Using Wolbachia to Eliminate Dengue: Will the Virus Fight Back?

ABSTRACT Recent field trials have demonstrated that dengue incidence can be substantially reduced by introgressing strains of the endosymbiotic bacterium Wolbachia into Aedes aegypti mosquito populations. This strategy relies on Wolbachia reducing the susceptibility of Ae. aegypti to disseminated infection by positive-sense RNA viruses like dengue. However, RNA viruses are well known to adapt to antiviral pressures. Here, we review the viral infection stages where selection for Wolbachia-resistant virus variants could occur. We also consider the genetic constraints imposed on viruses that alternate between vertebrate and invertebrate hosts, and the likely selection pressures to which dengue virus might adapt in order to be effectively transmitted by Ae. aegypti that carry Wolbachia. While there are hurdles to dengue viruses developing resistance to Wolbachia, we suggest that long-term surveillance for resistant viruses should be an integral component of Wolbachia-introgression biocontrol programs.


IMPACTS OF EVOLUTION ON WOLBACHIA AS A BIOCONTROL TOOL
The ability of Wolbachia to provide long term protection against DENV could be undermined by genome evolution of wMel, Ae. aegypti, and/or DENV. Evolution of wMel tracks slower than the mitochondrial genome of its natural host, D. melanogaster (27,28), and sequencing of wMel from Ae. aegypti collected 2 to 8 years postrelease in Queensland, Australia only rarely detected genetic polymorphisms (29). These studies suggest that the wMel genome is quite stable in Ae. aegypti, which will presumably aid in the continuation of its antiviral properties in this host.
Plausibly, evolution of the Ae. aegypti genome could attenuate wMel-mediated viral inhibition by adapting to the endosymbiont over time. Ford et al. selectively bred wMelinfected mosquitoes that either established high or low levels of viral RNA after DENV infection. They found the low and high DENV levels were linked to genomic variation in Ae. aegypti (30). However, the mosquito phenotypes that were less resistant to viral infection were also less fit, suggesting they would be unlikely to be selected in the field.
The stability of the Wolbachia-Ae. aegypti association has been demonstrated in Queensland (19,24) and Malaysia (31), where wMel and wAlbB, respectively, were introgressed into the Ae. aegypti population. Wolbachia has remained at a high frequency in these mosquito populations for up to a decade, and has retained its antiviral properties (31,32). Together, these studies indicate that the Wolbachia-Ae. aegypti relationship is unlikely to evolve rapidly in the field in a manner that quickly undermines the public health benefits of the Wolbachia introgression method.
In contrast to Wolbachia and Ae. aegypti, RNA viruses like DENV have much faster mutation rates. Viruses that accumulate mutations in the genome (variants) that can replicate in Wolbachia-carrying mosquitoes may be rapidly selected. These variants could be maintained in a Wolbachia-Ae. aegypti population provided they can replicate well within the human host. Thus, whether DENV will remain susceptible to the antiviral state created by wMel and wAlbB infection in Ae. aegypti remains a key question to be addressed (33,34). In this review, we examine the risk and potential mechanisms by which DENV resistance against Wolbachia might evolve and discuss how viral resistance to Wolbachia could be identified and managed operationally.

SELECTION AND EMERGENCE OF WOLBACHIA-RESISTANT VIRUS IN MOSQUITOES
The urban transmission cycle sees DENV circulate between human and mosquito hosts. Mosquitoes become infected with DENV when the insect takes an infectious blood meal from a viremic person. Since Wolbachia resides within mosquitoes, selective pressure for the virus population to overcome Wolbachia's antiviral properties will only be present in this part of the transmission cycle. While the emergence of viral resistance to antiviral therapeutics in humans is a relatively common phenomenon (35)(36)(37), selection pressures applied to DENV by Wolbachia are likely to differ in many ways. For instance, while antiviral drugs have a defined mode of action, the mode of action of Wolbachia appears to be broad and may be indirect (38). In addition, while therapeutics are administered at optimized concentrations and have well-defined pharmacological properties (39), Wolbachia abundance (density) cannot be easily controlled and varies between Wolbachia strains, individual mosquitoes, and host tissues (40)(41)(42)(43)(44)(45). Control of the levels of DENV inhibition within specific Ae. aegypti tissues appears to be complex and is not just associated with Wolbachia density (40,42,46). In this section we postulate how Wolbachia-resistant DENV variants may emerge, based on our current understanding of DENV infection, dissemination, and transmission in mosquitoes.
