Insect Viruses Research Articles

New clades of viruses infecting the obligatory biotroph Bremia lactucae representing distinct evolutionary trajectory for viruses infecting oomycetes.

Recent advances in high throughput sequencing (HTS) approaches allowed a broad exploration of viromes from different fungal hosts, unveiling a great diversity of mycoviruses with interesting evolutionary features. The word mycovirus historically applies also to viruses infecting oomycetes but most studies are on viruses infecting fungi, with less mycoviruses found and characterized in oomycetes, particularly in the obligatory biotrophs. We, here, describe the first virome associated to Bremia lactucae, the causal agent of lettuce downy mildew, which is an important biotrophic pathogen for lettuce production and a model system for the molecular aspects of the plant-oomycetes interactions. Among the identified viruses, we could detect (1) two new negative sense ssRNA viruses related to the yueviruses, (2) the first example of permuted RdRp in a virus infecting fungi/oomycetes, (3) a new group of bipartite dsRNA viruses showing evidence of recent bi-segmentation and concomitantly, a possible duplication event bringing a bipartite genome to tripartite, (4) a first representative of a clade of viruses with evidence of recombination between distantly related viruses, (5) a new open reading frame (ORF)an virus encoding for an RdRp with low homology to known RNA viruses, and (6) a new virus, belonging to riboviria but not conserved enough to provide a conclusive phylogenetic placement that shows evidence of a recombination event between a kitrinoviricota-like and a pisuviricota-like sequence. The results obtained show a great diversity of viruses and evolutionary mechanisms previously unreported for oomycetes-infecting viruses, supporting the existence of a large diversity of oomycetes-specific viral clades ancestral of many fungal and insect virus clades.

Nearly complete genome sequences of the first two identified Colorado potato beetle viruses

The Colorado potato beetle is one of the most devastating potato pests in the world. However, its viral pathogens, which might have potential in pest control, have remained unexplored. With high-throughput sequencing of Colorado potato beetle samples derived from prepupal larvae which died from an unknown infection, we have identified two previously unknown RNA viruses and assembled their nearly complete genome sequences. The subsequent genetic and phylogenetic analysis demonstrated that the viruses, tentatively named Leptinotarsa iflavirus 1 and Leptinotarsa solinvi-like virus 1, are the novel representatives of the Iflaviridae and Solinviviridae families, respectively. To the best of our knowledge, these are the first sequencing-confirmed insect viruses derived from Colorado potato beetle samples. We propose that Leptinotarsa iflavirus 1 may be associated with a lethal disease in the Colorado potato beetle.

Expansion and Storage of Insect Cells and Virus Stocks.

Healthy insect cell cultures are critical for any method described in this book, including making productive baculovirus banks, protein or AAV expression, and determining viral titers. This chapter describes cell maintenance in shake flasks using serum-free conditions and the expansion of virus stocks from a single plaque purified virus. Insect cells can be passaged over multiple generations, but as the cells may undergo changes over multiple passages, limiting the use of your cells to a defined number of passages such as 50 passages is recommendable. Baculovirus stocks once created using serum-free media are not very stable at 4-8°C. This chapter also includes a simple method to store cells from an early cell passage and your virus stock in liquid nitrogen.

Overexpression of Endoplasmic Reticulum Proteins from Arabidopsis thaliana in Baculovirus.

The overproduction of proteins of the endoplasmic reticulum (ER) of plant cells in prokaryotic heterologous gene expression system remains a technical challenge. Recent advances in genetically modified insect cell technology and virus engineering methods have paved the way to produce recombinant ER plant proteins, including those harboring posttranslational modifications, and therefore, to yield ER plant proteins that are natively folded and fully functional. The present contribution focuses on the baculovirus-expression system flashBAC, which overcomes certain technical hurdles found in other insect cell-based expression systems such as the generation of a bacmid and the negative selection of recombinant clones.

Plant Expression of Trans-Encapsidated Chimeric Viral Vaccines with Animal RNA Replicons: An Update.

