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The infectivity of virus particles from Wolbachia-infected Drosophila

BMC Microbiology volume 25, Article number: 25 (2025) Cite this article

Abstract

Viruses transmitted by arthropods pose a huge risk to human health. Wolbachia is an endosymbiotic bacterium that infects various arthropods and can block the viral replication cycle of several medically important viruses. As such, it has been successfully implemented in vector control strategies against mosquito-borne diseases, including Dengue virus. Whilst the mechanisms behind Wolbachia-mediated viral blocking are not fully characterised, it was recently shown that viruses grown in the presence of Wolbachia in some Dipteran cell cultures are less infectious than those grown in the absence of Wolbachia. Here, we investigate the breadth of this mechanism by determining if Wolbachia reduces infectivity in a different system at a different scale. To do this, we looked at Wolbachia’s impact on insect viruses from two diverse virus families within the whole organism Drosophila melanogaster. Drosophila C virus (DCV; Family Dicistroviridae) and Flock House virus (FHV; Famliy Nodaviridae) were grown in adult D. melanogaster flies with and without Wolbachia strain wMelPop. Measures of the physical characteristics, infectivity, pathogenicity, and replicative properties of progeny virus particles did not identify any impact of Wolbachia on either DCV or FHV. Therefore, there was no evidence that changes in infectivity contribute to Wolbachia-mediated viral blocking in this system. Overall, this is consistent with growing evidence that the mechanisms behind Wolbachia viral blocking are dependent on the unique tripartite interactions occurring between the host, the Wolbachia strain, and the infecting virus.

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Introduction

Viruses spread by arthropods (arboviruses) are a big risk to human health, accounting for 700,000 deaths annually [1]. Mosquito-borne viruses are a major contributor to this, with the last decade seeing the emergence and re-emergence of disease [2], highlighting the need for their control [3]. Targeting vector populations is an important means of protecting humans from these arthropod-borne viruses. One novel method currently being trialled around the world is the use of the bacterium Wolbachia pipientis [4, 5], referred to henceforth as Wolbachia. The presence of Wolbachia in mosquitoes can interfere with and block the viral infection cycle of arboviruses, including Dengue virus [6,7,8,9,10], Chikungunya virus [7, 11,12,13], Yellow Fever virus [13], Zika virus [14,15,16,17] and Sindbis virus [18]. Thereby impacting virus transmission to humans [19, 20].

Wolbachia, a Rickettsiales Alphaproteobacterium, is a common, intracellular, obligate endosymbiont of arthropods [21,22,23]. Infection with Wolbachia can impact hosts in different ways, from causing reproductive manipulation to interfering with host pathogens like viruses. The mechanisms underlying Wolbachia-mediated viral blocking and their contribution to the overall phenotype is an important area of research. The phenotype is likely a culmination of the complex tripartite interactions occurring between host-virus and Wolbachia strain.

Wolbachia-mediated pathogen interference has been recorded for numerous Wolbachia strains in a range of hosts against a range of pathogens (Reviewed in [24, 25]). Phenotypes range from no impact [7, 16, 26, 27] to interference, including both blocking [11, 28,29,30,31,32,33,34,35,36,37] and enhancement [38,39,40] of pathogens. The impact of Wolbachia on its hosts varies across different hosts, pathogens or Wolbachia strains, likely a consequence of unique interactions and genetic factors at play in each system [6, 29, 34, 41,42,43]. Despite this, Wolbachia’s blocking of positive-sense single-stranded RNA (+ ssRNA) viruses is broad. Even when Wolbachia is introduced from native Drosophila into mosquitoes, it protects against + ssRNA viruses [11, 13, 37, 44]. For example, wMelPop in D. melanogaster delays mortality in flies challenged with Drosophila C virus (DCV) and Flock House virus (FHV) [27, 45], and when established in non-native hosts, Aedes aegypti mosquitoes protects against Dengue (DENV; 11), Yellow Fever and Chikungunya virus [13]. This suggests the mechanisms of blocking against + ssRNA may be broadly acting and independent of host background. Most arboviruses belong to families that contain + ssRNA genomes thus, understanding if and what mechanisms underlie Wolbachia-mediated blocking of + ssRNA viruses is valuable.

One overarching hypothesis is that Wolbachia-mediated viral blocking is attributed to a Wolbachia-modified cellular environment that hinders viral infection cycles [25]. As an obligate intracellular endosymbiont, Wolbachia is reliant on its host for survival. To survive and replicate, Wolbachia interacts and modulates host processes and resources. For example, Wolbachia is known to modulate the host cytoskeleton (Reviewed in [46]) iron [47, e.g. 48], carbohydrates [49, 50], amino acids [e.g. 50, 51], nucleotides [e.g. 52], the endoplasmic reticulum [e.g. 53, 54] and lipid metabolism [e.g. 55, 56]. The host also responds to Wolbachia infection by producing reactive oxygen species [57, 58], enduring endoplasmic reticulum stress [54] and inducing the immune system [57, 59]. Similarly, viruses rely on some of these same essential host processes to complete their infection cycle. Wolbachia-mediated blocking of viral pathogens is thus likely to occur at the interface of these Wolbachia-modified host processes. Wolbachia-mediated viral interference has also been linked to Wolbachia density, with higher densities providing a greater level of blocking, consistent with increasing competition between Wolbachia and virus for host resources, although this is not always the case [11, 16, 29, 30, 34, 39, 41, 43, 60,61,62,63]. Wolbachia density can be dynamic and variable and seems to be a function of temperature, age, tissue, and genotype, and can be impacted by viruses within the system (Reviewed in [24, 43, 64]).

