Aedes aegypti (Diptera, Culicidae) is considered the major vector for Dengue infection outbreaks worldwide 1 . Aedes albopictus (Diptera, Culicidae) is a less efficient vector for this virus, although it was involved in Dengue outbreaks in Japan, Seychelles, Hawaii, and Reunion Island 2 . The recent invasion of this second vector into America, Europe, and Africa could increase the transmission of arboviruses in tropical as well as temperate regions 3 4 . Dengue virus (DENV) can cause several clinical forms, ranging from an asymptomatic disease to severe Dengue hemorrhagic fever (DHF) or Dengue shock syndrome 5 . The World Health Organization estimates that 2.5 billion people live in more than 100 endemic areas where DENV can be transmitted 5 . DENV dramatically expands each year into new territories 6 7 as a consequence of combined factors such as the rapid and easy mobility of human populations, the distribution of mosquito vectors, and the lack of herd immunity in unexposed populations 8 .

There are four serotypes of DENV (DENV-1 to DENV-4) that differ by their antigenic groups. Each group can be divided into three to five different genotypes. Therefore, it is difficult to take into consideration all these factors when trying to determine the mechanisms involved in the pathogenesis of the virus. Many researches have focused on DENV-2, which provokes the most severe form of Dengue in secondary infection 8 9 , but types 1 and 3 have recently been found as new emerged types in Europe and Africa 2 10 . The report of two autochthonous DENV-1 infected persons in metropolitan France in September 2010 is a recent example of the introduction and local transmission of DENV outside its traditional area 2 . Many studies have reported that DENV-1 and DENV-3 cause severe disease at primary infection while DENV-2 and DENV-4 are frequently involved in Dengue outbreaks at secondary infection 11 12 13 14 .

Because there is no available antiviral treatment or vaccine to cure or prevent DENV, other approaches are needed to fight and control the virus. A good understanding at the molecular level of the virus-mosquito interaction should help with the design of efficient strategies for the control of DENV. Mosquito cell infection is a part of the Dengue viruses' life cycle that is poorly understood. In this study, we compared the proteome of infected and non-infected cultures of the C6/36 Ae. albopictus cell line using two-dimensional differential in-gel electrophoresis (2D-DIGE) to examine modulated proteins of infected mosquito cells. We chose DENV-1 and DENV-3 as study models because of their remarkable capacities to induce severe disease with the first infection. Furthermore, travelers have more chance to develop DENV-1 or DENV-3 infection when they live in non-endemic areas and have never been in contact with the virus 2 10 15 . This could be due to the lack of herd immunity in this human population 16 .

DENV usually induces apoptosis in infected mammalian cells but causes less damage in mosquito cells 23 . The mechanisms involved in the interaction between DENV and insect cells have been described in recent studies 9 24 25 26 27 , but these only concern the DENV-2 virus serotype. DENV-1 and DENV-3 have been less studied than DENV-2 and DENV-4. DENV-1 and DENV-3 have been found to cause DHF at first infection 12 . This finding as well as the recent report of DENV-1 introduction in France 2 and the re-emergence of DENV-3 in Sudan 28 attract our interest in these two virus serotypes. In this study, we compared the proteomes of C6/36 Ae. albopictus cells infected and non-infected with DENV-1 and DENV-3.

Cells infected with DENV-1 and DENV-3 exhibited many proteins that were differentially expressed compared to the mock-infected cells. These data may reveal cellular stress due to viral infection. In response to this stress, mosquito cells utilize antioxidant reactions combined with increased energy production to cope with the virus. The up-regulation of chaperone proteins in infected cells might be due to stress in the endoplasmic reticulum (ER) induced by viral infection; ER is where proteins are synthesized. Only proteins that are correctly folded by ER chaperones are transported to the Golgi apparatus. Misfolded and unfolded proteins lead to ER stress. Eukaryotic cells up-regulate ER chaperones and establish the cytoprotective mechanism known as the unfolded protein response (UPR) to degrade the unfolded protein 29 . Molecular chaperones are involved in preventing both newly synthesized polypeptide chains and assembled subunits from aggregating into nonfunctional structures 30 . Chaperone proteins were shown to interfere with virus assembly and replication in Ae. albopictus cells infected with Mayaro virus, and thus protect the cells from injury during viral infection 31 . Furthermore, the expression of Hsp90 was required for the activity of hepatitis B virus reverse transcriptase 32 .

Interestingly, it has been reported that acute infection of mammalian cells with several types of viruses often results in the induction of heat-shock protein expression 33 . The up-regulation of chaperone proteins 24 hours after infection with DENV was previously reported 23 . These studies support our findings with infected cells, which show two up-regulated proteins involved in protein folding biological process: chaperonin-60kD (Q16PM9_AEDAE) and one putative uncharacterized protein identified by mass spectrometry as being from Culex quinequefasciatus (B0WBF6_CULQU). These proteins belong to the heat-shock protein families Hsp60 and Hsp70, respectively.