In this protocol, we outline how to produce a chimeric viral vaccine in a biosafety level 1 (BSL1) environment. An animal viral vector RNA encapsidated with tobacco mosaic virus (TMV) coat protein can be fully assembled in planta. Agrobacterium cultures containing each component are inoculated together into tobacco leaves and the self-assembled hybrid chimeric viral vaccine is harvested 4days later and purified with a simple PEG precipitation. The viral RNA delivery vector is derived from the BSL1 insect virus, Flock House virus (FHV), and replicates in human and animal cells but does not spread systemically. A polyethylene glycol purification protocol is also provided to collect and purify these vaccines for immunological tests. In this update, we also provide a protocol for in trans co-inoculation of a modified FHV protein A, which significantly increased the yield of in planta chimeric viral vaccine.

Leveraging insect viruses and genetic manipulation for sustainable agricultural pest control.

The potential of insect viruses in the biological control of agricultural pests is well-recognized, yet their practical application faces obstacles such as host specificity, variable virulence, and resource scarcity. High-throughput sequencing (HTS) technologies have significantly advanced our capabilities in discovering and identifying new insect viruses, thereby enriching the arsenal for pest management. Concurrently, progress in reverse genetics has facilitated the development of versatile viral expression vectors. These vectors have enhanced the specificity and effectiveness of insect viruses in targeting specific pests, offering a more precise approach to pest control. This review provides a comprehensive examination of the methodologies employed in the identification of insect viruses using HTS. Additionally, it explores the domain of genetically modified insect viruses and their associated challenges in pest management. The adoption of these cutting-edge approaches holds great promise for developing environmentally sustainable and effective pest control solutions. © 2023 Society of Chemical Industry.

Structure and energetics guide dynamic behaviour in a T = 3 icosahedral virus capsid

Although virus capsids appear as rigid, symmetric particles in experimentally determined structures; biochemical studies suggest a significant degree of structural flexibility in the particles. We carried out all-atom simulations on the icosahedral capsid of an insect virus, Flock House Virus, which show intriguing differences in the degree of flexibility of quasi-equivalent capsid subunits consistent with previously described biological behaviour. The flexibility of all the β and γ subunits of the protein and RNA fragments is analysed and compared. Both γA subunit and RNA fragment exhibit higher flexibility than the γB and γC subunits. The capsid shell is permeable to the bidirectional movement of water molecules, and the movement is heavily influenced by the geometry of the capsid shell along specific symmetry axes. In comparison to the symmetry axes along I5 and I3, the I2 axis exhibits a slightly higher water content. This enriched water environment along I2 could play a pivotal role in facilitating the structural transitions necessary for RNA release, shedding some light on the intricate and dynamic processes underlying the viral life cycle. Our study suggests that the physical characterization of whole virus capsids is the key to identifying biologically relevant transition states in the virus life cycle and understanding the basis of virus infectivity.

Processing of the 3C/D Region of the Deformed Wing Virus (DWV).

The deformed wing virus (DWV) belongs to the genus Iflavirus and the family Iflaviridae within the order Picornavirales. It is an important pathogen of the Western honey bee, Apis mellifera, causing major losses among honey bee colonies in association with the ectoparasitic mite Varroa destructor. Although DWV is one of the best-studied insect viruses, the mechanisms of viral replication and polyprotein processing have been poorly studied in the past. We investigated the processing of the protease-polymerase region at the C-terminus of the polyprotein in more detail using recombinant expression, novel serological reagents, and virus clone mutagenesis. Edman degradation of purified maturated polypeptides uncovered the C- and N-termini of the mature 3C-like (3CL) protease and RNA-dependent RNA polymerase (3DL, RdRp), respectively. Autocatalytic processing of the recombinant DWV 3CL protease occurred at P1 Q2118 and P1' G2119 (KPQ/GST) as well as P1 Q2393 and P1' S2394 (HAQ/SPS) cleavage sites. New monoclonal antibodies (Mab) detected the mature 3CL protease with an apparent molecular mass of 32 kDa, mature 3DL with an apparent molecular mass of 55 kDa as well as a dominant 3CDL precursor of 90 kDa in DWV infected honey bee pupae. The observed pattern corresponds well to data obtained via recombinant expression and N-terminal sequencing. Finally, we were able to show that 3CL protease activity and availability of the specific protease cleavage sites are essential for viral replication, protein synthesis, and establishment of infection using our molecular clone of DWV-A.