There is evidence that Wolbachia interferes with several stages of the virus infection cycle. Wolbachia-mediated interference has been observed at entry, translation, and transcription, which all occur during the early stages of the infection cycle [16, 65,66,67]. While the impact on later stages, such as viral packaging, assembly, exit, and resulting progeny virus, is less studied, some evidence suggests that exit/assembly is impacted [68]. A recent series of studies show that Wolbachia can reduce the infectivity of progeny virus particles and may, therefore, be impacting later stages of the viral replication cycle, such as viral assembly and exit [18, 69, 70]. Here, they showed that Wolbachia reduced the proportion of total virus particles that were infectious when collected from Aedes albopictus (Aa23- wAlbB, RML12-wMel, C710- wStri) and Drosophila melanogaster (JW18-wMel) cells after infection [18, 69, 70]. The viruses included in the study were the enveloped + ssRNA arboviruses: Sindbis virus (SINV; family Alphaviridae), Chikungunya virus (CHIKV; family Flaviviridae) and Zika virus (ZIKV; family Flaviviridae) [18, 69, 70]. Wolbachia reduced infectivity of related viruses’ alphaviruses (CHIKV and SINV) by several-fold while having a greater impact on distantly related ZIKV [69], reducing infectivity by four orders of magnitude. A different study reported saliva from ZIKV-exposed mosquitos with Wolbachia did not cause infection in naïve mosquitoes [71]. This could suggest that the virus in the saliva was less infectious. However, the presence of virus in these samples was not confirmed, so it could also have been that virus was just not present [71].

To expand the current understanding of Wolbachia’s impact on the infectivity of viruses and its potential contribution to Wolbachia-mediated viral blocking, we investigated this phenotype for two distinct viruses DCV (Genus Cripavirus, Family Dicistroviridae, Order Picornavirales) and FHV (Genus Alphanodavirus, Family Nodaviridae, Order Nodamuvirales) in whole Drosophila melanogaster. Our system differed biologically from previous studies in the following three ways: (i) hosts (mosquito vs. drosophila), (ii) scale (cell culture vs. whole organisms), and (iii) virus (arbovirus vs. insect-specific, non-enveloped vs. enveloped). We found that in whole Drosophila melanogaster hosts, the infectivity of DCV and FHV was not impacted by Wolbachia.

Methods

Drosophila melanogaster fly and cell stock maintenance

Drosophila melanogaster lines (w1118-wMelPop and w1118-wMelPopTet) were maintained on a cornmeal-dextrose agar media [72] at 25 °C ± 0.5 °C with a 12-hour light-dark cycle [73]. The Wolbachia-free w1118-wMelPopTet line was established by Tetracycline treatment of the Wolbachia-infected w1118-wMelPop line as described by [74]. Flies were reared off tetracycline for at least ten generations before experimentation.

Drosophila melanogaster S2 cells were maintained at 27 °C in complete Schneiders media with 1% Penicillin/Streptomycin, 10% FBS, and 1% glutamine (Gibco).

Viral stocks

Stocks of DCV (isolate EB) and FHV were grown in adult Drosophila (w1118). As is the standard for virus preparation, flies were collected when mortality was first observed (approximately 3–4 days post-infection (dpi)), after freezing at -20 °C, they were homogenised in phosphate buffer pH 7.2 (PB) in a TissueLyser II (Qiagen) with three 3 mm glass beads (Sigma) at 30 Hz for 90 s. Homogenate was centrifuged to remove cellular debris (5,000 rpm for 5 min). Virus stocks were then prepared by sucrose-gradient purification in PB and titred as described previously [29]. The unit of all virus titres is infectious units per mL (IU/mL), and virus stocks were stored at -20 °C.

Table 1 Table of the primer sequences used in this study
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DNA extraction

DNA extractions were conducted using the DNeasy® Blood and Tissue kit (Qiagen) as per the manufacturer’s protocol. In general, pools of five flies were homogenised in 140 µL of 1 X Phosphate Buffered Saline (PBS) with three 3 mm glass beads in a TissueLyser II (Qiagen) at 30 Hz for 90 s. Following extraction, DNA was recovered in 100 µL UltraPure™ water. An Epoch Microspot Spectrophotometer (BioTek) was used to quantify the DNA content and quality.

RNA extraction

Total RNA was extracted from pools of five flies or 100 µL of cell culture using TRIzol™ (Invitrogen) as per the manufacturer’s instructions. Flies were homogenised in 1 mL of TRIzol™ with three 3 mm glass beads in a tissue lyser (Qiagen) at 30 Hz for 90 s. Final RNA pellets were re-suspended in 12 µL of UltraPure™ water (Invitrogen). An Epoch Microspot Spectrophotometer (BioTek) was used to quantify the RNA content and quality.

DNAse treatment and cDNA synthesis

Two micrograms of RNA sample were used for RQ1 RNase-free DNase treatment (Promega) before cDNA synthesis using SuperScript® III Reverse Transcriptase Kit (Thermo Fisher Scientific Inc) and specific primers (Table 1). cDNA was generated by placing samples in a thermocycler using the following profile: 25 °C for 5 min, 50 °C for 60 min and 70 °C for 15 min. The cDNA samples were then diluted at least 1:5 (1:500 for samples with high levels of DCV and 1:3000 for FHV) with UltraPure™ water (Invitrogen) before being used for quantitative polymerase chain reaction (qPCR).

