Doxycycline cotherapy with albendazole relieves neural function damage in C57BL/6 and BALB/c mice infected with Angiostrongylus cantonensis.

BACKGROUND: We investigated the effects of combination therapy albendazole and doxycycline in Angiostrongylus cantonensis-infected mice during early and late treatment. MATERIALS AND METHODS: C57BL/6 and BALB/c mice were divided into five groups: (i) uninfected, (ii) infected with A. cantonensis, (iii) infected + 10 mg/kg albendazole, (iv) infected + 25mg/kg doxycycline, and (v) infected + 10 mg/kg albendazole + 25 mg/kg doxycycline. We administered drugs in both early treatments started at 7-day post infections (dpi) and late treatments (14 dpi) to A. cantonensis-infected C57BL/6 and BALB/c mice. To assess the impact of these treatments, we employed the Morris water maze test to evaluate spatial learning and memory abilities, and the rotarod test to measure motor coordination and balance in C57BL/6 mice. Additionally, we monitored the expression of the cytokine IL-33 and GFAP in the brain of these mice using western blot analysis. RESULTS: In this study, A. cantonensis infection was observed to cause extensive cerebral angiostrongyliasis in C57BL/6 mice. This condition significantly affected their spatial learning and memory abilities, as assessed by the Morris water maze test, as well as their motor coordination, which was evaluated using the rotarod test. Early treatment with albendazole led to favorable recovery outcomes. Both C57BL/6 and BALB/c mice express IL-33 and GFAP after co-therapy. The differences of levels and patterns of IL-33 and GFAP expression in mice may be influenced by the balance between pro-inflammatory and anti-inflammatory signals within the immune system. CONCLUSIONS: Combination therapy with anthelmintics and antibiotics in the early stage of A. cantonensis infection, in C57BL/6 and BALB/c mice resulted in the death of parasites in the brain and reduced the subsequent neural function damage and slowed brain damage and neurobehavior impairment. This study suggests a more effective and novel treatment, and drug delivery method for brain lesions that can decrease the neurological damage of angiostrongyliasis patients.

Life as a Vector of Dengue Virus: The Antioxidant Strategy of Mosquito Cells to Survive Viral Infection.

Dengue fever is a mosquito-borne viral disease of increasing global importance. The disease has caused heavy burdens due to frequent outbreaks in tropical and subtropical areas of the world. The dengue virus (DENV) is generally transmitted between human hosts via the bite of a mosquito vector, primarily Aedes aegypti and Ae. albopictus as a minor species. It is known that the virus needs to alternately infect mosquito and human cells. DENV-induced cell death is relevant to the pathogenesis in humans as infected cells undergo apoptosis. In contrast, mosquito cells mostly survive the infection; this allows infected mosquitoes to remain healthy enough to serve as an efficient vector in nature. Overexpression of antioxidant genes such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione S-transferase (GST), glutaredoxin (Grx), thioredoxin (Trx), and protein disulfide isomerase (PDI) have been detected in DENV2-infected mosquito cells. Additional antioxidants, including GST, eukaryotic translation initiation factor 5A (eIF5a), and p53 isoform 2 (p53-2), and perhaps some others, are also involved in creating an intracellular environment suitable for cell replication and viral infection. Antiapoptotic effects involving inhibitor of apoptosis (IAP) upregulation and subsequent elevation of caspase-9 and caspase-3 activities also play crucial roles in the ability of mosquito cells to survive DENV infection. This article focused on the effects of intracellular responses in mosquito cells to infection primarily by DENVs. It may provide more information to better understand virus/cell interactions that can possibly elucidate the evolutionary pathway that led to the mosquito becoming a vector.

Cell-to-Cell Spread of Dengue Viral RNA in Mosquito Cells.

