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1.
Proc Natl Acad Sci U S A ; 119(16): e2120048119, 2022 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-35412888

RESUMO

Heritable symbionts display a wide variety of transmission strategies to travel from one insect generation to the next. Parasitoid wasps, one of the most diverse insect groups, maintain several heritable associations with viruses that are beneficial for wasp survival during their development as parasites of other insects. Most of these beneficial viral entities are strictly transmitted through the wasp germline as endogenous viral elements within wasp genomes. However, a beneficial poxvirus inherited by Diachasmimorpha longicaudata wasps, known as Diachasmimorpha longicaudata entomopoxvirus (DlEPV), is not integrated into the wasp genome and therefore may employ different tactics to infect future wasp generations. Here, we demonstrated that transmission of DlEPV is primarily dependent on parasitoid wasps, since viral transmission within fruit fly hosts of the wasps was limited to injection of the virus directly into the larval fly body cavity. Additionally, we uncovered a previously undocumented form of posthatch transmission for a mutualistic virus that entails external acquisition and localization of the virus within the adult wasp venom gland. We showed that this route is extremely effective for vertical and horizontal transmission of the virus within D. longicaudata wasps. Furthermore, the beneficial phenotype provided by DlEPV during parasitism was also transmitted with perfect efficiency, indicating an effective mode of symbiont spread to the advantage of infected wasps. These results provide insight into the transmission of beneficial viruses among insects and indicate that viruses can share features with cellular microbes during their evolutionary transitions into symbionts.


Assuntos
Entomopoxvirinae , Interações entre Hospedeiro e Microrganismos , Simbiose , Tephritidae , Vespas , Animais , Evolução Biológica , Entomopoxvirinae/fisiologia , Tephritidae/virologia , Vespas/genética , Vespas/virologia
2.
Science ; 373(6554): 535-541, 2021 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-34326235

RESUMO

Interkingdom competition occurs between hymenopteran parasitoids and insect viruses sharing the same insect hosts. It has been assumed that parasitoid larvae die with the death of the infected host or as result of competition for host resources. Here we describe a gene family, parasitoid killing factor (pkf), that encodes proteins toxic to parasitoids of the Microgastrinae group and determines parasitism success. Pkfs are found in several entomopathogenic DNA virus families and in some lepidopteran genomes. We provide evidence of equivalent and specific toxicity against endoparasites for PKFs found in entomopoxvirus, ascovirus, baculovirus, and Lepidoptera through a mechanism that elicits apoptosis in the cells of susceptible parasitoids. This highlights the evolutionary arms race between parasitoids, viruses, and their insect hosts.


Assuntos
Entomopoxvirinae/fisiologia , Proteínas de Insetos/toxicidade , Lepidópteros/parasitologia , Lepidópteros/virologia , Proteínas Virais/toxicidade , Vespas/fisiologia , Animais , Apoptose , Evolução Biológica , Transferência Genética Horizontal , Genoma de Inseto , Interações Hospedeiro-Parasita , Proteínas de Insetos/química , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Vírus de Insetos/fisiologia , Larva/genética , Larva/parasitologia , Larva/virologia , Lepidópteros/genética , Lepidópteros/metabolismo , Nucleopoliedrovírus/fisiologia , Spodoptera/genética , Spodoptera/metabolismo , Spodoptera/parasitologia , Spodoptera/virologia , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/metabolismo , Vespas/crescimento & desenvolvimento
3.
PLoS Pathog ; 16(11): e1009069, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-33253317

RESUMO

Insects are known to host a wide variety of beneficial microbes that are fundamental to many aspects of their biology and have substantially shaped their evolution. Notably, parasitoid wasps have repeatedly evolved beneficial associations with viruses that enable developing wasps to survive as parasites that feed from other insects. Ongoing genomic sequencing efforts have revealed that most of these virus-derived entities are fully integrated into the genomes of parasitoid wasp lineages, representing endogenous viral elements (EVEs) that retain the ability to produce virus or virus-like particles within wasp reproductive tissues. All documented parasitoid EVEs have undergone similar genomic rearrangements compared to their viral ancestors characterized by viral genes scattered across wasp genomes and specific viral gene losses. The recurrent presence of viral endogenization and genomic reorganization in beneficial virus systems identified to date suggest that these features are crucial to forming heritable alliances between parasitoid wasps and viruses. Here, our genomic characterization of a mutualistic poxvirus associated with the wasp Diachasmimorpha longicaudata, known as Diachasmimorpha longicaudata entomopoxvirus (DlEPV), has uncovered the first instance of beneficial virus evolution that does not conform to the genomic architecture shared by parasitoid EVEs with which it displays evolutionary convergence. Rather, DlEPV retains the exogenous viral genome of its poxvirus ancestor and the majority of conserved poxvirus core genes. Additional comparative analyses indicate that DlEPV is related to a fly pathogen and contains a novel gene expansion that may be adaptive to its symbiotic role. Finally, differential expression analysis during virus replication in wasps and fly hosts demonstrates a unique mechanism of functional partitioning that allows DlEPV to persist within and provide benefit to its parasitoid wasp host.