Flexible changes to the Heliothis virescens ascovirus 3h (HvAV-3h) virion components affect pathogenicity against different host larvae species

ABSTRACT The pathogenicity of a virus to a specific host species is an inerratic and describable ability of a virus to cause infection but is generally shaped by a variety of abiotic and biotic factors. In this investigation, the variations in pathogenicity of Heliothis virescens ascovirus 3h (HvAV-3h) to five noctuid pests were assessed based on mass spectrometry analysis on the virion compositions. Twenty-nine common HvAV-3h proteins were shared across all hosts, and different flexible proteins were identified in the virions of each specific host. Different host proteins were identified as HvAV-3h virion-associated proteins, including different detoxification enzyme proteins. Furthermore, a relatively fixed relationship between viral replication and changes in host detoxification enzyme activity caused by deficiencies in various viral structural proteins was found in the host larvae using a correlation matrix analysis: the host larval carboxylesterase and cytochrome P450 monooxygenases generally had highly similar responses to the viruses blocked by different structural proteins’ antisera and their effects on viral DNA replication. Different interaction patterns for the virion structural proteins were found in different host larvae-produced virions, and the interactions between Spodoptera litura glutathione S-transferases and viral structural proteins were confirmed. The different host responses after viral infection could be the reason for the changes in viral pathogenicity, while the virus responses gradually adapted to the different hosts and there were flexible changes in the virion structures. 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.

The intriguing phenomenon of cross-kingdom infections of plant and insect viruses to fungi: Can other animal viruses also cross-infect fungi?

Fungi are highly widespread and commonly colonize multicellular organisms that live in natural environments. Notably, studies on viruses infecting plant-associated fungi have revealed the interesting phenomenon of the cross-kingdom transmission of viruses and viroids from plants to fungi. This implies that fungi, in addition to absorbing water, nutrients, and other molecules from the host, can acquire intracellular parasites that reside in the host. These findings further suggest that fungi can serve as suitable alternative hosts for certain plant viruses and viroids. Given the frequent coinfection of fungi and viruses in humans/animals, the question of whether fungi can also acquire animal viruses and serve as their hosts is very intriguing. In fact, the transmission of viruses from insects to fungi has been observed. Furthermore, the common release of animal viruses into the extracellular space (viral shedding) could potentially facilitate their acquisition by fungi. Investigations of the cross-infection of animal viruses in fungi may provide new insights into the epidemiology of viral diseases in humans and animals.

Complex interactions among insect viruses-insect vector-arboviruses.

Insects are the host or vector of diverse viruses including those that infect vertebrates, plants, and fungi. Insect viruses reside inside their insect hosts and are vertically transmitted from parent to offspring. The insect virus-host relationship is intricate, as these viruses can impact various aspects of insect biology, such as development, reproduction, sex ratios, and immunity. Arthropod-borne viruses (arboviruses) that cause substantial global health or agricultural problems can also be vertically transmitted to insect vector progeny. Multiple infections with insect viruses and arboviruses are common in nature. Such coinfections involve complex interactions, including synergism, dependence, and antagonism. Recent studies have shed light on the influence of insect viruses on the competence of insect vectors for arboviruses. In this review, we focus on the biological effects of insect viruses on the transmission of arboviruses by insects. We also discuss the potential mechanisms by which insect viruses affect the ability of hosts to transmit arboviruses, as well as potential strategies for disease control through manipulation of insect viruses. Analyses of the interactions among insect vectors, insect viruses and arboviruses will provide new opportunities for development of innovative strategies to control arbovirus transmission.

Virome characterization and identification of a putative parvovirus and poxvirus in bat ectoparasites of Yunnan Province, China

Ectoparasites found on bats are known to contain important microbes. However, the viruses hosted by these obligate parasites are understudied. This has led to the near oversight of the potential role of these ectoparasites in virus maintenance and transmission from bats to other interacting species and the environment. Here, we sampled bat ectoparasites parasitizing a diverse selection of bat species in the families Rhinolophidae, Vespertilionidae, Megadermatidae, Hipposideridae and Pteropodidae in Yunnan Province, China. We show that the ectoparasite prevalence was generally higher in male compared to female bats. Most ectoparasites were found to fall within the Nycteribiidae, Spinturnicidae and Streblidae bat ectoparasite families. We subsequently applied a non-biased sequencing of libraries prepared from the pooled ectoparasites, followed by an in-silico virus-centric analysis of the resultant reads. We show that ectoparasites hosted by the sampled families of bats are found to carry, in addition to a diverse set of phages, vertebrate and insect viruses in the families Aliusviridae, Ascoviridae, Chuviridae, Circoviridae, Flaviviridae, Hepadnaviridae, Hepeviridae, Herpesviridae, Iridoviridae, Marseilleviridae, Nairoviridae, Orthomyxoviridae, Parvoviridae, Poxviridae, Reoviridae, Retroviridae, and Rhabdoviridae. We further report a partial Parvovirus VP1/VP2 gene and partial Poxvirus ubiquitin-like gene predicted by two independent next generation sequencing data analysis pipelines. This study describes the natural virome of bat ectoparasites, providing a platform for understanding the role these ectoparasites play in the maintenance and spread of viruses to other animals.

