Archives of Virology https://doi.org/10.1007/s00705-017-3686-5

REVIEW

Enemies with benefts: mutualistic interactions of with lower eukaryotes

Shounak S. Jagdale1 · Rakesh S. Joshi1

Received: 8 July 2017 / Accepted: 6 November 2017 © Springer-Verlag GmbH Austria, part of Springer Nature 2018

Abstract Viruses represent some of the deadliest pathogens known to science. Recently they have been reported to have mutualistic interactions with their hosts, providing them direct or indirect benefts. The mutualism and symbiogenesis of such viruses with lower eukaryotic partners such as fungi, yeast, and insects have been reported but the full mechanism of interaction often remains an enigma. In many instances, these viral interactions provide resistance against several biotic and abiotic stresses, which could be the prime reason for the ecological success and positive selection of the hosts. These viruses modulate host metabolism and behavior, so both can obtain maximum benefts from the environment. They bring about micro- and macro- level changes in the hosts, benefting their adaptation, reproduction, development, and survival. These -host interactions can be bilateral or tripartite with a variety of interacting partners. Exploration of these interactions can shed light on one of the well-coordinated biological phenomena of co-evolution and can be highly utilized for various applications in agriculture, fermentation and the pharmaceutical industries.

Introduction that results in the formation of a new species is a theory of evolution called symbiogenesis [2]. A fusion of the virus Viruses are intracellular parasites, with capable with its host has been observed in interactions prevailing of directing their own replication. Classically they are clas- since ancient times, termed as viral symbiogenesis. Various sifed as non-cellular entities with an extrachromosomal mutualistic and symbiogenetic viruses of lower eukaryotes phase, without any essential function to their host [1]. Viral and their efects on host survival and adaptation are dis- pathogenicity is well studied through the various aspects of cussed here (Table 1). immunology, vaccine development, and genetic engineering. Recently, researchers have identifed mutualistic viruses of diferent organisms, but their replication and mechanism of Endophytic fungi and viruses interaction with the host still remains ambiguous. Discovery of such interactions has modifed the defnition of viruses, to Mycoviruses are dsRNA viruses that infect fungi. These account for mutualistic viruses, as ‘intracellular parasites, viruses are host dependent and their persistent infection with nucleic acids that are capable of directing their own leads to long-term transmission. In many cases, mycovi- replication, and are not cells’ [1]. ruses infect the host multiple times, thereby aiding the Mutualism is part of a very broad concept called symbio- host’s genetic variability, without causing any detrimental sis, which is an umbrella term that includes parasitism, com- efects [3]. Recently, symbiotic efects of mycoviruses on mensalism and mutualism – wherein both partners beneft various traits of endophytic fungi have been documented, from each other. The integration of two diferent organisms expounding a three-way symbiotic relationship amongst plants, fungi, and viruses [4]. In the case of a thermotol- erant panic grass, Dichanthelium lanuginosum, three-way Handling Editor: Robert H.A. Coutts. symbiosis has been described. The prime reason behind * Rakesh S. Joshi the growth of these plants in geothermal soils (65 °C) was [email protected]; [email protected] thought to be due to its association with a named protuberata [5]. However, the third partner 1 Institute of Bioinformatics and Biotechnology, Savitribai of this system was found to be a virus, the Curvularia Phule Pune University, Pune, Maharashtra 411007, India

Vol.:(0123456789)1 3 S. S. Jagdale, R. S. Joshi thermal tolerance virus (CThTV) which is responsible for (TauD) leads to enhanced synthesis of osmolytes and conferring thermal tolerance to the plant [Figure 1]. The osmoprotectants, namely glycine betaine and taurine [7, spherical virus, with a diameter of 27 nm, contains 2 RNA 10, 11]. In addition, an elevated level of melanin due to segments of 2.2 and 1.8 kb. Each of the 2 strands contain 2 overexpression of scytalone dehydratase (SCD), provides ORFs. ORFs of RNA1, namely ORF1a and b, overlap each protection from extreme temperature and radiation [7, 12]. other and show sequence similarity to RNA-dependent Plants colonized by CThTV-infected C. protuberata show RNA polymerases (RdRp). RNA2 ORFs show no sequence constitutive overexpression of osmolytes when compared similarity with sequences currently available. The verti- to normal plants [6]. However, the exact mechanism of this cal transmission of the virus takes place through conidi- virus mediated thermal tolerance in plants is still unclear. ospores [6]. Virus infected C. protuberata show a two-fold It was reported that C. protuberata could colonize increase in the expression of trehalose phosphate synthase various plants such as Oryza sp., Triticum sp., Solanum (TPS) leading to increased levels of an osmoprotectant - lycopersicum and Cucurbita pepo [13]. In S. lycoper- trehalose - that maintains protein and membrane integrity sicum, thermal tolerance is observed when associated under environmental stress conditions [7, 8]. Furthermore, with CThTV infected C. proturberata [6, 14]. Thus, this metabolic overexpression of mannitol, a potential osmo- mechanism of virus infection-mediated thermal protection protectant, is also observed in the hyphae [9]. Similarly, can be applied to other plants for the development of abi- upregulation of the homologs of betaine aldehyde dehy- otic stress tolerance. drogenase (BadH) and taurine catabolism dioxygenase

