Experimental Viral Infection in Adult Mosquitoes by Oral Feeding and Microinjection

Mosquito-borne viruses (MBVs), a heterogeneous group of RNA viruses, can persist in mosquito vectors and subsequently spread to vertebrate hosts¹. The clinically important MBVs are majorly distributed in four virus families, namely Flaviviridae, Togaviridae, Reoviridae, and Peribunyavividae^2,3. In recent decades, these viruses have been reported all across the globe, causing public health issues. As one of the most well-known MBVs, Dengue virus (DENV) has become the most prevalent emerging or re-emerging arbovirus in over 100 countries during the last 20 years⁴. Since the discovery of Zika virus (ZIKV) inland, almost all tropical and subtropical countries and territories of the continent have reported human ZIKV infections⁵. In order to assess the risk of virus transmission, numerous studies in recent years have focused on mosquito vector competence for these viruses^6,7. As a result, it is critical to effectively prevent and control vector-borne diseases.

Aedes aegypti (Ae. aegypti), one of the most easily reared mosquitoes in the laboratory, is an important vector of DENV, ZIKV, Chikungunya virus (CHIKV), and yellow fever virus (YFV)⁸. For a long time, Ae. aegypti was solely found in the African continent and in Southeast Asia, but in recent years it has colonized nearly all continents⁹. Furthermore, the global abundance of Ae. aegypti has been continuously growing, with an estimated 20% increase by the end of the century¹⁰. From 2004 to 2009 in China, there was an evident increase in Ae. aegypti vector competence for DENV due to higher day-to-day temperatures¹¹. The status of Ae. aegypti as the pathogenic vector has significantly risen in China. Consequently, to address these challenges, it is necessary to investigate the vector competence of Ae. aegypti‘s to transmit viruses.

As a haematophagous arthropod, the female mosquito pierces the skin of a vertebrate host and feeds on the blood. Mosquitoes do occasionally acquire viruses from virus-infected hosts and then transfer the viruses to a new host. As such, to determine vector competence, mosquitoes are fed an artificial bloodmeal containing arboviruses through a feeding system in the laboratory setting¹². Individual mosquitoes are separated into heads, bodies, and saliva secretions several days after infection. To measure virus infection, dissemination, and transmission rates, virus titers have been detected by quantitative reverse-transcription PCR (qRT-PCR) or plaque assay. However, not all mosquitoes develop midgut infections and the capacity to transfer a virus to the next host following blood feeding. It is linked with mosquitoes’ physiological barriers, which prevent pathogens from penetrating the body and play a vital role in their innate immunity¹³. The midgut barriers, particularly the midgut infection barrier (MIB) and midgut escape barrier (MEB), influence whether the virus could infect the vector systemically and the efficiency with which it spreads. It obstructs the analysis of other tissues’ infection, such as salivary glands which also exhibit salivary gland infection and escape barriers^13,14. To better characterize the infection of midguts and salivary glands in the vector, a detailed protocol for oral feeding and intrathoracic inoculation of arbovirus in Ae. aegypti is presented herein. This protocol might be applied to additional arbovirus infections in a variety of mosquito vectors, such as DENV and ZIKV infection in Aedes spp., and could prove to be a practicable procedure.