Evolutionary Ecology of Wolbachia Releases for Disease Control.

Wolbachia is an endosymbiotic Alphaproteobacteria that can suppress insect-borne diseases through decreasing host virus transmission (population replacement) or through decreasing host population density (population suppression). We contrast natural Wolbachia infections in insect populations with Wolbachia transinfections in mosquitoes to gain insights into factors potentially affecting the long-term success of Wolbachia releases. Natural Wolbachia infections can spread rapidly, whereas the slow spread of transinfections is governed by deleterious effects on host fitness and demographic factors. Cytoplasmic incompatibility (CI) generated by Wolbachia is central to both population replacement and suppression programs, but CI in nature can be variable and evolve, as can Wolbachia fitness effects and virus blocking. Wolbachia spread is also influenced by environmental factors that decrease Wolbachia titer and reduce maternal Wolbachia transmission frequency. More information is needed on the interactions between Wolbachia and host nuclear/mitochondrial genomes, the interaction between invasion success and local ecological factors, and the long-term stability of Wolbachia-mediated virus blocking.

ICTV Virus Taxonomy Profile: Phasmaviridae 2024.

Phasmaviridae is a family for negative-sense RNA viruses with genomes of about 9.7-15.8 kb. These viruses are maintained in and/or transmitted by insects. Phasmavirids produce enveloped virions containing three single-stranded RNA segments that encode a nucleoprotein (N), a glycoprotein precursor (GPC), and a large (L) protein containing an RNA-directed RNA polymerase (RdRP) domain. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Phasmaviridae, which is available at ictv.global/report/phasmaviridae.

The Interplay Between Viruses and RNAi Pathways in Insects.

As an overarching immune mechanism, RNA interference (RNAi) displays pathogen specificity and memory via different pathways. The small interfering RNA (siRNA) pathway is the primary antiviral defense mechanism against RNA viruses of insects and plays a lesser role in defense against DNA viruses. Reflecting the pivotal role of the siRNA pathway in virus selection, different virus families have independently evolved unique strategies to counter this host response, including protein-mediated, decoy RNA-based, and microRNA-based strategies. In this review, we outline the interplay between insect viruses and the different pathways of the RNAi antiviral response; describe practical application of these interactions for improved expression systems and for pest and disease management; and highlight research avenues for advancement of the field.

ICTV Virus Taxonomy Profile: Geminiviridae 2021.

The family Geminiviridae includes viruses with mono- or bipartite single-stranded, circular DNA genomes of 2.5-5.2 kb. They cause economically important diseases in most tropical and subtropical regions of the world. Geminiviruses infect dicot and monocot plants and are transmitted by insect vectors. DNA satellites are associated with some geminiviruses. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Geminiviridae which is available at ictv.global/report/geminiviridae.

Re-assessing the diversity of negative strand RNA viruses in insects.

The spectrum of viruses in insects is important for subjects as diverse as public health, veterinary medicine, food production, and biodiversity conservation. The traditional interest in vector-borne diseases of humans and livestock has drawn the attention of virus studies to hematophagous insect species. However, these represent only a tiny fraction of the broad diversity of Hexapoda, the most speciose group of animals. Here, we systematically probed the diversity of negative strand RNA viruses in the largest and most representative collection of insect transcriptomes from samples representing all 34 extant orders of Hexapoda and 3 orders of Entognatha, as well as outgroups, altogether representing 1243 species. Based on profile hidden Markov models we detected 488 viral RNA-directed RNA polymerase (RdRp) sequences with similarity to negative strand RNA viruses. These were identified in members of 324 arthropod species. Selection for length, quality, and uniqueness left 234 sequences for analyses, showing similarity to genomes of viruses classified in Bunyavirales (n = 86), Articulavirales (n = 54), and several orders within Haploviricotina (n = 94). Coding-complete genomes or nearly-complete subgenomic assemblies were obtained in 61 cases. Based on phylogenetic topology and the availability of coding-complete genomes we estimate that at least 20 novel viral genera in seven families need to be defined, only two of them monospecific. Seven additional viral clades emerge when adding sequences from the present study to formerly monospecific lineages, potentially requiring up to seven additional genera. One long sequence may indicate a novel family. For segmented viruses, cophylogenies between genome segments were generally improved by the inclusion of viruses from the present study, suggesting that in silico misassembly of segmented genomes is rare or absent. Contrary to previous assessments, significant virus-host codivergence was identified in major phylogenetic lineages based on two different approaches of codivergence analysis in a hypotheses testing framework. In spite of these additions to the known spectrum of viruses in insects, we caution that basing taxonomic decisions on genome information alone is challenging due to technical uncertainties, such as the inability to prove integrity of complete genome assemblies of segmented viruses.

