Insulin-mediated endothelin signaling is antiviral during West Nile virus infection.

IMPORTANCE: Arboviruses, particularly those transmitted by mosquitoes, pose a significant threat to humans and are an increasing concern because of climate change, human activity, and expanding vector-competent populations. West Nile virus is of significant concern as the most frequent mosquito-borne disease transmitted annually within the continental United States. Here, we identify a previously uncharacterized signaling pathway that impacts West Nile virus infection, namely endothelin signaling. Additionally, we demonstrate that we can successfully translate results obtained from D. melanogaster into the more relevant human system. Our results add to the growing field of insulin-mediated antiviral immunity and identify potential biomarkers or intervention targets to better address West Nile virus infection and severe disease.

A male-killing gene encoded by a symbiotic virus of Drosophila.

In most eukaryotes, biparentally inherited nuclear genomes and maternally inherited cytoplasmic genomes have different evolutionary interests. Strongly female-biased sex ratios that are repeatedly observed in various arthropods often result from the male-specific lethality (male-killing) induced by maternally inherited symbiotic bacteria such as Spiroplasma and Wolbachia. However, despite some plausible case reports wherein viruses are raised as male-killers, it is not well understood how viruses, having much smaller genomes than bacteria, are capable of inducing male-killing. Here we show that a maternally inherited double-stranded RNA (dsRNA) virus belonging to the family Partitiviridae (designated DbMKPV1) induces male-killing in Drosophila. DbMKPV1 localizes in the cytoplasm and possesses only four genes, i.e., one gene in each of the four genomic segments (dsRNA1-dsRNA4), in contrast to ca. 1000 or more genes possessed by Spiroplasma or Wolbachia. We also show that a protein (designated PVMKp1; 330 amino acids in size), encoded by a gene on the dsRNA4 segment, is necessary and sufficient for inducing male-killing. Our results imply that male-killing genes can be easily acquired by symbiotic viruses through reassortment and that symbiotic viruses are hidden players in arthropod evolution. We anticipate that host-manipulating genes possessed by symbiotic viruses can be utilized for controlling arthropods.

A novel transposable element-mediated mechanism causes antiviral resistance in Drosophila through truncating the Veneno protein.

Hosts are continually selected to evolve new defenses against an ever-changing array of pathogens. To understand this process, we examined the genetic basis of resistance to the Drosophila A virus in Drosophila melanogaster. In a natural population, we identified a polymorphic transposable element (TE) insertion that was associated with an ∼19,000-fold reduction in viral titers, allowing flies to largely escape the harmful effects of infection by this virulent pathogen. The insertion occurs in the protein-coding sequence of the gene Veneno, which encodes a Tudor domain protein. By mutating Veneno with CRISPR-Cas9 in flies and expressing it in cultured cells, we show that the ancestral allele of the gene has no effect on viral replication. Instead, the TE insertion is a gain-of-function mutation that creates a gene encoding a novel resistance factor. Viral titers remained reduced when we deleted the TE sequence from the transcript, indicating that resistance results from the TE truncating the Veneno protein. This is a novel mechanism of virus resistance and a new way by which TEs can contribute to adaptation.

Zika Virus Induces Sex-Dependent Metabolic Changes in Drosophila melanogaster to Promote Viral Replication.

