Arboviruses and co-infections with bacteria: CHIKV and DENV as examples

Arboviruses such as Dengue Virus (DENV), Chikungunya Virus (CHIKV) or Zika Virus (ZIKV) are arthropod borne viruses that are transmitted to humans via a relatively small number of mosquito species, in particular Aedes spp. and Culex spp., and only rarely does human-to-human transmission occur. Humans and other vertebrate animals may serve as a secondary host and in the absence of human-to-human transmission as a reservoir host. Indeed, uninfected (female) mosquitoes only become infected following a blood meal from an infected vertebrae. Following viral infection, the viral particle then needs to enter epithelial cells of the midgut where viral replication takes place, followed by the egress of viral particles into hemocoel and subsequent spread into the salivary glands, thus allowing the infection of humans and/or animals via an insect bite. Within the insect, both the salivary gland and the midgut are therefore natural barriers to viral replication.

Similar to the human gut, the insect gut contains an abundant bacterial flora that may inhibit or promote viral entry as well as viral replication, including obligate intracellular symbionts that may prevent viral replication. In the case of Aedes Agypti experimentally infected with DENV-serotype 2 (DENV-2), treatment with antibiotics reduces viral titres, and co-infection of midgut epithelial cells of Ae. aegypti larvae and adults with DENV-2 or CHIKV and Serrata odoroferia (a gram-negative, anaerobic, bacteria of the Enterobacteriaceae) increases susceptibility of Ae.aegypti to DENV-2 and CHIKV in the absence of increased viral replication suggesting that bacteria can at least facilitate viral entry. In the case of midgut epithelial cells co-infected with CHIKV and S.odoroferia, CHIKV has been shown to interact with two mitochondrial proteins, namely Hsp60 and Porin.

Furthermore,  the co-infection of Ae. aqypti but not Ae. notoscriptus  with both DENV and Wolbachia pipientis decreases viral replication, although the mechanism is not known. It has been speculated however that the infection of cells with Wolbachia induces the immune response, which not only targets bacteria but also DENV and -in Ae.albopictus- derived C6/36 cells also inhibits the replication of DENV and CHIKV as shown by decreased levels of viral RNA and viral titres.

                     CHIKV and Wolbachia Hsp60 protein

As discussed before, the infection of mouse embryonic fibroblast (MEF)  and HeLa cells with CHIKV induces the formation of autophagosomes via the ER /UPR and oxidative stress response which at least at the early stages of the replication cycle does not induce apoptosis and is required for the formation of viral replication centres in a p62/SQSTM-1, (human) NDP52 and LC3-C dependent manner. Since prolonged infection of both human and mouse cells may induce caspase-dependent apoptosis during later stages of the viral replication cycle due to a prolonged ER and/or oxidative stress response, it might be possible however that co-infection of C3/36 cells with Wolbachia may inhibit the ER stress response and thus prevent the formation of viral RCs. Since the infection of mosquitoe cells with Wolbachia alters the composition of the host cells miRNA expression both regulating the expression of cellular miRNAs and by expressing bacterial miRNAs it might be possible that cellular autophagy is inhibited via inhibiting the expression of autophagy related genes including Beclin-1.

Figure: CHIKV and the ER stress response

Figure: CHIKV proteins that induce the ER stress response, leading to different outcomes

Interestingly, the treatment of PBMCs but not lymphocytes derived from both asymptomatic endemic normal and chronic patients with purified recombinant Wolbachia Hsp60 (rWMhsp60) induces caspase-dependent apoptosis, which can be inhibited by autophagy. Apoptosis is induced by the activation of TLR-4 receptors at the cell surface and inducing the formation of reactive oxygen species (ROS) in mitochondria. As a result, mitochondrial membrane potential depolarizes, cytochrome C is released and subsequently downstream caspases (caspase-3 and -9) are activated, inducing caspase dependent cell death. In addition, recent research indicates that ROS also induces a variant of autophagy termed NETosis (Neutrophil extracellular traps), suggesting that Wolbachia and/or rWMHsp60 might induce cell death in the absence of classical apoptotic markers such as exposure of Phosphatidylserine. Activation of TLR-4 however may also induce autophagy via TRIF/MyD88 mediated cleavage of the Bcl-2/Beclin-1 complex and thus contributes to the formation of the phagosomes. Subsequently, bacterial induced autophagy may induce the cellular antibacterial response as well as promoting the presentation of antigens via the MHC Class II complex.

