Chikungunya virus (CHIKV) is an arthropod-borne virus from the Alphavirus genus that is transmitted to humans by Aedes spp. mosquitoes. First identified in Tanzania in the mid-1950s (Lo et al. 2012), CHIKV is endemic in sub-Saharan Africa, where it is maintained in a sylvatic cycle while causing recurrent epidemics in Asia, where only an urban cycle has been reported (Pialoux et al. 2007). Three different CHIKV genotypes have been identified: West African (WA), East–Central–South African, and Asian (Pialoux et al. 2007, Ng and Hapuarachchi 2010). Evolutionary studies suggest an African origin for CHIKV (Volk et al. 2010), belonging to the WA genotype (Chevillon et al. 2008).

Reemergence of CHIKV infection has been reported in many parts of the world (Rao 1971, Thaikruea et al. 1977): in the Indian Ocean islands and India (Schuffenecker et al. 2006, Vidya et al. 2007), and in European countries such as France, Germany, Italy, Norway, and Switzerland, which have experienced imported cases of CHIKV infection from people returning from endemic areas (WHO 2006, Pialoux et al. 2007, D'Ortenzio et al. 2009).

In December 2013, the World Health Organization reported the 1st local transmission of CHIKV in the Americas with cases of indigenous origin from the Caribbean island of St. Martin (CDC 2014). Since then, about 30 Caribbean countries have reported outbreaks of CHIKV with local transmissions (Arce and Nuñez 2014). In the USA, the 1st local transmission of CHIKV was in Florida (CDC 2014). In Mexico, the 1st case of CHIKV was confirmed in June 2014 (Rivera 2014), which proved to be imported; the 1st local transmission case was reported in September 2014 (Salud en Chiapas 2014).

The present study was carried out in San Marcos (16°38′ and 17°04′N, 99°11′ and 99°38′W), Guerrero, Mexico, with a total population of 12,268 habitants, a temperature range of 22–28°C, and approximately 50.12% RH (INEGI 2012).

The adult mosquitoes were collected with automatic backpacks and larvae by using a standard white enamel dipper attached to a handle of an appropriate length, and widemouthed pipette (eyedropper), in houses with a record of infected persons. Of the total 121 Ae. aegypti L. collected, 23 were males, 16 females, and 82 larvae. Adult mosquitoes were separated by gender, grouped into pools (1–11 specimens/pool), and stored in RNA solution before RNA extraction. The RNA was isolated from mosquitos using TRIZOL reagent according to the manufacturer's instructions (Invitrogen, Carlsbad, CA). The RNA samples were treated with DNase I (Invitrogen) for 10 min at 37°C to remove traces of genomic DNA. The RNA quality and integrity were assessed by Nanodrop (ThermoScientific, Waltham, MA) and electrophoretic methods, respectively.

A total of 2 μg of RNA was used for the reverse transcriptase reaction with a high-capacity cDNA Reverse transcription kit and random primers in 20-μl total volumes (Applied Biosystems, Foster City, CA). Polymerase chain reaction (PCR) amplification reactions were performed using 5 μl of cDNA as a template, 5 μM of each primer (sense [5′-TCACTCCCTGTTGGACTTGATAGA-3′] and antisense [5′-TTGACGAACAGAGTTAGGAACATACC-3′]) and the commercial GoTaq PCR master mix (Promega, Madison, WI), performed in a Biorad (Hercules, CA) T100 Thermal Cycler. The amplification program was used according to Niyas et al. (2010). The amplification products were visualized on 2.0% agarose gel stained with GELred nucleic acid stain 10,000× (Biotium, Hayward, CA) and visualized under ultraviolet light (Fig. 1). Amplified products were sequenced using Big Dye terminator v3.1 cycle sequencing kit (Applied Biosystems, Foster City, CA) and the same PCR primers. The reactions were analyzed in an ABI PRISM 3100 Genetic Analyzer using Sequencing Analysis Software v5.3 (Applied Biosystems). Electropherograms were analyzed using GeneStudio Pro software (GeneStudio, Inc., Suwanee, GA).

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A total of 121 Ae. aegypti samples (82 larvae, 23 males, and 16 females) were analyzed (Table 1). Of 20 pools, 7 tested positive for CHIKV by PCR. The PCR size (126 bp) corresponding to the suspected band (any alternatively spliced variants were detected; Fig. 1) was sequenced and the sequences did not show any nucleotide changes in the comparison with the reference sequence. In the current study, we used the pooled infrate program (Biggerstaff 2017); PooledInfRate: a Microsoft® Excel Add-In to compute prevalence estimates from pooled samples (Centers for Disease Control and Prevention, Fort Collins, CO) was considered for determining minimum infection rate (MIR) value. The MIR values calculated for the Ae. aegypti larval, male, and female pools were 21.50 (low limited [LL] 4.69–upper limited [UL] 83.18), 253.88 (LL 107.69–UL 599.70), and 64.80 (LL 3.92–UL 312.85), respectively.

Table 1. Field-collected mosquito samples (larval, male, and female) and viral tested results by minimum infection rate (MIR).

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This is the 1st field-collected mosquito report of CHIKV determined in larvae and 2nd in male and female samples of Ae. aegypti in Mexico.

Dzul et al. (2015) determined the presence of CHIKV by reverse transcriptase–polymerase chain reaction (RT-PCR) in adult stages of Ae. aegypti in the cities of Acapulco and Zihuatanejo, Guerrero, Mexico. In a total of 403 male mosquito pools, 2 were positive for CHIKV; and of 492 female mosquito pools, 16 were positive. For the city of Zihuatanejo, 10 pools of males were not positive; while of 21 female pools analyzed, 2 were positive. Unlike Dzul et al. (2015), we report positive CHIKV in females, males, and larvae. Agarwal et al. (2014) also determined (experimentally) vertical transmission of CHIKV in Ae. aegypti larvae (8 out of 30 pools positive) and adults (14 out of 33 pools positive). Meanwhile, Dottori et al. (2011) determined vertical transmission of CHIKV in 113 females (0.88%) of Aedes albopictus (Skuse) obtained from females, CHIKV-infected experimentally. In Orissa, India, Biswadeep et al. (2012) determined CHIKV in wild females of Ae. albopictus, but not in larvae and males; and wild larvae and adults of Ae. aegypti samples. In Castiglione Ravenna and Cervia, Italy, Bonilauri et al. (2008) determined CHIKV by RT-PCR in field-collected females of Ae. albopictus, whereas Zayeda et al. (2012) reported CHIKV in field-collected females of Ae. aegypti, but not in males, in Al Hodayda, Yemen. The majority of these studies indicate that vertical transmission is possible in Ae. aegypti and Ae. albopictus, but we could conclude that vertical transmission of CHIKV is a rare event.