Assuntos
Entomopoxvirinae/genética , Genoma Viral/genética , Genômica , Poxviridae/genética , Simbiose , Vespas/virologia , Animais , Entomopoxvirinae/fisiologia , Feminino , Masculino , Poxviridae/fisiologia , Replicação Viral/genética
4.
J Virol ; 94(8)2020 03 31.
Artigo em Inglês | MEDLINE | ID: mdl-32024779

RESUMO

For insects known as parasitoid wasps, successful development as a parasite results in the death of the host insect. As a result of this lethal interaction, wasps and their hosts have coevolved strategies to gain an advantage in this evolutionary arms race. Although normally considered to be strict pathogens, some viruses have established persistent infections within parasitoid wasp lineages and are beneficial to wasps during parasitism. Heritable associations between viruses and parasitoid wasps have evolved independently multiple times, but most of these systems remain largely understudied with respect to viral origin, transmission and replication strategies of the virus, and interactions between the virus and host insects. Here, we report a detailed characterization of Diachasmimorpha longicaudata entomopoxvirus (DlEPV), a poxvirus found within the venom gland of Diachasmimorpha longicaudata wasps. Our results show that DlEPV exhibits similar but distinct transmission and replication dynamics compared to those of other parasitoid viral elements, including vertical transmission of the virus within wasps, as well as virus replication in both female wasps and fruit fly hosts. Functional assays demonstrate that DlEPV is highly virulent within fly hosts, and wasps without DlEPV have severely reduced parasitism success compared to those with a typical viral load. Taken together, the data presented in this study illustrate a novel case of beneficial virus evolution, in which a virus of unique origin has undergone convergent evolution with other viral elements associated with parasitoid wasps to provide an analogous function throughout parasitism.IMPORTANCE Viruses are generally considered to be disease-causing agents, but several instances of beneficial viral elements have been identified in insects called parasitoid wasps. These virus-derived entities are passed on through wasp generations and enhance the success of the wasps' parasitic life cycle. Many parasitoid-virus partnerships studied to date exhibit common features among independent cases of this phenomenon, including a mother-to-offspring route of virus transmission, a restricted time and location for virus replication, and a positive effect of virus activity on wasp survival. Our characterization of Diachasmimorpha longicaudata entomopoxvirus (DlEPV), a poxvirus found in Diachasmimorpha longicaudata parasitoid wasps, represents a novel example of beneficial virus evolution. Here, we show that DlEPV exhibits functional similarities to known parasitoid viral elements that support its comparable role during parasitism. Our results also demonstrate unique differences that suggest DlEPV is more autonomous than other long-term viral associations described in parasitoid wasps.


Assuntos
Interações entre Hospedeiro e Microrganismos/fisiologia , Poxviridae/fisiologia , Simbiose , Vespas/virologia , Animais , Evolução Biológica , Entomopoxvirinae/genética , Entomopoxvirinae/fisiologia , Regulação Viral da Expressão Gênica , Genes Virais , Genoma Viral , Poxviridae/genética , Interferência de RNA , Fenômenos Fisiológicos Virais , Replicação Viral , Vírus , Venenos de Vespas
5.
Virus Genes ; 54(3): 438-445, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29666979

RESUMO

Amsacta moorei entomopoxvirus (AMEV) infects certain lepidopteran and orthopteran insects and is the most studied member of the genus Betaentomopoxvirus. It has been considered as a potential vector for gene therapy, a vector to express exogenous proteins and a biological control agent. One of its open reading frames, amv248, encodes a putative glycosyltransferase and is the only known attachment protein conserved in AMEV and chordopoxviruses. The ORF was successfully expressed and the protein was shown to bind soluble heparin, both in silico and in vitro. Our results also showed that, while viral infection was inhibited by soluble glycosaminoglycans (GAGs), GAG-deficient cells were more resistant to the virus. Finally, we revealed that amv248 encodes an active heparin-binding glycosyltransferase which is likely to have a key role in the initiation of infection by AMEV.