wMel and wAlbB Wolbachia strains provide incomplete protection against DENV. The wMel and wAlbB Wolbachia strains used in field releases have been rigorously tested in laboratory studies to determine their impacts on DENV infection dynamics in Ae. aegypti. Broadly speaking, these strains provide partial protection against fulminant DENV infection compared to mosquitoes without Wolbachia (46). Most important to the effectiveness of these strains in the field is their ability to both reduce the proportion of Ae. aegypti with infectious DENV in their saliva (22,23) and lengthen the extrinsic incubation period (time taken for mosquito saliva to become infectious following virus uptake in a blood meal), thus reducing the number of days in a mosquito's life span in which it can infect people (22,47). Nevertheless, Wolbachia-mediated viral inhibition is incomplete, such that a proportion of mosquitoes become infectious with DENV. For example, after feeding on blood from viremic dengue patients, infection was detected in the abdomen (53 to 61%) and saliva (6 to 12%) of wMel-carrying mosquitoes (22,23). Even at a population level, it has been estimated that introgression of wMel would not eliminate DENV in high-transmission settings indefinitely (6). Also of note, DENV-1 is marginally less inhibited by wMel than serotypes 2, 3, and 4 (22,23). Plausibly, a smoldering pattern of DENV replication and transmission could provide the opportunity for Wolbachia-resistant viruses to emerge and be selected (48).

PROCESS OF WOLBACHIA-RESISTANT VIRUS SELECTION
Within mosquito tissues, both wMel-carrying and wMel-free cells can be observed (42) and these cells are likely to possess different antiviral states. At the cellular level, Nainu et al. determined the antiviral effects of wMel to be cell-autonomous (i.e., viral protection is limited to Wolbachia-infected cells) (49). JW18 Drosophila cells with wMel were unable to protect Wolbachia-free JW18 cells from infection by Drosophila C Virus (DVC; Dicistroviridae, cripavirus) or Sindbis virus (SINV; Togaviridae, alphavirus) when cocultured in trans-wells separated by a porous membrane (49). Similarly, it seems that antiviral Wolbachia strains show a "superinfection exclusion-like phenotype," whereby cells that have Wolbachia prevent productive viral infection (50,51) and DENV and Wolbachia coinfected cells are rarely visualized in mosquito tissues and cell culture (50,52). These studies suggest that Wolbachia-free cells within mosquito tissues that can support productive virus infection may be the site where Wolbachia-resistant virus types may emerge, followed by their selection in Wolbachia-carrying cells.
After ingesting a blood meal from a viremic person, DENV replicates in the Ae. aegypti midgut. The virus must then traverse the midgut barrier, enter the hemolymph, and infect other tissues, reaching the salivary glands after ;7 days. Once the virus enters the mosquito's saliva it can be passed to a new host when the next blood meal is taken.
In blood-fed mosquitoes, only a small number of infectious units are thought to seed infection in the mosquito midgut (53,54). This reduction in virus population size, known as a population bottleneck, decreases the genetic diversity of the infective virus population (55,56). This event may cause low-frequency Wolbachia-resistant DENV variants already present in the incoming blood meal to be filtered out ( Fig. 1, step 1).
Replication of DENV in the midgut leads to the generation of viral variants because the virus lacks proofreading capacity. These variants may be unable to disseminate beyond the midgut if they have reduced competitive fitness (57) or are susceptible to immune mediators within the midgut and hemolymph (58) (Fig. 1, step 2).
