# Susceptibility of diverse sand fly species to Toscana virus

Magdalena Jancarova, Nikola Polanska, Adrien Thiesson, Frédérick Arnaud, Marketa Stejskalova, Marketa Rehbergerova, Alain Kohl, Barbara Viginier, Petr Volf, Maxime Ratinier

► To cite this version:

Magdalena Jancarova, Nikola Polanska, Adrien Thiesson, Frédérick Arnaud, Marketa Stejskalova, et al. Susceptibility of diverse sand fly species to Toscana virus. PLoS Neglected Tropical Diseases, 2025, 19 (5), pp.e0013031. 10.1371/journal.pntd.0013031. hal-05055919

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RESEARCH ARTICLE

# Susceptibility of diverse sand fly species to Toscana virus

Magdalena Jancarova¹*, Nikola Polanska¹, Adrien Thiesson², Frédérick Arnaud², Marketa Stejskalova¹, Marketa Rehbergerova¹, Alain Kohl³,⁴, Barbara Viginier², Petr Volf¹, Maxime Ratinier²

1 Laboratory of Vector Biology, Department of Parasitology, Faculty of Science, Charles University, Prague, Czech Republic, 2 IVPC UMR754, INRAE, Universite Claude Bernard Lyon 1, EPHE, Universite PSL, Lyon, France, 3 MRC-University of Glasgow Centre for Virus Research, Glasgow, United Kingdom, 4 Centre for Neglected Tropical Diseases, Departments of Tropical Disease Biology and Vector Biology, Liverpool School of Tropical Medicine, Liverpool, United Kingdom

* magdalena.jancarova@natur.cuni.cz

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## Abstract

Toscana virus (TOSV) is an emerging but neglected human pathogen currently circulating around the Mediterranean basin including North Africa. Human illness ranges from asymptomatic or mild flu-like syndromes to severe neurological diseases such as meningitis or meningoencephalitis. Despite its significant impact, understanding of TOSV transmission and epidemiology remains limited. Sand flies (Diptera: Phlebotominae), specifically *Phlebotomus perniciosus* and *Phlebotomus perfiliewi*, are believed to be the primary vectors of TOSV. However, the spread of TOSV to new geographical areas and its detection in other sand fly species suggest that additional species play a role in the circulation and transmission of this virus. This study investigated the vector competence of four sand fly species - *P. tobbi*, *P. sergenti*, *P. papatasi*, and *Sergentomyia schwetzi* - for two TOSV strains: 1500590 (TOSV A lineage) and MRS20104319501 (TOSV B lineage). Sand flies were orally challenged with TOSV via bloodmeals. None of the tested species showed susceptibility to the TOSV A strain. However, for TOSV B strain, *P. tobbi* demonstrated a high potential as a new vector, exhibiting high infection and dissemination rates. *P. sergenti* also showed some susceptibility to TOSV B, with the virus dissemination observed in all infected females. These findings suggests that *P. tobbi* and *P. sergenti* are new potential vectors for TOSV B. Given that *P. tobbi* and *P. sergenti* are the primary vectors of human leishmaniasis in the Balkans, Turkey and Middle East, their susceptibility to TOSV could have significant epidemiological consequences. On the other hand, *P. papatasi* and *S. schwetzi* appeared refractory to TOSV B infection. Refractoriness of *P. papatasi*, a highly anthropophilic species distributed from the Mediterranean to the Middle East and India, suggests that this species does not contribute to TOSV circulation.

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OPEN ACCESS

**Citation:** Jancarova M, Polanska N, Thiesson A, Arnaud F, Stejskalova M, Rehbergerova M, et al. (2025) Susceptibility of diverse sand fly species to Toscana virus. PLoS Negl Trop Dis 19(5): e0013031. https://doi.org/10.1371/journal.pntd.0013031

**Editor:** Jeremy V. Camp, Medizinische Universität Wien, AUSTRIA

**Received:** November 7, 2024

**Accepted:** April 1, 2025

**Published:** May 2, 2025

**Copyright:** © 2025 Jancarova et al. This is an open access article distributed under the terms of the [Creative Commons Attribution License](https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

**Data availability statement:** Data are openly available in the manuscript and [Supporting information](#).

**Funding:** This publication was supported by the European Virus Archive Global (EVA-GLOBAL) project that has received funding from the European Union's Horizon 2020 research and
innovation programme under grant agreement No 871029, the project National Institute of virology and bacteriology (Programme EXCELES, ID Project No. LX22NPO5103 - Funded by the European Union - Next Generation EU), and the INFRAVEC 2 (Research Infrastructures for the Control of Vector-Borne Diseases, H2020, no. 731060). This study was co-funded by European Commission grant 101057690 and UKRI grants 10038150 and 10039289, and is catalogued by the CLIMOS Scientific Committee as CLIMOS number CLIMOS023 (www.climos-project.eu). A.T. was a doctoral fellow of the École Doctorale de l'École Pratique des Hautes Études (ED472). A.K. was funded by the UK Medical Research Council (MC_UU_12014/8, and MC_UU_00034/4). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

**Competing interests:** The authors have declared that no competing interests exist.

## Author summary

Sand flies are blood-sucking insects that transmit various human pathogens, including parasitic protists of the genus *Leishmania* and several viruses, such as the phlebovirus Toscana (TOSV). TOSV infections typically result in flu-like symptoms, but in some patients, the infection can affect the central nervous system (CNS) or even be fatal. Due to climatic and environmental changes associated with human activity, it is likely that sand flies and the pathogens they transmit will spread beyond current boundaries. However, the specific sand fly species that transmit TOSV are not well understood. Therefore, it is essential to identify vectors that are involved in the circulation and transmission of the virus. In our study, we found that the range of potential TOSV vectors is broader than previously thought and that individual virus strains may influence vector competence. Specifically, we demonstrated that *Phlebotomus tobbi* and *P. sergenti*, two sand flies common in Mediterranean area and Middle East, are susceptible to TOSV and likely transmit this virus in nature.

## Introduction

*Phlebovirus toscanaense* (Toscana virus, TOSV) is an emerging yet neglected human pathogen (genus *Phlebovirus*, family *Phenuiviridae*, order *Haeravirales* [1]) that is transmitted by sand flies. Symptoms in humans vary from non-symptomatic to febrile illness to (occasionally fatal) central nervous system diseases. TOSV is considered to be one of the three most important causes of aseptic meningitis in the countries where this virus circulates [2]. TOSV has been reported in many countries around the Mediterranean basin, including Southern Europe (Italy, Spain, France, Portugal), South-Eastern Europe (Turkey, Cyprus), the Balkan peninsula (Greece, Croatia, Bosnia and Herzegovina, Kosovo, Bulgaria), North Africa (Morocco, Algeria, Tunisia) and Mediterranean islands (reviewed by [3,4]). Popescu et al. [5] described the occurrence of neuroinvasive TOSV infections in Romania.

TOSV is currently divided into three genetic lineages: A, B and C [3], however, the latter was only characterised based on partial sequences, and whole genome sequences or virus isolates have never been obtained [6]. Thus far, no biological differences between these different genetic lineages have been described with regards to host infection, clinical signs, or disease severity [2,5] though their geographical distribution may differ (reviewed by [7]). Despite the wide geographical distribution of this pathogen and the high incidence of summer meningitis and encephalitis around the Mediterranean basin [8], data on TOSV biology and epidemiology are very limited.

