microscopy and sub-particle reconstructions. At the template entry of RNA-dependent RNA polymerase (RdRp), the RNA-loading region gets flexible after uncoating resulting in the unloading of terminal genomic RNA and inactivity of transcription. However, upon adding transcriptional substrates, the RNA-loading region is recovered leading the RNAs loaded again. The priming loop in RdRp was found to play a critical role in regulating transcription, which hinders the elongation of transcript in virion and triggers the rearrangement of RdRp C-terminal domain (CTD) during elongation, resulting in splitting of template-transcript hybrid and opening of transcript exit. With the integration of these structures, a transcriptional model of reovirus with five states is proposed. Our structures illuminate the RdRp activation and regulation of the multilayered turreted reovirus.


Assuntos
RNA Viral , Reoviridae , Animais , Microscopia Crioeletrônica , RNA Viral/genética , Reoviridae/genética , RNA Polimerase Dependente de RNA/genética , Capsídeo , Mamíferos/genética
4.
Viruses ; 14(12)2022 Nov 26.
Artigo em Inglês | MEDLINE | ID: mdl-36560642

RESUMO

It has been previously shown that amino acid polymorphisms in reovirus proteins µ2 and λ1 are associated with differing levels of interferon induction. In the present study, viruses carrying these polymorphisms in either or both proteins, were further studied. The two viral determinants exert a synergistic effect on the control of ß-interferon induction at the protein and mRNA level, with a concomitant increase in RIG-I. In contrast, levels of phospho-Stat1 and interferon-stimulated genes are increased in singly substituted viruses but with no further increase when both substitutions were present. This suggests that the viral determinants are acting during initial events of viral recognition. Accordingly, difference between viruses was reduced when infection was performed with partially uncoated virions (ISVPs) and transfection of RNA recovered from early-infected cells recapitulates the differences between viruses harboring the different polymorphisms. Altogether, the data are consistent with a redundant or complementary role of µ2 and λ1, affecting either early disassembly or the nature of the viral RNA in the incoming viral particle. Proteins involved in viral RNA synthesis are thus involved in this likely critical aspect of the ability of different reovirus variants to infect various cell types, and to discriminate between parental and transformed/cancer cells.


Assuntos
Orthoreovirus , Reoviridae , Animais , Reoviridae/genética , Orthoreovirus/genética , Interferon beta/farmacologia , RNA Viral/genética , Mamíferos
5.
Viruses ; 14(12)2022 Nov 23.
Artigo em Inglês | MEDLINE | ID: mdl-36560608

RESUMO

Rice black-streaked dwarf virus (RBSDV) is the main pathogen causing maize rough dwarf disease (MRDD) in China. Typical enation symptoms along the abaxial leaf veins prevail in RBSDV-infected maize inbred line B73 (susceptible to RBSDV), but not in X178 (resistant to RBSDV). Observation of the microstructures of epidermal cells and cross section of enations from RBSDV-infected maize leaves found that the increase of epidermal cell and phloem cell numbers is associated with enation formation. To identify proteins associated with enation formation and candidate proteins against RBSDV infection, comparative proteomics between B73 and X178 plants were conducted using isobaric tags for relative and absolute quantitation (iTRAQ) with leaf samples at the enation forming stage. The proteomics data showed that 260 and 316 differentially expressed proteins (DEPs) were identified in B73 and X178, respectively. We found that the majority of DEPs are located in the chloroplast and cytoplasm. Moreover, RBSDV infection resulted in dramatic changes of DEPs enriched by the metabolic process, response to stress and the biosynthetic process. Strikingly, a cell number regulator 10 was significantly down-regulated in RBSDV-infected B73 plants. Altogether, these data will provide value information for future studies to analyze molecular events during both enation formation and resistance mechanism to RBSDV infection.


