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1.
Insect Sci ; 27(1): 22-32, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29704325

RESUMO

The Junonia coenia densovirus rapidly traverses the gut epithelium of the host lepidopteran without replicating in the gut cells. The ability of this virus to transcytose across the gut epithelium is of interest for the potential use of virus structural proteins as delivery vehicles for insecticidal peptides that act within the insect hemocoel, rather than in the gut. In this study, we used fall armyworm, Spodoptera frugiperda to examine the binding of the virus to brush border membrane vesicle proteins by two-dimensional ligand blot analysis. We also assessed the rate of flux of the primary viral structural protein, VP4 fused to eGFP with a proline-rich linker (VP4-P-eGFP) through the gut epithelium ex vivo in an Ussing chamber. The mechanisms involved with transcytosis of VP4-P-eGFP were assessed by use of inhibitors. Bovine serum albumin (BSA) and eGFP were used as positive and negative control proteins, respectively. In contrast to BSA, which binds to multiple proteins on the brush border membrane, VP4-P-eGFP binding was specific to a protein of high molecular mass. Protein flux was significantly higher for VP4-P-eGFP after 2 h than for albumin or eGFP, with rapid transcytosis of VP4-P-eGFP within the first 30 min. In contrast to BSA which transcytosed following clathrin-mediated endocytosis, the movement of VP4-P-eGFP was vesicle-mediated but clathrin-independent. The specificity of binding combined with the efficiency of transport across the gut epithelium suggest that VP4 will provide a useful carrier for insecticidal peptides active within the hemocoel of key lepidopteran pests including S. frugiperda.


Assuntos
Densovirus/fisiologia , Spodoptera/fisiologia , Transcitose/fisiologia , Proteínas Virais/fisiologia , Animais , Sistema Digestório/virologia , Fenômenos Fisiológicos do Sistema Digestório , Epitélio/fisiologia , Epitélio/virologia , Larva/crescimento & desenvolvimento , Larva/fisiologia , Larva/virologia , Spodoptera/crescimento & desenvolvimento , Spodoptera/virologia , Transcitose/genética
2.
Insect Biochem Mol Biol ; 112: 103202, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31422153

RESUMO

The microRNA (miRNA) pathway is an epigenetic mechanism that plays important roles in various biological processes including host-virus interactions by regulating gene expression of the host and/or the virus. Previously, we showed that the cellular microRNAome in Spodoptera frugiperda (Sf9) cells is modulated following Autographa californica multiple nucleopolyhedrovirus (AcMNPV) infection suggesting that miRNAs may contribute in the cellular antiviral immunity. Here, we investigated the role of core components of the miRNA pathway in Sf9-AcMNPV interaction. Gene expression analyses showed that the expression levels of Dicer-1 (Dcr1), Argonaute-1 (Ago1) and Exportin-5 (Exp5) increased following AcMNPV infection particularly at 16 h post infection (hpi). Ran expression levels, however, decreased in response to virus infection. The expression levels of cellular miRNAs, miR-184 and let-7, also diminished at the post infection times further confirming differential expression of the cellular miRNAs following AcMNPV infection. To determine the role of the miRNA pathway in the interaction, we silenced key genes in the pathway using specific dsRNAs. RNAi of Dcr1, Ago1 and Ran enhanced viral DNA replication and reduced the abundance of miR-184 and let-7 underscoring the importance of the miRNA pathway in antiviral immunity in Sf9 cells. Suppression of the miRNA pathway in mock and infected cells had no effect on Ran expression levels suggesting miRNA-independent downregulation of this gene after virus infection. In conclusion, our results suggest the antiviral role of the miRNA pathway in Sf9 cells against AcMNPV. To modulate this immune response, AcMNPV represses host miRNAs likely through downregulation of Ran to enhance its replication in the host cells.


Assuntos
MicroRNAs/metabolismo , Spodoptera/imunologia , Spodoptera/virologia , Animais , DNA Viral , Regulação da Expressão Gênica , Interações entre Hospedeiro e Microrganismos/genética , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , MicroRNAs/genética , Interferência de RNA , Células Sf9 , Spodoptera/genética , Replicação Viral
3.
Virology ; 533: 68-76, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31125854

RESUMO

Reoviruses are thought to replicate and assemble in special cytoplasmic structures called 'viroplasms'. However, little is known about the viroplasms of the insect reoviruses, the cypoviruses. To investigate the viroplasm of Dendrolimus punctatus cypovirus (DpCPV), all proteins encoded by the 10 genomic segments of DpCPV were expressed in Sf9 cells using the Bac-to-Bac system. The viral nonstructural protein NSP2 formed viroplasm-like dots which showed close apposition with the endoplasmic reticulum and were surrounded by intracellular membranes during transfection. Colocalization and coimmunoprecipitation assays showed that NSP2 interacts with 4 of 6 structural proteins and another 2 nonstructural proteins, while NSP1 only colocalized with VP4, and NSP3 did not colocalize with any structural protein. Immunoelectron microscopy revealed that NSP2 were nearby the endoplasmic reticulum and mitochondria, and viral particles were present in the electron-dense inclusions formed by NSP2. We proposed that NSP2 is responsible for forming the viroplasms structures of DpCPV.


