Flexible changes to the Heliothis virescens ascovirus 3h (HvAV-3h) virion components affect pathogenicity against different host larvae species.

IMPORTANCE: Different pathogenic processes of a virus in different hosts are related to the host individual differences, which makes the virus undergoes different survival pressures. Here, we found that the virions of an insect virus, Heliothis virescens ascovirus 3h (HvAV-3h), had different protein composition when they were purified from different host larval species. These "adaptive changes" of the virions were analyzed in detail in this study, which mainly included the differences of the protein composition of virions and the differences in affinity between virions and different host proteins. The results of this study revealed the flexible changes of viruses to help themselves adapt to different hosts. Also, these interesting findings can provide new insights to improve our understanding of virus adaptability and virulence differentiation caused by the adaptation process.

Enhanced virulence of genetically engineered Autographa californica nucleopolyhedrovirus owing to accelerated viral DNA replication aided by inserted ascovirus genes.

Genetic engineering technology is an ideal method to improve insecticidal efficiency by combining the advantages of different pathogenic microorganisms. Thus, six ascovirus genes were introduced into the genomic DNA of Autographa californica nucleopolyhedrovirus (AcMNPV) to possibly transfer the intrinsically valuable insecticidal properties from ascovirus to baculovirus. The viral budded virus (BV) production and viral DNA replication ability of AcMNPV-111 and AcMNPV-165 were significantly stronger than that of AcMNPV-Egfp (used as the wild-type virus in this study), whereas AcMNPV-33 had reduced ones. AcMNPV-111 and AcMNPV-165 also exhibited excellent insecticidal efficiency in the in vivo bioassays: AcMNPV-111 showed a 24.1% decrease in the LT50 value and AcMNPV-165 exhibited a 56.3% decrease in the LD50 value compared with AcMNPV-Egfp against the 3rd instar of Spodoptera exigua larvae, respectively. Furthermore, the size of the occlusion bodies (OBs) of AcMNPV-33, AcMNPV-111, and AcMNPV-165 were significantly increased compared to that of AcMNPV-Egfp. AcMNPV-111 and AcMNPV-165 had stable virulence against the 2nd to 4th instars tested larvae and higher OB yield than AcMNPV-Egfp in the 3rd and 4th instar larvae. Correlation and regression analyses indicated that it is better to use 5 OBs/larva virus to infect the 2nd instar larvae to produce AcMNPV-111 and 50 OBs/larva virus to infect the 3rd instar larvae to produce AcMNPV-165. The results of this study obtained recombinant viruses with enhanced virulence and exhibited a diversity of ascovirus gene function based on the baculovirus platform, which provided a novel strategy for the improvement of baculovirus as a biological insecticide.

Spodoptera litura larvae are attracted by HvAV-3h-infected S. litura larvae-damaged pepper leaves.

BACKGROUND: Herbivore-induced plant volatiles (HIPVs) are important self-defense outputs of pepper plants to resist insect pests. Ascoviruses are pathogenic to the larvae of most lepidopteran vegetable pests. However, whether Heliothis virescens ascovirus 3h (HvAV-3h)-infected Spodoptera litura larvae can change pepper leaf HIPVs is not well understood. RESULTS: Spodoptera litura larvae preferred S. litura-infested leaves, and this preference was stronger with longer duration of S. litura infestation. In addition, S. litura larvae significantly chose pepper leaves damaged by HvAV-3h-infected S. litura over the healthy pepper leaves. Results also showed that S. litura larvae preferred leaves mechanically damaged and treated with oral secretions from HvAV-3h infected-S. litura larvae in a simulation test. We captured the volatiles emitted by leaves under six treatments. Results showed that the volatile profile changed with the different treatments. Testing of volatile blends, prepared to the proportion released showed that the blend from simulated HvAV-3h-infected S. litura larvae-damaged plants was the most attractive to S. litura larvae. Further, we also found that some of the compounds significantly attracted S. litura larvae at specific concentrations. CONCLUSION: HvAV-3h-infected S. litura can alter the release of HIPVs in pepper plants and thus become more attractive to S. litura larvae. We speculate that this may be due to alterations in the concentration of some compounds (such as geranylacetone and prohydrojasmon) affecting the behavior of S. litura larvae. © 2023 Society of Chemical Industry.

Apoptosis or Antiapoptosis? Interrupted Regulated Cell Death of Host Cells by Ascovirus Infection In Vitro.

