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CES Medicina

Print version ISSN 0120-8705

CES Med. vol.33 no.3 Medellín Sep./Dec. 2019

http://dx.doi.org/10.21615/cesmedicina.33.3.2 

Investigación original

Natural infection of Aedes aegypti, Ae. albopictus and Culex spp. with Zika virus in Medellin, Colombia

Infección natural de Aedes aegypti, Ae. albopictus y Culex spp. con virus Zika en Medellín, Colombia

Juliana Pérez-Pérez1  , Raúl Alberto Rojo-Ospina2  , Enrique Henao3  , Paola García-Huertas4  , Omar Triana-Chavez5  , Guillermo Rúa-Uribe6 

1 Ingeniera biológica. MSc Biología. Facultad de Medicina. Universidad de Antioquia.

2 Ingeniero sanitario. MSc Salud Pública. Secretaría de Salud de Medellín.

3 Ingeniero sanitario. Especialista en Ingeniería ambiental. Secretaría de Salud de Medellín.

4 Bióloga. PhD en Biología. Universidad de Antioquia.

5 Biólogo. PhD en Biología.

6 Biólogo. PhD. Ciencias

Abstract

Introduction:

The Zika virus has generated serious epidemics in the different countries where it has been reported and Colombia has not been the exception. Although in these epidemics Aedes aegypti traditionally has been the primary vector, other species could also be involved in the transmission.

Methods:

Mosquitoes were captured with entomological aspirators on a monthly ba sis between March and September of 2017, in four houses around each of the 250 entomological surveillance traps installed by the Secretaria de Sa lud de Medellin (Colombia). Additionally, 70 Educational Institutions and 30 Health Centers were visited each month.

Results:

2 504 mosquitoes were captured and grouped into 1045 pools to be analyzed by RT-PCR for the detection of Zika virus. Twenty-six pools of Aedes aegypti, two pools of Ae. albopictus and one for Culex quinquefasciatus were positive for Zika virus.

Conclusion:

The presence of this virus in the three species and the abundance of these species in the different sectors of the city, suggests that the control strategies should be addressed to all species that could be potential vec tors until the vectorial role of Ae. albopictus and Culex quinquefasciatus is completely elucidated in the transmission of the Zika virus.

Key words: Zika, Aedes, Culex; Medellín

Resumen

Introducción:

El virus Zika ha generado serias epidemias en los diferentes países en donde se ha reportado, Colombia no ha sido la excepción. Aunque en estas epidemias el vector presuntamente involucrado ha sido Aedes aegypti, otras especies también podrían estar implicadas en su transmisión.

Meto dología:

en el marco del Programa de vigilancia virológica en mosquitos de la Secretaría de Salud de Medellín (Colombia).

Resultados:

entre marzo y septiembre de 2017 se capturaron 2 504 mosquitos en predios visitados durante los levantamientos de índices entomológicos. El material entomo lógico fue agrupado en 1 045 pooles para ser analizados por RT-PCR para la detección de virus Zika. Resultaron positivos para virus Zika 26 pooles de Aedes aegypti, dos pooles de Ae. albopictus y uno para Culex quinquefasciatus.

Conclusión:

la presencia de este virus en las tres especies y su abundancia en los distintos sectores de la ciudad sugieren que las estrategias de control deban dirigirse a todos los potenciales vectores, hasta tanto no se dilucide comple tamente el papel vectorial de Ae. albopictus y Cx. quinquefasciatus en la transmisión del virus Zika.

Palabras-clave: Zika, Aedes, Culex; Medellín

Introduction

The Zika virus is a Flavivirus that recently caused serious epidemics in Brazil, Colombia and other countries of the continent. Between August 2015 and December 2016, 106.000 Zika cases were notified in Colombia 1. After Brazil, Colombia has been the most-affected country, with the highest number of probable cases reported du ring Zika virus outbreak in South America 2. Although it has been widely reported that approximately 80% of people with the virus infection are asymptomatic, Zika virus infection during pregnancy is a cause of microcephaly and other congenital abnormalities in the developing fetus and newborn and it may also be a trigger for Guillain-Barré syndrome in adults 3,4. In Colombia, 48% of the infants and fetuses with microcephaly reported after epidemic were positive for Zika virus infection 5.