If a fit Wolbachia-resistant DENV variant is generated in the midgut, this virus would possess a selective advantage over wild-type viruses in mosquitoes that carry Wolbachia. Selection may occur if the variant could similarly infect both Wolbachia-carrying and Wolbachia-free cells, or if a variant evolves to specifically target Wolbachia-free cells. Mechanistically, DENV could specifically target Wolbachia-free cells by adapting its affinity for viral entry receptors (59) to those that are differentially expressed between Wolbachiafree and -carrying cells. Lu et al. showed that wAlbB infection modulates the expression of DENV attachment factors dystroglycan and tubulin in Aag2 cells (60). Another study showed that expression of the cell surface insulin receptor is modulated by wMel infection, reducing the susceptibility of mosquito cells to DENV and ZIKV (Zika virus) infection (61). While the insulin receptor is not a known entry receptor for DENV, this study illustrates that Wolbachia has the potential to modulate expression of cell membrane proteins and thereby alter the permissiveness of these cells to viral infection.
Preferential replication of Wolbachia-resistant DENV compared to wild-type virus would ultimately establish these fit viral variants in the salivary glands ( Fig. 1, step 3).
Onward transmission of Wolbachia-resistant DENV variants would be limited if they are unfit in the human host ( Fig. 1, step 4). This scenario would be considered a fitness trade-off, where fitness increases in one host (i.e., the mosquito) are counterbalanced by fitness losses in the second host (i.e., humans). Alternatively, if the variant can establish infection in the human host, onward transmission may occur.
Evolutionary processes that impact maintenance of DENV variants. Fitness trade-offs and population bottlenecks result in purifying selection, an evolutionary feature of DENV (62,63). In purifying selection, synonymous mutations, which do not cause amino acid changes, are more likely to be maintained than nonsynonymous mutations. Purifying selection purges deleterious variants from the transmission cycle, many of which are caused by nonsynonymous mutations since these mutations can impact protein stability, function, and viral replication (64). Arguably, nonsynonymous mutations in viral proteins might be more successful than synonymous changes in escaping the selective pressures imposed by Wolbachia. But these variants must still support efficient viral replication.
While purifying selection may slow the emergence of Wolbachia-resistant variants, it may not eliminate them. Wolbachia-resistant variants could accumulate over time, eventually becoming dominant in transmission cycles. Considering mosquito populations are large and their susceptibility to DENV infection can fluctuate, continued  (1) and are able to disseminate to distal tissues (2). Variants that are more resistant to the antiviral properties of Wolbachia may be selected, allowing the virus to replicate in Wolbachia-infected and -uninfected cells. These variants may possess a replicative advantage in disseminated sites of the mosquito with high Wolbachia density, such as the salivary gland (3). DENV variants that replicate efficiently in the mosquito might not always be infectious for humans (4), such that if a Wolbachia-resistant variant did infect a human, it may replicate poorly or be outcompeted by other variants that are better adapted for replication in humans.
monitoring for virus evolution in Wolbachia-carrying mosquitoes will be important in regions where Wolbachia-carrying Ae. aegypti have been established.
Certainly, compared to antiviral resistance events described for viruses that circulate in a single host, the sequential evolutionary speed bumps that DENV populations encounter are likely to delay Wolbachia-resistant viruses from emerging in transmission cycles.

WOLBACHIA IMPACTS ON THE SUBCELLULAR DENV INFECTIOUS CYCLE
Wolbachia is a complex organism thought to inhibit the infectious cycle of DENV within mosquito cells that carry the bacterium by numerous mechanisms (38). In the following section, we explore some of the proposed inhibitory mechanisms with the aim of speculating how DENV could evolve to bypass these on an intracellular level.
To determine the stage(s) of the DENV life cycle that are impacted by Wolbachia, the progression of viral infection has been tracked in insect cell lines artificially infected with Wolbachia. Consistently, it has been shown that viral replication is significantly reduced in mosquito or Drosophila cells when antiviral Wolbachia strains are present, and it is widely agreed that DENV (as well as other related flaviviruses and unrelated alphaviruses) are likely to be inhibited after virus entry, at an early stage in RNA replication, or perhaps at translation of the incoming viral RNA template (51,(65)(66)(67). It should be noted that, for practical reasons, many of the studies characterizing the impacts of Wolbachia at the cellular level have been performed using cell culture models. In whole mosquitoes, these effects may vary between tissues, as the virus encounters different Wolbachia densities, as well as cell type-specific effects during infection and dissemination.
