Horizontally transmitted parasitoid killing factor shapes insect defense to parasitoids.

Interkingdom competition occurs between hymenopteran parasitoids and insect viruses sharing the same insect hosts. It has been assumed that parasitoid larvae die with the death of the infected host or as result of competition for host resources. Here we describe a gene family, parasitoid killing factor (pkf), that encodes proteins toxic to parasitoids of the Microgastrinae group and determines parasitism success. Pkfs are found in several entomopathogenic DNA virus families and in some lepidopteran genomes. We provide evidence of equivalent and specific toxicity against endoparasites for PKFs found in entomopoxvirus, ascovirus, baculovirus, and Lepidoptera through a mechanism that elicits apoptosis in the cells of susceptible parasitoids. This highlights the evolutionary arms race between parasitoids, viruses, and their insect hosts.

Citizen science data opens multiple avenues for iridovirus research and prompts first detection of Invertebrate iridescent virus 31 in Australia.

From citizen science data we report the first records of blue to violet-colored oniscideans (Oniscidea: Isopoda), indicating potential invertebrate iridescent virus (IIV; Betairidovirinae: Iridoviridae) infection: in Africa, South America, and Oceania; and of the new hosts Armadillidium nasatum and Balloniscus sellowii. DNA sequencing of indigo Porcellio scaber confirms the presence of Invertebrate iridescent virus 31 in Australia. Beyond the Oniscidea, new, putative IIV hosts are identified: hoverfly pupae (Eristalinae), a tortrix moth larva (Phaecasiophora niveiguttana), and a millipede (Harpaphe haydeniana). In addition, the purported positive correlation between virion diameter and wavelength of iridescence is analyzed qualitatively for the first time.

Effects of several UV-protective substances on the persistence of the insecticidal activity of the Alphabaculovirus of Chrysodeixis chalcites (ChchNPV-TF1) on banana (Musa acuminata, Musaceae, Colla) under laboratory and open-field conditions.

Alphabaculovirus of Chrysodeixis chalcites (ChchNPV-TF1) has been investigated as a useful bioinsecticide against C. chalcites (Esper) (Lepidoptera: Noctuidae) in banana crops. This study investigated the effects of several substances on the persistence of ChchNPV-TF1 under field conditions in the Canary Islands. Natural photoprotective substances, such as moringa, cacao, green tea, benzopurpurine, charcoal, iron dioxide, benzimidazole, kaolinite, and bentonite, were first evaluated under laboratory conditions using a Crosslinker as UV light source at 200 J/cm2. The photoprotective substances were divided into three groups: low protection (0-8%; kaolinite), intermediate protection (48-62%; green tea, moringa, bentonite and cacao) and high protection (87-100%; charcoal, iron ioxide). Benzopurpurine and benzimidazole did not provide any photoprotective effects. Two of the substances that yielded the best results, 1% cacao and 1% charcoal, were selected for the open-field experiment in a banana plantation. The persistence of ChchNPV-TF1 OBs (occlusion bodies) on leaf surfaces with sunlight exposure was analysed by comparing the initial mortality of 2nd instar C. chalcites larvae with the mortality observed at various intervals postapplication. The mortality rates decreased over time in all treatments and were always higher in the UV-protective substance-treated parcels. The 1% charcoal treatment exhibited the highest protection in both the laboratory and field experiments. No specific interference of UV-protective substances on the maximum photochemical efficiency of banana plants was observed under field conditions.

Transcriptome sequencing reveals Cnaphalocrocis medinalis against baculovirus infection by oxidative stress.

