
==== Front
Rev Inst Med Trop Sao Paulo
Rev Inst Med Trop Sao Paulo
rimtsp
Revista do Instituto de Medicina Tropical de São Paulo
0036-4665
1678-9946
Instituto de Medicina Tropical de São Paulo

00227
10.1590/S1678-9946202466056
Original Article
Entomo-virological surveillance of Flavivirus in mosquitoes in Yucatan State, Mexico
https://orcid.org/0009-0000-0613-2395
Argaez-Sierra Diana Guadalupe conceived and designed the study All authors read and approved the final manuscript 1
https://orcid.org/0000-0002-3772-6740
Baak-Baak Carlos Marcial drafted the manuscript All authors read and approved the final manuscript 1
https://orcid.org/0000-0002-6681-1581
Garcia-Rejon Julian E. critically revised the manuscript for intellectual content All authors read and approved the final manuscript 1
https://orcid.org/0000-0003-3489-9520
Cetina-Trejo Rosa Carmina carried out the bioinformatics and laboratory analyses All authors read and approved the final manuscript 1
https://orcid.org/0000-0001-7676-3870
Tzuc-Dzul Julio C. carried out the bioinformatics and laboratory analyses All authors read and approved the final manuscript 1
https://orcid.org/0000-0001-7311-0858
Acosta-Viana Karla Y. critically revised the manuscript for intellectual content All authors read and approved the final manuscript 2
https://orcid.org/0000-0001-8128-9384
Chan-Perez José I. carried out the bioinformatics and laboratory analyses All authors read and approved the final manuscript 2
https://orcid.org/0000-0002-0363-0222
Cigarroa-Toledo Nohemi conceived and designed the study All authors read and approved the final manuscript 2
1 Universidad Autónoma de Yucatán, Centro de Investigaciones Regionales “Dr. Hideyo Noguchi”, Laboratorio de Arbovirología, Mérida, Yucatán, Mexico
2 Universidad Autónoma de Yucatán, Centro de Investigaciones Regionales “Dr. Hideyo Noguchi”, Laboratorio de Biología Celular, Mérida, Yucatán, Mexico
Correspondence to : Nohemi Cigarroa-Toledo Universidad Autónoma de Yucatán, Centro de Investigaciones Regionales “Dr. Hideyo Noguchi”, Laboratorio de Biología Celular, Calle 43 x 90, Nº 613, Col. Inalambrica, 97225, Mérida, Yucatán, Mexico; E-mail: nohemi.cigarroa@correo.uady.mx
CONFLICT OF INTERESTS: None.

23 9 2024
2024
66 e5621 1 2024
06 6 2024
https://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License
ABSTRACT

The genus Flavivirus (Family: Flaviviridae) comprises arboviruses with the capacity to infect humans and animals. It also integrates insect-specific viruses. This study aimed to identify Flavivirus in mosquitoes captured in 17 municipalities in Yucatan State, Mexico. The mosquitoes were caught in households from November 2021 to May 2022. A total of 4,321 adult mosquitoes from five species were caught. The most abundant were Culex quinquefasciatus (n = 3,563) and Aedes aegypti (n = 734). For molecular investigations, 600 female mosquitoes were split into groups of 10, mostly for species and site location. Reverse transcriptase polymerase chain reaction (RT-PCR) amplified a region of the NS5 gene to find the Flavivirus ribonucleic acids (RNA). A total of 24 pools that were positive for Flavivirus were detected in Ae. aegypti specimens and subsequently subjected to sequencing using the Sanger method. A total of 12 sequences matched the established quality criteria and were subsequently employed for sequence homology analysis. We found that one sequence corresponded to the Zika virus (ZIKV), and 11 sequences had sequence similarity with Phlebotomus-associated flavivirus (PAFV), an insect-specific virus (ISF). In conclusion, we found ZIKV in the Merida municipality, Yucatan State, which suggests that the virus is silently circulating. Phlebotomus-associated flavivirus is distributed in five municipalities in Yucatan State, Mexico. Future studies could focus on isolating this virus and studying its biological role within Ae. aegypti.