Assuntos
Oryza , Vírus de Plantas , Reoviridae , Proteômica , Zea mays , Plantas , Doenças das Plantas , Reoviridae/fisiologia
6.
Viruses ; 14(12)2022 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-36560814

RESUMO

Grasshoppers can swarm in the millions and destroy crops over wide areas, posing a major economic threat to agriculture. A wide range of insect-related viruses has recently been reported in the metagenomics of grasshoppers. Here, we identified and isolated a novel reovirus from grasshoppers, named Acrididae reovirus (ARV). The complete genome of ARV was composed of nine dsRNA segments. Phylogenetic analysis revealed that ARV formed a monophyletic lineage with unclassified insect-associated reoviruses and was sufficiently distinct from known genera of Reoviridae. ARV could replicate in its host Locusta migratoria and result in host death. Lower-dose ARV infection affected ovary development and resulted in a significant reduction in fecundity. The identification and characterization of a novel pathogenic reovirus could potentially promote the development of new biological control agents.


Assuntos
Gafanhotos , Orthoreovirus , Infecções por Reoviridae , Reoviridae , Animais , Filogenia , Orthoreovirus/genética , Infecções por Reoviridae/veterinária
7.
Viruses ; 14(11)2022 Nov 17.
Artigo em Inglês | MEDLINE | ID: mdl-36423155

RESUMO

Aquatic animal viruses infect and transmit in aquatic environments, causing serious harm to the aquaculture industry and a variety of wild aquatic animals. How are they affected by environmental factors and do they represent potential threat to mammalian heath or not? Here, the effects of environmental factors (ultraviolet radiation (UV), temperature, pH, and drying) and their threshold on five epidemic aquatic animal viruses infecting amphibians and bony fish, including Rana grylio virus (RGV), Andrias davidianus ranavirus (ADRV), Grass carp reovirus (GCRV), Paralichthys olivaceus rhabdovirus (PORV), and Scophthalmus maximus rhabdovirus (SMRV), were measured and compared in a fish cell line. The examination of virus titers after different treatment in fish cells showed that the two iridoviruses, RGV and ADRV, had a higher tolerance to all of the environmental factors, such as they only had a decay rate of 22-36% when incubated at 37 °C for 7 days. However, the rhabdovirus SMRV was sensitive to all of the factors, with a decay rate of more than 80% in most of the treatments; even a complete inactivation (100%) can be observed after drying treatment. To address the potential threat to mammals, infectivity and limitation factors of the five viruses in Baby hamster kidney fibroblast cells (BHK-21) were tested, which showed that three of the five viruses can replicate at a low temperature, but a high temperature strongly inhibited their infection and none of them could replicate at 37 °C. This study clarified the sensitivity or tolerance of several different types of aquatic animal viruses to the main environmental factors in the aquatic environment and proved that the viruses cannot replicate in mammalian cells at normal physiological temperature.


Assuntos
Ranavirus , Reoviridae , Rhabdoviridae , Animais , Raios Ultravioleta , Ranavirus/fisiologia , Urodelos , Mamíferos
8.
Int J Mol Sci ; 23(19)2022 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-36232671

RESUMO

Complement factor I (CFI), a complement inhibitor, is well known for regulating the complement system activation by degrading complement component 3b (C3b) in animal serum, thus becoming involved in innate defense. Nevertheless, the functional mechanisms of CFI in the complement system and in host-pathogen interactions are far from being clarified in teleost fish. In the present study, we cloned and characterized the CFI gene, CiCFI, from grass carp (Ctenopharyngodon idella) and analyzed its function in degrading serum C3b and expression changes after grass carp reovirus (GCRV) infection. The open reading frame of CiCFI was found to be 2121 bp, encoding 706 amino acids with a molecular mass of 79.06 kDa. The pairwise alignments showed that CiCFI shared the highest identity (66.9%) with CFI from Carassius gibelio and the highest similarity (78.7%) with CFI from Danio rerio. The CiCFI protein was characterized by a conserved functional core Tryp_SPc domain with the catalytic triad and substrate binding sites. Phylogenetic analysis indicated that CiCFI and the homologs CFIs from other teleost fish formed a distinct evolutionary branch. Similar with the CFIs reported in mammals, the recombinant CiCFI protein could significantly reduce the C3b content in the serum, demonstrating the conserved function of CiCFI in the complement system in the grass carp. CiCFI mRNA and protein showed the highest expression level in the liver. After GCRV infection, the mRNA expressions of CiCFI were first down-regulated, then up-regulated, and then down-regulated to the initial level, while the protein expression levels maintained an overall downward trend to the late stage of infection in the liver of grass carps. Unexpectedly, the protein levels of CiCFI were also continuously down-regulated in the serum of grass carps during GCRV infection, while the content of serum C3b proteins first increases and then returns to the initial level, suggesting a distinct role of CiCFI in regulating complement activation and fish-virus interaction. Combining our previous results that complement factor D, a complement enhancer, shows continuously up-regulated expression levels in grass carps during GCRV infection, and this study may provide the further essential data for the full picture of complex complement regulation mechanism mediated by Df and CFI of the grass carp during pathogen infection.