Fig. 1 Three-way symbiotic relationship amongst plant, Infection of CThTV C. proturberta endophytic fungus and a virus. D. lanuginosum shows enhanced thermal tolerance [65 °C]. This thermal tolerance is acquired due to the presence of a dsRNA virus CThTV resid- ing within an endophytic fungus C. proturberta

Increased Thermo tolereance

D. lanuginosum

1 3 Mutualism between viruses and lower eukaryotes

Mutualistic viruses of yeast ORF which encode the killer pre-protoxin. Satellite viruses parasitize the helper virus for the production of their cap- Over the course of evolution, various organisms have devel- sid proteins [20]. Subsequently, the toxins produced, act on oped tactics to overcome competition. For example, yeasts sensitive yeast colonies in a two-step process: The frst step such as Saccharomyces cerevisiae, Ustilago maydis and is energy independent wherein, the toxin binds to the cell Zygosaccharomyces bailii utilize viral assisted toxin pro- wall receptor of sensitive yeast. K1 and K2 bind to β-1,6- duction to kill competing yeast colonies, and are thus called D-glucan, whereas K28 binds to α-1,3-mannoprotein [21, ‘killer yeast’ [15]. The Saccharomyces cerevisiae 22]. The second step is energy dependent, where the toxin virus L-A (ScV-L-A) present in the yeast helps in the sta- translocates to the cytoplasmic membrane and interacts with ble maintenance and replication of satellite viruses namely a secondary receptor [Figure 2]. For K1, this receptor is a S. cerevisiae virus M1 (ScV-M1), ScV-M2 or ScV-M28. GPI-anchored plasma membrane protein Kre1P and for K28, Infection of ScV-M1, ScV-M2 or ScV-M28 in S. cerevi- it is cellular HDEL receptor Erd2P [23, 24]. Interaction of siae results in the production of toxins, namely K1, K2 and K1 with Kre1P results in the formation of an ion channel, K28 [16–19]. ScV-L-A and ScV-M viruses show cytoplas- disrupting the cytoplasmic membrane [25–27]. K28, after mically-inherited, symptomless infection of killer yeast. binding to α-1,3-mannoprotein, is taken up by endocyto- ScV-L-A has 2 ORFs which encode gag and pol. The virus sis and is targeted to early endosomal compartments. From capsid protein consists of 120 copies of gag and 2 copies of there, it enters the cell cytosol through the secretory pathway a gag-pol fusion protein. The satellite viruses have only 1 and blocks cellular DNA synthesis, arresting the cells in early S phase [28].

Sensitive yeast ScV-L-A

K1, K2 or ScV-M K28 toxin Erd2P Kre1P

Ion-channel formation in the ScV-M β-1-6 D-Glucan cytoplasm by K1 and toxin α-1-3- mannoprotein

K28 endocytosis

Block in DNA synthesis by K28 toxin Killer yeast

Fig. 2 Killer yeast mechanism of action. Killer yeast harbour the sat- lowed by binding to plasma membrane receptors Kre1P and Erd2P. ellite M viruses which parasitize the helper L-A virus for coat pro- Once inside the sensitive yeast cell, K1 toxin results in ion channel teins. M viruses then produce the toxins K1, K2 or K28 which act formation in the cytoplasmic membrane and K28 translocates to the on the sensitive yeast colonies in a 2-step process. Toxins frst bind nucleus blocking DNA synthesis to cell wall receptors β-1,6-D glucan and α-1,3-manonprotein fol-

