Detection of Zika and dengue viruses in wild-caught mosquitoes collected during field surveillance in an environmental protection area in São Paulo, Brazil

Karolina Morales Barrio-Nuevo; Mariana Sequetin Cunha; Adriana Luchs; Aristides Fernandes; Iray Maria Rocco; Luis Filipe Mucci; Renato Pereira de Souza; Antônio Ralph Medeiros-Sousa; Walter Ceretti-Junior; Mauro Toledo Marrelli

doi:10.1371/journal.pone.0227239

. 2020 Oct 16;15(10):e0227239. doi: 10.1371/journal.pone.0227239

Detection of Zika and dengue viruses in wild-caught mosquitoes collected during field surveillance in an environmental protection area in São Paulo, Brazil

Karolina Morales Barrio-Nuevo ¹, Mariana Sequetin Cunha ², Adriana Luchs ³, Aristides Fernandes ¹, Iray Maria Rocco ², Luis Filipe Mucci ⁴, Renato Pereira de Souza ², Antônio Ralph Medeiros-Sousa ¹, Walter Ceretti-Junior ¹, Mauro Toledo Marrelli ^1,^*

Editor: Humberto Lanz-Mendoza⁵

PMCID: PMC7567345 PMID: 33064724

Abstract

Species of the genus Flavivirus are widespread in Brazil and are a major public health concern. The country’s largest city, São Paulo, is in a highly urbanized area with a few forest fragments which are commonly used for recreation. These can be considered to present a potential risk of flavivirus transmission to humans as they are home simultaneously to vertebrate hosts and mosquitoes that are potential flavivirus vectors. The aim of this study was to conduct flavivirus surveillance in field-collected mosquitoes in the Capivari-Monos Environmental Protection Area (EPA) and identify the flavivirus species by sequence analysis in flavivirus IFA-positive pools. Monthly mosquito collections were carried out from March 2016 to April 2017 with CO₂-baited CDC light traps. Specimens were identified morphologically and grouped in pools of up to 10 individuals according to their taxonomic category. A total of 260 pools of non-engorged females were inoculated into C6/36 cell culture, and the cell suspensions were analyzed by indirect immunofluorescence assay (IFA) after the incubation period. IFA-positive pools were tested by qRT-PCR with genus-specific primers targeting the flavivirus NS5 gene to confirm IFA-positive results and sequenced to identify the species. Anopheles cruzii (19.5%) and Wyeomyia confusa (15.3%) were the most frequent vector species collected. IFA was positive for flaviviruses in 2.3% (6/260) of the sample pools. This was confirmed by qRT-PCR in five pools (83.3%). All five flavivirus-positive pools were successfully sequenced and the species identified. DENV serotype 2 (DENV-2) was detected in Culex spp. and Culex vaxus pools, while ZIKV was identified in An. cruzii, Limatus durhamii and Wy. confusa pools. To the best of our knowledge, detection of flavivirus species of medical importance has never previously been reported in these species of wild-caught mosquitoes. The finding of DENV-2 and ZIKV circulating in wild mosquitoes suggests the existence of an enzootic cycle in the area. In-depth studies of DENV-2 and ZIKV, including investigation of mosquito infection, vector competence and infection in sylvatic hosts, are needed to shed light on the transmission dynamics of these important viruses and the potential risk of future outbreaks of DENV-2 and ZIKV infections in the region.

Introduction

Over 700,000 deaths worldwide every year are caused by infections transmitted by blood-feeding arthropods, accounting for 17% of all infectious diseases [1, 2]. Examples of these arthropods include mosquitoes (Diptera: Culicidae), which are competent vectors for viruses of great epidemiological importance, as seen in recent major outbreaks and epidemics of Chikungunya-virus (CHIKV), Dengue-virus (DENV), Zika-virus (ZIKV) and Yellow Fever-virus (YFV) infections in Brazil [3, 4]. Arboviruses (arthropod-borne viruses) are found worldwide, and their emergence and reemergence usually manifest as infections with mild to severe clinical symptoms in humans and domestic animals, occasionally progressing to death. These diseases therefore have a considerable impact on public health and the economy of the affected region [5–7].

Among the arboviruses circulating in Brazil, members of genus Flavivirus (family Flaviviridae) are noteworthy as they are etiological agents of some of the most common viral infections and diseases in humans. In addition to DENV, ZIKV and YFV, several other flaviviruses of medical importance have been isolated in Brazil, including Bussuquara virus (BUSV), Cacicaporé virus (CPCV), Rocio virus (ROCV), Iguape virus (IGUV), Ilhéus virus (ILHV) and Saint Louis encephalitis virus (SLEV) [8–11]. Dengue is a reemerging disease in Brazil, with over 2 million confirmed cases and 702 recent deaths [12]. ZIKV has gained global attention as its geographic distribution has expanded dramatically from equatorial Africa and Asia to the Pacific Islands, South America and the Caribbean, causing many cases of neurological disorders and neonatal malformations [13–15].

Prevention and control of arboviral diseases require continuous surveillance and vector control measures. Investment in appropriate integrated surveillance measures should therefore be a priority in Brazil, especially considering the size of the country’s population. Integrated surveillance, which covers epidemiological, entomological, sanitary and laboratory-based surveillance, is essential for early detection of epidemics and for rapid, effective control measures [16]. Donalisio et al. [16] stress that investment in epidemiological, virological, vector and epizootic surveillance measures should be a priority in Brazil. In the absence of specific treatment and an effective vaccine, ongoing entomological and epidemiological surveillance should be strengthened and integrated to control and prevent these arboviral diseases [3].

The Capivari-Monos Environmental Protection Area (EPA), in the south of the city of São Paulo, Brazil, is a forest remnant close to urban areas (an urban area). Previous studies in the Atlantic Forest in Brazil have shown that forest fragments offer favorable conditions for mosquitoes that are vectors of viruses to shelter and proliferate [17–19]. As circulating flaviviruses in city forest fragments can potentially cause disease outbreaks by infecting visitors and residents in neighboring areas, and given the lack of information about flavivirus-infected mosquitoes in parks in the city of São Paulo, the aim of this study was to conduct flavivirus surveillance in field-collected mosquitoes in the Capivari-Monos EPA and identify the virus species by sequence analysis.