Zika is a member of the Flaviviridae virus family that poses some of the most significant global health risks, causing neurologic complications that range from sensory neuropathy and seizures to congenital Zika syndrome (microcephaly) in infants born to mothers infected during pregnancy. The recent outbreak of Zika virus (ZIKV) and its serious health threats calls for the characterization and understanding of Zika pathogenesis, as well as host antiviral immune functions. Although ZIKV has been associated with activating the RNA interference (RNAi) immune pathway and altering host metabolism, in-depth studies are still required to uncover the specifics of the complex host-virus interactions and provide additional insights into the molecular components that determine the outcome of this disease. Previous research establishes the fruit fly Drosophila melanogaster as a reliable model for studying viral pathogens, as it shares significant similarities with that of vertebrate animal systems. Here, we have developed an in vivo Drosophila model to investigate ZIKV-mediated perturbed metabolism in correlation to the RNAi central mediator Dicer-2. We report that ZIKV infection reprograms glucose and glycogen metabolism in Dicer-2 mutants to maintain efficient replication and successful propagation. Flies that exhibit these metabolic effects also show reduced food intake, which highlights the complicated neurological defects associated with ZIKV. We show that ZIKV infection significantly reduces insulin gene expression in Dicer-2 mutants, suggesting an insulin antiviral role against ZIKV and a direct connection to RNAi immunity. Moreover, we find that the insulin receptor substrate chico is crucial to the survival of ZIKV-infected flies. These observations are remarkably more severe in adult female flies compared to males, indicating possible sex differences in the rates of infection and susceptibility to the development of disease. Such findings not only demonstrate that metabolic alterations can be potentially exploited for developing immune therapeutic strategies but also that preventive measures for disease development may require sex-specific approaches. Therefore, further studies are urgently needed to explore the molecular factors that could be considered as targets to inhibit ZIKV manipulation of host cell metabolism in females and males.

Identification of Cellular Genes Involved in Baculovirus GP64 Trafficking to the Plasma Membrane.

The baculovirus envelope protein GP64 is an essential component of the budded virus and is necessary for efficient virion assembly. Little is known regarding intracellular trafficking of GP64 to the plasma membrane, where it is incorporated into budding virions during egress. To identify host proteins and potential cellular trafficking pathways that are involved in delivery of GP64 to the plasma membrane, we developed and characterized a stable Drosophila cell line that inducibly expresses the AcMNPV GP64 protein and used that cell line in combination with a targeted RNA interference (RNAi) screen of vesicular protein trafficking pathway genes. Of the 37 initial hits from the screen, we validated and examined six host genes that were important for trafficking of GP64 to the cell surface. Validated hits included Rab GTPases Rab1 and Rab4, Clathrin heavy chain, clathrin adaptor protein genes AP-1-2ß and AP-2µ, and Snap29. Two gene knockdowns (Rab5 and Exo84) caused substantial increases (up to 2.5-fold) of GP64 on the plasma membrane. We found that a small amount of GP64 is released from cells in exosomes and that some portion of cell surface GP64 is endocytosed, suggesting that recycling helps to maintain GP64 at the cell surface. IMPORTANCE While much is known regarding trafficking of viral envelope proteins in mammalian cells, little is known about this process in insect cells. To begin to understand which factors and pathways are needed for trafficking of insect virus envelope proteins, we engineered a Drosophila melanogaster cell line and implemented an RNAi screen to identify cellular proteins that aid transport of the model baculovirus envelope protein (GP64) to the cell surface. For this we developed an experimental system that leverages the large array of tools available for Drosophila and performed a targeted RNAi screen to identify cellular proteins involved in GP64 trafficking to the cell surface. Since viral envelope proteins are often critical for production of infectious progeny virions, these studies lay the foundation for understanding how either pathogenic insect viruses (baculoviruses) or insect-vectored viruses (e.g., flaviviruses, alphaviruses) egress from cells in tissues such as the midgut to enable systemic virus infection.

A Polydnavirus Protein Tyrosine Phosphatase Negatively Regulates the Host Phenoloxidase Pathway.

