Detection of Culex flavivirus and Aedes flavivirus nucleotide sequences in mosquitoes from parks in the city of São Paulo, Brazil

Highlights

• Mosquitoes known to be vectors of flaviviruses are present in parks in the city of São Paulo.

• First report of Culex flavivirus-related nucleotide sequences in the city of São Paulo.

• First report of Aedes flavivirus-related nucleotide sequences in Latin America.

• Surveillance for flaviviruses in parks in the city of São Paulo is recommended.

Abstract

The dengue viruses are widespread in Brazil and are a major public health concern. Other flaviviruses also cause diseases in humans, although on a smaller scale. The city of São Paulo is in a highly urbanized area with few green spaces apart from its parks, which are used for recreation and where potential vertebrate hosts and mosquito vectors of pathogenic Flavivirus species can be found. Although this scenario can contribute to the transmission of Flavivirus to humans, little is known about the circulation of members of this genus in these areas. In light of this, the present study sought to identify Flavivirus infection in mosquitoes (Diptera: Culicidae) collected in parks in the city of São Paulo. Seven parks in different sectors of the city were selected. Monthly mosquito collections were carried out in each park from March 2011 to February 2012 using aspiration and traps (Shannon and CD C-CO₂). Nucleic acids were extracted from the mosquitoes collected and used for reverse-transcriptase and real-time polymerase chain reactions with genus-specific primers targeting a 200-nucleotide region in the Flavivirus NS5 gene. Positive samples were sequenced, and phylogenetic analyses were performed. Culex and Aedes were the most frequent genera of Culicidae collected. Culex flavivirus (CxFV)-related and Aedes flavivirus (AEFV)- related nucleotide sequences were detected in 17 pools of Culex and two pools of Aedes mosquitoes, respectively, among the 818 pools of non-engorged females analyzed. To the best of our knowledge, this is the first report of CxFV and AEFV in the city of São Paulo and Latin America, respectively. Both viruses are insect- specific flaviviruses, a group known to replicate only in mosquito cells and induce a cytopathic effect in some situations. Hence, our data suggests that CxFV and AEFV are present in Culex and Aedes mosquitoes, respectively, in parks in the city of São Paulo. Even though Flavivirus species of medical importance were not detected, surveillance is recommended in the study areas because of the presence of vertebrates and mosquitoes that could act as amplifying hosts and vectors of flaviviruses, providing the required conditions for circulation of these viruses.

Introduction

Among the arthropod- borne viruses (arboviruses) circulating in Brazil, members of the Flavivirus genus (family Flaviviridae) are noteworthy as they cause the largest number of infections and diseases in humans (Travassos da Rosa et al., 1998). Bussuquara (BUSV), Cacicaporé (CPCV), Dengue (DENV) serotypes 1–4, Rocio (ROCV), Iguape (IGUV), Ilhéus (ILHV), Yellow fever (YFV) and Saint Louis encephalitis virus (SLEV) are all flaviviruses of medical importance that have been isolated in Brazil (Figueiredo, 2000, Figueiredo et al., 2008). Recently, Zika virus (ZIKV) emerged in the country (Campos et al., 2015, Vasconcelos, 2015, Zanluca et al., 2015).

Some of these viruses have caused outbreaks in many cities, especially the four serotypes of DENV. Since its re-emergence in 1982, DENV has spread throughout Brazil, and the four serotypes (DENV 1–4) now cause dengue epidemics in all five regions of the country, where the virus is transmitted by Aedes aegypti, an urban, domestic mosquito (Figueiredo, 1998, Figueiredo, 2000, Figueiredo, 2012, Figueiredo et al., 2008).

Transmission of YFV is restricted to some forested areas of the country and their surroundings, where the virus circulates among nonhuman primates and is transmitted by mosquitoes of the genera Haemagogus and Sabethes (Vasconcelos et al., 2005). Although the vaccine against the virus is efficient, the abundance in many Brazilian cities of Aedes aegypti, the vector of the virus in the urban cycle, makes the disease an important public health issue, especially because it has become more widespread since 1998 (Vasconcelos, 2003, Vasconcelos, 2010, Vasconcelos et al., 2003).

In addition to flaviviruses of medical importance, other members of the genus, known as insect-specific flaviviruses (ISFs), have also been detected in many countries and were recently reported in Brazil (Blitvich and Firth, 2015). These viruses have been observed to be unable to replicate in mammalian cells although they replicate and, in some situations, cause cytopathic effect (CPE) in mosquito cells (Stollar and Thomas, 1975, Sang et al., 2003, Crabtree et al., 2003, Blitvich and Firth, 2015).

The first ISF to be described was cell fusing agent virus (CFAV), which was isolated from an Aedes aegypti cell line (Stollar and Thomas, 1975, Cammisa-Parks et al., 1992) and was also found in mosquitoes from several countries (Cook et al., 2006, Kihara et al., 2007, Yamanaka et al., 2013, Hoshino et al., 2009, Espinoza-Gomez et al., 2011). Apart from CFAV, many other ISFs, including Culex flavivirus and Aedes flavivirus, have been isolated and characterized, as reviewed by Blitvich and Firth (2015).

Culex flavivirus (CxFV) is the most reported ISF to date. It has been found to infect different species of Culex mosquitoes in Japan and Indonesia (Hoshino et al., 2007), Guatemala (Morales-Betoulle et al., 2008), Mexico (Farfan-Ale et al., 2009, Farfan-Ale et al., 2010, Saiyasombat et al., 2010), the USA (Kim et al., 2009, Blitvich et al., 2009, Bolling et al., 2011, Crockett et al., 2012, Newman et al., 2011), Trinidad (Kim et al., 2009), Uganda (Cook et al., 2009), Brazil (Machado et al., 2012), China (Huanyu et al., 2012, An et al., 2012), Taiwan (Chen et al., 2013) and Argentina (Goenaga et al., 2014).

Aedes flavivirus (AEFV) was first isolated in Japan (Hoshino et al., 2009) and, after that, it has since been found in Italy (Calzolari et al., 2010a, Calzolari et al., 2010b, Calzolari et al., 2012, Roiz et al., 2012, Rizzo et al., 2014, Grisenti et al., 2015), the USA (Haddow et al., 2013) and Thailand (Bolling et al., 2015).

The city of São Paulo is in a highly urbanized area with over eleven million inhabitants. The parks in the city are the main green spaces and are used for recreational purposes. Vertebrates such as birds and rodents act as amplifying hosts for Flavivirus in these parks, and mosquitoes (Culicidae) that can be vectors of some arboviruses, in particular members of the genera Culex and Aedes, are also found there (Urbinatti et al., 2001, Laporta et al., 2006, Taipe-Lagos and Natal, 2003, Montes, 2005, Medeiros-Sousa et al., 2013, Medeiros-Sousa et al., 2015, Paula et al., 2015). Thus, it is reasonable to conclude that all the elements required for the Flavivirus transmission cycle can be found in parks in the city of São Paulo.

In light of this and given the medical importance of Flavivirus and the lack of information about the circulation of viruses of this genus in mosquitoes in parks in the city of São Paulo, the aim of this study was to identify Flavivirus-infected mosquitoes (Diptera: Culicidae) collected in these parks and to characterize the genetic diversity among the nucleotide sequences found.