qPCR

qPCR was carried out using SYBR Green qPCR SuperMix-UDG kit (Thermofisher) on either cDNA or gDNA with previously validated primers (Table 1). Two technical replicates of each sample were processed in a Rotor-gene®Q (Qiagen) under the following program: 50 °C for 2 min, 95 °C 2 min, followed by 40 cycles of 95 °C for 10 s, 58 °C (52 °C wsp primers) for 20 s and 72 °C for 20 s. Melt curve analysis between 65 °C and 95 °C confirmed the specificity of the resulting products. Controls in each run included a reaction with no reverse transcriptase control to ensure no contaminating DNA, no template to ensure no contamination in reagents and an inter-replicate control to ensure the data from independent runs were comparable. The following quality checks were applied to the data: (1) cut-off values of 30 for the take-off (Ct), (2) Amplification values above 1.4 and (3) standard deviation below 0.5 between technical replicates. Relative amounts of the target genes were calculated by normalising to the endogenous host gene RpL32 using the delta-delta comparative threshold method (ΔΔCT) [75, 76]. Analysis was conducted using the method outlined by QGene (Biotechniques) [77].

Screening for virus infection and Wolbachia infection

Fly and cell stocks were confirmed to be free from contamination with other viruses commonly used in the laboratory (DCV, FHV, Cricket paralysis virus (CrPV) and SINV). Viruses were screened by RT-PCR. The presence of Wolbachia in the w1118-wMelPop line was similarly confirmed by PCR of gDNA. The total RNA or gDNA was extracted from a pool of 5 unaged female flies or 100 µL of cell culture as previously described. PCR of the cDNA or gDNA template was conducted using MyTaq polymerase (Bioline) and virus (DCV, FHV, CrPV and SINV), Wolbachia surface protein (wsp) and Drosophila RpL32 primers (positive control for extraction quality; Table 1). A no-template control sample was also run for each primer pair. Reactions were run with the following cycle profile: 94 °C 5 min denature, followed by 35 cycles of 30 Sect. 94 °C denature, 30 Sect. 55 °C anneal, and 1 min 72 °C extension (Table 1). PCR products were visualised on a 1% agarose gel using SYBR safe.

The relative amount of Wolbachia was quantified as the normalised amount of wsp to Rpl32 in three randomly sampled populations using qPCR on the gDNA template, as previously mentioned (Fig S1).

Viral infection of flies for accumulation and survival assays

Aged (4–7 day) female Drosophila melanogaster w1118 flies with or without Wolbachia wMelPop were injected with 50.6 nL of Drosophila C virus or Flock House Virus at 108 IU/mL or 109 IU/mL. Carbon dioxide-anesthetized flies were injected in the upper lateral part of the abdomen using needles pulled from borosilicate glass capillaries (Drummond Scientific) and a Nanoject II microinjector (Drummond Scientific). Needles were pulled on the Narishige PC-10: Dual-Stage Glass Micropipette Puller with two heavy and one light weight and heater 1 set at 86 °C and heater 2 set at 68 °C. Mortality was scored daily, death on day one was considered needle stick associated, and the data from these flies were censored. Three independent cohorts of flies (n = 3) with two to three vials of 15–20 flies as technical replicates. Flies were also mock-injected with PBS as a negative control. For RNA accumulation measures, pools of five flies, separate from those used to score survival, were collected at each time point (DCV − 0, 6, 10, 24, 48, 72, and 96 hpi; FHV − 0, 72 hpi) and RNA was extracted and quantified as previously described.

Virus propagation

Figure 1A outlines the experimental process of generating progeny virus particles. Flies were maintained at a standardised density of approximately 200 flies. Three independent cohorts of female flies > 48 h old were used to generate three independent virus preparations for each DCV and FHV. Virus was grown in approximately 1200 w1118-wMelPop-Tet and 1200 w1118-wMelPop flies. Inoculated flies were maintained until flies started to succumb to virus-induced mortality (four dpi for flies without and seven dpi for flies with Wolbachia infected with DCV and five dpi for both flies with and without Wolbachia infected with FHV), at which point they were collected and frozen at -20 °C until purification as described below. The different collection times for DCV were to accommodate for the lower amount of virus that would be recovered from flies with Wolbachia.

Fig. 1
figure 1

Wolbachia has no visible impact on the structure of Drosophila C virus or Flock House virus progeny particles. (A) Schematic diagram of the process of producing progeny virus particles. Representative transmission electron micrographs of Drosophila C virus progeny W- virus (B) and W + virus (C) and FHV progeny W- virus (D) and W + virus (E) All virus samples were negatively stained with 2% uranyl acetate and placed on a Cu 400 homemade glow-discharged grid before being visualised. Images were taken at 300,000 X magnification, and the scale bar represents 100 nm

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Virus purification

Flies were homogenised in 15 µL/fly of PB with three 3 mm glass beads in a TissueLyser II (Qiagen) at 30 Hz for 2 min. Homogenate was frozen at -20 °C before being clarified by two rounds of centrifugation (4 °C, 5 min, 14,000 rpm) to remove the fly and cellular debris. Samples were kept on ice to reduce virus degradation. Virus was purified from the supernatant by ultracentrifugation on a sucrose-density gradient as previously described [29]. Briefly, the supernatant was diluted in PB and centrifuged through a 20% sucrose cushion (27,000 rpm, three h, 12 °C) in SW28 tubes using a SW28 swing rotor (Beckmann Coulter, Life Science). The pellet was resuspended in PB (4 °C overnight). The resuspended pellet was then centrifuged (14,000 rpm, 30 s, 4 °C) and run through another 20% sucrose cushion ultracentrifuge. The pellet was resuspended in PB (4 °C, overnight) before being ultracentrifuged through a 10–40% sucrose gradient (27,000 rpm, 2.5 h, 12 °C). One mL fractions of the gradient were collected, and viral protein was detected by 12% SDS-PAGE gel, stained with Coomassie Brilliant Blue G-250. Positive fractions were pooled, diluted with PB and ultracentrifuged (27,000 rpm, 3 h, 12 °C). The supernatant was removed, and the viral pellet was resuspended in 250 µL PB by vortexing and incubation (4 °C, overnight). The sample was briefly centrifuged (15,000 rpm, 30 s, 4 °C. before the supernatant was stored at -20 °C. Unless specified, all ultracentrifugations were in SW41 tubes (Beckmann Coulter, Life Science) using a SW41 swing rotor in an Optima L-80XP Ultracentrifuge (Beckmann Coulter, Life Science). The purity of virus samples was assessed by SDS-PAGE.

