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1.
Sci Rep ; 11(1): 16402, 2021 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-34385487

RESUMO

Ascoviruses are large dsDNA viruses characterized by the extraordinary changes they induce in cellular pathogenesis and architecture whereby after nuclear lysis and extensive hypertrophy, each cell is cleaved into numerous vesicles for virion reproduction. However, the level of viral replication and transcription in vesicles compared to other host tissues remains uncertain. Therefore, we applied RNA-Sequencing to compare the temporal transcriptome of Spodoptera frugiperda ascovirus (SfAV) and Trichoplusia ni ascovirus (TnAV) at 7, 14, and 21 days post-infection (dpi). We found most transcription occurred in viral vesicles, not in initial tissues infected, a remarkably novel reproduction mechanism compared to all other viruses and most other intracellular pathogens. Specifically, the highest level of viral gene expression occurred in hemolymph, for TnAV at 7 dpi, and SfAV at 14 dpi. Moreover, we found that host immune genes were partially down-regulated in hemolymph, where most viral replication occurred in highly dense accumulations of vesicles.


Assuntos
Ascoviridae/genética , Hemolinfa/virologia , Transcriptoma/genética , Tropismo/genética , Animais , Vírus de DNA/genética , DNA Viral/genética , Genoma Viral/genética , Fases de Leitura Aberta/genética , Reprodução/genética , Análise de Sequência de DNA/métodos , Spodoptera/genética , Vírion/genética , Replicação Viral/genética
2.
Virol Sin ; 36(5): 1036-1051, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-33830433

RESUMO

3h-31 of Heliothis virescens ascovirus 3h (HvAV-3h) is a highly conserved gene of ascoviruses. As an early gene of HvAV-3h, 3h-31 codes for a non-structural protein (3H-31) of HvAV-3h. In the study, 3h-31 was initially transcribed and expressed at 3 h post-infection (hpi) in the infected Spodoptera exigua fat body cells (SeFB). 3h-31 was further inserted into the bacmid of Autographa californica nucleopolyhedrovirus (AcMNPV) to generate an infectious baculovirus (AcMNPV-31). In vivo experiments showed that budded virus production and viral DNA replication decreased with the expression of 3H-31, and lucent tubular structures were found around the virogenic stroma in the AcMNPV-31-infected SeFB cells. In vivo, both LD50 and LD90 values of AcMNPV-31 were significantly higher than those of the wild-type AcMNPV (AcMNPV-wt) in third instar S. exigua larvae. An interesting finding was that the liquefaction of the larvae killed by the infection of AcMNPV-31 was delayed. Chitinase and cathepsin activities of AcMNPV-31-infected larvae were significantly lower than those of AcMNPV-wt-infected larvae. The possible regulatory function of the chitinase and cathepsin for 3H-31 was further confirmed by RNAi, which showed that larval cathepsin activity was significantly upregulated, but chitinase activity was not significantly changed due to the RNAi of 3h-31. Based on the obtained results, we assumed that the function of 3H-31 was associated with the inhibition of host larval chitinase and cathepsin activities, so as to restrain the hosts in their larval stages.


Assuntos
Ascoviridae , Quitinases , Animais , Ascoviridae/genética , Catepsinas/genética , Quitinases/genética , Replicação do DNA , DNA Viral , Larva , Spodoptera , Replicação Viral
3.
Insect Sci ; 28(2): 472-484, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32243720

RESUMO

Melanization is an important innate immune defense mechanism of insects, which can kill invading pathogens. Most pathogens, for their survival and reproduction, inhibit the melanization of the host. Interestingly, our results suggested that after infection with Heliothis virescens ascovirus 3h (HvAV-3h), the speed of melanization in infected Spodoptera exigua larval hemolymph was accelerated and that the phenoloxidase (PO) activity of hemolymph in larvae infected with HvAV-3h increased significantly (1.20-fold at 96 hpi, 1.52-fold at 120 hpi, 1.23-fold at 144 hpi, 1.12-fold at 168 hpi). The transcription level of the gene encoding S. exigua prophenoloxidase-1 (SePPO-1 gene) was upregulated dramatically in the fat body during the middle stage of infection. In addition, when melanization was inhibited or promoted, the replication of HvAV-3h was inhibited or promoted, respectively. In conclusion, infection with HvAV-3h can markedly induce melanization in the middle stage of infection, and melanization is helpful for HvAV-3h viral replication.