Ascoviruses are insect-specific viruses thought to utilize the cellular apoptotic processes of host larvae to produce numerous virion-containing vesicles. In this study, we first determined the biochemical characteristics of ascovirus-infected, in vitro-cultured insect cells and the possible antiapoptotic capacity of ascovirus-infected insect cells. The results indicated that the ascovirus infection in the first 24 h was different from the infection from 48 h to the later infection stages. In the early infection stage, the Spodoptera exigua host cells had high membrane permeability and cleaved gasdermin D (GSDMD) but uncleaved Casp-6 (SeCasp-6). In contrast, the later infection stage had no such increased membrane permeability and had cleaved SeCasp-6. Four different chemicals were used to induce apoptosis at different stages of ascovirus infection: hydrogen peroxide (H2O2) and actinomycin D (ActD) had similar effects on the ascovirus-infected cells, whereas cMYC inhibitors and tumor necrosis factor alpha (TNF-α) plus SM-164 apoptosis inducers (T/S) had similar effects on infected cells. The former two inducers inhibited viral DNA replication in most situations, while the latter two inducers inhibited viral DNA replication in the early stage of infection but promoted viral DNA replication in the later infection stage. Furthermore, immunoblotting assays verified that T/S treatment could increase the expression levels of viral major capsid protein (MCP) and the host inhibitor of apoptosis protein (SeIAP). Coimmunoprecipitation assays revealed interaction between SeIAP and SeCasps, but this interaction was disturbed in ascovirus-infected cells. This study details the in vitro infection process of ascovirus, indicating the utilization of pyroptosis for antiapoptosis cytopathology. IMPORTANCE Clarifying the relationship between different types of viral infections and host regulation of cell death (RCD) can provide insights into the interaction between viruses and host cells. Ascoviruses are insect-specific viruses with apoptosis-utilizing-like infection cytopathology. However, RCD does not only include apoptosis, and while in our previous transmission electron microscopic observations, ascovirus-infected cells did not show typical apoptotic characteristics (unpublished data), in this study, they did show increased membrane permeability. These results indicate that the cytopathology of ascovirus infection is a complex process in which the virus manipulates host RCD. The RCD of insect cells is quite different from that of mammals, and studies on the former are many fewer than those on the latter, especially in the case of RCD in lepidopteran insects. Our results will lay a foundation for understanding the RCD of lepidopteran insects and its function in the process of insect virus infection.

An Ascovirus Utilizes Different Types of Host Larval Regulated Cell Death Mechanisms To Produce and Release Vesicles.

Ascoviruses are insect-specific viruses that are thought to utilize the cellular apoptotic processes of host larvae to produce numerous virion-containing vesicles. In this study, we monitored the in vivo infection processes of Heliothis virescens ascovirus 3h (HvAV-3h) to illustrate the regulated cell death (RCD) of host cells. Transmission electron microscopic observations did not reveal any morphological markers of apoptosis in the fat bodies or hemocytes of HvAV-3h-infected Helicoverpa armigera or Spodoptera exigua larvae. However, several hemocytes showed the morphological criteria for necrosis and/or pyroptosis. Further in vitro biochemical tests were performed to confirm the RCD type of host cells after infection with HvAV-3h. Different morphological characteristics were found between the early (prior to 24 hours post-infection, [hpi]) and later (48 to 120 hpi) stages in both HvAV-3h infected larval fat bodies and hemocytes. In the early stages, the virions could only be found in several adipohemocytes, and the fat bodies were cleaving their contained lipid inclusions into small lipid dots. In the later stage, both fat bodies and hemocytes were filled with numerous virions. According to the morphological characteristics of HvAV-3h infected larval fat bodies or hemocytes, the pathogenic characteristics and infection patterns of HvAV-3h in the host larvae were described, and the systematic pathogenic mode of ascovirus infection was refined in this study. This study details the complete infection process of ascoviruses, which provides insights into the relationship between a pathogenesis of an insect virus and the RCD of different host tissues at different stages of infection. IMPORTANCE Viruses and other pathogens can interrupt host cellular apoptosis to gain benefits, such as sufficient resources and a stable environment that enables them to complete their replication and assembly. It is unusual for viruses to code proteins with homology to caspases, which are commonly recognized as apoptosis regulators. Ascoviruses are insect viruses with special cytopathology, and they have been hypothesized to induce apoptosis in their host larvae via coding a caspase-like protein. This enables them to utilize the process of cellular apoptosis to facilitate vesicle formation and replication. However, our previous studies revealed different trends. The fat bodies and hemocytes of Heliothis virescens ascovirus 3h (HvAV-3h)-infected larvae did not show any morphological markers of apoptosis but did display necrosis and/or pyroptosis morphological characteristics. The pathogenic characteristics and infection patterns of HvAV-3h in the host larvae were described, which can help us understand the relationship between the pathogenesis of an insect virus and host RCD.