Material and methods

Study area and mosquito sampling

The study was approved by the Ethics Committee of the University of São Paulo, Brazil, and collection permits were obtained in the Brazilian Biodiversity Authorization and Information System (SISBIO: Number: 44740–3).

Surveillance was conducted in the Capivari-Monos EPA, an area extending over 251 km² in the Atlantic Forest in the extreme south of the city of São Paulo where sustainable use of natural resources is practiced (Fig 1). Representing around one-sixth of the area of the whole municipality and bordering on the Serra do Mar State Park, Capivari-Monos EPA extends over the first hills and ocean slopes toward the upper reaches of Serra do Mar range, at altitudes varying from 740 to 800 m above sea level. It has a super-humid, tropical, ocean climate with average annual temperatures of around 19°C and rainfall of between 1,600 and 2,200 mm. The vegetation is dense, tropical, montainous forest made up of Atlantic Forest remnants with different degrees of conservation, varying from well-conserved original forest to areas that have undergone a process of regeneration since 1950 and others that have been degraded recently as a result of rural and especially urban expansion. The district of Engenheiro Marsilac, in the Parelheiros region, lies in the EPA and has around 10,000 inhabitants, most of whom are low-income settlers. The population density is approximately 41 inhabitants per km² [20, 21], and the area is home to the non-human primate (NHP) Alouatta guariba clamitans [22].

Fig 1 — Collection sites are numbered as follows: (1) Embura village, (2) Marsilac village, (3) Transition zone, (4) Wild area. Green represents dense tropical forest. Grey represents areas where there is human activity (villages, roads and rural properties). The map was created using QGIS v2.18.9 (http://www.qgis.org).

Mosquito collections were carried out monthly from March 2016 to April 2017 in forested areas in Engenheiro Marsilac with different levels of anthropogenic intervention. Specimens were collected in (1) Embura—a village surrounded by small farms and the EPA forest (23° 53.036′ S/46° 44.485′ W); (2) Marsilac—a village surrounded by the EPA forest and near a railway line (23° 54.395′ S/46° 42.486′ W); (3) Transition zone—private property near Marsilac village constituting a transitional area between a rural environment and the EPA forest (23° 54.556′ S/46° 42.167′ W); and (4) Wild area—private property in the EPA forest next to a waterfall with a visitation area (23° 56.378′ S/46° 41.659′ W) (Fig 1). In each collection area two CO₂-baited CDC light traps [23] with Lurex3^™ were installed, one in the tree canopy (>10 m) and another at ground level. All the traps were set up early in the afternoon and removed after 18 hours of exposure.

Specimens were carried alive to the Entomology in Public Health Laboratory at the School of Public Health, University of São Paulo (LESP/FSP/USP), where they were morphologically identified on a specially designed chill table with a stereo microscope and the dichotomous keys described by Consoli & Lourenço-de-Oliveira [24] and Forattini [25]. Non-engorged females were grouped in pools of up to 10 individuals according to their taxonomic category and place and date of collection, giving a total of 260 pools. The pools were then transported in dry ice to the Vector-borne Diseases Laboratory, Adolfo Lutz Institute, and stored at -70°C until use.

Detection of flaviviruses

The 260 pools were macerated individually in tubes containing 1 mL of 1.8% bovine albumin and antibiotics (100 units/mL of penicillin and 100 μL/mL of streptomycin) and centrifuged at 2,500 rpm for 10 min. The supernatant was stored at -70°C until inoculation into C6/36 cell culture (Aedes albopictus clone). The cultures were incubated for nine days at 28°C with L-15 medium containing 2% FBS, penicillin (100 units/mL) and streptomycin (100 μg/mL). After the incubation period, the cells were then scraped off the tube and the cell suspensions were spotted onto a glass slide, which was air dried, fixed with acetone and used for an Indirect Immunofluorescence Assay (IFA) with in-house anti-flavivirus polyclonal antibody and FITC-labeled anti-mouse IgG (whole molecule) antibody (Sigma-Aldrich, St. Louis, MO, USA) [26, 27]. The slides were examined under an epifluorescence microscope.

Identification of flavivirus species

Samples positive for flaviviruses in IFA were analyzed by real-time reverse transcription polymerase chain reaction (qRT-PCR) to confirm the result and sequenced to identify the species. Viral RNA was isolated from 140 μL aliquots of supernatants from IFA-positive cell cultures which had been stored at -70°C using the QIAamp^® Viral RNA Mini Kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions and tested using the pan-flavi qRT-PCR assay described previously by Patel et al. [28]. Positive qRT-PCR amplicons (~260 bp) were sequenced directly with the primers Flavi S and Flavi AS2 [28] and BigDye Kit v3.1 (Applied Biosystems, Inc., Foster City, CA, USA). Dye-labeled products were sequenced on an ABI 3130 sequencer (Applied Biosystems, Inc., Foster city, CA, USA). Chromatograms were edited manually with Sequencer 4.7 software and screened with the Basic Local Alignment Search Tool (BLASTn and BLASTx). Sequences generated in this way and a set of cognate sequences of DENV and ZIKV available in GenBank (S1 Table) were aligned with the BioEdit sequence alignment editor (version 7.0.5.2) [29]. Neighbor Joining (NJ) trees were constructed with the Kimura 2-parameter model in MEGA 6.0 and 1,000 bootstrap replicates [30]. Reference ZIKV and DENV sequences were added to the corresponding tree so that species identity could be confirmed. The nucleotide (nt) sequences were deposited in GenBank under accession numbers MK134005, MK134006, MK371391, MK371392 and MK371393.

Results

In total, 878 specimens of mosquitoes belonging to 37 taxa (11 genera) were sampled, of which 99.8% were female and 0.2% male. Most of the specimens were collected in the canopy (54.1%), 41.1% at ground level and for 4.8% information on stratification was not available. The species Anopheles (Kerteszia) cruzii, (171 specimens), Wyeomyia (Prosopolepis) confusa (134), Culex (Culex) spp. (109), Limatus durhamii (61), Wyeomyia (Phoniomyia) theobaldi (58) and Culex (Melanoconion) vaxus (36) were the most common species collected. More mosquitoes (61.4%) were collected in the wild area than in the transition zone (14.9%), Embura (13.1%) and Marsilac (8.7%) (S2 Table).

