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.

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.

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.

ICTV Virus Taxonomy Profile: Ascoviridae.

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.

Characterization and Growing Development of Spodoptera exigua (Lepidoptera: Noctuidae) Larvae Infected by Heliothis virescens ascovirus 3h (HvAV-3h).

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.

Imperfection works: Survival, transmission and persistence in the system of Heliothis virescens ascovirus 3h (HvAV-3h), Microplitis similis and Spodoptera exigua.

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.

Effects of an ascovirus (HvAV-3e) on diamondback moth, Plutella xylostella, and evidence for virus transmission by a larval parasitoid.

Ascoviruses (AVs) are pathogenic to lepidopteran larvae, and most commonly attack species in the Noctuidae. The unique pathology includes cleavage of host cells into virion-containing vesicles which leads to the milky white colouration of the hemolymph as opposed to the clear hemolymph of healthy larvae. Recently, we showed that a Heliothis virescens AV (HvAV-3e) isolate is able to induce disease in Crocidolomia pavonana F. (Lepidoptera: Crambidae), affecting feeding, growth and survival of infected larvae. In this study, we investigated the effect of different variants of HvAV-3e on diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae) larvae, another non-noctuid host. In hemolymph inoculation bioassays fourth instar larvae showed a significant dose response to each of the HvAV-3e variants and significant differences between the virulence of the three variants were detected. Both second and fourth instars were readily infected with the virus and infected individuals demonstrated significant reductions in food consumption and growth. The majority of infected individuals died at the larval or pupal stage and individuals which developed into adults were usually deformed, less fecund than non-infected controls and died shortly after emergence. In transmission studies, Diadegmasemiclausum (Hymenoptera: Ichneumonidae), a key parasitoid of diamondback moth, infected healthy host larvae during oviposition following previous attack of HvAV-3e infected hosts. In choice tests D. semiclausum did not discriminate between infected individuals but host infection had no detectable impact on the development of immature D. semiclausum or on subsequent adults.

A lipase-like gene from Heliothis virescens ascovirus (HvAV-3e) is essential for virus replication and cell cleavage.

A unique feature of ascovirus infection is cleavage of host cells into virus containing vesicles. It has been suggested that the virus induces apoptosis, either by expression of a caspase or other means, which is then diverted toward vesicle formation. There is little known about the mechanism of vesicle formation. Recent genome sequences of three ascoviruses indicated the presence of several putative open reading frames coding for proteins that could be involved in lipid metabolism. These proteins may play a role in rearrangement of membranes in infected host cells leading to formation of vesicles. Here, we analyzed a lipase-like gene (ORF19) from Heliothis virescens ascovirus (HvAV-3e) expressed from 8 h after infection and essential for virus replication and cell cleavage. In addition, ORF19 knock down by RNA interference inhibited virus replication indicating that the gene is indispensable for HvAV-3e replication. However, under enzymatic assays tested, we did not detect any lipase or esterase activity from ORF19.

The role of actin filaments in ascovirus replication and pathology.

Ascoviruses (AVs) are insect viruses transmitted by parasitoid wasps. The unique pathology in host cells upon AV infection includes enlargement, blebbing and cleavage of host cells into virus-containing vesicles that are important in dissemination of the virus. The mechanism of pathogenesis and vesicle formation is largely unknown. Here, we explored involvement of actin filaments in virus entry, replication and pathology. The results suggested that entry of Heliothis virescens ascovirus-3e (HvAV-3e) leads to rearrangement of the actin cytoskeleton. After HvAV-3e infection, actin filaments were found in foci rather than in a homogenous distribution within the cytoplasm. Actin filaments were also found concentrating around blebs and vesiculation areas of the cell cortex following infection. Destabilization of filamentous actin by cytochalasin D did not inhibit entry or replication of the virus but affected vesiculation and pathology associated with HvAV-3e infection. These observations suggested that actin may not be required for virus entry and replication but essential for virus pathology, mainly vesicle formation.

Ascoviruses: superb manipulators of apoptosis for viral replication and transmission.