Assuntos
Carpas , Doenças dos Peixes , Infecções por Reoviridae , Reoviridae , Aminoácidos/metabolismo , Animais , Carpas/genética , Carpas/metabolismo , Ativação do Complemento , Complemento C3b , Fator D do Complemento/genética , Fator I do Complemento/genética , Fator I do Complemento/metabolismo , Inativadores do Complemento , Proteínas de Peixes/metabolismo , Regulação da Expressão Gênica , Mamíferos/metabolismo , Filogenia , RNA Mensageiro/genética , Reoviridae/fisiologia , Infecções por Reoviridae/genética , Infecções por Reoviridae/veterinária
9.
Arch Razi Inst ; 77(2): 615-622, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-36284984

RESUMO

Colorectal cancer is the fourth leading cause of cancer-related deaths that has significantly increased over the past three decades. New therapeutic approaches, such as oncolytic viruses, have become very imperative recently to destroy cancer cells. The use of mesenchymal stem cells (MSCs) secretome that is produced in response to variant conditions involves different paracrine molecules secretion that has therapeutic potential in several chronic diseases. Mesenchymal stem cells and their derivatives are employed as regenerative medicine; nevertheless, there is ambiguity in the function of these cells in the control of malignancy. This study aimed to examine the apoptotic effect of secretomes derived from MSCs affected by encompassing oncolytic reoviruses. Mesenchymal stem cells were cultured after separation from abdominal adipose tissue of BALB/c mice. After three passages, the cells were infected by reovirus at the multiplicity of infection of 1 plaque-forming unit per cell. Uninfected and infected secretomes with reovirus were collected separately. The colorectal cancer CT26 cells were confronted with uninfected secretome, infected secretions, reovirus as a positive control, and Dulbecco's Modified Eagle Medium/High Glucose as negative control separately. Finally, apoptosis and necrosis were evaluated by flow cytometry. The infected secretome with reovirus was capable to induce apoptosis more than the uninfected secretome in CT26. However, the supernatant of reovirus infected cells was more capable to induce cell death, in comparison to the infected secretome. Infected MSCs with oncolytic reovirus produced a type of condition media that enhanced apoptosis induction and could have a therapeutic effect on cancer cells. Nonetheless, tumoral cells confronted with the oncolytic reovirus showed more capability in inducing apoptosis in CT26 cells. As a result, the use of oncolytic virus and infected secretome are more effective than uninfected secretome in inducing apoptosis.


Assuntos
Neoplasias Colorretais , Células-Tronco Mesenquimais , Vírus Oncolíticos , Reoviridae , Doenças dos Roedores , Camundongos , Animais , Secretoma , Reoviridae/fisiologia , Vírus Oncolíticos/fisiologia , Células-Tronco Mesenquimais/metabolismo , Neoplasias Colorretais/terapia , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/veterinária , Glucose/metabolismo , Doenças dos Roedores/metabolismo
10.
Viruses ; 14(10)2022 10 14.
Artigo em Inglês | MEDLINE | ID: mdl-36298813

RESUMO

Rice (Oryza sativa L.) is one of the major staple foods for global consumption. A major roadblock to global rice production is persistent loss of crops caused by plant diseases, including rice blast, sheath blight, bacterial blight, and particularly various vector-borne rice viral diseases. Since the late 19th century, 19 species of rice viruses have been recorded in rice-producing areas worldwide and cause varying degrees of damage on the rice production. Among them, southern rice black-streaked dwarf virus (SRBSDV) and rice black-streaked dwarf virus (RBSDV) in Asia, rice yellow mottle virus (RYMV) in Africa, and rice stripe necrosis virus (RSNV) in America currently pose serious threats to rice yields. This review systematizes the emergence and damage of rice viral diseases, the symptomatology and transmission biology of rice viruses, the arm races between viruses and rice plants as well as their insect vectors, and the strategies for the prevention and control of rice viral diseases.