Transmission electron microscopy of DCV particles

The purified virus was negatively stained with 2% uranyl acetate placed on a Cu 400 homemade glow-discharged grid and visualised by transmission electron microscopy (JEOL JEM-1011 TEM).

Measuring infectivity of progeny W- and W + virus particles

As it is difficult to count virus particles, the concentration of RNA was used as a proxy to estimate viral genome numbers and, therefore, the relative amount of virus. An Epoch Microplate Spectrophotometer (BioTek) was used to take three independent readings of the pure virus preparations to determine the amount of RNA. This machine uses spectrophotometry to measure the absorbance of the purified viral preparation as light passes through it. Nucleic acids absorb light most strongly at 260 nm, and an absorbance ratio at 260 nm to 280 nm of 2.0 is generally considered indicative of pure RNA. The amount of viral RNA (in nanograms) was then determined based on this absorbance measurement. The size of the genomes for DCV (9264 nucleotides(nt) ssRNA; GenBank accession number NC_001834) and FHV (RNA1 3107 nt, RNA2 1400 nt, Total = 4507 nt ssRNA; GenBank accession numbers, RNA 1 = NC_004146.1, RNA 2 = NC_004144.1) and the average measured amount of RNA (ng) was used to calculate the number of total RNA genome copies using the NEB calculator [78]. Note that for FHV, which has a bipartite RNA genome, the combined length of the two RNA particles (4507 nt) was used for the calculation. Assuming that all RNA in the sample was encapsidated viral RNA and that one RNA genome copy was equivalent to one virus particle, this gave the number of total virus particles present. Unless otherwise stated, equivalent numbers of total virus particles were used by creating samples that had the same concentration of total virus (1012 total virus particles/mL) and using the same quantity of this concentrated sample for the following experiments.

The number of infectious particles was calculated by tissue culture infectious dose 50 (TCID50) using Drosophila melanogaster S2 cells. Briefly, a 96-well plate was seeded with 1 × 106 cells/well and then infected with serial 1:10 dilutions of the viral preparations. Cytopathic effects were scored six dpi for FHV and seven dpi for DCV, and the Reed-Muench Method [79] was used to calculate the concentration (IU/mL). The concentration was calculated for each of the three virus preparations.

The infectivity was measured as the ratio of the number of infectious particles over the number of total particles.

Measuring pathogenicity of progeny W- and W + virus particles in flies

To measure the pathogenicity of the progeny W- and W + virus (DCV and FHV), their ability to cause mortality in naïve w1118-wMelPop-Tet flies was assessed. Aged female w1118-wMelPop-Tet flies were injected with virus diluted to 1012 virus particles/mL in PBS as previously described. Three biological replicates (independent preparations of virus) were injected into approximately 10–15 flies per vial in 1–2 vials (technical replicates). Flies were injected with PBS as a negative control. FHV and DCV injections were done on separate days. Survival was scored, as previously mentioned.

Measuring the ability of W- and W + virus particles to replicate in Drosophila cells

To determine if Wolbachia impacted the ability of progeny DCV to replicate, viral RNA accumulation was measured through time in Drosophila melanogaster S2 cells. Virus was used to infect 1 × 106 cells at 0.001 and 100 total particles per cell for DCV and FHV, respectively. The inoculating virus was removed after two hours, and cells were washed with 1X PBS, after which the PBS was replaced with 1000 µL of fresh complete media. Cells were sprayed down and 250 µl were collected at 2, 4, 6, 10, 24, 48 and 72 hpi. Cells were pelleted at 5,000 xg for 5 min. The supernatant was removed, and pellets were resuspended in 1 mL of TRIzol™. Samples were stored at -20 °C for RNA extraction as described above. This was repeated for each of the separate virus preparations (n = 3).

Statistical analysis

All statistical analyses were performed using R Statistical Software (v 4.3.0) [80]. Associated datasets and code can be found as part of the supplementary material, a list of the packages and associated references can be found in the S1 Table. In all instances, the best fit statistical model (with or without factors and their interactions) was determined using the Akaike information coefficient [81].

To determine if fly survival was different between treatments (flies with Wolbachia vs. flies without Wolbachia, or flies injected with W- virus vs. flies injected with W + virus), a Cox’s proportional hazard mixed-effect model was fit (R package coxme [82, 24]). In this model, Treatment was fit as a categorical fixed effect while the Biological and replicate vials were fit as random effects Surv(Days, Survival) ~ Treatment + (1 | BiolRep/TechRep). If the model without the random Biological or Technical effects did not fulfil the assumptions of proportional hazards, assessed using the function cox.zph [84], we used a survival regression (survreg) function (R package survival [85]). This function fits a parametric survival model, and if this gave us qualitatively similar results to the Cox mixed effects model, deviations from the assumptions were determined to have had a negligible impact on the conclusions, and thus the results from coxme were reported [86, 87].

