Arboviruses, arthropod-borne viruses, are of great epidemiological and socioeconomic importance worldwide due to their involvement in febrile and neurological diseases that affect a significant part of the global population. Dengue virus (DENV) stands out for causing the most prevalent infection, especially in tropical and subtropical countries. In recent decades, its incidence has grown exponentially worldwide, with more than 4 billion people at risk of infection in these regions. Therefore, epidemiological surveillance and vector control play a fundamental role in the effective control of these diseases. The efficient spread of arboviruses, such as DENV, is facilitated primarily by the Aedes aegypti mosquito, widely recognized as a highly competent vector. Its marked anthropophilic behavior and close association with urban and peridomestic environments contribute significantly to this ability. The biological characteristics of Aedes mosquitoes promote their rapid adaptation to new habitats and their extensive geographic dispersal, often related to human interventions such as unplanned urbanization, global trade and climate change. As a result, this vector species has become a significant threat, compromising the effectiveness of preventive measures and the operational implementation of control programs, as well as the management of associated disease outbreaks. Likewise, other mosquitoes of the family Culicidae have also been implicated in the transmission of pathogenic viruses in both urban and rural cycles. However, in rural areas at risk of zoonotic disease transmission to humans, endemic vectors continue to be studied. Advances in next-generation sequencing (NGS) technologies have revolutionized entomovirological surveillance strategies by enabling the detection of (re)emerging viruses and the discovery of new viral sequences. These advances have led to the identification of a representative group of viruses known as insect-specific viruses (ISVs), which naturally infect arthropods but cannot replicate in vertebrate cells or infect humans. Some of these ISVs have been observed to form a stable, species-specific core virome for each mosquito species, suggesting their ability to influence host biology. Specifically, these ISVs can modulate viral agent transmission and vector competence, making them a crucial component for the development of new arbovirus control strategies. Colombia, a tropical country, offers optimal conditions for the development and adaptation of various mosquito species, which creates an environment conducive to the transmission of epidemiologically important arboviruses. Cases of arbovirus-borne diseases, especially dengue, have been prevalent in the country due to their high estimated incidence. Since 2016, co-circulation of other arboviruses such as chikungunya (CHIKV) and Zika (ZIKV) has been observed. This phenomenon is attributed to Colombia's geographical characteristics, favorable conditions for viral transmission and the widespread presence of the vector in most municipalities of the country. In this context, molecular epidemiology studies of insect-borne arboviruses are essential for understanding viral dynamics and anticipating possible epidemic outbreaks. However, most of these However, most of these investigations have focused on the analysis of samples from infected patients, and few studies have been conducted on vector samples. This limitation has possibly hindered a comprehensive and efficient control of this type of disease. Currently, one of the main challenges in the study of arboviruses is to understand the diversity of arboviruses during their life cycle in the vector agent. In addition, it is crucial to describe the viral communities associated with mosquito vectors, which could provide a more precise understanding of vector competence. This approach would provide a comprehensive understanding of arbovirus transmission dynamics, contribute to high-resolution epidemiological surveillance, and facilitate the design of efficient control and prevention strategies. Therefore, the general objective of this doctoral thesis was to Characterize arboviruses of public health importance and the virome in Colombian mosquitoes using a metagenomic approach, which was broken down into three specific objectives: 1. To analyze the frequency of arbovirus infection and molecular identification of the species of Aedes mosquitoes in different departments of Colombia. 2. To characterize the Aedes aegypti virome with natural infection by dengue virus (DENV) through metagenomic analysis. To describe the transmission dynamics and viral composition in mosquito populations (Diptera: Culicidae) in strategic rural ecosystems. Each of these objectives is associated with a chapter within this research work. The first chapter shows that the use of molecular techniques in entomovirological surveillance is a powerful tool for the early detection of arboviruses, thus facilitating decision-making in control and prevention strategies. During the period 2020-2021, an analysis was conducted on Ae. aegypti mosquitoes collected in various regions of Colombia, where a high frequency of infection and natural co-infection by dengue virus serotypes (DENV-1, DENV-2 and DENV-3) was observed. In turn, the presence of Chikungunya virus (CHIKV) was less frequent and Zika virus (ZIKV) was not detected. These results reaffirm the predominant role of Ae. aegypti in dengue transmission in Colombia and point to a decrease in the circulation of other arboviruses following significant