Assuntos
Corpos de Inclusão Viral/virologia , Reoviridae/metabolismo , Spodoptera/virologia , Proteínas não Estruturais Virais/metabolismo , Animais , Ligação Proteica , Reoviridae/genética , Células Sf9 , Proteínas não Estruturais Virais/genética
4.
Virol Sin ; 34(4): 423-433, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31037643

RESUMO

As specific pathogens of noctuid pests, including Spodoptera exigua, S. litura, Helicoverpa armigera, and Mythimna separata, ascoviruses are suitable for the development of bioinsecticides. In this study, the infectivity of Heliothis virescens ascovirus 3j (HvAV-3j) on insect and mammalian cells was evaluated. HvAV-3j infection induced drastic morphological changes in Sf9, HzAM1, SeFB, and HaFB cells, including swelling and detachment. Notably, the latter phenomena did not occur in HvAV-3j-inoculated mammalian cells (HEK293, 7402, HePG2, PK15, ST, and TM3). MTT assays indicated that HvAV-3j inhibited the growth of host insect cells from the 6th hpi, but no effects were detected in the HvAV-3j-inoculated mammalian cells. Furthermore, viral DNA replication, gene transcription, and protein expression were investigated, and the results consistently suggested that HvAV-3j viruses were not able to replicate their genomic DNA, transcribe, or express their proteins in the non-target vertebrate cells. The HvAV-3j genes were only transcribed and expressed in the four insect cell lines. These results indicated that HvAV-3j was infectious to cells derived from S. frugiperda, S. exigua, H. armigera, and H. zea but not to cells derived from human, pig, and mouse, suggesting that ascoviruses are safe to non-target vertebrate cells.


Assuntos
Ascoviridae/genética , Ascoviridae/fisiologia , Interações entre Hospedeiro e Microrganismos , Replicação Viral , Animais , Replicação do DNA , DNA Viral/genética , Células HEK293 , Humanos , Larva/virologia , Camundongos , Mariposas/virologia , Fases de Leitura Aberta , Filogenia , Medição de Risco , Células Sf9 , Spodoptera/virologia , Suínos
5.
Arch Virol ; 164(6): 1677-1682, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30955090

RESUMO

We describe an unexpected feature observed for the heterologous expression of the Thyrinteina arnobia cypovirus polyhedrin from a recombinant baculovirus infection in different insect cell lines. The in cellulo-formed crystals varied in size and shape depending on the cell line. Crystals formed in Trichoplusia ni-derived cells were cubic (0.1-2 µm) and localized in both the nucleus and cytoplasm, whereas those formed in Spodoptera frugiperda-derived cells were ovate and ellipsoidal (0.1-3 µm) and also localized in both the nucleus and cytoplasm. The molecular basis for differences in the morphology, size, and location of cypovirus occlusion bodies is unclear, and cellular proteins might play a role in their formation and location.


Assuntos
Baculoviridae/genética , Proteínas de Matriz de Corpos de Inclusão/metabolismo , Proteínas Recombinantes/metabolismo , Reoviridae/metabolismo , Spodoptera/citologia , Animais , Baculoviridae/metabolismo , Linhagem Celular , Núcleo Celular/metabolismo , Núcleo Celular/virologia , Cristalização , Citoplasma/metabolismo , Citoplasma/virologia , Microscopia Eletrônica de Varredura , Proteínas de Matriz de Corpos de Inclusão/genética , Reoviridae/genética , Células Sf9 , Spodoptera/virologia
6.
Mol Immunol ; 108: 89-101, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30784767

RESUMO

Polydnaviruses (PDVs) are obligatory symbionts found in thousands of endoparasitoid species and essential for successful parasitism. The two genera of PDVs, ichnovirus (IV) and bracovirus (BV), use different sets of virulence factors to ensure successful parasitization of the host. Previous studies have shown that PDVs target apoptosis, one of the innate antiviral responses in many host organisms. However, IV and BV have been shown to have opposite effects on this process. BV induces apoptosis in host cells, whereas some IV proteins have been shown to have anti-apoptotic activity. The different biological contexts in which the assays were performed may account for this difference. In this study, we evaluated the interplay between apoptosis and the ichnovirus HdIV from the parasitoid Hyposoter didymator, in the HdIV-infected hemocytes and fat bodies of S. frugiperda larvae, and in the Sf9 insect cell line challenged with HdIV. We found that HdIV induced cell death in hemocytes and fat bodies, whereas anti-apoptotic activity was observed in HdIV-infected Sf9 cells, with and without stimulation with viral PAMPs or chemical inducers. We also used an RT-qPCR approach to determine the expression profiles of a set of genes known to encode key components of the other main antiviral immune pathways described in insects. The analysis of immune gene transcription highlighted differences in antiviral responses to HdIV as a function of host cell type. However, all these antiviral pathways appeared to be neutralized by low levels of expression for the genes encoding the key components of these pathways, in all biological contexts. Finally, we investigated the effect of HdIV on the general antiviral defenses of the lepidopteran larvae in more detail, by studying the survival of S. frugiperda co-infected with HdIV and the entomopathogenic densovirus JcDV. Coinfected S. frugiperda larvae have increased resistance to JcDV at an early phase of infection, whereas HdIV effects enhance the virulence of the virus at later stages of infection. Overall, these results reveal complex interactions between HdIV and its cellular environment.