To determine the impact that the treatment (flies with Wolbachia vs. flies without Wolbachia; flies injected with W- or W + virus) and time had on viral RNA levels, an ANOVA was fit to the log10 mean normalised expression of DCV RNA to RpL32. If it did not meet the assumption of normality, data were first rank transformed [88]. If the interaction between time and treatment was significant, post hoc comparisons were made (R package emmean [89]). Where comparisons were only made between treatments for one-time point, a Welch’s T-test (R package stat [90], R package rstatix [91]) with no assumptions of equal variance was conducted.

To determine the impact that Wolbachia had on the infectivity of W- and W+, a binomial general linear model with no interaction (infectivity ratio ~ Wolbachia + virus (DCV or FHV)) was fit. To meet the assumptions of the general linear model, the ratio of the log10 infectious progeny particles to the log10 total number of virus particles was used as the measure of infectivity.

Results

Wolbachia increased host survival and reduced accumulation of viral RNA of Drosophila C virus and Flock House virus

To confirm the viral blocking phenotype of wMelPop against DCV and FHV, survival and associated accumulation of viral RNA were monitored following virus challenge in flies with and without Wolbachia. Survival of flies with Wolbachia was increased when challenged with DCV (median survival of six days compared with ten days; Fig. 2A) and FHV (median survival of seven days compared with fifteen days; Fig. 2C). Flies with Wolbachia were found to be less likely to succumb to virus-induced mortality for both DCV (coxme DCV, p-value < 0.0001, Hazard ratio (HR) = 0.09, HR 95% CI [ 0.06, 0.14], se = 0.23, z = -10.49; S1 Output) and FHV (coxme FHV, p-value < 0.0001, HR = 0.005, HR 95% CI [ 0.001, 0.024], se = 0.7, z = -7.29; S2 Output). Thus, as seen previously [27, 45], Wolbachia increased the survival of flies when challenged with either DCV or FHV (Fig. 2A and C).

Fig. 2
figure 2

Wolbachia increases survival in flies, which is linked to a reduction in the accumulation of viral RNA during infection with Drosophila C virus or Flock House Virus. A. C. Survival and B. D. accumulation of viral RNA as measured by RT-qPCR in Drosophila melanogaster flies with (red) or without (black)Wolbachia following infection 50.6 nL of Drosophila C virus (DCV) A. and B. or Flock House Virus (FHV) C. and D. at 108 IU/mL or 109 IU/mL respectively in phosphate-buffered saline (PBS). A. and C. Kaplan-Meier survival curves and 95% confidence intervals (shaded (n = 3). Flies with and withoutWolbachiawere also mock-infected with PBS as a negative control (dashed lines). B. and D. Log10 transformed mean normalised expression of DCV or FHV genome relative to host gene RpL32 (n = 6). Lines representing LOESS regression and associated confidence interval (shaded). Time points where DCV RNA was lower in flies with Wolbachia are signified by *

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To determine if the increased survival time was associated with changes in viral RNA, accumulation of viral RNA in flies was measured through time. Wolbachia had a significant impact (p-value < 0.0001, df = 1, F = 126.5), as did time (p-value < 0.0001, df = 6, F = 140.6) and the interaction between Wolbachia and time (p-value < 0.0001, df = 6, F = 5.1) on the relative amount of DCV RNA (Fig. 2B; S3 Output). Results from post-hoc comparisons showed that less viral RNA had accumulated in flies with Wolbachia 10, 24, 48, 72 and 96 h post-infection (hpi; Fig. 2B). As expected, there was no difference in the abundance of FHV RNA 0 h post-infection (Welch T-test, P = 0.67, df = 7.7, t = 0.45; Fig. 2D), suggesting the inoculum was equivalent. However, by 72 hpi, the abundance of FHV RNA was lower in flies with Wolbachia (mean = 0.09) than in flies without Wolbachia (mean = 1.8; Welch T-test, p-value = 0.0005, df = 6.9, t. = 6.1; Fig. 2D; S4 Output). Thus, the increase in survival of flies with Wolbachia was associated with a reduction in the accumulation of viral RNA for both DCV and FHV. Overall, this suggests that Wolbachia is interfering with the accumulation of virus in the host, which is associated with a reduction in viral-related pathology.

Wolbachia had no visible impact on the structure of Drosophila C virus or Flock House virus progeny particles

To measure the impact Wolbachia has on progeny virus particles, it was first necessary to grow and purify the viruses from flies with and without Wolbachia (Process summarised in Fig. 1A). The progeny virus particles produced in flies with Wolbachia and without Wolbachia are referred to henceforth as W + and W – virus, respectively. To qualitatively determine if Wolbachia impacted the overall morphology of progeny virus, they were visualised using transmission electron microscopy (TEM; Fig. 1). Results show that the progeny DCV W- (Fig. 1B) and W+ (Fig. 1C) virus particles approximated 28–30 nm in diameter and were of a similar shape, characteristic of DCV [92]. Similarly, progeny FHV W- (Fig. 1D) and W+ (Fig. 1E) virus particles approximated 30–32 nm in diameter and were of a similar shape, characteristic of FHV [93, 94].

While overall, the morphology of the progeny virus particles is similar, the level of staining was variable. Stain-permeable particles are seen in all samples and could indicate an artifact of the staining process (Fig S2).

