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.

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.

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.

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.

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.

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.

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.

In Vitro Infectious Risk Assessment of Heliothis virescens ascovirus 3j (HvAV-3j) toward Non-target Vertebrate Cells.

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.

Genomic analysis of a novel isolate Heliothis virescens ascovirus 3i (HvAV-3i) and identification of ascoviral repeat ORFs (aros).

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%.

An ascovirus isolated from Spodoptera litura (Noctuidae: Lepidoptera) transmitted by the generalist endoparasitoid Meteorus pulchricornis (Braconidae: Hymenoptera).

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.

Response analysis of host Spodoptera exigua larvae to infection by Heliothis virescens ascovirus 3h (HvAV-3h) via transcriptome.

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.

A rapid method for titration of ascovirus infectivity.

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.

Transcriptome Analysis of the Spodoptera frugiperda Ascovirus In Vivo Provides Insights into How Its Apoptosis Inhibitors and Caspase Promote Increased Synthesis of Viral Vesicles and Virion Progeny.

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 ascovirus1a (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.

Selection of reference genes for expression analysis using RT-qPCR in the dissemination system of Heliothis virescens ascovirus 3 h (HvAV-3h).

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.