Assuntos
Ascoviridae/fisiologia , Mariposas/imunologia , Replicação Viral , Animais , Larva/crescimento & desenvolvimento , Larva/imunologia , Larva/virologia , Mariposas/crescimento & desenvolvimento , Mariposas/virologia
4.
J Virol ; 94(9)2020 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-32075926

RESUMO

Ascoviruses are large, enveloped DNA viruses that induce remarkable changes in cellular architecture during which the cell is partitioned into numerous vesicles for viral replication. Previous studies have shown that these vesicles arise from a process resembling apoptosis yet which differs after nuclear lysis in that mitochondria are not degraded but are modified by the virus, changing in size, shape, and motility. Moreover, infection does not provoke an obvious innate immune response. Thus, we used in vivo RNA sequencing to determine whether infection by the Spodoptera frugiperda ascovirus 1a (SfAV-1a) modified expression of host mitochondrial, cytoskeletal, and innate immunity genes. We show that transcripts from many mitochondrial genes were similar to those from uninfected controls, whereas others increased slightly during vesicle formation, including those for ATP6, ATP8 synthase, and NADH dehydrogenase subunits, supporting electron microscopy (EM) data that these organelles were conserved for virus replication. Transcripts from 58 of 106 cytoskeletal genes studied increased or decreased more than 2-fold postinfection. More than half coded for mitochondrial motor proteins. Similar increases occurred for innate immunity transcripts and their negative regulators, including those for Toll, melanization, and phagocytosis pathways. However, those for many antimicrobial peptides, such as moricin, increased more than 20-fold. In addition, transcripts for gloverin-3, spod_x_tox, Hdd23, and lebocin, also antimicrobial, increased more than 20-fold. Interestingly, a phenoloxidase inhibitor transcript increased 12-fold, apparently to interfere with melanization. SfAV-1a destroys most fat body cells by 7 days postinfection, so innate immunity gene transcripts apparently occur in remaining cells in this tissue and possibly other major tissues, namely, epidermis and tracheal matrix.IMPORTANCE Ascoviruses are large DNA viruses that infect insects, inducing a cellular pathology that resembles apoptosis but which differs by causing enormous cellular hypertrophy followed by cleavage of the cell into numerous viral vesicles for replication. Previous EM studies suggest that mitochondria are important for vesicle formation. Transcriptome analyses of Spodoptera frugiperda larvae infected with SfAV-1a showed that mitochondrial transcripts were similar to those from uninfected controls or increased slightly during vesicle formation, especially for ATP6, ATP8 synthase, and NADH dehydrogenase subunits. This pattern resembles that for chronic disease-inducing viruses, which conserve mitochondria, differing markedly from viruses causing short-term viral diseases, which degrade mitochondrial DNA. Though mitochondrial transcript increases were low, our results demonstrate that SfAV-1a alters host mitochondrial expression more than any other virus. Regarding innate immunity, although SfAV-1a destroys most fat body cells, certain immunity genes were highly upregulated (greater than 20-fold), suggesting that these transcripts may originate from other tissues.


Assuntos
Ascoviridae/genética , Mitocôndrias/genética , Replicação Viral/genética , Animais , Ascoviridae/metabolismo , Perfilação da Expressão Gênica , Imunidade Inata/genética , Larva/virologia , Mitocôndrias/metabolismo , Análise de Sequência de RNA , Spodoptera/genética , Spodoptera/metabolismo , Transcriptoma , Proteínas Virais/genética , Replicação Viral/fisiologia
5.
Pest Manag Sci ; 76(3): 1048-1059, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31515935