Wolbachia had no impact on the infectivity of Drosophila C virus or Flock House virus progeny particles

After purification of progeny virus (W + and W – virus; Fig. 1A), their infectivity was measured. Infectivity was measured as the ratio of the number of infectious virus particles over the total number of virus particles (Fig. 3). The total number of virus particles in each purified viral preparation was calculated by quantifying the amount of RNA by spectrophotometry and assuming that the RNA present corresponded to full viral genomes. This measure was used as a proxy as virus particles could not be counted directly. The number of infectious units (IU) was estimated using TCID50. Wolbachia had no statistically significant impact on the infectivity of progeny DCV (t-test, t = -1.2, df = 3.5, p-value = 0.3; S5 Output) or FHV virus particles (t-test, t = -2.6, df = 3.1 p-value = 0.08; S5 Output). Therefore, there is no evidence that Wolbachia impacts the infectivity of DCV or FHV particles. Whilst there are only three replicates, if further samples were added there may be an impact in the opposite direction to what we expected to explain antiviral effects which is more obvious for FHV.

Fig. 3
figure 3

Wolbachia has no impact on the infectivity of DCV or FHV progeny particles. The infectivity ratio of progeny W- virus (black) and progeny W + virus (red) for DCV and FHV. The Infectivity of progeny virus particles is represented as the ratio of the log10 transformed number of virus particles that are infectious over the log10 transformed total number of progeny virus particles. The total number of virus particles in each prep was measured by spectrophotometry, assuming that all virus samples were pure, and RNA present was viral and encapsidated. The number of infectious virus numbers was estimated using tissue culture infectious dose 50 (TCID50). The line represents the mean (n = 3), and the error bars are the standard error of the mean. Infectivity was not significantly different between W- and W + virus (DCV t-test, t = -1.2, df = 3.5, p-value = 0.3; FHV t-test, t = -2.6, df = 3.1 p-value = 0.08)

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To compare the measure of infectivity here to those in the literature, the specific infectivity was calculated as the reciprocal of the data presented in Fig. 3 (number of total particles per infectious particle). Data for both DCV and FHV are summarised in Table 2 and represent the same data as Fig. 3. When comparing the specific infectivity of these viruses to others in the literature, they fall within the range of variation previously reported. The specific infectivity for FHV (Table 2) was within one order of magnitude of that measured by others (Table S1, [95]). This difference may represent the variation known to exist between populations of the same virus or differences in measuring infectious and total virus particles.

Table 2 Specific infectivity of progeny virus
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Wolbachia had no impact on the pathogenicity of progeny Drosophila C virus or Flock House virus in flies

Next, we investigated whether Wolbachia impacted the ability of progeny virus to cause mortality in future infections. This was investigated by monitoring the survival of flies without Wolbachia following infection with the same number of total progeny virus particles (W + virus or W- virus; Process summarised in Fig. 4A). There was no statistically significant difference between the survival of flies injected with W- or W + DCV virus, with median survival occurring six dpi ( p-value = 0.7, Hazard ratio (HR) = 0.9, HR 95% CI [ 0.5, 1.6], se = 0.3, z = -0.39; Fig. 4B; S6 Output). Similarly, there was no statistically significant difference between the survival of flies injected with W- or W + FHV virus (coxme, p-value = 0.25, Hazard ratio (HR) = 0.84, HR 95% CI [0.62, 1.13], se = 0.15, z = -1.16; Fig. 4C; S7 Output) with median survival for both occurring five-six dpi. Overall, Wolbachia did not appear to impact the pathogenicity of progeny DCV or FHV.

Fig. 4
figure 4

Wolbachia has no impact on the pathogenicity of progeny Drosophila C virus or Flock House virus in flies. (A) Schematic outlining how pathogenicity was measured. B and C Kaplan-Meier survival curves and associated confidence intervals (shaded) of Drosophila melanogaster following infection with 50.6 nl of 1012 total virus particles/mL in phosphate buffered saline of W- (black) or W+ (red) Drosophila C virus (DCV) B. or Flock House virus (FHV) C (n = 3). There was no statistical difference between the survival of flies injected with W- or W + DCV virus (coxme, p-value = 0.7, Hazard ratio (HR) = 0.9, HR 95% CI [ 0.5, 1.6], se = 0.3, z = -0.39) or between survival of flies injected with W- or W + FHV virus (coxme, p-value = 0.25, Hazard ratio (HR) = 0.84, HR 95% CI [0.62, 1.13], se = 0.15, z = -1.16)

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Wolbachia had no impact on the replicative dynamics of Drosophila C virus or Flock House virus in Drosophila cells

To determine if Wolbachia impacted the replicative properties of progeny virus that may not have been detected by the measure of infectivity, the dynamics of the viral replication cycle were investigated (Processed summarised in Fig. 5A). These dynamics were investigated within the cell culture model. Following virus challenge with the same number of total virus particles, the accumulation of viral RNA was measured across a three-day time course in S2 Drosophila cells. While samples were collected at two and four hpi, DCV was not detectable in most samples until six hpi. Results showed that time had a statistically significant impact on the level of DCV RNA (p-value < 0.001, df = 4, F = 477.3; Fig. 5B; S8 Output), but the type of virus W- or W + did not (p-value = 0.2, df = 1, F = 1.4; Fig. 5B; S8 Output). The interaction between the type of virus and time was also not significant (p-value = 0.8, df = 4, F = 0.4; Fig. 5B; S8 Output). Similarly, time had a statistically significant impact on the level of viral FHV RNA (p-value < 0.001, df = 4, F = 86.9; Fig. 5C; S9 Output), but the type of virus W- or W + did not (p-value = 0.39, df = 1, F = 0.8; Fig. 5C; S9 Output). The interaction between the type of virus FHV W- or W + and time was also not significant (p-value = 0.55, df = 4, F = 0.78; Fig. 5B; S9 Output). Therefore, Wolbachia does not appear to impact the replicative dynamics of progeny DCV or FHV.