Polydnavirus (PDV) is a parasitic factor of endoparasitic wasps and contributes greatly to overcoming the immune response of parasitized hosts. Protein tyrosine phosphatases (PTPs) regulate a wide variety of biological processes at the post-transcriptional level in mammals, but knowledge of PDV PTP action during a parasitoid−host interaction is limited. In this study, we characterized a PTP gene, CvBV_12-6, derived from Cotesia vestalis bracovirus (CvBV), and explored its possible regulatory role in the immune response of the host Plutella xylostella. Our results from qPCR show that CvBV_12-6 was highly expressed in hemocytes at an early stage of parasitization. To explore CvBV_12-6 function, we specifically expressed CvBV_12-6 in Drosophila melanogaster hemocytes. The results show that Hml-Gal4 > CvBV_12-6 suppressed the phenoloxidase activity of hemolymph in D. melanogaster, but exerted no effect on the total count or the viability of the hemocytes. In addition, the Hml-Gal4 > CvBV_12-6 flies exhibited decreased antibacterial abilities against Staphylococcus aureus. Similarly, we found that CvBV_12-6 significantly suppressed the melanization of the host P. xylostella 24 h post parasitization and reduced the viability, but not the number, of hemocytes. In conclusion, CvBV_12-6 negatively regulated both cellular and humoral immunity in P. xylostella, and the related molecular mechanism may be universal to insects.

Innovative Toolbox for the Quantification of Drosophila C Virus, Drosophila A Virus, and Nora Virus.

Quantification of viral replication underlies investigations into host-virus interactions. In Drosophila melanogaster, persistent infections with Drosophila C virus, Drosophila A virus, and Nora virus are commonly observed in nature and in laboratory fly stocks. However, traditional endpoint dilution assays to quantify infectious titers are not compatible with persistently infecting isolates of these viruses that do not cause cytopathic effects in cell culture. Here we present a novel assay based on immunological detection of Drosophila C virus infection that allows quantification of infectious titers for a wider range of Drosophila C virus isolates. We also describe strand specific RT-qPCR assays for quantification of viral negative strand RNA produced during Drosophila C virus, Drosophila A virus, and Nora virus infection. Finally, we demonstrate the utility of these assays for quantification of viral replication during oral infections and persistent infections with each virus.

Wolbachia reduces virus infection in a natural population of Drosophila.

Wolbachia is a maternally transmitted bacterial symbiont that is estimated to infect approximately half of arthropod species. In the laboratory it can increase the resistance of insects to viral infection, but its effect on viruses in nature is unknown. Here we report that in a natural population of Drosophila melanogaster, individuals that are infected with Wolbachia are less likely to be infected by viruses. By characterising the virome by metagenomic sequencing and then testing individual flies for infection, we found the protective effect of Wolbachia was virus-specific, with the prevalence of infection being up to 15% greater in Wolbachia-free flies. The antiviral effects of Wolbachia may contribute to its extraordinary ecological success, and in nature the symbiont may be an important component of the antiviral defences of insects.

Heterogeneity in the Response of Different Subtypes of Drosophila melanogaster Midgut Cells to Viral Infections.

Single-cell RNA sequencing (scRNA-seq) offers the possibility to monitor both host and pathogens transcriptomes at the cellular level. Here, public scRNA-seq datasets from Drosophila melanogaster midgut cells were used to compare the differences in replication strategy and cellular responses between two fly picorna-like viruses, Thika virus (TV) and D. melanogaster Nora virus (DMelNV). TV exhibited lower levels of viral RNA accumulation but infected a higher number of cells compared to DMelNV. In both cases, viral RNA accumulation varied according to cell subtype. The cellular heat shock response to TV and DMelNV infection was cell-subtype- and virus-specific. Disruption of bottleneck genes at later stages of infection in the systemic response, as well as of translation-related genes in the cellular response to DMelNV in two cell subtypes, may affect the virus replication.

Wolbachia-Conferred Antiviral Protection Is Determined by Developmental Temperature.