Assuntos
Entomopoxvirinae/genética , Glicosiltransferases/genética , Animais , Linhagem Celular , Glicosiltransferases/química , Glicosiltransferases/metabolismo , Heparina/metabolismo , Estrutura Molecular , Fases de Leitura Aberta , Ligação Proteica
6.
Virus Res ; 248: 31-38, 2018 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-29471050

RESUMO

Entomopoxviruses are an important group of viruses infecting only insects. They belong to Poxviridae which infect both invertebrates and vertebrates, including humans. Protein kinases are known to have roles at virus morphogenesis, host selectivity, the regulation of cell division and apoptosis in some vertebrate poxviruses. In this study, 2 protein kinases (PKs) (AMV153 and AMV197) of Amsacta moorei entomopoxvirus (AMEV) were investigated for the interactions among 230 viral proteins using yeast two-hybrid system (Y2H). For this purpose, two protein kinases and 230 viral genes were cloned into the bait and prey vectors, respectively. Bait vectors were introduced into Saccharomyces cerevisiae AH109. Expression of the bait genes were confirmed by western blot analysis. Both yeast strains of bait were transformed individually with each prey clone and grown on a selective medium (minimal synthetic defined) to determine the protein-protein interactions between bait and prey proteins. Transformations identified totally 16 interactions among AMEV protein kinases and all viral proteins of which 5 belong to AMV153 and 11 belong to AMV197. One of the five interactions detected for AMV153 protein kinase is self-association. Its other four interactions are with two virus entry complex proteins (AMV035 and AMV083), a membrane protein (AMV165) and a subunit of RNA polymerase (AMV230). The other protein kinase, AMV197, interacted with two virus entry complex proteins (AMV035 and AMV083) as AMV153, a caspase-2 enzyme (AMV063), a Holliday junction resolvase (AMV162), a membrane protein (AMV165), a subunit of RNA polymerase (AMV230) and five other hypothetical proteins (AMV026, AMV040, AMV062, AMV069, AMV120) encoded by AMEV genome. Glutathione S-transferase (GST) pull-down assay was used to confirm all interactions described by Y2H analysis. In addition, the theoretical structures of the two of 16 interactions were interpreted by docking analysis. Consistent with Y2H and pull down assays, docking analysis also showed the interactions of AMV063 with AMV153 and AMV197. Detected interactions of the AMEV viral proteins with viral protein kinases could lead to the understanding of the regulation of the viral activities of interacted viral proteins.


Assuntos
Entomopoxvirinae/fisiologia , Insetos/virologia , Infecções por Poxviridae/virologia , Mapeamento de Interação de Proteínas , Proteínas Quinases/metabolismo , Proteínas Virais/metabolismo , Animais , Biblioteca Gênica , Modelos Moleculares , Plasmídeos/genética , Ligação Proteica , Conformação Proteica , Mapeamento de Interação de Proteínas/métodos , Proteínas Quinases/química , Proteínas Quinases/genética , Técnicas do Sistema de Duplo-Híbrido , Proteínas Virais/química , Proteínas Virais/genética
7.
Virus Res ; 243: 25-30, 2018 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-29020603

RESUMO

Amsacta moorei entomopoxvirus (AMEV), the most studied member of the genus Betaentomopoxvirus, was initially isolated from Red Hairy caterpillar larvae, Amsacta moorei. According to genome sequence and previous studies it was shown that amv248 encodes a putative glycosyltransferase that is the only conserved attachment protein in betaentomopoxviruses. Transcriptional analysis of the amv248 gene by RT-PCR and qPCR showed that transcription starts at 6h post infection (hpi). Also, transcription was not affected by a DNA replication inhibitor but was severely curtailed by a protein synthesis inhibitor. These results indicate that amv248 belongs to the intermediate class of gene expression. 5' and 3' untranslated regions analysis revealed that transcription initiates at position -126 relative to the translational start site, and ends between 50 and 83 bases after the stop codon. To narrow down the size and location of the gene's promoter, the upstream region as well as several different sized deletions thereof were generated and cloned upstream of a luciferase reporter gene. The constructs were used to measure the Firefly and Renilla luciferase activities in dual assays. The results showed that luciferase activity decreased when bases -198 to -235 of amv248 upstream region were missing. Sequence analysis among the intermediate gene promoters of AMEV showed that TTTAT(T/A)TT(T/A)2TTA is possibly a common motif, however, further investigations are needed to confirm this conclusion.