Polyubiquitin protein of Aedes aegypti as an interacting partner of dengue virus envelope protein.

Dengue virus (DENV) is an arbovirus that comprises four antigenically different serotypes. Aedes aegypti (Diptera: Culicidae) acts as the principal vector for DENV transmission, and vector control is crucial for dengue fever epidemic management. To design effective vector control strategies, a comprehensive understanding of the insect vector and virus interaction is required. Female Ae. aegypti ingests DENV during the acquisition of a blood meal from an infected human. DENV enters the insect midgut, replicates inside it and reaches the salivary gland for transmitting DENV to healthy humans during the subsequent feeding cycles. DENV must interact with the proteins present in the midgut and salivary glands to gain entry and accomplish successful replication and transmission. Ae. aegypti midgut cDNA library was prepared, and yeast two-hybrid screening was performed against the envelope protein domain III (EDIII) protein of DENV-2. The polyubiquitin protein was selected from the various candidate proteins for subsequent analysis. Polyubiquitin gene was amplified, and the protein was purified in a heterologous expression system for in vitro interaction studies. In vitro pull-down assay presented a clear interaction between polyubiquitin protein and EDIII. To further confirm this interaction, a dot blot assay was employed, and polyubiquitin protein was found to interact with DENV particles. Our results enable us to suggest that polyubiquitin plays an important role in DENV infection within mosquitoes.

Viral Prevalence and Genomic Xenology in the Coevolution of HzNV-2 (Nudiviridae) with Host Helicoverpa zea (Lepidoptera: Noctuidae).

Insect viruses have been described from numerous lineages, yet patterns of genetic exchange and viral prevalence, which are essential to understanding host-virus coevolution, are rarely studied. In Helicoverpa zea, the virus HzNV-2 can cause deformity of male and female genitalia, resulting in sterility. Using ddPCR, we found that male H. zea with malformed genitalia (agonadal) contained high levels of HzNV-2 DNA, confirming previous work. HzNV-2 was found to be prevalent throughout the United States, at more than twice the rate of the baculovirus HaSNPV, and that it contained several host-acquired DNA sequences. HzNV-2 possesses four recently endogenized lepidopteran genes and several more distantly related genes, including one gene with a bacteria-like sequence found in both host and virus. Among the recently acquired genes is cytosolic serine hydroxymethyltransferase (cSHMT). In nearly all tested H. zea, cSHMT contained a 200 bp transposable element (TE) that was not found in cSHMT of the sister species H. armigera. No other virus has been found with host cSHMT, and the study of this shared copy, including possible interactions, may yield new insights into the function of this gene with possible applications to insect biological control, and gene editing.

Co-Occurrence of Wing Deformity and Impaired Mobility of Alates with Deformed Wing Virus in Solenopsis invicta Buren (Hymenoptera: Formicidae).

Deformed wing virus (DWV), a major honey bee pathogen, is a generalist insect virus detected in diverse insect phyla, including numerous ant genera. Its clinical symptoms have only been reported in honey bees, bumble bees, and wasps. DWV is a quasispecies virus with three main variants, which, in association with the ectoparasitic mite, Varroa destructor, causes wing deformity, shortened abdomens, neurological impairments, and colony mortality in honey bees. The red imported fire ant, Solenopsis invicta Buren, is one of the most-invasive and detrimental pests in the world. In this study, we report the co-occurrence of DWV-like symptoms in S. invicta and DWV for the first time and provide molecular evidence of viral replication in S. invicta. Some alates in 17 of 23 (74%) lab colonies and 9 of 14 (64%) field colonies displayed deformed wings (DWs), ranging from a single crumpled wing tip to twisted, shriveled wings. Numerous symptomatic alates also exhibited altered locomotion ranging from an altered gait to the inability to walk. Deformed wings may prevent S. invicta alates from reproducing since mating only occurs during a nuptial flight. The results from conventional RT-PCR and Sanger sequencing confirmed the presence of DWV-A, and viral replication of DWV was confirmed using a modified strand-specific RT-PCR. Our results suggest that S. invicta can potentially be an alternative and reservoir host for DWV. However, further research is needed to determine whether DWV is the infectious agent that causes the DW syndrome in S. invicta.