Wolbachia is a maternally transmitted bacterium that is widespread in arthropods and filarial nematodes and confers strong antiviral protection in Drosophila melanogaster and other arthropods. Wolbachia-transinfected Aedes aegypti mosquitoes are currently being deployed to fight transmission of dengue and Zika viruses. However, the mechanism of antiviral protection and the factors influencing are still not fully understood. Here, we show that temperature modulates Wolbachia-conferred protection in Drosophila melanogaster. Temperature after infection directly impacts Drosophila C virus (DCV) replication and modulates Wolbachia protection. At higher temperatures, viruses proliferate more and are more lethal, while Wolbachia confers lower protection. Strikingly, host developmental temperature is a determinant of Wolbachia-conferred antiviral protection. While there is strong protection when flies develop from egg to adult at 25°C, the protection is highly reduced or abolished when flies develop at 18°C. However, Wolbachia-induced changes during development are not sufficient to limit virus-induced mortality, as Wolbachia is still required to be present in adults at the time of infection. This developmental effect is general, since it was present in different host genotypes, Wolbachia variants, and upon infection with different viruses. Overall, we show that Wolbachia-conferred antiviral protection is temperature dependent, being present or absent depending on the environmental conditions. This interaction likely impacts Wolbachia-host interactions in nature and, as a result, frequencies of host and symbionts in different climates. Dependence of Wolbachia-mediated pathogen blocking on developmental temperature could be used to dissect the mechanistic bases of protection and influence the deployment of Wolbachia to prevent transmission of arboviruses. IMPORTANCE Insects are often infected with beneficial intracellular bacteria. The bacterium Wolbachia is extremely common in insects and can protect them from pathogenic viruses. This effect is being used to prevent transmission of dengue and Zika viruses by Wolbachia-infected mosquitoes. To understand the biology of insects in the wild, we need to discover which factors affect Wolbachia-conferred antiviral protection. Here, we show that the temperature at which insects develop from eggs to adults can determine the presence or absence of antiviral protection. The environment, therefore, strongly influences this insect-bacterium interaction. Our work may help to provide insights into the mechanism of viral blocking by Wolbachia, deepen our understanding of the geographical distribution of host and symbiont, and incentivize further research on the temperature dependence of Wolbachia-conferred protection for control of mosquito-borne disease.

Baculovirus infection affects caterpillar chemoperception.

Baculoviruses are double-stranded DNA entomopathogenic viruses that infect predominantly insects of the order Lepidoptera. Research in the last decade has started to disentangle the mechanisms underlying the insect-virus interaction, particularly focusing on the effects of the baculovirus infection in the host's physiology. Among crucial physiological functions, olfaction has a key role in reproductive tasks, food source detection and enemy avoidance. In this work, we describe that Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) induces expression changes in some odorant receptors (ORs) - the centrepiece of insect's olfaction - when infecting larvae from its natural host Spodoptera exigua (Lepidoptera: Noctuidae). Different ORs are up-regulated in larvae after SeMNPV infection, and two of them, SexiOR35 and SexiOR23, were selected for further functional characterization by heterologous expression in empty neurons of Drosophila melanogaster coupled to single-sensillum recordings. SexiOR35 appears to be a broadly tuned receptor able to recognise multiple and different chemical compounds. SexiOR23, although correctly expressed in Drosophila neurons, did not display any significant response to a panel of 58 stimuli. Behavioural experiments revealed that larvae infected by SeMNPV exhibit altered olfactory-driven behaviour to diet when it is supplemented with the plant volatiles linalool or estragole, two of the main SexiOR35 ligands, supporting the hypothesis that viral infection triggers changes in host perception through changes in the expression level of specific ORs.

cGAS-like receptors sense RNA and control 3'2'-cGAMP signalling in Drosophila.