outbreaks in 2015 and 2016. Additionally, analysis of the genetic diversity of Ae. aegypti, performed by sequencing of the COI molecular marker, revealed the predominance of lineage I in the country. This lineage is widely distributed in the Americas and is associated with areas of high dengue incidence. The findings of this study underscore the importance of detecting arbovirus infections and co-infections in Aedes mosquitoes, as well as the identification of vector species and the analysis of their population dynamics using molecular techniques. This information is crucial for establishing early warning systems that support the timely implementation of intervention measures in the event of arbovirus emergence or reemergence. In the second chapter, virome characterization of Aedes aegypti mosquitoes was carried out using samples that were previously identified in Chapter 1 as naturally infected by DENV (serotypes DENV-1 and DENV-2), as well as samples from mosquitoes that tested negative for viral infection (DENV-negative). For this, a viral enrichment method followed by metagenomic sequencing using Oxford Nanopore Technologies (ONT) technology was employed. This approach allowed identification of the predominant ISVs in these populations. Significant variation in the abundance of certain species and families of ISVs was observed in response to natural infection by the different DENV serotypes. These variations may be attributed to the complex mechanisms of interaction between ISVs and arboviruses within the mosquito vector. In addition, a differential presence of Phasi-Charoen-like phasivirus (PCLV) was found in DENV-infected samples. This ISV has been previously described as part of the core virome in Aedes mosquitoes. Phylogenetic analyses of PCLV revealed associations with sequences from several regions worldwide, especially highlighting similarities with sequences from Brazil and Guadeloupe, suggesting a shared evolutionary relationship with these hosts. The results of this study not only improve our understanding of viral diversity in mosquito vectors, but also provide crucial information on possible interactions between ISVs and arboviruses. This information is fundamental for the design of effective control and prevention strategies for Ae. aegypti-borne diseases. The third chapter initially investigated the frequency of Flavivirus infection and feeding preferences in mosquito species inhabiting a rural savanna ecosystem in the Orinoquia of Colombia. A viral metagenomic analysis was then performed focusing on the mosquito species of greatest abundance and public health relevance, including genera such as Ochlerotatus, Culex, Limatus, Mansonia, Psorophora and Sabethes. The region studied is characterized by minimal human interference and limited anthropogenic disturbance. The results showed the presence of West Nile virus (WNV), a pathogen of medical importance, in mosquitoes of the Culex browni species. This finding highlights the potential impact of human activities on ecosystems sensitive to anthropogenic interventions. In addition, feeding patterns demonstrated that most mosquito species exhibit generalist behavior. Similarly, the presence of several insect-specific viruses (ISVs) and a common group of these viruses was identified in all mosquito species analyzed. This suggests that environmental and habitat factors could influence the composition of viral communities in these insects, especially among species cohabiting local ecosystems with similar ecological characteristics. The results obtained highlight the importance of maintaining and expanding entomovirological surveillance studies, especially in areas with minimal human intervention. These findings support the high probability of potentially pathogenic viruses spreading among human and animal populations in contexts of deforestation and environmental change. Furthermore, these findings highlight the need to better understand the viral composition of mosquitoes in diverse ecosystems. Studying the influence of local rural habitat conditions on the structure and composition of mosquito vector virome could provide a stronger basis for the development of effective vector control strategies and the prevention of future vector-borne disease outbreaks. In summary, this doctoral thesis expands the knowledge on surveillance and detection of arboviruses in Aedes mosquitoes in urban areas and in other mosquito vectors in rural areas of Colombia, being the first study to use ONT technology in insects and viruses in the country. In addition, this research delves into the importance of insect-specific viruses (ISVs) in vector biology and dynamics. Using molecular tools, NGS sequencing and metagenomic analysis, the composition of the Ae. aegypti virome was described, highlighting how the presence of DENV-1 and DENV-2 serotypes influences the structure of ISVs in this mosquito. Likewise, the feeding habits and viral communities in mosquitoes of the family Culicidae in strategic rural regions were characterized, revealing the dynamics of flaviviruses and shared viral species -ISVs- in mosquitoes of eastern Colombia. This study underscores the importance of integrating entomovirological surveillance into public health systems to better understand arbovirus transmission and its potential impact on human and animal health. Likewise, this research establishes a solid foundation for future explorations of mosquito-host-virus interactions in their natural habitats, as well as their implications for vectorial competence.