Overcoming Wolbachia-induced host effects that contribute to viral inhibition. Both DENV and Wolbachia are known to alter their host environment. Wolbachia is present in the mosquito throughout its life cycle, and it is therefore likely that some of the Wolbachia-induced host changes interfere with essential stages of virus infection. Identifying how DENV is restricted will help us to determine how viral resistance may emerge against Wolbachia. Relevant host cell modifications induced by Wolbachia can be grouped into 3 main categories: host cell structural modifications, altered nutrient homeostasis, and induction of host immune/stress responses. Lindsey et al. provide a comprehensive review discussing the various ways Wolbachia may induce these changes and how they may impact on viral pathogens (38). While it is possible that DENV may adapt to overcome a specific antiviral factor that drives these modifications (either Wolbachia-or host-cell derived), we have kept our discussion broad, since it is not known which viral/antiviral factor interaction(s) is responsible for inhibiting DENV. Additionally, viral inhibition is probably induced by the collective contribution of several Wolbachia-induced host modifications (38). As such, several points in the DENV life cycle may be simultaneously under selective pressure when Wolbachia is present. While it is unlikely that a single mutation in the viral genome may allow complete viral resistance to emerge, it is possible that particular mutations may allow the virus to overcome one or some of these effects, reducing the overall impact of Wolbachia in inhibiting viral transmission. Here, we will focus on three subcellular modifications that are likely to be critical for Wolbachia to induce its antiviral effect, and consider whether viruses could adapt to overcome these pressures.
(i) Altered lipid homeostasis. wMel and wAlbB infection of Ae. aegypti imparts minor costs on host fitness (41,68,69). Genomic studies of multiple Wolbachia strains show it must source a variety of amino acids and lipid complexes from its host to complement its own limited metabolic pathways (70). Several groups have examined the hypothesis that Wolbachia may alter the lipid profile of host cells, disrupting the requirements for productive DENV infection. Koh et al. (71) examined the lipid profile in whole wMel-Ae. aegypti and DENV-infected Wolbachia-free Ae. aegypti (intrathoracically injected with DENV-3). They reported that DENV infection of mosquitoes induced a lipid profile distinct from mosquitoes carrying wMel, suggesting that DENV-3 and Wolbachia are not in direct competition for lipids. In mosquitoes coinfected with DENV-3 and wMel, they found that DENV modulation of host lipids dominates the changes normally induced by Wolbachia. However, it is important to note that intrathoracic DENV infections are known to overwhelm the effects of Wolbachia and may not represent the virus-Wolbachia relationship in a natural infection (68). Furthermore, analysis of the lipidome in whole mosquitoes may mask smaller, tissue-specific lipid changes induced by Wolbachia.
Manokaran et al. (72) also attempted to define the lipid changes that occur when wMel and/or DENV is present in Ae. aegypti. Using the Aag2 Ae. aegypti-derived cell line, they identified acyl-carnitines (a class of lipids involved in energy production) as specifically upregulated by DENV and ZIKV, but downregulated in the presence of wMel, including in wMel-Aag2 following viral infection. This may suggest that wMel and DENV are in fact competing for some lipids. The acyl-carnitine inhibitor etomoxir reduced DENV levels in Ae. aegypti without wMel, supporting an in vivo role for this lipid. It is possible that this shift in acyl-carnitines occurs in only a subset of mosquito cell types, which could explain why it was not observed by Koh et al. Other studies have also shown that supplementing or chemically modulating host lipid profiles in mosquito cell lines or Drosophila that carry various antiviral Wolbachia strains reduces the antiviral effectiveness of Wolbachia (73)(74)(75). This suggests that regardless of whether viruses are competing with Wolbachia for the same lipids, lipids are likely to contribute in some way to the antiviral state imposed by Wolbachia.
Flaviviruses are highly dependent on cholesterol and other lipids for virion entry and exit, and formation of modified endoplasmic reticulum (ER) membranes for viral RNA replication (vesicle packets) (76)(77)(78). DENV infection also causes an accumulation of acyl-carnitines in the midgut of Ae. aegypti, suggesting the virus may divert energy to better support its own replication (79).
Perhaps Wolbachia-modulated lipid levels change the cholesterol content of cellular membranes to impair intracellular trafficking or formation of membrane-associated replication complexes, or reduce energy availability for DENV replication (Fig. 2). Further work is needed to determine if these hypotheses hold true and whether DENV can adapt to overcome these cellular changes.