Flaviviruses were detected by IFA in 2.3% (6/260) of the sample pools. However, only five IFA-positive pools of non-engorged females (83.3%) were confirmed by qRT-PCR. All five positive pools were successfully sequenced, and the species identified with an ~200 bp long NS5 fragment. DENV serotype 2 (DENV2) was detected in Cx. spp. and Cx. vaxus pools, while ZIKV was detected in An. cruzii, Li. durhamii and Wy. confusa pools. The flavivirus-positive pools came from two distinct areas in the EPA: the wild area and the transition zone (Table 1).

Table 1. Flavivirus-positive samples detected in pools of mosquitoes from the Capivari-Monos EPA, São Paulo, Brazil, 2016–2017.

Pool code (Sample)	Collection date	Location	Taxon	Number of mosquitoes per pool (N)	Forest stratum	Flavivirus species	Isolate	GenBank accession no.
168 14P-F	October 2016	Wild area	Cx. spp.	6	Canopy	DENV-2	Flavi-168-DENV-2	MK134006
143 9P-C	February 2017	Wild area	Cx. vaxus	4	Canopy	DENV-2	Flavi-143-DENV-2	MK134005
148 11P-A	February 2017	Transition zone	Li. durhamii	6	Ground	ZIKV	Flavi-148- ZIKV	MK371391
157 14P-A2	October 2016	Wild area	An. cruzii	10	Canopy	ZIKV	Flavi-157- ZIKV	MK371393
150 12P-A	February 2017	Transition zone	Wy. confusa	2	Canopy	ZIKV	Flavi-150- ZIKV	MK371392

Open in a new tab

Descriptions of flavivirus-positive pools showing pool code, collection date, collection site, taxon, number of specimens, stratification, viral RNA identified, isolate and GenBank accession number.

The NJ trees constructed include the isolates sequenced in this study (in bold) and reference sequences from GenBank. The trees confirm the classification of Flavi-143 and Flavi-168 as DENV-2 since the sequences formed well-supported monophyletic groups with their corresponding reference isolates (Fig 2). The two isolates in the present study share an nt identity of 94.1%. Genetic analysis of the partial NS5 gene revealed that the DENV-2 isolates identified here are more closely related to those isolated in humans in Cuba in 1981, China in 2015 and Papua New Guinea in 1944 (93.3–97.6% identity) than to the Brazilian human BID-V2377 isolated in 2000 (88.6–93.7% identity). Genetic analysis of the partial NS5 gene sequences revealed that the three Brazilian ZIKV isolates exhibited high nt identity (97.0–98.1%) with other ZIKV isolates detected in the Americas since 2015, including isolates from humans, Aedes mosquitoes and NHPs, and 99.6% nt identity with each other. The three ZIKV lineages (America, Asia and Africa) can be clearly distinguished in Fig 3.

Fig 2 — Reference DENV-1, DENV-2, DENV-3 and DENV-4 isolates were retrieved from GenBank. The species and accession number of each isolate are indicated. The scale indicates the number of divergent nucleotide residues. Percentage bootstrap values are shown at the branch node.

Fig 3 — Reference ZIKV sequences were retrieved from GenBank. The species and accession number of each isolate are indicated. The scale indicates the number of divergent nucleotide residues. Percentage bootstrap values are shown at the branch nodes.

Discussion

The ecological networks that connect flaviviruses to their vectors and hosts are varied, complex and poorly understood. However, the ecology and epidemiology of these viruses can be better understood through mosquito-based flavivirus surveillance [31]. In the present study, we have described the detection of DENV-2 and ZIKV in Cx. spp, Cx. vaxus, Li. durhamii, An. cruzii and Wy. confusa captured in the Capivari-Monos EPA during the period 2016 to 2017. DENV-2 and ZIKV are known to have a great impact on human health [32–34], particularly in urban centers, and several studies have reported the finding of mosquitoes that are considered important flavivirus vectors in fragments of the Atlantic Forest in various areas of the city [18, 35–37] and state [4, 38, 39] of São Paulo. To the best of our knowledge, the detection of flavivirus species of medical importance has never been previously reported in wild-caught mosquitoes in the area in which the present study was carried out.

Culex vaxus appears to retain behavioral characteristics typical of wild mosquitoes as it has been shown to adapt poorly to areas with reduced forest [40]. Although information about this species is still quite scarce, it is known to have eclectic feeding habits and to be able to participate in sylvatic arbovirus cycles [24, 25]. Furthermore, viruses have been detected in some species of the genus Culex collected in wild environments [24]. Culex mosquitoes are vectors of a considerable number of flaviviruses that occur in Brazil, such as West Nile virus, Saint Louis Encephalitis virus and Ilheus virus [24, 41, 42]. Although some studies have demonstrated replication of DENV-2 in species of the genus Culex, the general consensus is that Cx. spp. are not biological vectors of dengue viruses [43]. However, Guedes et al [44] demonstrated recently that Cx. quinquefasciatus is a competent vector for ZIKV.

Anopheles cruzii is generally restricted to the Brazilian coast and has a distribution similar to the original distribution of the Atlantic Forest [25]. The high abundance of this species is directly related to the high availability of natural breeding sites but can also be attributed to the species’ opportunistic behavior and eclectic feeding habits [24, 25]. An. cruzii is commonly associated with Plasmodium transmission in humans and simians in the Atlantic Forest. Its feeding source includes NHPs [45–47] and although usually associated with malaria, the species has been found infected with arboviruses. Epelboin et al. [48] pointed out that An. cruzii is also involved in O’nyong-nyong virus (Alphavirus) transmission, and Cunha et al. [42] recently reported the finding of An. cruzii naturally infected with the Iguape virus (genus Flavivirus) in field samples collected in Juquitiba, São Paulo, in 1994. In addition, two species of the genus Anopheles, An. coustani and An. gambiae, were found naturally infected with ZIKV in Africa [48].

Wyeomya confusa and Li. durhamii belong to tribe Sabethini and have close phylogenetic relationships [24, 25]. The former is an opportunistic species and frequently bites humans [25]. Although it is usually sylvatic, Wy. confusa was collected here in an environment with greater anthropic interventions (Embura) and in a transition zone. While this species has already been found infected with arboviruses in wild environments, there is virtually no information about its medical importance [25]. Li. durhamii is the species of tribe Sabethini best adapted to anthropic environments [24] and there have been reports of this mosquito carrying Guama, Tucunduba and Maguari viruses (orthobunyaviruses) [49]. Intriguingly, ZIKV was detected in these three different species of sylvatic mosquitoes in the Capivari-Monos EPA.