Assuntos
Entomopoxvirinae/enzimologia , Glicosiltransferases/genética , Mariposas/virologia , Transcrição Genética , Proteínas Virais/genética , Animais , Entomopoxvirinae/classificação , Entomopoxvirinae/genética , Entomopoxvirinae/isolamento & purificação , Glicosiltransferases/metabolismo , Larva/virologia , Regiões Promotoras Genéticas , Proteínas Virais/metabolismo
8.
Sci Rep ; 7(1): 13522, 2017 10 19.
Artigo em Inglês | MEDLINE | ID: mdl-29051595

RESUMO

Microbial parasitism, infection, and symbiosis in animals often modulate host endocrine systems, resulting in alterations of phenotypic traits of the host that can have profound effects on the ecology and evolution of both the microorganisms and their hosts. Information about the mechanisms and genetic bases of such modulations by animal parasites is available from studies of steroid hormones. However, reports involving other hormones are scarce. We found that an insect virus, a betaentomopoxvirus, encodes a juvenile hormone acid methyltransferase that can synthesize an important insect hormone, the sesquiterpenoid juvenile hormone. Phylogenetic analysis suggested that this gene is of bacterial origin. Our study challenges the conventional view that functional enzymes in the late phase of the juvenile hormone biosynthesis pathway are almost exclusive to insects or arthropods, and shed light on juvenoid hormone synthesis beyond Eukaryota. This striking example demonstrates that even animal parasites having no metabolic pathways for molecules resembling host hormones can nevertheless influence the synthesis of such hormones, and provides a new context for studying animal parasite strategies in diverse systems such as host-parasite, host-symbiont or host-vector-parasite.


Assuntos
Entomopoxvirinae/genética , Hormônios Juvenis/biossíntese , Metamorfose Biológica/genética , Metiltransferases/genética , Sequência de Aminoácidos , Animais , Hemolinfa/metabolismo , Interações Hospedeiro-Parasita/genética , Insetos/crescimento & desenvolvimento , Larva/metabolismo , Larva/virologia , Legionella/genética , Metiltransferases/classificação , Metiltransferases/metabolismo , Filogenia , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência
9.
Virus Res ; 215: 25-36, 2016 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-26820433

RESUMO

Insect-born entomopoxviruses (Fam: Poxviridae) are potentially important bio-pesticide against insect pests and expression vectors as well as vectors for transient human gene therapies including recombinant viral vaccines. For these reasons, it is necessary to understand the regulatory genes functions to improve its biotechnological potential. Here, we focused on the characterization of serine/threonine (Ser/Thr; ORF AMV197) protein kinase gene from the Amsacta moorei entomopoxvirus (AMEV), the type species of the genus Betaentomopoxvirus. Transcription of the parental and an amv197-null recombinant AMEV was compared by whole-genome gene expression microarray analysis. Blast2GO analysis reflected a broad diversity of upregulated and downregulated genes. Results showed that expression levels of 102 genes (45%) out of 226 tested genes changed significantly in the recombinant AMEV infected cells. Of these transcripts, 72 (70.58%) were upregulated and 30 (29.41%) were downregulated throughout the infection period. Genes involved in DNA repair, replication and nucleotide metabolism, transcription and RNA modification, and protein modification were mostly upregulated at different times in cells infected with the recombinant virus. Furthermore, transcription of all studied cellular genes including metabolism of apoptosis (Nedd2-like caspase, hemolin and elongation factor-1 alpha (ef1a) gene) was downregulated in the absence of amv197. Quantitative real time reverse transcription-PCR confirmed viral transcriptional changes obtained by microarray. The results of this study indicated that the product of amv197 appears to affect the transcriptional regulation of most viral and many cellular genes. Further investigations are, however, needed to narrow down the role of AMV197 throughout the infection process.


Assuntos
Entomopoxvirinae/genética , Deleção de Genes , Perfilação da Expressão Gênica , Regulação Viral da Expressão Gênica , Proteínas Quinases/metabolismo , RNA Mensageiro/análise , Análise em Microsséries , Proteínas Quinases/genética
10.
J Gen Virol ; 97(1): 225-232, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26499185

