Nikos Vasilakis Lab

Our focus is to understand the mechanisms that allow zoonotic, sylvatic arthropod-borne viruses to jump species boundaries and cause human disease. DENV are the most important arboviral pathogens of humans. An estimated 400 million infections occur every year in tropical and subtropical regions, where nearly a third of the global population is at risk. DENV comprise of four epidemiologically similar yet phylogenetic distinct viruses. Phylogenetic studies of sylvatic DENV suggest that these are these represent the ancestral population from which human strains have emerged. Similarly my recent research has also shown that sylvatic DENV has the capability to cause human infection and disease, and can readily reemerge in the future unless effective vaccination is developed and sustained.

Critically, however, although understanding the ecology and epidemiology of sylvatic DENV is essential to understanding and predicting emergence, it has been largely ignored to date. Currently, little is known of the distribution and ecology of sylvatic DENV in Asia, and human infections have been both rarely and poorly characterized. In addition, the ecological contact between sylvatic cycles and human populations is largely unexplored, and the range of human disease caused by sylvatic DENV strains is unknown. Through preliminary fieldwork and clinical surveillance conducted in Sarawak, we have recently discovered that sylvatic DENV currently circulates in Sarawak and spills over into humans to cause disease, including hemorrhagic fever. Moreover one sylvatic DENV strain we isolated from such a spillover event is so highly divergent from the four currently recognized serotypes that it represents a fifth serotype (DENV-5). The identification of DENV-5 and detection of additional sylvatic lineages could have profound implications for development of vaccines and diagnostics, and underscores the need for a comprehensive investigation of sylvatic DENV diversity, transmission and spillover in Southeast Asia. Sylvatic DENV cycles in Borneo contain novel DENV variants that could potentially spill over into humans, thereby undermining arbovirus control efforts and potentially launching these new viruses into global transmission.

However the spillover frequency and disease incidence in humans exposed to the sylvatic cycle is not known. Identifying the ecological and genetic drivers of DENV emergence from the sylvatic transmission cycle in its geographical “cradle of emergence” is of major importance for public health. Therefore, advances in understanding DENV emergence and predicting locations and times with the greatest potential for emergence will require comprehensive, prospective epidemiological and ecological studies in enzootic locations such as Asia. Such studies may ultimately lead to the identification of additional distinct lineages of DENV that have not yet caused disease in humans. The urgent need for effective tetravalent DENV vaccination heightens the need for understanding the ease with which the sylvatic strains can reemerge to reinitiate urban transmission cycles

Since June 2017, we have also established another field site in the Brazilian Amazon near the city of Manaus, where the main focus is to investigate the potential of Zika establishing an enzootic transmission cycle in the Americas.

The development of new sequencing technologies, for example next generation sequencing (NGS), has resulted in an unprecedented bonanza of virus discovery and an explosion of taxonomic dilemmas. Classic serologic methods of virus isolation and characterization are limited in identifying novel and/or host-restricted viruses. Leveraging the vast repertoire of uncharacterized viruses in the World Reference Collection of Emerging Viruses and Arboviruses (WRCEVA) and existing collaborations in Southeast Asia, Latin America and within our national borders over the past 6 years the laboratory was instrumental in the discovery of several new taxa of viruses, including the negeviruses, the mesoniviruses, the identification and classification of new rhabdovirus genera as well as several viruses of restricted host range. Furthermore, using electron microscopy as well as serologic and phylogenetic methods, their their taxonomic assignments were assessed and determined. Collectively our research has led to important advances in our understanding of the vast virus diversity of naturally occurring viral symbionts of mosquitoes and how these may potentially contribute in reducing their vector competence for selected arboviral pathogens.

Arboviruses (e.g. WNV, DENV, JEV, YFV, etc.) and the diseases they cause are major public health problems in regions of the world where they occur. The two most used methods to control these diseases are vaccination and application of pesticides. Vaccines, if available, are effective in controlling human disease, but do not eliminate the pathogenic arboviruses because most arboviruses are maintained in zoonotic cycles independent of humans. The application of pesticides is designed to reduce the density of a vector population, diminishing the level of virus transmission and indirectly reducing the incidence of infection and illness. But like vaccines, pesticides must be applied indefinitely, or the vectors and their associated diseases will reemerge. Heavy application of pesticides can be beneficial during epidemics but is not sustainable long-term because of the cost, public opposition, environmental toxicity and the development of resistance by the target vectors.

Recently, a third control option has been considered; manipulation of the insect microbiome to reduce the vector competence of mosquitoes for selected arbovirus pathogens. During the past 4 years the laboratory has identified and characterized two new taxa of mosquito specific viruses (MSVs), as well as several others within established taxa that include arboviruses, including flaviviruses. Mosquito-specific flaviviruses (MSFs) belong in two distinct clades within the genus flavivirus. One is phylogenetically distinct from the main flavivirus group that includes vertebrate arboviral pathogens (tick- and mosquito-borne). This clade includes MSFs such as cell fusing agent (CFAV), Culex flavivirus (CxFV), Aedes flavivirus (AeFV), Kamiti River (KRV), Quang Binh (QBV), and Nakiwogo (NAKV) virus. The second clade falls within the mosquito-borne clade of flavivirus pathogens and is closely related to medically important viruses such as WNV, DENV and YFV. This MSV clade includes Aripo (ARPV), Nounane (NANV), Nhumirim (NHUV), Donggang (DGV), Long Pine Key (LPKV), marisma (MMV) and other viruses.

Using LPKV and MMV as models the laboratory is currently exploring methods to create a population of mosquitoes that is infected with LKPV with reduced capacity to transmit human pathogenic arboviruses. The factors that determine the narrow host range of mosquito-specific viruses (using LKPV and MMV as models) are poorly understood and mechanisms such as attachment and entry, incompatibility with host cell factors, or temperature sensitivity have been suggested. Gaining insights into the elements that determine this host range will improve our understanding of MSF-vector interactions and evolution, and this knowledge can be leveraged to manipulate specific natural viral symbionts in mosquitoes, such as LPKV, to reduce their ability to transmit arboviral pathogens of public health importance.

While the molecular interactions between Aedes and dengue virus (Flaviviridae) have been studied and characterized to a significant detail over the past decade, the molecular interactions between Chikungunya virus (CHIKV) and yellow fever virus (YFV) with this vector remain largely unknown, despite the public health importance. The mosquito’s innate immune system is of particular interest since it has been shown to modulate mosquito resistance to virus infection and could thereby be used to develop novel disease control strategies.

Recently it has for the first time shown that the JAK-STAT immune pathway can be engineered to render Ae. aegypti more resistant to DENV. However, past studies to evaluate and develop dengue control strategies based on blocking the virus in the mosquito have largely ignored the fact that CHIKV and YFV are frequently transmitted by the same vector in sympatry with DENV, and may thus not affect the transmission of the other viruses. Our laboratory in partnership with Dr. Dimopoulos of John Hopkins University has developed a research program, which is designed to close this gap by comparatively addressing the molecular responses of Ae. aegypti to infection with DENV, CHIKV, and YFV, as well as the more specialized function of the mosquito’s innate immune pathways (main focus on Toll, IMD, and JAK/STAT) in controlling virus infection.

This project will explore transgenic strategies for developing mosquitoes that are resistant multiple viruses. The outcomes of this project will provide further insights on how mosquito immune response to the three viruses relate and which immune pathway and infection-responsive genes are likely to control infection.