Assuntos
Imunidade , Polydnaviridae/fisiologia , Spodoptera/imunologia , Spodoptera/virologia , Animais , Apoptose , Sobrevivência Celular , Corpo Adiposo/citologia , Corpo Adiposo/virologia , Hemócitos/citologia , Hemócitos/virologia , Imunidade/genética , Larva/citologia , Larva/virologia , RNA de Cadeia Dupla/metabolismo , Células Sf9 , Ativação Transcricional/genética
7.
PLoS One ; 14(2): e0209937, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30735528

RESUMO

Spodoptera exempta nucleopolyhedrovirus (SpexNPV) is a viral pathogen of the African armyworm, Spodoptera exempta (Lepidoptera: Noctuidae), a significant agricultural pest of cereal crops in Africa. SpexNPV has been evaluated as a potential insecticide for control of this pest and has served as the subject of research on baculovirus pathology and transmission. Occlusion bodies (OBs) of SpexNPV isolate 244.1 were examined, and the nucleotide sequence of the genome was determined and characterized. SpexNPV-244.1 OBs consisted of irregular polyhedra with a size and appearance typical for alphabaculoviruses. Virions within the polyhedra contained 1-8 nucleocapsids per unit envelope. The SpexNPV-244.1 genome was comprised of a 129,528 bp circular sequence, in which 139 ORFs were annotated. Five homologous regions (hrs) consisting of a variable number of 28-bp imperfect palindromes were identified in the genome. The genome sequence contained the 38 core genes of family Baculoviridae, as well as three ORFs unique to the SpexNPV sequence and one ORF that was apparently acquired by horizontal gene transfer with a betabaculovirus ancestor. Phylogenetic inference with core gene amino acid sequence alignments placed SpexNPV-244.1 in a lineage containing alphabaculoviruses of Spodoptera frugiperda and Spodopotera exigua which in turn is part of a larger group of alphabaculoviruses from the subfamily Noctuinae in the lepidopteran family Noctuidae. Kimura-2-parameter pairwise nucleotide distances indicated that SpexNPV-244.1 represented a different and previously unlisted species in the genus Alphabaculovirus. Gene parity plots indicated that the gene order of SpexNPV-244.l was extensively collinear with that of Spodoptera exigua NPV (SeMNPV). These plots also revealed a group of 17 core genes whose order was conserved in other alpha- and betabaculoviruses.


Assuntos
Baculoviridae/genética , Spodoptera/virologia , África , Animais , Sequência de Bases , Produtos Agrícolas/parasitologia , DNA Viral/genética , Genoma Viral , Filogenia , Sequenciamento Completo do Genoma
8.
J Virol ; 93(6)2019 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-30626676

RESUMO

The Sf9 and Sf21 cell lines derived from ovarian tissues of the wide-host-range phytophagous lepidopteran Spodoptera frugiperda are widely used for research and commercial-scale production of recombinant proteins. These cell lines are chronically infected with a rhabdovirus (Sf-RV) that does not cause any overt cytopathic effects. We demonstrate that wild populations of S. frugiperda in the eastern United States and Caribbean are infected with genetically diverse strains of Sf-RV and that this virus is also capable of infecting cells of Spodoptera exigua, Heliothis subflexa, and Bombyx mori Feeding studies demonstrated the ability of S. frugiperda larvae to deposit Sf-RV onto human-consumed vegetables during feeding. Although no evidence for replication in two species of plant cells was detected, subcellular localization studies demonstrated that the Sf-RV nucleocapsid was targeted to plasmodesmata, while two forms of the accessory protein were differentiated on the basis of their ability to localize to nuclei. Collectively, the results from this study suggest that environmental exposure of humans to Sf-RV is likely to be commonplace and frequent, but its inability to replicate in plant or human cells suggests that there is no substantial risk to human health.IMPORTANCE Insect-derived cell lines are widely used commercially for the production of vaccines and protein-based pharmaceuticals. After decades of safe and beneficial use, it was a surprise to the biotechnology industry to discover an endemic rhabdovirus in Sf9 cells. This discovery was made possible only by the substantial advancements in DNA sequencing technologies. Given the public health concerns associated with many rhabdovirus species, several initiatives were undertaken to establish that Spodoptera frugiperda rhabdovirus (Sf-RV) does not pose a threat to humans. Such actions include the generation of cell lines that have been cleared of Sf-RV. Given that Sf9 is derived from a moth whose larvae feed on human-edible foods, we explored the prevalence of Sf-RV in its wild and lab-grown populations, as well as its ability to be deposited on food items during feeding. Collectively, our data suggest that there is no overt risk from exposure to Sf-RV.