RESUMO

Insect viruses are among the most important pathogens of lepidopteran insects. Virus-infected larvae often show developmental defects including a prolonged larval period and a failure to pupate, but the mechanisms by which insect viruses regulate host development need further investigation. In this study, the regulation of host endocrinology by a lepidopteran entomopoxvirus (EPV), Mythimna separata EPV (MySEV), was examined. When fourth instar M. separata were inoculated with MySEV occlusion bodies, pupation was prevented and the insects died during the final (sixth) larval instar. Liquid chromatography-MS analysis revealed that juvenile hormone (JH) titres in the haemolymph of MySEV-infected sixth instars were higher than those in mock-infected larvae. JH esterase (JHE) activity was also examined by kinetic assay using a colorimetric substrate. The level of JHE activity in the haemolymph of MySEV-infected larvae was generally lower than that found in mock-infected larvae. In contrast, ecdysteroid titre in the haemolymph of final-instar MySEV-infected larvae was lower than that found in mock-infected larvae when measured by radioimmunoassay. A statistically significant difference in the release of ecdysteroids from prothoracic glands (PGs) that were dissected from MySEV- or mock-infected sixth instar Day 3 larvae was not found following prothoracicotropic hormone (PTTH) exposure. Our results indicate that the release of ecdysteroids was reduced not by infection of the PGs by MySEV, but by reduced PTTH production from the brain. Taken together our study suggests that EPVs retard host development by both reducing ecdysone titre and maintaining status quo levels of JH by preventing its metabolism.


Assuntos
Ecdisteroides/análise , Entomopoxvirinae/fisiologia , Interações Hospedeiro-Patógeno , Hormônios Juvenis/análise , Lepidópteros/virologia , Animais , Cromatografia Líquida , Esterases/análise , Hemolinfa/química , Larva/crescimento & desenvolvimento , Larva/virologia , Lepidópteros/crescimento & desenvolvimento , Espectrometria de Massas
11.
Acta Parasitol ; 60(3): 462-5, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26204184

RESUMO

The importance of pathogens in the population dynamics of Ips typographus remains a subject of ongoing debate. The main objective of our experiment was to compare the pathogen infection levels of individuals overwintering in bark with the levels of individuals from the same population captured with pheromone traps and thereby to determine primary answers as to whether it can be confirmed that pathogenic organisms affect the flight ability of bark beetles or their ability to leave their places of overwintering. A total of 402 I. typographus individuals were analyzed at a study location under limited management. Three pathogens were confirmed to be present: the gregarine Gregarina typographi, the virus ItEPV, and the microsporidium Nosema typographi. Infection levels of Gregarina typographi and ItEPV were the same in beetles collected at places of overwintering and in those beetles collected in pheromone traps within the immediate vicinity. As these pathogens infect the host's intestine, the tendency to leave the places of overwintering is apparently not diminished. A similar analysis and comparison of pathogens located in the fat body might bring different results, as our study only detected N. typographi in a single dissected adult spruce bark beetle.


Assuntos
Apicomplexa/isolamento & purificação , Entomopoxvirinae/isolamento & purificação , Nosema/isolamento & purificação , Gorgulhos/microbiologia , Gorgulhos/parasitologia , Animais , Gorgulhos/virologia
12.
Viruses ; 7(4): 1960-74, 2015 Apr 13.
Artigo em Inglês | MEDLINE | ID: mdl-25871928

RESUMO

Organisms from diverse phylogenetic origins can thrive within the same ecological niches. They might be induced to evolve convergent adaptations in response to a similar landscape of selective pressures. Their genomes should bear the signature of this process. The study of unrelated virus lineages infecting the same host panels guarantees a clear identification of phyletically independent convergent adaptation. Here, we investigate the evolutionary history of genes in the accessory genome shared by unrelated insect large dsDNA viruses: the entomopoxviruses (EPVs, Poxviridae) and the baculoviruses (BVs). EPVs and BVs have overlapping ecological niches and have independently evolved similar infection processes. They are, in theory, subjected to the same selective pressures from their host's immune responses. Their accessory genomes might, therefore, bear analogous genomic signatures of convergent adaption and could point out key genomic mechanisms of adaptation hitherto undetected in viruses. We uncovered 32 homologous, yet independent acquisitions of genes originating from insect hosts, different eukaryotes, bacteria and viruses. We showed different evolutionary levels of gene acquisition convergence in these viruses, underlining a continuous evolutionary process. We found both recent and ancient gene acquisitions possibly involved to the adaptation to both specific and distantly related hosts. Multidirectional and multipartite gene exchange networks appear to constantly drive exogenous gene assimilations, bringing key adaptive innovations and shaping the life histories of large DNA viruses. This evolutionary process might lead to genome level adaptive convergence.