1 3 S. S. Jagdale, R. S. Joshi

It is critical for the killer yeast to be immune to these tox- hemocytes, creating favorable conditions for the survival of ins. Killer yeast immunity against these toxins involves the wasp eggs and larval development [39, 40]. The Cotesia con- K1 toxin precursor, which acts as a competitive inhibitor of gregrata (CcBV) contains 156 genes; 27 the active toxin by saturating the plasma membrane receptor. genes encoding protein tyrosine phosphatases and 6 for pro- In the case of K28 toxin, the active toxin taken up by killer teins with ankyrin repeat motifs from the IκB family, which cells binds to the pre-protoxin present in the cytosol. This inhibit the immune responses. Four genes code for cysteine- protein complex is then ubiquitinated and degraded by the rich cysteine knot motif proteins, similar to the teratocyte proteasome machinery [29]. Both helper and satellite viruses secreted protein 14 (TSP 14) which inhibits the translation are dependent on the expression of several yeast chromo- of storage proteins in the hosts, resulting in developmental somal genes. Amongst these, MAK3 encodes an N-acetyl- defects. Furthermore, 3 genes code for cystantin superfamily transferase which acetylates the capsid proteins allowing cysteine protease inhibitors with immunosuppressive roles their self-assembly [30]. MAK10 which encodes N-acetyl- [41]. transferase, and PET18 that encodes thiamin metabolism PDVs and parasitoid wasps have co-evolved over 73 mil- protein, are necessary for the stabilization and replication of lion years [42]. Their phenomenal symbiogenic interaction viral particles [31]. Furthermore, SEC genes are required for blurs the boundaries that make them two separate entities extracellular protein secretion [32]. KEX encoded proteases, [43]. The strong antagonistic efect of PDVs on wasps’ Kex1p and Kex2p are necessary for the pre-protoxin pro- insect hosts, results in a favorable condition for wasp’s eggs cessing [33]. This clearly suggests a mutualistic relationship survival and larval development, which could be the prime between these viruses and yeast. Other than Saccharomyces, reason for wasp-PDV symbiogenesis. yeasts like Hanseniaspora uvarum and U. maydis also show In contrast, recurrent infection by PDVs has triggered the presence of killer viruses. The toxins generated by these horizontal gene transfer (HGT) between the virus and lepi- viruses show a broad spectrum antimycotic efect [15]. dopteran host, which is benefcial to the wasp’s host. HGT There are many applications of these viral toxins in the of two genes, BV2-5 and BLL-2, has been observed in Spo- fermentation and pharmaceutical industries. During wine doptera sp., providing it resistance against baculoviruses fermentation, killer yeasts are used to eradicate contaminat- such as nuclear polyhedrosis virus (NPV), which are used ing yeasts [34]. As the killer toxins selectively target the cell as biopesticides. BV2-5 interferes with baculovirus motil- wall components of yeasts and fungi, they also have poten- ity and replication while BLL-2, a bracoviral homolog of tial uses in the topical treatment of fungal diseases. Recently C-lectin, blocks baculoviral infection in insects [44]. studied toxins, wicaltin and zygocin produced by Williopsis californica and Z. bailii respectively, showed efective anti- fungal activity [35, 36]. Other than fungi, these kinds of Mutualistic viruses of insects mutualistic interactions are also observed in insects, as will be discussed later. Mutualistic viruses of parasitoid wasps include ascoviruses and reoviruses. In Diadromus pulchellus, the Diadromus pulchellus ascovirus 4 (DpAV4) genome is maintained in Symbiogenic viruses of insects the wasp cell nuclei as an episome, which does not integrate into the host genome. This virus is ellipsoid in shape, about Viruses show diverse and dynamic interactions with vari- 220 nm long and 150 nm wide with a genome size of 116 ous insects. Symbiogenesis between (PDVs) kb. As the wasp deposits eggs in the host, Acrolepiopsis and endoparasitoid wasps is well studied. PDVs are dsDNA assectella, virions are also injected. Post infection, viral rep- viruses which have prevailed across generations as provi- lication begins in synchrony with the wasp egg development. ruses meaning they are integrated in the host genome. In DpAV4 afects host metabolism and inhibits melanization, several species of braconid and ichneumonid wasps these protecting the wasp eggs from encapsulation [45]. Addi- are called and , respectively. These tionally, D. pulchellus reovirus 2 (DpRV2) also has similar wasps are primary endoparasites of coleopterans, dipterans, efects on A. assectella [46]. In the case of the spotted lady and lepidopterans. They oviposit in insect larvae, trigger- beetle Coleomegilla maculata, Dinocampus coccinellae is ing the innate immune response of the larvae resulting in the primary endoparasitic wasp. In the wasp, a ssRNA virus encapsulation of the eggs by larval hemocytes. PDVs sup- Dinocampus coccinellae paralysis virus (DcPV), is present press the insect immune response and help the wasp eggs’ within large vesicles in the cellular lining of the female wasp survival [37]. PDVs replicate in the calyx of the oviduct of oviduct. The virus genome is about 10 kb in size and has the female wasp and get transmitted to the wasp host insect one large ORF which encodes a precursor polyprotein. The during oviposition [38]. Upon infection, viral gene expres- N-terminal part of the polyprotein encodes viral structural sion results in morphological alteration and apoptosis of host proteins, whereas the C-terminus encodes an RdRp. Female