Ascoviruses are members of a recently described new family (Ascoviridae) of large double-stranded DNA viruses that attack immature stages of insects belonging to the order Lepidoptera, in which they cause a chronic, fatal disease. Ascoviruses have several unusual characteristics not found among other viruses, the most novel of which are their transmission by endoparasitic wasps and a unique cytopathology that resembles apoptosis. Cell infection induces apoptosis and in some species is associated with synthesis of a virus-encoded executioner caspase and several lipid-metabolizing enzymes. Rather than leading directly to cell death, synthesis of viral proteins results in the rescue of developing apoptotic bodies that are converted into large vesicles in which virions accumulate and continue to assemble. In infected larvae, millions of these virion-containing vesicles begin to disperse from infected tissues 48-72 h after infection into the blood, making it milky white, a major characteristic of the disease. Circulation of virions and vesicles in the blood facilitates mechanical transmission by parasitic wasps. Although ascoviruses appear to be very common, only five species are currently recognized, with the type species being the Spodoptera frugiperda ascovirus 1a. Ascovirus virions are large, enveloped, typically bacilliform or reniform in shape, and, depending on the species, have genomes that range from 119 to 186 kbp. Molecular phylogenetic evidence indicates that ascoviruses evolved from iridoviruses (family Iridoviridae) that attack lepidopteran larvae and are likely the evolutionary source of ichnoviruses (family Polydnaviridae), which assist endoparasitic hymenopterans in overcoming the defense responses of their insect hosts. Thus, as other molecular evidence suggests that iridoviruses evolved from phycodnaviruses (family Phycodnaviridae), an evolutionary pathway is apparent from phycodnaviruses via iridoviruses and ascoviruses to ichnoviruses.

P64, a novel major virion DNA-binding protein potentially involved in condensing the Spodoptera frugiperda Ascovirus 1a genome.

We recently identified 21 structural proteins in the virion of Spodoptera frugiperda ascovirus 1a (SfAV1a), a virus with a large, double-stranded DNA genome of 157 kbp, which attacks species of the lepidopteran family Noctuidae. The two most abundant virion proteins were the major capsid protein and a novel protein (P64) of 64 kDa that contained two distinct domains not known previously to occur together. The amino-terminal half of P64 (residues 1 to 263) contained four repeats (a recently recognized motif with an unknown function) of a virus-specific two-cysteine adaptor. Adjoined to this, the carboxy-terminal half of P64 (residues 279 to 455) contained 14 copies of a highly basic, tandemly repeated motif rich in arginine and serine, having an 11- to 13-amino-acid consensus sequence, SPSQRRSTS(V/K)(A/S)RR, yielding a predicted isoelectric point of 12.2 for this protein. In the present study, we demonstrate by Southwestern analysis that SfAV1a P64 was the only virion structural protein that bound DNA. Additional electrophoretic mobility shift assays showed that P64 bound SfAV1a as well as non-SfAV1a DNA. Furthermore, we show through immunogold labeling of ultrathin sections that P64 is a component of virogenic stroma and appears to be progressively incorporated into the SfAV1a DNA core during virion assembly. As no other virion structural protein bound DNA and no basic DNA-binding proteins of lower mass are encoded by the SfAV1a genome or were identified by proteomic analysis, our results suggest that P64's function is to condense the large genome of this virus and assist in packaging this genome into its virion.

Effects of Heliothis virescens ascovirus (HvAV-3e) on a novel host, Crocidolomia pavonana (Lepidoptera: Crambidae).