KEYWORDS

Aedes aegypti
Zika virus
Phlebotomus-associated flavivirus
Insect-specific flaviviruses
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pmcINTRODUCTION

Arboviruses (a term derived from arthropod-borne viruses) are a diverse group of viruses that are potentially infectious to humans and animals when they are transmitted by arthropod vectors (mosquitoes, ticks, sandflies, or biting midges) 1 . More than 600 arboviruses have been discovered; these are integrated into six families. Most arboviruses that cause human diseases belong to the families Flaviviridae, Togaviridae, and Peribunyaviridae 2 . The studies on Flavivirus in arthropods have concentrated on those that are associated with diseases in vertebrates. This emphasis stems partly from the fact that hosts who are vertebrates typically show more visible signs of infection 1 . The genus Flavivirus encompasses 53 species of viruses and contains those transmitted between hematophagous arthropods and vertebrate hosts, viruses with no known vector (NKV), and insect-specific flavivirus (ISF) 3-5 . Flaviviruses that infect vertebrates can be categorized into two groups: mosquito-borne flaviviruses (MBFVs), which include dengue virus, Zika virus, and West Nile virus 5 ; and tick-borne flaviviruses (TBFVs), which include tick-borne encephalitis virus and Powassan virus 6 . The viruses with no known vector have been isolated in nature from bats, rodents, and occasionally humans, but never from wild-caught or laboratory-inoculated arthropods or arthropod cell cultures. Viruses with no known vector can be categorized into two groups: flaviviruses linked with bats and flaviviruses linked with rodents 3,7 . Infections from the group encompassing insect-specific-flaviviruses show no visible signs of infection in the arthropods they infect. It also does not infect animals or vertebrate cell cultures; however, viruses such as the Aedes flavivirus (AEFV) can produce a mild cytopathic effect in mosquito C6/36 cell 4,5 .

In nature, mosquitoes are the primary source of ISFs. However, they have also been identified in bees, cockroaches, chironomids, and sandflies, suggesting the potential for infection in other arthropods 5 . A number of these viruses have been unintentionally identified by the use of generic primer sets, specifically those that are intended to detect a segment of the non-structural protein NS5 found in flaviviruses 8-11 . Insect-specific flaviviruses can be classified into two categories: classical ISFs (cISFs) and dual-host affiliated ISFs (dISFs). The cISFs were the first to be discovered as a clade different from the others 4 . The cell-fusing agent virus (CFAV) represents the group 12 . An interesting fact is that the dISFs are phylogenetically related to the mosquito-borne flaviviruses, despite their inability to replicate in vertebrate cells 4,5 .

In Mexico, there is active circulation of the dengue virus, with occasional occurrences of the Zika virus 13 . During monitoring of infectious flaviviruses in vertebrates and mosquitoes, the ISFs Culex flavivirus (CxFV) and T’Ho virus have been frequently detected in Cx. quinquefasciatus 14-17 . In field or experimental studies, it has been shown that ISFs show the capacity to modulate or enhance the co-infection, replication, and transmission of flavivirus 18-20 . In an endemic focus on West Nile virus (WNV) transmission in Chicago city, United States, it was discovered that 40% of Culex mosquitoes who were WNV-positive were also CxFV-positive, demonstrating that both viruses can co-infect mosquitoes in nature 18 . In a controlled laboratory study, it was demonstrated that the Nhumirim virus (NHUV) can inhibit the replication of the West Nile virus in Cx. quinquefasciatus mosquitoes when they are infected with both NHUV and WNV 19 . Similarly, it was observed that NHUVs suppress the viral replication of Zika virus up to 100,000 times and dengue virus-2 up to 10,000 times in Aedes albopictus cells 20 .

Prior to the coronavirus pandemic, Mexico showed 501,600 recorded cases of dengue from 2007 to 2020, and the virus was linked to 1,230 deaths 13 . However, the incidence of diseases spread by vectors, such as dengue, significantly decreased during the peak of SARS-Cov-2 transmission 21 . Our hypothesis was that the flaviviruses were circulating despite the underreporting of cases. Therefore, this study aimed to conduct entomo-virological surveillance of flaviviruses in mosquitoes from households in Yucatan State, Mexico, after the critical period of the pandemic.