(ii) Disruption of intracellular membranes. Studies examining wMel in a Drosophiladerived cell line have shown that Wolbachia is intimately associated with host cell membranes. wMel is contained within and around ER and Golgi-derived vesicles, causing regions of these organelles to swell (80)(81)(82). Given that specific remodeling of these organelles is required by DENV for replication and maturation, it seems likely that their disruption by Wolbachia could impair the establishment of viral infection. Work from Bhattacharya et al. has shown that the small amount of virus produced from insect cells carrying the wMel strain of Wolbachia has reduced infectivity and/or replication capacity in mammalian cells (51,67). This would be consistent with disrupted ER/Golgi structures, which are strictly required for viral RNA replication and the production and maturation of envelope proteins for flaviviruses (Fig. 2). In this scenario, perhaps disruption of viral RNA replication events could lead to the production of defective interfering viral particles (viruses that contain substantial deletions in their genomes) and/or perturbed ER/Golgi organelles may not allow the correct maturation and processing of viral envelope proteins, i.e., events which could reduce the infectivity of any viral particles produced.
Notably, while alphaviruses replicate and form virions in quite distinct regions of the cell compared to flaviviruses, alphaviruses are still dependent on their replication complexes forming in association with ER membranes, and trafficking and maturation of their envelope proteins through the ER and Golgi secretory pathway (83). Thus, disruption of these organelles could potentially similarly impact the two virus families.
It is yet to be determined whether wMel or other antiviral Wolbachia strains similarly occupy these organelles in vivo in Ae. aegypti, but it is certainly a compelling hypothesis for a mechanism that may contribute to Wolbachia's antiviral activity.
If Wolbachia is colonizing regions of the ER and Golgi, preventing typical establishment of DENV replication complexes and virus-specific remodeling events at these organelles, then perhaps the virus could adapt to replicate in regions unaffected by Wolbachia, or else could adapt to bud from the plasma membrane like alphaviruses. Given the intricate association of DENV with these organelles, from viral replication to virion formation and budding, it seems that these adjustments would take an enormous number of compensatory mutations arising across interacting viral proteins, before functional virus would emerge.
(iii) Changes to the host cell cytoskeleton. Other studies in Drosophila have revealed that Wolbachia utilizes microtubules and actin to support its localization, particularly in the Drosophila oocyte. This may allow the endosymbiont to persist throughout Drosophila development and to pass from generation to generation (84)(85)(86). Furthermore, Wolbachia has been shown to secrete the actin bundler protein WalE1. Overexpression of WalE1 in transgenic flies leads to an increase in Wolbachia titer, suggesting Wolbachia may manipulate actin to modulate its own replication (87). For DENV, each aspect of the virus life cycle, including entry, intracellular transport, replication, and egress is intimately tied to the host cell cytoskeleton. For example, DENV entry is dependent on actin filament integrity (88,89), while organelle remodeling and formation of vesicle packets are associated with changes in the cytoskeleton structure, including reorganization of the intermediate filament vimentin-critical for DENV replication (90,91). A situation where Wolbachia modulates the cytoskeleton to disrupt DENV trafficking into cells and/or formation of vesicle packets would be consistent with Wolbachia restricting DENV at a stage prior to RNA replication (Fig. 2) (51, 65, 66). (1) Virus uptake occurs through clathrin-mediated endocytosis, and the viral genome is delivered following fusion of the viral and mature-endosomal membranes. (2) Replication of viral RNA (red) is restricted in Wolbachia-carrying cells and so is vesicle packet formation on ER membranes. This could be due to disturbance of ER and Golgi apparatus membranes due to (3) occupation/disruption by Wolbachia (green). (4) Altered cellular lipid content, e.g., increased cholesterol storage (yellow) or reduced acyl-carnitines, may restrict trafficking of membrane-bound vesicles and/or lower energy resources for virus production. Similarly, Wolbachia-induced alterations of the host cell cytoskeleton (5) may interfere with trafficking of endosomes and/or ER and Golgi vesicles required for movement of incoming virions and the maturation of daughter virions.