Cyclic GMP-AMP synthase (cGAS) is a cytosolic DNA sensor that produces the second messenger cG[2'-5']pA[3'-5']p (2'3'-cGAMP) and controls activation of innate immunity in mammalian cells1-5. Animal genomes typically encode multiple proteins with predicted homology to cGAS6-10, but the function of these uncharacterized enzymes is unknown. Here we show that cGAS-like receptors (cGLRs) are innate immune sensors that are capable of recognizing divergent molecular patterns and catalysing synthesis of distinct nucleotide second messenger signals. Crystal structures of human and insect cGLRs reveal a nucleotidyltransferase signalling core shared with cGAS and a diversified primary ligand-binding surface modified with notable insertions and deletions. We demonstrate that surface remodelling of cGLRs enables altered ligand specificity and used a forward biochemical screen to identify cGLR1 as a double-stranded RNA sensor in the model organism Drosophila melanogaster. We show that RNA recognition activates Drosophila cGLR1 to synthesize the novel product cG[3'-5']pA[2'-5']p (3'2'-cGAMP). A crystal structure of Drosophila stimulator of interferon genes (dSTING) in complex with 3'2'-cGAMP explains selective isomer recognition, and 3'2'-cGAMP induces an enhanced antiviral state in vivo that protects from viral infection. Similar to radiation of Toll-like receptors in pathogen immunity, our results establish cGLRs as a diverse family of metazoan pattern recognition receptors.

Two cGAS-like receptors induce antiviral immunity in Drosophila.

In mammals, cyclic GMP-AMP (cGAMP) synthase (cGAS) produces the cyclic dinucleotide 2'3'-cGAMP in response to cytosolic DNA and this triggers an antiviral immune response. cGAS belongs to a large family of cGAS/DncV-like nucleotidyltransferases that is present in both prokaryotes1 and eukaryotes2-5. In bacteria, these enzymes synthesize a range of cyclic oligonucleotides and have recently emerged as important regulators of phage infections6-8. Here we identify two cGAS-like receptors (cGLRs) in the insect Drosophila melanogaster. We show that cGLR1 and cGLR2 activate Sting- and NF-κB-dependent antiviral immunity in response to infection with RNA or DNA viruses. cGLR1 is activated by double-stranded RNA to produce the cyclic dinucleotide 3'2'-cGAMP, whereas cGLR2 produces a combination of 2'3'-cGAMP and 3'2'-cGAMP in response to an as-yet-unidentified stimulus. Our data establish cGAS as the founding member of a family of receptors that sense different types of nucleic acids and trigger immunity through the production of cyclic dinucleotides beyond 2'3'-cGAMP.

Endogenous piRNA-guided slicing triggers responder and trailer piRNA production from viral RNA in Aedes aegypti mosquitoes.

In the germline of animals, PIWI interacting (pi)RNAs protect the genome against the detrimental effects of transposon mobilization. In Drosophila, piRNA-mediated cleavage of transposon RNA triggers the production of responder piRNAs via ping-pong amplification. Responder piRNA 3' end formation by the nuclease Zucchini is coupled to the production of downstream trailer piRNAs, expanding the repertoire of transposon piRNA sequences. In Aedes aegypti mosquitoes, piRNAs are generated from viral RNA, yet, it is unknown how viral piRNA 3' ends are formed and whether viral RNA cleavage gives rise to trailer piRNA production. Here we report that in Ae. aegypti, virus- and transposon-derived piRNAs have sharp 3' ends, and are biased for downstream uridine residues, features reminiscent of Zucchini cleavage of precursor piRNAs in Drosophila. We designed a reporter system to study viral piRNA 3' end formation and found that targeting viral RNA by abundant endogenous piRNAs triggers the production of responder and trailer piRNAs. Using this reporter, we identified the Ae. aegypti orthologs of Zucchini and Nibbler, two nucleases involved in piRNA 3' end formation. Our results furthermore suggest that autonomous piRNA production from viral RNA can be triggered and expanded by an initial cleavage event guided by genome-encoded piRNAs.

Forward genetics in Wolbachia: Regulation of Wolbachia proliferation by the amplification and deletion of an addictive genomic island.