Insight into the regulation of the Nrf2 pathway in response to ascovirus infection in Spodoptera exigua.

BACKGROUND: Ascoviruses are a type of entomopathogenic microorganism with high biological pest control potential and are expected to contribute to the natural control of lepidopteran pests. However, knowledge of the molecular mechanism underlying the biocidal activity of ascovirus on its host insects remains limited. RESULTS: In this study, the relative enzyme activity of superoxide dismutase and peroxidase, as well as the expression level of Spodoptera exigua peroxidase (SePOD), were found to be significantly increased at 6 h post infection with Heliothis virescens ascovirus 3h (HvAV-3h). H2 O2 accumulation and enhanced expression of NADPH Oxidase (SeNOX) were also observed. In addition, Nuclear Factor erythroid 2-Related Factor 2 (SeNrf2) and muscle aponeurosis fibromatosis (SeMaf) were overexpressed following infection with HvAV-3h. Silencing of SeNrf2 decreased the expression of SePOD, whereas the mortality of SeNrf2-silenced larvae and viral genome copy number also increased. Further RNA interference of SeNOX significantly decreased expression of SeNrf2 and SePOD and therefore increased the mortality and viral genome copy number of the ascovirus-infected host. CONCLUSION: The HvAV-3h activated Nrf2/ARE pathway of S. exigua and reactive oxygen species were found to respond to ascovirus infection by regulating alterations in antioxidant enzyme genes mediated by the host Nrf2/ARE pathway. These findings enhance our knowledge of ascovirus-host interactions and lay the foundation for the application of ascoviruses in biological pest control. © 2022 Society of Chemical Industry.

Host Cytoskeleton Gene Expression Is Correlated with the Formation of Ascovirus Reproductive Viral Vesicles.

Ascoviruses are large DNA viruses that primarily infect lepidopteran larvae. They differ markedly from other plant or animal viruses by initiating replication in the nucleus, then inducing nuclear lysis followed by extensive cellular hypertrophy and subsequent cleavage of the entire enlarged cell into numerous viral vesicles. Most progeny virions are assembled in these vesicles as they circulate in the hemolymph. Here, we report transcriptome studies of host cytoskeletal genes in larvae infected with ascoviruses from 6 h to 21 days post-infection (dpi). We focused on the cabbage looper, Trichoplusia ni, infected with the Trichoplusia ni ascovirus (TnAV), along with supporting studies on the fall armyworm, Spodoptera frugiperda, infected with the Spodoptera frugiperda ascovirus (SfAV). In T. ni, many cytoskeleton genes were upregulated at 48 hours post-infection (hpi), including 29 tubulins, 21 actins, 21 dyneins, and 13 kinesins. Mitochondrial genes were upregulated as much as two-fold at 48 hpi and were expressed at levels comparable to controls in both T. ni and S. frugiperda, even after 21 dpi, when several cytoskeleton genes remained upregulated. Our studies suggest a temporal correlation between increases in the expression of certain host cytoskeletal genes and viral vesicle formation. However, these results need confirmation through functional genetic studies of proteins encoded by these genes.

Early in vivo transcriptome of Trichoplusia ni ascovirus core genes.

Ascoviruses are large double-stranded DNA insect viruses that destroy the nucleus and transform each cell into 20 or more viral vesicles for replication. In the present study we used RNA-sequencing to compare the expression of Trichoplusia ni ascovirus 6a1 (TnAV-6a1) core genes during the first week of infection, with emphasis on the first 48 h, comparing transcript levels in major somatic tissues (epidermis, tracheal matrix and fat body), the sites infected initially, with those of the haemolymph, where viral vesicles circulate and most replication occurs. By 48 h post-infection (p.i.), only 26 genes were expressed in somatic tissues at ≥5 log2 reads per kilobase per million, whereas in the haemolymph 48 genes were expressed at a similar level by the same time. Early and high expression of TnAV caspase-2-like gene occurred in all tissues, implying it is required for replication, but that it is probably not associated with apoptosis induction, which occurs in infections of Spodoptera frugiperda ascovirus 1 a (SfAV-1a), the ascovirus type species. Other highly expressed viral genes at 48 h p.i. in viral vesicles included a dynein-like beta chain and lipid-modifying enzymes, suggesting their importance to vesicle formation and growth as well as virion synthesis. Finally, as occurs in SfAV expression, we found bicistronic and tricistronic mRNA messages produced by TnAV.