Assuntos
Hemípteros , Oryza , Vírus de Plantas , Reoviridae , Animais , Doenças das Plantas , Insetos Vetores , Ásia , África
11.
Subcell Biochem ; 99: 525-552, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36151388

RESUMO

The members of the family Reoviridae (reoviruses) consist of 9-12 discrete double-stranded RNA (dsRNA) segments enclosed by single, double, or triple capsid layers. The outer capsid proteins of reoviruses exhibit the highest diversity in both sequence and structural organization. By contrast, the conserved RNA-dependent RNA polymerase (RdRp) structure in the conserved innermost shell in all reoviruses suggests that they share common transcriptional regulatory mechanisms. After reoviruses are delivered into the cytoplasm of a host cell, their inner capsid particles (ICPs) remain intact and serve as a stable nanoscale machine for RNA transcription and capping performed using enzymes in ICPs. Advances in cryo-electron microscopy have enabled the reconstruction at near-atomic resolution of not only the icosahedral capsid, including capping enzymes, but also the nonicosahedrally distributed complexes of RdRps within the capsid at different transcriptional stages. These near-atomic resolution structures allow us to visualize highly coordinated structural changes in the related enzymes, genomic RNA, and capsid protein during reovirus transcription. In addition, reoviruses encode their own enzymes for nascent RNA capping before RNA releasing from their ICPs.


Assuntos
Reoviridae , Capsídeo/metabolismo , Proteínas do Capsídeo/química , Microscopia Crioeletrônica , RNA de Cadeia Dupla/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , RNA Polimerase Dependente de RNA/genética , Reoviridae/genética , Reoviridae/metabolismo
12.
Front Immunol ; 13: 956587, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36091067

RESUMO

Grass carp reovirus (GCRV) is the most pathogenic double-stranded (ds) RNA virus among the isolated aquareoviruses. The molecular mechanisms by which GCRV utilizes host factors to generate its infectious compartments beneficial for viral replication and infection are poorly understood. Here, we discovered that the grass carp ADP ribosylation factor 1 (gcARF1) was required for GCRV replication since the knockdown of gcARF1 by siRNA or inhibiting its GTPase activity by treatment with brefeldin A (BFA) significantly impaired the yield of infectious viral progeny. GCRV infection recruited gcARF1 into viral inclusion bodies (VIBs) by its nonstructural proteins NS80 and NS38. The small_GTP domain of gcARF1 was confirmed to be crucial for promoting GCRV replication and infection, and the number of VIBs reduced significantly by the inhibition of gcARF1 GTPase activity. The analysis of gcARF1-GDP complex crystal structure revealed that the 27AAGKTT32 motif and eight amino acid residues (A27, G29, K30, T31, T32, N126, D129 and A160), which were located mainly within the GTP-binding domain of gcARF1, were crucial for the binding of gcARF1 with GDP. Furthermore, the 27AAGKTT32 motif and the amino acid residue T31 of gcARF1 were indispensable for the function of gcARF1 in promoting GCRV replication and infection. Taken together, it is demonstrated that the GTPase activity of gcARF1 is required for efficient replication of GCRV and that host GTPase ARF1 is closely related with the generation of VIBs.


Assuntos
Carpas , Proteínas Monoméricas de Ligação ao GTP , Orthoreovirus , Reoviridae , Fator 1 de Ribosilação do ADP/genética , Aminoácidos , Animais , Anticorpos Antivirais , Guanosina Trifosfato , Corpos de Inclusão Viral , Reoviridae/fisiologia
13.
Front Immunol ; 13: 969517, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36159797