RESUMO

BACKGROUND: Calcineurin (CaN) is involved in numerous cellular processes and Ca2+ -dependent signal transduction pathways. According to our previous transcriptome studies, thousands of host larval (Spodoptera exigua) transcripts were downregulated after the infection of Heliothis virescent ascovirus 3h (HvAV-3h), while the Spodoptera exigua calcineurin genes (SeCaNs) were significantly upregulated. To understand the regulation of SeCaNs in S. exigua larvae during the infection of HvAV-3h, the functions of CaN subunit A (SeCaN-SubA) and CaN binding protein (SeCaN-BP) were analysed. RESULTS: The in vitro assays indicated that the bacterial expressed SeCaN-SubA is an acid phosphatase, but no phosphatase activity was detected with the purified SeCaN-BP. The transcription level of SeCaN-SubA was upregulated after HvAV-3h infection and the CaN activity was significantly increased after HvAV-3h infection in S. exigua larvae. Interestingly, the SeCaN-BP transcripts were only detectable in the HvAV-3h infected larvae. Further immunoblotting results consistently agree with those obtained by qPCR, indicating that the infection of HvAV-3h causes the upregulated expression of SeCaN-SubA and the appearance of SeCaN-BP. An interaction between the cleaved SeCaN-SubA and SeCaN-BP was detected by co-immunoprecipitation assays, and the expression of SeCaN-BP in Spodoptera frugiperda-9 (Sf9) cells can help to increase the CaN activity of SeCaN-SubA. Further investigations with CaN inhibitors suggested that HvAV-3h. Further investigations with CaN inhibitors suggested that the inhibition on host larval CaN activity can also inhibit the viral replication of HvAV-3h. CONCLUSION: The increase in CaN activity caused by HvAV-3h infection might be due to the upregulation of SeCaN-SubA and the induced expression of SeCaN-BP, and increased CaN activity is essential for ascoviral replication. © 2019 Society of Chemical Industry.


Assuntos
Ascoviridae , Animais , Calcineurina , Larva , Spodoptera
6.
Virol Sin ; 35(2): 134-142, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31784872

RESUMO

So far, ascoviruses have only been identified from Lepidoptera host insects and their transmission vectors-endoparasitic wasps. Here, we reported the first finding of a complete novel ascovirus genome from a Diptera insect, Dasineura jujubifolia. Initially, sequence fragments with homology to ascoviruses were incidentally identified during metagenomic sequencing of the mitochondria of D. jujubifolia (Cecidomyiidae, Diptera) which is a major pest on Ziziphus jujuba. Then a full circular viral genome was assembled from the metagenomic data, which has an A+T percentage of 74% and contains 142,600 bp with 141 open reading frames (ORFs). Among the 141 ORFs, 37 were conserved in all sequenced ascoviruses (core genes) including proteins predicted to participate in DNA replication, gene transcription, protein modification, virus assembly, lipid metabolism and apoptosis. Multi-gene families including those encode for baculovirus repeated open reading frames (BROs), myristylated membrane proteins, RING/U-box E3 ubiquitin ligases, and ATP-binding cassette (ABC) transporters were found in the virus genome. Phylogenetic analysis showed that the newly identified virus belongs to genus Toursvirus of Ascoviridae, and is therefore named as Dasineura jujubifolia toursvirus 2 (DjTV-2a). The virus becomes the second reported species of the genus after Diadromus pulchellus toursvirus 1 (DpTV-1a). The genome arrangement of DjTV-2a is quite different from that of DpTV-1a, suggesting these two viruses separated in an early time of evolution. The results suggest that the ascoviruses may infect a much broader range of hosts than our previous knowledge, and shed lights on the evolution of ascoviruses and particularly on that of the toursviruses.