Hepatitis A virus and the origins of picornaviruses.

Hepatitis A virus (HAV) remains enigmatic, despite 1.4 million cases worldwide annually. It differs radically from other picornaviruses, existing in an enveloped form and being unusually stable, both genetically and physically, but has proved difficult to study. Here we report high-resolution X-ray structures for the mature virus and the empty particle. The structures of the two particles are indistinguishable, apart from some disorder on the inside of the empty particle. The full virus contains the small viral protein VP4, whereas the empty particle harbours only the uncleaved precursor, VP0. The smooth particle surface is devoid of depressions that might correspond to receptor-binding sites. Peptide scanning data extend the previously reported VP3 antigenic site, while structure-based predictions suggest further epitopes. HAV contains no pocket factor and can withstand remarkably high temperature and low pH, and empty particles are even more robust than full particles. The virus probably uncoats via a novel mechanism, being assembled differently to other picornaviruses. It utilizes a VP2 'domain swap' characteristic of insect picorna-like viruses, and structure-based phylogenetic analysis places HAV between typical picornaviruses and the insect viruses. The enigmatic properties of HAV may reflect its position as a link between 'modern' picornaviruses and the more 'primitive' precursor insect viruses; for instance, HAV retains the ability to move from cell-to-cell by transcytosis.

Insects and their pathogens in a changing climate.

The complex nature of climate change-mediated multitrophic interaction is an underexplored area, but has the potential to dramatically shift transmission and distribution of many insects and their pathogens, placing some populations closer to the brink of extinction. However, for individual insect-pathogen interactions climate change will have complicated hard-to-anticipate impacts. Thus, both pathogen virulence and insect host immunity are intrinsically linked with generalized stress responses, and in both pathogen and host have extensive trade-offs with nutrition (e.g., host plant quality), growth and reproduction. Potentially alleviating or exasperating these impacts, some pathogens and hosts respond genetically and rapidly to environmental shifts. This review identifies many areas for future research including a particular need to identify how altered global warming interacts with other environmental changes and stressors, and how consistent these impacts are across pathogens and hosts. With that achieved we would be closer to producing an overarching framework to integrate knowledge on all environmental interplay and infectious disease events.

New Technologies for Studying Negative-Strand RNA Viruses in Plant and Arthropod Hosts.

The plant viruses in the phylum Negarnaviricota, orders Bunyavirales and Mononegavirales, have common features of single-stranded, negative-sense RNA genomes and replication in the biological vector. Due to the similarities in biology, comparative functional analysis in plant and vector hosts is helpful for understanding host-virus interactions for negative-strand RNA viruses. In this review, we will highlight recent technological advances that are breaking new ground in the study of these recalcitrant virus systems. The development of infectious clones for plant rhabdoviruses and bunyaviruses is enabling unprecedented examination of gene function in plants and these advances are also being transferred to study virus biology in the vector. In addition, genome and transcriptome projects for critical nonmodel arthropods has enabled characterization of insect response to viruses and identification of interacting proteins. Functional analysis of genes using genome editing will provide future pathways for further study of the transmission cycle and new control strategies for these viruses and their vectors.

The Insect Virome: Opportunities and Challenges.