One of the criteria used to determine whether sylvatic cycles of arboviruses are occurring is the presence of a sufficient number of susceptible NHPs and competent mosquitoes that feed on NHPs [50]. The finding of arboviruses in remote forest-dwelling mosquitoes is considered reliable evidence of a sylvatic cycle [32, 50]. Flaviviruses with known sylvatic cycles include DENV serotypes 1–4, ZIKV in parts of Africa and Asia [15, 48, 51–55] and YFV in South America and Africa [56]. In Brazil, the YFV cycle is sustained by NHPs and Haemagogus spp. mosquitoes [56]. As for a sylvatic cycle of ZIKV, this virus was detected in periurban Callithrix monkeys in the state of São Paulo during a human outbreak [57], but to date no field studies on wild-caught mosquitoes have detected ZIKV, and a study using colonized Sabethes cyaneus mosquitoes showed only a low vector competence for this virus [58].

The detection of DENV-2 and ZIKV in these sylvatic mosquitoes must be treated with caution. Identification of DENV-2 and ZIKV in sylvatic mosquitoes may not necessarily be associated with natural infection and may not indicate that these mosquitoes play a role in the transmission of flaviviruses as the virus detected could have originated from blood remnants in mosquitoes that had blood fed a few days before being caught. To better elucidate the existence of a sylvatic cycle of DENV-2 and ZIKV in Brazil, further studies on the infectivity and vector competence of wild-caught species of mosquitoes [24, 25, 44] are required, as well as investigations into the presence of arboviruses in different species of Brazilian NHPs.

The frequency of species of the genus Flavivirus detected in this study (2.6%) was similar to that observed in other studies in Italy (2.3%) [59], China (5.1%) [60] and Brazil (2.8%; 4.0%) [19, 61]. Cunha et al. [4] found a frequency of 0.29% of mosquitoes infected with YFV during the 2016–2017 outbreak in the state of São Paulo. Detection of flavivirus RNA in mosquitoes is not an easy task as detection of viruses in mosquitoes requires that large samples of specimens be screened. Gu et al. [62] showed that to detect arboviruses in mosquito populations with low levels of infection requires samples with more than 1,600 individuals for a high probability (0.8) of detection. Sample size is thus a key determinant of arbovirus detection in mosquitoes, making intensified mosquito surveillance essential to detect arboviral activity [62].

Phylogenetic analysis based on the partial NS5 gene showed that the Brazilian DENV-2 and ZIKV sequences clustered together with sequences from distinct continents, suggesting that the flaviviruses circulating in the Capivari-Monos EPA are genetically related to those circulating worldwide. While there is no evidence that a particular variant is spreading in the study area, it should be stressed that the phylogenetic tree in the present study was generated with short sequences (~200 bp). Phylogenetic reconstructions based on longer genomic sequences might provide more robust, informative data.

Although DENV-2 and ZIKV are considered urban and periurban arboviruses [13, 14, 34, 63], in the present study they were identified in a conserved wild area. In fact, this is the first study to have detected DENV-2 and ZIKV in mosquitoes captured in a conservation unit in the city of São Paulo. The finding of DENV-2 and ZIKV circulating in the Capivari-Monos EPA suggests an enzootic cycle in the area. Aedes aegypti, the main vector of these two flaviviruses of medical importance in the Americas [16, 64], was not found in the study area, corroborating previous findings [17]. Nevertheless, the Parelheiros region (where the study area is) has registered autochthonous dengue cases in the last few years, and these have spread progressively towards Engenheiro Marsilac [17]. Other studies carried out in urban parks in São Paulo also failed to find Ae. aegypti [19, 65, 66], highlighting the anthropophilic nature of this species. A search for DENV and ZIKV in NHPs together with mosquito and virological field surveillance are needed to investigate the potential role of mosquito species circulating in EPAs and whether urban arboviruses can establish an enzootic cycle in the city of São Paulo.

Conclusion

DENV-2 and ZIKV were found in Culex, Anopheles, Limatus and Wyeomyia mosquitoes in the Capivari-Monos EPA, confirming the presence and activity of these two potentially medically important flaviviruses in this conservation unit. Flavivirus surveillance in field-caught mosquitoes is essential for continuous monitoring of virus activity and for an understanding of the ecology and epidemiology of species of this genus. In addition, in-depth studies of DENV-2 and ZIKV, including vector competence and molecular studies, are needed to shed light on the transmission dynamics of these two arboviruses and the potential risk of future epidemic outbreaks in the study region.

Supporting information

S1 Table. Reference sequences of dengue and Zika viruses from GenBank.

Sequences of dengue and Zika viruses from GenBank aligned to construct a phylogenetic tree by country of origin, isolate, year, origin of isolated material, genome sequence and GenBank accession number.

(DOCX)

Click here for additional data file.^{(15.1KB, docx)}

S2 Table. Mosquito species collected in the Capivari-Monos Environmental Protection Area (EPA).

The table shows the number of individuals at each study point, forest stratum, sex and number of pools. Mosquitoes collected from March 2016 to April 2017.

(DOCX)

Click here for additional data file.^{(19KB, docx)}

Acknowledgments

We would like to express our gratitude to the following staff of the Superintendency for the Control of Endemic Diseases, São Paulo Zoonosis Control Center, and the School of Public Health, São Paulo University: Dr. Ana Maria Ribeiro de Castro Duarte, João Carlos do Nascimento, Paulo Frugoli dos Santos, Luis Milton Bonafé, Antônio Waldomiro de Oliveira, Laércio Molinari, Gabriel Marcelino Neto, Luiz Sposito Jr, Renildo Souza Teixeira, Daniel Pagotto Vendrami, Laura Cristina Multini, Gabriela Cristina de Carvalho, Ramon Wilk da Silva, Rafael de Oliveira Christe, Eduardo Evangelista de Souza, Amanda Alves Camargo and Ana Leticia da Silva de Souza.

Data Availability

All data generated or analyzed during this study are included in the manuscript.

Funding Statement

This work was funded by the São Paulo Research Foundation (FAPESP: grants nos. 2014/50444-5 and 2014/10919-4) and the National Council for Scientific and Technological Development, Brazil (CNPq: grant no. 301466/2015-7).