Assuntos
Ascoviridae/genética , Dípteros/virologia , Genoma Viral , Fases de Leitura Aberta , Filogenia , Animais , Ascoviridae/classificação , DNA Viral/genética , Metagenômica , Mitocôndrias/genética , Replicação Viral
7.
Virus Genes ; 55(5): 688-695, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31236766

RESUMO

The open reading frame 117 (3h-117) of Heliothis virescens ascovirus 3h (HvAV-3h), which is a conserved coding region present in all completely sequenced ascovirus members, was characterized in this study. By RT-PCR detection, 3h-117 transcription began at 6-h post-infection (hpi) and remained stable until 168 hpi in HvAV-3h-infected Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) larvae. In addition, 3h-117 putatively encodes a 21.5-kDa protein (3H-117) predicted to be a CTD-like phosphatase. Western blot analysis using a prepared rabbit polyclonal antibody specific to 3H-117 showed that the product could be detected at 24 hpi, which remained stably detectable until 168 hpi. The same analysis also demonstrated that the 3H-117 protein localized in the virions of HvAV-3h. Immunofluorescence analysis showed that at 24 hpi, 3H-117 was mainly located in the nuclei of H. armigera larval fat body cells and later spread into the cytoplasm. In summary, our results indicate that 3H-117 is a structural protein of HvAV-3h.


Assuntos
Ascoviridae/crescimento & desenvolvimento , Lepidópteros/virologia , Transcrição Genética , Proteínas Estruturais Virais/biossíntese , Animais , Ascoviridae/química , Ascoviridae/genética , Western Blotting , Perfilação da Expressão Gênica , Larva/virologia , Coelhos , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Estruturais Virais/genética , Vírion/química
8.
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
9.
J Gen Virol ; 100(2): 301-307, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30540243

RESUMO

Ascoviruses are enveloped, circular, double-stranded DNA viruses that can effectively control the appetite of lepidopteran larvae, thereby reducing the consequent damage and economic losses to crops. In this study, the virion of a sequenced Heliothis virescens ascovirus 3i (HvAV-3i) strain was used to perform proteomic analysis using both in-gel and in-solution digestion. A total of 81 viral proteins, of which 67 were associated with the virions, were identified in the proteome of HvAV-3i virions. Among these proteins, 23 with annotated functions were associated with DNA/RNA metabolism/transcription, virion assembly, sugar and lipid metabolism, signalling, cellular homoeostasis and cell lysis. Twenty-one viral membrane proteins were also identified. Some of the minor 'virion' proteins identified may be non-virion contaminants of viral proteins synthesized during replication, identified by more recent and highly sensitive methods. The extensive identification of the ascoviral proteome will establish a foundation for further investigation of ascoviral replication and infection.


Assuntos
Ascoviridae/química , Proteoma/análise , Proteínas Virais/análise , Vírion/química , Biologia Computacional , Proteômica
10.
Arch Virol ; 163(10): 2849-2853, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-29948385

RESUMO

Ascoviruses are circular double-stranded DNA viruses that infect insects. Herein we sequenced and analyzed the genome of the previously unrecorded ascovirus isolate Heliothis virescens ascovirus 3i (HvAV-3i). The genome size is 185,650 bp with 181 hypothetical open reading frames (ORFs). Additionally, definition based on ascovirus repeated ORFs (aros) is proposed; whereby the 29 aros from all sequenced Ascoviridae genomes are divided into six distinct groups. The topological relationship among the isolates of Heliothis virescens ascovirus 3a is (HvAV-3f, {HvAV-3h, [HvAV-3e, (HvAV-3g, HvAV-3i)]}) with every clade well supported by a Bayesian posterior probability of 1.00 and a Bootstrap value of 100%.


Assuntos
Ascoviridae/genética , Ascoviridae/isolamento & purificação , Fases de Leitura Aberta , Spodoptera/virologia , Animais , Ascoviridae/classificação , Genoma Viral , Genômica , Hemolinfa/virologia , Larva/virologia , Filogenia
11.
J Invertebr Pathol ; 155: 55-63, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29778741

RESUMO

Spodoptera exigua (Lepidoptera: Noctuidae) larvae were used to analyze the regulation of lipid, protein and carbohydrate metabolism in host larvae infected with the Heliothis virescens ascovirus 3h (HvAV-3h). Using histological sections, significant pathological changes were found in the fat bodies of infected larvae from 24 h to 72 h post-infection (hpi). The lipid and protein contents of the infected larvae were significantly higher than those of the uninfected larvae, while the carbohydrate content of the infected larvae was significantly lower than that of the mock-infected larvae. The selected primary metabolite metabolism-associated genes showed different expression patterns. Further co-relationship analysis of the gene expression level and content changes of primary metabolites indicated the following: the acyl-CoA dehydrogenase and fatty acid synthase genes were closely associated with lipid metabolism, and the hexokinase and the glycogen synthase gene expression levels were related to carbohydrate metabolism, while the aminopeptidase N and the protein disulfide isomerase gene expression levels were not correlated with protein metabolism. These results indicate that the HvAV-3h virus stimulates host larval lipid and protein syntheses and inhibits carbohydrate synthesis.