The insect virome is composed of a myriad of viruses. Both field populations and laboratory colonies of insects harbour diverse viruses, including viruses that infect the insect itself, viruses of microbes associated with the insect, and viruses associated with ingested materials. Metagenomics analysis for identification of virus-derived sequences has allowed for new appreciation of the extent and diversity of the insect virome. The complex interactions between insect viruses and host antiviral immune pathways (RNA interference and apoptosis), and between viruses and other members of the microbiome (e.g. Wolbachia) are becoming apparent. In this chapter, an overview of the diversity of viruses in insects and recent virus discovery research for specific insects and insect-derived cell lines is provided. The opportunities and challenges associated with the insect virome, including the potential impacts of viruses on both research and insect management programs are also addressed.

miRNA Modulation of Insect Virus Replication.

The outcome of virus infection in insects is impacted by regulation of both host and virus gene expression. A class of small RNAs called microRNAs (miRNA) have emerged as important regulators of gene expression that can influence the outcome of virus infection. miRNA regulation occurs at a comparatively late stage of gene expression, allowing for rapid control and fine-tuning of gene expression levels. Here we discuss the biogenesis of miRNAs from both host and virus genomes, the interactions that lead to regulation of gene expression, and the miRNA-mRNA interactions that lead to either antivirus or provirus consequences in the course of virus infection in insects.

Sensing Viral Infections in Insects: A Dearth of Pathway Receptors.

Insects, the most diverse group of animals, can be infected by an extraordinary diversity of viruses. Among them, arthropod-borne viruses can be transmitted to humans, while bee and silkworm viruses cause important economic losses. Like all invertebrates, insects rely solely on innate immunity to counter viral infections. Protein-based mechanisms, involving restriction factors and evolutionarily conserved signaling pathways regulating transcription factors of the NF-kB and STAT families, participate in the control of viral infections in insects. In addition, RNA-based responses play a major role in the silencing of viral RNAs. We review here our current state of knowledge on insect antiviral defense mechanisms, which include conserved as well as adaptive, insect-specific strategies. Identification of the innate immunity receptors that sense viral infection in insects remains a major challenge for the field.

Dicistrovirus-Host Molecular Interactions.

Members of the family Dicistroviridae are small RNA viruses containing a monopartite positive-sense RNA genome. Dicistroviruses mainly infect arthropods, causing diseases that impact agriculture and the economy. In this chapter, we provide an overview of current and past research on dicistroviruses including the viral life cycle, viral translational control mechanisms, virus structure, and the use of dicistrovirus infection in Drosophila as a model to identify insect antiviral responses. We then delve into how research on dicistrovirus mechanisms has yielded insights into ribosome dynamics, RNA structure/function and insect innate immunity signaling. Finally, we highlight the diseases caused by dicistroviruses, their impacts on agriculture including the shrimp and honey bee industries, and the potential use of dicistroviruses as biopesticides. Although knowledge of the mechanisms underlying dicistrovirus virus-host interactions is limited, the establishment of the first infectious clone should accelerate the discovery of new mechanistic insights into dicistrovirus infections and pathogenesis.

ICTV Virus Taxonomy Profile: Nudiviridae.

Members of the family Nudiviridae are large dsDNA viruses with distinctive rod-shaped nucleocapsids and circular genomes of 96-232 kbp. Nudiviruses have been identified from a diverse range of insects and crustaceans and are closely related to baculoviruses. This is a summary of the International Committee on Taxonomy of Viruses Report on the taxonomy of the family Nudiviridae, which is available at ictv.global/report/nudiviridae.

ICTV Virus Taxonomy Profile: Chrysoviridae.

Members of the family Chrysoviridae are isometric, non-enveloped viruses with segmented, linear, dsRNA genomes. There are 3-7 genomic segments, each of which is individually encapsidated. Chrysoviruses infect fungi, plants and possibly insects, and may cause hypovirulence in their fungal hosts. Chrysoviruses have no known vectors and lack an extracellular phase to their replication cycle; they are transmitted via intracellular routes within an individual during hyphal growth, in asexual or sexual spores, or between individuals via hyphal anastomosis. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of the family Chrysoviridae, which is available at ictv.global/report/chrysoviridae.