Figure: Binding of rWMhsp60 to TLR-4 stimulates NETosis and/or caspase dependent apoptosis

Figure: WMHsp60 might activate TLR-4 induced formation of ROS and thus promotes the degradation
viral RC

Treatment of cells derived from endemic normal but not chronic patients with Rapamycin and purified rWMhsp60 however induces not only the formation of autophagosomes but also prevents apoptosis; if autophagic flux is inhibited has not been demonstrated, although the results suggest that this is the case since TLR-4 localises inside LC3 positive vesicles. In any case, these results suggest that C3/36 cells infected with CHIKV may undergo apoptosis in the presence of Wolbachia or at least if expressing WMhsp60. Alternatively, Wolbachia derived Hsp60 might sequester CHIKV viral particles and/or CHIKV viral proteins, thus inhibiting release of the viral genome, viral assembly, and/or viral egress. Whilst this has not been investigated yet, it is known that in cells derived from the midgut epithelium and infected with both CHIKV and Wolbachia, CHIKV binds to Hsp60. Given that in cells treated rWMhsp60 apoptosis is induced by depolarization of the mitochondrial membrane, the induction of autophagy by rapamycin might induce mitophagy; consequently CHIKV (bound to mitochondrial Hsp60) might be degraded by mitophagy. In addition, the co-infection with CHIKV may promote the presentation of both viral and bacterial antigens via the MHC Class II complex and thus promote the induction of the immune response.

It should be noted however that Hsp60 is also located at the cell surface and rWMhsp60 has been reported to interact with cell surface TLR-4, leading to the activation of a pro-apoptotic cell signaling pathway including the induction of oxidative stress signaling and the activation of caspase-3. The induction of autophagy by Rapamycin relocalises TLR-4 to late endosomes thus preventing the activation of the pathways. Analogous to Rapamycin treatment, the induction of the ER stress response by CHIKV might relocalise TLR-4 in rWMHsp60 treated mosquito cells as well, thus inhibiting antiviral as well as antibacterial signaling pathways.

The problem is that any effect on the autophagic flux has not been evaluated and consequently any effect on the stability on the viral RC is only hypothetic. If autophagic flux however increases in Rapamycin treated and Wolbachia infected cells, then the co-infection with CHIKV might induce the degradation of not only TLR-4 (and thus protect cells from Wolbachia induced apoptosis) but also of the viral RC (and thus decrease viral replication) particularly at early stages of viral replication during formation of the RC.

In conclusion, Wolbachia might either sequester viral particles and/or viral proteins via WMhsp60 or alternatively CHIKV induced formation of autophagosomes might facilitate the degradation of TLR-4 and viral RC, thus not only preventing apoptosis (and senescence) but also leading to reduced viral replication. Additionally, inducing the degradation of viral RCs and/or TLR-4 following the infection of Wolbachia containing cells might also degrade Wolbachia itself since the induction of autophagy has also been shown to degrade Wolbachia in C3/36 and PC35 cells.