Comprehensive identification of protein orthologs in the family Ascoviridae facilitates an understanding of phylogenomics, protein conservation, and phosphorylation.

Analysis of orthology is important for understanding protein conservation, function, and phylogenomics. In this study, we performed a comprehensive analysis of gene orthology in the family Ascoviridae based on identification of 366 protein homologue groups and phylogenetic analysis of 34 non-single-copy proteins. Our findings revealed 90 newly annotated proteins, five newly identified core proteins for the family Ascoviridae, and 14 core proteins for the genus Ascovirus. A phylogenomic tree of 11 Ascoviridae members was constructed based on a concatenation of 35 of the 45 ortholog groups. In combination with phosphoproteomic results and conservation estimations, 30 conserved phosphorylation sites on 17 phosphoproteins were identified from a total of 176 phosphosites on 57 phosphoproteins from Heliothis virescens ascovirus 3h (HvAV-3h), providing potential research targets for investigating the role of these protein in the regulation of viral infection. This study will facilitate genome annotation and comparison of further Ascoviridae members as well as functional genomic investigations.

Sequences Encoding a Novel Toursvirus Identified from Southern and Northern Corn Rootworms (Coleoptera: Chrysomelidae).

Sequences derived from a novel toursvirus were identified from pooled genomic short read data from U.S. populations of southern corn rootworm (SCR, Diabrotica undecimpunctata howardi Barber) and northern corn rootworm (NCR, Diabrotica barberi Smith & Lawrence). Most viral sequences were identified from the SCR genomic dataset. As proteins encoded by toursvirus sequences from SCR and NCR were almost identical, the contig sets from SCR and NCR were combined to generate 26 contigs. A total of 108,176 bp were assembled from these contigs, with 120 putative toursviral ORFs identified indicating that most of the viral genome had been recovered. These ORFs included all 40 genes that are common to members of the Ascoviridae. Two genes typically present in Ascoviridae (ATP binding cassette transport system permeases and Baculovirus repeated open reading frame), were not detected. There was evidence for transposon insertion in viral sequences at different sites in the two host species. Phylogenetic analyses based on a concatenated set of 45 translated protein sequences clustered toursviruses into a distinct clade. Based on the combined evidence, we propose taxonomic separation of toursviruses from Ascoviridae.

Comparisons of pathogenic course of two Heliothis virescens ascovirus isolates (HvAV-3i and HvAV-3j) in four noctuid (Lepidoptera) pest species.

Ascoviruses are fatal double-stranded DNA viruses with a special pathogenesis in which cells are converted into vesicles with virions. Several closely related ascovirus isolates that shared more than 90% genomic DNA identity showed different pathogenic courses in previous studies. To investigate the pathogenic differences between the related ascovirus isolates, Heliothis virescens ascovirus 3i (HvAV-3i) and Heliothis virescens ascovirus 3j (HvAV-3j) were used to inoculate four noctuid pest species (Helicoverpa armigera, Mythimna separata, Spodoptera frugiperda, and Spodoptera litura), and the pathogenic indexes were recorded. The mortality of HvAV-3i infected H. armigera and S. frugiperda was approximately 60%, while the other HvAV-infected larvae had mortality rates above 90%. The maximum lethal dilution ratios of HvAV-3i in H. armigera, M. separata, S. frugiperda, and S. litura were 1.90 × 107, 1.90 × 103, 1.90 × 108, and 1.90 × 104 viral genome DNA copies/mL, respectively, while the ratios of HvAV-3j were 8.22 × 106, 8.22 × 102, 8.22 × 105, and 8.22 × 103 viral genome DNA copies/mL, respectively. Extended larval survival time was found in the HvAV-infected larvae; median survival time of the HvAV-infected larvae ranged from 13 to 19 days. An additional larval instar was found in HvAV-infected M. separata, S. frugiperda, and S. litura. Larval growth and food intake were significantly inhibited from 2 days post-infection (dpi) in the tested H. armigera, S. frugiperda, and S. litura after infection with HvAV-3i or HvAV-3j. The detoxification enzyme activity of host larvae was influenced after infection with HvAVs, and two different regulation patterns were detected, one in infected H. armigera and M. separata and the other in S. frugiperda and S. litura. The results obtained in this study provide insights into the pathogenic characteristics of ascoviruses.

