Novel Genetic and Molecular Tools for the Investigation and Control of Dengue Virus Transmission by Mosquitoes

Curr Trop Med Rep (2014) 1:21–31 DOI 10.1007/s40475-013-0007-2 VIRAL TROPICAL MEDICINE (CM BEAUMIER, SECTION EDITOR) Novel Genetic and Molecular Tools for the Investigation and Control of Dengue Virus Transmission by Mosquitoes Alexander W. E. Franz & Rollie J. Clem & A. Lorena Passarelli Published online: 10 January 2014 # Springer International Publishing AG 2014 Abstract Aedes aegypti is the principal vector of dengue virus (DENV) throughout the tropical world. This anthropophilic mosquito species needs to be persistently infected with DENV before it can transmit the virus through its saliva to a new vertebrate host. In the mosquito, DENV is confronted with several innate immune pathways, among which RNA interference is considered the most important. The Ae. aegypti genome project opened the doors for advanced molecular studies on pathogen–vector interactions, including genetic manipulation of the vector for basic research and vector control purposes. Thus, Ae. aegypti has become the primary model for studying vector competence for arboviruses at the molecular level. Here, we present recent findings regarding DENV–mosquito interactions, emphasizing how innate immune responses modulate DENV infections in Ae. aegypti. We also describe the latest advancements in genetic manipulation of Ae. aegypti and discuss how this technology can be used to investigate vector transmission of DENV at the molecular level and to control transmission of the virus in the field. A. W. E. Franz (*) Department of Veterinary Pathobiology, 303 Connaway Hall, College of Veterinary Medicine, University of Missouri, Columbia, MO 65211, USA e-mail: R. J. Clem : A. L. Passarelli Molecular, Cellular, and Developmental Biology Program, Division of Biology, 116 Ackert Hall, Kansas State University, Manhattan, KS 66506, USA R. J. Clem e-mail: A. L. Passarelli e-mail: Keywords Dengue virus . Mosquito . Aedes aegypti . Aedes albopictus . Virus transmission . Innate immunity . RNA interference . Toll . JAK-STAT . Apoptosis . Transgenesis . Transposon . Site-specific recombination . Promoter . Gene-knockout . Gene expression . Homing endonuclease . TALEN . Zinc finger nuclease . Wolbachia . RIDL . Population replacement . Effector gene . Viral tropical medicine Introduction The global epidemiology of dengue virus (Flaviviridae; Flavivirus; dengue virus 1-4; [DENV1-4]) depends on the presence of two mosquito vectors, Aedes (Stegomyia) aegypti (L.) and Aedes (Stegomyia) albopictus (Skuse). Ae. aegypti is the principal DENV vector in urban environments of tropical countries [1]. This mosquito species is of African origin and breeds in tropical regions of Africa, Asia, Australia, SouthPacific, the Middle East, and the Americas [2]. Ae. aegypti exemplifies a peridomestic, anthropophilic day biter. In contrast, Ae. albopictus is a zoophilic, catholic biter, which easily adapts to peridomestic environments in temperate regions [3, 4]. During the last few decades, Ae. albopictus has undergone a dramatic expansion in its geographic distribution [5•]. However, due to lower viral infection and transmission rates, Ae. albopictus is considered to be a less important vector for DENV than Ae. aegypti. The DENV disease cycle between the mosquito vector and the human host requires persistent infection of the mosquito [6]. DENV typically causes disease symptoms in the human host, but there is no apparent pathology associated with the infection of the mosquito [7•]. DENV is acquired by female mosquitoes while they orally ingest a viremic bloodmeal from a human host. A correlation exists between disease severity of the host, high DENV titer in the blood and the level of 22 infection of the mosquito vector [8]. The ingested bloodmeal enters the midgut lumen to be digested, and virions enter midgut epithelial cells and replicate [9]. Virus then spreads cell-to-cell to form infection foci in the midgut epithelium [10]. At 4–7 days post-infection, DENV starts disseminating from the midgut to secondary tissues such as muscle, nerve, fat body, ovary, hemocytes, and eventually salivary glands, which typically become infected between 10 and 14 days post-infectious bloodmeal. Once the salivary glands are infected, DENV is transmitted to a new human host through release of virion-containing mosquito saliva during feeding. The extrinsic incubation period (EIP) is defined as the time period between initial infection of the vector and appearance of virus in the saliva [11]. The EIP can vary according to virus strain, mosquito strain, and virus titer in the mosquito. The midgut and salivary glands constitute physical barriers that DENV needs to overcome before it can be transmitted [12]. A midgut infection barrier (MIB) can prevent the virus from infecting the mosquito midgut, while a midgut escape barrier (MEB) allows DENV to productively infect midgut epithelial cells, but prevents the virus from disseminating from the midgut. The presence of MIB and MEB depend on specific virus strain–mosquito strain combinations [13, 14]. Although higher midgut infection rates have been observed for DENV in Ae. albopictus, dissemination rates were significantly lower in this mosquito species compared to Ae. aegypti [5•]. Vertical transmission of DENV by Ae. aegypti and Ae. albopictus through a transovarial route has been demonstrated in several studies [15, 16]. Transovarial transmission rates (percentage of infected females transmitting virus to their progeny) of up to 13 % for Ae. aegypti and 11–41 % for Ae. albopictus have been reported [17, 18]. Vertical transmission of DENV strongly affects its etiology, because it allows the virus to be maintained among mosquitoes during interepidemic periods, i.e. during dry seasons or winter seasons, when there is only little active horizontal DENV transmission. Molecular Interactions Between DENV and Innate Immune Pathways of Ae. aegypti During infection of a mosquito, DENV is confronted with several innate immune pathways such as RNA interference (RNAi), Toll, and JAK-STAT, which modulate DENV infection. As with MIB and MEB, the effectiveness of these pathways in inhibiting DENV replication likely varies depending on the virus and mosquito strains in question. RNAi RNAi has been considered the most important antiviral innate immune pathway in Ae. aegypti [7•]. The RNAi pathway in mosquitoes follows the same principle as described in great detail for Drosophila [19–21]. The genome of Ae. aegypti Curr Trop Med Rep (2014) 1:21–31 encodes key gene homologs of the three small RNA (miRNA, siRNA, and piRNA) regulatory pathways [22, 23]. Initial studies showed that the siRNA pathway in Ae. aegypti could be triggered to silence DENV replication. Mosquitoes infected with recombinant Sindbis virus (Togaviridae; Alphavirus; [SINV]) expressing ~300 nt anti-sense cDNAs complementary to the genomes of DENV1, 2, 3, or 4 silenced DENV replication in serotype-specific manner [24]. Dicer2 of the siRNAi pathway (...truncated)