Apoptosis, autophagy and unfolded protein response pathways in Arbovirus replication and pathogenesis

Arboviruses are pathogens that widely affect the health of people in different communities around the world. Recently, a few successful approaches toward production of effective vaccines against some of these pathogens have been developed, but treatment and prevention of the resulting diseases remain a major health and research concern. The arbovirus infection and replication processes are complex, and many factors are involved in their regulation. Apoptosis, autophagy and the unfolded protein response (UPR) are three mechanisms that are involved in pathogenesis of many viruses. In this review, we focus on the importance of these pathways in the arbovirus replication and infection processes. We provide a brief introduction on how apoptosis, autophagy and the UPR are initiated and regulated, and then discuss the involvement of these pathways in regulation of arbovirus pathogenesis.

There are currently 534 viruses listed in the International Catalogue of Arboviruses, of which 214 are known to be, or are probably associated with arthropods, 287 viruses are reported to be possible arboviruses and 33 are considered to probably not be, or definitely not be, arboviruses. In total, 134 of the 534 arboviruses have been reported to cause illness in humans ( Refs 7,8).
Arboviruses have a global distribution but the majority circulate in tropical areas where climatic conditions are favourable for year-round transmission. Arboviruses usually circulate within enzootic cycles involving wild or domestic animals with relatively few human infections (Ref. 9). Birds and rodents are the main reservoir hosts and mosquitoes and ticks are most often the vectors for the most important arboviruses (Table 1). 'Spill-over' of arboviruses from enzootic cycles to humans by enzootic or 'bridge vectors' can occur, under the appropriate ecological conditions. For most arboviruses, humans are deadend or incidental hosts; however, there are several viruses such as dengue, yellow fever and chikungunya that primarily infect people during outbreaks and then begin to use humans as amplification sources (Ref. 9). Figure 1 illustrates the various mechanisms by which humans are infected by zoonotic and nonzoonotic arboviruses (Ref. 10). †These authors contributed equally to this work.
Arboviruses have been causing human disease for at least a thousand years but during recent decades some have newly emerged or re-emerged and a few have increased in importance because of human population expansion and increased urbanization, increased trade or travel and global climate change ( Refs 2,9,36). Arthropod-borne viruses have been a serious public health concern, with viruses such as dengue (DEN) and yellow fever viruses causing millions of infections annually, while emerging arboviruses, such as West Nile, Japanese encephalitis (JE) and Chikungunya viruses (CHIKV) have significantly increased their geographical ranges in recent years (Refs 9,37,38,39).
From a public health point of view, those arboviruses that produce viremia in humans and cause major mosquito-borne epidemics are most important (Ref. 40). Figure 2 shows world geographical distribution of the most important vector-born arboviruses. In the following section we will discuss some of the most common arbovirus-induced diseases.

Common arbovirus-induced diseases
Dengue/dengue haemorrhagic fever The dengue viruses (DENV) are the only arboviruses that are fully adapted to the human host and its environment, thus eliminating the need for an enzootic transmission cycle (Refs 52,53). Consequently, in recent years, transmission has increased in urban and semiurban areas and has caused a major international public health concern (Refs 54, 55). DEN is now endemic in more than 100 countries in Africa, the  Routes of transmission and human exposure to zoonotic arboviruses. Infectious agents may be transmitted to humans by direct contact with infected animals, mechanical vectors or intermediate hosts. Arboviruses are maintained in mosquito-monkey, mosquito-rodent, mosquitobird, mosquito-pig, mosquito-horse and mosquito-human cycles. The enzootic cycle occurs in the region where humans intrude into the natural foci of infection. The rural epizootic cycle is involved among domestic animals and mosquitos, and amplified in the presence of intermediate hosts, which result in representing a large reservoir of viruses and severe spillover effect to dead-end hosts. In urban settings, viruses are transmitted between humans and the mosquito vectors in an urban epidemic cycle, using humans for amplification (Ref. 10).