Assuntos
Baculoviridae/genética , Entomopoxvirinae/genética , Evolução Molecular , Transferência Genética Horizontal , Insetos/virologia , Adaptação Biológica , Animais , Baculoviridae/fisiologia , DNA Viral/química , DNA Viral/genética , Entomopoxvirinae/fisiologia , Dados de Sequência Molecular , Seleção Genética , Análise de Sequência de DNA
13.
Intervirology ; 58(1): 41-8, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25591507

RESUMO

OBJECTIVES: Lipolytic genes have been investigated in several viral genomes, and some of them show enzyme activity which can be used for various functions including the production of DNA replication metabolites, rescue from endosomes, and membrane fusion. Amsacta moorei entomopoxvirus (AMEV) replicates in nearly the entire insect body, especially in the adipose tissue. One of the open reading frames (ORFs) in the AMEV genome, amv133, encodes a putative lipase enzyme. In this study, we therefore investigate the enzyme activity of amv133. METHODS: amv133 was aligned with known lipase genes and their homologs in entomopoxviruses. Expressed proteins were partially purified and assayed for lipase, esterase and protease. RESULTS: We found that amv133 contains all the domains required for a functional lipase enzyme and that it shows a significant similarity with homologs in other entomopoxviruses. Since there is a similarity of the catalytic triad between lipases and serine proteases, we also investigated the protease activity of amv133. Lipase, esterase and protease assays showed that amv133 encodes a functional esterase enzyme with protease activity. CONCLUSION: The current data show that amv133 is a conserved gene in all entomopoxvirus genomes sequenced so far and might contribute greatly to degrading the lipids or proteins and hence improve the virus infection.


Assuntos
Entomopoxvirinae/enzimologia , Esterases/genética , Esterases/metabolismo , Proteínas Virais/metabolismo , Sequência de Aminoácidos , Animais , Entomopoxvirinae/genética , Entomopoxvirinae/metabolismo , Esterases/química , Genes Virais , Genoma Viral , Insetos/virologia , Lipase/genética , Dados de Sequência Molecular , Fases de Leitura Aberta , Peptídeo Hidrolases/metabolismo , Alinhamento de Sequência , Proteínas Virais/química , Proteínas Virais/genética
14.
Arch Virol ; 159(10): 2541-7, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-24796553

RESUMO

The open reading frame (ORF) amv133 of Amsacta moorei entomopoxvirus, encodes a putative lipase gene. Its temporal expression pattern was characterized by RT-PCR and found to start at 6 h postinfection (h p.i.) and reach a maximum level at 48 h p.i. While the ORF has a late promoter motif, the inhibition of viral DNA synthesis by Ara-C failed to inhibit transcription, but a general inhibitor of protein synthesis prevented its transcription, indicating that amv133 is an intermediate gene. 5'-RACE analysis showed that transcription was initiated at position -77 relative to the translational start site. To determine the size of the promoter, several truncations were generated and cloned upstream of the firefly luciferase reporter gene. The resulting constructs were tested in a dual assay. A fragment that contained 115 bp relative to the transcription start site exhibited optimum promoter length.


Assuntos
Entomopoxvirinae/genética , Lipase/genética , Fases de Leitura Aberta/genética , Proteínas Virais/genética , Regiões 5' não Traduzidas , Animais , Antivirais/farmacologia , Sequência de Bases , Linhagem Celular , Citarabina/farmacologia , Replicação do DNA/efeitos dos fármacos , DNA Viral/genética , Regulação Viral da Expressão Gênica , Genes Virais , Dados de Sequência Molecular , Mariposas/virologia , Infecções por Poxviridae , Regiões Promotoras Genéticas , Biossíntese de Proteínas/efeitos dos fármacos , Sítio de Iniciação de Transcrição , Transcrição Genética/efeitos dos fármacos
15.
J Invertebr Pathol ; 118: 12-7, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24603041

RESUMO

Mythimna separata entomopoxvirus (MySEV), of the genus Betaentomopoxvirus, was found to replicate in High Five™ cells. The infected cells produced many occlusion bodies and were hypertrophied but did not lyse. Following infection at a multiplicity of infection of 0.1, titers of extracellular virus reached a plateau 3-4days post infection at 25°C and were estimated at ca. 3×10(5) plaque-forming units per ml in TC-100 or TMN-FH media, both of which contained fetal bovine serum (FBS). Serum free medium, Express Five® SFM, also supported virus replication in High Five™ cells, but the titers were approximately one-tenth of those grown in TC-100 or TMN-FH media containing FBS. Using High Five™ cells, a recombinant MySEV was successfully constructed using homologous recombination. This study opens an avenue to the evaluation of entomopoxvirus gene functions using reverse genetic approaches with in vitro and in vivo hosts.