To date, vertebrates infected by TOSV appear to be dead-end hosts that do not play a crucial role in TOSV circulation [3]. Viremia in humans is low and transient (24–36 hours) [9] and dogs do not develop significant viremia after experimental infection [10]. Anti-TOSV antibodies or/and viral RNA were found in humans [11–13],
livestock, horses, dogs and cats [14–18], bats [19] as well as birds [20,21]. However, there are only two studies describing isolation of the virus from non-human hosts; the bat *Pipistrellus kuhli* (brain) [22] and birds (pigeons, mallards, partridges) [23]. Overall, the life cycle of TOSV in nature remains unclear.

It is generally accepted that TOSV is transmitted by phlebotomine sand flies [3,24–27]. Sand flies (Diptera: *Psycho-didae*) are hematophagous insects known as vectors of human and animal pathogens, such as protozoan parasites (namely *Leishmania* sp.), bacteria (e.g., *Bartonella bacilliformis*) and viruses [28,29]. Both male and female sand flies feed on natural sugar sources, such as plant sap or honeydew, but females also feed on vertebrate blood to obtain proteins and nutrients necessary for egg development [28,29]. Until now, only two sand fly species, *Phlebotomus perniciosus* and *P. perfiliewi* are considered as vectors of TOSV. However, the seroprevalence of TOSV in humans and animals in Eastern Mediterranean countries, where *P. perniciosus* is not present and *P. perfiliewi* is rare, suggests the involvement of other sand fly species in the circulation of the virus [3]. Interestingly, TOSV was detected in *Sergentomyia minuta* in Marseille [30], *P. tobbi* in Cyprus [31], *P. neglectus* in Croatia [16], *P. longicuspis* and *P. sergenti* in Morocco [32–34] and in *P. major*, *P. papatasi* and *S. dentata* in Turkey [35]. However, the role of these sand fly species in TOSV transmission remains to be elucidated as virus detection does not mean necessary transmission.

The two main objectives of this study were to identify novel TOSV vectors and to determine whether distinct TOSV strains differ in their interactions with the insect vector. This information is crucial not only to fill scientific knowledge gaps but also to better understand the ecology and epidemiology of TOSV. Moreover, due to climate change, animal migration and human activities, sand flies expanded to new areas along with the pathogens that they transmit [36]. Therefore, this knowledge is essential to assess the risk of TOSV expansion and implement adequate transmission control strategies.

# Materials and methods

## Sand flies

Four sand fly species were used for experimental infections: *P. tobbi*, *P. sergenti*, *P. papatasi* (all three colonies originating from Turkey) and *S. schwetzi* (colony originating from Ethiopia). All colonies have been established for many generations at the Laboratory of Vector Biology at Charles University, Prague and tested for the presence of phleboviruses and *Wolbachia* sp.. All colonies' tests were negative. More detailed information on the establishment and maintenance of sand fly colonies was published elsewhere [37]. Three-to-seven-day-old females were used in all experiments and were maintained at 26°C with 50% sucrose after infection.

## Cell culture

All experiments were done with either VeroE6 cells (obtained from Philippe Marianneau, Unité de virologie - ANSES Lyon, France) or BSR cells (BHK21 clone, kindly provided by K. K. Conzelmann) [38]. Cells were cultivated in Dulbecco's Modified Eagle medium with high glucose, L-glutamine, sodium pyruvate and phenol-red (DMEM) supplemented with 10% foetal bovine serum (FBS) and 1% penicillin-streptomycin (P/S), in 75 cm² plastic tissue culture flasks with filtered screw cap, placed horizontally in a CO₂ incubator (5% CO₂, 37°C).

## Virus

Experimental infections of sand flies were performed with TOSV strain 1500590 (Lineage A, named TOSV A: kindly provided by Philippe Marianneau, GenBank Accession No: MT032306, MT032307 and MT032308) and strain MRS2010-4319501 (Lineage B, called TOSV B; purchased at European Virus Archive collection, GenBank Accession No: KC776214, KC776215 and KC776216) [39,40]. TOSV stocks were produced by infecting BSR cells, cultivated in DMEM supplemented with 4% FBS, with either TOSV A or TOSV B. Supernatants were collected when cytopathic effect (CPE) was evident (usually 4 days post-infection, d.p.i.) and clarified by centrifugation at 500g for 5 min at 4°C. Viral
suspensions were subsequently deposited at the top of 20% sucrose cushion (w/v, diluted in PBS) and ultracentrifugated at 124,000g for 3 hours at 4°C. Pellets were resuspended in PBS and stored at -80°C. Viral stock titers were determined by plaque-forming assays using veroE6 cells. Briefly, virus stocks were diluted in a 10-fold manner and incubated on VeroE6 cells for 2 hours at 37°C with 5% CO₂. Cells were washed twice with PBS and overlayed with a volume-to-volume mixture of 2.5% ultrapure agarose solution (w/v) and 2X minimal essential medium (MEM 2X), supplemented with 4% FBS. Cells were incubated at 37°C, 5% CO₂ for 6 days to allow viral plaques to grow to visible sizes, before being fixed using 4% formaldehyde solution and stained using crystal violet solution. Viral plaques were then counted, and viral titers are expressed as PFU/ml (plaque-forming unit per ml).

## Sand fly infections

All infectious experiments were performed in the BSL2 laboratory. In each experiment, three groups of 120 sand fly females were used: a first group for blood feeding without the virus as control group, a second group for infection with TOSV lineage A and a third group for infection with TOSV B. All experimental infections were carried out in a glove box; firstly, female sand flies from the control group were allowed to feed through the chick skin membrane on heat-inactivated ram blood (LabMediaServis s.r.o.) for 90-120 min (more detailed information about feeding system was published elsewhere [37]). Subsequently, groups for infectious bloodmeal were fed one by one through the chick skin membrane on heat-inactivated ram blood containing an infectious dose of TOSV A or TOSV B (approx. 10⁶ PFU/ml) of blood in the same mode as for control group without virus. Subsequently, non-fed sand fly females were removed by a battery-powered aspirator. All experiments were done within 3 years. The results were consistent.

## Sand fly dissections and sample processing

After blood feeding, two blood fed females from each group were homogenized individually by a crushing pestle in 1 ml of crushing medium (DMEM, 4% FBS, amphotericin B 2.5 g/ml, nystatin 100 U/ml, gentamycin 50 g/ml, penicillin-streptomycin 50 g/ml), aliquoted into two samples (each 500 µl) and stored at -80°C as a control of the feeding process (at day 0 post infection - D0 p.i.), to see if the virus stayed infectious during the feeding process. All sand fly species tested were kept at 26°C and humidity above 70%. Short-living females of *P. tobbi* were dissected on days 4 and 8 post infection (D4, D8 p.i.), while females of *P. sergenti*, *P. papatasi*, and *S. schwetzi* were also dissected on day 14 (D14 p.i.). Additionally, due to the high survival rate of *S. schwetzi*, an extra dissection was performed on day 18 post infection (D18 p.i.). Sand fly females were anesthetized on ice and they were dissected in the glove box under a binocular microscope, in a drop of PBS to head with attached salivary glands (H) and rest of the body (B) to determine TOSV presence. These body parts were homogenized as described above, aliquoted to 500 µl) and stored at -80°C. Thus, for each sample - the body of one sand fly female or the head of one sand fly female, we had two aliquots (the first aliquot for TCID₅₀ and the second for quantitative PCR - qPCR, see below), each with a volume of 500 µl. Every sample has a unique code for species - letter ("T" - *P. tobbi*, "E" - *P. sergenti*, "P" - *P. papatasi*, "C" - *S. schwetzi*) and sample numbers are listed in S1 Table.