There are no specific anti-viral treatments for Yellow fever, and the primary interventions are supportive care. Vaccination is the most important strategy to prevent Yellow fever. The current vaccine is highly effective and provides immunity within 30    Viruses and autophagy, apoptosis and unfolded protein response (UPR) Many viruses hijack host cell responses for their own benefit and use them as complementary mechanisms for replication and infection. Some of the most important host mechanisms that are usually affected by viral infection are pathways involved in cell death and cellular responses against environmental stress. These mechanisms include apoptosis (i.e. programmed cell death I), autophagy (programmed cell death II) and UPR. These mechanisms play essential functions in regulating cell fate and are important for normal cellular functions. In addition, these mechanisms are tightly regulated and can affect each other. They are usually interconnected and also 'cross-talk' with each other. We will briefly review the general concepts of apoptosis, autophagy and UPR and explain their cross-talk and regulatory mechanisms. We will then focus on the role of apoptosis, autophagy and UPR in arbovirus replication and infection and then describe different possible therapeutic approaches for arboviruses by discussing the involvement of apoptosis, autophagy and how they may determine therapeutic strategies.
An overview of autophagy, apoptosis and UPR  110,111). However, macroautophagy is the major regulated catabolic mechanism by which the bulk of damaged cytoplasmic proteins and organelles are sequestered within an autophagosome ( Refs 112,113,114). In this review, we will focus on macroautophagy (referred to herein as 'autophagy'). The first step in autophagy (see Fig. 3) involves formation and expansion of a double-membrane structure, which is called the 'isolation membrane' or 'phagophore'. The edges of this membrane eventually fuse to form a new double membrane-bound vacuole, known as the autophagosome that sequesters the cytoplasmic cargo. The autophagolysosome is formed by fusion of the autophagosome with a lysosome and lysosomal contents are degraded by hydrolytic enzymes ( Refs 115,116,117,118). As a result of degradation, nucleotides, amino acids and free fatty acids (FFAs) are generated and then reused for energy metabolism, macromolecular production and biosynthesis (Refs 119, 120). It is assumed that the different steps in macroautophagy are mediated by autophagy-related genes (ATG), which encode proteins involved in autophagy (Refs 121, 122). These proteins have been classified into five different functional categories: (i) a protein serine/threonine kinase complex that responds to upstream events such as target of rapamycin (TOR) kinase (Atg1/ULK1, Atg13 and Atg17); (ii) a lipid kinase group that controls vesicle nucleation (Atg6/Beclin1, Atg14, Vps34/ PI3KC3 and Vps15); (iii) two ubiquitin-like conjugation pathways that stimulate vesicle expansion (the Atg8 and Atg12 conjugation systems); (iv) a recycling pathway that is required for disassembly of Atg proteins (Atg2, Atg9, Atg18); and (v) vacuolar permeases that permit the efflux of amino acids from the degradative compartment (Atg22) (Refs 93, 119). The mammalian TOR (mTOR) kinase acts as a negative regulator of autophagy and is a central controller of cell growth, aging and proliferation ( Refs 123,124). Under starvation conditions, inhibited mTOR induces autophagy through  Graphic representation of autophagy. Autophagy is a process for the degradation and recycling of damaged or unnecessary cellular compartments, which has several tightly regulated steps including induction, nucleation, expansion and completion, fusion and degradation. The mTOR is known as the key regulator of autophagy induction and can be suppressed by ULK1, leading to trigger VPS34-Beclin 1-class III PI3-kinase complex. Several different membrane pools contribute to the formation of the phagophore. The Atg proteins (Atg2, Atg9, Atg18) are essential for phagophore formation. The ATG and LC3 conjugation system also contribute in autophagosome membrane formation and elongation. The autophagolysosome then is formed by fusion of the autophagosome with a lysosome to degrade and reuse the compounds. ATG, autophagyrelated genes; mTOR, mammalian target of rapamycin.
phosphorylation of the Ulk1-Atg13-FIP200-Atg101 complex (Refs 125,126), leading to localization of Ulk1/2 and Atg13 to the autophagic isolation membrane ( Refs 127,128). During the initiation step of autophagy, Beclin 1 interacts with Vps34, which contributes to Atg protein recruitment and autophagosome nucleation (Refs 129,130). Interaction with various Beclin-1-interacting proteins facilitates the coordination of these events (Ref. 131). LC3, the mammalian ortholog of Atg8, is cleaved by Atg4 and then conjugated to the polar head of phosphatidylethanolamine (PE) to generate LC3-II, which is necessary during the elongation step of autophagy ( Refs 132,133). Hence, the autophagosome is regulated in response to the Beclin-1/Vps34/UVRAG complex, known as the maturation step ( Refs 134,135,136). An overview of autophagy is summarised in Figure 3.