MATERIALS AND METHODS

Study area

The entomo-virological study was conducted in 17 municipalities located in Yucatan State (Figure 1). Yucatan State is located on the Yucatan Peninsula in Mexico and shares borders with Quintana Roo State and Campeche State (20°50’0” N, 89°0’0” W). The rainy season (May to October) records an average rainfall of 1,000 millimeters and an average temperature of 27.58 °C. The dry season extends from November to April, with an average precipitation of 300 millimeters and an average temperature of 25.18 °C 22 .

Figure 1 Municipalities of Yucatan State, Mexico, that were visited to capture mosquitoes. The green star represents the municipality with mosquitoes positive for Phlebotomus-associated flavivirus, the purple star represents the pool positive for Zika, and the red circles are the municipalities with mosquitoes negative for Flavivirus.

Design and capture of adult mosquitoes

A cross-sectional study was conducted. In each location, seven to 22 households were visited. The houses were chosen based on their convenience. Sampling was carried out in areas where flaviviruses circulation and mosquito abundance were previously reported 22-25 . Mosquito capture was performed from November 2021 to May 2022 with the aid of a Propack-type backpack-mounted aspirator (John W. Hock Company, Gainesville, US). Every room in each house was subjected to mosquito trapping, with a focus on dark locations such as behind furniture, within closets, and on curtains. Backyards were also included, particularly the shady parts (tool sheds and pet shelters). Adult mosquitoes were placed in plastic containers labeled with an identification number, collection date, and site sample. Mosquitoes caught were transported alive to the Arbovirologia laboratory at the Universidad Autonoma de Yucatan and identified up to species using stereomicroscopes and taxonomic keys 26 .

RNA extraction and molecular assays

To perform flaviviruses detection tests, the mosquito species with the highest abundance of females were selected, including the Ae. aegypti (n = 260), Cx. quinquefasciatus (n = 328), and Ae. albopictus (n = 12). A female mosquito was put into each Eppendorf microtube, and then 50 µL of the DNA/RNA ShieldTMpreserver (Zymo Research) was added. Then, the samples were stored at −80 °C until they were used in molecular analyses. Afterwards, each mosquito was mechanically homogenized with sterile pestles, and 50 µL of Eagle’s minimal essential medium (Invitrogen, Carlsbad, CA, USA). Homogenates were centrifuged at 10,000 × g for 7 min. In total, 15 µL of each individual supernatant was collected to create pools of 10 mosquitoes (150 µL). Female mosquitos were pooled according to species and capture site.

Total RNA was extracted from each pool (aliquot 120 µL) using a QIAamp Viral RNAkit (Qiagen, catalog Nº 52906. Valencia, CA, US) following the manufacturer’s protocols. Complementary DNA (cDNA) was synthesized with 650 ng of total ARN samples using a commercial kit ADNc RevertAid H Minus First Strand (Thermo Scientific). The semi-nested pan-flavivirus PCR protocol described by Scaramozzino et al. 11 was followed to detect flavivirus RNA. The primers cFD2 (5’-GTGTCCCAGCCGGCGGTGTCATCAGC-3’), MAMD (5’-AACATGATGGGRAARAGRGARAA-3’), and FS778 (5’-AARGGHAGYMCDGCHATHTGGT-3’) were used to amplify a ~250 nucleotide region of the NS5 gene 11 . For a 25-µL PCR reaction, 3 µL of ADNc, 2 µL of MgCl2 at a concentration of 25 mM, 2.5 µL of 5X reaction buffer, 0.2 µL of dNTPs, 0.5 µL of dNTPs, 1.5 U of Taq polymerase (Invitrogen), and 0.5 µL of each primer at a concentration of 10 µM were added, with the rest of the volume being filled with nuclease-free water. Negative (molecular biology-grade water) and positive controls (DENV-2) were included in each PCR. Thermal cycling conditions consisted of denaturation at 94 °C for 5 min, hybridization at 35 cycles at 94 °C for 1 min, 54 °C for 1 min, and 72 °C for 1 min, followed by a final elongation at 72 °C for 5 min. Amplicons were visualized on 1.5% agarose gels with 0.5 µg/mL ethidium bromide and visualized on a Doc™ XR+ Gel Documentation System. Bands of interest were cut from the gel and purified using a Zymoclean DNA recovery kit (Zymo Research, Irvine, CA, US) following the manufacturer’s protocols. The purified products were sequenced using a 3500xL genetic analyzer (Applied Biosystems, Foster City, CA, US) in the Synthesis Unit and a Sequencing Unit from the Institute of Biotechnology, UNAM, Mexico.