Fig. 5
figure 5

Wolbachia has no impact on the replicative dynamics of progeny DCV or FHV within Drosophila cells. A. Schematic of the experimental set-up. B. Accumulation of viral RNA of progeny Drosophila C virus grown in flies with W + virus (red) and without W- virus (black)Wolbachia wMelPop. Drosophila melanogaster S2 cells without Wolbachia were infected with either W- or W + virus at an MOI 0.001 total virus particles to cells. Viral RNA levels were measured at various times post-infection. Data represents log10 transformed mean normalised expression of B. DCV (n = 6) or C. FHV genome relative to host gene RpL32 (n = 3). Lines represent LOESS regression and confidence interval (shaded). B. Time had a statistically significant impact on the level of DCV RNA (p-value < 0.0001, df = 4, F = 477.3), but the type of virus W- or W + did not (p-value = 0.2, df = 1, F = 1.4) The interaction between the type of virus and time was also not significant (p-value = 0.8, df = 4, F = 0.4). C. Time had a statistically significant impact on the level of viral FHV RNA (p-value < 0.001, df = 4, F = 86.9), but the type of virus W- or W + did not (p-value = 0.39, df = 1, F = 0.8). The interaction between the type of virus FHV W- or W + and time was also not significant (p-value = 0.55, df = 4, F = 0.78)

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Discussion

Arboviruses are a huge health concern. One of the most common arboviruses, Dengue virus (DENV, Flavivirdae), transmitted by Aedes mosquitoes, causes an estimated 40,000 deaths annually [1, 96]. Wolbachia interferes and blocks + ssRNA viruses in insects, including Drosophila and mosquitoes, as has been successfully used as a form of vector control for DENV [20]. While not fully characterised, a variety of mechanisms have been identified that contribute to this blocking (Reviewed in 24, 25). Recently, a series of studies identified that Wolbachia reduces viral particle infectivity. Contrastingly, we found no evidence that Wolbachia (wMelPop) impacts the infectivity, physical characteristics, pathogenicity, or replicative properties of DCV or FHV grown in Drosophila melanogaster. Therefore, reduced infectivity does not contribute to the overall Wolbachia (wMelPop)-mediated blocking of DCV or FHV in Drosophila melanogaster. These contrasting results may reflect differences between systems, encompassing both biological and technical factors, used to observe the impact of Wolbachia on viral infectivity (Systems are summarised in S3 Table). Some of these differences include (i) hosts, (ii) scale (cell culture vs. whole organisms) and (iii) virus (arbovirus vs. insect-specific, non-enveloped vs. enveloped).

Variation in the impact of Wolbachia on host-pathogen interactions is well documented. Unique and multi-faceted tripartite host-pathogen-Wolbachia interactions likely account for this variance, suggesting that mechanisms may vary across different hosts, pathogens and Wolbachia strain system combinations [6, 29, 34, 41, 42]. The overall outcome of Wolbachia interference could be influenced by each of the unique entities involved in these interactions. Wolbachia-mediated blocking is known to vary between Wolbachia strains, with some strains showing more inhibition than others [12, 15, 29, 30, 34, 45, 62, 97]. Recent evidence suggests in some instances that this may be due to underlying mechanistic differences [98]. Wolbachia also differentially impacts different viruses, likely a consequence of viruses’ unique infection cycle strategies. For example, Wolbachia-mediated blocking of flaviviruses but not alphaviruses is linked to cholesterol and may reflect differences in the cholesterol requirements of these viruses [99]. Intriguingly, the magnitude of Wolbachia-mediated changes to viral infectivity varies across different Wolbachia strains and viruses, with more closely related viruses being impacted to a similar extent when compared to more distantly related [69]. Lastly, it is possible that the host could impact whether Wolbachia mediates a change in the infectivity of the virus. However, it is known that the host genome does not have a great influence on the magnitude of Wolbachia-mediated blocking [97] and some strains, such as wMelPop, can interfere with viruses across multiple hosts [11, 13, 37, 44, 45].

Wolbachia-mediated blocking occurs broadly against + ssRNA viruses. While Wolbachia may impact phenomena common to all these viruses, that does not mean it cannot also impact things that are unique to individual viruses. This may partially explain the contrasting results here. Wolbachia-mediated reduction of viral infectivity is linked to changes in viral RNA methylation patterns [70]; however, these changes may not occur or play a role in the blocking of DCV or FHV. Wolbachia can alter host methylation [100, 101] and could non-discriminately target viral RNA. Previous results show that changes in 5-methylcytosine (5mC) patterns on the viral RNA of SINV are thought to be induced by Wolbachia and, at least in part, contribute to the reduced infectivity of SINV particles [70]. Previously, 5mC patterns on SINV RNA were shown to be different between viral RNA that is intracellular and that which is packed into a virion [102, 103], suggesting in this instance that Wolbachia-mediated dysregulation of these patterns could impact both genome replication and assembly. These changes in 5mC patterns have been attributed to the function of a host protein, DNA methyltransferase 2 (Dnmt2) [70]. Independent of Wolbachia, Dnmt2 interacts with DCV and may cause 5mC DCV RNA modifications that lead to suppression of infection [104]. Thus, there is potential for Wolbachia to interact with DCV through this pathway. Interestingly, Dnmt2 expression, and therefore methylation type genome modification, is not linked to Wolbachia-mediated blocking of DCV or FHV [34]. It should be noted, while un-tested, Wolbachia could still induce post-translational modification or initiate translocation of Dnmt2 proteins [101, 105] and cause changes to DCV or FHV RNA. It is also important to note that the function and regulation of Dnmt2 are different between Drosophila and mosquito species [18, 106]. Given the data presented and the lack of evidence so far of Dnmt2 involvement, it is unlikely that Wolbachia impacts the 5mC of DCV or FHV, or if it does, it is in a way that does not impact their infectivity, e.g., not on encapsidated viral RNA.