Wolbachia is one of the most prevalent bacterial endosymbionts, infecting approximately 40% of terrestrial arthropod species. Wolbachia is often a reproductive parasite but can also provide fitness benefits to its host, as, for example, protection against viral pathogens. This protective effect is currently being applied to fight arboviruses transmission by releasing Wolbachia-transinfected mosquitoes. Titre regulation is a crucial aspect of Wolbachia biology. Higher titres can lead to stronger phenotypes and fidelity of transmission but can have a higher cost to the host. Since Wolbachia is maternally transmitted, its fitness depends on host fitness, and, therefore, its cost to the host may be under selection. Understanding how Wolbachia titres are regulated and other aspects of Wolbachia biology has been hampered by the lack of genetic tools. Here we developed a forward genetic screen to identify new Wolbachia over-proliferative mutant variants. We characterized in detail two new mutants, wMelPop2 and wMelOctoless, and show that the amplification or loss of the Octomom genomic region lead to over-proliferation. These results confirm previous data and expand on the complex role of this genomic region in the control of Wolbachia proliferation. Both new mutants shorten the host lifespan and increase antiviral protection. Moreover, we show that Wolbachia proliferation rate in Drosophila melanogaster depends on the interaction between Octomom copy number, the host developmental stage, and temperature. Our analysis also suggests that the life shortening and antiviral protection phenotypes of Wolbachia are dependent on different, but related, properties of the endosymbiont; the rate of proliferation and the titres near the time of infection, respectively. We also demonstrate the feasibility of a novel and unbiased experimental approach to study Wolbachia biology, which could be further adapted to characterize other genetically intractable bacterial endosymbionts.

Rift Valley fever virus detection in susceptible hosts with special emphasis in insects.

Rift Valley fever phlebovirus (RVFV, Phenuiviridae) is an emerging arbovirus that can cause potentially fatal disease in many host species including ruminants and humans. Thus, tools to detect this pathogen within tissue samples from routine diagnostic investigations or for research purposes are of major interest. This study compares the immunohistological usefulness of several mono- and polyclonal antibodies against RVFV epitopes in tissue samples derived from natural hosts of epidemiologic importance (sheep), potentially virus transmitting insect species (Culex quinquefasciatus, Aedes aegypti) as well as scientific infection models (mouse, Drosophila melanogaster, C6/36 cell pellet). While the nucleoprotein was the epitope most prominently detected in mammal and mosquito tissue samples, fruit fly tissues showed expression of glycoproteins only. Antibodies against non-structural proteins exhibited single cell reactions in salivary glands of mosquitoes and the C6/36 cell pellet. However, as single antibodies exhibited a cross reactivity of varying degree in non-infected specimens, a careful interpretation of positive reactions and consideration of adequate controls remains of critical importance. The results suggest that primary antibodies directed against viral nucleoproteins and glycoproteins can facilitate RVFV detection in mammals and insects, respectively, and therefore will allow RVFV detection for diagnostic and research purposes.

Drosophila as a Model for Infectious Diseases.

The fruit fly, Drosophila melanogaster, has been used to understand fundamental principles of genetics and biology for over a century. Drosophila is now also considered an essential tool to study mechanisms underlying numerous human genetic diseases. In this review, we will discuss how flies can be used to deepen our knowledge of infectious disease mechanisms in vivo. Flies make effective and applicable models for studying host-pathogen interactions thanks to their highly conserved innate immune systems and cellular processes commonly hijacked by pathogens. Drosophila researchers also possess the most powerful, rapid, and versatile tools for genetic manipulation in multicellular organisms. This allows for robust experiments in which specific pathogenic proteins can be expressed either one at a time or in conjunction with each other to dissect the molecular functions of each virulent factor in a cell-type-specific manner. Well documented phenotypes allow large genetic and pharmacological screens to be performed with relative ease using huge collections of mutant and transgenic strains that are publicly available. These factors combine to make Drosophila a powerful tool for dissecting out host-pathogen interactions as well as a tool to better understand how we can treat infectious diseases that pose risks to public health, including COVID-19, caused by SARS-CoV-2.