Sequence analysis

Sequences were analyzed and manually edited using the Bioedit v.7.0.9 software and the Mega v.7 software 27 . The consensus sequences were compared to other sequences from the GenBank database using the Basic Local Alignment Search Tool of the National Library of Medicine. To determine a hit, the flaviviruses-positive PCR sequences were assessed based on specific criteria, including a total coverage of over 90%, an identity of over 97%, and a minimum length of 223 base pairs. The sequences generated in the present study were deposited in GenBank.

Phylogenetic analysis

The phylogenetic analysis was performed using the molecular evolutionary genetics analysis (MEGA) v.11 software 28 . The NS5 sequences of viruses representing the Flavivirus genus were used. The selection of ZIKV from the American, Asian, and African lineages was obtained from Cabral et al. 29 and Shen et al. 30 . The sequences of the cISF, dISF, and NKV groups were obtained from the works of Huang et al. 31 and Rizzo et al. 32 . The tree was complemented with the PAFV sequences from Saudi Arabia (MN294940, MN294941, and MN294942) obtained from the National Center for Biotechnology Information (NCBI). In addition, all samples from this study were included. Sequences were aligned using the Clustal W v.2.10 software (EMBL-EBI, Hinxton, Cambridgeshire, UK). Multiple alignment under default parameters and end fragments that were not shared by most of the analyzed sequences were removed.

The phylogenetic reconstruction in MEGA 5 was performed by employing a statistical method based on the Maximum Likelihood method 33 . After analyzing the best tree model, the Kimura 2 (K2) + G + I model was selected. The phylogeny test was performed with the Bootstrap method with 1,000 replications.

The minimum infection rate

The minimum infection rate (MIR) was estimated as: (number of positive pools/total specimens tested) × 1,000. The MIR is a parameter used to estimate the probability of finding a viral infection in mosquitoes.

RESULTS

Adult mosquito collections

A total of 4,321 mosquitoes of five species were captured in 164 households across 17 municipalities in Yucatan State, Mexico (Table 1). Of these, 88 (53.66%) were mosquito-infested. The most common mosquitoes were Cx. quinquefasciatus (n = 3,563) and Ae. aegypti (n = 734) (Table 1). The adult population of Cx. quinquefasciatus consisted of 1,338 females and 2,225 males. Aedes aegypti adults were 35.83% female (263 out of 734) and 64.17% male (471 out of 734). The least abundant were Aedes albopictus (n = 18), Aedes taeniorhynchus (n =5), and Culex iolambdis (n =1) (Table 1).

Table 1 Households visited and the abundance of female mosquitoes collected by municipality, Yucatan State, Mexico.

Collection site	Nº positive households/total	Cx. quinquefasciatus	Ae. aegypti	Ae. albopictus	Ae. taeniorhynchus	Cx. iolambdis	
Merida municipality	14/14	68	96	1	1	0	
Celestun municipality	22/22	239	13	0	4	1	
Ekmul municipality	12/18	237	78	1	0	0	
Tetiz municipality	4/8	138	12	0	0	0	
Kinchil municipality	2/8	90	0	0	0	0	
Valladolid municipality	1/7	6	0	12	0	0	
Chemax municipality	2/8	16	0	0	0	0	
Tinum municipality	1/8	5	1	0	0	0	
Uman municipality	4/8	26	4	0	0	0	
Tahmek municipality	3/8	56	1	0	0	0	
Homun municipality	6/8	89	17	0	0	0	
Hocaba municipality	2/8	170	4	0	0	0	
Cuzama municipality	3/7	24	1	0	0	0	
Sotuta municipality	1/7	74	33	1	0	0	
Yaxcaba municipality	5/8	88	0	1	0	0	
Tizimin municipality	3/10	1	0	0	0	0	
Calotmul municipality	3/7	11	3	0	0	0	
Total	88/164	1,338	263	16	5	1	

Detection of Flavivirus in mosquitoes

In total, 600 female mosquitoes were divided into 60 pools, which corresponded to Ae. aegypti (n = 26 pools), Cx. quinquefasciatus (n = 33 pools), and Ae. albopictus (1 pool). A total of 24 pools of Flavivirus-positive PCR were detected in Ae. aegypti and subsequently subjected to sequencing using the Sanger method. In total, 12 sequences matched the established quality criteria for assembly (forward and reverse) and were subsequently employed for sequence similarity searches. Culex quinquefasciatus and Ae. albopictus did not exhibit any positive Flavivirus results.