Assuntos
Entomopoxvirinae/genética , Técnicas de Introdução de Genes/métodos , Genes Virais/genética , Lepidópteros/virologia , Animais , Linhagem Celular , Microscopia Eletrônica de Transmissão , Reação em Cadeia da Polimerase , Transfecção
16.
Virology ; 452-453: 95-116, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24606687

RESUMO

Complete genome sequence of Anomala cuprea entomopoxvirus, which belongs to the genus Alphaentomopoxvirus, including its terminal hairpin loop sequences, is reported. This is the first genome sequence of Alphaentomopoxvirus reported, and hairpin loops in entomopoxviruses have not previously been sequenced. The genome is 245,717 bp, which is smaller than had previously been estimated for Alphaentomopoxvirus. The inverted terminal repeats are quite long, and experimental results suggest that one genome molecule has one type of hairpin at one end and another type at the other end. The genome contains unexpected ORFs, e.g., that for the ubiquitin-conjugating enzyme E2 of eukaryotes. The BIR and RING domains found in a single ORF for an inhibitor of apoptosis in baculoviruses and entomopoxviruses occurred in two different, widely separated ORFs. Furthermore, an ORF in the genome contains a serpin domain that was previously found in vertebrate poxviruses for apoptosis inhibition but not in insect viruses.


Assuntos
Apoptose , Replicação do DNA , Entomopoxvirinae/genética , Genoma Viral , Insetos/citologia , Sequências Repetidas Invertidas , Infecções por Poxviridae/veterinária , Proteínas Virais/metabolismo , Animais , Sequência de Bases , DNA Viral/química , DNA Viral/genética , DNA Viral/metabolismo , Entomopoxvirinae/química , Entomopoxvirinae/fisiologia , Insetos/virologia , Dados de Sequência Molecular , Infecções por Poxviridae/fisiopatologia , Infecções por Poxviridae/virologia , Proteínas Virais/química , Proteínas Virais/genética
17.
Pest Manag Sci ; 70(1): 46-54, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23424042

RESUMO

BACKGROUND: Entomopoxviruses (EVs) form two types of inclusion body: spheroids, which contain virions, and spindles, which do not. The authors tested whether the spindles from a coleopteran EV, Anomala cuprea EV (ACEV), enhanced the insecticidal activity of a commercial Bacillus thuringiensis (Bt) formulation and the susceptibility of scarabaeid pest species in Japan to the virus's spheroids, to assess whether ACEV inclusion bodies are potential biological control agents for pest insects. RESULTS: Peroral inoculation with both ACEV spindles and the Bt toxin only or the complete Bt formulation shortened the survival and increased the mortality of treated insects compared with those of insects inoculated with Bt without the spindles (8-38 h of decrease in LT50 values among assays). ACEV showed high infectivity to a major scarabaeid pest species in Japanese sugar cane fields. CONCLUSION: The results suggest that spindles or the constituent protein fusolin can be used as a coagent with Bt formulations, and that fusolin coexpression with a Bt toxin in crops might improve the insecticidal efficacy. In addition, the spheroids are potential biocontrol agents for some scarabaeid pests that are not easy to control because of their underground habitation.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/farmacologia , Química Farmacêutica/métodos , Besouros/efeitos dos fármacos , Endotoxinas/química , Endotoxinas/farmacologia , Entomopoxvirinae/química , Proteínas Hemolisinas/química , Proteínas Hemolisinas/farmacologia , Corpos de Inclusão/química , Controle Biológico de Vetores/métodos , Animais , Toxinas de Bacillus thuringiensis , Entomopoxvirinae/metabolismo , Corpos de Inclusão/metabolismo , Controle Biológico de Vetores/instrumentação
18.
J Virol ; 87(14): 7992-8003, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23678178

