**1. Alphaviruses**

Viruses belonging to the *Alphavirus* genus can be found in an ecological but not taxonomic group called arboviruses (an acronym for "arthropod-borne viruses" [1]). These viruses are transmitted by a hematophagous arthropod to a vertebrate host during a blood meal; in the case of alphaviruses, the predominant vectors are mosquitoes [2].

Alphaviruses are small, enveloped viruses of approximately 70 nm of diameter. The positive-sense, single-strand RNA contains two open reading frames (ORFs) that encode four non-structural proteins (nsp1–4) and five structural proteins (capsid, E3, E2, 6K, and E1) [3]. Alphaviruses include approximately 30 members, and infection results in clinical symptoms range from mild to severe [3]. Historically, alphaviruses were divided into New World and Old World alphaviruses, following their global distribution, evolution, pathogenicity, tissue, and cellular tropism or interactions with respective hosts. Old World alphaviruses (chikungunya virus (CHIKV); Sindbis virus (SINV); Semliki Forest virus (SFV); Ross River virus (RRV), etc.) were mainly found in Asia, Africa, and Europe, while New World alphaviruses (Eastern, Western, Venezuelan, and Equine Encephalitis Viruses (EEVs); Mayaro virus (MAYV)) were found in North and South America. However, with the global spreading of these viruses and their vectors, this division between New and Old World has become obsolete. Alphaviruses are now divided into three categories: aquatic viruses, arthritogenic viruses, and encephalitic viruses [4,5]. Infections by arthritogenic viruses in humans are characterized by rashes, fever, joint and muscle pain, and encephalitis for some of them (e.g., SINV, SFV). In some cases, incapacitating arthralgia and myalgia can last for months or years after infection (e.g., CHIKV, RRV, MAYV). Encephalitic virus infections are characterized by debilitating febrile disease and encephalomyelitis, leading to death in some cases (e.g., EEEV, VEEV) [3,6].

In mammals, skin cells are the first cells targeted by an arbovirus, such as an alphavirus, when inoculated by an infected mosquito. They are not clearly defined for each alphavirus but may be dermal fibroblasts [7], dermal dendritic cells, enterocytes or keratinocytes [8,9]—they constitute the first line of defense. Viruses will then reach other organs, such as joints, muscles [10] or the brain, where they will trigger pathology through induced cell death in the acute phase or long-lasting inflammation during the chronic phase [6].

In mosquito vectors, the arboviral infection is persistent and lasts the insect's whole life. In comparison with their e ffect in the vertebrate host, alphaviruses do not seem to cause significant pathology in the mosquito vector. Even if some fitness costs have been described for some arboviruses [11–13], many other studies have concluded that arboviral infection is mainly silent and that mosquito vectors are tolerant to arboviruses [14,15]. From the oral acquisition of a viremic bloodmeal to the transmission to a new uninfected vertebrate host, alphaviruses replicate in arthropod cells and must cope with antiviral pathways. In mosquito, the first limit threshold to cross is the gu<sup>t</sup> epithelium (i.e., midgut barrier), where the virus replicates to join the hemocoel (blood-containing body cavity) thus allowing viral spread to the whole body. To be transmitted again through blood feeding, the virus must penetrate the basal lamina of the salivary glands (salivary gland barrier) to join the acinar cells and replicate inside [16].

Interestingly, among the approximately 112 mosquito genera, the *Aedes* and *Culex* genera (such as *Aedes Albopictus*, *Aedes Aegypti*, *Culex Quinquefasciatus* or *Culex Pipiens*) seem to be the main vectors able to transmit viruses to humans [17]. Indeed, some mosquitoes may bite preferentially animals or may not be viral-transmission competent. As will be described below, part of the competence is linked to the di fferent tissue barriers that can be crossed or not during the viral dissemination in the mosquito; this depends on innate immune response and cell death regulation in the infected cells [15,17,18].