Assuntos
Especificidade de Hospedeiro/fisiologia , Rhabdoviridae/fisiologia , Spodoptera/virologia , Animais , Linhagem Celular , Humanos , Insetos/virologia , Larva/metabolismo , Larva/virologia , Plantas/virologia , Proteínas Recombinantes/metabolismo , Rhabdoviridae/metabolismo , Células Sf9 , Spodoptera/metabolismo , Proteínas Virais/metabolismo
9.
J Gen Virol ; 100(4): 669-678, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30694169

RESUMO

Wild-type ODVs (Wt) have an intact ODV entry complex in their envelope and are orally infectious towards insect larvae (left panel). In the absence of Ac108 (mut ac108), the stable core is still present but nevertheless fails to form an entry complex, affecting the ODV oral infectivity (right panel). The components of the core complex are depicted in yellow and the loosely associated components are depicted in red. PIF7 is depicted in green as its affinity with the complex is currently not known.Baculoviruses orally infect insect larvae when they consume viral occlusion bodies (OBs). OBs consist of a crystalline protein matrix in which the infectious virus particles, the occlusion-derived viruses (ODVs), are embedded. The protein matrix dissolves in the alkaline environment of the insect's midgut lumen. The liberated ODVs can then infect midgut endothelial cells through the action of at least nine different ODV-envelope proteins, called per os infectivity factors (PIFs). These PIF proteins mediate ODV oral infectivity, but are not involved in the systemic spread of the infection by budded viruses (BVs). Eight of the known PIFs form a multimeric complex, named the ODV entry complex. In this study, we show for Autographa californica multiple nucleopolyhedrovirus that mutation of the ac108ORF abolishes the ODV oral infectivity, while production and infectivity of the BVs remains unaffected. Furthermore, repair of the ac108 mutant completely recovered oral infectivity. With an HA-tagged repair mutant, we were able to demonstrate by Western analysis that the Ac108 protein is a constituent of the ODV entry complex, where the formation was abolished in the absence of this protein. Based on these results, we conclude that ac108 encodes a per os infectivity factor (PIF9) that is also an essential constituent of the ODV entry complex.


Assuntos
Baculoviridae/metabolismo , Baculoviridae/patogenicidade , Proteínas Virais/metabolismo , Fatores de Virulência/metabolismo , Animais , Linhagem Celular , Sistema Digestório/virologia , Células Endoteliais/virologia , Insetos/virologia , Larva/virologia , /patologia , Células Sf9 , Spodoptera/virologia , Proteínas do Envelope Viral/metabolismo , Vírion/metabolismo , Vírion/patogenicidade , Internalização do Vírus
10.
Mol Pharm ; 16(3): 1406-1411, 2019 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-30608704

RESUMO

The bile salt export pump (BSEP) is the primary canalicular transporter responsible for the secretion of bile acids from hepatocytes into bile canaliculi, and inhibition of this transporter has been associated with drug-induced liver injury (DILI). A common variant (rs2287622; p.V444A) in the gene encoding BSEP has been associated with an increased risk of cholestatic DILI. Although p.444V BSEP (reference) and p.444A BSEP (variant) do not differ in their transport kinetics of taurocholic acid (TCA), transport of the more abundant glycocholic acid (GCA) has not been investigated. Importantly, differences in the susceptibility of p.444V and p.444A BSEP to inhibition by drugs causing cholestatic DILI have not been investigated. To address these issues, the transport kinetics of GCA were evaluated by incubating membrane vesicles expressing either p.444V or p.444A BSEP with GCA over a range of concentrations (1, 10, 25, 50, and 100 µM). The abilities of commonly used cholestatic medications to inhibit the transport of TCA and GCA by the reference and variant proteins were compared. Resulting data indicated that GCA transport kinetics for reference and variant BSEP followed Michaelis-Menten kinetics and were not statistically different [ Vmax values of 1132 ± 246 and 959 ± 256 pmol min-1 (mg of protein)-1, respectively, and Km values of 32.7 ± 18.2 and 45.7 ± 25.5 µM, respectively]. There were no statistically significant differences between the reference and variant BSEP in the inhibition of TCA or GCA transport by the cholestatic drugs tested. In conclusion, differential inhibition of TCA or GCA transport cannot account for an association between the variant BSEP and the risk for cholestatic DILI due to the drugs tested.


