*3.2. ROS after the Establishment of Infection in Hosts*

While ROS are an important component of defense by the host against the pathogen, ROS are also generated during the establishment of a pathogen within a host and continue to be produced throughout the progression of the disease, for example, flaviviruses are known to induce the production of ROS, which are linked to apoptosis and are, thus, involved in the killing of infected cells together with the pathogen and eventually support the survival of the remaining non-infected cells. Through the production of ROS, infected individuals battle against pathogens to eliminate invading microbes in the early stage of invasion to prevent the progression of damage to the adjacent cells caused by the pathogens themselves and by the triggered inflammatory insults as well (Figure 1A) [57,58]. Therefore, this cellular response could affect the vectorial capacity of the arthropods. However, the coevolution of the arthropods with the pathogens carried by them has led to their coadaptation with each other's immune responses (Figure 1B). Pathogens have devised various protective shields to evade host responses to ensure their transmission, colonization, and survival in a hostile environment within vertebrate hosts, until either the recovery or the death of the host [49,50,59].

During dengue virus (DENV) infection, apoptosis is the usual outcome [60]. Apoptosis is usually brought about by the production of viral proteins, which disrupts the function of the endoplasmic reticulum (ER), resulting in the accumulation of misfolded and unfolded proteins. The presence of these misfolded and unfolded proteins activates the unfolded protein response (UPR). Even with the UPR, the mitigation of the effect of ER stress may not be addressed within a specific time and will still result in apoptosis [61,62]. However, in a mosquito cell line, it was found that mosquito cells were neither severely damaged nor subjected to apoptosis, rather the infection persisted in this setting, and ROS were detected. Interestingly, a p53 paralogue was upregulated during infection. The p53 is a transcription

factor that selectively transcribes the catalase gene, which alleviates ROS accumulation within the cells, therefore reducing the rate of apoptosis (Figure 1B). In experiments that reduced the expression of the *p53* gene, ROS accumulated in the infected cells [60]. In *Ae. aegypti*, knockdown of the catalase gene also resulted in reduced oviposition and lifespan with H2O2 challenge and reduced virus titer in the midgut upon infection with DENV [63]. Aside from the catalase activity, glutathione S-transferase (GST) activity was also higher in DENV-infected cells. GST suppression resulted in an earlier release of superoxide ions and higher cell mortality. Interestingly, this upregulation was not observed in mammalian cells infected with the same virus, indicating that this phenomenon may be limited to only mosquito cells [64]. Besides GST activity, an additive anti-apoptotic activity was observed due to the upregulation of the inhibitor of apoptosis (IAP) [61]. ROS and oxidative stress are also believed to be controlled by the proper refolding of misfolded proteins, and this is usually achieved through the production of the X-box binding protein 1 (XBP1), which is presumed to be a critical transcription factor for various chaperones, including the *BiP/GRP 78* mRNA [62]. In contrast, West Nile virus, another flavivirus, also induces ROS production. However, the exact mechanism of this ROS production is still unknown. Mosquito cells infected with this virus-induced upregulation of Nrf2 and NRF1-mediated antioxidant genes, eventually result in elevated reduced glutathione (GSH) levels. This ultimately increased the oxidative capacity of the cells to withstand the oxidative stress elicited by the virus infection [65].

In contrast, transfection with the nonstructural protein 1 of the flavivirus, e.g., tickborne encephalitis virus, induces oxidative stress in HEK293T cells and activates the antioxidant defense of these cells [66]. Moreover, in tick cells, the knockdown of the antioxidant *GST* molecule with subsequent infection of the Langat virus, another member of Flaviviridae, resulted in increased mortality, decreased proliferation, and decreased viral titer [67]. Furthermore, infection of LGTV in tick cells indicates a possible correction of the protein folding as seen by the upregulation of chaperone proteins, specifically heat shock proteins (HSPs) 90 and 70 [68,69]. These HSPs help in the refolding of misfolded proteins or are related to the degradation of terminally misfolded proteins to prevent protein aggregation, thereby creating an anti-apoptotic environment within cellular niches [58,70]. This corrective response was also observed in *Anaplasma phagocytophilum* infection in ticks and tick cells, wherein HSP20, HSP70, and HSP90 expressions have been upregulated [58,71,72]. Metabolomics also indicates that terminally misfolded proteins tend to prevent ER stress and apoptosis. Accordingly, *HSP70* knockdown decreases *Ana. phagocytophilum* titer in ticks [71].

Furthermore, *Ana. marginale* infection upregulated genes closely related to the generation of antioxidants. Simultaneous knockdown of catalase, glutathione peroxidase, and thioredoxin together with oxidative resistance 1 gene favored the colonization of *Ana. marginale* in BME26 cells, strongly supporting that the oxidant response is involved in the control of infection and the maintenance of cell survival [56]. Additionally, mitochondrial ROS production also increases in response to *Ana. phagocytophilum* to control the parasite. Conversely, to maintain the parasite's fitness and maintain the infection, other alternative ROS production and apoptosis pathways are also inhibited [73].

Another group of molecules that are also upregulated during infection are selenoproteins. They are a group of proteins that both catalyze and regulate several redox reactions [38]. It has been proposed that pathogens can induce the production of selenoproteins that not only allow their proliferation and transmission but also play a key role in pathogen acquisition. In *Borrelia burgdorferi* infection, Salp25D, a tick selenoprotein, is utilized against the oxidative stress from the inflammatory process at the biting site [32]. Knockdown of selenoprotein M reduces the titer of *Ana. marginale* and inhibits the development of the infective stage of *Ana. marginale* [74]. Selenoprotein P (SelP) is upregulated in the salivary glands of the *Ri. parkeri*–infected ticks to ameliorate oxidative stress during feeding. Furthermore, the knockdown of *SelP* genes also reduced the transovarial transmission of pathogens [52]. In addition to the direct control of ROS through antioxidant

enzymes, the generation of ROS is also controlled by regulating free cations such as iron, which augments ROS productions. Ferritins that sequester free iron are upregulated in *Dermacentor variabilis* during *Escherichia coli* infection [13]. Bacterial iron-binding proteins could also sequester iron in the blood meal, which is expressed in the infective stage of *Ana. phagocytophilum* [75].