### **2. Cell Death in Mammals**

Cell death pathways can be divided into two opposite processes: accidental cell death (ACD) and regulated cell death (RCD). If ACD is a consequence of a severe and rapid injury (osmotic forces, pH variations, lytic viral replication), RCD is based on dedicated molecular machinery, implying that it can be modulated by pharmacological, genetic or infectious interventions [19].

Regulated cell death occurs under two di fferent circumstances. The first one is programmed cell death (PCD) [20], which occurs during embryonic development or in the event of tissue homeostasis. The second one regroups di fferent RCD pathways that occur following an external or internal, prolonged, and intense stress event. This contributes to tissue homeostasis and protection by eliminating useless or potentially dangerous cells (i.e., malignant or infected cells).

Dying cells present di fferent and well described morphological features that have been used so far to classify cell death processes into three main types [19]: apoptosis, autophagy-dependent cell death, and necrosis. Apoptosis is characterized by chromatin condensation, nuclear fragmentation, cytoplasmic shrinkage, membrane blebbing, and the formation of "apoptotic bodies", which are subsequently destroyed by professional or non-surrounding phagocytes. Autophagy-dependent cell death is essentially defined by its distinctive features of extensive cytoplasm vacuolization, leading to phagocytosis and degradation by lysosomes. Necrosis is mainly characterized by swelling, plasma membrane disruption, and cytoplasmic content e fflux in the extracellular environment, without evident phagocytosis or lysosomal degradation by the neighboring cells.

Intuitively, necrosis is associated to ACD, but it is nowadays clear that some RCD can also lead to non-apoptotic cell death as recently described in necroptosis [21], pyroptosis [22], and ferroptosis [23].

We will focus here on the cell death pathways that have been shown to be involved in antiviral response so far, without considering subtypes such as attachment dependence (i.e., anoïkis) and entotic cell death, parthanos, etc. For complete reviews see References [19,24].

### *2.1. Intrinsic Apoptosis*

"Intrinsic apoptosis is a form of RCD initiated by perturbations of the intracellular or extracellular microenvironment, demarcated by mitochondrial outer membrane permeabilization (MOMP) and precipitated by executioner caspases, mainly caspase 3 (CASP3)" (Nomenclature Committee on Cell Death (NCCD) [19]). It can be induced by numerous dysregulations including DNA damage, endoplasmic reticulum (ER) stress, reactive oxygen species (ROS) overload or infection. The main characteristic is that cells still present plasma membrane integrity and metabolic activity, leading, in vivo, to the removal of apoptotic bodies by surrounding phagocytic cells that recognize phosphatidylserine (PS) at the cell surface. In vitro, unless the cultured cells present phagocytic capacities, apoptosis usually ends by a "secondary necrosis", exposing damaged plasma membrane [25].

The decisive step of intrinsic apoptosis is the irreversible and extensive MOMP, leading to the release of numerous pro-apoptotic factors contained in the intermembrane space [26]. Mitochondrial outer membrane permeabilization is controlled by a family of 20 pro- or anti-apoptotic proteins: the B cells lymphoma 2 (Bcl-2) family proteins, which share one to four Bcl-2 homology domains (BH1 to BH4) [27]. All of them are finely regulated at the transcriptional and/or post-translational level (degradation, phosphorylation, localization, oligomerization, etc.) in order to integrate the extracellular or intracellular signals, which will potentially lead to apoptosis.