A positive pool of Ae. aegypti for the Zika virus (GenBank ID: OR701943) was found in a residence in Merida municipality, Yucatan State, Mexico. The Plebotomus-associated flavivirus was found in Ae. aegypti from Celestun municipality (GenBank ID: OR636207), Ekmul municipality (GenBank IDs: OR636209 - OR636212), Homun municipality (GenBank ID: OR636214), Merida municipality (GenBank IDs: OR636204 - OR636206, and OR636208), and Sotuta municipality (GenBank ID: OR636213), of Yucatan State, Mexico (Figure 1).

Phylogenetic tree

In total, one out of the 12 Flaviviridae-positive pools in Ae. aegypti showed an NS5 sequence that was 96.74% the same as the ZIKV strain from Mexico (MT507050.1). In the phylogenetic tree, this sequence was clustered within the Asian lineage’s ZIKV (OR701943) (Figure 2). The following 11 sequences showed nucleotide identity (97.07–99.5%) with a Phlebotomus-associated flavivirus (MN294942.1) from Saudi Arabia (2020). In the phylogenetic tree, these sequences were clustered within classical ISFs (Figure 2).

Figure 2 Maximum-likelihood phylogenetic tree of 70 flaviviruses. The tree is based on 219-nt partial NS5 nucleotide sequences. The ML method used the Kimura 2 (K2) + G + I model to generate the tree. Nodes’ numbers indicate bootstrap support values. Labels include the GenBank accession number, corresponding virus name, host, collection site, and date. Sequences identified in this study are marked with blue circles. Zika virus (ZIKV) lineage, classical Insect-specific flaviviruses (cISFs), dual-host-affiliated Insect-specific flaviviruses (dISFs), and viruses with no known vector (NKV).

The minimal infection rate (MIR) for Flavivirus was determined to be 40, whereas the Zika virus displayed a MIR of 1.66 and the PAFV displayed a MIR of 18.33.

DISCUSSION

During the COVID-19 pandemic in Mexico, a significant reduction in the incidence of vector-borne diseases (VBD) was noted. The increased demand for resources dedicated to monitoring and diagnosing COVID-19 may have had an impact on the underreporting of VBD cases 21 . The dengue virus was not identified in our study. However, we found the ZIKV in the mosquito Ae. aegypti from November 2021 to May 2022. The finding suggests that the ZIKV was in silent circulation during this period. It is important to acknowledge that this arbovirus exhibited periods of heightened transmission from 2015 to 2017. Currently, instances linked to this particular virus are sporadically documented 13,21 . The estimated MIR for ZIKV was 1.66. The value is similar to that reported for the ZIKV identified in Ae. aegypti captured in cemeteries in Merida municipality, Yucatan State, Mexico 24 . However, the value is low compared to the MIR of the dengue virus from Ae. aegypti captured from households and schools in the same location 23,25 .

In Mexico, ZIKV RNA has been identified in humans, pigs, and the mosquitoes Ae. aegypti, Ae. albopictus, Aedes epactius, Ae. taeniorhynchus, Cx. coronator, Cx. erraticus, Cx. lactator, Cx. nigripalpus, Cx. quinquefasciatus, Cx. tarsalis, Cx. thriambus, Culiseta inornata, and Cs. particeps 24,34-36 .

In Mexico, the dengue virus holds significant importance as a flavivirus, mostly due to its impact on human morbidity and mortality. Moreover, since the reemergence of dengue in Mexico in 1978, there has been a continuous occurrence of outbreaks associated with the four serotypes (DENV 1–4) 13 . Nevertheless, a circulation of flaviviruses in hosts different from humans has been reported within the country. Serological and molecular evidence has been documented, indicating the presence of the West Nile virus and the St. Louis encephalitis virus in birds 16 . The Apoi virus and Modoc virus, which are flaviviruses that specifically infect rodents and present no known arthropod vectors, have been detected in peridomestic rodents within the urban area of Merida municipality, Yucatan State, Mexico 37 .