## Virus detection by end point dilution assay (TCID₅₀)

Ninety-six well plates were filled with 100 µl DMEM medium supplemented with 4% FBS and 1% P/S except for the first row, where 111 µl of each sample were added. All samples were tested in quadruplicates and for each series of experiments females fed with non-infected blood were also used as a negative control. Serial dilution of the sample was done by transferring 11 µl from one line to the other until the last line where 11 µl was removed and discarded. Subsequently, 100 µl of VeroE6 cells (concentration at 4x10⁴ cells/ml) were added to each well. Plates were incubated for 5 days in a CO₂ incubator (5% CO₂, 37°C) and then the presence of CPE caused by the virus was assessed under an inverted microscope. Wells showing CPE were scored as positives when wells with no apparent CPE were considered negatives. In each endpoint dilution assay, two types of controls were included: wells containing only cells and wells with homogenate
from blood-fed females in the control group. This allowed for a clear detection of the CPE of TOSV on the cell monolayer. Viral titers were calculated by using Reed and Muench's method and expressed as tissue culture infectious dose 50 per millilitre (TCID₅₀/ml) [41].

## Isolation of TOSV RNA and cDNA synthesis from experimental samples

Detection of TOSV RNA by qPCR in the experimental samples was carried out to determine viral RNA levels. This method was used to support the data from TCID₅₀ and all the positive samples and samples on the detection limit of the TCID₅₀ assay were tested, as well as some randomly chosen negative samples from TCID₅₀. From these analysed samples, TOSV RNA was isolated from a 500 µl aliquot using QiaAmp Viral RNA Mini Kit (Qiagen). The 500 µl was divided by half and 1000 µl AVL buffer with acyl carrier RNA added to each aliquot and incubated for 10 min at room temperature. RNA isolation steps were carried out according to the manufacturer's protocol and the RNA was eluted in two steps each with 40 µl AVE buffer (1 min, 8000 rpm). Two protocols were used for cDNA synthesis - one to produce positive strands and one for negative strands. cDNA synthesis was performed by using QuantiTect Reverse Transcription kit (Qiagen) by using 12 µl of RNA samples. cDNAs specific to the TOSV negative strand of the S segment were obtained with primer STOS-50F primer, cDNA specific to the TOSV positive strand of the S segment were synthesised using primer STOS-138R [42]. cDNA samples were stored at -20°C until used.

## Preparation of the TOSV RNA standard curve for quantitative PCR

For RNA standard production, DNA templates were produced by PCR on plasmids containing either TOSV A or TOSV B antigenome of S segment using CloneAmp Hifi PCR Premix (Takara) and qPCR_S_TOSV_RT (ACACAGAGAT-TCCCGTG) and qPCR_S_TOSV_T7_rev (TAATACGACTCACTATAGGGCCATGAGCATCAGCAATRGTGG) primers. To enable the production of the genomic viral RNA, the T7 RNA polymerase promoter was added at the 5' termini of qPCR_S_TOSV_T7_rev primer (underline sequence). Briefly, the PCR mixture (final volume 25 µl) contained 12.5 µl of Clone Amp Premix, 1 µl forward primer (10 µM), 1 µl reverse primer (10 µM), 8.5 µl of PCR-grade H₂O, and 2 µl of plasmid (5 ng/µL). PCR, 30 cycles: 10 sec 98°C, 10 sec 55°C, and 5 sec 72°C. PCR products (25 µl) were then digested with 0.5 µl DpnI enzyme (NEB) for 1 hour at 37°C. Subsequently, digested products were loaded on horizontal gel electrophoresis on 1.5% agarose gel (35 min, 100 V) and then visualised. The digested product was cut from the gel and cleaned up by the NucleoSpin Gel and PCR Clean-up kit (Macherey-Nagel) and DNA concentration was measured on NanoDrop One (ThermoFisher Scientific).

The purified DNA template was used to produce single-stranded negative sense TOSV S-segment RNA with the T7 RiboMAX (Promega) production system. Briefly, the reaction of 20 µl contained: 10 µl of RiboMAX Express T7 2X Buffer, 2 µl of Enzyme Mix, T7 Express, 125 ng of linear DNA template, and filled up to 20 µl by PCR-grade H₂O. RNA was produced in a PCR thermocycler at 37°C for 30 min and then cooled down. RNA was then purified using the QIAamp Viral RNA Mini kit (Qiagen). The purified RNA was cleaned from template DNA contaminations by using the TURBO DNA-free Kit (Invitrogen). Before DNase treatment, RNA was incubated for 10 min at 65°C followed by 5 min on ice to increase DNase efficiency. DNase treatment was followed according to the manufacturer's protocol. Briefly, 50 µl reaction contains 5 µl of 10X TURBO DNase Buffer, 30 µl of RNA produced with T7 RiboMAX, and 14 µl by PCR-grade H₂O. This mixture was incubated for 30 min at 37°C, and then 0.5 µl of TURBO DNase Enzyme (2 Units/µl) were added to the reaction mixture, which was subsequently incubated for another 30 min at 37°C. After this, DNase inactivation buffer was added to the reaction mixture for 5 minutes at room temperature and the mix was centrifugated for 2 minutes at 10,000 g. The supernatant was collected to obtain pure RNA.

The concentration and purity of RNA were checked on NanoDrop One (A260/A280) and the number of RNA copies was calculated according to the formula:
$$RNA copy number/ = \frac{[RNA]g/}{N \times 340} \times 6.02 \times 10^{23}$$

N = RNA fragment length in bp

340 = Molecular weight of 1 bp

$6.02 \times 10^{23} = Avogadro number$

Finally, the RNA was aliquoted in the working solutions with a concentration of $10^8$ RNA copies/ µl which were stored at -80°C until used and diluted as the standard curve for qPCR.

## Virus detection by quantitative PCR

Quantitative PCR was performed on the LightCycler 480 Real-Time PCR System (Roche) using the QuantiTect Probe PCR kit (Qiagen) in a total volume of the reaction of 10 µl in the LightCycler 480 Multiwell Plate 384 white (Roche). Each reaction contained 5 µl qPCR master mix, 0.4 µl forward primer (10 µM), 0.4 µl reverse primer (10 µM), 0.1 µl probe, 2.1 µl PCR-grade H₂O, and 2 µl cDNA template (obtained by the transcription either positive or negative strand of S segment of TOSV RNA). Gene specific primers and probes for the TOSV S-segment were used [42]. The qPCR conditions were as follows: enzyme activation 15 min at 95°C and 45 cycles of denaturation (94°C, 15 sec) and annealing with extension step (60°C, 1 min), followed by a cooling step (40°C, 10 min). The results were analysed in LightCycler 480 SW 1.5.1 using “absolute quantification 2nd derivative maximum for all samples” analyses. The samples and the calibration curve were measured every time in triplicate, the obtained Cp (crossing point) was averaged, and the standard deviation was calculated. If one Cp of the reactions did not correspond with the other two from triplicate, this reaction was manually excluded from the analysis and only a duplicate was used for the calculation of the final Cp of the sample. Subsequently, the genome copy number (RNA copies/µl) was counted from obtained Cp values and the standard curve for every sample. All TCID₅₀ positive samples (both heads and bodies of one sand fly female), along with those on detection limit of TCID₅₀ (lower than $1.78 \times 10^2$ TCID₅₀/ml, “Q” in S1 Table), were tested by qPCR. For some samples both 500 µl aliquots (one for TCID₅₀ and the second for qPCR) were used for retesting of viral titer by TCID₅₀, thus there was nothing left for qPCR (S1 Table).