1 3 Mutualism between viruses and lower eukaryotes wasp oviposit in the lady beetle and the larva develops inside armigera is one of the most cosmopolitan crop pests. Chemi- the beetle’s body. After 20 days, the larva egresses and spins cal pesticides, genetically modifed crops, and the use of a cocoon between the beetle’s legs. At this time, the beetle viruses like baculoviruses as biopesticides are common prac- gets paralyzed and becomes the unwitting protector of the tices for H. armigera control. Recently, H. armigera was cocoon [47]. This phenomenon is termed as ‘Bodyguard found to show an increased resistance against major biope- Behavior’, which is due to the wasp symbiont DcPV [Fig- sticides due to the presence of H. armigera densovirus-2 ure 3]. Inside the beetle, the DcPV population is very low (HaDV-2), previously known as H. armigera densovirus-1 during the early wasp-egg developmental period. The DcPV (HaDNV1). HaDV-2 is a dsDNA virus classifed within the population only starts increasing post wasp-egg hatching. family , transmitted vertically through eggs. Just before the larvae egress, DcPV infects the glial cells The genome of the virus is about 5 kb in size and contains of the beetle. Immediately after egression, vacuolization of 3 ORFs. ORF1 and 2 encode for non-structural (NS) pro- glial cells, axonal swelling, and phagosomal activity in the teins similar to helicase and NS2, respectively. ORF3 on CNS results in neuronal degeneration consequently altering the other hand, encodes a structural protein VP. Insects the behavior of the host beetle. Apart from this, suppression infected with the virus show an increase in lifespan, weight of the beetle’s viral immune response is also observed dur- and fecundity as well as enhanced resistance to cry1Ac and ing infection. After pupation, the viral load is cleared from H. armigera nucleopolyhedrovirus infection [Figure 4]. This the beetle’s body and neuronal restoration is initiated by a suggests that HaDV-2 is a mutualistic virus, but its mecha- still unknown mechanism [48]. nism of interaction is still unknown [49]. Some plant viruses Apart from wasps, mutualistic viruses are found in many also show similar efects on their insect vectors. Feeding other insects. A polyphagous lepidopteran Helicoverpa of thrip, Frankliniella occidentalis, on plants leads to the

(Day 0) Oviposition No detectable viral titer in eggs

Latent viruses in wasp oviduct (Day 5) Healthy beetle (Day 35) (Day 0) Viral replication in larvae (Day 13)

Neuronal restoration and Immune- Adult wasp Larval development recovery suppression (Day 25) (Day 35)

(Day 21) Viral transmission to the Neuronal beetle cells degeneration Viral clearance by immune (Day 20) system

Viral infection of the glial Pupation cells

Fig. 3 Bodyguard behavior in spotted lady beetle. D. coccinellae ovipos- in tremors and paralysis in the beetle. The larva then uses the paralyzed its in the healthy beetle. The viral titer of DcPV is undetectable in eggs beetle as its bodyguard and produces a cocoon below the beetle. The adult but it increases with the development of the larvae. Just before egression, wasp leaves the cocoon after development. The beetle’s immune system the virus infects the glial cells causing neuronal degradation which results then clears the viral load and progressive neuronal restoration takes place