Dengue virus (DENV) is an important mosquito-borne arbovirus that is particularly prevalent in tropical and subtropical areas of the world. The virus is generally ingested with a blood meal, replicates in host tissues, and disseminates into salivary glands for transmission to the next host. Membrane-bound vacuoles carrying DENV particles have been documented in mosquito cells and play a role in the cell-to-cell transmission of DENV2. C189 is one member of the tetraspanin family and generally increases its expression as one component of the vacuoles (C189-VCs) within C6/36 cells infected with DENV2. In the present study, we have further demonstrated via sucrose gradient centrifugation as well as magnetic immune isolation (MI) that the RNA of DENV2 was eventually carried by C189-VCs. In addition, viral RNA was shown to spread from donor to recipient cells in a coculture assay even when 20 mM NH4Cl was added to inhibit virus replication in the culture. In an alternate assay using the transwell system, viral RNA was only detected in recipient cells in the absence of 40 mM NH4Cl, suggesting that cell-cell contact is required for the intercellular spread of DENV2. In turn, the formation of viral synapse (VS) derived from aggregates of viral particles was frequently observed at sites of cell contact. Taken together, the formation of C189-VCs in C6/36 cells is induced by DENV2 infection, which may serve as a vehicle for transferring virions and also viral RNA to neighboring cells by cell-to-cell transmission after cell-cell contact. This finding provides insight into the understanding of viral spread between mosquito cells. It may also elucidate the benign persistent infection in mosquito cells and efficient dissemination of DENV infection within a mosquito vector.

Dengue outbreaks and the geographic distribution of dengue vectors in Taiwan: A 20-year epidemiological analysis.

Dengue fever is an important mosquito-borne viral infectious disease that mostly occurs in tropical and subtropical areas of the world. According to epidemiological data from the Center for Disease Control of Taiwan, more than 98.62% of outbreaks of indigenous total dengue cases were reported in the southern part of Taiwan. Southern Taiwan is an aggregate area encompassing Tainan, Kaohsiung, and Pingtung, all of which are located below the Tropic of Cancer (23º35'N). With a few exceptions, dengue outbreaks mainly occur in southern Taiwan which is highly associated or overlaps with the prevalence of Aedes aegypti. A.aegypti is presumed to be absent from the northern part of Taiwan, while Aedes albopictus breeds in areas throughout the island. According a collection of 20 years of epidemiological data from Taiwan, the inability of A. aegypti to survive the winter weather in northern Taiwan may account for its restricted geographical distribution and that of dengue outbreaks it transmits. A.aegypti, unlike temperate strains of A. albopictus, lacks embryonic diapause signaled by a short photoperiod which thus reduces its cold-hardiness. Therefore it is intolerant of low temperatures that frequently accompany rains and unable to survive during winter in the northern part of Taiwan.

A novel p53 paralogue mediates antioxidant defense of mosquito cells to survive dengue virus replication.

Mosquito cells allow dengue viruses (DENVs) to undergo replication without causing serious deleterious effects on the cells, leading to advantages for dissemination to other cells. Despite this, increased accumulation of reactive oxygen species (ROS) is usually detected in C6/36 cells with DENV2 infection as shown in mammalian cells. Uniquely, oxidative stress caused by the ROS is alleviated by eliciting antioxidant defense which leads to protection of mosquito cells from the infection. In the present study, a novel p53 paralogue (p53-2) was identified and proved to be regulated in C6/36 cells with DENV2 infection. With a gene-knockdown technique, p53-2 was demonstrated to transcribe catalase which plays a critical role in reducing ROS accumulation and the death rate of infected cells. Ecologically, a higher survival rate of mosquito cells is a prerequisite for prosperous production of viral progeny, allowing infected mosquitoes to remain healthy and active for virus transmission.

XBP1-Mediated BiP/GRP78 Upregulation Copes with Oxidative Stress in Mosquito Cells during Dengue 2 Virus Infection.

Dengue viruses (DENVs) cause dengue fever which is an important mosquito-borne disease in tropical areas. Generally, DENV does not cause cellular damage in mosquito cells. However, alterations in cytosolic calcium ions ([Ca2+]cyt) and the mitochondrial membrane potential (MMP), as well as accumulated reactive oxygen species (ROS), including superoxide anions (O2∙-) and hydrogen peroxide (H2O2), can be detected in C6/36 cells with DENV2 infection. Evident upregulation of BiP/GRP78 also appeared at 24 h postinfection in DENV2-infected C6/36 cells. As expression of BiP/GRP78 mRNA was reduced when the transcription factor X-box-binding protein-1 (XBP1) was knocked down in C6/36 cells, it demonstrated that BiP/GRP78 is the target gene regulated by the XBP1 signal pathway. We further demonstrated that the expression and splicing activity of XBP1 were upregulated in parallel with DENV2 infection in C6/36 cells. In C6/36 cells with BiP/GRP78 overexpression, oxidative stress indicators including [Ca2+]cyt, MMP, O2∙-, and H2O2 were all pushed back to normal. Taken together, DENV2 activates XBP1 at earlier stage of infection, followed by upregulating BiP/GRP78 in mosquito cells. This regulatory pathway contributes a cascade in relation to oxidative stress alleviation. The finding provides insights into elucidating how mosquitoes can healthily serve as a vector of arboviruses in nature.