Cnaphalocrocis medinalis granulovirus (CnmeGV) is a potential microbial agent against the rice leaffolder. Innate immunity is essential for insects to survive pathogenic infection. Therefore, to clarify the immune response of Cnaphalocrocis medinalis to the viral colonization, the gene expression profile of C. medinalis infected with CnmeGV was constructed by RNA-seq. A total of 8,503 differentially expressed genes (DEGs) were found including 5,304 up-regulated and 3,199 down-regulated unigenes. Gene enrichment analysis indicated that these DEGs were mainly linked to protein synthesis and metabolic process as well as ribosome and virus-infection pathways. Specifically, a significantly up-regulated PiggyBac-like transposon gene was identified suggested that the enhancement of transposon activity is related to host immunity. Further, the DEGs encoding oxidative stress related genes were identified and validated by RT-qPCR. Overall, 9 antioxidant enzyme genes and 4 antioxidant protein genes were up-regulated, and the extensive glutathione S-transferase genes were down-regulated. Our results provide a basis for understanding the molecular mechanisms of baculovirus action and oxidative stress response in C. medinalis and other insects.

Structures of diverse poxin cGAMP nucleases reveal a widespread role for cGAS-STING evasion in host-pathogen conflict.

DNA viruses in the family Poxviridae encode poxin enzymes that degrade the immune second messenger 2'3'-cGAMP to inhibit cGAS-STING immunity in mammalian cells. The closest homologs of poxin exist in the genomes of insect viruses suggesting a key mechanism of cGAS-STING evasion may have evolved outside of mammalian biology. Here we use a biochemical and structural approach to discover a broad family of 369 poxins encoded in diverse viral and animal genomes and define a prominent role for 2'3'-cGAMP cleavage in metazoan host-pathogen conflict. Structures of insect poxins reveal unexpected homology to flavivirus proteases and enable identification of functional self-cleaving poxins in RNA-virus polyproteins. Our data suggest widespread 2'3'-cGAMP signaling in insect antiviral immunity and explain how a family of cGAS-STING evasion enzymes evolved from viral proteases through gain of secondary nuclease activity. Poxin acquisition by poxviruses demonstrates the importance of environmental connections in shaping evolution of mammalian pathogens.

Genome Analysis of a Novel Clade b Betabaculovirus Isolated from the Legume Pest Matsumuraeses phaseoli (Lepidoptera: Tortricidae).

Matsumuraeses phaseoli is a Lepidopteran pest that primarily feeds on numerous species of cultivated legumes, such as Glycine and Phaseolus. It is widely distributed in northeast Asia. A novel granulovirus, designated as Matsumuraeses phaseoli granulovirus (MaphGV), was isolated from pathogenic M. phaseoli larvae that dwell in rolled leaves of Astragalus membranaceus, a Chinese medicinal herb. In this study, using next-generation sequencing, we report the complete genome of MaphGV. MaphGV genome comprises a double-stranded DNA of 116,875 bp, with 37.18% GC content. It has 128 hypothetical open reading frames (ORFs). Among them, 38 are baculovirus core genes, 18 are lepidopteran baculovirus conserved genes, and 5 are unique to Baculoviridae. MaphGV has one baculovirus repeat ORF (bro) and three inhibitors of apoptosis proteins (iap), including a newfound iap-6. We found two atypical baculoviral homologous regions (hrs) and four direct repeats (drs) in the MaphGV genome. Based on phylogenetic analysis, MaphGV belongs to Clade b of Betabaculovirus and is closely related to Cydia pomonellagranulovirus (CpGV) and Cryptophlebia leucotretagranulovirus (CrleGV). This novel baculovirus discovery and sequencing are invaluable in understanding the evolution of baculovirus and MaphGV may be a potential biocontrol agent against the bean ravaging pest.

A new nodavirus-negative Trichoplusia ni cell line for baculovirus-mediated protein production.