In this study, we identified a Phlebotomus-associated flavivirus that was distributed across five municipalities in Yucatan State. The Phlebotomus-associated flavivirus was found for the first time in Algeria in a sample of Phlebotomus perniciosus 38 , from which it derives its name. It is the sole ISF that was not named after a mosquito genus. In subsequent investigations, the virus was discovered in Ae. aegypti from Saudi Arabia 39 . The importance of ISF in nature has not yet been elucidated; however, it has been suggested that, in situations of co-infection, they may suppress the viral replication of the ZIKV virus and dengue virus 20 . However, evidence also points towards positive interactions between WNV and the ISF Culex flavivirus 18 . An important limitation is that no study has been able to isolate the virus, so its full genome is unknown. However, the evidence of Phlebotomus-associated flavivirus in Ae. aegypti of Yucatan State provides an opportunity for future studies to perform the isolation and sequencing to better understand its function.

In the current investigation, flaviviruses were not detected in Cx. quinquefasciatus and Ae. albopictus. In Cx. quinquefasciatus from Yucatan State, researchers have found the Zika virus 35,36 as well as viruses that are specific to mosquitoes, namely the Culex flavivirus, the T’Ho virus, and the Houston virus 10,15,17 . For Ae. albopictus from Yucatan State, there are no reports of flavivirus, and the only ISF report is of AEFV from Japan in 2009 40 .

CONCLUSION

In conclusion, we found 24 pools of PCR-positive Flavivirus, and 12 sequences were assembled during entomo-virological surveillance of Flavivirus. The ZIKV was detected in a mosquito pool in an urban area, suggesting that the virus may be silently circulating in Merida municipality, Yucatan State, Mexico. Most of the flavivirus-positive samples were identified as Phlebotomus-associated flavivirus, which shows a wide distribution in Yucatan State and may belong to the virome of Ae. aegypti mosquitoes. Future studies should isolate the PAFV to obtain the complete genome and study the function of the species in a mosquito cell line. Therefore, it could be used in competitive superinfection experiments to determine whether PAFV increases or controls the spread of medically important viruses, such as DENV, which is endemic in Mexico.

ACKNOWLEDGMENTS

We would like to thank the residents of the houses for their kindness in letting us enter their houses and capture the mosquitoes.