Virulence of Japanese Encephalitis Virus Genotypes I and III, Taiwan.

The virulence of genotype I (GI) Japanese encephalitis virus (JEV) is under debate. We investigated differences in the virulence of GI and GIII JEV by calculating asymptomatic ratios based on serologic studies during GI- and GIII-JEV endemic periods. The results suggested equal virulence of GI and GIII JEV among humans.

PERK Signal-Modulated Protein Translation Promotes the Survivability of Dengue 2 Virus-Infected Mosquito Cells and Extends Viral Replication.

Survival of mosquitoes from dengue virus (DENV) infection is a prerequisite of viral transmission to the host. This study aimed to see how mosquito cells can survive the infection during prosperous replication of the virus. In C6/36 cells, global protein translation was shut down after infection by DENV type 2 (DENV2). However, it returned to a normal level when infected cells were treated with an inhibitor of the protein kinase RNA (PKR)-like ER kinase (PERK) signaling pathway. Based on a 7-Methylguanosine 5'-triphosphate (m7GTP) pull-down assay, the eukaryotic translation initiation factor 4F (eIF4F) complex was also identified in DENV2-infected cells. This suggests that most mosquito proteins are synthesized via canonical cap-dependent translation. When the PERK signal pathway was inhibited, both accumulation of reactive oxygen species and changes in the mitochondrial membrane potential increased. This suggested that ER stress response was alleviated through the PERK-mediated shutdown of global proteins in DENV2-infected C6/36 cells. In the meantime, the activities of caspases-9 and -3 and the apoptosis-related cell death rate increased in C6/36 cells with PERK inhibition. This reflected that the PERK-signaling pathway is involved in determining cell survival, presumably by reducing DENV2-induced ER stress. Looking at the PERK downstream target, α-subunit of eukaryotic initiation factor 2 (eIF2α), an increased phosphorylation status was only shown in infected C6/36 cells. This indicated that recruitment of ribosome binding to the mRNA 5'-cap structure could have been impaired in cap-dependent translation. It turned out that shutdown of cellular protein translation resulted in a pro-survival effect on mosquito cells in response to DENV2 infection. As synthesis of viral proteins was not affected by the PERK signal pathway, an alternate mode other than cap-dependent translation may be utilized. This finding provides insights into elucidating how the PERK signal pathway modulates dynamic translation of proteins and helps mosquito cells survive continuous replication of the DENV2. It was ecologically important for virus amplification in mosquitoes and transmission to humans.

The p53 gene with emphasis on its paralogues in mosquitoes.

The p53 gene is highly important in human cancers, as it serves as a tumor-suppressor gene. Subsequently, two p53 homologues, i.e., p73 and p63, with high identity of amino acids were identified, leading to construction of the p53 family. The p53 gene is highly important in human cancer because it usually transcribes genes that function by causing apoptosis in mammalian cells. In contrast, p63 and p73 tend to be more important in modulating development than inducing cell death, even though they share similar protein structures. Relatively recently, p53 was also identified in mosquitoes and many other insect species. Uniquely, its structure lacks the sterile alpha motif domain which is a putative protein-protein interaction domain and exclusively exists at the C-terminal region in p73 and p63 in mammals. A phylogenetic analysis revealed that the p53 gene derived from mosquitoes is composed of two paralogues, p53-1 and p53-2. Of these, only p53-2 is responsively upregulated by dengue 2 virus (DENV2) in C6/36 cells which usually survive the infection. This indicates that the p53 gene is closely related to DENV infection in mosquito cells. The specific significance of p53-2's involvement in cell survival from virus-induced stress is described and briefly discussed in this report.

Honoring antiparasitics: The 2015 Nobel Prize in Physiology or Medicine.

Protozoa and helminths are the two main groups that cause parasitic diseases with a broad spectrum of clinical symptoms. Protozoa are unicellular organisms like the malaria parasite Plasmodium, which is responsible for the majority of deaths associated with parasitic infections. Helminths are alternative parasites that can produce debilitating diseases in hosts, some of which result in chronic infections. The discovery of effective therapeutic drugs is the key to improving health in regions of poverty and poor sanitation where these parasites usually occur. It is very encouraging that the 2015 Nobel Prize in Physiology or Medicine was awarded to Youyou Tu as well as William C. Campbell and Satoshi Õmura for their considerable contributions in discovering artemisinin and avermectin, respectively. Both drugs revolutionized therapies for filariasis and malaria, significantly reducing by large percentages their morbidity and mortality.