Cell lines derived from Trichoplusia ni (Tn) are widely used as hosts in the baculovirus-insect cell system (BICS). One advantage of Tn cell lines is they can produce recombinant proteins at higher levels than cell lines derived from other insects. However, Tn cell lines are persistently infected with an alphanodavirus, Tn5 cell-line virus (TnCLV), which reduces their utility as a host for the BICS. Several groups have isolated TnCLV-negative Tn cell lines, but none were thoroughly characterized and shown to be free of other adventitious viruses. Thus, we isolated and extensively characterized a new TnCLV-negative line, Tn-nodavirus-negative (Tn-NVN). Tn-NVN cells have no detectable TnCLV, no other previously identified viral contaminants of lepidopteran insect cell lines, and no sequences associated with any replicating virus or other viral adventitious agents. Tn-NVN cells tested negative for >60 species of Mycoplasma, Acholeplasma, Spiroplasma, and Ureaplasma. Finally, Tn-NVN cells grow well as a single-cell suspension culture in serum-free medium, produce recombinant proteins at levels similar to High Five™ cells, and do not produce recombinant glycoproteins with immunogenic core α1,3-fucosylation. Thus, Tn-NVN is a new, well-characterized TnCLV-negative cell line with several other features enhancing its utility as a host for the BICS.

A novel cypovirus found in a betabaculovirus co-infection context contains a poxvirus immune nuclease (poxin)-related gene.

The cassava hornworm Erinnyis ello ello (Lepidoptera: Sphingidae) is an important pest in Brazil. This insect feeds on host plants of several species, especially Manihot esculenta (cassava) and Hevia brasiliensis (rubber tree). Cassava hornworm outbreaks are quite common in Brazil and can cause great impact over crop production. Granulare and polyhedral-shaped occlusion bodies (OBs) were observed in extracts of dead E. ello larvae from rubber-tree plantations by light and scanning electron microscopy (SEM), suggesting a mixed infection. The polyhedral-shaped OB surface revealed indentations that resemble those found in cypovirus polyhedra. After OB nucleic acid extraction followed by cDNA production and Illumina deep-sequencing analysis, the results confirmed for the presence of a putative novel cypovirus that carries ten segments and also a betabaculovirus (Erinnyis ello granulovirus, ErelGV). Phylogenetic analysis of the predicted segment 1-enconded RdRP showed that the new cypovirus isolate is closely related to a member of species Cypovirus 2, which was isolated from Inachis io (Lepidoptera: Nymphalidae). Therefore, we named this new isolate Erinnyis ello cypovirus 2 (ErelCPV-2). Genome in silico analyses showed that ErelCPV-2 segment 8 (S8) has a predicted amino acid identity of 35.82 % to a hypothetical protein of betabaculoviruses. This putative protein has a cGAMP-specific nuclease domain related to the poxvirus immune nucleases (poxins) from the 2',3'-cGAMP-degrading enzyme family.

Efficient entry of budded virions of Autographa californica multiple nucleopolyhedrovirus into Spodoptera frugiperda cells is dependent on dynamin, Rab5, and Rab11.

Autographa californica multiple nucleopolyhedrovirus (AcMNPV), a member of the Alphabaculovirus genus of the family Baculoviridae, is an enveloped double-stranded DNA virus. Budded virions (BVs) of AcMNPV enter host cells via clathrin-mediated endocytosis. However, the route of functional intracellular trafficking of AcMNPV BVs during entry is not well established. In the current study, we found that entering BVs were colocalized mainly with cellular Rab5 and Rab11. Expression of dominant-negative (DN) Rab5 and Rab11 or RNAi-mediated down regulation of these two cellular transcripts significantly reduced BVs entry into but not egress from Spodoptera frugiperda cells (Sf9), whereas similar treatments for Rab4 and Rab7 had no apparent effect on virus infection. Combined with data from RNAi knockdowns of dynamin, and dynasore inhibition assays, our results support a model in which AcMNPV BVs enter permissive host cells by clathrin-mediated endocytosis, followed by de-envelopment of BVs predominantly within early and maturing endosomes rather than within late endosomes. Additionally, Rab11 suppression studies suggest the Rab11-dependent recycling endosomal pathway is involved in virion entry.

Identification of a Conserved Prophenoloxidase Activation Pathway in Cotton Bollworm Helicoverpa armigera.