RESUMO

SERPINA1, a member of the serine protease inhibitor family, plays a role in viral infection and inflammation by regulating the activities of serine and cysteine proteases. To date, there have been no reports on the immune function of SERPINA1 in fishes. In this study, we first cloned the serpina1 gene of grass carp (Ctenopharyngodon idellus) and found that it could respond rapidly to the infection of Grass carp reovirus (GCRV), and overexpression of serpina1 could enhance the antiviral response of CIK cells. A polyclonal antibody of SERPINA1 was prepared, and the protein interacting with SERPINA1 was screened by CoIP/MS in grass carp hepatopancreas tissue. It was found that SERPINA1 interacted with coagulation factor 2 (CF2) and could degrade it in a dose-dependent manner. In addition, overexpression of cf2 contributed to the infection of GCRV in CIK cells, whereas co-expression of serpina1 and cf2 in grass carp reduced the copy number of GCRV in cells. The results showed that grass carp SERPINA1 could inhibit GCRV infection by degrading CF2. This study proposes that SERPINA1 can inhibit viral infection through interaction with the coagulation factor, providing new insights into the molecular mechanism of SERPINA1's antiviral function.


Assuntos
Carpas , Cisteína Proteases , Doenças dos Peixes , Infecções por Reoviridae , Reoviridae , Animais , Antivirais/uso terapêutico , Fatores de Coagulação Sanguínea/metabolismo , Cisteína Proteases/metabolismo , Infecções por Reoviridae/veterinária , Serina/metabolismo , Inibidores de Serino Proteinase/uso terapêutico
14.
J Virol ; 96(19): e0117522, 2022 10 12.
Artigo em Inglês | MEDLINE | ID: mdl-36102647

RESUMO

The frequent outbreak of grass carp hemorrhagic disease caused by grass carp reovirus (GCRV), especially the mainly prevalent type II GCRV (GCRV-II), has seriously affected the grass carp culture in China. However, its pathogenic mechanism is still far from clear. In this study, the GCRV-II outer capsid protein VP35 was used as bait to capture interacting partners from Ctenopharyngon idellus kidney (CIK) cells, and heat shock protein 90 (Hsp90) was selected and confirmed interacting with VP35 through the C-terminal domain of Hsp90. Knockdown of Hsp90 or inhibition of Hsp90 activity suppressed GCRV-II proliferation, demonstrating that Hsp90 is an essential factor for GCRV-II proliferation. The confocal microscopy and flow cytometry showed that Hsp90 localized at both membrane and cytoplasm of CIK cells. The entry of GCRV-II into CIK cells was efficiently blocked by incubating the cells with Hsp90 antibody or by pretreating the virus with recombinant Hsp90 protein. Whereas overexpression of Hsp90 in CIK cells, grass carp ovary (GCO) cells, or 293T cells promoted GCRV-II entry, indicating that the membrane Hsp90 functions as a receptor of GCRV-II. Furthermore, Hsp90 interacted with clathrin and mediated GCRV-II entry into CIK cells through clathrin endocytosis pathway. In addition, we found that the cytoplasmic Hsp90 acted as a chaperone of VP35 because inhibition of Hsp90 activity enhanced VP35 polyubiquitination and degraded VP35 through the proteasome pathway. Collectively, our data suggest that Hsp90 functions both as a receptor for GCRV-II entry and a chaperone for the maturation of GCRV-II VP35, thus ensuring efficient proliferation of GCRV-II. IMPORTANCE Identification of viral receptors has always been the research hot spot in virus research field as receptor functions at the first stage of viral infection, which can be designed as efficient antiviral drug targets. GCRV-II, the causative agent of the grass carp epidemic hemorrhagic disease, has caused tremendous losses in grass carp culture in China. To date, the receptor of GCRV-II remains unknown. This study focused on identifying cellular receptor interacting with the GCRV-II outer capsid protein VP35, studying the effects of their interaction on GCRV-II proliferation, and revealing the underlying mechanisms. We demonstrated that Hsp90 acts both as a receptor of GCRV-II by interacting with VP35 and as a chaperone for the maturation of VP35, thus ensuring efficient proliferation of GCRV-II. Our data provide important insights into the role of Hsp90 in GCRV-II life cycle, which will help understand the mechanism of reovirus infection.