RESUMO

Poxviruses are nucleocytoplasmic large DNA viruses encompassing two subfamilies, the Chordopoxvirinae and the Entomopoxvirinae, infecting vertebrates and insects, respectively. While chordopoxvirus genomics have been widely studied, only two entomopoxvirus (EPV) genomes have been entirely sequenced. We report the genome sequences of four EPVs of the Betaentomopoxvirus genus infecting the Lepidoptera: Adoxophyes honmai EPV (AHEV), Choristoneura biennis EPV (CBEV), Choristoneura rosaceana EPV (CREV), and Mythimna separata EPV (MySEV). The genomes are 80% AT rich, are 228 to 307 kbp long, and contain 247 to 334 open reading frames (ORFs). Most genes are homologous to those of Amsacta moorei entomopoxvirus and encode several protein families repeated in tandem in terminal regions. Some genomes also encode proteins of unknown functions with similarity to those of other insect viruses. Comparative genomic analyses highlight a high colinearity among the lepidopteran EPV genomes and little gene order conservation with other poxvirus genomes. As with previously sequenced EPVs, the genomes include a relatively conserved central region flanked by inverted terminal repeats. Protein clustering identified 104 core EPV genes. Among betaentomopoxviruses, 148 core genes were found in relatively high synteny, pointing to low genomic diversity. Whole-genome and spheroidin gene phylogenetic analyses showed that the lepidopteran EPVs group closely in a monophyletic lineage, corroborating their affiliation with the Betaentomopoxvirus genus as well as a clear division of the EPVs according to the orders of insect hosts (Lepidoptera, Coleoptera, and Orthoptera). This suggests an ancient coevolution of EPVs with their insect hosts and the need to revise the current EPV taxonomy to separate orthopteran EPVs from the lepidopteran-specific betaentomopoxviruses so as to form a new genus.


Assuntos
Entomopoxvirinae/genética , Evolução Molecular , Genoma Viral/genética , Mariposas/virologia , Filogenia , Sequência de Aminoácidos , Animais , Composição de Bases/genética , Sequência de Bases , Canadá , China , Entomopoxvirinae/classificação , Genômica , Japão , Funções Verossimilhança , Modelos Genéticos , Dados de Sequência Molecular , Fases de Leitura Aberta/genética , Alinhamento de Sequência , Análise de Sequência de DNA , Especificidade da Espécie
19.
J Gen Virol ; 94(Pt 8): 1876-1887, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23620379

RESUMO

CF-70-B2 cells derived from the spruce budworm (Choristoneura fumiferana) undergo apoptosis when infected with Amsacta moorei entomopoxvirus (AMEV), as characterized by membrane blebbing, formation of apoptotic bodies, TdT-mediated dUTP nick-end labelling (TUNEL) staining, condensed chromatin and induction of caspase-3/7 activity. The apoptotic response was reduced when cells were infected with UV-inactivated AMEV, but not when infected in the presence of the DNA synthesis inhibitor, cytosine ß-d-arabinofuranoside. Hence, only pre-DNA replication events were involved in inducing the antiviral response in CF-70-B2 cells. The virus eventually overcame the host's antiviral response and replicated to high progeny virus titres accompanied by high levels of caspase-3/7 activity. The CF-70-B2 cells were less productive of progeny virus in comparison to LD-652, a Lymantria dispar cell line routinely used for propagation of AMEV. At late stages of infection, LD-652 cells also showed characteristics of apoptosis such as oligosomal DNA fragmentation, TUNEL staining, condensed chromatin and increased caspase-3/7 activity. Induction of apoptosis in LD-652 cells was dependent on viral DNA replication and/or late gene expression. A significantly reduced rate of infection was observed in the presence of general caspase inhibitors Q-VD-OPH and Z-VAD-FMK, indicating caspases may be involved in productive virus infection.


Assuntos
Apoptose , Entomopoxvirinae/patogenicidade , Lepidópteros/virologia , Animais , Caspase 3/metabolismo , Caspase 7/metabolismo , Linhagem Celular , Membrana Celular/patologia , Fragmentação do DNA , Marcação In Situ das Extremidades Cortadas
20.
J Invertebr Pathol ; 112 Suppl: S138-41, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22841637

RESUMO

Insect cell lines have been initiated since the 1930s and were used to replicate insect baculoviruses as well as arboviruses. Since the latter group of viruses cause serious diseased in man and equines, efforts were expended to characterize the viruses in the new cell lines in attempts to understand the replication cycle at the cellular and molecular levels. Soon it was realized that insect baculoviruses have a potential as viable alternatives to chemicals in the control of agricultural and forest insect pests. The cell lines provided excellent tools to understand the molecular biology of baculoviruses before wide-scale use in the field. During these investigastions, it came to light that baculoviruses can be exploited as vectors for the expression of exogenous proteins and vaccines. The amenability of the virus to genetic modifications and the increasing numbers of permissive cell lines opened new avenues in protein expression. However, not all baculoviruses were able to replicate in cell lines. Indeed, there are no cell lines permissive to viruses belonging to the genera Gammabaculvirus and Deltabaculovirus. Some entomopoxviruses have been replicated in a few cell lines and this paper reports the replication of an entomopoxvirus from the spruce budworm in a homologous cell line.


Assuntos
Técnicas de Cultura de Células/métodos , Entomopoxvirinae/fisiologia , Mariposas/virologia , Replicação Viral , Animais , Linhagem Celular , Células Cultivadas
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