This virus is transmitted to humans mainly by the bite of infected Aedes mosquitoes, but there is also evidence of infection by sexual transmission and congenital cases 6,7.

The first detection of the Zika virus in mosquitoes was in Aedes africanus8. Then, the epidemic in the rural area of the Yap Islands in 2007 was attributed to Ae. hensilli9 and Ae. aegypti was the main vector in the French Polynesia outbreak of 2013 10.

Although Ae. aegypti is considered the most important vector for the Zika virus trans mission to humans, Ae. albopictus was associated as vector in the Gabon outbreak (Central Africa) in 2007 11 and recent studies have found the virus in Culex mos quitoes 12,13.

Despite the natural infection of mosquitoes with Zika virus has been reported, few studies have been carried out 14,15,16,17. The purpose of this study was to surveillance the presence of Zika virus in field-collected mosquitoes Ae. aegypti, Ae. albopictus, and Culex spp during a year after the epidemic in Colombia.

Methods

The study area was the city of Medellín (Antioquia - Colombia) which is the second largest in the country. Mosquitoes were captured with entomological aspirators on a monthly basis between March and September of 2017, in four houses in around each of the 250 entomological surveillance traps installed by the Secretaria de Salud de Medellin. Additionally, 70 Educational Institutions and 30 Health Centers were visited each month. Live mosquitoes were transported to the Medical Entomology Laboratory of the Faculty of Medicine of Universidad de Antioquia for taxonomic identification. Pools were formed containing one to 10 mosquitoes, which were analyzed by reverse transcription polymerase chain reaction (RT-PCR) for the detection of Zika virus.

Ribonucleic acid (RNA) extraction was performed using the commercial kit RNeasy Mini Kit® (Qiagen). Each pool was mechanically macerated following the protocol recommended by Qiagen. One-step RT-PCR was performed with the Luna Universal One-Step RT-qPCR kit ® (Biolabs). Each 10μl reaction contained 1 μL of RNA, 1X enzyme mix, 1X reaction mix and 0.4 μM of each of the primers ZIKF-5’-CCTTGGATTCTTGAACGAGGA-3’ and ZI KR-5’-AGAGCTTCATTCTCCAGATCAA-3’ 18. These primers amplified a specific region of the NS5 gene of the virus of approximately 191 bp.

For the RT-PCR the following thermal profile was used: reverse transcription at 55°C for 10 min, initial denaturation at 95°C for 1 min, followed by 40 cycles at 95°C for 10s, 56°C for 30s and 72°C for 20s. Negative controls for extraction and amplifica tion were included in each reaction, and virus RNA from the supernatant of infected cells was included as a positive control. The controls were processed simultaneously and under the same conditions as the samples. The amplification products were analyzed on 2.5% agarose gels in 0.5X Tris Borate EDTA (TBE), stained with Ethidium Bromide (0.6μg/ml) and visualized under (ultraviolet) UV light.

To confirm the results of the pools, positive samples were randomly selected and se quenced in Macrogen (Seoul, Korea). Forward and reverse sequences were manually edited and aligned using the BioEdit v.7.2.5 program. The sequences obtained were compared with a reference sequence (KY785466.1) from GenBank, which belongs to a Colombian isolate of the Zika virus, and were subsequently compared with the Na tional Center for Biotechnology Information (NCBI) database using the Blastn algori thm to estimate the identity percentages with reported sequences of the Zika virus. This was a descriptive study conducted with full compliance with ethical standards of research. We explained the experimental procedure to the community and ob tained consent from residents to collect mosquitoes. This study was classified as risk-free research according to resolution number 8340 (1993) from the Ministerio de Salud y Protección Social of Colombia.

Results

A total of 2504 mosquitoes were analyzed (Ae. aegypti, Ae. albopictus and Culex spp). A subset of mosquitoes was dissected to separate the abdomen section and the secondary tissues such as the head and thorax to identify the disseminated form of the virus. From the 1 045 pools evaluated, 2.8 % were found to be positive for Zika virus (Table 1).