In mammals, only three of them (Bax, Bak, and Bok) have been described as able to form pores in the mitochondrial outer membrane (MOM) and other cellular membranes after oligomerization. These proteins are thus considered as "e ffectors" that can be activated, transcriptionally or post-translationally, after a cellular stress to induce MOMP [28,29]. Moreover, a pool of BH3-only proteins, described as "activators" promotes MOMP induction by interacting with Bax and Bak, allowing the conformational changes necessary for pore formation. These proteins can be post-translationally modified (e.g., Bid, cleaved in the pro-apoptotic truncated form "t-Bid") [30,31] or transcriptionally activated (e.g., PUMA, Noxa and Bim). In particular, the transcription factor p53 represents one of the links between DNA damage or oxidative stress and intrinsic apoptosis. Indeed, after a stress signal, post-translational modifications induce p53 stabilization and translocation in the nucleus. p53 will induce pro-apoptotic Bcl-2 family proteins transcription (i.e., Bax, Bak, PUMA, and Noxa) [32,33]. In the absence of stress conditions, other members of the Bcl-2 family (Bcl-2, Bcl-Xl, Bcl-W, Mcl-1, and Bfl-1) constantly block MOMP [34]. They contain all four BH domains and are inserted in the MOM or ER membrane, interacting with and inhibiting the e ffectors members (Bax, Bak, and Bok) or the BH3-only activators (PUMA, Noxa, Bim, and tBid) [35,36]. In addition, these anti-apoptotic proteins have been shown to regulate Ca2+ homeostasis in the ER [37,38] and cellular redox equilibrium [39,40]. Finally, it has been shown that some BH3-only proteins (Bad, Bmf, and Hrk) carry out their pro-apoptotic e ffect without interacting with the e ffector proteins but by inhibiting the pro-survival ones [41].

Interestingly, it is now clear that mitochondria and ER are physically connected, forming platforms called mitochondria associated (ER) membranes (MAMs) [42]. Mitochondria associated membranes regulate numerous cellular processes such as calcium (Ca<sup>2</sup>+) homeostasis, autophagy, lipid metabolism, apoptosis, and the rapid exchange of biological molecules [43]. They are involved in inflammasome formation and activation and participate in the antiviral response through the mitochondrial antiviral protein (MAV)/RNA sensors (retinoic acid-inducible gene I (RIG-I) or melanoma di fferentiation-associated protein 5 (MDA5)) complex activation [44].

Mitochondrial outer membrane permeabilization induces the release of intermembrane space elements, among which cytochrome C (CYC), Omi, and DIABLO (also called SMAC) [45–47]. Furthermore, following MOMP, the mitochondrial transmembrane potential (ΔΨm) is usually lost, mostly due to CYC release in the cytosol and the consequent stop of the respiratory chain [48].

In the cytosol, the association of CYC with Apaf1 and pro-caspase 9 (CASP9) forms a complex called apoptosome that will activate CASP9 in an ATP-dependent process [45]. In turn, the activated CASP9 will then activate the executioner caspases (i.e., mainly CASP3 and -7) that are involved in the final cellular destruction: poly (ADP-ribose) polymerase (PARP) cleavage, DNA fragmentation, PS exposure, apoptotic bodies formation [49–52]. Omi and DIABLO enhance cell death by inhibiting the inhibitor of apoptosis protein (IAP) family, which includes XIAP, c-IAP1, and c-IAP2. XIAP is constitutively bound to executioner CASP3 and -7 and, thus, blocks their activity [53,54]. c-IAP1 and c-IAP2, for their part, are two E3 ubiquitin ligases. They upregulate the CASP8 inhibitor c-Flip, induce caspases' degradation through ubiquitination, and promote NF-κB pro-survival pathway through receptor interacting serine/threonine kinase 1 (RIPK1) ubiquitination. These functions have mostly been maintained throughout evolution, from insects to mammals [55–57] (Figure 1).

**Figure 1.** Apoptosis and necroptosis in mammals. PM: plasma membrane, ER: endoplasmic reticulum, MAM: mitochondria-associated membranes, CYC: cytochrome c, IAP: inhibitors of apoptosis proteins, RIPK1/3: receptor interacting serine/threonine kinase 1/3, Ub: uiquitin, p: phosphorylation, MLKL: mixed-lineage kinase domain-Like, FADD: Fas-associated protein with death domain, TRADD: tumor necrosis factor receptor super family (TNFR1)-associated death domain protein, TLR3/4: Toll-like receptor.

Finally, executioner caspases can positively or negatively regulate the emission of multiple damage-associated molecular patterns (DAMPs) by dying cells, including immunostimulatory [58] as well as immunosuppressive [59] factors.