Functional analysis of the baculovirus per os infectivity factors 3 and 9 by imaging the interaction between fluorescently labelled virions and isolated midgut cells.

Baculovirus occlusion-derived viruses (ODVs) contain ten known per os infectivity factors (PIFs). These PIFs are crucial for midgut infection of insect larvae and form, with the exception of PIF5, an ODV entry complex. Previously, R18-dequenching assays have shown that PIF3 is dispensable for binding and fusion with midgut epithelial cells. Oral infection nevertheless fails in the absence of PIF3. PIF9 has not been analysed in much depth yet. Here, the biological role of these two PIFs in midgut infection was examined by monitoring the fate of fluorescently labelled ODVs when incubated with isolated midgut cells from Spodoptera exigua larvae. Confocal microscopy showed that in the absence of either PIF3 or PIF9, the ODVs bound to the brush borders, but the nucleocapsids failed to enter the cells. Finally, we discuss how the results obtained for PIF3 with dequenching assays and confocal microscopy can be explained by a two-phase fusion process.

Discovery and characterization of a novel insect-specific reovirus isolated from Psammotettix alienus.

A novel double-stranded RNA (dsRNA) virus designated Psammotettix alienus reovirus (PARV) was found in the leafhopper Psammotettix alienus in China. Spherical particles approximately 70 nm in diameter arranged in a crystalline array were observed in the salivary gland tissues of infected leafhoppers by transmission electron microscopy. Some viral particles were also encased in tubules, similar to those of previously described reoviruses. Whole-genome sequencing revealed that the dsRNA genome of PARV consists of 29 569 nucleotides (nt) divided into 10 segments ranging from 4403 to 1476 nt, with low G+C content (29.5-36.5 %). All segments contained conserved terminal sequences (5'AAC…GUCA3') and specific panhandle structures formed by inverted terminal repeats in the noncoding regions. Phylogenetic analysis based on the deduced RNA-dependent RNA polymerase (RdRp) revealed that PARV was in the fijivirus clade, but in a monophyletic lineage with an unassigned insect reovirus (Hubei insect virus 2, HBIV-2), although PARV and HBIV-2 are distinct enough to represent a new group within the genus Fijivirus. Biological assays showed that PARV infects P. alienus but not wheat plants, implying that it is a new insect-specific reovirus in the leafhopper. Given these features, PARV should be considered as a new species in the genus Fijivirus, family Reoviridae.

Baculovirus Per Os Infectivity Factor Complex: Components and Assembly.

Baculovirus entry into insect midgut cells is dependent on a multiprotein complex of per os infectivity factors (PIFs) on the envelopes of occlusion-derived virions (ODVs). The structure and assembly of the PIF complex are largely unknown. To reveal the complete members of the complex, a combination of blue native polyacrylamide gel electrophoresis, liquid chromatography-tandem mass spectrometry, and Western blotting was conducted on three different baculoviruses. The results showed that the PIF complex has a molecular mass of ∼500 kDa and consists of nine PIFs, including a newly discovered member (PIF9). To decipher the assembly process, each pif gene was knocked out from the Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) genome individually by use of synthetic baculovirus technology, and the impact on PIF complex formation was investigated. Deletion of pif8 resulted in the formation of an ∼400-kDa subcomplex. Deletion of pif0, -4, -6, -7, or -9 resulted in a subcomplex of ∼230 kDa, but deletion of pif1, -2, or -3 abolished formation of any complex. Taken together, our data identified a core complex of ∼230 kDa, consisting of PIF1, -2, and -3. This revised the previous knowledge that the core complex was about 170 kDa and contained PIF1 to -4. Analysis of the PIF complex in cellular fractions suggested that it is assembled in the cytoplasm before being transported to the nucleus and subsequently incorporated into the envelopes of ODVs. Only the full complex, not the subcomplex, is resistant to proteolytic attack, indicating the essentiality of correct complex assembly for oral infection.IMPORTANCE Entry of baculovirus into host insects is mediated by a per os infectivity factor (PIF) complex on the envelopes of occlusion-derived viruses (ODVs). Knowledge of the composition and structure of the PIF complex is fundamental to understanding its mode of action. By using multiple approaches, we determined the complete list of proteins (nine) in the PIF complex. In contrast to previous knowledge in the field, the core complex is revised to ∼230 kDa and consists of PIF1 to -3 but not PIF4. Interestingly, our results suggest that the PIF complex is formed in the cytoplasm prior to its transport to the nucleus and subsequent incorporation into ODVs. Only the full complex is resistant to proteolytic degradation in the insect midgut, implying the critical role of the entire complex. These findings provide the baseline for future studies on the ODV entry mechanism mediated by the multiprotein complex.