Melanization is a prominent insect humoral response for encapsulation of and killing invading pathogens. It is mediated by a protease cascade composed of a modular serine protease (SP), and clip domain SPs (cSPs), which converts prophenoloxidase (PPO) into active phenoloxidase (PO). To date, melanization pathway in cotton bollworm Helicoverpa armigera, an important agricultural pest, remains largely unclear. To biochemically reconstitute the pathway in vitro, the putative proteases along with modified proteases containing the factor Xa cleavage site were expressed by Drosophila S2 cell expression system. Purified recombinant proteins were used to examine their role in activating PPO. It is revealed that cascade is initiated by a modular SP-SP41, followed by cSP1 and cSP6. The three-step SP41/cSP1/cSP6 cascade could further activate PPO, and the PO activity was significantly enhanced in the presence of two cSP homologs (cSPHs), cSPH11 and cSPH50, suggesting the latter are cofactors for PPO activation. Moreover, baculovirus infection was efficiently blocked by the reconstituted PPO activation cascade, and the effect was boosted by cSPH11 and cSPH50. Taken together, we unraveled a conserved PPO activation cascade in H. armigera, which is similar to that exists in lepidopteran biochemical model Manduca sexta and highlighted its role in antagonizing viral infection.

Biopesticide synergy when combining plant flavonoids and entomopathogenic baculovirus.

Four crop plants known to be hosts for the lepidopteran Trichoplusia ni (soybean, green bean, cotton, and cabbage) were treated with the biopesticide AfMNPV baculovirus in a dosage response assay. Treated soybean had, on average, a 6-fold increase in virus activity compared with the other crops. Leaf trichomes on soybeans were not found to be responsible for the observed increase of insecticidal activity. Three flavonoid compounds (daidzein, genistein, and kaempferol) were uniquely found only in the soybean crop, and were not detected in cotton, cabbage, or green bean plant matter. The individual flavonoid compounds did not cause T ni. mortality in no-virus assays when incorporated into artificial insect diet. The combination of the three flavonoid compounds at leaf level concentrations significantly increased baculovirus activity in diet incorporation assays. When the daidzein, genistein, and kaempferol were added to artificial diet, at 3.5-6.5 × leaf level concentrations, virus activity increased 1.5, 2.3, and 4.2-fold for each respective flavonoid. The soybean flavonoid compounds were found to synergistically improve baculovirus activity against T. ni.

Baculovirus ODV-E66 degrades larval peritrophic membrane to facilitate baculovirus oral infection.

ODV-E66 is a major envelope proteins of baculovirus occlusion derived virus (ODV) with chondroitinase activity. Here, we studied the roles of ODV-E66 during Helicoverpa armigera nucleopolyhedrovirus (HearNPV) primary infection. ODV-E66 is a late viral protein dispensable for BV production and ODV morphogenesis. Deletion of odv-e66 had a profound effect on HearNPV oral infectivity in 4th instar larvae with a 50% lethal concentration (LC50) value of 26 fold higher than that of the repaired virus, compared to in 3rd instar larvae. Calcofluor white, an agent which destroys the peritrophic membrane (PM), could rescue the oral infectivity of odv-e66 deleted HearNPV, implying the PM may be the target of ODV-E66. In vitro assays showed HearNPV ODV-E66 has chondroitinase activity. Electron microscopy demonstrated that odv-e66 deletion alleviated the damage to the PM caused by HearNPV infection. These data suggest an important role of ODV-E66 in the penetration of the PM during oral infection.

Where the baculoviruses lead, the caterpillars follow: baculovirus-induced alterations in caterpillar behaviour.

