**1. Introduction**

In recent years, due to anthropogenic actions, including deforestation, disorganized urbanization, and globalization, arboviruses have emerged as a major challenge to global health [1, 2]. The arboviruses (arthropod borne viruses) are transmitted to

humans through the bite of infected hematophagous insects, causing febrile diseases [3], with a broad variety of clinical manifestations, ranging from the absence of symptoms to the severe hemorrhagic and encephalitic disorders [4, 5]. The arbovirus vectors include different species of mosquitoes, flies, and ticks. The most worldwide prevalent arboviruses encompass Dengue Virus (DENV), Yellow Fever Virus (YFV), West Nile Virus (WNV), and Zika Virus (ZIKV) from Flaviviridae family and *Flavivirus* genus, and Chikungunya Virus (CHIKV) from family Togaviridae and genus *Alphavirus* [6], which are transmitted through mosquitoes belonging to the Culicidae family, mainly from *Culex* and *Aedes* genera [2].

Arbovirus transmissions occur principally in tropical and subtropical areas, since the presence of vector mosquitoes is associated, mainly, with hot and humid environments, fundamental requirements for their reproduction [7]. Recently, with the global warming and increase in international traveling, the dispersion of arthropod vectors is rising, especially mosquitoes of the genus *Aedes*, and the dissemination of arboviruses is reaching regions considered nonendemic [8–10]. Besides, mosquitoes' high genetic plasticity enables its adaptation to colder environments and international tourism to tropical and subtropical endemic areas, which contributes to arbovirus vectors' global dissemination [11, 12].

Dengue is the most prevalent arbovirus and is responsible for an estimate of 390 million annual cases worldwide [13], and 3.9 billion people, living in 128 countries, are on risk of infection [14]. Since 2010, dengue cases have been reported in nonendemic countries in Europe, including France, Croatia, and Portugal, where in 2012, an outbreak occurred with more than 2000 reported cases. In this period, 10 other European countries were affected by dengue fever. Further, among European travelers returning from low incoming endemic countries, dengue fever is the most diagnosed disease, after malaria [10, 15]. In 2016, more than 3.34 million cases of dengue were reported in American countries, Southeast Asia, and the Western Pacific. Only in the Americas, approximately 2.38 million people were affected, with 1032 deaths, including Brazil, responsible for almost 1.5 million of the reported cases [15]. In addition to Dengue, in Brazil, a South American country with high international touristic activity, according to the Ministry of Health, occurred 216,207 cases of Zika fever, another important emerging *Flavivirus* [16], and 691 cases of yellow fever with 220 confirmed deaths, only in 2016 [17]. This panel entails the flaviviruses negative impact on public health and also causes the economic burden, having direct and indirect consequences [18]. Direct costs include hospitalization, medications, diagnostic tests, vector control, training of professionals, and health surveillance. Among indirect costs are loss of worker productivity and profits, and interference in trade and tourism in affected areas.

There are no effective vaccines available for Dengue, Zika, and Chikungunya fevers [19], and the control of these arboviruses is exclusively implemented by chemical arthropod vector elimination [1]. Therefore, active searching for vectors is necessary to prevent the circulation of known arboviruses. Detection of arboviruses occurs only after detection of human cases, which causes delay in the disease and vector dissemination controls. Thus, to prevent emergency and reemergence of arboviruses, very early detection of vectors and arbovirus and the understanding of their diversity and infection cycle are of great importance. These strategies include also the identification of factors related to the dispersion and entrance of arboviruses in previous indene areas and the identification of wild animal natural reservoirs [20]. Considering the current difficulties in detecting silent circulation of arboviruses and also in obtaining samples from arthropod vectors, human and animal febrile cases, principally in the forests areas, the use of sentinels could be an alternative surveillance approach. Hematophagous insects are present in different wild natural and urban environments, being an excellent group of animal to be used

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**Table 1.**

*Novel Single Hematophagous Insect RNA Detection Method Supports Its Use as Sentinels…*

park using CDC traps, in order to detect RNA from flaviviruses.

as sentinel. Thus, in this work, the single-insect nucleic acid extraction method [21] was evaluated in hematophagous dipterans collected in a Brazilian municipal public

A total of 106 insects of the order Diptera were analyzed, of which 100 were collected in the Municipal Park of the city of Marília—São Paulo, and six were specimens of *Aedes aegypti*, RED strain, of which five were experimentally infected with DENV2. More detailed information on the specimens used in this study is shown in **Table 1**. The protocol of collection and transport of arthropods in the Municipal Park of the city of Marília-São Paulo was authorized by the Biodiversity Authorization and Information System (SISBIO), of the Chico Mendes Institute for Biodiversity Conservation (ICMBio), Brazilian Ministry of Environment (MMA), under number 64603-1 (10/18/2018). *Ae. aegypti* strain RED specimens infected with DENV2, maintained in a biosafety level 2 (BSL-2) insectary facility in Institute of Biomedical Sciences from University of São Paulo, were gently donated by Margareth de Lara Capurro Guimarães at the Department of Parasitology of the Institute of Biomedical Sciences of the University of São Paulo (USP), of which five were submitted to infection by DENV-2, while one sample was not submitted to infection and was used as a negative control for DENV. These insects were previously used for purposes that are not related to this study and had approval and

This work did not involve collection of human samples, total or partial, and specimens or tissue samples from vertebrate animals and/or embryos. In addition,

For RNA extraction, each dipteran was digested for 16 h at 56°C, inserted in 200 μL of a lysis buffer composed of 200 mM Tris-HCl (pH 7.5), 250 mM NaCl, 25 mM EDTA (pH 8.0), 0.5% of SDS, and 400 μg/mL of proteinase K as described for DNA extraction [21]. Before digestion, ethanol from insects stored in 80% ethanol was removed after washing twice with 1 mL of 1× PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 1.8 mM KH2PO4). Following the incubation period,

**Collection local, data Number of** 

USP-SP, March 2003 6

**specimens**

**method**

−20 °C

Anopheles (Culicidae) Ethanol 80% Marília-SP, December 2017 3 Phlebotomus (Psychodidae) Ethanol 80% Marília-SP, December 2017 2 Ceratopogonidae Ethanol 80% Marília-SP, November 2017 3 Cecidomyiidae Ethanol 80% Marília-SP, December 2017 1 Culicinae (Culicidae) Ethanol 80% Marília-SP, June 2017 91

*DOI: http://dx.doi.org/10.5772/intechopen.92071*

**2. Materials and methods**

**2.1 Specimens and ethical aspects**

permissions needed in their own respective study.

no threatened or protected species were collected.

**2.2 Single-insect nondestructive RNA extraction**

**Taxon Storage** 

*Aedes aegypti* (Culicidae) Frozen at

*Specimens of dipteran included in the study.*

*Novel Single Hematophagous Insect RNA Detection Method Supports Its Use as Sentinels… DOI: http://dx.doi.org/10.5772/intechopen.92071*

as sentinel. Thus, in this work, the single-insect nucleic acid extraction method [21] was evaluated in hematophagous dipterans collected in a Brazilian municipal public park using CDC traps, in order to detect RNA from flaviviruses.