Assuntos
Ascoviridae , Metabolismo dos Carboidratos , Metabolismo dos Lipídeos , Proteínas/metabolismo , Spodoptera/virologia , Animais , Spodoptera/metabolismo
12.
J Gen Virol ; 99(4): 574-584, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29517480

RESUMO

The family Ascoviridae is a recently described virus family whose members are transmitted by parasitoids and cause chronic and lethal infections in lepidopteran insects. Little is known about the biology and ecology of ascoviruses, and few isolates have been found outside the United States. We report here the isolation of a new ascovirus variant from Spodoptera litura in Japan. Full genome sequence and phylogenetic analyses showed that this virus was closely related to variants in Heliothis virescens ascovirus-3a, and it was named HvAV-3j. HvAV-3j has a DNA genome of 191 718 bp, with 189 putative ORFs and a GC content of 45.6 %, and is highly similar to HvAV-3h, which was isolated in China. In a field survey, the endoparasitoid Meteorus pulchricornis caused a high percentage of parasitization in populations of S. litura larvae, and under laboratory conditions M. pulchricornis was able to transmit HvAV-3j from infected to uninfected larvae by oviposition. Meteorus pulchricornis is thus likely to be a major vector for HvAV-3j transmission in Japan. This species is recognized here for the first time as a vector of ascoviruses that parasitizes a range of host species that extends across families.


Assuntos
Ascoviridae/isolamento & purificação , Mariposas/virologia , Spodoptera/virologia , Vespas/virologia , Animais , Ascoviridae/classificação , Ascoviridae/genética , Ascoviridae/fisiologia , Composição de Bases , Feminino , Japão , Larva/virologia , Masculino , Mariposas/parasitologia , Fases de Leitura Aberta , Filogenia , Vespas/fisiologia
13.
Sci Rep ; 8(1): 5367, 2018 03 29.
Artigo em Inglês | MEDLINE | ID: mdl-29599494

RESUMO

Heliothis virescens ascovirus 3 h (HvAV-3h), a dsDNA insect virus, belonging to the family Ascoviridae, can infect caterpillars of several Noctuidae species by ovipositing parasitoid wasps. In order to provide a comprehensive overview of the interactive responses of host larvae after infection by the ascovirus, a transcriptome analysis of Spodoptera exigua to HvAV-3h was conducted from 6 to 168 hours post infection (hpi). Approximately 101.64 Gb of RNA sequencing (RNA-seq) data obtained from infected and uninfected S. exigua larvae were used to perform a de novo transcriptome assembly, which generated approximately 62,258 S. exigua unigenes. Using differential gene expression analysis, it was determined that the majority of host transcripts were down-regulated beginning at 6 hpi and continuing throughout the infection period, although there was an increase in up-regulated unigene number during the 12 to 72 hpi stage. It is noteworthy that the most abundantly enriched pathways in KEGG annotation were Metabolism terms, indicating that the host larval metabolic mechanisms were highly influenced post HvAV-3h infection. In addition, the host cuticle protein encoding unigenes were highly down-regulated in most of the situations, suggesting that the host larval cuticle synthesis were inhibited by the viral infection.