Apoptosis
There are two main functionally distinct pathways for apoptosis induction (Fig. 4): the extrinsic and the intrinsic mitochondrial pathways ( Refs 137,138,139). Caspases are involved in most of the apoptotic processes and are activated by ligation of death receptors [tumour necrosis factor receptor (TNFR), Fas, TNF-related apoptosis-inducing ligand (TRAIL)] or release of specific proteins from the mitochondria (Refs 140, 141). However, accumulating evidence suggests that the two pathways are intimately intertwined (Refs 138, 142), which will be described in the next sections. The extrinsic apoptosis cascade is stimulated after the binding of cell surface receptors to their ligands, resulting in Fas-associated protein with death domain (FADD)-dependent activation of initiator caspases, namely caspase-8, and subsequently caspase-3 and -7 (Refs 143, 144). As a consequence, effector caspases (i.e. caspase-3 and caspase-7) are dimerized and activated and, once active they can cause apoptosis (Refs 141, 145). The mitochondrial apoptotic death mechanism integrates various extracellular stimuli including drugs, nutrients and radiation and also different intracellular stimuli such as oxidative stress, oncogene expression, endoplasmic reticulum (ER) stress and DNA damage ( Refs 146,147). The apoptotic signals in this pathway converge on the mitochondria to release apoptogenic proteins such as cytochrome c, apoptosisinducing factor (AIF), Smac/DIABLO, Omi/HtrA2 and mitochondrial endonuclease G ( Refs 148,149,150,151). The Bcl-2 family of proteins serve as important regulators of the release of these mitochondrial proteins that can be divided into two classes: (i) antiapoptotic members (e.g. Bcl-2 and Bcl-xL); and (ii) proapoptotic members (e.g. Bax, Bak, Bid, Bad, Noxa, Puma and others) ( Refs 152,153). Up-regulation of proapoptotic proteins or down-regulation of antiapoptotic proteins can cause an increase in permeability of the mitochondrial membrane, which later promotes release of cytochrome c and other proteins into the cytosol ( Refs 151,154,155,156). In the presence of deoxyadenosine triphosphate (dATP), the released cytochrome c interacts with Apaf-1 and caspase-9 and forms a ternary complex, leading to activation of caspase-3 and then apoptosis ( Refs 142,157,158). In addition, p53 plays a stimulating role in intrinsic apoptosis induction ( Refs 159,160,161). Thus, the two direct p53 transcriptional targets Noxa and Puma can mediate the pro-apoptotic activity of Bax and Bak, and thereby promote apoptosis ( Refs 162,163).
It is widely accepted that there is cross-talk between the two extrinsic and intrinsic pathways, such that activity in one pathway interferes with signalling steps in the other pathway (Ref. 141).
The pro-apoptotic cytochrome c-releasing factor Bid is positioned to serve as a link between the extrinsic death receptor pathway and the intrinsic pathway (Ref. 154). Cleavage of the BID protein in the cytoplasm by caspase-8 causes Bid to localise in the cytosol while truncated Bid translocates to the mitochondria and activates the mitochondrial pathway after apoptosis induction through death receptors, and can be used to amplify the apoptotic signal (Ref. 164). Although Bid is a downstream target of caspase-8 in the extrinsic apoptotic pathway, it also acts as ligand for Bax and Bak, causing caspase-9 activation (Refs 154, 165). Caspase-9 activation proteolytically activates downstream caspases (e.g. caspases-3,-6,-7), which, in turn, can result in apoptosis ( Refs 166,167).