Distinct expression of interferon-induced protein with tetratricopeptide repeats (IFIT) 1/2/3 and other antiviral genes between subsets of dendritic cells induced by dengue virus 2 infection.

Dengue virus (DENV) infection is an emerging public health hazard threatening inhabitants of the tropics and sub-tropics. Dendritic cells (DCs) are one of the major targets of DENV and the initiators of the innate immune response against the virus. However, current in vitro research on the DENV-DC interaction is hampered by the low availability of ex vivo DCs and donor variation. In the current study, we attempted to develop a novel in vitro DC model using immature DCs derived from the myeloid leukaemia cell line MUTZ-3 (IMDCs) to investigate the DENV-DC interaction. The IMDCs morphologically and phenotypically resembled human immature monocyte-derived dendritic cells (IMMoDCs). However, the permissiveness of IMDCs to DENV2 was lower than that of IMMoDCs. RT-PCR arrays showed that a group of type I interferon (IFN) -inducible genes, especially IFIT1, IFITM1, and IFI27, were significantly up-regulated in IMMoDCs but not in IMDCs after DENV2 infection. Further investigation revealed that IFIT genes were spontaneously expressed at both transcriptional and protein levels in the naive IMDCs but not in the naive IMMoDCs. It is possible that the poor permissiveness of IMDCs to DENV2 was a result of the high basal levels of IFIT proteins. We conclude that the IMDC model, although less permissive to DENV2, is a useful platform for studying the suppression mechanism of DENV2 and we expand the knowledge of cellular factors that modulate DENV2 infection in the human body.

Involvement of Tetraspanin C189 in Cell-to-Cell Spreading of the Dengue Virus in C6/36 Cells.

Dengue virus (DENV) is naturally transmitted by mosquitoes to humans, infecting cells of both hosts. Unlike in mammalian cells, DENV usually does not cause extremely deleterious effects on cells of mosquitoes. Despite this, clustered progeny virions were found to form infection foci in a high density cell culture. It is thus interesting to know how the virus spreads among cells in tissues such as the midgut within live mosquitoes. This report demonstrates that cell-to-cell spread is one way for DENV to infect neighboring cells without depending on the "release and entry" mode. In the meantime, a membrane-bound vacuole incorporating tetraspanin C189 was formed in response to DENV infection in the C6/36 cell and was subsequently transported along with the contained virus from one cell to another. Knockdown of C189 in DENV-infected C6/36 cells is shown herein to reduce cell-to-cell transmission of the virus, which may be recovered by co-transfection with a C189-expressing vector in DENV-infected C6/36 cells. Moreover, cell-to-cell transmission usually occurred at the site where the donor cell directly contacts the recipient cell. It suggested that C189 is crucially involved in the intercellular spread of progeny viral particles between mosquito cells. This novel finding presumably accounts for the rapid and efficient infection of DENV after its initial replication within tissues of the mosquito.

Heat shock cognate protein 70 isoform D is required for clathrin-dependent endocytosis of Japanese encephalitis virus in C6/36 cells.

Japanese encephalitis virus (JEV), one of encephalitic flaviviruses, is naturally transmitted by mosquitoes. During infection, JEV generally enters host cells via receptor-mediated clathrin-dependent endocytosis that requires the 70 kDa heat-shock protein (Hsp70). Heat-shock cognate protein 70 (Hsc70) is one member of the Hsp70 family and is constitutively expressed; thus, it may be expressed under physiological conditions. In C6/36 cells, Hsc70 is upregulated in response to JEV infection. Since Hsc70 shows no relationship with viruses attaching to the cell surface, it probably does not serve as the receptor according to our results in the present study. In contrast, Hsc70 is evidently associated with virus penetration into the cell and resultant acidification of intracellular vesicles. It suggests that Hsc70 is highly involved in clathrin-mediated endocytosis, particularly at the late stage of viral entry into host cells. Furthermore, we found that Hsc70 is composed of at least three isoforms, including B, C and D; of these, isoform D helps JEV to penetrate C6/36 cells via clathrin-mediated endocytosis. This study provides relevant evidence that sheds light on the regulatory mechanisms of JEV infection in host cells, especially on the process of clathrin-mediated endocytosis.

