Genome analysis of Heliothis virescens ascovirus 3h isolated from China

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

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,HzAMl,SeFB,and HaFB cells,including swelling and detachmen 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 nontarget vertebrate cells.

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(Hv AV-3h)is a highly conserved gene of ascoviruses.As an early gene of Hv AV-3h,3h-31 codes for a non-structural protein(3H-31)of Hv AV-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(Se FB).3h-31 was further inserted into the bacmid of Autographa californica nucleopolyhedrovirus(Ac MNPV)to generate an infectious baculovirus(Ac MNPV-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 Ac MNPV-31-infected Se FB cells.In vivo,both LD50and LD90values of Ac MNPV-31 were significantly higher than those of the wild-type Ac MNPV(Ac MNPV-wt)in third instar S.exigua larvae.An interesting finding was that the liquefaction of the larvae killed by the infection of Ac MNPV-31 was delayed.Chitinase and cathepsin activities of Ac MNPV-31-infected larvae were significantly lower than those of Ac MNPV-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.

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.

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 exigiia 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

ldentifying 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 B1k/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 II,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.