UPR
The ER contains an extensive network of tubules, sacs and cisternae, which extend from the cell plasma membrane through the cytoplasm and to the nuclear envelop through a continuous connected network ( Refs 168,169). The ER is the main sub-cellular compartment involved in proper folding of proteins and their maturation. Approximately one-third of the total proteins are synthesised in the ER. Many different perturbations can alter the function of the ER leading to unfolding or misfolding of proteins in the ER. This condition is referred to as ER stress ( Refs 169,170). The ER creates a series of adaptive mechanisms to prevent cell death complications and these together are referred to as the UPR ( Refs 170,171). The UPR can be involved in the secretory pathway leading to restoration of protein folding homeostasis. However, if there is too much stress on the ER, and the ER cannot cope with this stress, it will eventually lead to cell death (Ref. 172). The UPR also plays an important role in maintaining cellular homeostasis of specialised secretory cells such as pancreatic beta cells, salivary glands and plasma B cells (Ref. 170). It is becoming increasingly evident from animal models that UPR has several functions that are not directly linked to protein folding including inflammation, energy control and lipid and cholesterol metabolism (Ref. 170). The existence of UPR was first reported by Kozatsumi et al. more than 25 years ago (Ref. 173). They showed that glucose regulated proteins (GRPs) that are associated with the ER are upregulated upon sensing the presence of unfolded or misfolded proteins in the ER (Ref. 173). While the mechanisms and signalling events behind it were not known at the time, today we have a much better understanding of the UPR and how these events are regulated in the ER at the molecular level. ER stress response signals are constantly monitored by three main classes of sensors. These include inositol requiring enzyme 1 alpha (IRE-1α) and IRE-1β, protein kinase RNA like ER kinase (PERK) and activating transcription factor 6 (ATF6; both α and β isoforms) (Fig. 5). In normal healthy cells these sensors are in an inactive state.
IRE1. This is a type I transmembrane protein receptor having an N-terminal ER luminal-sensing domain. The cytoplasmic C-terminal region contains both an endoribonuclease domain and a Ser/Thr kinase domain (Ref. 169). There are two homologues of IRE1: IRE1α and IRE1β. Activation of IRE1 involves dissociation from Grp78, followed by dimerization, oligomerization and trans-autophosphorylation, which leads to conformational changes and activation of its   FIGURE 5. Graphic representation of ER stress and virus replication. ER stress is enhanced in the viral infected cells and activates UPR proteins (e.g. PERK, ATF6, and IRE1). Activated PERK leads to induce ATF4 via phosphorylation of eIF2α, causing attenuation of translation and inducing genes encoding CHOP. Upon IRE1 activation, TRAF2 and sXBPmRNA1 splicing are initiated in the cytoplasm, subsequently leading to activation of UPR target genes. The degradation of ATF6 is increased through recruitment of ATF6, a UPR sensor. ATF6 translocates to the Golgi and is cleaved to a nucleus targeting form that promotes expression of UPR-responsive genes. The consequences of UPR activation are necessary for viral replication and pathogenesis. ATF, activating transcription factor; CHOP, C/EBP homologous protein; ER, endoplasmic reticulum; IRE1, inositol-requiring enzyme; PERK, protein kinase RNA like ER kinase; UPR, unfolded protein response. 177 (Refs 169, 183). The role of Nrf2 as a pro-survival factor is further shown by the fact that cells devoid of Nrf2 display increased sensitivity to cell death via apoptosis after ER stress (Refs 169, 183). The overall UPR signalling pathway is shown in Figure 5.

The role of autophagy in arbovirus replication
Although autophagy was initially proposed as a physiological cellular response to environmental stress followed by virus amplification, increasing evidence now indicates that several viruses may use autophagy as a survival strategy to support their life cycle, which is known as 'pro-viral autophagy' (Refs 131, 138, 184, 185) (Fig. 6). Virus-induced induction of autophagy seems to be associated with replication/ translation of many arboviruses like DENV, JEV, CHIKV, rotavirus, and epizootic haemorrhagic disease virus (EHDV, an orbivirus) ( Refs 186,187,188,189,190,191). The results that were obtained by monitoring LC3 lipidation in JEV-infected NT-2 cells, a pluripotent human testicular embryonal carcinoma cell line treated with Rapamycin and 3-methyladenine, revealed that there was a direct relationship between autophagy and viral replication The results were confirmed using an Atg5 Although the function or functions of autophagy in promoting virus replication are not completely understood, experimental evidence suggests that there are multiple autophagy pro-viral mechanisms, including serving as a scaffold for viral replication, contributing to viral entry, regulation of lipid metabolism, suppressing innate immune responses and preventing cell death (Ref. 196). A group of arboviruses including DENV, and JEV may need to invoke autophagy components such as the autophagosome, amphisome and autolysosome to: (i) serve as a scaffold for viral replication; and (ii) escape from the immune system ( Refs 187,197,198,199). The amphisomes play major roles in DENV entry and localisation of viral translation/replication constituents (Ref. 199). DENV-2 needs pre-lysosomal fusion vacuoles (amphisomes) while DENV-3 interacts with both amphisomes and autophagolysosomes as the sites for their viral translation/replication complexes (composed of viral RNA and proteins) (Ref. 199). Poliovirus and CHIKV also stimulate autophagosome formation as a site for aggregation of viral translation/replication complexes ( Refs 188,189,200). After DENV and JEV induce autophagy, the presence of viral replication/translation complexes in both the autophagosome and the endosome suggests an auxiliary role for autophagosome-endosome fusion in viral entry (Refs 187,201). Autophagy can regulate lipid metabolism (lipophagy) through modulating the degradation of triglycerides that have accumulated in cytosolic lipid droplets (Ref. 202). Lipid droplet usage as an energy source is another autophagy-mediated pro-viral mechanism that is used for DENV replication (Ref. 203). Thus, lipid droplets are sequestered in autophagosomes and delivered to lysosomes for degradation to generate FFAs from triglycerides (Ref. 203). The released FFAs are imported to mitochondria and they undergo β-oxidation to produce ATP for viral replication (Ref. 203).