Cell type-dependent RNA recombination frequency in the Japanese encephalitis virus.

Japanese encephalitis virus (JEV) is one of approximately 70 flaviviruses, frequently causing symptoms involving the central nervous system. Mutations of its genomic RNA frequently occur during viral replication, which is believed to be a force contributing to viral evolution. Nevertheless, accumulating evidences show that some JEV strains may have actually arisen from RNA recombination between genetically different populations of the virus. We have demonstrated that RNA recombination in JEV occurs unequally in different cell types. In the present study, viral RNA fragments transfected into as well as viral RNAs synthesized in mosquito cells were shown not to be stable, especially in the early phase of infection possibly via cleavage by exoribonuclease. Such cleaved small RNA fragments may be further degraded through an RNA interference pathway triggered by viral double-stranded RNA during replication in mosquito cells, resulting in a lower frequency of RNA recombination in mosquito cells compared to that which occurs in mammalian cells. In fact, adjustment of viral RNA to an appropriately lower level in mosquito cells prevents overgrowth of the virus and is beneficial for cells to survive the infection. Our findings may also account for the slower evolution of arboviruses as reported previously.

Discriminable roles of Aedes aegypti and Aedes albopictus in establishment of dengue outbreaks in Taiwan.

Aedes aegypti and Aedes albopictus were reported to be significant as vectors of dengue fever. In Taiwan, the latter is distributed throughout the island while the former appears only south of the Tropic of Cancer; i.e., 23.5°N. In the past decade, there were five outbreaks with over 1000 cases of dengue fever in Taiwan. Without exception, these outbreaks all occurred in the south where the two Aedes mosquitoes are sympartic. According to the Center for Disease Control of Taiwan, imported cases are thought to provide the seeds of dengue outbreaks every year. Mostly, the number of imported cases is greater in northern island, probably due to a larger population of travelers and imported workers from endemic countries. Looking at the example in 2002, northern, central, and southern parts of Taiwan reported 28, 11, and 13 imported cases, respectively. However, 54, 21, and 5309 total cases were confirmed in the corresponding regions over the entire year, indicating a significant skew of case distributions. A hypothesis is thus inspired that the existence of Ae. aegypti is a prerequisite to initiate a dengue outbreak, while participation of Ae. albopictus expands or maintains the scale until the de novo herd immunity reaches high level.

Apoptosis of Ascogregarina taiwanensis (Apicomplexa: Lecudinidae), which failed to migrate within its natural host.

Sexual reproduction of Ascogregarina taiwanensis (Apicomplexa: Lecudinidae), a parasite specific to the mosquito Aedes albopictus, in Malpighian tubules is initiated by the entry of the trophotozoites developed in the midgut shortly after pupation (usually <5 h). However, only a low proportion of trophozoites are able to migrate; others end up dying. In this study, we demonstrated that those trophozoites that failed to migrate eventually died of apoptosis. Morphological changes such as shrinkage, chromatin aggregations and formation of blunt ridges on the surface were seen in moribund trophozoites. In addition, DNA fragmentation of trophozoites isolated from the midgut of pupae was demonstrated by the presence of DNA ladders, Annexin V staining and TUNEL assays. Detection of caspase-like activity suggests that apoptosis of those trophozoites may have occurred through a mechanism of an intrinsic or mitochondrial-mediated pathway. Although apoptosis has been observed in various protozoan species, it is not clear how apoptosis in single-celled organisms might result from evolution by natural selection. However, we speculate that apoptosis may regulate the parasite load of A. taiwanensis within its natural mosquito host, leading to an optimized state of the survival rate for both parasite and host.

Additive protection by antioxidant and apoptosis-inhibiting effects on mosquito cells with dengue 2 virus infection.

Cytopathic effects (CPEs) in mosquito cells are generally trivial compared to those that occur in mammalian cells, which usually end up undergoing apoptosis during dengue virus (DENV) infection. However, oxidative stress was detected in both types of infected cells. Despite this, the survival of mosquito cells benefits from the upregulation of genes related to antioxidant defense, such as glutathione S transferase (GST). A second defense system, i.e., consisting of antiapoptotic effects, was also shown to play a role in protecting mosquito cells against DENV infection. This system is regulated by an inhibitor of apoptosis (IAP) that is an upstream regulator of caspases-9 and -3. DENV-infected C6/36 cells with double knockdown of GST and the IAP showed a synergistic effect on activation of these two caspases, causing a higher rate of apoptosis (> 20%) than those with knockdown of each single gene (-10%). It seems that the IAP acts as a second line of defense with an additional effect on the survival of mosquito cells with DENV infection. Compared to mammalian cells, residual hydrogen peroxide in DENV-infected C6/36 cells may signal for upregulation of the IAP. This novel finding sheds light on virus/cell interactions and their coevolution that may elucidate how mosquitoes can be a vector of DENV and probably most other arboviruses in nature.

