Experimental evaluation of sand fly collection and storage methods for the isolation and molecular detection of Phlebotomus-borne viruses

Maria Elena Remoli, Gioia Bongiorno, Claudia Fortuna, Antonella Marchi, Riccardo Bianchi, Cristina Khoury, Maria Grazia Ciufolini, Marina Gramiccia

Several viruses have been recently isolated from Mediterranean phlebotomine sand flies; some are known to cause human disease while some are new to science. To monitor the Phlebotomus-borne viruses spreading, field studies are in progress using different sand fly collection and storage methods. Two main sampling techniques consist of CDC light traps, an attraction method allowing collection of live insects in which the virus is presumed to be fairly preserved, and sticky traps, an interception method suitable to collect dead specimens in high numbers, with a risk for virus viability or integrity. Sand flies storage requires a "deep cold chain" or specimen preservation in ethanol. In the present study the influence of sand fly collection and storage methods on viral isolation and RNA detection performances was evaluated experimentally. Specimens of laboratory-reared Phlebotomus perniciosus were artificially fed with blood containing Toscana virus (family Bunyaviridae, genus Phlebovirus). Various collection and storage conditions of blood-fed females were evaluated to mimic field procedures using single and pool samples. Isolation on VERO cell cultures, quantitative Real time-Retro-transcriptase (RT)-PCR and Nested-RT-PCR were performed according to techniques commonly used in surveillance studies. Live engorged sand flies stored immediately at -80 °C were the most suitable sample for phlebovirus identification by both virus isolation and RNA detection. The viral isolation rate remained very high (26/28) for single dead engorged females frozen after 1 day, while it was moderate (10/30) for specimens collected by sticky traps maintained up to 3 days at room temperature and then stored frozen without ethanol. Opposed to viral isolation, molecular RNA detection kept very high on dead sand flies collected by sticky traps when left at room temperature up to 6 days post blood meal and then stored frozen in presence (88/95) or absence (87/88) of ethanol. Data were confirmed using sand fly pools. While the collection and storage methods investigated had not much impact on the ability to detect viral RNA by molecular methods, they affected the capacity to recover viable viruses. Consequently, sand fly collection and handling procedures should be established in advance depending on the goal of the surveillance studies.