DENV and Wolbachia: inhibition of viral entry v. protection of WMHsp60 induced apoptosis

In the case of DENV, the viral NS1 protein has been reported to localize to vesicular structures containing the viral NS5, NS3, NS2A, NS2B, NS4A and NS1 proteins as well as dsRNA and the viral RNA dependent RNA Polymerase (RdRP) in infected Vero and C6/36 cells. In subsequent studies, it was shown that the formation of viral RC -similar to other positive strand viruses such as Poliovirus and Coronaviruses- is dependent on autophagy as evidenced by decreased viral replication in autophagy deficient MEF cells, knockdown of autophagy related genes, or treatment with 3-Methyladenine (3-MA) of DENV-2 or DENV-3 infected human Pre-Basophils/Mast KU812, Huh7 and HepG2 cells as well as increased viral replication in DENV-3 infected HepG2 cells treated with Rapamycin. DENV RC however do not represent classical autophagosomes but rather invaginations of ER cisternae that may mature into amphisomes as a result of the fusion of endosomes with autophagosomes as indicated by the co-localization of dsRNA with mannose-6-phosphate receptor (MPR) as well as LC3. In addition, DENV inhibits Torin-1 induced, starvation induced and basal autophagic flux in Huh7 cells as evidenced by the use of a mCherry-LC3 reporter plasmid whilst inducing the proteasomal degradation of p62/SQSTM-1, suggesting that the formation of viral RC indeed might be not entirely dependent on autophagy related proteins. The induction of autophagy by DENV however is required for the degradation of lipid droplets and the release of lipids thus stimulating lipid metabolism via selective autophagy.

Figure: DENV induction of lipophagy - connection to mitophay?

Similar to other viruses, DENV therefore has been postulated to induce the formation of autophagosome or autophagosome-like structures early in the replication cycle whilst inhibiting autophagic flux later in the replication cycle.

In the case of mosquito cells, C3/36 cells infected with DENV support the findings obtained from mammalian cells is so far as the viral RC are in close proximity to the ER and coated with ribosomes (similar to the co-localization of dsRNA with L28 in DENV-2 infected HepG2 cells)  and similar to mammalian cells, cellular fatty acid synthase is redistributed to the viral RC (by the viral NS3 protein and dependent on Rab18).

The degradation of lipid droplets by DENV induced autophagy might therefore be conserved in both mammalian and mosquito cells. In the case of mosquito cells infected with both Wolbachia and DENV however, DENV might prevent cells from WMhsp60 induced apoptosis and senescence by either sequestering TLR-4 in amphisomes and/or degradation of TLR-4 at least early in infection whereas later in infection due to the inhibition of autophagic flux TLR-4 might accumulate in late endosomes or amphisomes.

However, this might only apply if the infection of cells with DENV occurs prior the infection with Wolbachia since Wolbachia has been reported to prevent the entry of DENV and other Flavivirus' into cells by sequestering cholesterol. In contrast, Serratia odorifera might increase viral entry by increasing prohibitin on the cell surface. Mitochondria localized prohibitin has also been shown to inhibit mitophagy, but in the absence of data it is not possible to discern if Serratia odorifera induces merely the redistribution of either HSP60 and/or Prohibitin or alternatively promotes the redistribution of mitochondria to sites of viral replication. The pore forming ShlA toxin from Serratia marcesens has been shown to induce autophagy in CHO cells, a potential homologoue in Serratia odorifera might increase DENV replication by facilitating the fusion of lipid droplets with autophagosomes and lysosomes.

               Lipophagy: autophagy of lipid droplets

Autophagy has been shown to regulate the metabolism of lipid droplets that are storage areas of triglycerides (TG) and cholesterol both in the absence and presence of viral replication. The induction of autophagy correlates with the decrease of triglyceride levels since the fusion of lipid droplets (LD) with autophagosomes and subsequent lysosomal fusion induces the release of free fatty acids in a process commonly referred to as lipophagy which requires both lipases and the small Rab7GTPase. Free fatty acids released by lipophagy generate ATP by mitochondrial β-oxidation and thus contribute  to the maintenance of  the cellular energy homeostasis. In the context of viral replication, Brome Mosaic Virus replication is dependent on specific localised lipid compositions whereas West Nile Virus induces the synthesis and redistribution of cholesterol to sites of viral replication. In the case of Hepatitis C Virus (HCV) -arguably the most prominent virus that utilises lipid droplets for viral replication- assembly of viral particles takes place at lipid droplets and the release of viral particles is dependent on the very low-density lipid secretion pathway.

