2. Toscana virus experiments

Lyophilized TOSV aliquots (strain MRS2010, lineage B, Genbank ID: KC776214 KC776215 KC776216) were provided by the laboratory UVE (Unité des Virus Emergents, Marseille, France). Vero E6 cells were grown in monolayers in Minimum Essential Medium (MEM, Gibco) complemented with 7% heat-inactivated Fetal Bovin Serum (FBS, Eurobio Scientific), 1% L-glutamine (Gibco) and 1% penicillin-streptomycin (Gibco) at 37°C with 5% CO₂. Stocks of TOSV were obtained by dissolving the lyophilisates in pure water, and used for infecting Vero E6 cells at 0.1 multiplicity of infection (MOI). After five days of post infection, supernatant was collected. TOSV stocks at a concentration of 4.2 x 10⁶ 50% tissue culture infective dose (TCID₅₀/ml), at passage 2, were aliquoted and stored at -80°C.

Experimental infections of females Ph. perniciosus with TOSV were performed in enhanced biosafety level 3 (BSL-3) laboratory (IRD Vectopôle, Montpellier, France). Experimental infections were realized as previously described for Leishmania infection [35] modified for TOSV. Twenty-four hours prior to the experiment, 5 to 9-day-old females from the colony were starved by deprivation of sugar meal. Before infection, approximately 100 females were transferred into a feeding pot and stored in the BSL-3 climate chamber under standard conditions (26 ± 1°C, 80% rh) to acclimatize. Approximately 10% males were added to mate and stimulate females to take blood [29]. Glass feeders were fill with heat-inactivated rabbit blood and covered with a chick skin membrane. For infected batches, TOSV (2^nd passage of viral culture) was added at a final concentration of 10² TCID₅₀/ml (dose 1), 10⁴ TCID₅₀/ml (dose 2) or 10⁶ TCID₅₀/ml (dose 3). For control batch (control), the same volume of MEM culture medium, without virus, was added. The complete system was set up in the climate chamber where the feeders were connected to a 37°C circulating water bath and fixed to the feeding pots (Fig 1). Feeding was performed for six hours, with reduced brightness, and regular manual blood mix to avoid clotting. Six hours of blood feeding was chosen as TOSV remains infectious and at the same amount in the blood meal under these conditions as previously described [17]. As engorged females are fragile, it is recommended to handle them after the first 24 hours of digestion [29]. Thus, they were sorted on ice the day after blood meal. Males and non-engorged females were killed and removed. All the experiments were replicated twice.

rh: relative humidity.

a. Infection dynamics

Engorged females were placed by 30 in cardboard boxes in climate chamber, under standard conditions, with a daily intake of cotton pads soaked in 50% organic sugar solution. Three groups were set up for this experiment, infected with dose 1, dose 2 and dose 3 of TOSV. Between 10 to 30 individuals were dissected in three parts: head, body (thorax and abdomen), wings and legs, every two days for 15 days (Fig 2), and stored at -80°C for further TOSV detection and quantification analysis. In order to detect virus released by the females, the sugar cotton pads were collected daily, and stored at -80°C. One cotton per day at two, four, six, eight and 10 days post infection (dpi) with dose 3 only (as a proof of concept, to ensure the viral detection) were analyzed with the same RT-qPCR protocol as the sand fly samples.

b. Impact of infection on life-history traits

To investigate the potential impact of TOSV infection on sand fly life-history traits, vector survival and fecundity traits, which are two important traits in the virus transmission, were measured. For this experiment, engorged females were individually dispatched in small egg-laying pots filled with a 1cm layer of plaster of Paris, pooled by group in plastic boxes containing moistened sand and placed in climate chamber under standard conditions [33]. A pool of sand flies was set aside in a cardboard box for TOSV infection verification by RT-qPCR. Groups were set up for this experiment, control with uninfected individuals, and two tests with individuals infected by different TOSV doses (10⁴ and 10⁶ TCID₅₀/ml). Life history traits were measured daily and individually for each female: death date, oviposition date, eggs laid number, egg-hatching date and larvae number. Immature stages were only monitored up to first instar larva, due to potential cannibalism between larvae [36], and larvae were killed directly after hatching.

To measure infection impact on female survival, the experiment was performed with a different batch of females. After blood feeding, engorged females were transferred to cardboard boxes with a maximum of 30 individuals per box in climate chamber and maintained under standard conditions (26 ± 1°C, 80% relative humidity (rh), 14h: 10h light: dark cycle). Five blood-fed females were killed, dissected and stored at -80°C for RT-qPCR analysis directly after oral infection to measure the initial amount of virus ingested. The survival time of two female groups, fed with (i) healthy blood (control) and (ii) infected blood with the highest dose of TOSV (dose3), was measured every day until the death of all individuals. In order to check the potential impact on survival, this experiment was only realized with the highest virus dose. To prevent the fungi development which could affect the survival of the sand flies, dead females were removed and the sugar source was changed every day.

Viral load was analyzed by RT-qPCR. Entire female or each part (head, body, wings and legs) were individually crushed in 300μl of MEM (enriched with 1% penicillin–streptomycin, 1% (200mM) l-glutamine, 1% kanamycin, and 3% amphotericin B (Gibco)). Sand fly tissues were homogenized by a Tissuelyser II (Qiagen) using 3mm tungsten carbide beads (Qiagen), and centrifuged at 10000 rpm for 5min. A volume of 200μl homogenate supernatant was used for nucleic acid extraction with the QIAcube (Qiagen) machine, using Virus Extraction Mini Kit (Qiagen). The RT-qPCR assays were performed with a SuperScript III Platinum One-Step RT-qPCR Kit with ROX (Invitrogen—Thermo Fisher Scientific) on a QuantStudio 12K Flex thermocycler (ThermoFisher). A volume of 5μl of RNA was added to 20μl of mix containing 12.5μl of 2X Reaction Mix, 0.5μl of Superscript III RT/Platinum Taq Mix, and primers and probes STOS at 10μM [37]. Negative (pure water) and positive controls (at 4.81 x 10⁴ RNA in vitro transcribed copies/ml as described by Beckert & Masquida [38]) were included in each RT-qPCR run. A standard curve was carried out for absolute quantification of TOSV RNA through RT-qPCR (S1 Fig). This standardised RT-qPCR protocol was used to identify the number of viral particles per body part, assuming that the number of cells per body part is consistent from one sand fly to another.

TOSV titers were determined by end-point dilution assay. Tenfold dilutions were used to infect confluent Vero E6 cells in a 96-well plate in MEM (5% FBS, 1% penicillin-streptomycin, 1% kanamycin, 3% amphotericin B (Gibco)) at 37°C and 5% CO₂. Wells were classified as positive (cytopathic effect) versus negative (no cytopathic effect) at five dpi and TCID₅₀/ml was calculated according to the Reed-Muench method [39].