Consistent with this study, evidence from virus evolution experiments in the presence of Wolbachia shows that DCV infects hosts to the same extent after several passages, suggesting infectivity is not impacted [107]. Contrastingly, DENV-3 could not establish infection and produced less viral RNA after evolution in the presence of Wolbachia, suggesting infectivity may be impacted [108]. These two contrasting results, in conjunction with the contrasting findings of this study, are consistent with growing evidence that some mechanisms that contribute to Wolbachia-mediated blocking may be system-specific [98, 99].

The major technical difference between this and the initial study on Wolbachia’s impact on viral infectivity is that we used a whole organism model rather than cell culture. While mechanisms of Wolbachia-mediated blocking are unlikely to vary between these models, the ability to resolve the impacts of mechanisms might. There is evidence that Wolbachia-mediated blocking is cell-autonomous [11, 109]. If Wolbachia reduces the infectivity of viruses co-infecting the same cell, then cells without Wolbachia may still have the capacity to produce infectious unchanged virus particles. Therefore, the proportion of cells with Wolbachia would impact the probability of virus encountering it and being impacted. This is in line with evidence that Wolbachia density is associated with the level of viral blocking [11, 16, 29, 30, 34, 41, 43, 60,61,62]. In whole organisms, the probability of a virus encountering a cell with Wolbachia is also impacted by tissue tropism of both the virus and Wolbachia [41], although not always [63]. In cells, the lack of differentiated tissues and, thus, physical barriers would increase the chance of virus being exposed to cells with Wolbachia. Therefore, the observable impact that Wolbachia has on viral infectivity may vary from whole organisms to cell culture, with the impacts being more pronounced in cell culture when measured for the same Wolbachia-host-pathogen pairing.

Wolbachia appears to have no impact on the infectivity of either FHV or DCV grown in Drosophila melanogaster files, suggesting that this does not contribute to Wolbachia-mediated viral blocking in this system. This evidence contrasts previous findings that Wolbachia reduces viral infectivity of SINV, ZIKA and CHIKV in cell culture, suggesting this impact may be system-dependent. Multiple mechanisms likely contribute to Wolbachia-mediated blocking, and what is observed in functional assays is the culmination of all those mechanisms. While there may be some mechanisms that are consistent across systems, infectivity does not appear to be one of those. These results add to the growing evidence that the mechanisms behind Wolbachia-mediated viral blocking are complex and multifactorial, a product of unique interactions between hosts, viruses and Wolbachia. This is important to consider in the context of utilising Wolbachia for the biocontrol of arboviruses in different host species.

Data availability

The dataset and code used to conduct statistical analysis supporting this article are available from GitHub repository https://github.com/AngeliqueKAsselin/Asselin-BMC-Microbiology-2024.

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Acknowledgements

The authors thank Lou Brilliant and acknowledge the facilities, and the scientific and technical assistance, of the Microscopy Australia Facility at the Centre for Microscopy and Microanalysis (CMM), The University of Queensland for imaging of TEM samples. We thank members of the lab, Daniel Chew and Tyson Thomson for support and useful comments. We also thank Andrew Letten and Katrina McGuigan for guidance on statistical analysis.

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    Angelique Asselin & Karyn Johnson

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CRediT (Contributor Roles Taxonomy) Conceptualization AA, KJ; Data curation AA; Formal analysis AA; Funding acquisition KJ; Investigation AA, KJ; Methodology AA, KJ; Project administration KJ; Resources KJ; Software AA; Supervision KJ; Validation AA; Visualization AA; Writing – original draft AA; Writing – review & editing AA, KJ. All authors read and approved the final manuscript.

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12866_2024_3722_MOESM1_ESM.docx

Supplementary Material 1: Figure S1. Relative Wolbachia abundance in three randomly sampled populations of w1118-wMelPop flies used for this study. Relative abundance was measured as the Wolbachia surface protein gene copies normalised to the endogenous RpL32 copies in gDNA of homogenised adult flies. Normalisation was conducted using the delta-delta comparative threshold method (ΔΔCT), giving a proxy for Wolbachia genome copies per Drosophila genome copies. Each bar represents a different cohort of flies sampled. Wolbachia surface protein was not detected in w1118-wMelPop-Tet, confirming the absence of infection. Figure S2. Representative transmission electron micrographs of virus particles. (A) DCV W-, (B) DCV W+, (C) FHV W-, (D) FHV W+. The staining of viral particles was variable. Black arrows indicate examples of stain-permeable particles, and white arrows indicate examples of particles that are less stain-permeable. Stain-permeable particles were seen in all samples. S1 Table: List of packages used and associated references. S2 Table: Specific infectivity of viruses from the literature. S3 Table: Different systems in which Wolbachia’s impact on viral infectivity has been investigated. S1 Output: Analysis for Fig. 2A. S2 Output: Analysis for Fig. 2C. S3 Output: Analysis for Fig. 2B. S4 Output: Analysis for Fig. 2D. S5 Output: Analysis for Fig. 3. S6 Output: Analysis for Fig. 4B. S7 Output: Analysis for Fig. 4C. S8 Output: Analysis for Fig. 5B. S9 Output: Analysis for Fig. 5C.

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Asselin, A., Johnson, K. The infectivity of virus particles from Wolbachia-infected Drosophila. BMC Microbiol 25, 25 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12866-024-03722-6

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