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The simultaneous transmission of chikungunya virus (CHIKV) and dengue viruses (DENV) has been a major public health concern because of their sympatric distribution and shared mosquito vectors. Groups of Aedes aegypti (L.) and Aedes albopictus (Skuse) were orally infected with 1.5 × 10(5) PFU/ml of CHIKV and 3.2 × 10(6) FFU/ml of DENV-2 simultaneously or separately in inverse orders and evaluated for dissemination and transmission by qRT-PCR. Simultaneous dissemination of both viruses was detected for all groups in Ae. aegypti and Ae. albopictus while cotransmission of CHIKV and DENV-2 only occurred at low rates after sequential but not simultaneous infection.

The Flavivirus genus is in the family Flaviviridae and is comprised of more than 70 viruses. These viruses have a broad geographic range, circulating on every continent except Antarctica. Mosquito-borne flaviviruses, such as yellow fever virus, dengue virus serotypes 1–4, Japanese encephalitis virus, and West Nile virus are responsible for significant human morbidity and mortality in affected regions. This review focuses on what is known about flavivirus-mosquito interactions and presents key data collected from the field and laboratory-based molecular and ultrastructural evaluations.

Arthropod-borne viruses (arboviruses) may infect and replicate in haematophagus insects and arachnids when the arthropods take a bloodmeal from an infected vertebrate. Following an appropriate incubation period, the arthropod may transmit the amplified virus to susceptible vertebrates when the arthropod takes a subsequent bloodmeal. Of more than 500 recognised arboviruses, almost 100 cause a wide variety of illnesses in humans, livestock, and/or wildlife species ranging from subclinical infection to mild febrile disease, haemorrhagic fever, encephalitis or death. Arboviruses have wide geographic distributions which are largely dependent upon their vectors, and different arboviruses can be found on all continents including Antarctica. Since very few specific antiviral therapies or arbovirus vaccines exist, control focuses primarily on vector elimination and education on arthropod avoidance. Arbovirus prevalence has increased dramatically during the past 3 decades, with viral emergence and re-emergence driven by urbanisation, increased international travel and commercial transportation, and expanding vector distribution. Key Concepts: Arboviruses infect and replicate in insects and ticks and may be transmitted during blood feeding to susceptible vertebrates, including humans. ‘Arbovirus’ is an ecological term, and the recognised arboviruses are found in the families Togaviridae, Flaviviridae, Bunyaviridae, Orthomyxoviridae, Rhabdoviridae, Reoviridae, and Asfarviridae. Most arboviruses are single-stranded RNA viruses. Infection with an arbovirus may be asymptomatic or may cause fever, haemorrhage, encephalitis, with or without sequelae and death. Currently licensed human vaccines only exist for yellow fever virus (YFV), Japanese encephalitis virus (JEV) and tick-borne encephalitis virus (TBEV). Arbovirus prevalence in specific regions fluctuates, but emergence and re-emergence has caused an increase in human infections over the past 30 years. Keywords: arboviruses; arthropods; fever; rash; encephalitis; haemorrhagic

Introduction Approximately 100 million confirmed infections and 20,000 deaths are caused by Dengue virus (DENV) outbreaks annually. Global warming and rapid dispersal have resulted in DENV epidemics in formally non-endemic regions. Currently no consistently effective preventive measures for DENV exist, prompting development of transgenic and paratransgenic vector control approaches. Production of transgenic mosquitoes refractory for virus infection and/or transmission is contingent upon defining antiviral genes that have low probability for allowing escape mutations, and are equally effective against multiple serotypes. Previously we demonstrated the effectiveness of an anti-viral group I intron targeting U143 of the DENV genome in mediating trans-splicing and expression of a marker gene with the capsid coding domain. In this report we examine the effectiveness of coupling expression of ΔN Bax to trans-splicing U143 intron activity as a means of suppressing DENV infection of mosquito cells. Results Targeting the conserved DENV circularization sequence (CS) by U143 intron trans-splicing activity appends a 3’ exon RNA encoding ΔN Bax to the capsid coding region of the genomic RNA, resulting in a chimeric protein that induces premature cell death upon infection. TCID50-IFA analyses demonstrate an enhancement of DENV suppression for all DENV serotypes tested over the identical group I intron coupled with the non-apoptotic inducing firefly luciferase as the 3’ exon. These cumulative results confirm the increased effectiveness of this αDENV-U143-ΔN Bax group I intron as a sequence specific antiviral that should be useful for suppression of DENV in transgenic mosquitoes. Annexin V staining, caspase 3 assays, and DNA ladder observations confirm DCA-ΔN Bax fusion protein expression induces apoptotic cell death. Conclusion This report confirms the relative effectiveness of an anti-DENV group I intron coupled to an apoptosis-inducing ΔN Bax 3’ exon that trans-splices conserved sequences of the 5’ CS region of all DENV serotypes and induces apoptotic cell death upon infection. Our results confirm coupling the targeted ribozyme capabilities of the group I intron with the generation of an apoptosis-inducing transcript increases the effectiveness of infection suppression, improving the prospects of this unique approach as a means of inducing transgenic refractoriness in mosquitoes for all serotypes of this important disease.