The innate antiviral immune response
The innate antiviral response is initiated by binding of pattern recognition receptors (PRRs), retinoic acidinducible gene (RIG) and Melanoma differentiationassociated protein 5 (MDA5) to intracellular viral pathogen associated molecular patterns (PAMPs)  Graphic proviral functions of autophagy. There are five possible mechanisms for modulating viral replication by autophagy. Amphisome formation is thought to be beneficial for viral cellular entry and replication. Induction of autophagosome formation is also important for some virus' replication. Furthermore, viruses initiate autophagy to benefit from lipid droplets as an energy source during viral replication. Free fatty acids are liberated from lipid droplets during autophagy to produce ATP. Viruses also stimulate autophagy to subvert immune responses by selectively degrading key regulatory molecules. Another mechanism is that viruses promote their replication by prolonging cell survival and suppressing cell death. The mechanistic details related to proviral functions of autophagy are discussed in the text. With attention to the cross-talk between autophagy and apoptosis, it is becoming apparent that autophagy postpones apoptosis and promotes CHIKV propagation by inducing the IRE1α-XBP-1 pathway in conjunction with ROS-mediated mTOR inhibition (Ref. 211). A schematic representation of autophagy and arbovirus replication is summarised in Figure 6.

The role of apoptosis in arbovirus replication
To date, several investigations have been carried out on the importance of apoptosis in different virus infections, pathogenesis and replication, but many issues are still unclear and under debate ( Refs 212,213,214). As summarised in Figure 7, a number of arboviruses such as Sindbis virus, WNV and JEV seem to use apoptosis as a virulence factor to promote their own pathogenesis (215,216,217). Each of these viruses has specific targets and biochemical-induced mechanisms during virus-induced programmed cell death. The observations suggest that Sindbis virusinduced apoptosis plays an important role in Sindbis virus pathogenesis and mortality (Ref. 215). After entry of Sindbis virus into the host cell and subsequent formation of Sindbis virus double-stranded RNA intermediates, dsRNA-dependent protein kinase (PKR) recognises these particles (Refs 218, 219, 220). PKR blocks cellular translation through eIF2a phosphorylation, which later can inhibit Mcl-1 (anti-apoptotic Bcl2 family protein) biosynthesis (Ref. 221). PKR also controls c-Jun N-terminal kinases (JNK) through IRS1 phosphorylation and later activates 14-3-3 (Ref. 222). Thus, 14-3-3 affects the accessibility of substrates (e.g., Bad) to kinases and serves to localise kinases to their substrates, thereby leading to release of Bad and disruption of the complex between antiapoptotic Bcl2 family proteins, Bcl-xl and Bak. Both Bad and Bik can displace Bak from Mcl-1, which results in Bak oligomerization and cytochrome c release, and subsequent induction of apoptosis (Ref. 222). CHIKV triggers the apoptosis machinery and uses apoptotic blebs to evade immune responses and facilitate its dissemination by infecting neighboring cells (Ref. 223). CHIKV infection can induce apoptotic cell death via at least two apoptotic pathways: the intrinsic pathway, which has been reported to be involved in virus replication and results in activation of caspase-9, and the extracellular pathway, which is dependent on the induction of cell surface or soluble death effector ligands that activate caspase-8. Thus, both pathways activate caspase-3 and finally induce cell death, and this facilitates virus release and spread (Ref. 211). The replication of Crimean-Congo haemorrhagic fever virus (CCHFV), an arbovirus from the family Bunyaviridae, is associated with the death receptor pathway of apoptosis. Up-regulation of proapoptotic proteins (i.e. BAX and HRK) and novel components of the ER stress-induced apoptotic pathways (i.e. PUMA and Noxa) have also been shown in a CCHFV-infected hepatocyte cell line, which suggests a link between CCHFV replication, ER stress and apoptotic pathways. Notably, differential high levels of transcription factors, such as