Assuntos
Membro 11 da Subfamília B de Transportadores de Cassetes de Ligação de ATP/genética , Ácidos e Sais Biliares/metabolismo , Colagogos e Coleréticos/uso terapêutico , Colestase/tratamento farmacológico , Polimorfismo de Nucleotídeo Único , Membro 11 da Subfamília B de Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Animais , Baculoviridae , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Colagogos e Coleréticos/farmacologia , Dipiridamol/farmacologia , Eritromicina/farmacologia , Ácido Glicocólico/antagonistas & inibidores , Ácido Glicocólico/metabolismo , Cetoconazol/farmacologia , Cinética , Proteínas de Membrana Transportadoras/metabolismo , Células Sf9 , Transdução de Sinais/efeitos dos fármacos , Spodoptera/virologia , Ácido Taurocólico/antagonistas & inibidores , Ácido Taurocólico/metabolismo , Vesículas Transportadoras/metabolismo
11.
J Virol ; 93(3)2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30429334

RESUMO

Alphabaculoviruses are lepidopteran-specific nucleopolyhedroviruses that replicate within the nucleus; however, the anterograde transport of the nucleocapsids of these viruses, which is an obligatory step for progeny virion production, is not well understood. In the present study, a unique Alphabaculovirus gene with unknown function, namely, the Autographa californica multiple nucleopolyhedrovirus (AcMNPV) ac51 gene, was found to be required for efficient nuclear egress of AcMNPV nucleocapsids. Our results indicate that ac51 is a late gene, and Ac51 protein was detectable from 24 to 72 h postinfection using an antibody raised against Ac51. Ac51 is distributed in both the cytoplasm and nuclei of infected cells. Upon ac51 deletion, budded virion (BV) production by 96 h posttransfection was reduced by approximately 1,000-fold compared with that of wild-type AcMNPV. Neither viral DNA synthesis nor viral gene expression was affected. Ac51 was demonstrated to be a nucleocapsid protein of BVs, and ac51 deletion did not interrupt nucleocapsid assembly and occlusion-derived virion (ODV) formation. However, BV production in the supernatants of transfected cells during a viral life cycle was substantially decreased when ac51 was deleted. Further analysis showed that, compared with wild-type AcMNPV, ac51 deletion decreased nucleocapsid egress, while the numbers of nucleocapsids in the nuclei were comparable. Deletion of ac51 also eliminated the virulence of AcMNPV in vivo Taken together, our results support the conclusion that ac51 plays an important role in the nuclear egress of nucleocapsids during BV formation and is essential for the in vivo virulence of AcMNPV.


Assuntos
Núcleo Celular/virologia , Proteínas do Nucleocapsídeo/metabolismo , Nucleocapsídeo/metabolismo , Spodoptera/virologia , Liberação de Vírus , Replicação Viral , Transporte Ativo do Núcleo Celular , Animais , Vírion , Virulência
12.
Sci Rep ; 8(1): 17817, 2018 12 13.
Artigo em Inglês | MEDLINE | ID: mdl-30546025

RESUMO

Heliothis zea nudivirus-1 (HzNV-1) is an insect virus that can induce both lytic and latent infections in various insect cell lines. During latent infection, several microRNAs (miRNAs) are produced from persistency-associated gene 1 (pag1) as the only detectable HzNV-1 transcript. Previous studies have shown that the pag1 gene suppresses the immediate-early gene hhi1 and promotes host switching into a latent infection via miRNAs derived from pag1. Although other functions of the miRNAs derived from pag1 have not yet been elucidated, several studies have suggested that miRNAs encoded from latency-associated genes can regulate histone-associated enzymes. Because pag1 is a noncoding transcript, it potentially regulates host chromatin structure through miRNAs upon infection. Nevertheless, the exact mechanism by which pag1 alters viral infections remains unknown. In this study, we found that the pag1-encoded miRNA miR-420 suppresses expression of the histone modification-associated enzyme su(var)3-9. Therefore, this miRNA causes histone modification to promote HzNV-1 infection. These results suggest that HzNV-1 may directly influence epigenetic regulation in host cells through interactions with pag1 miRNAs to promote lytic infection. This study provides us with a better understanding of both the HzNV-1 infection pathway and the relationship between viral miRNAs and epigenetic regulation.


Assuntos
Epigênese Genética , Regulação Viral da Expressão Gênica , Histonas/metabolismo , Proteínas de Insetos/metabolismo , MicroRNAs/biossíntese , RNA Viral/biossíntese , Spodoptera , Animais , Metilação , Células Sf9 , Spodoptera/metabolismo , Spodoptera/virologia , Proteínas Virais/metabolismo
13.
Viruses ; 10(10)2018 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-30262719