## Statistical analysis and data visualisation

Samples above the TCID₅₀ quantification limit of the TCID₅₀ assays, and samples on or below the TCID₅₀ quantification limit (at least one well positive in the first row) with confirmed presence of TOSV RNA by qPCR, were considered TOSV positive. For data representation, samples below quantification limit were arbitrarily assigned a titer value corresponding to half of the value of the quantification limit (i.e., $8.9 \times 10^1$ TCID₅₀/ml). Data visualisation and statistical analysis were performed using R and RStudio softwares [43] (version 4.3.3 and 2023.12.1+402 respectively) and ggplot2 (https://CRAN.R-project.org/package=ggplot2) and rstatix packages (https://CRAN.R-project.org/package=rstatix). Pairwise comparisons of proportions tests were used as statistical analysis to compare infection and dissemination rates between TOSV A and TOSV B blood fed sand flies.

## Results

### Infection of *P. tobbi* by TOSV

We first assessed the vector competence of *P. tobbi* for TOSV A and TOSV B strains through blood feeding. This experiment was repeated five times and data were pooled as this species does not feed very readily on an artificial feeding system under laboratory conditions. Together 55 and 77 females were dissected and tested for infection and dissemination of TOSV A and TOSV B, respectively. In total, 25 and 30 females were tested at D4 and D8 p.i. for TOSV A infection and dissemination (Fig 1A). None of these females were positive except at D0 p.i. where the mean value of viral titers was
determined at 6.13x10⁴ TCID₅₀/ml (Fig 1B); proving that blood contained infectious TOSV A. In the case of TOSV B, at D4 p.i., 28 out of 41 females tested positive for TOSV by TCID₅₀ assay (infection rate of 68.3%) and 16 also showed a disseminated infection (57.1%) (Fig 1A). At D8 p.i., 19 out of 36 females were positive for TOSV B (infection rate of 52.8%) and 18 females exhibited a disseminated infection (94.7%) (Fig 1A). In the analyzed bodies, infectious viral titers at D4 p.i. and D8 p.i. ranged from 3.16x10³ to 5.41x10⁵ TCID₅₀/ml and 1.00x10³ to 1.26x10⁶ TCID₅₀/ml, respectively (Fig 1B). For the head fraction, viral titers ranged between 1.78x10² and 1.78x10³ TCID₅₀/ml at D4 p.i., and 1.00x10³ and 3.16x10⁴ TCID₅₀/ml at D8 p.i. (Fig 1B).

Detection of TOSV negative-strand RNA by qPCR was used to confirm the results above. A total of 39 females (6 TOSV A, 33 TOSV B) at D4 p.i., and 20 females (5 TOSV A, 15 TOSV B) were tested at D8 p.i. The negative strand RNA of TOSV B was detected in 29 and 12 females at D4 and D8 p.i., respectively. Interestingly, TOSV A RNA was present in 2 females (6.3x10³ and 8.5x10¹ RNA copies/µl) at D4 p.i. while none was detected at D8 p.i.. (Fig 1C).

To further confirm TOSV replication in sand flies, molecular detection of positive-strand viral RNA was also performed, as antigenome is produced during virus replication. Among TOSV-positive sand flies, 40 females were tested for

A

<table><thead><tr><th rowspan="2">Species</th><th rowspan="2">Virus</th><th colspan="3">IR (%)</th><th colspan="2">DR (%)</th><th colspan="2">DI/R (%)</th></tr><tr><th>D0</th><th>D4</th><th>D8</th><th>D4</th><th>D8</th><th>D4</th><th>D8</th></tr></thead><tbody><tr><td rowspan="2">P. tobbi</td><td>TOSV-A</td><td>100 (4/4)</td><td>0 (0/25)</td><td>0 (0/30)</td><td>0 (0/25)</td><td>0 (0/30)</td><td>-</td><td>-</td></tr><tr><td>TOSV-B</td><td>100 (7/7)</td><td>68.3 (28/41)</td><td>52.8 (19/36)</td><td>39 (16/41)</td><td>50 (18/36)</td><td>57.1 (16/28)</td><td>94.7 (18/19)</td></tr></tbody></table>

B

<figure><img src="image_4.png" /></figure>

<figure><img src="image_5.png" /></figure>

**Fig 1. Susceptibility of *P. tobbi* to TOSV infection.** *P. tobbi* sand flies were orally exposed to bloodmeal with either TOSV A or TOSV B (10⁶ PFU/ml) and collected at D4 and D8 p.i. to assess the presence of TOSV in bodies or heads with salivary glands. (A) Summary values of infection rate (IR), dissemination rate (DR) and dissemination rate among infected sand flies (DI/R). (B) Infectious viral titers were obtained by TCID₅₀ limiting-dilution assays in body and head homogenates. Each dot represents a single sand fly. Light grey dashed lines indicate the threshold of virus detection (1.78x10² TCID₅₀/ml). Statistical significance between infection and dissemination rate was determined using pairwise comparisons of proportions tests: p<0.001 (***) and p<0.0001 (****). (C) Viral genome copy numbers were determined by qPCR and plotted against infectious viral titers. Light grey dashed lines indicate the threshold of virus quantification by TCID₅₀ dilution assay (1.78x10² TCID₅₀/ml).

https://doi.org/10.1371/journal.pntd.0013031.g001
TOSV-positive strand RNA (28 at D4 p.i. and 12 at D8 p.i.). At D4 p.i., 20 females were positive (74.1%), while 11 were at D8 p.i. (91.7%), demonstrating that TOSV is indeed replicating in infected females. Detailed information about the viral titers, Cp values and genome copy number, and the presence of positive strands of RNA are shown in S1 Table.

Taken together, these data confirm that TOSV A does not infect *P. tobbi*, whereas TOSV B is capable of infection, replication and dissemination in this species. This information is consistent between both TCID$_{50}$ and molecular methods.

## Infection of *P. sergenti* by TOSV

The experiment with *P. sergenti* was repeated ten times and data were pooled. Here, 68 and 153 successfully blood fed females were dissected and tested for infection and dissemination with TOSV A and TOSV B, respectively. Out of all tested females, only 1 was positive for TOSV A at D4 p.i. albeit with disseminated infection (Fig 2A). Other positive females were only detected at D0 p.i., with a mean viral titer of $4.37 \times 10^5$ TCID$_{50}$/ml (Fig 2B). For TOSV B 45, 65 and 43 *P. sergenti* females were tested at D4, D8 and D14 p.i., respectively. At D4 p.i., 3 out of 45 females were positive (Fig 2A); 1 female defecated without visible blood remnants in the midgut and viral loads were measured at $2.15 \times 10^6$ TCID$_{50}$/ml in

### A

<table><thead><tr><th rowspan="2">Species</th><th rowspan="2">Virus</th><th colspan="4">IR (%)</th><th colspan="3">DR (%)</th><th colspan="3">DI/R (%)</th></tr><tr><th>D0</th><th>D4</th><th>D8</th><th>D14</th><th>D4</th><th>D8</th><th>D14</th><th>D4</th><th>D8</th><th>D14</th></tr></thead><tbody><tr><td rowspan="2">P. sergenti</td><td>TOSV-A</td><td>87.5 (7/8)</td><td>2.3 (1/44)</td><td>0 (0/19)</td><td>0 (0/5)</td><td>2.3 (1/44)</td><td>0 (0/19)</td><td>0 (0/5)</td><td>100 (1/1)</td><td>-</td><td>-</td></tr><tr><td>TOSV-B</td><td>93.3 (14/15)</td><td>6.7 (3/45)</td><td>6.2 (4/65)</td><td>9.3 (4/43)</td><td>2.2 (1/45)</td><td>6.2 (4/65)</td><td>9.3 (4/43)</td><td>33.3 (1/3)</td><td>100 (4/4)</td><td>100 (4/4)</td></tr></tbody></table>