Assuntos
Proteínas do Capsídeo , Doenças dos Peixes , Proteínas de Choque Térmico , Infecções por Reoviridae , Reoviridae , Animais , Anticorpos Antivirais/metabolismo , Proteínas do Capsídeo/metabolismo , Carpas/virologia , Proliferação de Células , Clatrina/metabolismo , Doenças dos Peixes/virologia , Proteínas de Choque Térmico/metabolismo , Chaperonas Moleculares/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Receptores Virais/metabolismo , Reoviridae/fisiologia , Infecções por Reoviridae/veterinária
15.
J Virol ; 96(18): e0091022, 2022 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-36094315

RESUMO

Reassortment, or genome segment exchange, increases diversity among viruses with segmented genomes. Previous studies on the limitations of reassortment have largely focused on parental incompatibilities that restrict generation of viable progeny. However, less is known about whether factors intrinsic to virus replication influence reassortment. Mammalian orthoreovirus (reovirus) encapsidates a segmented, double-stranded RNA (dsRNA) genome, replicates within cytoplasmic factories, and is susceptible to host antiviral responses. We sought to elucidate the influence of infection multiplicity, timing, and compartmentalized replication on reovirus reassortment in the absence of parental incompatibilities. We used an established post-PCR genotyping method to quantify reassortment frequency between wild-type and genetically barcoded type 3 reoviruses. Consistent with published findings, we found that reassortment increased with infection multiplicity until reaching a peak of efficient genome segment exchange during simultaneous coinfection. However, reassortment frequency exhibited a substantial decease with increasing time to superinfection, which strongly correlated with viral transcript abundance. We hypothesized that physical sequestration of viral transcripts within distinct virus factories or superinfection exclusion also could influence reassortment frequency during superinfection. Imaging revealed that transcripts from both wild-type and barcoded viruses frequently co-occupied factories, with superinfection time delays up to 16 h. Additionally, primary infection progressively dampened superinfecting virus transcript levels with greater time delay to superinfection. Thus, in the absence of parental incompatibilities and with short times to superinfection, reovirus reassortment proceeds efficiently and is largely unaffected by compartmentalization of replication and superinfection exclusion. However, reassortment may be limited by superinfection exclusion with greater time delays to superinfection. IMPORTANCE Reassortment, or genome segment exchange between viruses, can generate novel virus genotypes and pandemic virus strains. For viruses to reassort their genome segments, they must replicate within the same physical space by coinfecting the same host cell. Even after entry into the host cell, many viruses with segmented genomes synthesize new virus transcripts and assemble and package their genomes within cytoplasmic replication compartments. Additionally, some viruses can interfere with subsequent infection of the same host or cell. However, spatial and temporal influences on reassortment are only beginning to be explored. We found that infection multiplicity and transcript abundance are important drivers of reassortment during coinfection and superinfection, respectively, for reovirus, which has a segmented, double-stranded RNA genome. We also provide evidence that compartmentalization of transcription and packaging is unlikely to influence reassortment, but the length of time between primary and subsequent reovirus infection can alter reassortment frequency.


Assuntos
Coinfecção , Genoma Viral , Reoviridae , Superinfecção , Animais , Genoma Viral/genética , RNA de Cadeia Dupla , Vírus Reordenados/genética , Reoviridae/genética , Superinfecção/genética
16.
PLoS Pathog ; 18(9): e1010641, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-36099325

RESUMO

Reoviridae virus family members, such as mammalian orthoreovirus (reovirus), encounter a unique challenge during replication. To hide the dsRNA from host recognition, the genome remains encapsidated in transcriptionally active proteinaceous core capsids that transcribe and release +RNA. De novo +RNAs and core proteins must repeatedly assemble into new progeny cores in order to logarithmically amplify replication. Reoviruses also produce outercapsid (OC) proteins µ1, σ3 and σ1 that assemble onto cores to create highly stable infectious full virions. Current models of reovirus replication position amplification of transcriptionally-active cores and assembly of infectious virions in shared factories, but we hypothesized that since assembly of OC proteins would halt core amplification, OC assembly is somehow regulated. Kinetic analysis of virus +RNA production, core versus OC protein expression, and core particles versus whole virus particle accumulation, indicated that assembly of OC proteins onto core particles was temporally delayed. All viral RNAs and proteins were made simultaneously, eliminating the possibility that delayed OC RNAs or proteins account for delayed OC assembly. High resolution fluorescence and electron microscopy revealed that core amplification occurred early during infection at peripheral core-only factories, while all OC proteins associated with lipid droplets (LDs) that coalesced near the nucleus in a µ1-dependent manner. Core-only factories transitioned towards the nucleus despite cycloheximide-mediated halting of new protein expression, while new core-only factories developed in the periphery. As infection progressed, OC assembly occurred at LD-and nuclear-proximal factories. Silencing of OC µ1 expression with siRNAs led to large factories that remained further from the nucleus, implicating µ1 in the transition to perinuclear factories. Moreover, late during infection, +RNA pools largely contributed to the production of de-novo viral proteins and fully-assembled infectious viruses. Altogether the results suggest an advanced model of reovirus replication with spatiotemporal segregation of core amplification, OC complexes and fully assembled virions.