Japanese encephalitis virus induces matrix metalloproteinase-9 expression via a ROS/c-Src/PDGFR/PI3K/Akt/MAPKs-dependent AP-1 pathway in rat brain astrocytes.

BACKGROUND: Japanese encephalitis virus (JEV) infection is a major cause of acute encephalopathy in children, which destroys central nervous system (CNS) cells, including astrocytes and neurons. Matrix metalloproteinase (MMP)-9 has been shown to degrade components of the basal lamina, leading to disruption of the blood-brain barrier (BBB) and to contribute to neuroinflammatory responses in many neurological diseases. However, the detailed mechanisms of JEV-induced MMP-9 expression in rat brain astrocytes (RBA-1 cells) are largely unclear. METHODS: In this study, the effect of JEV on expression of MMP-9 was determined by gelatin zymography, western blot analysis, RT-PCR, and promoter assay. The involvement of AP-1 (c-Jun and c-Fos), c-Src, PDGFR, PI3K/Akt, and MAPKs in these responses were investigated by using the selective pharmacological inhibitors and transfection with siRNAs. RESULTS: Here, we demonstrate that JEV induces expression of pro-form MMP-9 via ROS/c-Src/PDGFR/PI3K/Akt/MAPKs-dependent, AP-1 activation in RBA-1 cells. JEV-induced MMP-9 expression and promoter activity were inhibited by pretreatment with inhibitors of AP-1 (tanshinone), c-Src (PP1), PDGFR (AG1296), and PI3K (LY294002), and by transfection with siRNAs of c-Jun, c-Fos, PDGFR, and Akt. Moreover, JEV-stimulated AP-1 activation was inhibited by pretreatment with the inhibitors of c-Src, PDGFR, PI3K, and MAPKs. CONCLUSION: From these results, we conclude that JEV activates the ROS/c-Src/PDGFR/PI3K/Akt/MAPKs pathway, which in turn triggers AP-1 activation and ultimately induces MMP-9 expression in RBA-1 cells. These findings concerning JEV-induced MMP-9 expression in RBA-1 cells imply that JEV might play an important role in CNS inflammation and diseases.

Autoimmunity-related demyelination in infection by Japanese encephalitis virus.

Japanese encephalitis (JE) virus is the most common cause of epidemic viral encephalitis in the world. The virus mainly infects neuronal cells and causes an inflammatory response after invasion of the parenchyma of the brain. The death of neurons is frequently observed, in which demyelinated axons are commonly seen. The mechanism that accounts for the occurrence of demyelination is ambiguous thus far. With a mouse model, the present study showed that myelin-specific antibodies appeared in sera, particularly in those mice with evident symptoms. Meanwhile, specific T cells proliferating in response to stimulation by myelin basic protein (MBP) was also shown in these mice. Taken together, our results suggest that autoimmunity may play an important role in the destruction of components, e.g., MBP, of axon-surrounding myelin, resulting in demyelination in the mouse brain after infection with the JE virus.

Antioxidant defense is one of the mechanisms by which mosquito cells survive dengue 2 viral infection.

Dengue viruses (DENVs) generally induce apoptosis in mammalian cells but cause only minor damage in mosquito cells. To find genes involved in determining the cell fate, datasets derived from expressed sequence tags (ESTs) of C6/36 cells with and without infection were established. Of overexpressed genes found in infected dataset, chaperone proteins were validated significantly upregulated in C6/36 cells at 24 hpi. It suggests that DENV-2 in mosquito cells activates the unfolded protein response to cope with endoplasmic reticular stress. Changes in the mitochondrial membrane potential and generation of superoxide provided further evidence that DENV-2 induces oxidative stress in both C6/36 and BHK-21 cells. Significant elevation of glutathione S-transferase (GST) activity was shown in infected C6/36, but not BHK-21, cells, while suppression of GST produced superoxide at 36 hpi and increased the cell death rate at 48 hpi. This indicates that mosquito cells protect themselves against viral infection through antioxidant defenses.