Warmer temperatures reduce the transmission of a virus in a gregarious forest insect.

Understanding how climate warming will influence species interactions is a key question in ecology and predicting changes in the prevalence of disease outbreaks is particularly challenging. Ectotherms are likely to be more influenced by climatic changes as temperature governs their growth, feeding, development, and behavior. We test the hypothesis that pathogen transmission and host mortality will increase at warmer temperatures using a cyclic forest insect, the western tent caterpillar (WTC), Malacosoma californicum pluviale, and its baculovirus. The virus causes population declines at peak host density. WTC are gregarious and clustering is predicted to increase the risk of within family infection; however, how temperature influences this has not been examined. We investigated the impact of temperature on different components of the transmission process in order to pinpoint the possible mechanisms involved. In the laboratory, leaf consumption increased linearly with rising temperature between 15 and 30°C. Insects died more rapidly from virus infection as temperature increased, but this did not translate into differences in the production of viral transmission stages. To examine the influence of temperature on virus transmission, we created a temperature difference between two greenhouses containing potted red alder trees, Alnus rubra. The cooler greenhouse (mean 19.5°C) was roughly similar to ambient temperatures in the field, while the warmer greenhouse was 10°C higher (mean 29°C). As predicted, both larval movement and feeding were higher at the warmer temperature, while the likelihood of the preinfected, inoculum larvae dying on the tents was twice as high in the cooler greenhouse. This resulted in increased virus mortality and a higher transmission parameter under cooler conditions. Therefore, we suggest that, contrary to our prediction, the reduced movement of infected larvae at colder temperatures increased the risk of infection in these gregarious insects and had a greater impact on virus transmission than the increased activity of the susceptible larvae in warmer conditions. Long-term population data from the field, however, show no relationship between temperature and infection levels, suggesting that local changes in virus transmission might not scale up to population infection levels.

Mining Public Data to Investigate the Virome of Neglected Pollinators and Other Floral Visitors.

This study reports the virome investigation of pollinator species and other floral visitors associated with plants from the south of Bahia: Aphis aurantii, Atrichopogon sp., Dasyhelea sp., Forcipomyia taiwana, and Trigona ventralis hoozana. Studying viruses in insects associated with economically important crops is vital to understand transmission dynamics and manage viral diseases that pose as threats for global food security. Using literature mining and public RNA next-generation sequencing data deposited in the NCBI SRA database, we identified potential vectors associated with Malvaceae plant species and characterized the microbial communities resident in these insects. Bacteria and Eukarya dominated the metagenomic analyses of all taxon groups. We also found sequences showing similarity to elements from several viral families, including Bunyavirales, Chuviridae, Iflaviridae, Narnaviridae, Orthomyxoviridae, Rhabdoviridae, Totiviridae, and Xinmoviridae. Phylogenetic analyses indicated the existence of at least 16 new viruses distributed among A. aurantii (3), Atrichopogon sp. (4), Dasyhelea sp. (3), and F. taiwana (6). No novel viruses were found for T. ventralis hoozana. For F. taiwana, the available libraries also allowed us to suggest possible vertical transmission, while for A. aurantii we followed the infection profile along the insect development. Our results highlight the importance of studying the virome of insect species associated with crop pollination, as they may play a crucial role in the transmission of viruses to economically important plants, such as those of the genus Theobroma, or they will reduce the pollination process. This information may be valuable in developing strategies to mitigate the spread of viruses and protect the global industry.

Exploring viral infections in honey bee colonies: insights from a metagenomic study in southern Brazil.