Ascoviruses are double-stranded DNA viruses which cause fatal disease in lepidopteran host larvae. They induce a unique pathology, causing cleavage of host cells into virion-containing vesicles. With the single exception of Diadromus pulchellus ascovirus, all ascoviruses have been exclusively reported from the Noctuidae. To investigate whether Heliothis virescens AV (HvAV-3e) has a broader host range at the family level, larvae of Crocidolomia pavonana F. (Lepidoptera: Crambidae), a major pest of brassica crops in tropical and sub-tropical regions of the Old World and Australasia, were inoculated with HvAV-3e. Larvae were readily infected by the ascovirus and feeding, growth and survival were significantly affected. However, the milky white discolouration of the haemolymph which is characteristic of ascovirus infection in noctuid hosts was not apparent. In further contrast to infected noctuid host larvae that do not develop to the pupal stage, a significant proportion of infected C. pavonana larvae pupated but all were killed at this stage. Thus, C. pavonana appears to be a semi-permissive host of the ascovirus, the presence of such hosts in the field might be an explanation for the conundrum for the ascovirus-noctuid-wasp relationship, helping explain the persistence of the ascovirus.

An insect virus-encoded microRNA regulates viral replication.

MicroRNAs (miRNAs) are small ( approximately 22 nucleotides) noncoding RNAs which play an essential role in gene regulation and affect a wide range of processes, including development, differentiation, and oncogenesis. Here we report the identification of the first miRNA from an insect virus, derived from the major capsid protein (MCP) gene in Heliothis virescens ascovirus (HvAV) (HvAV-miR-1). Although MCP was abundantly expressed at all time points 24 h after infection, HvAV-miR-1 expression was strictly regulated and specifically detected from 96 h postinfection. HvAV-miR-1 expression coincided with a marked reduction of the expression of HvAV DNA polymerase I, which is a predicted target. Ectopic expression of full-length and truncated versions of MCP retaining the miRNA sequence significantly reduced DNA polymerase I transcript levels and inhibited viral replication. Our results indicate that HvAV-miR-1 directs transcriptional degradation of DNA polymerase I and regulates HvAV replication. These findings are congruent with recent reports that miR-BART-2 regulates Epstein-Barr virus DNA polymerase expression and suggest that virus-encoded miRNA regulation of virus replication may be a general phenomenon.

A caspase-like gene from Heliothis virescens ascovirus (HvAV-3e) is not involved in apoptosis but is essential for virus replication.

Ascoviruses (AVs) are double-stranded DNA viruses causing a fatal disease in lepidopteran host larvae. A unique feature of AV infection is cleavage of host cells into membrane bound vesicles containing the virions. A recent study showed that a caspase from Spodoptera frugiperda AV (SfAV) is directly involved in initiation of apoptosis and eventually cell cleavage. Results shown here indicate that Heliothis virescens AV does not induce apoptosis in host cells. HvAV codes for a caspase-like protein but no apoptosis was observed when the gene was expressed in vitro. RNAi studies indicated that the gene is essential for virus replication.

Genomic sequence of Spodoptera frugiperda Ascovirus 1a, an enveloped, double-stranded DNA insect virus that manipulates apoptosis for viral reproduction.

Ascoviruses (family Ascoviridae) are double-stranded DNA viruses with circular genomes that attack lepidopterans, where they produce large, enveloped virions, 150 by 400 nm, and cause a chronic, fatal disease with a cytopathology resembling that of apoptosis. After infection, host cell DNA is degraded, the nucleus fragments, and the cell then cleaves into large virion-containing vesicles. These vesicles and virions circulate in the hemolymph, where they are acquired by parasitic wasps during oviposition and subsequently transmitted to new hosts. To develop a better understanding of ascovirus biology, we sequenced the genome of the type species Spodoptera frugiperda ascovirus 1a (SfAV-1a). The genome consisted of 156,922 bp, with a G+C ratio of 49.2%, and contained 123 putative open reading frames coding for a variety of enzymes and virion structural proteins, of which tentative functions were assigned to 44. Among the most interesting enzymes, due to their potential role in apoptosis and viral vesicle formation, were a caspase, a cathepsin B, several kinases, E3 ubiquitin ligases, and especially several enzymes involved in lipid metabolism, including a fatty acid elongase, a sphingomyelinase, a phosphate acyltransferase, and a patatin-like phospholipase. Comparison of SfAV-1a proteins with those of other viruses showed that 10% were orthologs of Chilo iridescent virus proteins, the highest correspondence with any virus, providing further evidence that ascoviruses evolved from a lepidopteran iridovirus. The SfAV-1a genome sequence will facilitate the determination of how ascoviruses manipulate apoptosis to generate the novel virion-containing vesicles characteristic of these viruses and enable study of their origin and evolution.

