**1. Introduction**

Zika virus (ZIKV) is an emerging arthropod-borne *Flavivirus*, transmitted mainly by mosquitoes of the genus *Aedes*, but the ZIKV infection could also be produced by sexual contact or vertical transmission from mother to child [1,2]. ZIKV was first isolated in 1947 from the blood of a sentinel Rhesus monkey No. 766, stationed in the Zika forest in Uganda. Again, in 1948, ZIKV was isolated in the same forest from a pool of *Aedes (Ae) africanus* mosquitoes. Thereafter, serological and entomological data indicated that ZIKV circulates actively in East and West Africa and South-East Asia. In 2007, ZIKV caused an outbreak of relatively mild disease characterized by rash, arthralgia, and conjunctivitis on Yap Island in the Southwestern Pacific Ocean. This was the first time that the virus was detected outside of Africa [3].

Later, a ZIKV epidemic in Brazil was present in 2015 and spread rapidly throughout South and Central America in 2016. The Pan American Health Organization (PAHO) has received reports of more than 7.5 × 10<sup>5</sup> cases of Zika in 84 cities or territories in America [3,4]. The ZIKV infection during pregnancy can cause fetal loss, microcephaly, and other brain abnormalities that are classified as congenital Zika syndrome [5,6]. Further, severe forms of encephalopathies, meningoencephalitis, myelitis, uveitis, autoimmunity (Guillain-Barré syndrome), and severe thrombocytopenia have been associated with ZIKV infection [7,8]. The pathogenic mechanisms that give rise to severe forms of Zika are still unclear, and to date, no safe vaccine or specific antiviral treatments for ZIKV infection are available [9]. A rapid and successful expansion of ZIKV has occurred due to the high virulence of circulating strains, immunologically susceptible populations, and the wide distribution of its vectors [10,11].

*Ae. aegypti* and *Ae. albopictus* mosquitoes are the primary vectors of several *Flavivirus* such as ZIKV and dengue virus (DENV) [12]. Female mosquitoes acquire the virus from an infected host during feeding, it undergoes replication in the gu<sup>t</sup> and disseminates to the salivary glands, and the virus is released into the saliva, where it is transmitted to the host during subsequent feeding [13,14]. Cime et al. (2015) reported that *Ae. aegypti* saliva plays an important role during DENV transmission to the host cells. Likewise, they detected an enhanced viral infection of mammalian cells in the presence of mosquito salivary gland extract [15]. However, the mechanisms in the transmission of *Flavivirus* from vector to host are not entirely understood [16]. In human hosts, monocytes, macrophages, endothelial vascular cells, and central nervous system cells are identified as main ZIKV target cells [17–19]. During di fferentiation or activation, cells release extracellular vesicles (EVs) [20]. EVs are considered crucial mediators of intercellular communication and play a role in the pathophysiology of inflammation-associated disorders [21].

EVs are a heterogeneous group of particles naturally released by the cells, delimited by a lipid bilayer, and cannot replicate. The classification proposed by the International Society of Extracellular Vesicles (ISEV) has established that EVs can be distinguished by their biogenesis. Vesicles are derived from the plasma membrane (microparticles [MPs]) and are also derived from endosomal maturation (exosomes). Further, they di ffer in size, where the MPs (> 200 nm) are grouped as large EVs (lEVs), and the exosomes (< 200 nm) are grouped as small EVs (sEVs) [22]. These EVs can be identified by the presence of di fferent membrane markers (phosphatidylserine [PS] in lEVs or tetraspanins in sEVs) or by their internal content, since they transport active biomolecules (proteins and di fferent types of RNA) capable of modifying the response of the cells with which they interact [22,23].

Small EVs are formed as intraluminal vesicles within multivesicular bodies during the endosome maturation process and released into the extracellular space through highly specialized cellular secretory pathways [24]. During the infectious process by some RNA viruses such as flaviviruses, the viral replication cycle and the biogenesis of sEVs can converge, so di fferent viral components (antigens, genomes, or complete viruses) can be part of the internal content, being potential vehicles for viral transmission, evasion of the host's immune response, and the enhancement of pathophysiological processes by promoting the spread of the pathogen to immunologically privileged sites [25,26]. Therefore, sEVs are considered a new, alternative mechanism that is e fficient for viral spread [27]. Large EVs are formed by cytoskeleton rearrangemen<sup>t</sup> and released from the plasma membrane after the cell activation process [24]. In blood circulation, MPs facilitate cell–cell interaction and induce di fferent responses associated with inflammation, thrombosis, or vascular dysfunction [28]. Virus-infected cells secrete lEVs that may contain viral proteins and RNAs [29]. Little is known about the EV participation function in the vector–human host interaction during the flaviviruses transmission-infection processes. Recently, Vora et al. (2018) reported that DENV-infected mosquito cells release EVs that contain infectious DENV RNA and proteins, favoring viral transmission from the vector to human keratinocytes and endothelial cells (ECs) [16]. Likewise, Reyes-Ruiz et al. (2019) reported that sEVs from DENV-infected mosquito cells have homologous proteins to human CD9 tetraspanin, containing virion-like particles inside them [30].

To date, the participation of EVs released from *Aedes* mosquito cells during the ZIKV infection process has not been described. This study aimed to evaluate the potential role of EVs from ZIKV-infected C6/36 cells in viral-element, cell-to-cell transmission to the main host's target cells (monocytes and endothelial vascular cells) as well in the naïve cellular behavior modification. EVs from ZIKV-infected mosquito cells were then isolated by differential ultracentrifugation, characterized by nanoparticle tracking analysis, identified by transmission electron microscopy, and subject to phosphatidylserine (MPs) and tetraspanin CD63 (exosomes) detection by cytofluorometry assays. The isolated sEVs were purified by using paramagnetic beads coated with anti-CD63 antibodies, thus demonstrating their endosomal origin. The possible modification of cellular behavior mediated by ZIKV C6/36 EVs was evaluated by using different cell activation assays. Our results support that EVs (small and large) from ZIKV-infected *Aedes* mosquito cells modify host cells responses, which could be implicated in the pathogenic mechanisms associated with the progression to severe forms of the disease.