Assuntos
Reoviridae , Animais , Proteínas do Capsídeo/metabolismo , Linhagem Celular , Cicloeximida , Cinética , Mamíferos , RNA Viral/genética , Reoviridae/genética , Reoviridae/metabolismo , Proteínas Virais , Montagem de Vírus
17.
Microb Pathog ; 173(Pt A): 105790, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36170950

RESUMO

Turkey arthritis reovirus (TARV) has been established as a cause of lameness in meat type turkeys in the past decade. However, no information is available on the age susceptibility of TARV or its transmission dynamics. We conducted this study to determine the age at which turkey poults are susceptible to TARV infection and whether infected birds can horizontally transmit the virus to their non-infected pen mates (sentinels). Five groups of turkeys were orally inoculated with TARV (∼106 TCID50/ml) at 2, 7, 14, 21 and 28 days of age (DOA). Two days after each challenge, four uninfected sentinel turkeys of equal age were added to the virus-inoculated groups. At one- and two-weeks post infection, turkeys from each group, including two sentinels, were euthanized followed by necropsy. Inoculated birds in all age groups had TARV replication in the intestine and gastrocnemius tendon with no statistically significant variation at p < 0.5. Furthermore, the inoculated birds at different age groups showed consistently high gastrocnemius tendon histologic lesion scores while birds in the 28-days-old age group had numerically lower lesion scores at 14 days post inoculation (dpi). The sentinels, in turn, also showed virus replication in their intestines and tendons and histologic lesions in gastrocnemius tendons. The findings indicate that turkeys at the age of 28 days or less are susceptible to infection with TARV following oral challenge. It was also found that TARV-infected birds could transmit the infection to naïve sentinel turkeys of the same age.


Assuntos
Artrite , Doenças das Aves Domésticas , Infecções por Reoviridae , Reoviridae , Animais , Perus , Anticorpos Antivirais
18.
Fish Shellfish Immunol ; 129: 52-63, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-35995370

RESUMO

Integrins are α-ß heterodimeric cell receptors that can bind the protein components of pathogens, and play crucial roles in mammalian immune responses, but the immune functions mediated by integrins remains largely unknown in teleost fish. In this study, an integrin αvß3 (GCαvß3) originally assembled by αv (GCαv) and ß3 (GCß3) subunits, was identified from a teleost fish grass carp Ctenopharyngodon idella. The pairwise alignment analyses showed that the amino acid sequences of GCαv and GCß3 shared high similarity (75.2-95.1%) and identity (58.6-90.7%) with their homologs from other vertebrates. Both GCαv and GCß3 harbored the conserved protein domains and motifs, and were clustered in fish branch of the phylogenetic tree containing the counterparts from various vertebrates. Co-immunoprecipitation displayed that GCß3 could interact with the grass carp reovirus (GCRV) outer capsid protein VP5. Two incubation experiments revealed that the interaction of GCRV or VP5 proteins with GCß3 could induce the expressions of type I interferons (IFNs) including IFN2 and IFN3 in grass carp ovary cell line. The functional analysis demonstrated that GCαvß3 served as a receptor of viral protein components to be involved in antiviral immunity as human integrin αvß3 did. In addition, both GCαv and GCß3 were significantly upregulated in various tissues of grass carp after GCRV infection. This study might provide fundamental basis for understanding the molecular characteristics and immune functions of GCαvß3, and offer a new insight into the antiviral immune mechanism specific to the integrins in grass carp.