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PLoS One. doi: 10.1371/journal.pone.0227239.r001

Decision Letter 0

George Dimopoulos

9 Jan 2020

PONE-D-19-34539

Zika and dengue viruses infecting wild-caught mosquitoes in an environmental protection area in Brazil

PLOS ONE

Dear Dr Marrelli,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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PLOS ONE

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Reviewer #1: Partly

Reviewer #2: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: N/A

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Overall, I find this study to be sound and interesting, but not enough detail provided about the methods or the findings for it to be accepted without revisions. The authors describe several times that they identified mosquitoes "infected" with virus, but due to the lack of details about the IFA assay which appears to be the only assay used that might demonstrate infectivity, from just the data provided this statement is inaccurate. More information and data regarding the IFA must be provided, including relevant information around positive and negative controls, and how samples were determined to be positive, before samples could be suggested to be "infected". For instance, if the IFA is focus forming and the number of infectious virions in each mosquito pool could be quantified, this would add strength to the conclusion that the mosquitoes were "infected". Otherwise, the authors must rephrase and restructure their discussion to reflect the limited findings of virus RNA in inoculated cell culture samples. Please see attached document for additional comments.

Reviewer #2: Barrio-Nuevo and colleagues present a surveillance study of flaviviruses in a natural area in Sao Paolo. They report isolation and partial sequencing of ZIKV and DENV from this area, which suggests sylvatic perpetuation of these agents in this region. The work appears to have been conducted and analyzed in a technically acceptable manner and the conclusions are justified by the data. Moreover, this work contributes to our knowledge about flavivirus lifecycles in Brazil.

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Attachment

Submitted filename: Comments on PONE-D-19-34539.pdf

Click here for additional data file.^{(113.3KB, pdf)}

PLoS One. 2020 Oct 16;15(10):e0227239. doi: 10.1371/journal.pone.0227239.r002

Author response to Decision Letter 0

20 Feb 2020

To the Editor

Plos One

Dear Dr. Dimopoulos

Thank you for reviewing our manuscript. We have made substantial changes to the scientific note to address the points made by the reviewers. We believe the changes proposed by the reviewers have improved the article significantly.

Sincerely,

Mauro Marrelli

Answers: Reviewer #1

We are grateful to the reviewer for his/her very useful comments. All the suggestions and criticisms have been addressed.

Query 1

Line 44 – “Mosquitoes are competent vectors for viruses…”

Query 2

Line 46 – no hyphens between virus names

Query 3

Line 67 – “a priority” not “priorities”

Answer: We thank the reviewer for pointing out these mistakes and have made the necessary corrections to the manuscript.

Query 4

Line 127 – The Gubler et al reference contains many different types of virus detection and therefore the exact methods you used are not clear from that reference. Please describe the methods of your IFA assay.

Answer: A description of the methods used in the IFA has been added.

Query 5

Line 127 – Is this a focus-forming assay? Is it possible to quantify the number of foci and determine how many infectious virus particles were in the mosquito pool? Were positive controls used to compare to positive results? How was “positive” determined for the IFA? What about negative controls for antibody background staining? Please clarify these points, it will improve confidence in the findings of this study.

Answer: The IFA is not a focus-forming assay. After inoculation of the C6/36 cells with the mosquito pools, the virus replicated (this is why we kept it for 9 days, which is the mean time for dengue virus replication in Vero cell lines). In the IFA the cytoplasm of the cells was fluorescent, showing that the virus was able to replicate in this cell line and was viable, confirming viral isolation. We used the 17DD Yellow Fever virus as positive control, and a non-inoculated tube as negative control.

Query 6

Line 131 – What quantification method did you use to determine the amount of viral RNA in your qRT-PCR assay? What was the basis of your standard curve?

Answer: We did not quantify the amount of RNA. We followed the approach described in the original article by Patel et al. (2013), who determined that positive results are those with Ct values lower than 39.7. This information has been added in the text.

Query 7

Line 137 – are these primers the Flavi S and Flavi AS2 primers used for sequencing (Line 140)? If not please clarify which primers were used and provide sequences

Answer: Yes, we used the same primers, as stated in lines 151-152.

"The qRT-PCR amplicons (~260bp) were directly sequenced with the BigDyeTM kit v3.1 (Applied Biosystems, Inc., Foster City, CA, USA) and Flavi S and Flavi AS2 primers [26].

Query 8

Line 162 – is this also how many were positive by the IFA? Meaning, all the IFA positives were also positive by qRT-PCR?

Answer: Yes. All isolates were positive by qRT-PCR.

Query 9

Line 162 – Since this is a qRT-PCR assay, please report the quantities of viral RNA found in the supernatants of the C6/36 cells. This will help determine if the virus from the inoculated mosquito pool was infectious and replicated in the C6/36 cells.

Answer: We did not quantify RNA in the supernatants of the C6/36 cells. The result is based on visual fluorescence of the cell cytoplasm where viruses replicated. As we were able to isolate Dengue and Zika viruses from the mosquito pools, we can assume that these viruses were viable, and therefore infectious.

Query 10

Line 162 – The qRT-PCR protocol you used, as described by Patel et al, is very sensitive (10-100 genome copies). Why not determine the presence of viral RNA directly in the mosquito pools themselves?

Answer: We used isolation of virus in C6/36 cells to avoid false positive results, which could be a problem using only the very sensitive qRT-PCR protocol described by Patel. A further advantage of isolation in relation to the qRT-PCR protocol is that it allowed us to prove that the virus is able to replicate in C6/36 cells. In addition, the virus-isolation approach we used is cheaper.

Query 11

Line 206 – Technically this study doesn’t demonstrate that the mosquitoes are infected with the viruses, but that the mosquitoes contain infectious virions (if the IFA is indeed a focus forming assay). Just because there is no visible blood does not mean these mosquitoes have not blood fed recently and that some infectious virus could remain even if it isn’t infecting the mosquitoes themselves. Clarify language here and in other areas where “infected” is used

Answer: We thank the reviewer for pointing this out. Indeed, these viruses may have come from a recent human blood meal. However, we showed that the mosquitoes were infected as the virus was viable and could replicate in C6/36 cells.