Inf DENV-2 infected Huh 7.5 cells, the induction of autophagy not only protects cells from apoptosis induced by the UPR pathway but also decreases TG levels and LD area by approx. 35% (leading to a reduction of approx. 70% of the LD volume) as measured by oil red staining and EM respectively at 48 hrs p.i. .Treatment of infected cells with either 3-MA or transfection with siRNA targeting Beclin-1 or ATG12 prior infection results in a marked increase in LD compared to mock treated cells, indicating that indeed not only the induction of autophagy is necessary for DENV-2 induced lipophagy. Accordingly, lipophagy should be inhibited at later stages of the replication cycle. Rather than inducing the formation of autophagosomes however, DENV-2 might rather promote the fusion of autophagosomes with LD as indicated by results showing that the number of lysosomes is not altered in DENV-2 infected Huh 7.5 cells but that the number of structures positive for both Bodipy 493/503 and Lysotracker increases.  Using a novel inhibitor, SAR-405, which targets the Vps34 kinase should clarify if DENV-2 does induce the formation of mitophagosomes akin to HCV or alternatively promotes the fusion of existing autophagosomes independent of de novo formed mitophagosomes. So far however DENV NS4A has not been shown to exhibit mitochondrial localisation in infected Huh 7 cells. The free fatty acids generated by the degradation of TG however are not utilised to generate ATP but also required for the formation of viral particles; free fatty acids are recruited to viral RC via an interaction between the viral DENV NS3 protein localised at the ER and free fatty acids in a Rab18GTPase dependent manner. In addition to mitophagy, lipid droplets may also be degraded by autophagy.

Figure: DENV and ZIKV induce autophagy and/or mitophagy and lipophagy

Recent evidence however suggests that in Aag-2 cells derived from Aedes Aqypti, both the infection with bacteria and with Sindbis Virus (SINV) or DENV increases the number of LD, suggesting that the induction of LD synthesis induces the antiviral and antibacterial response. It should be noted however that the authors did not evaluate lipophagy and/or the co-localisation of LD with LC3 in these cells. 

Similar to Vero cells transfected with the Polyomavirus BK Angnoprotein or Polyomavirus BK infected cells, LD in DENV-2 infected cells are localised in close proximity to the ER, which is in accordance with the site of viral replication, but similar to DENV, an accumulation of mitoplysophagsomes has not been demonstrated for Polyomavirus. .

In the case of co-infection of cells with DENV-2 and Serratia odorifera,  mitochondria might  relocated to the site of viral replication and thus increase the availability of ATP provided if the bacterial HSP60 and/or Prohibitin localises to the mitochondria of the host cell and interacts with viral proteins localised at the ER. If these mitochondria are susceptible to depolarisation and undergo mitophagy remains also to be seen.

Finally in my opinion, DENV might increase replication of ZIKV by increasing the turnover of LD, thus providing a favourable environment for ZIKV replication.\
In summary, both Wolbachia and Serratia odorifera might play an important role in regulating the replication of CHIKV, DENV, and potentially ZIKV in mosquito cells both by decreasing and increasing viral titres. Further research is however need to establish the nature of these interactions.

Further reading

Clem RJ (2016). Arboviruses and apoptosis: the role of cell death in determining vector competence. The Journal of general virology PMID: 26872460


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Ana Beatriz Ferreira Barletta, Liliane Rosa Alves, Maria Clara L. Nascimento Silva, Shuzhen Sim, George Dimopoulos, Sally Liechocki, Clarissa M. Maya-Monteiro & Marcos H. Ferreira Sorgine
Emerging role of lipid droplets in Aedes aegypti immune response against bacteria and Dengue virus

Scientific Reports 6:19928