Horizontal Transfer of a Retrotransposon from the Rice Planthopper to the Genome of an Insect DNA Virus.

Horizontal transfer of genetic materials between virus and host has been frequently identified. Three rice planthoppers, Laodelphax striatellus, Nilaparvata lugens, and Sogatella furcifera, are agriculturally important insects because they are destructive rice pests and also the vector of a number of phytopathogenic viruses. In this study, we discovered that a small region (∼300 nucleotides [nt]) of the genome of invertebrate iridescent virus 6 (IIV-6; genus Iridovirus, family Iridoviridae), a giant DNA virus that infects invertebrates but is not known to infect planthoppers, is highly homologous to the sequences present in high copy numbers in these three planthopper genomes. These sequences are related to the short interspersed nuclear elements (SINEs), a class of non-long terminal repeat (LTR) retrotransposons (retroposons), suggesting a horizontal transfer event of a transposable element from the rice planthopper genome to the IIV-6 genome. In addition, a number of planthopper transcripts mapped to these rice planthopper SINE-like sequences (RPSlSs) were identified and appear to be transcriptionally regulated along the different developmental stages of planthoppers. Small RNAs derived from these RPSlSs are predominantly 26 to 28 nt long, which is a typical characteristic of PIWI-interacting RNAs. Phylogenetic analysis suggests that IIV-6 acquires a SINE-like retrotransposon from S. furcifera after the evolutionary divergence of the three rice planthoppers. This study provides further examples of the horizontal transfer of an insect transposon to virus and suggests the association of rice planthoppers with iridoviruses in the past or present.IMPORTANCE This study provides an example of the horizontal transfer event from a rice planthopper genome to an IIV-6 genome. A small region of the IIV-6 genome (∼300 nt) is highly homologous to the sequences presented in high copy numbers of three rice planthopper genomes that are related to the SINEs, a class of retroposons. The expression of these planthopper SINE-like sequences was confirmed, and corresponding Piwi-interacting RNA-like small RNAs were identified and comprehensively characterized. Phylogenetic analysis suggests that the giant invertebrate iridovirus IIV-6 obtains this SINE-related sequence from Sogatella furcifera through a horizontal transfer event in the past. To the best of our knowledge, this is the first report of a horizontal transfer event between a planthopper and a giant DNA virus and also is the first evidence for the eukaryotic origin of genetic material in iridoviruses.

Antigenicity and Immunogenicity of Differentially Glycosylated Hepatitis C Virus E2 Envelope Proteins Expressed in Mammalian and Insect Cells.