FUNDING: None.
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REFERENCES

1 Young PR Arboviruses: a family on the move Hilgenfeld R Vasudevan SG editors Dengue and Zika: control and antiviriral treatment strategies Singapore Springer 2018 1 10
2 Centers for Disease Control and Prevention Arbovirus catalog cited 2024 June 10 https://wwwn.cdc.gov/arbocat/
3 Billoir F De Chesse R Tolou H De Micco P Gould EA De Lamballerie X Phylogeny of the genus flavivirus using complete coding sequences of arthropod-borne viruses and viruses with no known vector J Gen Virol 2000 81 781 790 10675416
4 Blitvich BJ Firth AE Insect-specific flaviviruses: a systematic review of their discovery, host range, mode of transmission, superinfection exclusion potential and genomic organization Viruses 2015 7 1927 1959 25866904
5 Calisher CH Higgs S The discovery of arthropod-specific viruses in hematophagous arthropods: an open door to understanding the mechanisms of arbovirus and arthropod evolution? Annu Rev Entomol 2018 63 87 103 29324047
6 Kemenesi G Bányai K Tick-borne flaviviruses, with a focus on Powassan virus Clin Microbiol Rev 2019 32 e00106-17 30541872
7 Blitvich BJ Firth AE A review of flaviviruses that have no known arthropod vector Viruses 2017 9 154 28635667
8 Huhtamo E Moureau G Cook S Julkunen O Putkuri N Kurkela S et al Novel insect-specific flavivirus isolated from northern Europe Virology 2012 433 471 478 22999256
9 Ravanini P Huhtamo E Ilaria V Crobu MG Nicosia AM Servino L et al Japanese encephalitis virus RNA detected in Culex pipiens mosquitoes in Italy Euro Surveill 2012 17 20221
10 Cigarroa-Toledo N Baak-Baak CM Cetina-Trejo RC Cordova-Fletes C Martinez-Nuñez MA Talavera-Aguilar LG et al Complete genome sequence of Houston virus, a newly discovered mosquito-specific virus isolated from Culex quinquefasciatus in Mexico Microbiol Resour Announc 2018 7 e00808-18 30533617
11 Scaramozzino N Crance JM Jouan A DeBriel DA Stoll F Garin D Comparison of Flavivirus universal primer pairs and development of a rapid, highly sensitive heminested reverse transcription-PCR assay for detection of flaviviruses targeted to a conserved region of the NS5 gene sequences J Clin Microbiol 2001 39 1922 1927 11326014
12 Stollar V Thomas VL An agent in the Aedes aegypti cell line (Peleg) which causes fusion of Aedes albopictus cells Virology 1975 64 367 377 806166
13 Baak-Baak CM Cigarroa-Toledo N Pinto-Castillo JF Cetina-Trejo RC Torres-Chable O Blitvich BJ et al Cluster analysis of dengue morbidity and mortality in Mexico from 2007 to 2020: implications for the probable case definition Am J Trop Med Hyg 2022 106 1515 1521 35292593
14 Briese T Loroño-Pino MA Garcia-Rejon JE Farfan-Ale JA Machain-Williams C Dorman KS et al Complete genome sequence of T'Ho virus, a novel putative flavivirus from the Yucatan Peninsula of Mexico Virol J 2017 14 110
15 Charles J Tangudu CS Hurt SL Tumescheit C Firth AE Garcia-Rejon JE et al Detection of novel and recognized RNA viruses in mosquitoes from the Yucatan Peninsula of Mexico using metagenomics and characterization of their in vitro host ranges J Gen Virol 2018 99 1729 1738 30412047
16 Farfán-Ale JA Loroño-Pino MA García-Rejón JE Hovav E Powers AM Lin M et al Detection of RNA from a novel West Nile-like virus and high prevalence of an insect-specific Flavivirus in mosquitoes in the Yucatan Peninsula of Mexico Vector-Borne Zoonotic Dis 2009 80 85 95
17 Farfan-Ale JA Lorono-Pino MA Garcia-Rejon JE Soto V Lin M Staley M et al Detection of flaviviruses and orthobunyaviruses in mosquitoes in the Yucatan Peninsula of Mexico in 2008 Vector-Borne Zoonotic Dis 2010 10 777 783 20370430
18 Newman CM Cerutti F Anderson TK Hamer GL Walker ED Kitron UD et al Culex flavivirus and West Nile virus mosquito coinfection and positive ecological association in Chicago, United States Vector Borne Zoonotic Dis 2011 11 1099 1105 21254845
19 Goenaga S Kenney JL Duggal NK Delorey M Ebel GD Zhang B et al Potential for co-infection of a mosquito-specific Flavivirus, Nhumirim virus, to Block West Nile Virus transmission in mosquitoes Viruses 2015 7 5801 5812 26569286