1 3 S. S. Jagdale, R. S. Joshi

H. defensa are less susceptible to A. ervi [54]. Other than Increased A. pisum, this anti-parasitoid role of H. defensa has also resistance AP against NPV been observed in other aphid species due to SE infection and Cry1AC [55, 56]. APSEs are dsDNA viruses with a capsid morphol- ogy similar to that of viruses classifed within the Podoviri- Cry1AC NPV Infection to dae AP insect with [57]. SE contains proteinaceous toxins required by HaDV-2 A. pisum for defense against the wasp’s ofspring. There are 7 APSE variants based on the eukaryotic toxins and bacterial lysis genes that they encode. On the basis of these toxins, they are distinguishable into 3 groups: cytolethal distending toxin subunit B (cdtB) homolog (APSE 2, 6, and H. armigera 7), Shiga-like toxin (stx) (APSE 1, 4, and 5), and toxin con- taining YD-repeat (ydp) (APSE 3) [58]. CdtB has DNase activity which disrupts actively dividing cells, while Shiga- like toxins have an N-glycosidase activity that prevents pro- tein synthesis in eukaryotes, by rRNA cleavage [59, 60]. The exact functioning of ydp toxins is still enigmatic. In pea aphids, loss of APSEs results in numerous deleterious efects on aphid ftness by slowing development, causing weight loss, delaying the onset of reproduction, and reduc- ing fecundity. This loss also results in increased titers of H. defensa leading to lethal efect on aphid growth and reproduction, thereby hampering the aphids’ lifecycle [61]. Fig. 4 Mutualistic relationship between H. armigera and HaDV-2. H. Therefore, APSEs keep a check on the population of pri- armigera larvae are susceptible to HaNPV and cry1Ac toxin. When mary endosymbionts. Recently, it has been observed that HaDV-2 is present in the insect, the larvae become resistant to the APSEs are also associated with another bacterial symbiont baculovirus and cry1Ac. The insect also shows increased ftness in known as Arsenophonus which plays an important role in the presence of HaDV-2 parasite control [62]. The molecular mechanism of APSEs interactions in Arsenophonus is still under investigation. transmission of tomato spotted wilt virus. Larvae feeding on Aside from these, many more three-way interactions are such virus-infected plants show rapid development, as com- likely to exist in nature. However, the high degree of com- pared to those feeding on non-infected plants [50]. Similarly, plexity of these interactions makes them onerous to detect aphids Micromyzus kalimpongensis transmit cardamom and study (Table 1). bushy dwarf virus, which causes foorkey disease in large Another example of insect-virus mutualism is the rela- cardamom. This virus belongs to the genus of the tionship between the parasitic mite Varroa destructor virus family Nanoviridae. M. kalimpongensis carrying the and the deformed wing virus (DMV). V. destructor is an virus show a signifcant increase in the fecundity, longevity ectoparasite of honey bees, which attaches to the bee’s body and growth rate during nymphal instar [51]. and feeds on its hemolymph [63]. The mites transmit the Similar to a three-way interaction in fungus-plant-virus, viral pathogen DMV to the honey bees. Upon infection, this some insects also exhibit a three-way relationship between virus suppresses the humoral and cellular immune response, the host insect, primary endosymbiotic bacteria, and a bac- which results in phenotypic abnormalities such as damaged teriophage. An example of this relationship is the defen- appendages, stubby wings, and paralysis of the legs [64–66]. sive symbiosis in aphids Acyrthosiphon pisum, the primary It is hypothesized that the presence of the virus results in symbiont bacteria Hamiltonella defensa, and a secondary increased ftness and in turn, fertility in mites [67]. In a symbiont phage A. pisum secondary endosymbiont (APSE) few cases, viruses have shown a more complex interaction [52]. One of the most predominant enemies of A. pisum is between antagonism and mutualism with lower eukaryotic a wasp Aphidius ervi, which lays eggs in the aphid. Along organisms. For example, in Drosophila melanogaster, Dros- with oviposition, it injects the aphid with venom which ophila C virus (DCV) shows an antagonistic efect in young degrades the aphid’s reproductive system [53]. The larvae fies, but mutualistic efects in adults. DCV infection through subsequently feed and develop inside the aphid. When the ingestion boosts the reproductive capacity and decreases the larvae are fully developed, they feed on the remnants of development time of Drosophila [68]. A detailed analysis the aphid and pupate within the hardened cuticle, called a of these interactions will help to understand the host-virus mummy. It has been observed that A. pisum infected with co-evolution.

1 3 Mutualism between viruses and lower eukaryotes

Table 1 Mutualistic viruses of eukaryotes Organism Virus Virus family Virus genus Genome Virus function References