Assuntos
Ascoviridae/genética , Interações entre Hospedeiro e Microrganismos , Spodoptera/genética , Spodoptera/virologia , Animais , Sequência de Bases/genética , Larva/genética , Larva/virologia , Análise de Sequência de RNA/métodos , Transcriptoma
14.
J Virol Methods ; 255: 101-106, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29454017

RESUMO

Ascoviruses are a recently described family and the traditional plaque assay and end-point PCR assay have been used for their titration. However, these two methods are time-consuming and inaccurate to titrate ascoviruses. In the present study, a quick method for the determination of the titer of ascovirus stocks was developed based on ascovirus-induced apoptosis in infected insect cells. Briefly, cells infected with serial dilutions of virus (10-2-10-10) for 24 h were stained with trypan blue. The stained cells were counted, and the percentage of nonviable cells was calculated. The stained cell rate was compared between virus-infected and control cells. The minimum-dilution group that had a significant difference compared with control and the maximum-dilution group that had no significant difference were selected and then compared each well of the two groups with the average stained cell rate of control. The well was marked as positive well if the stained cell rate was higher than the average stained cell rate of control wells; otherwise, the well was marked as negative wells. The percentage of positive wells were calculated according to the number of positive. Subsequently, the virus titer was calculated through the method of Reed and Muench. This novel method is rapid, simple, reproducible, accurate, and less material-consuming and eliminates the subjectivity of the other procedures for titrating ascoviruses.


Assuntos
Ascoviridae/genética , Infecções por Vírus de DNA/virologia , DNA Viral , Carga Viral/métodos , Animais , Linhagem Celular , Insetos
15.
J Virol ; 91(23)2017 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-28956762

RESUMO

Ascoviruses are double-stranded DNA (dsDNA) viruses that attack caterpillars and differ from all other viruses by inducing nuclear lysis followed by cleavage of host cells into numerous anucleate vesicles in which virus replication continues as these grow in the blood. Ascoviruses are also unusual in that most encode a caspase or caspase-like proteins. A robust cell line to study the novel molecular biology of ascovirus replication in vitro is lacking. Therefore, we used strand-specific transcriptome sequencing (RNA-Seq) to study transcription in vivo in third instars of Spodoptera frugiperda infected with the type species, Spodoptera frugiperda ascovirus 1a (SfAV-1a), sampling transcripts at different time points after infection. We targeted transcription of two types of SfAV-1a genes; first, 44 core genes that occur in several ascovirus species, and second, 26 genes predicted in silico to have metabolic functions likely involved in synthesizing viral vesicle membranes. Gene cluster analysis showed differences in temporal expression of SfAV-1a genes, enabling their assignment to three temporal classes: early, late, and very late. Inhibitors of apoptosis (IAP-like proteins; ORF016, ORF025, and ORF074) were expressed early, whereas its caspase (ORF073) was expressed very late, which correlated with apoptotic events leading to viral vesicle formation. Expression analysis revealed that a Diedel gene homolog (ORF121), the only known "virokine," was highly expressed, implying that this ascovirus protein helps evade innate host immunity. Lastly, single-nucleotide resolution of RNA-Seq data revealed 15 bicistronic and tricistronic messages along the genome, an unusual occurrence for large dsDNA viruses.IMPORTANCE Unlike all other DNA viruses, ascoviruses code for an executioner caspase, apparently involved in a novel cytopathology in which viral replication induces nuclear lysis followed by cell cleavage, yielding numerous large anucleate viral vesicles that continue to produce virions. Our transcriptome analysis of genome expression in vivo by the Spodoptera frugiperda ascovirus shows that inhibitors of apoptosis are expressed first, enabling viral replication to proceed, after which the SfAV-1a caspase is synthesized, leading to viral vesicle synthesis and subsequent extensive production of progeny virions. Moreover, we detected numerous bicistronic and tricistronic mRNA messages in the ascovirus transcriptome, implying that ascoviruses use other noncanonical translational mechanisms, such as internal ribosome entry sites (IRESs). These results provide the first insights into the molecular biology of a unique coordinated gene expression pattern in which cell architecture is markedly modified, more than in any other known eukaryotic virus, to promote viral reproduction and transmission.


