We are using SINV as a model to study the effect of inducing or inhibiting apoptosis on the ability of mosquito cells to permit arbovirus replication. Arboviruses usually do not induce apoptosis in mosquito cell lines; however, there are reports of cytopathic effects resembling apoptosis in arbovirus-infected mosquitoes, leading to the question of whether apoptosis could be an anti-viral response in certain tissues or in some arbovirus-mosquito combinations. The effects of apoptosis on arbovirus replication have not been previously investigated. In this study we have characterized the effects of expressing apoptotic regulatory genes on cell viability and virus replication in the mosquito cell line C6/36.
The genetic factors that govern susceptibility to arbovirus infection in mosquitoes are poorly understood. One pathway that increasingly appears to be important in regulating the level of virus replication in mosquitoes is RNA interference (RNAi) (Campbell et al., 2008
, Keene et al., 2004
, Sanchez-Vargas et al., 2004
). Besides RNAi, there are other pathways that are also likely to be involved in mosquito anti-viral immunity, but at this time little evidence exists in this area. Transcript levels of members of the Toll and JNK pathways, as well as several serpin genes, were shown to be altered following SINV infection of Ae. aegypti
(Sanders et al., 2005
), suggesting that known innate immune pathways may be stimulated by virus infection in mosquitoes. In addition, reducing or activating Toll pathway signaling has effects on dengue virus replication in Ae. Aegypti
(Xi et al., 2008
). Finally, heat shock protein cognate 70B of Anopheles gambiae
is upregulated by o’nyong-nyong virus infection, and that silencing of this gene results in higher levels of o’nyong-nyong replication in An. gambiae
mosquitoes (Sim et al., 2007
Apoptosis is another attractive candidate anti-viral response in mosquitoes, given its importance in other virus-host systems (Clem, 2007
, Hay & Kannourakis, 2002
). It has been postulated that there are at least three barriers to successful infection and dissemination of arboviruses in mosquitoes: the midgut infection barrier (the ability to establish infection and replicate in midgut epithelium), the midgut escape barrier (the ability to penetrate the midgut and establish replication in other tissues), the salivary gland infection barrier (the ability to infect salivary glands), and the salivary gland escape barrier (the ability to enter the salivary gland lumen) (Black et al., 2002
). A successful apoptotic response in the midgut or salivary gland could thus limit the ability of a virus to replicate and be disseminated.
In this study, we expressed the IAP antagonists Mx and Rpr and the caspase inhibitor P35 to either purposely induce or inhibit apoptosis during SINV infection. While SINV normally causes non-lytic, persistent infection in mosquito cell lines, expression of Mx or Rpr from SINV caused apoptosis in C6/36 cells, as determined by cell morphology, caspase activity, and DNA fragmentation. Expression of P35, on the other hand, inhibited apoptosis induced by ActD treatment This result, together with the fact that P35 is a broad-spectrum caspase inhibitor which inhibits apoptosis in a wide variety of situations (Clem, 2007
), suggests that this virus could be used to test the effect of inhibiting apoptosis on vector competence in mosquitoes. The viruses expressing P35 still induced apoptosis in BHK cells, despite expressing P35. The reason for this is unclear, but it may be because SINV induces apoptosis rapidly in BHK cells, perhaps before sufficient amounts of P35 can be expressed from the subgenomic promoter. In a previous report, SINV-mediated expression of another caspase inhibitor, CrmA, inhibited apoptosis in BHK cells (Nava et al., 1998
), but different strains of SINV and BHK cells were used.
The two SINV expression systems used in this study, MRE and TE, differ from each other in their ability to replicate in cultured cells, and in their ability to infect mosquitoes following a blood meal. TE is derived from a laboratory strain of SINV that is well adapted to replication in cultured cells. As a consequence, we observed higher levels of foreign gene expression in C6/36 cells with TE-based viruses, and we also saw that TE viruses expressing Mx or Rpr caused apoptosis faster than their MRE-based counterparts. Higher levels of virus replication were also observed for the TE-based viruses than for the MRE-based viruses when a foreign gene insert was present in the genome, although MRE without any additional insert replicated at equivalent levels to TE in either BHK or C6/36 cells. MRE, on the other hand, is derived from a field isolate of SINV, and has higher oral infectivity in mosquitoes than TE (Foy et al., 2004
). It will thus be interesting to determine how purposely inducing or inhibiting apoptosis affects the infectivity and dissemination of these viruses in mosquitoes following infection via a blood meal.
Neither induction nor inhibition of apoptosis had significant effects on the initial burst of replication of SINV in C6/36 cells. This may be in part due to the expression of these foreign genes from the viral subgenomic promoter, which is not expressed until after the viral genome has been replicated. In mammalian cells, SINV also replicates to high titers in spite of the apoptosis that is typically associated with infection, and blocking apoptosis does not have a significant effect on the levels of replication (Nava et al., 1998
). However, cells that were infected by viruses expressing Mx or Rpr died after the initial burst of replication, and thus were not able to maintain high levels of virus replication over time. In an infected mosquito, this could be an important factor in determining vector competence. Sustained virus replication is presumably required for virus escape from the midgut and dissemination to other tissues, including the salivary glands. Therefore, if infected cells die after producing a burst of initial virus replication, virus dissemination may be adversely affected. In addition, other mechanisms may operate in vivo
to limit virus replication. For example, early and rapid recognition of apoptotic cells by phagocytic cells (hemocytes) could result in enhanced clearance of infected cells and destruction of newly formed virus before it is able to bud from the infected cell. Thus, apoptosis could have a negative effect on the ability of SINV to productively infect and be transmitted by mosquitoes.
It is generally thought that arbovirus infection has little or no negative consequences for mosquito vectors in terms of cytopathology or decreased fecundity or life span. However, there have been reports of cytopathic effects in mosquitoes infected with arboviruses (including West Nile virus and several alphaviruses) including observations of apoptosis occurring in midgut or salivary gland (Bowers et al., 2003
, Girard et al., 2005
, Mims et al., 1966
, Weaver et al., 1992
, Weaver et al., 1988
), as well as negative effects on mosquito life span (Cooper et al., 2000
). It is likely that, if apoptosis has a negative effect on vector competence, there would be little apoptosis observed in successful virus-vector combinations. In these situations, the virus may either actively inhibit apoptosis or avoid inducing apoptosis altogether. Thus, apoptosis may be more likely to occur in mosquitoes which do not have the ability to vector a particular virus, and which can mount a successful anti-viral response. To date the role of apoptosis in determining viral vector competence in mosquitoes has not been studied experimentally. The recombinant viruses characterized in this study will be useful tools to study the effects of apoptosis on determining the outcome of arbovirus infection in vivo
in mosquito vectors.