Heliothis virescens ascovirus 3h blocks the cell cycle of Spodoptera exigua fat body cells at G2 /M phase by downregulating cyclin B1 and cyclin-dependent kinase 1.

Ascoviruses are double-stranded DNA viruses that are pathogenic to noctuid larvae. In vitro infection causes the cells to fail to replicate and proliferate normally. However, the molecular mechanisms are unclear. In this study, the transmission electron microscopy data of infected-Spodoptera exigua (Hübner) fat body cells (SeFB, IOZCAS-SpexII-A cells) showed that virions were internalized in phagocytic vesicles, but not in the nucleus. FACS of cell-cycle progression was performed in SeFB cells infected with Heliothis virescens ascovirus 3h (HvAV-3h). The cell cycle phase distributions of the SeFB cells were G1 = 29.52 ± 1.10%, S = 30.33 ± 1.19%, and G2 /M = 40.06 ± 0.75%. The cell culture doubling time was approximately 24 h. The G1 , S, and G2 /M phases were each approximately 8 h. The unsynchronized or synchronized cells were arrested at G2 /M phase after infection with HvAV-3h. Our data also showed that cells with more than 4N DNA content appeared in the HvAV-3h-treated group. While the mRNA levels of cyclin B1 , cyclin H, and cyclin-dependent kinase 1 (CDK1) were downregulated after HvAV-3h infection, the mRNA expression levels of cyclin A, cyclin D, and cyclin B2 were not significantly changed. Western blotting results showed that the expression of cyclin B1 and CDK1 in infected SeFB cells within 24 h postinfection (hpi), and HvAV-3h infection inhibited the expression of cyclin B1 and CDK1 at 12-24 hpi. Overall, these data implied that HvAV-3h infection leads to an accumulation of cells in the G2 /M phases by downregulating the expression of cyclin B1 and CDK1.

Extended in vivo transcriptomes of two ascoviruses with different tissue tropisms reveal alternative mechanisms for enhancing virus reproduction in hemolymph.

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.

3H-31, A Non-structural Protein of Heliothis virescens ascovirus 3h, Inhibits the Host Larval Cathepsin and Chitinase Activities.

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.

Melanization induced by Heliothis virescens ascovirus 3h promotes viral replication.

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.

Novel strategies for the biocontrol of noctuid pests (Lepidoptera) based on improving ascovirus infectivity using Bacillus thuringiensis.

Identifying novel biocontrol agents and developing new strategies are urgent goals in insect pest biocontrol. Ascoviruses are potential competent insect viruses that may be developed into bioinsecticides, but this aim is impeded by their poor oral infectivity. To improve the per os infectivity of ascovirus, Bacillus thuringiensis kurstaki (Btk) was employed as a helper to damage the midgut of lepidopteran larvae (Helicoverpa armigera, Mythimna separata, Spodoptera frugiperda, and S. litura) in formulations with Heliothis virescens ascovirus isolates (HvAV-3h and HvAV-3j). Btk and ascovirus mixtures (Btk/HvAV-3h and Btk/HvAV-3j) were fed to insect larvae (3rd instar). With the exception of S. frugiperda larvae, which exhibited low mortality after ingesting Btk, the larvae of the other tested species showed three types of response to feeding on the formulas: type I, the tested larvae (H. armigera) were killed by Btk infection so quickly that insufficient time and resources remained for ascoviral invasion; type II, both Btk and the ascovirus were depleted by their competition, such that neither was successfully released or colonized the tissue; type III, Btk was eliminated by the ascovirus, and the ascovirus achieved systemic infection in the tested larvae. The feeding of Btk/ascovirus formulas led to a great reduction in larval diet consumption and resulted in a significant decrease in the emergence rate of H. armigera, M. separata, and S. litura larvae, which suggested that the formulas exerted marked oral control effects on both the contemporary individuals and the next generation of these tested pest species.

Mitochondrial and Innate Immunity Transcriptomes from Spodoptera frugiperda Larvae Infected with the Spodoptera frugiperda Ascovirus.

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.

Altering of host larval (Spodoptera exigua) calcineurin activity in response to ascovirus infection.

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.

Genome Analysis of Dasineura jujubifolia Toursvirus 2, A Novel Ascovirus.

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.

3H-117, a structural protein of Heliothis virescens ascovirus 3h (HvAV-3h).

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.