Replication of Heliothis virescens ascovirus in insect cell lines.

Ascoviruses (AVs) infect larvae of various insect pests belonging to the family Noctuidae. The result of AV infection in the hosts is cleavage of infected cells into vesicles, a unique feature of AV infection. Since insect cell lines facilitate the study of virus life cycles, attempts were made to analyze Heliothis virescens AV (HvAV3e) infection in several cell lines and compare cell pathology to larval infection. In this study, replication and cytopathological effects of HvAV3e on four different cell lines were investigated. HvAV3e replication was confirmed in three noctuid cell lines from Spodoptera frugiperda (Sf9) and Helicoverpa zea (BCIRL-Hz-AM1 and FB33). However, the virus did not replicate in the non-noctuid insect cell line from Pieris rapae (Pieridae). Despite replication of the virus in the three permissive cell lines, the cytopathological effects of the virus were significantly different from that of larval infection.

Characteristics of pathogenic and mutualistic relationships of ascoviruses in field populations of parasitoid wasps.

Ascoviruses are disseminated among larvae in lepidopteran populations by parasitic wasps during oviposition. Ascovirus relationships with these wasps vary from pathogenic to mutualistic, and experimentally can be shown possibly to be commensal non-pathogenic virus having little or no effect. Most ascoviruses are pathogens that female wasps vector mechanically. Other ascoviruses have a more intimate relationship with their wasp vectors in that their genome is stably maintained in all wasp nuclei through several generations by vertical transmission. In this relationship, these viruses are mutualistic, enhancing the successful development of the wasp larvae by suppressing lepidopteran defence mechanisms. The DpAV4 ascovirus is a mutualist in certain Diadromus wasps but is pathogenic or not when vectored by other species of this genus. These various biologies suggest that ascovirus/wasp relationships depend on wasp regulatory factors that control virus replication. Thus, certain ascoviruses can potentially have either a pathogenic, mutualistic, or non-pathogenic relationship with a specific wasp vector, the type of relationship being dependent upon the species system in which the relationship evolved. Finally, because ascoviruses appear to be related to ichnoviruses (Polydnaviridae), the DpAV4/Diadromus system constitutes a possible interesting intermediate between the pathogenic ascoviruses and symbiotic viruses that evolved to be ichnoviruses.

Commensal and mutualistic relationships of reoviruses with their parasitoid wasp hosts.

During evolution, certain endoparasitoid wasps have developed mechanisms to suppress the defence systems of their hosts. For this purpose, these species, all of which belong to the families Ichneumonidae and Braconidae, inject various kinds of virus-like particles. The most studied of these particles are classified as polydnaviruses (family Polydnaviridae) which are symbiotic viruses. Over the past decade, it has also been shown that several wasp species harbour reoviruses (family Reoviridae), and that two of these suppress host defence, allowing the development of the parasitoid eggs. In this paper, we summarize the key features of these viruses and their relationships with their wasp hosts. Five reoviruses are known that appear to be non-pathogenic for the wasps. Three of these, McRVLP, HeRV, OpRVLP, use their wasp hosts as vectors, and do not appear to be involved in host defence suppression. The fourth, DpRV-1, is a commensal reovirus detected in most field populations of the wasp, Diadromus pulchellus. This reovirus is always found associated with an ascovirus, DpAV-4a, which is indispensable for host immune suppression. Although DpRV-1 has not been shown to directly increase D. pulchellus parasitic success, it may contribute to this success by retarding DpAV-4a replication in the wasp. The fifth reovirus, DpRV-2, occurs in a specific population of D. pulchellus in which DpRV-1 and DpAV-4 are absent. This virus has a mutualistic relationship with its wasp host, as its injection by females during oviposition is essential for host immunosuppression. Interestingly, these viruses belong to several different reovirus genera.