Baculoviruses are well-known for altering the behaviour of their caterpillar hosts by inducing hyperactivity (enhanced locomotion) and/or tree-top disease (climbing to elevated positions before death). These features, along with the genomic small size of baculoviruses compared to non-viral parasites and the at hand techniques for producing mutants, imply that baculoviruses are excellent tools for unravelling the molecular mechanisms underlying parasitic alteration of host behaviour. Baculoviruses can be easily mutated, allowing an optimal experimental setup in comparative studies, where for instance host gene expression can be compared between insects infected with wild-type viruses or with mutant viruses lacking genes involved in behavioural manipulation. Recent studies have revealed the first insight into the underlying molecular pathways that lead to the typical behaviour of baculovirus-infected caterpillars and into the role of light therein. Since host behaviour in general is mediated through the host's central nervous system (CNS), a promising future step will be to study how baculoviruses regulate the neuronal activity of the host.

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.

Providence virus: An animal virus that replicates in plants or a plant virus that infects and replicates in animal cells?

INTRODUCTION: Emerging viral diseases, most of which are zoonotic, pose a significant threat to global health. There is a critical need to identify potential new viral pathogens and the challenge is to identify the reservoirs from which these viruses might emerge. Deep sequencing of invertebrate transcriptomes has revealed a plethora of viruses, many of which represent novel lineages representing both plant and animal viruses and little is known about the potential threat that these viruses pose. METHODS: Providence virus, an insect virus, was used to establish a productive infection in Vigna unguiculata (cowpea) plants. Providence virus particles purified from these cowpea plants were used to infect two mammalian cell lines. FINDINGS: Here, we present evidence that Providence virus, a non-enveloped insect RNA virus, isolated from a lepidopteran midgut cell line can establish a productive infection in plants as well as in animal cells. The observation that Providence virus can readily infect both plants and mammalian cell culture lines demonstrates the ability of an insect RNA virus to establish productive infections across two kingdoms, in plants and invertebrate and vertebrate animal cell lines. CONCLUSIONS: The study highlights the potential of phytophagous insects as reservoirs for viral re-assortment and that plants should be considered as reservoirs for emerging viruses that may be potentially pathogenic to humans.

The dual life of ichnoviruses.

Ichnoviruses (IVs) are mutualistic, double-stranded DNA viruses playing a key role in the successful parasitism of thousands of endoparasitoid wasp species. IV particles are produced exclusively in the female wasp reproductive tract. They are co-injected along with the parasitoid egg into caterpillar hosts upon parasitization. The expression of viral genes by infected host cells leads to an immunosuppressive state and delayed development of the host, two pathologies that are critical to the successful development of the wasp egg and larva. Ichnovirus is one of the two recognized genera within the family Polydnaviridae (polydnaviruses or PDVs), the other genus being Bracovirus (BV), associated with braconid wasps. IVs are associated with ichneumonid wasps belonging to the subfamilies Campopleginae and Banchinae; attempts to identify IV particles in other ichneumonid subfamilies have so far been unsuccessful. Functional studies targeting IV genes expressed in parasitized hosts, along with investigations of the molecular mechanisms responsible for viral morphogenesis in the female wasp, have resulted in a better understanding of the biology of these atypical viruses.

Biological and molecular characterization of two Anticarsia gemmatalis multiple nucleopolyhedrovirus clones exhibiting contrasting virulence.

Baculovirus natural populations are known to be genetically heterogeneous and such genotypic diversity could have implications in the performance of biocontrol agents. The Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV) has been widely used to control the velvetbean caterpillar, Anticarsia gemmatalis, in Brazil. In the present work, morphological and molecular analyses as well as the biological activity of AgMNPV genotypes derived from a Brazilian field isolate (AgMNPV-79) were carried out. The existence of genotypic variants in the population was confirmed by DNA restriction analysis. Although difference in virulence was observed among the variants, the most (Ag79-01) and the least (AgL-16) virulent clones do not show any morphological and cytopathological changes when compared to the most studied isolate (AgMNPV-2D). The complete genome analysis of the two viral clones showed the presence of single open reading frames (ORFs) of the pe-38 and he65 genes, which contrasts with the two split ORFs present in the genome of the AgMNPV-2D isolate. The viral clone AgL-16 has many variations in the ie-2 and pe-38 genes, which are transcription regulatory genes responsible for the regulation of viral early gene expression during insect cell infection. Furthermore, other genes showed alterations like the odv-e56, which have an essential role in the maturation and envelopment of the ODVs, and bro-a and bro-b genes which were fused to form a single ORF. For the Ag79-01, although the total number of single nucleotide variants (SNVs) was more prominent in the pe-38 gene, its genome showed very few modifications in comparison to the AgMNPV-2D genome.