Query 12

Line 258 – Guedes et al do not demonstrate ZIKV replication in Cx. quinquefasciatus – a closer look at the titers of virus RNA in those mosquitoes will show RNA levels for ZIKV remaining stagnant over time and do not increase at any point measured. Clarify language

Answer: We respectfully disagree with the reviewer. The article by Guedes et al. [Guedes DRD et al. Zika virus replication in the mosquito Culex quinquefasciatus in Brazil. Emerg Microbes Infect. 2017; doi: 10.1038/emi.2017.59.] does in fact demonstrate that ZIKV replicated and was detected in the salivary glands and saliva of artificially fed Cx. quinquefasciatus mosquitoes.

Query 13

Line 262 – Your study has absolutely not demonstrated this. You have found DENV/ZIKV RNA in cell culture samples that are positive for Flaviviruses by IFA. This indicates that pools of these mosquitoes may contain virions that can infect C6/36 cells, but does not indicate that these virions are infecting the mosquitoes in the pool, or that they would be disseminated in the mosquito saliva to new hosts. This sentence greatly misrepresents your findings and should be rewritten.

Answer:

We agree in part with the reviewer. Our results show that the virus was infecting the mosquitoes, was viable and could replicate in C6/36. But we agree that the vector competence needs to be clarified and have modified the sentence accordingly.

Query 14

Line 281 – Once again, you have not found these mosquitoes to be “infected”. Clarify language

Answer: As before, we agree in part with the reviewer and have therefore modified the sentence as requested.

Query 15

Line 293 – I’m not sure one citation constitutes this mosquito being found “frequently… in natural and artificial breeding sites”. Possibly clarify this sentence or find more references

Answer: We agree and have modified the sentence accordingly.

Query 16

Line 295 – The sentence “This mosquito bites actively…” should have citations

Answer:

Thank you for pointing this out. We have accordingly added the reference.

Attachment

Submitted filename: ReplytoReviewers_rev.docx

Click here for additional data file.^{(19.3KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0227239.r003

Decision Letter 1

Humberto Lanz-Mendoza

6 May 2020

PONE-D-19-34539R1

Zika and dengue viruses infecting wild-caught mosquitoes in an environmental protection area in Brazil

PLOS ONE

Dear Dr Marrelli,

==============================

1) The authors need to make clear the methodology, including positive and negative controls.

2) It is fundamental to determine if the mosquitoes were infected. The presence of the virus in mosquitoes extracts is not an indicator of infection.

3) The authors force the conclusion that these mosquitoes might be vectors, but there is a long way to go before this can be concluded.

We would appreciate receiving your revised manuscript by Jun 20 2020 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

Humberto Lanz-Mendoza

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #3: (No Response)

Reviewer #4: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #3: Partly

Reviewer #4: Partly

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #3: N/A

Reviewer #4: N/A

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #3: Yes

Reviewer #4: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #3: Yes

Reviewer #4: Yes

**********

6. Review Comments to the Author

Reviewer #3: The present study investigated the potential presence of arboviruses (more specifically flaviviruses) in wild caught mosquito species in a Environmental Reserve in Sao Paulo, Brazil. Although the findings are important for surveillance purposes, one cannot conclude that these mosquitoes species are vectors for these arboviruses. Although the authors claim they only used "non-engorged" females, there is a possibility that these mosquitoes had traces of blood in their midgut.

The authors emphasise in several instances that these mosquitoes are infected and therefore could be vectors of flaviviruses, which is not appropriate. This has to be worked throughout the manuscript.

Reviewer #4: The paper “Zika and dengue viruses infecting wild-caught mosquitoes in an environmental protection area in Brazil” describes the ZIKV and DENV infection in a C6/36 cell line, from inoculation with extracts pools (10) of wild collected mosquitoes in Brazil. Flavivirus detection was carried out by IFA and identification by qRT-PCR and sequencing. The results are interesting, but for this reviewer additional information is needed and some queries must be clarified.

Line 119. “Detection of flaviviruses” This methodology is 1/3 of the whole Materials and Methods section, but no results are provided (it is surprising because only five samples were positive). This reviewer consider it so important to the readers, mainly because the comparison between positive samples with positive and negative controls.

Line 131. “…primary antibody a polyclonal anti-St Louis encephalitis virus antibody...” I have the concern about if such antibody is able to detect any flavivirus (cross-reaction), if so, a reference should be provided (Gluber et al., used a monoclonal antibody against DENV).

Line 135. Why YFV was used as positive control? Were C6/36 infected with YFV and then performed the IFA? Again, the polyclonal anti-St Louis encephalitis virus antibody also recognize YFV (reference)? Was the IFA signal in YFV similar to problem samples?

Please clarify.

Line 138. “Positive flavivirus samples were analyzed...” Which positive samples (IFA)? Please clarify.

Line 166-167. Full genus name must be wrote at first time it appears if the main body text.

Line 178. Table 1. It is not clear in the table if N (number of specimens) correspond to the amount of mosquitoes (number) or number of analyzed pools. Mainly because in the first column, the key number looks to indicate single pools. If so, there are pools from 2 to 10 mosquitos. Please clarify; it is so important to interpret de results.

Line 235-237. I have the concern regarding the “infected mosquitoes” interpretation. I afraid the results based in the methodology cannot conclude that mosquitoes were infected. No evaluations on virus dissemination (in mosquitoes) were performed. The infection of C6/36 cells with mosquito pool extracts could be the result of the presence of virus in remaining blood of mosquitos that fed blood some days previous to be cached. In addition, it is so intriguing that flavivirus naturally infecting were identified only in mosquitoes that were not previously incriminated as flavivirus vectors (as authors mention). It had been interesting the evaluation (molecular) in well-known mosquito vectors of DENV and ZIKV pools collected in the study area.

Line 336-339. Same comment regarding infected mosquitoes, so difficult to conclude this. In addition, here is mentioned by authors that the five positive mosquito species were the most abundant species in the area. Could be this the reason to which DENV and ZIKV were detected in those mosquitoes? Please explain.

Finally, the “infecting” word in the title should be changed, may be by “detected in”.

**********

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Reviewer #3: No

Reviewer #4: No

PLoS One. 2020 Oct 16;15(10):e0227239. doi: 10.1371/journal.pone.0227239.r004

Author response to Decision Letter 1

20 Jun 2020

Dr. Humberto Lanz-Mendoza

Academic Editor

PLOS ONE

Thank you for sending the evaluation of our manuscript. We very much appreciate the comments received from the Reviewers. We have made substantial changes to the manuscript, essentially addressing the points made by the reviewers. Accordingly, we believe that we have significantly improved the article. The changes are summarized in the reply to reviewers.