Cells require a chaperone function to prevent or correct misfolded or unfolded proteins created by environmental stress. These chaperone proteins aid protein folding in different ways. Hsp60 is a mitochondrial chaperone that generally assists the carrying and refolding of proteins from the cytosol to mitochondria 34 . Many Hsp70 chaperones could surround an unfolded substrate to stabilize it and prevent aggregation until the unfolded molecule folds properly, after which the Hsp70 chaperones will lose affinity for the molecule and diffuse away 34 35 . Hsp60 and Hsp70 could induce mitochondrial UPR in the same way as the ER stress response 29 .

The modulation of chaperone-associated proteins could protect cells from apoptosis, as does the overexpression of calreticulin, which is a Ca²⁺-binding chaperone protein 36 . These findings suggest that the activation of these two chaperone proteins in infected cells due to oxidative stress could induce UPR to cope with ER or mitochondrial stress 23 29 . In turn, the increase of chaperone proteins may also be due to the subversion of the cell by the virus in order to complete viral replication. Indeed, it has been demonstrated that flavivirus infection activates the key transcription factor of the UPR and take advantage of this cellular response to alleviate virus-induced cytotoxicity 37 .

Chen et al. 23 further demonstrated that mosquito cells use antioxidant mechanisms to survive DENV infection. The changes in mitochondrial membrane potential and the generation of superoxide confirm that DENV induces oxidative stress in C6/36 cells 23 . This supports our results showing the overexpression in infected cells of quinone oxidoreductase, which is increasingly recognized as the major contributor to reactive oxygen species formation 38 . NADH quinone oxidoreductase catalyses NADH to NAD⁺, reduces ubiquinone, and transports protons across the inner mitochondrial membrane. This enzyme complex also reduces O₂ to superoxide, which causes cellular oxidative stress 38 . This means that mosquito cells use the oxido-reduction mechanism to protect themselves against DENV viral infection.

Another interesting result of our study is that five enzymes involved in the glycolysis pathway were up-regulated. Increased glucose uptake and glycolytic enzyme activity due to viral infection have been found in other studies 39 40 . The induction of glycolytic proteins involved in energy production is also found in CHIKV and DENV-2 infections 9 . Ritter et al. 18 suggested that the increased activity of glycolysis was due to the breakdown of the mitochondrial membrane, which decreased ATP concentration. As a result, the glycolysis pathway was activated to compensate for the lack of energy 18 . Recent studies have demonstrated more multifaceted functions of glycolytic enzymes such as GAPD and enolase. Both of these acquired non-glycolytic functions in transcriptional regulation. Moreover, GAPD might play a role as regulator or indicator of apoptosis 41 . Glycolysis is also reported to be the main source of energy production in Trypanosoma; this finding could represent a new drug target against parasites 42 43 . In addition, it has been reported that GAPDH binds to the minus-strand RNAs of Japanese encephalitis virus and the subcellular localization of GAPDH changed upon JEV infection, suggesting that GAPDH may play a role during the virus life cycle 44 . These evidences suggest that blocking the glycolysis pathway could constitute a new tool in the fight against mosquito-borne diseases via their vector control. The increase in glycolytic enzymes could also be a result of the arrest at the G1 cell cycle due to UPR.

The modulation of protein expression found in our study might be the strategy of the virus to overcome host pathways to facilitate survival at the expense of the host. Further studies are needed to understand the mechanism by which these proteins are induced during viral infection. Virus could stimulate the transcription and translation of some host products for their survival, but the mechanism is still unknown. The modulation of protein expression could also be the mosquito's response to the viral infection. It would be interesting in a further study, to determine whether a similar response occurs in cells infected with another virus or if the observed modulation of protein expression is specifically induce in response to DENV infection. Mosquitoes generate oxido-reduction stress to cope with the virus and stimulate the glycolysis pathway to prevent cellular damage. Thus, it might be beneficial to understand the proteome of infected cells to develop an anti-pathogen approach. However, these proteomics studies need to be complemented by studies using RNAi gene silencing to allow the characterization of modulated genes in vivo in mosquito tissues. Improvements in our knowledge on mosquito cell systems will be important to decipher the infection process of dengue virus in human.

Dengue Virus: DENV; Aedes: Ae; Japanese encephalitis virus: JEV; DHF: Dengue Hemorrhagic Fever; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; UPR: Unfolded protein response; PLOD: Procollagen-lysine 2-oxyglutarate 5-dioxygenase; HSP: Heat shock protein; MALDI-TOF MS: Matrix assisted laser desorption ionisation time-of-flight mass spectrometry.

The authors declare that they have no competing interests.

Conceived and designed the experiments: SP, DM. Performed the experiments: SP, PS, MS. Analyzed the data: SP, PS, DM. Wrote the paper: SP, DM. Corrected the manuscript: VC, FT. All authors read approved the final version of the MS