RESUMO

The Autographa californica multiple nucleopolyhedrovirus (AcMNPV) ac111 gene is highly conserved in lepidopteran-specific baculoviruses, and its function in the AcMNPV life cycle is still unknown. To investigate the function of ac111, an ac111-knockout AcMNPV (vAc111KO) was constructed through homologous recombination in Escherichia coli. Viral growth curve analysis and plaque assays showed that the deletion of ac111 had no effect on infectious budded virion production. Quantitative real-time polymerase chain reaction analysis confirmed that viral DNA replication was unaffected in the absence of ac111. Electron microscopy revealed that the ac111 deletion did not affect nucleocapsid assembly, occlusion-derived virion formation, or the embedding of occlusion-derived virions into the occlusion bodies. However, in vivo bioassays showed that although the deletion of ac111 did not affect the per os infectivity of AcMNPV in Spodoptera exigua larvae, it led to an approximately five-fold reduction in infectivity of AcMNPV in Trichoplusia ni larvae, and vAc111KO took approximately 21 h longer to kill Trichoplusia ni larvae than the wild-type viruses. Taken together, our results demonstrated that although ac111 is not essential for virus replication in vitro, it plays an important role in the per os infectivity of AcMNPV in a host-dependent manner.


Assuntos
DNA Viral/genética , /patogenicidade , Proteínas Virais/genética , Replicação Viral/genética , Animais , Replicação do DNA , DNA Viral/metabolismo , Técnicas de Inativação de Genes , Interações Hospedeiro-Patógeno , Larva/virologia , Lepidópteros/virologia , Nucleocapsídeo/fisiologia , Células Sf9 , Spodoptera/virologia , Proteínas Virais/metabolismo , Vírion/fisiologia
14.
Appl Microbiol Biotechnol ; 102(23): 10139-10146, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30238142

RESUMO

The baculovirus Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) infects lepidopteran invertebrates as natural hosts, although it also has been used as display vector for vaccine development. In this work, we evaluated the effectiveness of repetitive doses of AcMNPV-based vectors on the cytotoxic immune response specific to the capsid-displayed heterologous antigen ovalbumin (OVA). Our results demonstrate that baculovirus vectors induce a boosting effect in the cytotoxic immune response to OVA, making possible to recover the levels obtained in the primary response. Moreover, mice preimmunized with wild-type baculovirus showed a complete lack of antigen-specific CD8 cytotoxic T lymphocytes (CTLs) that may be related to the presence of antibodies directed to baculoviral surface proteins, particularly to GP64. However, baculovirus was able to induce the innate immune response in spite of a previous response against this vector, although some quantitative differences reflect a distinct activation of the immune cells in prime and boost. This is the first report in which the novel capsid display strategy is evaluated in prime-boost schemes to improve efficient CTL responses.


Assuntos
Proteínas do Capsídeo/imunologia , Capsídeo/imunologia , Vacinação , Animais , Antígenos Virais/imunologia , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular , Citocinas/sangue , Feminino , Interações entre Hospedeiro e Microrganismos/imunologia , Imunidade Inata , Camundongos , Camundongos Endogâmicos C57BL , Ovalbumina/imunologia , Spodoptera/imunologia , Spodoptera/virologia , Linfócitos T Citotóxicos/imunologia
15.
J Virol ; 92(23)2018 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-30209166

RESUMO

The baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is a large double-stranded DNA (dsDNA) virus that encodes approximately 156 genes and is highly pathogenic to a variety of larval lepidopteran insects in nature. Oral infection of larval midgut cells is initiated by the occlusion-derived virus (ODV), while secondary infection of other tissues is mediated by the budded virus (BV). Global viral gene expression has been studied in detail in BV-infected cell cultures, but studies of ODV infection in the larval midgut are limited. In this study, we examined expression of the ∼156 AcMNPV genes in Trichoplusia ni midgut tissue using a transcriptomic approach. We analyzed expression profiles of viral genes in the midgut and compared them with profiles from a T. ni cell line (Tnms42). Several viral genes (p6.9, orf76, orf75, pp31, Ac-bro, odv-e25, and odv-ec27) had high expression levels in the midgut throughout the infection. Also, the expression of genes associated with occlusion bodies (polh and p10) appeared to be delayed in the midgut in comparison with the cell line. Comparisons of viral gene expression profiles revealed remarkable similarities between the midgut and cell line for most genes, although substantial differences were observed for some viral genes. These included genes associated with high level BV production (fp-25k), acceleration of systemic infection (v-fgf), and enhancement of viral movement (arif-1/orf20). These differential expression patterns appear to represent specific adaptations for virus infection and transmission through the polarized cells of the lepidopteran midgut.IMPORTANCE Baculoviruses such as AcMNPV are pathogens that are natural regulators of certain insect populations. Baculovirus infections are biphasic, with a primary phase initiated by oral infection of midgut epithelial cells by occlusion-derived virus (ODV) virions and a secondary phase in which other tissues are infected by budded-virus (BV) virions. While AcMNPV infections in cultured cells have been studied extensively, comparatively little is known regarding primary infection in the midgut. In these studies, we identified gene expression patterns associated with ODV-mediated infection of the midgut in Trichoplusia ni and compared those results with prior results from BV-infected cultured cells, which simulate secondary infection. These studies provide a detailed analysis of viral gene expression patterns in the midgut, which likely represent specific viral strategies to (i) overcome or avoid host defenses in the gut and (ii) rapidly move infection from the midgut, into the hemocoel to facilitate systemic infection.