Wolbachia and Virus Alter the Host Transcriptome at the Interface of Nucleotide Metabolism Pathways.

Wolbachia is a maternally transmitted bacterium that manipulates arthropod and nematode biology in myriad ways. The Wolbachia strain colonizing Drosophila melanogaster creates sperm-egg incompatibilities and protects its host against RNA viruses, making it a promising tool for vector control. Despite successful trials using Wolbachia-transfected mosquitoes for dengue control, knowledge of how Wolbachia and viruses jointly affect insect biology remains limited. Using the Drosophila melanogaster model, transcriptomics and gene expression network analyses revealed pathways with altered expression and splicing due to Wolbachia colonization and virus infection. Included are metabolic pathways previously unknown to be important for Wolbachia-host interactions. Additionally, Wolbachia-colonized flies exhibit a dampened transcriptomic response to virus infection, consistent with early blocking of virus replication. Finally, using Drosophila genetics, we show that Wolbachia and expression of nucleotide metabolism genes have interactive effects on virus replication. Understanding the mechanisms of pathogen blocking will contribute to the effective development of Wolbachia-mediated vector control programs.IMPORTANCE Recently developed arbovirus control strategies leverage the symbiotic bacterium Wolbachia, which spreads in insect populations and blocks viruses from replicating. While this strategy has been successful, details of how this "pathogen blocking" works are limited. Here, we use a combination of virus infections, fly genetics, and transcriptomics to show that Wolbachia and virus interact at host nucleotide metabolism pathways.

Genetic determinants of antiviral immunity in dipteran insects - Compiling the experimental evidence.

The genetic basis of antiviral immunity in dipteran insects is extensively studied in Drosophila melanogaster and advanced technologies for genetic manipulation allow a better characterization of immune responses also in non-model insect species. Especially, immunity in vector mosquitoes is recently in the spotlight, due to the medical impact that these insects have by transmitting viruses and other pathogens. Here, we review the current state of experimental evidence that supports antiviral functions for immune genes acting in different cellular pathways. We discuss the well-characterized RNA interference mechanism along with the less well-defined JAK-STAT, Toll, and IMD signaling pathways. Furthermore, we highlight the initial evidence for antiviral activity observed for the autophagy pathway, transcriptional pausing, as well as piRNA production from endogenous viral elements. We focus our review on studies from Drosophila and mosquito species from the lineages Aedes, Culex, and Anopheles, which contain major vector species responsible for virus transmission.

Dissecting genetic and sex-specific sources of host heterogeneity in pathogen shedding and spread.

Host heterogeneity in disease transmission is widespread but precisely how different host traits drive this heterogeneity remains poorly understood. Part of the difficulty in linking individual variation to population-scale outcomes is that individual hosts can differ on multiple behavioral, physiological and immunological axes, which will together impact their transmission potential. Moreover, we lack well-characterized, empirical systems that enable the quantification of individual variation in key host traits, while also characterizing genetic or sex-based sources of such variation. Here we used Drosophila melanogaster and Drosophila C Virus as a host-pathogen model system to dissect the genetic and sex-specific sources of variation in multiple host traits that are central to pathogen transmission. Our findings show complex interactions between genetic background, sex, and female mating status accounting for a substantial proportion of variance in lifespan following infection, viral load, virus shedding, and viral load at death. Two notable findings include the interaction between genetic background and sex accounting for nearly 20% of the variance in viral load, and genetic background alone accounting for ~10% of the variance in viral shedding and in lifespan following infection. To understand how variation in these traits could generate heterogeneity in individual pathogen transmission potential, we combined measures of lifespan following infection, virus shedding, and previously published data on fly social aggregation. We found that the interaction between genetic background and sex explained ~12% of the variance in individual transmission potential. Our results highlight the importance of characterising the sources of variation in multiple host traits to understand the drivers of heterogeneity in disease transmission.