If Wolbachia disrupts DENV entry via endocytosis, could DENV entry adapt to occur in a pH-independent manner, at the cell surface? There have been reports that alphaviruses, including SINV, may be able to enter both by receptor-mediated endocytosis and at the plasma membrane (92,93). This would require mutations to accumulate in the viral envelope protein that allow fusion activation (conformational changes in the envelope proteins that drive the merging of viral and host cell membranes) to occur at a neutral and acidic pH.
In fact, pH-independent entry has been described in laboratory experiments for flaviviruses and related hepaciviruses. Endosomal fusion activation events for these viruses are crucially controlled by specific histidine residues within the viral fusion protein (94)(95)(96). Boo et al. demonstrated that substitution of histidine with positively charged arginine enhanced entry of hepatitis C virus (hepacivirus) at neutral pH (94).

VIRUS FAMILIES THAT ARE RESISTANT TO WOLBACHIA
Perhaps another way we can consider how viral resistance may arise against Wolbachia is to examine the life cycle of viruses that are not inhibited by this endosymbiont. There are several reports that negative-sense RNA viruses, including bunyaviruses, are not inhibited by Wolbachia. The insect-specific virus Phasi Charoen-like virus (Bunyaviridae) can replicate effectively as both a persistent infection and following an acute challenge in the Ae. aegypti-derived cell line Aag2 coinfected with wMel or wMelPop Wolbachia strains (97,98).
Bunyaviruses typically have three negative-sense RNA segments that are bound to multiple copies of the viral polymerase (L) and nucleoprotein (N), encased in a lipid bilayer. Similar to flaviand alphaviruses, bunyaviruses are internalized via clathrinmediated endocytosis, and transcription and translation are closely coupled, occurring in association with the rough ER (see reference 99 for a review on bunyavirus replication). However, the replication strategy for these viruses differs substantially to flaviand alphaviruses, since the incoming viral RNA must be transcribed to a positive-sense RNA (generating either an mRNA for translation or a positive-sense replicative intermediate), with the replicative intermediate copied again to generate the negative-sense progeny viral RNAs. Interestingly, these progeny viral RNAs may associate with newly formed L and N proteins in a structure called the viral tube before budding through the Golgi, where it collects its membrane and envelope proteins (100). Perhaps this distinct RNA replication and assembly strategy, whereby shorter viral RNAs are protected by L and N proteins at each stage, enables bunyaviruses to persist in the presence of Wolbachia.

INVESTIGATING EVOLUTION OF WOLBACHIA-RESISTANT VIRUSES
Further studies into the evolution of DENV in the presence of Wolbachia may direct us toward the mechanisms that underlie viral inhibition by indicating the regions of the genome that are under selective pressure. This in turn may allow us to predict the likelihood of these mutations arising in the field. To do this, we can use a laboratory setting to push conditions to favor viral sequence diversity. By continually passaging DENV in an invertebrate host with Wolbachia (whole insects or cell culture), we can remove the purifying selection usually associated with host alternation in order to broaden the repertoire of viral sequences being maintained over time.
Such studies have been reported by two groups. One study passaged the RNA virus DCV through whole D. melanogaster with a native wMelCS infection (a strain closely related to wMel) over 10 passages (101). The other study passaged DENV-3 ten times in Ae. aegypti-derived Aag2 cells artificially infected with wMel (102). In both studies, the viruses replicated over time when consistently challenged by Wolbachia. However, these viruses grew to substantially lower titers and had no replicative advantage over those passaged in Wolbachia-free cells. Notably, no studies have yet examined viral passaging in the presence of wAlbB.
While these are very artificial evolutionary experiments, they show that, in the laboratory, RNA viruses do not develop fit viral variants with resistance to wMel in a short time frame.

DETECTION AND MANAGEMENT OF WOLBACHIA-RESISTANT DENV
If a fit DENV variant that is able to replicate in Wolbachia-carrying mosquitoes were to establish itself in a transmission cycle, how would it be identified and how would we mitigate the impact? In regions such as Yogyakarta, Indonesia, where local transmission of DENV has ceased in areas where Wolbachia has been introgressed into Ae. aegypti populations (26), viral resistance could be suspected if persistent local DENV transmission chains were reported in areas of Wolbachia establishment. Since loss of Wolbachia-mediated virus inhibition could occur due to changes in the virus, Wolbachia, or the Ae. aegypti host, it would be essential to first determine the underlying cause(s) of the transmission events.