Assuntos
Sistema Digestório/metabolismo , Perfilação da Expressão Gênica , Larva/genética , RNA Viral/genética , Spodoptera/genética , Proteínas Virais/genética , Animais , Sistema Digestório/virologia , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Larva/metabolismo , Larva/virologia , Spodoptera/metabolismo , Spodoptera/virologia , Proteínas Virais/metabolismo
16.
J Invertebr Pathol ; 158: 16-23, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30189196

RESUMO

Plants damaged by herbivore feeding can induce defensive responses that reduce herbivore growth. The slow-growth, high-mortality hypothesis postulates that these non-lethal plant defenses prolong the herbivore's period of susceptibility to natural enemies, such as predators and parasitoids. While many juvenile animals increase their disease resistance as they grow, direct tests of the slow-growth, high-mortality hypothesis in the context of plant-herbivore-pathogen interactions are lacking. Caterpillars increase their resistance to lethal baculoviruses as they develop within and across instars, a phenomenon termed developmental resistance. Progression of developmental resistance can occur through age-related increases in systemic immune functioning and/or midgut-based resistance. Here, we examined the slow-growth, high-mortality hypothesis in the context of developmental resistance of caterpillars to baculoviruses. Intra-stadial (within-instar) developmental resistance of the fall armyworm, Spodoptera frugiperda, to an oral inoculum of the baculovirus SfMNPV increased more rapidly with age when larvae were fed on non-induced foliage than foliage that was induced by jasmonic acid (a phytohormone that up-regulates plant anti-herbivore defenses). The degree of developmental resistance observed was attributable to larval weight at the time of virus inoculation. Thus, slower growth on induced plants prolonged the window of larval susceptibility to the baculovirus. Developmental resistance on induced and non-induced plants was absent when budded virus was injected intrahemocoelically bypassing the midgut, suggesting that developmental resistance was gut-based. Addition of fluorescent brightener, which weakens midgut-based resistance mechanisms to oral virus challenge, abolished developmental resistance. These results highlight the impact of plant defenses on herbivore growth rate and consequences for disease risk.


Assuntos
Ciclopentanos/imunologia , Resistência à Doença/imunologia , Oxilipinas/imunologia , Imunidade Vegetal/imunologia , Spodoptera/imunologia , Spodoptera/virologia , Animais
17.
PLoS One ; 13(8): e0202598, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30133523

RESUMO

A new isolate of the Spodoptera frugiperda granulovirus, SfGV ARG, was completely sequenced and analyzed. The SfGV ARG genome is 139,812 bp long and encodes 151 putative open reading frames. Of these ORFs, 56 were found in betabaculoviruses, 19 of which are present only in GVs closely related to SfGV. Seven ORFs found homologs in this small GV group and also in noctuid NPVs. ORF066 codes a 74 amino acid protein, overlapped with nudix gene, with several homologs in baculovirus, found by tblastn search. Comparison with the genome of the Colombian isolate SfGV VG008 resulted in SfGV being 1101 bp smaller and lacking a homologue of VG008 ORF084, which codes for Lef-7. However, we found that ORF051 shows remote homology to Lef-7 proteins. Moreover, analysis of ORF051 along with Lef-7 proteins coded by a group of noctuid specific GVs and NPVs indicated that Lef-7 proteins coded by these viruses include three F-box domains in contrast to the single one reported for AcMNPV Lef-7. SfGV ARG genome also contains a split photolyase as a distinct feature not found in VG008. BlastX analysis revealed that a complete photolyase is coded considering a putative frameshift in a poly-A tract, which resembles known slippery sequences involved in programmed ribosome frameshifting.


Assuntos
Genômica , Granulovirus/genética , Spodoptera/genética , Proteínas Virais/genética , Sequência de Aminoácidos/genética , Animais , Baculoviridae/genética , Proteínas F-Box/genética , Genoma Viral/genética , Fases de Leitura Aberta/genética , Filogenia , Análise de Sequência de DNA , Spodoptera/virologia
18.
Insect Biochem Mol Biol ; 101: 24-31, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30075239