CHOP, which are activated through ER stress, are present in hepatocytes following CCHFV replication (Ref. 224). In this study, it was shown that the over-expression of IL-8, an apoptosis inhibitor, during CCHFV infection was independent from apoptotic pathways. However, in other studies, a positive correlation was detected between IL-8 induction and DENV infection ( Refs 224,225,226). In contrast to Sindbis virus, CHIKV and CCHFV replication in infected cells have been proposed to be necessary for apoptosis induction, as demonstrated by the use of UV-inactivated viral particles ( Refs 227,228,229). The replication of Flaviviruses (e.g. WNV, JEV and DENV) can be limited by virus-induced programmed cell death at the early stage of virus infection. These viruses might block or delay apoptosis via activating several cell survival pathways, such as PI3K/ Akt signalling, to improve their replication rate ( Refs 227,230). Blocking PI3K (using LY294002 and wortmannin) showed that the induction of apoptosis might be a result of p38 MAPK activation and did not affect JEV and DENV viral particle production (Ref. WNV can initiate apoptosis through caspases-3 and -12 and p53 after several rounds of replication and it is noteworthy that initial viral dose exerts an influence on kinetics of WNV-induced cell death ( Refs 228,233,234,235). After some RNA virus infections, expression of multiple miRNAs in host cells might have either a positive or negative effect on virus replication. One such cellular miRNA, Hs_154, limits WNV replication by inducing apoptosis through inhibition of two anti-apoptotic proteins like CCCTC binding factor (CTCF) and EGFR-co-amplified and overexpressed protein (ECOP) (Refs 227, 236). JEV, an RNA virus, may induce ROS-mediated ASK1-ERK/p38 MAPK activation and thus lead to initiation of apoptosis (Ref. 237). In mouse neuroblastoma cells (line N18) infected with ultraviolet-inactivated JEV (UV-JEV), replication-incompetent JEV virions induced cell death through a ROS-dependent and NF-kB-mediated pathway (Ref. 238). Initial suppression of UV-JEVinduced cell death, followed by co-infection with active or inactive JEV, showed that JEV may trigger cell survival signalling to modify the cell environment for timely virus production (Ref. 238). NS1 ′ protein, a neuroinvasiveness factor that is only produced by the JEV serogroup of Flaviviruses during their replication, was introduced as a caspase substrate in virusinduced apoptosis; however, use of a caspase inhibitor had no effect on virus replication (Ref. 239). Empirical evidence showed that JEV can affect Bcl-2 expression to increase anti-apoptotic response rather than anti-viral effect to enhance virus persistence and reach equilibrium between replication and cell death (Ref. 240). Numerous in vitro studies have confirmed that DENV can induce apoptosis in a wide variety of mammalian cells including endothelial cells, hepatocytes, mast cells, monocytes, dendritic cells and neuroblastoma cells, but the mechanisms are not completely understood. Dendritic cells are believed to be the primary DENV targets that play central roles in supporting active replication during virus pathogenesis. However, a recent study reported that DENV replication in monocyte-derived dendritic cells (mdDCs) was positively correlated with pro-inflammatory cytokine secretion such as TNFα and apoptosis (Ref. 241). To achieve high replication in macrophages, hepatoma and dendritic cells, DENV may subvert apoptosis by inhibiting NF-kB in response to TNFα stimulation ( Refs 242,243). Interaction between DENV capsid protein and the hepatoma cell line (Huh7)  Apoptosis has also been extensively linked to reovirus replication. BTV induces apoptosis in three mammalian cell lines but not in insect cell lines that were tested. BTV-mediated apoptosis involved activation of NF-kB and required virus uncoating and exposure to both outer capsid proteins VP2 and VP5 (Ref. 249). Apoptosis was mediated by both intrinsic and caspase-dependent extrinsic pathways (Ref. 250). African horse sickness virus (AHSV), another orbivirus, also induced apoptosis in mammalian BHK-21 cells but not in insect KC cells, through activation of caspase-3 (Ref. 251).