Viruses of endophytic fungi D. lanuginosum CThTV Unassigned Unassigned dsRNA Thermal tolerance of the fungus and the associated [6, 14] S. lycopersicum [Mycovirus] plants C. proturberta Viruses of killer yeasts S. cerevisiae ScV-L-A Totivirus dsRNA Helper virus [15] ScV-M1 Unassigned Unassigned dsRNA A satellite virus. Encodes toxin protein K1 which causes disruption of the cytoplasmic membrane ScV-M2 Unassigned Unassigned dsRNA A satellite virus. Encodes toxin protein K2 which causes disruption of the cytoplasmic membrane ScV-M28 Unassigned Unassigned dsRNA A satellite virus. Encodes toxin protein K28. Blocks DNA synthesis and causes cell cycle arrest in S-phase U. maydis UmV-P1 Unassigned Unassigned dsRNA Encodes toxin protein KP1 which causes disruption of [69] the cytoplasmic membrane UmV-P4 Unassigned Unassigned dsRNA Encodes toxin protein KP4 which blocks calcium [70] uptake of the cell UmV-P6 Unassigned Unassigned dsRNA Encodes toxin protein KP6 which causes disruption of [29] the cytoplasmic membrane H. uvarum HuV-L Unassigned Unassigned dsRNA Helper virus [35] HuV-M Unassigned Unassigned dsRNA Satellite killer virus Z. bailli ZbV-L Unassigned Unassigned dsRNA Helper virus [37] ZbV-M Unassigned Unassigned dsRNA A satellite virus. Encodes toxin protein zygocin which causes disruption of the cytoplasmic membrane W. californica WcV-M Unassigned Unassigned dsRNA A satellite virus. Encodes toxin protein wicaltin which [71] causes disruption of cell wall disruption and inhibi- tion of cell wall regeneration Viruses of insects Braconid wasps Bracovirus Polydnaviridae Bracovirus dsDNA Suppression of the host immune response resulting in [72] generation of favorable conditions for the wasp larval development Ichneumonid wasps Polydnaviridae Ichnovirus dsDNA Suppression of the host immune response resulting in generation of favorable conditions for the wasp larval development D. pulchellus DpAV4 Ascovirus dsDNA Inhibition of melanisation in host insect [45] D. pulchellus DpRV2 Unassigned dsRNA Inhibition of melanisation in host insect [46] D. coccinellae DcPV Ifaviridae Ifavirus +ssRNA Neuronal degeneration in beetle causing bodyguard [47, 48] behavior H. armigera HaDV-2 Parvoviridae Unassigned dsDNA Increased ftness of the host. Resistance against bio- [49] pesticides cry1Ac toxin and HaNPV F. occidentalis TSWV Tospoviridae Orthoto- -ssRNA Rapid insect larval development [50] spovirus M. kalimpongensis CBDV Nanoviridae Babuvirus ssDNA Increased fecundity, longevity and growth rate in nym- [51] phal development A. pisum APSE Unassigned dsDNA Production of toxins against the endoparasitic wasp [52] H. defensa A. ervi. Increased aphid ftness. Keeps the primary Arsenophonus endosymbiont population in check V. destructor DWV Ifaviridae Ifavirus +ssRNA Suppression of humoral and cellular response in honey [67] bees causing phenotypical abnormalities. Increased ftness of the mite D. melanogaster DCV +ssRNA Increased reproductive capacity and decreased develop- [68] ment time in adults

1 3 S. S. Jagdale, R. S. Joshi

Future prospects and conclusion various omics, more and more viral mutualistic interac- tions are coming to light. Along with the genomic studies, The current knowledge of virus-host mutualism is lim- the efect of these viruses on the organisms’ proteome and ited, due to the complexity of these interactions. In many metabolome has to be studied to understand these interac- cases, due to a high degree of co-evolution, it is difcult tions to their fullest extent. to distinguish between the virus and the host. Sequencing Acknowledgements techniques used to study the organization of the PDVs’ Authors acknowledge Dr. Tuli Dey, Dr. Rohan Khadilkar and Dr. Sneha Bansode for their critical comments. Authors genome can be extrapolated to diferent organisms in order also acknowledge Ms. Yoshita Bhide and Ms. Shriya Lele for their to reveal the presence of mutualistic viruses and elabo- editorial assistance. rate their genomic organization [73]. Once identifed, a detailed understanding of viral mutualism at the genetic, Author contributions The concept was developed and articulated by transcriptional and translational level is required to delin- SSJ and RSJ. Manuscript was written and edited by SSJ and RSJ. eate the role of viruses in the hosts’ life-cycle. It is clear Funding Financial support is provided by the research grant from that many insects are dependent on endosymbiotic bacte- Department of Science and Technology, Government of India under ria such as Hamiltonella, Wolbachia, and Arsenophonus. ECR/2015/000502 Grant and Savitribai Phule Pune University, Pune However, the recently observed viral mutualism has added 411007, Maharashtra India. another layer of complexity to the understanding of the ecological dynamics of mutualism. 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