RESUMO

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is a well-known virus in the Baculoviridae family. Presence of the p35 gene in the AcMNPV genome as a suppressor of the short interfering RNA (siRNA) pathway is a strong reason for the importance of the siRNA pathway in the host cellular defense. Given that, here we explored the roles of Dicer-2 (Dcr2) and Argonaute 2 (Ago2) genes, key factors in the siRNA pathway in response to AcMNPV infection in Spodoptera frugiperda Sf9 cells. The results showed that the transcript levels of Dcr2 and Ago2 increased in response to AcMNPV infection particularly over 16 h post infection suggesting induction of the siRNA pathway. Reductions in the expression levels of Dcr2 and Ago2 by using specific dsRNAs in Sf9 cells modestly enhanced production of viral genomic DNA which indicated their role in the host antiviral defense. Using deep sequencing, our previous study showed a large number of small reads (siRNAs of ∼20 nucleotides) from AcMNPV-infected Sf9 cells that were mapped to some of the viral genes (hot spots). Down-regulation of Dcr2 in Sf9 cells resulted in enhanced expression levels of the selected virus hotspot genes (i.e. ORF-9 and ORF-148), while the transcript levels of virus cold spots (i.e. ORF-18 and ORF-25) with no or few siRNAs mapped to them did not change. Overexpression of AcMNPV p35 as a suppressor of RNAi and anti-apoptosis gene in Sf9 cells increased virus replication. Also, replication of mutant AcMNPV lacking the p35 gene was significantly increased in Sf9 cells with reduced transcript levels of Dcr2 and Ago2, highlighting the antiviral role of the siRNA pathway in Sf9 cells. Together, our results demonstrate that Dcr2 and Ago2 genes contribute in efficient antiviral response of Sf9 cells towards AcMNPV, and in turn, the AcMNPV p35 suppresses the siRNA pathway, besides being an antiapoptotic protein.


Assuntos
Proteínas Argonauta/genética , Genoma Viral , Interações Hospedeiro-Patógeno , Ribonuclease III/genética , Spodoptera/virologia , Proteínas Virais/genética , Animais , Proteínas Argonauta/antagonistas & inibidores , Proteínas Argonauta/imunologia , Regulação da Expressão Gênica , Proteínas de Insetos/antagonistas & inibidores , Proteínas de Insetos/genética , Proteínas de Insetos/imunologia , /metabolismo , RNA Mensageiro/antagonistas & inibidores , RNA Mensageiro/genética , RNA Mensageiro/imunologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/imunologia , Ribonuclease III/antagonistas & inibidores , Ribonuclease III/imunologia , Células Sf9 , Transdução de Sinais , Spodoptera/genética , Spodoptera/imunologia , Spodoptera/metabolismo , Proteínas Virais/metabolismo , Replicação Viral
19.
J Immunol Methods ; 459: 81-89, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29894745

RESUMO

A bi-cistronic baculovirus expression vector was constructed to facilitate the expression, detection, and isolation of the hemagglutinin (HA) fragment HA1 of H6N1 avian influenza virus (AIV) in an insect and a culture of its cells. In this construct, the GP67sp signal peptide promoted the secretion of the recombinant protein into the culture medium, and improved protein expression and purification. Enhanced green fluorescent protein, co-expressed through an internal ribosome entry site, served as a visible reporter for protein expression detection. The hemolymph of Spodoptera litura larvae infected with the bi-cistronic baculovirus was collected for the purification of the recombinant HA1, which was found to be glycosylated, and monomeric and trimeric forms of the recombinant HA1 were identified. Proteins expressed in both the cell culture and larvae served as effective subunit vaccines for the production of antiserum against HA. The antiserum recognized the H6 subtype of AIV but not the H5 subtype.


Assuntos
Baculoviridae/genética , Vetores Genéticos , Glicoproteínas de Hemaglutininação de Vírus da Influenza/biossíntese , Animais , Linhagem Celular , Feminino , Expressão Gênica , Proteínas de Fluorescência Verde/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Vírus da Influenza A/classificação , Larva/virologia , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Spodoptera/citologia , Spodoptera/virologia
20.
Mol Genet Genomics ; 293(5): 1265-1277, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-29923069

RESUMO

The classical baculovirus display system (BDS) has often recruited fields including gene delivery, gene therapy, and the genetic engineering of vaccines, as it is capable of presenting foreign polypeptides on the membranes of recombinant baculovirus through a transmembrane protein. However, classical BDS's high cost, complicated operation, low display efficiency and its inability to simultaneously display multiple gene products impede its practicality. In this study, we present a novel and highly efficient display system based on ires-dependent gp64 for rescuing gp64-null Bacmid of baculovirus construction without affecting the viral replication cycle, which we name the baculovirus multigene display system (BMDS). Laser scanning confocal microscopy demonstrated that eGFP, eYFP, and mCherry were translocated on the membrane of Spodoptera frugiperda 9 cell successfully as expected. Western blot analysis further confirmed the presence of the fluorescent proteins on the budded, mature viral particles. The results showed the display efficiency of target gene on cell surface is fourfold that of classical BDS. In addition, a recombinant baculovirus displaying three kinds of fluorescent proteins simultaneously was constructed, thereby demonstrating the effectiveness of BMDS as a co-display system.


Assuntos
Baculoviridae/genética , Técnicas de Visualização da Superfície Celular/métodos , Spodoptera/virologia , Animais , Regulação Viral da Expressão Gênica/genética , Vírus dos Insetos/genética , Spodoptera/genética , Vacinas/genética
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