When apoptotic programmed cell death acts as a barrier against viral replication, previous research has revealed that some arboviruses can delay or block apoptosis to elevate their replication and dissemination. Moreover, viral replication of some arboviruses occurs following the presence of viral-induced apoptosis. However, the exact mechanisms whereby viruses modulate apoptosis in different mammalian cells need to be more extensively studied.

Arboviruses and UPR
The scientific literature related to the role of UPR in arbovirus pathogenesis is limited. Here, we review some of the arboviruses and the UPR pathways they elicit to aid replication. WNV is a neurotropic arbovirus that emerged as a pathogen of serious concern in the North American population. People infected with WNV are affected by severe neurological diseases such as meningitis, encephalitis and poliomyelitis (Ref. 233). WNV activates multiple UPR pathways leading to transcriptional and translational activation of several UPR target genes (Ref. 233). Of the three UPR pathways, the XBP1 pathway was shown to be non-essential for WNV replication and it was replaced by other pathways. ATF6 was degraded by the proteasome and PERK transiently phosphorylated eIF2α and induced the pro-apoptotic protein CHOP (Ref. 233). WNV-infected cells showed signs of apoptotic cell death including induction of growth arrest, activation of caspase-3 and activation of poly (ADPribose) polymerase (PARP). WNV titer levels were also significantly increased when grown in a CHOP −/− deficient mouse embryo fibroblast (MEF) cell line but not in wild type MEF cells (Ref. 233). This evidence showed that WNV activates the UPR, and a host mechanism to counteract WNV infection involved activation of CHOP-dependent cell death (Ref. 233). In another study, the WNV Kunjin strain activated UPR signalling upon infection in mammalian cells (Ref. 252). UPR ATF6/IRE1 pathways were activated by this strain. However, there was no significant phosphorylation of eIF2α indicating that the UPR PERK pathway was not activated (Ref. 252). The Kunjin strain nonstructural proteins, NS4A and NA4B, were potent inducers of UPR. Moreover, sequential removal of NS4A hydrophobic domains decreased UPR activation but increased interferon gamma-mediated signalling (Ref. 252). These results show that WNV Kunjin strain activates UPR signalling and hydrophobic residues of WNV nonstructural proteins regulate the UPR signalling cascade. The role of ATF6 signalling in WNV replication is poorly understood. Results from the same group showed that ATF6 signalling is required for WNV replication by promoting cell survival and inhibition of the innate immune response (Ref. 253). ATF6-deficient cells showed a decrease in protein and virion production when infected with WNV Kunjin strain. These cells also demonstrated increased eIF2a phosphorylation and CHOP transcription, but these events were absent in infected control cells (Ref. 253). In contrast, IRE I-deficient cells do not show any discernible differences when compared with IRE I-positive cells upon infection (Ref. 253). These results also demonstrate that, in the absence of ATF6, other UPR signalling cascades such as PERK and IRE1 pathways cannot activate or enhance virus production, indicating that ATF6 is required for viral replication. However, it has also been shown that both ATF6 and IRE I are required for signal transducer and activator of transcription (STAT) I phosphorylation, showing that ATF6 is required for inhibition of innate immune response (Ref. 253). The arboviruses CHIKV and Sindbis also cause frequent epidemics of febrile illness and long-term arthralgic sequelae that affect the lives of millions of people each year (Ref. 254). These viruses replicate in infected patients and also in mammalian cells indicating that they have certain control over the UPR of the host system. Analysis of these viral infections in mammalian cells shows that CHIKV specifically activates the ATF-6 and IRE-1 branches of the UPR pathway and suppresses the PERK pathway (Ref. 254). CHIKV nonstructural protein 4 (nsp4) expression in mammalian cells suppresses eIF2α phosphorylation that regulates the PERK pathway (Ref. 254). These results provide insight on the replication of CHIKV in mammalian cells by regulating the host UPR mechanism. However, experimental findings with Sindbis virus show that it induced uncontrolled UPR, which is reflected by failure to induce synthesis of ER chaperones, followed by increased phosphorylation of eIF2α and activation of CHOP leading to premature cell death (Ref. 254). In another study, it was reported that the UPR XBP1 pathway was activated when neuoroblastoma N18 cells were infected with the arboviruses JEV and DENV (Ref. 255). This was evidenced by splicing of XBP1 mRNA and activation of downstream genes ERDJ4, EDEM1 and p58. Reduction of XBP1 by small interfering RNA had no effect on cellular susceptibility to the two viruses but enhanced cellular apoptosis (Ref. 255). Overall, these results suggest that both encephalitis and DENV trigger the XBPI signalling pathway and take advantage of this cellular response to alleviate virus induced cytotoxicity (Ref. 255). According to another group, DENV infection of A547 ovarian cancer cells elicited the UPR signalling response (Ref. 256). This was demonstrated by phosphorylation of eIF2α. It was also shown that different serotypes of DENV, such as ATF6 and IRE1, activate other UPR pathways. These results show that different DENV serotypes have the capacity to modulate different UPR pathways. They also demonstrated that de-phosphorylation of eIF2α by a drug called solubrinal reduced virus infection. This unique report showed that the same virus could activate all three UPR pathways ( Refs 256,257).