### B

<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /><figcaption>**Fig 2. Susceptibility of *P. sergenti* to TOSV infection.** *P. sergenti* sand flies were orally exposed to bloodmeal with either TOSV A or TOSV B ($10^6$ PFU/ml) and collected at D4, D8 and D14 p.i. blood meal to analyse bodies and heads with salivary glands for the presence of TOSV. (A) Summary values of infection rate (IR), dissemination rate (DR) and dissemination rate among infected sand flies (DI/R). (B) Infectious viral titers were obtained by TCID$_{50}$ limiting-dilution assays in body and head homogenates. Each dot represents a single sand fly. Light grey dashed lines indicate the threshold of virus detection ($1.78 \times 10^2$ TCID$_{50}$/ml). (C) Viral genome copy numbers were determined by qPCR and plotted against Infectious viral titers. Light grey dashed lines indicate the threshold of virus quantification by TCID$_{50}$ dilution assay ($1.78 \times 10^2$ TCID$_{50}$/ml).</figcaption></figure>

https://doi.org/10.1371/journal.pntd.0013031.g002
the body and 5.62 x10⁴ TCID₅₀/ml in the head (Fig 2B), indicating dissemination of TOSV B. The 2 remaining females still had visible blood residues and detection of infection was at the limit of the TCID₅₀ method, but it was confirmed by qPCR, without disseminated infection. At D8 and D14 p.i., 4 out of 65 and 4 out of 43 females were positive by the TCID₅₀ assay, respectively, and showed disseminated infection (Fig 2A). For the bodies, the viral loads ranged from 5.62x10⁴ to 1.78x10⁶ TCID₅₀/ml at D8 p.i. while, at D14 p.i., they were even higher – 3.16x10⁵ to 2.20x10⁶ TCID₅₀/ml (Fig 2B). The viral loads in heads at D8 and D14 p.i. ranged from 3.16x10³ to 3.16x10⁴ TCID₅₀/ml and 5.62x10³ to 5.62x10⁴ TCID₅₀/ml, respectively (Fig 2B).

Subsequently, a total of 15 females (4 TOSV A, 9 TOSV B) at D4 p.i., 15 females (4 TOSV A, 13 TOSV B) at D8 p.i., and 14 females (3 TOSV A, 11 TOSV B) at D14 p.i. were tested by qPCR for the presence of negative strand TOSV RNA. The negative strand RNA of TOSV B was detected in 4, 7, and 6 females at D4, D8 and D14 p.i., respectively. In comparison, negative strands of TOSV A RNA were detected in 2 females at both D4 and D8 p.i. (negative by TCID₅₀). Like *P. tobbi*, data from qPCR correlate with viral titers (Fig 2C).

All TOSV-positive females were tested for positive-strand viral RNA (4 at D4 p.i., 4 at D8 p.i. and 4 at D14 p.i.). TOSV antigenome was then detected in 3, 1 and 4 females at D4, D8 and D14 p.i. respectively (75%, 25% and 100%). All results are summarised in S1 Table.

This suggest that *P. sergenti* seems to be susceptible to TOSV B infection when both TCID50 and molecular assays confirmed virus infection, replication and dissemination. Its role in the circulation of TOSV A needs to be further clarified in the future.

## Infection of *P. papatasi* by TOSV

In two independent experiments, 82 (21 at D4 p.i., 25 at D8 p.i., 36 at D14 p.i.) and 95 (20 at D4 p.i., 25 at D8 p.i., 50 at D14 p.i.) *P. papatasi* females were assessed following infection assay with TOSV A and TOSV B, respectively (Fig 3A). All dissected and tested females were negative for both TOSV A and TOSV B except at D0 p.i. (demonstrating that blood meals contained infectious viral particles), with a mean viral titer of 2.46x10⁵ TCID₅₀/ml for TOSV B and 1.60x10⁵ TCID₅₀/ml for TOSV A (Fig 3B).

As performed previously, randomly selected samples were tested for the presence of negative strand TOSV RNA using qPCR. 27 females were tested: 9 at D4 p.i. (3 females for TOSV A, 6 females for TOSV B), 9 at D8 p.i. (3 females for TOSV A, 6 females for TOSV B), and 9 at D14 p.i. (3 females for TOSV A, 6 females for TOSV B). Only 1 female had a positive body for TOSV A RNA at D4 p.i., whereas RNA of TOSV B was detected in the bodies of 2 females at D14 p.i. These 3 samples were also positive for TOSV positive-strand RNA, however all measured Cp values were high (from 38.1 to 39.3). Results are summarized in S1 Table. Importantly, these results suggest that neither TOSV A and TOSV B can disseminate in *P. papatasi*.

## Infection of *S. schwetzi* by TOSV

Five feeding experiments were performed with *S. schwetzi* and 135 (53 females for TOSV A, 82 females for TOSV B) females were dissected and tested by TCID₅₀ assay (Fig 4A). Sand fly females (4 for TOSV A, 6 for TOSV B) were all positive at D0 p.i., where the mean value of viral titers was 1.74 x10⁵ and 1.12x10⁵ TCID₅₀/ml for TOSV A and TOSV B, respectively. 53 sand fly females were tested after infection with TOSV A, from which only one female clearly showed a positive body, while 4 others, with visible remnants of the blood meal in the midgut, were at the detection limit of TCID₅₀ assay at D4 p.i. In the case of TOSV B, 2 out of 32 females were positive at D4 p.i., although with low viral loads (1.78x10³ and 5.62x10² TCID₅₀/ml) in the body (Fig 4B). These 2 individuals had visible blood meal remnants in the midgut and no viral disseminated infection was observed. Furthermore, the 28, 10 and 12 females tested at D8, D14 and D18 p.i. respectively were all negative for TOSV by TCID₅₀ assay. Detection of negative strand TOSV RNA was performed on 30 females (8 from TOSV A and 22 from TOSV B). TOSV A RNA was detected in 2 female bodies at D4 p.i., of which 1 was
A

<table><thead><tr><th rowspan="2">Species</th><th rowspan="2">Virus</th><th colspan="4">IR (%)</th><th colspan="3">DR (%)</th></tr><tr><th>D0</th><th>D4</th><th>D8</th><th>D14</th><th>D4</th><th>D8</th><th>D14</th></tr></thead><tbody><tr><td rowspan="2">P. papatasi</td><td>TOSV-A</td><td>100 (4/4)</td><td>0 (0/21)</td><td>0 (0/25)</td><td>0 (0/36)</td><td>0 (0/21)</td><td>0 (0/25)</td><td>0 (0/36)</td></tr><tr><td>TOSV-B</td><td>100 (4/4)</td><td>0 (0/20)</td><td>0 (0/25)</td><td>0 (0/50)</td><td>0 (0/20)</td><td>0 (0/25)</td><td>0 (0/50)</td></tr></tbody></table>

B

<figure><img src="image_8.png" /><figcaption>**Fig 3. Susceptibility of *P. papatasi* to TOSV infection.** *P. papatasi* sand flies were orally exposed to blood meal with either TOSV A or TOSV B (10⁶ PFU/ml) and collected at D4, D8 and D14 p.i. to analyse body and head with salivary glands for TOSV. (A) Summary values of infection rate (IR), dissemination rate (DR) among infected sand flies. (B) Infectious viral titers were obtained by TCID₅₀ limiting-dilution assays in body and head homogenates. Each dot represents a single sand fly. Light grey dashed lines indicate the threshold of virus quantification (1.78x10² TCID₅₀/ml).</figcaption></figure>

https://doi.org/10.1371/journal.pntd.0013031.g003

also positive for positive strand RNA. 4 females (3 from D4 p.i. and 1 from D14 p.i.) had bodies with detectable negative strand TOSV B RNA as well as for positive-strand RNA. However, all measured TOSV B RNA copy numbers were low (from 9.8x10³ to 3.4x10³ RNA copies/µl) and the Cp values for positive strand RNA were high (from 32.5 to 38.4). Results are summarised in S1 Table.

