**5. Conclusions**

*Dengue Fever in a One Health Perspective*

of field collected specimens.

distribution and/or with *Culex* species and strains.

(**Figure 7**).

also by *Aedes* mosquitoes, as traditionally accepted [38]. The single insect RNA extraction method facilitates the identification of arboviruses and also the blood source ingurgitated by a dipteran through molecular techniques, since each individual insect feeds on few vertebrates [35] (**Figure 8**). Furthermore, even after nucleic acid extraction, the taxonomic position of the dipteran can be reevaluated

*Flavivirus* diagnosis of 100 culicids collected in the city of Marilia, from São Paulo Brazilian State, based on NS5, revealed the occurrence of 19 positive specimens (19%) to CxFV. These viruses are part of the insect-specific flavivirus (ISF) group and have a wide geographic distribution, encompassing tropical and temperate regions, in various insect groups [39]. There is evidence that ISF infection may suppress or raise the rate of *in vitro* and/or *in vivo* replication of medical important flaviviruses [40]. A study conducted with Palm Creek virus (PCV), another ISF, originally found in Northern Australia, demonstrated that previously infected insect cells were suppressed for WNV and Murray Valley Encephalitis Virus replication, two important human arboviruses [41]. Virus replication suppression by ISF is also associated with mosquito strains [42], as demonstrated for WNV infection in two different lineages of *C. quinquefasciatus*. The mosquitoes isolated in Honduras presented increased WNV transmission rate when infected with CxFV. The ISF Nhumirim virus (NHUV), first isolated in the Pantanal region of Brazil, was evaluated in the growth and replication rates of ZIKV in insect cells [43]. They found that both in the previously inoculated cells and in cells coinoculated with NHUV, growth, and replication rates of ZIKV were significantly reduced. Also, in this work, the rates of ZIKV infection in *Ae. aegypti* infected with NHUV were significantly reduced, but the transmission rate was maintained. According to Romo (2018), ISFs can be used as models to understand the mechanisms involved in virus interference infection process, which may be used to reduce or suppress infections of important human pathogens in arthropod virus vectors. The methodology described in this work enables investigations of interactions among medical important flavivirus and ISFs at natural mosquito's population level through the use

It is believed that *Culex flaviviruses* may be specific to the culicid species and also to the region from which it is obtained [44]. In order to investigate the specificity of CxFV from Marilia, a phylogenetic reconstruction analysis was performed using homologous partial NS5 sequences of CxFV isolates from China, Africa, Argentina, and two Brazilian isolates (one in Mato Grosso and the other in São José of Rio Preto, São Paulo). The nucleotide and amino acid sequences corresponding to the *Flavivirus* partial NS5 protein varied little among then (about 0–3%). Two Neighbor-Joining-based phylogenetic trees constructed with partial NS5 nucleotide (**Figure 5**) and amino acid (**Figure 6**) homologous sequences showed the clustering of flaviviruses in two main branches, one formed by human pathogens (DENV, YFV, and ZIKV), and the other formed by the CxFV. The first branch is formed by taxa that share the same capacity to infect mammalian vertebrate cells, including humans, and present the *Ae. aegypti* as the main vector [45, 46]. The second branch is formed by organisms that differ in origin and time of collection. However, in the tree obtained with the nucleotide sequences, it is possible to observe a clade formed by all the records obtained from Marília, except for the specimen identified by number 48, which was closest to the CxFV of China. This result was different when the tree was constructed with amino acid sequences, where there was no geographical clustering of CxFV. Perhaps, the molecular marker employed in this work is not appropriate to investigate *CxFV* geographical specificity, and more studies are needed to explore the association of their genetic variability with geographical

**178**

The single dipteran RNA extraction technique described in this work permits the use of hematophagous insects as sentinels to detect arboviruses, preserving the chitinous skeleton of the insect and guaranteeing the subsequent morphological studies. The possibility to obtain RNA from a single dipteran also makes possible the investigation of infectious agent's vector capability and the identification of the ingurgitated blood meal source, enabling the description of arthropod alimentary habit and an indication of which vertebrates may be implicated in a virus life cycle. The method also opens the possibility for constant arbovirus surveillance, which can be used to prevent and control epidemics that affect millions of people each year. The presence of CxFV in *C. flavivirus* vectors may interfere with replication, transmission, and infection rates of arboviruses of medical importance, and the method described facilitates natural population studies.