Initiation of UPR signalling is critical for cell survival and also for viral replication. All the above results show that arboviruses induce UPR signalling upon infection in mammalian cells. However, the UPR pathways that are activated upon infection with various arboviruses are not the same. Even different strains of the same virus activate different UPR pathways. These results suggest that specific virusinduced UPR pathway usage depends on the type of viral strain used. In vitro studies using ectopicallyexpressed arbovirus nonstructural proteins alone in mammalian cells showed that the proteins themselves can elicit the UPR response. Mutations of certain hydrophobic residues in nonstructural proteins reduced the UPR signalling response. These results indicate that composition of viral nonstructural proteins can determine the type of UPR pathway to be elicited and the extent of UPR response. Viral nonstructural proteins often undergo mutation; thus, more studies are needed to understand the role of arbovirus nonstructural proteins in inducing UPR. The role these viruses may play in UPR in the invertebrate insect cells is even less defined. Thus, induction of UPR signalling by viruses is one important facet, and equally important is how these viruses respond to anti-viral therapy. Do these viruses use the UPR pathways to decrease the effectiveness of anti-viral therapies? This is also one of the main questions to be answered. Thus, in conclusion, a significant amount of research is needed to investigate the pathogenesis of arboviruses and their relationship with UPR signalling. These studies can provide us with better antiviral therapeutics to control arbovirus replication by addressing various mechanisms of virus propagation.

Conclusion
Arbovirus infections lead to serious health issues in many parts of the world. To date, there is no treatment for most arbovirus infections and vaccines have been recently developed for only a few of these arboviruses. Therefore, finding a way to increase the efficiency of current therapeutic approaches to arbovirus infections will improve health conditions in many areas of the world. As has been discussed in this review, arbovirus infection can stimulate apoptosis, autophagy, or UPR in infected cells or organs. Activation of these pathways usually interferes with arbovirus replication and infection processes. Therefore, modulating these pathways may be a part of future strategies to combat arbovirus infections.
Apoptosis, autophagy and UPR have been widely investigated in many diseases including cancer, cardiovascular diseases and pulmonary diseases. Many inhibitors and inducers of these pathways have been developed to improve treatment protocols in these diseases. Since apoptosis, autophagy and UPR are tightly interconnected with each other and usually affect each other, it is critical to find out which pathway is the dominant one in the arbovirus infection process and how it regulates viral infection and replication in the infected cells. It is very important to identify the extent of apoptosis, autophagy, and UPR alterations in virus infected cells. After identifying these changes it would be very important to address how induction/inhibition of these pathways would modulate virus replication, and production of active viral particle in infected cells. As an example, we can modulate UPR using inducers (thapsigargin) or inhibitors (PERK GSK inhibitor, IRE1 inhibitor) and find out how these treatments effect arbovirus replication. These findings would provide better opportunities to use the modulation of these pathways for better designing therapeutic strategies and controlling viral infection. If this question can be clearly answered, induction or inhibition of these pathways may represent a novel enhanced treatment or prevention strategy against arbovirus infections.