Table 1 Number of mosquitoes collected and number of analyzed pools for detection of Zika virus 

The sequences obtained presented identity values greater than 96%. The nucleotide sequence of the Cx. quinquefasciatus pool showed high identity percentage with the isolates from Haiti (MF384325.1), Brazil (MF376166.1) and Colombia (MF574587.1) (Figure 1).

The MF384325.1 sequence correspond to isolate from Haiti, MF376166.1 sequence correspond to isolate from Brazil; and MF574587.1 sequence to isolate from Colombia.

Figure 1 Nucleotide sequence alignment of Zika virus found in Cx. quinquefasciatus 

On the other hand, the nucleotide sequences of Ae. albopictus and Ae. aegypti pre sented identity percentages around 98% with isolates from Colombia (MF574587.1, MF574588.1 and KY785466.1) (Figure 2).

The MF574587.1, MF574588.1 and KY785466.1 sequences correspond to isolates from Colombia

Figure 2 Nucleotide sequence alignment of Zika virus found in Ae. albopictus and Ae. Aegypti 

Discussion

This is the first report of natural infection of Zika virus in Aedes and Culex mos quitoes in Colombia. Similar results for Aedes mosquitoes have been registered in Malaysia 14 and recently in Senegal 15, Brazil 17 and Mexico 19. For the case of Cx. quinquefasciatus, there is controversy with the incrimination of this mosquito as Zika vector. Some laboratory research has shown that this species is not com petent to transmit the virus 17,20,21,22, but recent studies have concluded that Cx. quinquefasciatus could have a role in the transmission. In Brazil, the presence of the virus was detected in mosquitoes captured in the areas of incidence of the di sease and in laboratory conditions, artificially-fed Cx. quinquefasciatus mosquitoes were able to replicate the virus in the midgut, salivary glands and saliva 13. These results are similar to those previously reported in China 12.

It is important to consider that, according to the feeding habits of Cx. quinquefasciatus, it is possible that our results are due to recent feeds on viraemic hosts. Therefore, future studies are necessary to determine the vectorial competence of this species in the transmission of the Zika virus.

Regarding the infection with Zika virus in Ae. albopictus, it has been previously demons trated that this species is a competent vector for the virus under laboratory condi tions 23,24, and is considered the main vector in some places like Gabon 11 and Sinagapur 25.

Our results do not allow the incrimination as vector of Ae. albopictus in the transmission of zika. However, finding it naturally infected in head and thorax, highly suggests that it plays a role in the transmission of this arbovirosis. Therefore, the monitoring of this species should be considered in anti-vector programs and subsequent studies should be conducted to determine its vector role in the transmission of Zika and new arboviruses.

Conclusion

The results obtained in this study evidence the importance of the virological survei llance of mosquitoes and the need to incorporate it into public control programs. This also implies a redesign of the surveillance and control strategies for arbovirosis, due to the differences in the bionomies of these three mosquito species. These findings show the need to monitor of virus in mosquitoes in other areas of the country, which allow to determine the vectorial role of these species in Colombia.

Acknowledgements

Many people and Institutions have contributed to this research. We are grateful for the support by COLCIENCIAS (project code: 111574455690. Contract number 634-2017) and the Secretaria de Salud de Medellin for the support and assistance with field work during this project. Also, we recognize the assistance of the Grupo Entomología Médica (GEM) and Grupo de Biología y Control de Enfermedades Infecciosas (BCEI) of the Universidad of Antioquia for the contributions to this work. Finally, and most importantly, we would like to thank the people of Medellín for welcoming us in to their homes to conduct this sampling work.

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Forma de citar: Pérez-Pérez J, Rojo-Ospina RA, Henao E, García-Huertas P, Triana-Chavez O, Rúa-Uribe G. Natural infection of Aedes aegypti, Ae. albopictus and Culex spp. with Zika virus in Medellin, Colombia. Rev CES Med 2019. 33(3): 175-181

Received: March 28, 2018; Revised: June 28, 2019; Accepted: July 05, 2019

Conflict of interest

The authors declare that they have no conflict of interests

Creative Commons License This is an open-access article distributed under the terms of the Creative Commons Attribution License