Abstract
Fourteen pools of Aedes aegypti larvae collected within the urban area of Culiacán, Sinaloa, were analyzed by RT-PCR. The results demonstrate, for the first time, the vertical infection of serotype-2 dengue virus (DENV-2) in Sinaloa, Mexico, suggesting that Ae. aegypti acts as a natural reservoir of DENV-2 in this region.
Introduction
I
Vertical infection of DENV-2 in Aedes aegypti.
Materials and Methods
Three hundred eight Ae. aegypti larvae were collected from September 2015 to December 2016 in Culiacán, Sinaloa, and identified according to the criteria of Farajollahi and Price (2013). RNA was extracted from 14 pools of larvae (15–28) (SV Total RNA Isolation System Kit; Promega, USA) and a reverse transcription reaction was performed afterward (GoScript Reverse Transcription System; Promega). A PCR was performed using D1 and D2 consensus primers that amplifies a fragment of 511-bp for DENV (Lanciotti et al. 1992) and 3 μL of complementary DNA under the following conditions: 94°C for 4 min, 35 cycles (94°C for 1 min, 55°C for 1 min, and 72°C for 1 min), and 72°C for 5 min. A second amplification (seminested multiplex PCR) was then performed using 1 μL from the initial amplification reaction and D1 primer with the DENV type-specific primers TS1–TS4 for DENV 1–4 (Lanciotti et al. 1992), under the following conditions: 95°C for 1 min, 25 cycles (95°C for 30 s, 60°C for 30 s, and 72°C for 30 s), and 71°C for 2 min. The amplicon was purified (SV Gel and PCR Clean-Up System; Promega) and sequenced in Macrogen™ (Korea). The sequences were analyzed using Blast and CLC Sequence Viewer 7. Minimum infection rate (MIR) for DENV as MIR = (PCR positive pools)/(total number of individuals tested) (1000) was calculated.
Results
Two of the pools, collected during a dengue outbreak in November 2015, tested positive (fragment of 119-bp) for DENV-2 (Fig. 1B). The MIR was 6.49. The sequences obtained showed 98% similarity with the reported sequences of DENV-2 outbreak (GenBank acc. nos. KU094070.1, KM587709.1, KJ189370.1, and KJ189308.1) (Fig. 1C).
Discussion
VI may be a determinant of the intensity and risk of DENV transmission in areas where dengue cases occur during nonepidemic periods (Joshi et al. 2002). This study reports the presence of DENV-2 in Ae. aegypti larvae collected in Culiacan during a dengue outbreak, demonstrating, for the first time, the VI of the virus in the state of Sinaloa. The MIR reported in this study is consistent with the results found in Oaxaca, Mexico (Günther et al. 2007). Along with other reports for Mexico (Ibáñez-Bernal et al. 1997, Sánchez-Rodriguez et al. 2004, Günther et al. 2007), these results suggests that Aedes acts as a natural reservoir of DENV. Further studies are needed on VI to gather the necessary information to implement efficient vector control measures to reduce the incidence of the disease.
Conclusions
This study demonstrates the VI of DENV-2 in Ae. aegypti in Culiacan Sinaloa, Mexico, which suggests that the mosquito may be a natural reservoir of DENV.
Footnotes
Acknowledgments
The authors thank INAPI and PROFAPI-UAS for the support provided.
Author Disclosure Statement
No competing financial interests exist.