To summarize, TOSV TCID₅₀ positive or on detection threshold of TCID₅₀ samples were detected at D4 p.i., in addition to females with blood meal residues, but not at any later days p.i.. However, positive RNA TOSV strands were detected in three TCID₅₀-negative females, implying replication, but no dissemination.

Discussion

The circulation of arboviruses in nature relies on complex interrelationships among the virus, vector and vertebrate hosts, that are influenced by several factors [44]. In this study, we focused on vector competence. For arbovirus transmission by mosquitoes, the following steps are necessary: (i) infection of gut cells, (ii) virus dissemination from the midgut to secondary tissues and (iii) infection of salivary glands followed by virus release into saliva. Arboviruses thus must overcome the following barriers to be transmitted: (i) midgut infection barrier (MIB), (ii) midgut escape barrier (MEB), (iii) salivary gland infection barrier (SGIB) and (iv) salivary glands escape barrier (SGEB) [44–47]. Other important factors include genetic
A

<table><thead><tr><th rowspan="2">Species</th><th rowspan="2">Virus</th><th colspan="6">IR (%)</th><th colspan="3">DR (%)</th><th>DI/R (%)</th></tr><tr><th>D0</th><th>D4</th><th>D8</th><th>D14</th><th>D18</th><th>D4</th><th>D8</th><th>D14</th><th>D18</th><th>D4</th></tr></thead><tbody><tr><td rowspan="2">S. schwetzi</td><td>TOSV-A</td><td>100 (4/4)</td><td>17.9<br>(5/28)</td><td>0 (0/21)</td><td>0 (0/4)</td><td>-</td><td>0 (0/28)</td><td>0 (0/21)</td><td>0 (0/4)</td><td>-</td><td>0 (0/5)</td></tr><tr><td>TOSV-B</td><td>100 (6/6)</td><td>6.3 (2/32)</td><td>0 (0/28)</td><td>0 (0/10)</td><td>0 (0/12)</td><td>0 (0/32)</td><td>0 (0/28)</td><td>0 (0/10)</td><td>0 (0/12)</td><td>0 (0/2)</td></tr></tbody></table>

B

<figure><img src="image_9.png" /><figcaption>**Fig 4. Susceptibility of S. schwetzi to TOSV infection.** S. schwetzi sand flies were orally exposed to blood meal with either TOSV A or TOSV B (10⁶ PFU/ml) and collected at D4, D8, D14 and 18 p.i. to analyse their body and head with salivary glands for the presence of TOSV. (A) Summary values of infection rate (IR), dissemination rate (DR) and dissemination rate among infected sand flies (DI/R). (B) Infectious viral titers were obtained by TCID₅₀ limiting-dilution assays in body and head homogenates. Each dot represents a single sand fly. Light grey dashed lines indicate the threshold of virus quantification (1.78x10² TCID₅₀/ml).</figcaption></figure>

https://doi.org/10.1371/journal.pntd.0013031.g004

factors [48,49], infection dose [50–52], immunity [53–55], gut microbiome [56–58], co-infections [59,60] and temperature [61–63].

Here, we assessed the vector competence of four sand fly species: *P. tobbi*, *P. sergenti*, *P. papatasi* and *S. schwetzi* for genetically distinct TOSV strains from lineage A and B. We used two methods, TCID₅₀ assay and qPCR because viral RNA level is not always directly correlated to the infectivity of virus. According to our results, the molecular detection is more sensitive compared to the TCID₅₀ assay, however, the benefit of this assay is that it can detect infectious viral particles. Altogether, we observed a good correlation between viral RNA quantities and titers.

TOSV B appeared to be more successful in terms of infection and dissemination in sand flies than TOSV A at least for the strains tested. *P. tobbi* appeared to be the most susceptible species. To our knowledge, only TOSV of lineage A has been detected in *P. tobbi* [31]. However, this could be due to bias in field trapping methodology, such as testing in pools based on location and date of collection without specifying the species, combined with the limited sequence data available for sand flies. In the Eastern Mediterranean area, where two main TOSV lineages circulate, *P. tobbi* is indeed present [3]. Thus, it may only be a matter of time before lineage B is detected in this species. Our experimental results and the spread of TOSV over the last decade suggest that *P. tobbi* plays a role in the circulation and transmission of this virus, warranting
further research on this vector-TOSV combination. Moreover, *Phlebotomus tobbi* is the major vector of *Leishmania infantum*, *L. donovani* and their hybrids, causing visceral and cutaneous leishmaniasis in the Balkans, Turkey, Cyprus and Middle East [64–66]. There are currently almost no information or experimental research on the co-infection of TOSV and *Leishmania*, as well as their potential interaction in sand flies. Future studies are needed to explore this relationship. This study paves the way for further research in this area.

In the case of *P. sergenti*, a strong midgut barrier seems to prevent the dissemination of TOSV B strain, with a significantly lower infection rate than *P. tobbi* (61% vs 7.2%). However, when the virus did overcome these barriers, it disseminated to the head. Infection conditions for *P. tobbi* and *P. sergenti* may of course be different under natural conditions. In nature, females may blood feed repeatedly during one gonotrophic cycle, increasing vector competence as described for dengue, Zika and chikungunya viruses in mosquitoes [67]. It has been shown that infectious blood meal followed by a non-infectious blood meal increases the dissemination rate compared to single-fed females, probably due to blood meal-induced microperforations in the basal lamina surrounding the midgut epithelium that increases the probability of virus escape [67]. *Phlebotomus sergenti* is a highly anthropophilic sand fly and the primary vector of *Leishmania tropica* causing cutaneous leishmaniasis in Magreb area, Middle East, Turkey and Crete [36,68]. Thus, its susceptibility to TOSV has important epidemiological consequences.

To definitively confirm these two sand fly species as new TOSV vectors according to World Health Organization (WHO) criteria [69], transmission experiments between sand flies and laboratory animal models or detection of TOSV from saliva would be necessary. However, conducting such experiments with sand flies presents significant challenges. They are considerably more fragile than mosquitoes, exhibiting lower feeding and survival rates under laboratory conditions. This was evident in our experiments, where, for example, *P. tobbi* did not survive beyond D8 p.i.. Furthermore, individuals that survived the initial blood feeding were highly reluctant to feed again, making it nearly impossible to obtain a sufficient sample size for the transmission experiments.

*Sergentomyia schwetzi* is refractory to TOSV B strain infection. Only 2 positive females at D4 p.i. were positive, likely related to blood meal residues in the midgut. None of the females tested at later time points was positive for TOSV. We tested this species since TOSV RNA has been previously detected in *S. minuta* in France [30] and *S. schwetzi* is its closest relative species, colonized in the laboratory, and willing to blood feed on an artificial system. Although *S. schwetzi* could be different than *S. minuta* in terms of TOSV vector competence, we believed that it was important to determine if this species, found in Africa, could play a role in TOSV transmission [11, 33, 70, 71]. Our data suggest that *S. schwetzi* is not a TOSV competent vector.

Similarly, negative results were obtained for *P. papatasi* which also appears to be resistant to TOSV infection. *Phlebotomus papatasi* is widespread across Europe, Africa and Asia and is a well-known peridomestic and anthropophilic sand fly, recognized for its aggressive behaviour [36]. In recent years, its range has expanded northward into new regions, including the Balkans and Eastern Europe, up to Moldova [72,73]. Therefore, our finding that this species does not contribute to TOSV circulation has important epidemiological implications for all areas where *P. papatasi* is present.

