Susceptibility of Aedes albopictus and Culex quinquefasciatus to Japanese encephalitis virus

Abstract

Background: Japanese encephalitis virus (JEV) is the principal cause of mosquito-borne encephalitis in human populations within Asia. If introduced into new geographic areas, it could have further implications for public and animal health. However, potential mosquito vectors for virus transmission have not been fully investigated. The Asian tiger mosquito, Aedes albopictus, has emerged in Europe and is now expanding its geographical range into more northerly latitudes. Culex quinquefasciatus, although absent from Europe, has been detected in Turkey, a country with territory in Europe, and could act as a vector for JEV in other regions. To assess the risk of these invasive species acting as vectors for JEV and therefore potentially contributing to its geographical expansion, we have investigated the vector competence of Ae. albopictus and Cx. quinquefasciatus.

Methods: Two colonised lines of Ae. albopictus (Italy and Spain) and a line of Cx. quinquefasciatus (Tanzania) were compared for susceptibility to infection by oral feeding with JEV strain SA-14, genotype III at 10⁶ PFU/ml and maintained at 25 °C. Specimens were processed at 7 and 14 days post-inoculation (dpi). Rates of infection, dissemination and transmission were assessed through detection of viral RNA by real-time polymerase chain reaction (RT-PCR) in mosquito body, legs and saliva, respectively, at each time point. Where possible, infection and dissemination were confirmed by immunohistochemical (IHC) detection of the JEV envelope protein.

Results: Aedes albopictus from Italy showed no susceptibility to infection with JEV strain SA-14. Conversely, Ae. albopictus colonised in Spain was susceptible and 100% of infected mosquitoes that were subjected to saliva screening expressed viral RNA at 14 dpi. Culex quinquefasciatus was highly susceptible to infection as early as 7 dpi and 50% of infected mosquitoes that were subjected to saliva screening expressed viral RNA at 14 dpi. Infection and dissemination were confirmed in Cx. quinquefasciatus by IHC detection of JEV envelope protein in both the mid-gut and salivary glands.

Conclusions: Aedes albopictus from two different locations in Europe range from being susceptible to JEV and capable of transmission through to being resistant. Culex quinquefasciatus also appears highly susceptible; therefore, both species could potentially act as vectors for JEV and facilitate the emergence of JEV into new regions.

Keywords: Emerging infectious disease; Invasive mosquitoes; Japanese encephalitis; Mosquito; Vector competency; Zoonosis.

Fig. 1

Boxplot comparing the cycle threshold (ct) values for Ae. albopictus and Cx. quinquefasciatus from RNA extractions of specimens infected with Japanese encephalitis virus and maintained at 25 °C. Culex quinquefasciatus ct values were significantly lower compared to Ae. albopictus, suggesting that quantity of viral RNA was higher in these samples. Significance (P < 0.05) denoted by a double asterisk (**)

Fig. 2

Japanese encephalitis virus infection at 25 °C of posterior midgut epithelial cells in Cx. quinquefasciatus. a Head (H), thorax (T), abdomen (Ab). b Intracytoplasmic immunolabelling in the distal lobes of salivary gland, defined by the presence of secretory masses (SM); intense antigen labelling particularly in the basal region of the epithelium (red arrow). c Antigen labelling in the apical ciliated cells (arrow) and basal epithelial cells (arrowhead) of the posterior midgut; lumen of the midgut (L). Scale bar: 500 µm (a); 20 µm (b, c)