Effects of a Covert Infection with Phthorimaea operculella granulovirus in Insect Populations of Phthorimaea operculella.

Virus infections of insects can easily stay undetected, neither showing typical signs of a disease, nor being lethal. Such a stable and most of the time covert infection with Phthorimaea operculella granulovirus (PhopGV) was detected in a Phthorimaea operculella laboratory colony, which originated from Italy (Phop-IT). This covert virus (named PhopGV-R) was isolated, purified and characterized at the genetic level by full genome sequencing. Furthermore, the insect colony Phop-IT was used to study the crowding effect, double infection with other PhopGV isolates (CR3 and GR1), and co-infection exclusion. An infection with a second homologous virus (PhopGV-CR3) activated the covert virus, while a co-infection with another virus isolate (PhopGV-GR1) led to its suppression. This study shows that stable virus infections can be common for insect populations and have an impact on population dynamics because they can suppress or enable co-infection with another virus isolate of the same species.

Elucidating the genetic diversity of Phthorimaea operculella granulovirus (PhopGV).

Twelve complete genome sequences of Phthorimaea operculella granulovirus (PhopGV) isolates from four different continents (Africa, South America, Asia and Europe) were analysed after Illumina next-generation sequencing (NGS). The isolates have a circular double-stranded DNA genome that is 118 355 to 119 177 bp in length and all of them encode 130 open reading frames (ORFs). Analysis of single-nucleotide polymorphisms (SNPs) revealed a unique set of SNP positions for every tested isolate. The genome sequences of the investigated PhopGV isolates were classified into a new system of four (1-4) groups according to the presence of group-specific SNPs as well as insertions and deletions. These genome groups correlated with phylogenetic lineages inferred from minimum-evolution trees of the whole-genome consensus nucleotide sequences. All members of group 3 originated from the Mediterranean area, whereas the geographical origin and the group assignment did not correlate for isolates belonging to genome groups 1, 2 or 4. The high degree of coverage facilitated the determination of variant nucleotide frequencies. We conclude that the geographical isolates of PhopGV are genetically highly similar. On the other hand, they were rarely genetically homogenous and in most cases appeared to be mixtures of multiple genotypes.

New Method for Differentiation of Granuloviruses (Betabaculoviruses) Based on Real-Time Polymerase Chain Reaction (Real-Time PCR).

Baculoviridae is a highly diverse family of rod-shaped viruses with double-stranded DNA. To date, almost 100 species have had their complete genomic sequences deposited in the GenBank database, a quarter of which comprises granuloviruses (GVs). Many of the genomes are sequenced using next-generation sequencing, which is currently considered the best method for characterizing new species, but it is time-consuming and expensive. Baculoviruses form a safe alternative to overused chemical pesticides and therefore there is a constant need for identifying new species that can be active components of novel biological insecticides. In this study, we have described a fast and reliable method for the detection of new and differentiation of previously analyzed granulovirus species based on a real-time polymerase chain reaction (PCR) technique with melting point curve analysis. The sequences of highly conserved baculovirus genes, such as granulin and late expression factors 8 and 9 (lef-8 and lef-9), derived from GVs available to date have been analyzed and used for degenerate primer design. The developed method was tested on a representative group of eight betabaculoviruses with comparisons of melting temperatures to allow for quick and preliminary granulovirus detection. The proposed real-time PCR procedure may be a very useful tool as an easily accessible screening method in a majority of laboratories.