Assuntos
Ascoviridae/patogenicidade , Perfilação da Expressão Gênica/métodos , Spodoptera/virologia , Proteínas Virais/genética , Animais , Ascoviridae/genética , Caspases/genética , Regulação Viral da Expressão Gênica , Proteínas Inibidoras de Apoptose , Família Multigênica , Análise de Sequência de RNA/métodos , Vírion/genética , Replicação Viral
16.
Sci Rep ; 7(1): 7045, 2017 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-28765578

RESUMO

Ascoviruses are double-stranded DNA viruses that mainly infect noctuid larvae, and are transmitted by the parasitoid wasp Microplitis similis Lyle. Ascovirus-parasitoids wasp-noctuid insects constitute the dissemination system. Selection of suitable reference genes for the dissemination system could play an important role in elucidating the pathogenic molecular mechanisms of ascovirus. Unfortunately, such studies on potential reference genes in the dissemination system of ascoviruses are lacking. In the present study, we evaluated 11 candidate reference genes: ß-actin1 (ACT1), ß-actin2 (ACT2), elongation factor 1 (EF1), elongation factor 2 (EF2), ribosomal protein L10 (L10), ribosomal protein L17A (L17A), superoxide dismutase (SOD), 28S ribosome (28S), Tubulin (TUB) and 18S ribosome (18S). The samples were originally from various virus concentrations and points-in-time of experimental treatments using RefFinder and four algorithms. The results showed that EF1 was the most stable internal gene in S. exigua and M. similis and that EF2 was the most stable in the IOZCAS-Spex-II-A cell line, and the stability of reference genes were confirmed via the expression levels of two inhibitor of apoptosis-like (iap-like) genes from Heliothis virescens ascovirus 3 h (HvAV-3h). This study provides a crucial basis for future research that explores the molecular mechanisms of the pathogenesis of ascoviruses.


Assuntos
Ascoviridae/genética , Perfilação da Expressão Gênica/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Padrões de Referência , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Animais , Perfilação da Expressão Gênica/normas , Himenópteros/virologia , Lepidópteros/virologia , Reação em Cadeia da Polimerase em Tempo Real/normas , Reação em Cadeia da Polimerase Via Transcriptase Reversa/normas
17.
Virol Sin ; 32(2): 147-154, 2017 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-28382574

RESUMO

No ascovirus isolated from China has been sequenced so far. Therefore, in this study, we aimed to sequence the genome of Heliothis virescens ascovirus 3h (HvAV-3h) using the 454 pyrosequencing technology. The genome was found to be 190,519-bp long with a G+C content of 45.5%. We also found that it encodes 185 hypothetical open reading frames (ORFs) along with at least 50 amino acids, including 181 ORFs found in other ascoviruses and 4 unique ORFs. Gene-parity plots and phylogenetic analysis revealed a close relationship between HvAV-3h and three other HvAV-3a strains and a distant relationship with Spodoptera frugiperda ascovirus 1a (SfAV-1a), Trichoplusia ni ascovirus 6a (TnAV-6a), and Diadromus pulchellus ascovirus 4a (DpAV-4a). Among the 185 potential genes encoded by the genome, 44 core genes were found in all the sequenced ascoviruses. In addition, 25 genes were found to be conserved in all ascoviruses except DpAV-4a. In the HvAV-3h genome, 24 baculovirus repeat ORFs (bros) were present, and the typical homologous repeat regions (hrs) were absent. This study supplies information important for understanding the conservation and functions of ascovirus genes as well as the variety of ascoviral genomes.


Assuntos
Ascoviridae/genética , Ascoviridae/isolamento & purificação , Lepidópteros/virologia , Animais , Composição de Bases , China , Análise por Conglomerados , DNA Viral/química , DNA Viral/genética , Ordem dos Genes , Genoma Viral , Fases de Leitura Aberta , Filogenia , Análise de Sequência de DNA , Homologia de Sequência
18.
J Gen Virol ; 98(1): 4-5, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-28218573

RESUMO

The family Ascoviridae includes viruses with circular dsDNA genomes of 100-200 kbp characterized by oblong enveloped virions of 200-400 nm in length. Ascoviruses mainly infect lepidopteran larvae and are mechanically transmitted by parasitoid wasps in which they may also replicate. Most known members belong to the genus Ascovirus, except one virus, that of the genus Toursvirus, which replicates in both its lepidopteran and parasitoid vector hosts. Ascoviruses cause high mortality among economically important insect pests, thereby controlling insect populations. This is a summary of the current International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of the Ascoviridae, which is available at www.ictv.global/report/ascoviridae.