Before assuming that a virus has evolved resistance to Wolbachia, it would be prudent to ensure Wolbachia has not been substantially reduced in density or frequency within a mosquito population, e.g., due to exposure to very high temperatures (103). It would also be important to rule out adaptation of the mosquito host or Wolbachia, which may allow the mosquito population to become permissive to DENV infection (30). This could be done by challenging wild-caught Wolbachia-carrying Ae. aegypti with a blood-meal spiked with laboratory viruses previously shown to be inhibited by that Wolbachia strain.
To determine if viral resistance is the underlying cause of the transmission events, laboratory Wolbachia-carrying Ae. aegypti colonies could be infected with circulating virus isolates from the region. Measuring the replication/transmission of these viruses in laboratory-reared mosquitoes, alongside previously published Wolbachia-sensitive laboratory viral strains, would determine if the DENV genotypes circulating in the community were better able to overcome the inhibitory effects of Wolbachia. Sequencing of the circulating DENV isolates from both human and mosquito hosts over the course of an outbreak and comparison with recent historical isolates may provide insight into the genetic changes that may have led to viral resistance. Viral resistance against an introgressed Wolbachia strain could be managed using various strategies. One option is to not alter the existing mosquito population, as it is unlikely that Wolbachia-carrying mosquitoes would be more susceptible to DENV than wild-type mosquitoes. Initially, it is likely that only one DENV genotype would be resistant to the antiviral properties of wMel or wAlbB, and Wolbachia may still protect against all other genotypes/serotypes. Over time, the resistant genotype would likely become dominant, and in this scenario supplementary interventions may be of benefit. Releases of mosquitoes that carry a reproductively incompatible Wolbachia strain could be performed to remove an existing strain or to replace it as long as viral resistance does not extend to all Wolbachia strains. Management of viral resistance could also be achieved through the use of complementary interventions, such as vaccines or vector control strategies that are based on gene drive and/or population suppression. While many of these complementary methods are still undergoing development and evaluation, initial reports indicate they show potential for future implementation (14,104,105).

CONCLUDING REMARKS
With a body of evidence now demonstrating that Wolbachia-Ae. aegypti introgression methods can substantially reduce the burden of dengue in areas of endemicity, it is expected that application of this technology will undergo a major expansion in coming years (17,19,24,26). The intention is that this will lead to long-term control or local elimination of human-pathogenic arboviruses. Achieving long-term suppression in the field would be dependent upon the evolutionary stability of the Wolbachia, Ae. aegypti, and DENV tripartite interaction. Wolbachia and Ae. aegypti evolve slowly compared to DENV, and Wolbachia-carrying mosquitoes collected years after release have so far retained their antiviral profile. Yet the rapid mutation rate of RNA viruses suggests it is inevitable that viruses like DENV will eventually adapt to Wolbachia's selective pressure and become resistant to the intervention. The question is, how long will this take?
There is no precedent for an antiviral intervention like Wolbachia, and we cannot be certain how viruses will adapt upon continued exposure to this endosymbiont. In the field, DENV will repeatedly face the selective pressures imposed by Wolbachia, but the genetic diversity generated and maintained by the virus will be limited by the need for the virus to infect a range of mosquito tissues, while also maintaining competence in the human host. In addition, since the mode of action of Wolbachia appears broad, it is most likely that multiple mutations across the viral genome will be necessary to allow the virus to adapt to this unique cellular landscape.
While we have focused on factors that may affect the development of viral resistance to Wolbachia-introgression methods, these considerations are also highly relevant to any gene drive/replacement technology where the virus and host will coexist in a long-term evolutionary relationship. Finally, as Wolbachia-based biocontrol methods increase in scope and longevity, monitoring for the emergence of viral resistance to Wolbachia should remain a critical component of these programs.

ACKNOWLEDGMENTS
This work was supported by National Health and Medical Research Council, Australia, Ideas Grant 1182432 (J.E.F.), Program Grant 1132412 (C.P.S.), and Investigator Grant 1173928 (C.P.S.).
We thank Patrick Lane (ScEYEnce Studios ) for graphical enhancement of the figures.