The decline in honey bee colonies in different parts of the world in recent years is due to different reasons, such as agricultural practices, climate changes, the use of chemical insecticides, and pests and diseases. Viral infections are one of the main causes leading to honey bee population declines, which have a major economic impact due to honey production and pollination. To investigate the presence of viruses in bees in southern Brazil, we used a metagenomic approach to sequence adults' samples of concentrated extracts from Apis mellifera collected in fifteen apiaries of six municipalities in the Rio Grande do Sul state, Brazil, between 2016 and 2017. High-throughput sequencing (HTS) of these samples resulted in the identification of eight previously known viruses (Apis rhabdovirus 1 (ARV-1), Acute bee paralysis virus (ABPV), Aphid lethal paralysis virus (ALPV), Black queen cell virus (BQCV), Bee Macula-like virus (BeeMLV), Deformed wing virus (DWV), Lake Sinai Virus NE (LSV), and Varroa destructor virus 3 (VDV-3)) and a thogotovirus isolate. This thogotovirus shares high amino acid identities in five of the six segments with Varroa orthomyxovirus 1, VOV-1 (98.36 to 99.34% identity). In contrast, segment 4, which codes for the main glycoprotein (GP), has no identity with VOV-1, as observed for the other segments, but shares an amino acid identity of 34-38% with other glycoproteins of viruses from the Orthomyxoviridae family. In addition, the putative thogotovirus GP also shows amino acid identities ranging from 33 to 41% with the major glycoprotein (GP64) of insect viruses of the Baculoviridae family. To our knowledge, this is the second report of a thogotovirus found in bees and given this information, this thogotovirus isolate was tentatively named Apis thogotovirus 1 (ATHOV-1). The detection of multiple viruses in bees is important to better understand the complex interactions between viruses and their hosts. By understanding these interactions, better strategies for managing viral infections in bees and protecting their populations can be developed.

Bioreactor Production Process of Spodoptera frugiperda multiple nucleopolyhedrovirus Biopesticide.

Spodoptera frugiperda (fall armyworm) is one of the most important maize pests in the world and the baculovirus Spodoptera frugiperda multiple nucleopolyhedrovirus (SfMNPV), a natural pathogen of this pest, has been used as a biopesticide for its control. At present, in vivo strategies at the commercial scale are employed by multiplying the virus in the host insect in biofactory facilities; however, in vitro large-scale production is an interesting alternative to overcome the limitations of baculoviruses massal production. This study aimed to develop the process of the SfMNPV in vitro production by evaluating the effects of different multiplicities of infection (MOI) and nutritional supplements, morphological and molecular analysis of the infection on the growth of Sf9 cells and virus production. The Bioreactor Stirred Tank Reactor (STR) approach with glutamine-supplemented Sf-900 III serum free culture medium, combined with the MOI of 1.0, showed the best viral production performance, with a specific productivity above 300 occlusion bodies (OBs)/cell and volumetric productivity of 9.0 × 1011 OBs/L.

Sublethal granulovirus infections in the tomato leafminer,Tuta absoluta: transmission and population‐level effects

AbstractGranuloviruses are insect viruses commonly used as biological insecticides for pest control in agriculture. Most pest control programs rely on the most virulent strains and doses to cause acute mortality events. However, we hypothesize that sublethal infections are key to maintaining persistent epidemics that regulate pest populations in the long‐term. The tomato leafminer (TLM),Tuta absoluta(Meyrick) (=Phthorimaea absolutaMeyrick) (Lepidoptera: Gelechiidae), is one of the most limiting pests of greenhouse and field tomatoes. Previous efforts have identified a strain ofPhthorimaea operculellagranulovirus (PhopGV), coded VG013, associated with field populations of the TLM, which causes significant acute mortalities and is being used as active ingredient of a biological insecticide. The aim of this study was to determine the effect of sublethal infections and transmission mechanisms of PhopGV‐VG013 on TLM populations. Six viral doses were tested for their virulence and effects on TLM development rate, and intrinsic population growth rate. A sublethal dose was selected to compare the density‐dependent mortality of TLM populations and vertical transmission was examined by testing for PhopGV in the offspring of surviving infected individuals. Horizontal transmission was assessed by testing for PhopGV in frass of infected larvae, and the infection of virus‐free larvae confined with infected larvae in the same leaf. We found that sublethal doses of PhopGV have slight effects on TLM population growth rates, and none on the strength of density‐dependent mortality. Although we found for the first time that PhopGV‐VG013 is transmitted to approximately 7.5% of the offspring of individuals that survive the virus challenge, we found no evidence suggesting horizontal transmission from sublethal infections. Our results show that sublethal infections of highly virulent ganulovirus strains may favor long‐term pathogen persistence but have a limited role in host population regulation.