A different situation has been observed with TOSV A. Various TOSV strains belonging to lineage A have been repeatedly detected in sand flies caught in the field, either from non-specified pools [70,74] or from pools of *S. dentata*, *P. papatasi*, *Phlebotomus* sp. [35], *P. tobbi*, *P. perfiliewi* s.l. [31], *P. perniciosus* [75] or individually from *P. major* s. l. [35]. However, in this study, the TOSV A strain tested did not appear to have much infectivity in any of the tested species; as females from D4 to D14 were completely negative. Whether this is due to the virus strain *per se*, its passage history of the virus, or genuine biological factors, remains to be determined. However, it cannot be excluded that infections detected in nature may be accidental. The development of a reverse genetics system for TOSV may be key to identify the viral determinant, if any, favouring TOSV B infection and dissemination ins and flies compared to TOSV A [76].

Overall, our results suggest that besides *P. perniciosus* and *P. perfiliewi*, other important anthropophilic sand fly species, particularly *P. tobbi* and possibly *P. sergenti*, play a role in TOSV transmission. To our knowledge, this is the first
successful viral infection of these species in a laboratory setting. Nevertheless, confirmation of TOSV transmission to the host is still required for the definitive confirmation of these species as TOSV vectors.

**Supporting information**

**S1 Table.** Samples that were analyzed in this study. The first sheet contains samples of *P. tobbi*, while the subsequent sheets contain samples of *P. sergenti*, *P. papatasi*, and *S. schwetzi*, respectively. The columns in the table indicate the following: the number of blood feeding experiment of each sand fly species, the specific code of each blood feeding experiment, the specific code of the sample, and the number of the sample with an explanation of which body part of sand fly it is (H - head with salivary glands, B - the rest of the body). The subsequent columns provide: information about the strain of TOSV (either TOSV A or TOSV B) that was used for blood feeding, the result of the TCID<sub>50</sub> assays, dissemination of TOSV in the sand fly (positive both head and body), the virus titers obtained by the TCID<sub>50</sub> assay (TCID<sub>50</sub>/ml), and notes about the results of the TCID<sub>50</sub> assay. The final six columns present the data from quantitative PCR (qPCR): the mean (from technical triplicate) crossing point (Cp) value of negative TOSV strand RNA measured by qPCR, standard deviation (STD) of the Cp value of TOSV negative strand RNA, the genome copy number counted from the sample Cp value of TOSV negative strand RNA and standard curve, STD of the genome copy number, the mean (from technical triplicate) Cp value of the TOSV positive strand RNA measured by qPCR and STD of the Cp value of the TOSV positive strand RNA. (XLSX)

**Acknowledgments**

**Disclaimer**

The contents of this publication are the sole responsibility of the authors and do not necessarily reflect the views of the European Commission, the Health and Digital Executive Agency, or UKRI. Neither the European Union nor granting authority nor UKRI can be held responsible. CLIMOS forms part of the Climate Change and Health Cluster comprising six Horizon Europe projects: BlueAdapt, CATALYSE, CLIMOS, HIGH Horizons, IDAlert, and TRIGGER.

**Author contributions**

**Conceptualization:** Magdalena Jancarova, Nikola Polanska, Frédérick Arnaud, Alain Kohl, Petr Volf, Maxime Ratinier.

**Formal analysis:** Adrien Thiesson.

**Funding acquisition:** Petr Volf, Maxime Ratinier.

**Investigation:** Magdalena Jancarova, Nikola Polanska, Adrien Thiesson, Marketa Stejskalova, Marketa Rehbergerova, Barbara Viginier.

**Methodology:** Magdalena Jancarova, Nikola Polanska, Barbara Viginier.

**Project administration:** Magdalena Jancarova.

**Visualization:** Adrien Thiesson.

**Writing – original draft:** Magdalena Jancarova, Nikola Polanska.

**Writing – review & editing:** Frédérick Arnaud, Alain Kohl, Petr Volf, Maxime Ratinier.

**References**

1. Kuhn JH, Brown K, Adkins S, de la Torre JC, Digiaro M, Ergünay K, et al. Promotion of order *Bunyavirales* to class *Bunyaviricetes* to accommodate a rapidly increasing number of related polyploviricotine viruses. J Virol. 2024;98(10):e0106924. https://doi.org/10.1128/jvi.01069-24 PMID: 39303014

2. Charrel RN, Bichaud L, de Lamballerie X. Emergence of Toscana virus in the mediterranean area. World J Virol. 2012;1(5):135-41. https://doi.org/10.5501/wjv.v1.i5.135 PMID: 24175218
3. Ayhan N, Prudhomme J, Laroche L, Bañuls A-L, Charrel RN. Broader Geographical Distribution of Toscana Virus in the Mediterranean Region Suggests the Existence of Larger Varieties of Sand Fly Vectors. Microorganisms. 2020;8(1):114. https://doi.org/10.3390/microorganisms8010114 PMID: 31947561

4. Keskek Turk Y, Ergunay K, Kohl A, Hughes J, McKimmie CS. Toscana virus - an emerging Mediterranean arbovirus transmitted by sand flies. J Gen Virol. 2024;105(11):002045. https://doi.org/10.1099/jgv.0.002045 PMID: 39508743

5. Popescu CP, Cotar Al, Dinu S, Zaharia M, Tardei G, Ceausu E, et al. Emergence of Toscana Virus, Romania, 2017–2018. Emerg Infect Dis. 2021;27(5):1482.

6. Punda-Polić V, Mohar B, Duh D, Bradarić N, Korva M, Fajs L, et al. Evidence of an autochthonous Toscana virus strain in Croatia. J Clin Virol. 2012;55(1):4–7. https://doi.org/10.1016/j.jcv.2012.06.006 PMID: 22795597

7. Ayhan N, Charrel RN. An update on Toscana virus distribution, genetics, medical and diagnostic aspects. Clin Microbiol Infect. 2020;26(8):1017–23.

8. Charrel RN, Gallian P, Navarro-Mari J-M, Nicoletti L, Papa A, Sánchez-Seco MP, et al. Emergence of Toscana virus in Europe. Emerg Infect Dis. 2005;11(11):1657–63. https://doi.org/10.3201/eid1111.050869 PMID: 16318715

9. Hemmersbach-Miller M, Parola P, Charrel RN, Paul Durand J, Brouqui P. Sandfly fever due to Toscana virus: an emerging infection in southern France. Eur J Intern Med. 2004;15(5):316–7. https://doi.org/10.1016/j.ejim.2004.05.006 PMID: 15450990

10. Muñoz C, Ayhan N, Ortuño M, Ortiz J, Gould EA, Maia C, et al. Experimental Infection of Dogs with Toscana Virus and Sandfly Fever Sicilian Virus to Determine Their Potential as Possible Vertebrate Hosts. Microorganisms. 2020;8(4):596. https://doi.org/10.3390/microorganisms8040596 PMID: 32326097

11. Alkan C, Allal-Ikhlef AB, Alwassouf S, Baklouti A, Piorkowski G, de Lamballerie X, et al. Virus isolation, genetic characterization and seroprevalence of Toscana virus in Algeria. Clin Microbiol Infect. 2015;21(11):1040.e1–9. https://doi.org/10.1016/j.cmi.2015.07.012 PMID: 26235198

12. Marchi S, Trombetta CM, Kistner O, Montomoli E. Seroprevalence study of Toscana virus and viruses belonging to the Sandfly fever Naples anti-genic complex in central and southern Italy. J Infect Public Health. 2017;10(6):866–9. https://doi.org/10.1016/j.jiph.2017.02.001 PMID: 28237695

13. Christova I, Panayotova E, Trifonova I, Taseva E, Gladnishka T, Ivanova V. Serologic evidence of widespread Toscana virus infection in Bulgaria. J Infect Public Health. 2020;13(2):164–6. https://doi.org/10.1016/j.jiph.2019.07.008 PMID: 31401037