Yours sincerely,

Mauro Toledo Marrelli and coworkers

Editor Comments

1) The authors need to make clear the methodology, including positive and negative controls.

ANSWER: IFA positive and negative controls issue was addressed. The “Detection of Flavivirus” topic was rewritten in order to avoid misunderstanding.

2) It is fundamental to determine if the mosquitoes were infected. The presence of the virus in mosquitos’ extracts is not an indicator of infection.

ANSWER: We agree with the comment. The statement was worked throughout the manuscript.

3) The authors force the conclusion that these mosquitoes might be vectors, but there is a long way to go before this can be concluded.

ANSWER: We agree with the comment. Following reviewer’s #3 suggestion, the conclusion that these mosquitoes could act as vectors for Flavivirus is too much speculative based on our data. The statement was worked throughout the manuscript and some new references were included.

Review Comments to the Author

Reviewer #3: The present study investigated the potential presence of arboviruses (more specifically flaviviruses) in wild caught mosquito species in an Environmental Reserve in Sao Paulo, Brazil. Although the findings are important for surveillance purposes, one cannot conclude that these mosquito’s species are vectors for these arboviruses. Although the authors claim they only used "non-engorged" females, there is a possibility that these mosquitoes had traces of blood in their midgut.

The authors emphasis in several instances that these mosquitoes are infected and therefore could be vectors of flaviviruses, which is not appropriate. This has to be worked throughout the manuscript.

ANSWER: Thank you for comments. We agreed with the reviewer remark, concluding that these mosquitoes could act as vectors is too much speculative based on our data. The statement was worked throughout the manuscript.

Line 119. “Detection of flaviviruses” This methodology is 1/3 of the whole Materials and Methods section, but no results are provided (it is surprising because only five samples were positive). This reviewer considers it so important to the readers, mainly because the comparison between positive samples with positive and negative controls.

ANSWER: Positive and negative controls were used only for IFA analysis as a control of the reaction per se and recommended for diagnosis proposes. The “Detection of Flavivirus” topic was rewritten in order to avoid misunderstanding. Detection of low levels of mosquito infections requires large samples (greater than 1,600 individuals) for a high probability (0.8) of detection (Gu et al. Am J Trop Med Hyg. 2004). The low detection rate obtained was included in the discussion topic.

ANSWER: Thank you for your observation. The topic was rewritten in order to avoid misunderstanding and a recent study conducted by Cunha et al (2020) was included as reference.

Please clarify.

ANSWER: YFV is flavivirus, therefore able to be recognized by anti-St Louis. 17D YFV vaccine strain was used because it is a well-known standard flaviviruses strain and can be used within the established biosafety standards for flavivirus studies. C6/36 cells were inoculated with 17D YFV vaccine strain (separately from the study samples) in order to be used as a positive control for the IFA reaction. In parallel, C6/36 cells culture per se was used as negative control for the IFA reaction. This approach has been used for decades in the flavivirus routine surveillance system conducted by Vector-born Disease Laboratory.

Line 138. “Positive flavivirus samples were analyzed...” Which positive samples (IFA)? Please clarify.

ANSWER: We clarified this issue in the text.

Line 166-167. Full genus name must be writing at first time it appears if the main body text.

ANSWER: Thank you for your observation. We corrected that throughout the text.

ANSWER: Table 1 was rewritten in order to clarify the interpretation.

ANSWER: Thank you for your observation. We agreed with the reviewer remark, concluding that these mosquitoes could act as vectors is too much speculative based on our data. The statement was worked throughout the manuscript. Your suggestion to evaluate DENV and ZIKV in well-known mosquito vectors in the same surveyed area is extremely valuable and will be carefully take in consideration for future studies.

ANSWER: The most abundant mosquito species were often collected and, for sure, the more specimens sampled of a given species, the greater the chance that some viral detection will occur. However, the chances of arbovirus detection, in specially infection in mosquito populations are very low (Gu W, Novak RJ. Short report: Detection probability of arbovirus infection in mosquito populations. Am. J. Trop. Med. Hyg. 2004; 71:636-38). Furthermore, the fact of detecting infection cases is not directly related to transmission, as this phenomenon depends on the vectorial capacity of each species. This is related to conditions that extrapolate abundance, such as the rate of bites, the competence, and capacity of the pathogen to multiply in the organism of the vector, among others. So, yes, it is a possibility. However, we do not know the role of peridomestic and sylvatic mosquitoes in Flavivirus cycle in Brazil. Viral isolation from mosquitoes collected in the wild, as made here, is a rare event, and therefore our results highlight the importance of performing entomological studies and viral detection in mosquitoes.

Finally, the “infecting” word in the title should be changed, may be by “detected in”.

ANSWER: Changed.

PLoS One. doi: 10.1371/journal.pone.0227239.r005

Decision Letter 2

Humberto Lanz-Mendoza

3 Aug 2020

PONE-D-19-34539R2

Detection of Zika and dengue viruses in wild-caught mosquitoes during field surveillance conducted in an environmental protection area, São Paulo, Brazil

PLOS ONE

Dear Dr. Marrelli,

==============================

The manuscript requires proofread.

Please carefully review your manuscript.

Please ensure that your decision is justified on PLOS ONE’s publication criteria and not, for example, on novelty or perceived impact.

==============================

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We look forward to receiving your revised manuscript.

Kind regards,

Humberto Lanz-Mendoza

Academic Editor

PLOS ONE

Additional Editor Comments (if provided):

The manuscript requires proofread.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #3: (No Response)

Reviewer #4: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #3: Yes

Reviewer #4: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #3: N/A

Reviewer #4: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #3: Yes

Reviewer #4: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #3: No

Reviewer #4: Yes

**********

6. Review Comments to the Author

Reviewer #3: Review PONE-D-19-34539_R2

Although what I have raised on my first review around the incrimination of vectors have been soften by the authors, I have the impression that, in many sections of the manuscript, they copied and pasted all the suggestions, which made the text flow very confusing and, therefore not suitable for publication in the current format. It definitely needs to go through a thorough proofreading and language review before it can be accepted.

Some examples of needed modifications/ corrections are shown below:

Rephrasing sentences on lines 21 and 23.

Line 25 Through and not throughout.

Several instances when mosquitos x mosquitoes are used.

Arbovirus is mentioned on line 50 and only explained on line 54.

Line 52-52. Please correct grammar here.