Assuntos
Carpas , Doenças dos Peixes , Interferon Tipo I , Infecções por Reoviridae , Reoviridae , Animais , Antivirais , Proteínas do Capsídeo , Carpas/genética , Carpas/metabolismo , Proteínas de Peixes/química , Humanos , Integrina alfaVbeta3/genética , Mamíferos/metabolismo , Filogenia , Reoviridae/fisiologia
19.
Fish Shellfish Immunol ; 128: 148-156, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-35921937

RESUMO

Autophagy impacts the replication cycle of many viruses. Grass Carp Reovirus (GCRV) is an agent that seriously affects the development of the grass carp aquaculture industry. The role of autophagy in GCRV infection is not clearly understood. In this study, we identified that GCRV infection triggered autophagy in CIK cells, which was demonstrated by transmission electron microscopy, the conversion of LC3B I to LC3B II and the level of autophagy substrate p62. Furthermore, we found that GCRV infection activated Akt-mTOR signaling pathway, and the conversion of LC3B I to LC3B II was increased by inhibiting mTOR with rapamycin (Rap) but decreased by activating Akt with insulin. We then assessed the effects of autophagy on GCRV replication. We found that inducing autophagy with Rap promoted GCRV proliferation but inhibiting autophagy with 3 MA or CQ inhibited GCRV replication in CIK cells. Moreover, it was found that enhancing Akt-mTOR activity by insulin, GCRV VP7 protein and viral titers of GCRV were decreased. Collectively, these results indicated that GCRV infection induced autophagy involved in GCRV replication via the Akt-mTOR signal pathway. Thus, new insights into GCRV pathogenesis and antiviral treatment strategies are provided.


Assuntos
Carpas , Doenças dos Peixes , Insulinas , Orthoreovirus , Infecções por Reoviridae , Reoviridae , Animais , Antivirais/farmacologia , Autofagia , Insulinas/farmacologia , Insulinas/uso terapêutico , Proteínas Proto-Oncogênicas c-akt , Infecções por Reoviridae/metabolismo , Infecções por Reoviridae/veterinária , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/genética , Replicação Viral
20.
Biol Open ; 11(9)2022 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-36017723

RESUMO

The δ-endotoxin Cry4Aa from Bacillus thuringiensis israelensis (Bti) has insecticidal characteristics specific to insects of the order Diptera. Although Cry4Aa has shown potential as an effective proteinaceous pesticide against mosquitoes, it has an ultraviolet (UV)-intolerant property that limits its outdoor use. Our previous research showed that protein microcrystal polyhedra from Bombyx mori cypovirus can encapsulate diverse foreign proteins and maintain long-term protein activity under hostile environmental conditions, including UV irradiation. In this study, we report the development of polyhedra encapsulating the Cry4Aa insecticidal activity domain by using a modified baculovirus expression system. We confirmed the oral intake of recombinant polyhedra introduced into the experimental environment by the larvae of a mosquito, Aedes albopictus, and delivery of encapsulated proteins into the digestive tract. The polyhedra encapsulating partial Cry4Aa showed mosquito larvicidal activity during incubation of larvae with 50% lethal-dose value of 23.717×104 cubes for 10 Aedes albopictus larvae in 1 ml water. In addition, polyhedra showed a specific property to reduce the impact of UV-C irradiation on the activity of encapsulated partial Cry4Aa, thus demonstrating the effectiveness of encapsulating Bti δ-endotoxins inside polyhedra to increase the availability of proteinaceous pesticides for outdoor use for mosquito control.


Assuntos
Aedes , Bacillus thuringiensis , Praguicidas , Reoviridae , Aedes/metabolismo , Animais , Bacillus thuringiensis/química , Bacillus thuringiensis/metabolismo , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/farmacologia , Endotoxinas/química , Endotoxinas/metabolismo , Proteínas Hemolisinas/metabolismo , Proteínas Hemolisinas/farmacologia , Larva/metabolismo , Praguicidas/metabolismo , Praguicidas/farmacologia , Reoviridae/metabolismo , Água/metabolismo
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