20 Romo H Kenney JL Blitvich BJ Brault AC Restriction of Zika virus infection and transmission in Aedes aegypti mediated by an insect-specific flavivirus Emerg Microbes Infect 2018 7 181
21 Cigarroa-Toledo N Jiménez-Coello M García-Rejón JE Chan-Pérez JI Acosta-Viana KY Ortega-Pacheco A et al Impacto indirecto de COVID-19 en las tasas de incidencia de enfermedades transmitidas por vector en México Gac Med Mex 2022 158 359 364
22 Baak-Baak CM Cigarroa-Toledo N Arana-Guardia R Chim WA Orilla JA Machain-Williams C et al Mosquito fauna associated with Aedes aegypti (Diptera: Culicidae) in Yucatán State of Southeastern México, and checklist with new records Florida Entomol 2016 99 703 709
23 Garcia-Rejon JE Loroño-Pino MA Farfán-Ale JA Flores-Flores LF López-Uribe MP Najera-Vazquez MR et al Mosquito infestation and dengue virus infection in Aedes aegypti females in schools in Merida, Mexico Am J Trop Med Hyg 2011 84 489 496 21363990
24 Garcia-Rejon JE Ulloa-Garcia A Cigarroa-Toledo N Pech-May A Machain-Williams C Cetina-Trejo RC et al Study of Aedes aegypti population with emphasis on the gonotrophic cycle length and identification of arboviruses: implications for vector management in cemeteries Rev Inst Med Trop Sao Paulo 2018 60 e44 30133604
25 Garcia-Rejon J Loroño-Pino MA Farfan-Ale JA Flores-Flores L Rosado-Paredes ED Rivero-Cardenas N et al Dengue virus-infected Aedes aegypti in the home environment Am J Trop Med Hyg 2008 79 940 950 19052309
26 Carpenter SJ La Casse WJ Mosquitoes of North America (North of Mexico) Berkeley University of California Press 1974
27 Kumar S Stecher G Tamura K MEGA7: Molecular Evolutionary Genetics Analysis version 7.0 for bigger datasets Mol Biol Evol 2016 33 1870 1874 27004904
28 Tamura K Stecher G Kumar S MEGA11: Molecular Evolutionary Genetics Analysis version 11 Mol Biol Evol 2021 38 3022 7 33892491
29 Cabral GB Ferreira JL Souza RP Cunha MS Luchs A Figueiredo CA et al Simple protocol for population (Sanger) sequencing for Zika virus genomic regions Mem Inst Oswaldo Cruz 2018 113 38 34 29185594
30 Shen S Shi J Wang J Tang S Wang H Hu Z et al Phylogenetic analysis revealed the central roles of two African countries in the evolution and worldwide spread of Zika virus Virol Sin 2016 31 118 130 27129451
31 Huang Y Zhang H Li X Zhao L Cai D Wang S et al In vitro and in vivo characterization of a new strain of mosquito Flavivirus derived from culicoides Viruses 2022 14 1298 35746769
32 Rizzo F Cerutti F Ballardini M Mosca A Vitale N Radaelli MC et al Molecular characterization of flaviviruses from field-collected mosquitoes in northwestern Italy, 2011-2012 Parasit Vectors 2014 7 395
33 Tamura K Peterson D Peterson N Stecher G Nei M Kumar S MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods Mol Biol Evol 2011 28 2731 2739 21546353
34 Correa-Morales F González-Acosta C Mejía-Zúñiga D Huerta H Pérez-Rentería C Vazquez-Pichardo M et al Surveillance for Zika in Mexico: naturally infected mosquitoes in urban and semi-urban areas Pathog Glob Health 2019 113 309 314 31902313
35 Nunez-Avellaneda D Cetina-Trejo RC Zamudio-Moreno E Baak-Baak C Cigarroa-Toledo N Reyes-Solis G et al Evidence of Zika virus infection in pigs and mosquitoes, Mexico Emerg Infect Dis J 2021 27 574 577
36 Lopez-Apodaca LI Zarza H Zamudio-Moreno E Nuñez-Avellaneda D Baak-Baak CM Reyes-Solis GC et al Molecular survey of Zika virus in the animal-human interface in traditional farming Front Vet Sci 2022 9 1057686
37 Cigarroa-Toledo N Talavera-Aguilar LG Baak-Baak CM García-Rejón JE Hernandez-Betancourt S Blitvich BJ et al Serologic evidence of Flavivirus infections in peridomestic rodents in merida, Mexico J Wildl Dis 2016 52 168 172 26540177
38 Moureau G Ninove L Izri A Cook S De Lamballerie X Charrel RN Flavivirus RNA in phlebotomine sandflies Vector Borne Zoonotic Dis 2010 10 195 197 19492949
39 Fang Y Tambo E Xue JB Zhang Y Zhou XN Khater EI Detection of DENV-2 and insect-specific flaviviruses in mosquitoes collected From Jeddah, Saudi Arabia Front Cell Infect Microbiol 2021 11 626368
40 Hoshino K Isawa H Tsuda Y Sawabe K Kobayashi M Isolation and characterization of a new insect flavivirus from Aedes albopictus and Aedes flavopictus mosquitoes in Japan Virology 2009 391 119 129 19580982