Assuntos
Ascoviridae/classificação , Animais , Ascoviridae/genética , Ascoviridae/fisiologia , Ascoviridae/ultraestrutura , Insetos/virologia , Larva/virologia
19.
J Econ Entomol ; 109(5): 2020-6, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27551150

RESUMO

Spodoptera exigua (Hübner) is a very serious worldwide pest capable of causing severe economic losses in numerous agricultural crops. The need for an effective, highly virulent, pathogenic microorganism for use as a biological control agent against S. exigua larvae is particularly important. Heliothis virescens ascovirus 3 h (HvAV-3h)-containing hemolymph with a titer of 9.58 × 10(12) genome copies per ml was used to inoculate S. exigua larvae per os with a 1.06 × 10(10) dosage per larva for the first- to second instar and 9.58 × 10(9) genome copies per larva for the third- to fifth instars. Intrahemocoelic injections were also used with a dosage of 1.53 × 10(9) genome copies per larva for third- to fifth instar. The postinjection mortality, body weight, and food intake of the S. exigua larvae were observed and recorded. The corrected mortality rates for the first- through fifth instar inoculated per os were 21.88 ± 0.98, 22.22 ± 4.00, 8.89 ± 4.01, 6.66 ± 3.33, and 8.89 ± 2.94%, respectively. The early instars were significantly easier to infect with virus compared to the later instar. The corrected mortality of the third, fourth, and fifth instars inoculated by injection was 96.58 ± 3.42, 98.83 ± 1.17, and 97.78 ± 2.22%, respectively. Compared to the healthy larval population, survival time of the diseased larval population was considerably extended. In addition, food intake was greatly reduced, and the body weight remained fairly constant in the third- and fourth instar. The body weight declined in the fifth instar corresponding to a reduction in food intake.


Assuntos
Ascoviridae/fisiologia , Agentes de Controle Biológico/farmacologia , Spodoptera/crescimento & desenvolvimento , Spodoptera/virologia , Animais , Larva/crescimento & desenvolvimento , Larva/virologia , Controle Biológico de Vetores
20.
Sci Rep ; 6: 21296, 2016 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-26878829

RESUMO

Ascoviruses are insect-specific large DNA viruses that mainly infect noctuid larvae, and are transmitted by parasitoids in the fields. Heliothis virescens ascovirus 3h (HvAV-3h) has been recently isolated from Spodoptera exigua, without parasitoid vector identified previously. Here we report that Microplitis similis, a solitary endoparasitoid wasp, could transmit HvAV-3h between S. exigua larvae in the laboratory. When the female parasitoid wasp acquired the virus and served as a vector, the period of virion viability on the ovipositor was 4.1 ± 1.4 days. Infected host larvae were still acceptable for egg laying by parasitoids, and the parasitoids thereafter transmitted virus to healthy hosts. Virus acquisition occurred only from donor hosts between 3 and 9 days post infection. The peak of virus acquisition (80.9 ± 6.3%) was found when M. similis wasps oviposited in larvae that had been inoculated with the virus 7 days previously. When virus infection of the host took place during the life cycle of the parasitoid wasp, it caused 1- to 4-day-old immature parasitoids death in the host, whilst a small proportion of 5- to 6-day-old and the majority of 7-day-old parasitoids larvae survived from the virus-infected hosts. Viral contamination did not reduce the life span or fecundity of female M. similis.


Assuntos
Ascoviridae/fisiologia , Spodoptera/parasitologia , Spodoptera/virologia , Viroses/transmissão , Vespas/parasitologia , Vespas/virologia , Animais , Feminino , Interações Hospedeiro-Parasita , Insetos Vetores , Larva/parasitologia , Larva/virologia , Estágios do Ciclo de Vida , Masculino , Oviposição , Temperatura
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