Line 212 – previously reported.

Line 225 – Plasmodium in italics.

Line 250 - Please rephrase this sentence. And has errors like “cached”.

Line 281 – species and not specie.

Line 286 – Please rephrase this sentence. Virus “are present”

Reviewer #4: In this second review, authors addressed all comments previously indicated. This reviewer consider them have done a good job by changing the title, reorganizing some data, discussion and conclusions. This reviewer consider that this new version of the manuscript is suitable for its publication.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #3: No

Reviewer #4: No

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

PLoS One. 2020 Oct 16;15(10):e0227239. doi: 10.1371/journal.pone.0227239.r006

Author response to Decision Letter 2

8 Sep 2020

Dr. Humberto Lanz-Mendoza

Academic Editor

Plos One

Dear Dr. Lanz-Mendoza

Thank you very much for reviewing our manuscript and for the many suggestions, which have enabled us to greatly improve the text. We agree with the changes proposed by Reviewer#3 and are glad that Reviewer #4 has approved the new version of our manuscript.

We have made the changes to the manuscript to address the points made by Reviewer#3, and we hope the article is now suitable for publication in Plos One.

Sincerely,

Mauro Marrelli

Answers: Reviewer #3

Reviewer comment: Although what I have raised on my first review around the incrimination of vectors have been soften by the authors, I have the impression that, in many sections of the manuscript, they copied and pasted all the suggestions, which made the text flow very confusing and, therefore not suitable for publication in the current format. It definitely needs to go through a thorough proofreading and language review before it can be accepted.

Answer: We thank the reviewer for pointing out these mistakes. We agree that the text was very confused and have made the necessary modifications to the manuscript in order to make it clearer and more focused. In addition, the text has been completely proofread by a native English speaker.

Reviewer comment: Some examples of needed modifications/ corrections are shown below:

Answer: We have made the modifications pointed out by the reviewer and have carried out a thorough review of the entire manuscript.

Rephrasing sentences on lines 21 and 23.

Answer: We have rephrased these sentences accordingly. Thank you for pointing this out.

Line 25 Through and not throughout.

Answer: We have corrected this.

Several instances when mosquitos x mosquitoes are used.

Answer: We have now used the term mosquitoes throughout the text. We are grateful to the reviewers for pointing this out.

Arbovirus is mentioned on line 50 and only explained on line 54.

Answer: We have modified the sentence accordingly.

Line 52-52. Please correct grammar here

Answer: We have rephrased the sentence accordingly.

Line 212 – previously reported.

Answer: We have corrected this.

Line 225 – Plasmodium in italics.

Answer: We have corrected this

Line 250 - Please rephrase this sentence. And has errors like “cached”.

Answer: We have corrected this. Thank you for pointing out this mistake.

Line 281 – species and not specie.

Answer: We have corrected this.

Line 286 – Please rephrase this sentence. Virus “are present”

Answer: We have rephrased this sentence.

PLoS One. doi: 10.1371/journal.pone.0227239.r007

Decision Letter 3

Humberto Lanz-Mendoza

30 Sep 2020

Detection of Zika and dengue viruses in wild-caught mosquitoes collected during field surveillance in an environmental protection area in São Paulo, Brazil

PONE-D-19-34539R3

Dear Dr. Marrelli,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Humberto Lanz-Mendoza

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

PLoS One. doi: 10.1371/journal.pone.0227239.r008

Acceptance letter

Humberto Lanz-Mendoza

5 Oct 2020

PONE-D-19-34539R3

Detection of Zika and dengue viruses in wild-caught mosquitoes collected during field surveillance in an environmental protection area in São Paulo, Brazil

Dear Dr. Marrelli:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

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on behalf of

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Academic Editor

PLOS ONE

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Table. Reference sequences of dengue and Zika viruses from GenBank.

(DOCX)

Click here for additional data file.^{(15.1KB, docx)}

S2 Table. Mosquito species collected in the Capivari-Monos Environmental Protection Area (EPA).

The table shows the number of individuals at each study point, forest stratum, sex and number of pools. Mosquitoes collected from March 2016 to April 2017.

(DOCX)

Click here for additional data file.^{(19KB, docx)}

Attachment

Submitted filename: Comments on PONE-D-19-34539.pdf

Click here for additional data file.^{(113.3KB, pdf)}

Attachment

Submitted filename: ReplytoReviewers_rev.docx

Click here for additional data file.^{(19.3KB, docx)}

Data Availability Statement

All data generated or analyzed during this study are included in the manuscript.

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PERMALINK

Detection of Zika and dengue viruses in wild-caught mosquitoes collected during field surveillance in an environmental protection area in São Paulo, Brazil

Karolina Morales Barrio-Nuevo

Mariana Sequetin Cunha

Adriana Luchs

Aristides Fernandes

Iray Maria Rocco

Luis Filipe Mucci

Renato Pereira de Souza

Antônio Ralph Medeiros-Sousa

Walter Ceretti-Junior

Mauro Toledo Marrelli

Roles

Abstract

Introduction

Material and methods

Study area and mosquito sampling

Fig 1. Capivari-Monos EPA in the extreme south of the city of São Paulo, SP, Brazil.

Detection of flaviviruses

Identification of flavivirus species

Results

Table 1. Flavivirus-positive samples detected in pools of mosquitoes from the Capivari-Monos EPA, São Paulo, Brazil, 2016–2017.

Fig 2. Neighbor-Joining (NJ) phylogenetic tree of partial NS5 gene sequences (generated in MEGA 6.0) of DENV-2 isolates detected in Cx. ssp and Cx. vaxus in the Capivari-Monos EPA, São Paulo, Brazil, 2016–2017.

Fig 3. Neighbor-Joining (NJ) phylogenetic tree (generated in MEGA 6.0) of partial NS5 gene sequences of ZIKV isolated from Li. durhamii, An. cruzii and Wy. confusa in the Capivari-Monos EPA, São Paulo, Brazil, 2016–2017.

Discussion

Conclusion

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

George Dimopoulos

Roles

Author response to Decision Letter 0

Decision Letter 1

Humberto Lanz-Mendoza

Roles

Author response to Decision Letter 1

Decision Letter 2

Humberto Lanz-Mendoza

Roles

Author response to Decision Letter 2

Decision Letter 3

Humberto Lanz-Mendoza

Roles

Acceptance letter

Humberto Lanz-Mendoza

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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