The development of a prophylactic vaccine for hepatitis C virus (HCV) remains a global health challenge. Cumulative evidence supports the importance of antibodies targeting the HCV E2 envelope glycoprotein to facilitate viral clearance. However, a significant challenge for a B cell-based vaccine is focusing the immune response on conserved E2 epitopes capable of eliciting neutralizing antibodies not associated with viral escape. We hypothesized that glycosylation might influence the antigenicity and immunogenicity of E2. Accordingly, we performed head-to-head molecular, antigenic, and immunogenic comparisons of soluble E2 (sE2) produced in (i) mammalian (HEK293) cells, which confer mostly complex- and high-mannose-type glycans; and (ii) insect (Sf9) cells, which impart mainly paucimannose-type glycans. Mass spectrometry demonstrated that all 11 predicted N-glycosylation sites were utilized in both HEK293- and Sf9-derived sE2, but that N-glycans in insect sE2 were on average smaller and less complex. Both proteins bound CD81 and were recognized by conformation-dependent antibodies. Mouse immunogenicity studies revealed that similar polyclonal antibody responses were generated against antigenic domains A to E of E2. Although neutralizing antibody titers showed that Sf9-derived sE2 induced moderately stronger responses than did HEK293-derived sE2 against the homologous HCV H77c isolate, the two proteins elicited comparable neutralization titers against heterologous isolates. Given that global alteration of HCV E2 glycosylation by expression in different hosts did not appreciably affect antigenicity or overall immunogenicity, a more productive approach to increasing the antibody response to neutralizing epitopes may be complete deletion, rather than just modification, of specific N-glycans proximal to these epitopes.IMPORTANCE The development of a vaccine for hepatitis C virus (HCV) remains a global health challenge. A major challenge for vaccine development is focusing the immune response on conserved regions of the HCV envelope protein, E2, capable of eliciting neutralizing antibodies. Modification of E2 by glycosylation might influence the immunogenicity of E2. Accordingly, we performed molecular and immunogenic comparisons of E2 produced in mammalian and insect cells. Mass spectrometry demonstrated that the predicted glycosylation sites were utilized in both mammalian and insect cell E2, although the glycan types in insect cell E2 were smaller and less complex. Mouse immunogenicity studies revealed similar polyclonal antibody responses. However, insect cell E2 induced stronger neutralizing antibody responses against the homologous isolate used in the vaccine, albeit the two proteins elicited comparable neutralization titers against heterologous isolates. A more productive approach for vaccine development may be complete deletion of specific glycans in the E2 protein.

Host Range and Population Survey of Spodoptera frugiperda Rhabdovirus.

The Sf9 and Sf21 cell lines derived from ovarian tissues of the wide-host-range phytophagous lepidopteran Spodoptera frugiperda are widely used for research and commercial-scale production of recombinant proteins. These cell lines are chronically infected with a rhabdovirus (Sf-RV) that does not cause any overt cytopathic effects. We demonstrate that wild populations of S. frugiperda in the eastern United States and Caribbean are infected with genetically diverse strains of Sf-RV and that this virus is also capable of infecting cells of Spodoptera exigua, Heliothis subflexa, and Bombyx mori Feeding studies demonstrated the ability of S. frugiperda larvae to deposit Sf-RV onto human-consumed vegetables during feeding. Although no evidence for replication in two species of plant cells was detected, subcellular localization studies demonstrated that the Sf-RV nucleocapsid was targeted to plasmodesmata, while two forms of the accessory protein were differentiated on the basis of their ability to localize to nuclei. Collectively, the results from this study suggest that environmental exposure of humans to Sf-RV is likely to be commonplace and frequent, but its inability to replicate in plant or human cells suggests that there is no substantial risk to human health.IMPORTANCE Insect-derived cell lines are widely used commercially for the production of vaccines and protein-based pharmaceuticals. After decades of safe and beneficial use, it was a surprise to the biotechnology industry to discover an endemic rhabdovirus in Sf9 cells. This discovery was made possible only by the substantial advancements in DNA sequencing technologies. Given the public health concerns associated with many rhabdovirus species, several initiatives were undertaken to establish that Spodoptera frugiperda rhabdovirus (Sf-RV) does not pose a threat to humans. Such actions include the generation of cell lines that have been cleared of Sf-RV. Given that Sf9 is derived from a moth whose larvae feed on human-edible foods, we explored the prevalence of Sf-RV in its wild and lab-grown populations, as well as its ability to be deposited on food items during feeding. Collectively, our data suggest that there is no overt risk from exposure to Sf-RV.