14. Navarro-Marí JM, Palop-Borrás B, Pérez-Ruiz M, Sanbonmatsu-Gámez S. Serosurvey study of Toscana virus in domestic animals, Granada, Spain. Vector Borne Zoonotic Dis. 2011;11(5):583–7. https://doi.org/10.1089/vbz.2010.0065 PMID: 20925529

15. Alwassouf S, Maia C, Ayhan N, Coimbra M, Cristovao JM, Richet H, et al. Neutralization-based seroprevalence of Toscana virus and sandfly fever Sicilian virus in dogs and cats from Portugal. J Gen Virol. 2016;97(11):2816–23. https://doi.org/10.1099/jgv.0.000592 PMID: 27589865

16. Ayhan N, Alten B, Ivovic V, Martinkovic F, Kasap OE, Ozbel Y, et al. Cocirculation of Two Lineages of Toscana Virus in Croatia. Front Public Health. 2017;5:336. https://doi.org/10.3389/fpubh.2017.00336 PMID: 29312917

17. Pereira A, Ayhan N, Cristóvão JM, Vilhena H, Martins Â, Cachola P, et al. Antibody Response to Toscana Virus and Sandfly Fever Sicilian Virus in Cats Naturally Exposed to Phlebotomine Sand Fly Bites in Portugal. Microorganisms. 2019;7(9):339. https://doi.org/10.3390/microorganisms8090339 PMID: 31514266

18. Sellali S, Lafri I, Hachid A, Ayhan N, Benbetka C, Medrouh B, et al. Presence of the sandfly-borne phlebovirus (Toscana virus) in different bio-geographical regions of Algeria demonstrated by a microneutralisation-based seroprevalence study in owned dogs. Comp Immunol Microbiol Infect Dis. 2022;88:101861. https://doi.org/10.1016/j.cimid.2022.101861 PMID: 35926272

19. Ayhan N, López-Roig M, Monastiri A, Charrel RN, Serra-Cobo J. Seroprevalence of Toscana Virus and Sandfly Fever Sicilian Virus in European Bat Colonies Measured Using a Neutralization Test. Viruses. 2021;13(1):88. https://doi.org/10.3390/v13010088 PMID: 33440618

20. Hacioglu S, Dincer E, Isler CT, Karapinar Z, Ataseven VS, Ozkul A, et al. A Snapshot Avian Surveillance Reveals West Nile Virus and Evidence of Wild Birds Participating in Toscana Virus Circulation. Vector Borne Zoonotic Dis. 2017;17(10):698–708. https://doi.org/10.1089/vbz.2017.2138 PMID: 28832259

21. Ayhan N, Rodríguez-Teijeiro JD, López-Roig M, Vinyoles D, Ferreres JA, Monastiri A, et al. High rates of antibodies against Toscana and Sicilian phleboviruses in common quail *Coturnix coturnix* birds. Front Microbiol. 2023;13:1091908. https://doi.org/10.3389/fmicb.2022.1091908 PMID: 36687574

22. Verani P, Ciufolini MG, Caciolli S, Renzi A, Nicoletti L, Sabatinelli G, et al. Ecology of viruses isolated from sand flies in Italy and characterized of a new *Phlebovirus* (Arabia virus). Am J Trop Med Hyg. 1988;38(2):433–9. https://doi.org/10.4269/ajtmh.1988.38.433 PMID: 3128131

23. Hacioglu S, Ozkul A. Do birds play a role in the transmission of Toscana virus? Initial isolation results from birds in northernmost Türkiye. Zoonoses Public Health. 2024;71(3):225–35. https://doi.org/10.1111/zph.13100 PMID: 38041213

24. Ciufolini MG, Maroli M, Verani P. Growth of two phleboviruses after experimental infection of their suspected sand fly vector, *Phlebotomus perniciosus* (Diptera: Psychodidae). The Am J Trop Med Hyg. 1985;34(1):174–9.

25. Tesh RB. Studies of the biology of *Phleboviruses* in sandflies. Yale Univ. New Haven Conn. School of Medicine; 1987.

26. Tesh RB, Modi GB. Maintenance of Toscana virus in *Phlebotomus perniciosus* by vertical transmission. Am J Trop Med Hyg. 1987;36(1):189–93. https://doi.org/10.4269/ajtmh.1987.36.189 PMID: 3028194
27. Tesh RB, Lubroth J, Guzman H. Simulation of arbovirus overwintering: survival of Toscana virus (Bunyaviridae: Phlebovirus) in its natural sand fly vector *Phlebotomus perniciosus*. Am J Trop Med Hyg. 1992;47(5):574-81.

28. Killick-Kendrick R. The biology and control of phlebotomine sand flies. Clin Dermatol. 1999;17(3):279-89. https://doi.org/10.1016/S0738-081X(99)00046-2 PMID: 10384867

29. Maroli M, Feliciangeli MD, Bichaud L, Charrel RN, Gradoni L. Phlebotomine sandflies and the spreading of leishmaniases and other diseases of public health concern. Med Vet Entomol. 2013;27(2):123-47. https://doi.org/10.1111/j.1365-2915.2012.01034.x PMID: 22924419

30. Charrel RN, Izri A, Temmam S, De Lamballerie X, Parola P. Toscana virus RNA in *Sergentomyia minuta* flies. Emerg Infect Dis. 2006;12(8):1299.

31. Ergunay K, Kasap OE, Orsten S, Oter K, Gunay F, Yoldar AZA, et al. Phlebovirus and Leishmania detection in sandflies from eastern Thrace and northern Cyprus. Parasit Vectors. 2014;7:575. https://doi.org/10.1186/s13071-014-0575-6 PMID: 25499083

32. Es-Sette N, Ajaoud M, Bichaud L, Hamdi S, Mellouki F, Charrel RN, et al. *Phlebotomus sergenti* a common vector of *Leishmania tropica* and Toscana virus in Morocco. J Vector Borne Dis. 2014;51(2):86-90. https://doi.org/10.4103/0972-9062.134785 PMID: 24947214

33. Es-sette N, Ajaoud M, Anga L, Mellouki F, Lemrani M. Toscana virus isolated from sandflies, Morocco. Parasit Vectors. 2015;8:205. https://doi.org/10.1186/s13071-015-0826-1 PMID: 25886511

34. Daoudi M, Calzolari M, Boussaa S, Bonilauri P, Torri D, Romeo G, et al. Identification of Toscana virus in natural population of sand flies (Diptera: Psychodidae) from Moroccan leishmaniasis foci. J Infect Public Health. 2022;15(4):406-11. https://doi.org/10.1016/j.jiph.2022.03.007 PMID: 35339013

35. Özbel Y, Oğuz G, Arserim SK, Erişöz Kasap Ö, Karaoglu B, Yilmaz A, et al. The initial detection of Toscana virus in phlebotomine sandflies from Turkey. Med Vet Entomol. 2020;34(4):402-10.

36. Dvorak V, Shaw J, Volf P. Parasite biology: the vectors. Bruschi F, Gradoni L, editors. The Leishmaniases: Old Neglected Tropical Diseases. Cham: Springer International Publishing; 2018.

37. Volf P, Volfova V. Establishment and maintenance of sand fly colonies. J Vector Ecol. 2011;36 Suppl 1:S1-9. https://doi.org/10.1111/j.1948-7134.2011.00106.x PMID: 21366760

38. Buchholz UJ, Finke S, Conzelmann KK. Generation of bovine respiratory syncytial virus (BRSV) from cDNA: BRSV NS2 is not essential for virus replication in tissue culture, and the human RSV leader region acts as a functional BRSV genome promoter. J Virol. 1999;73(1):251-9. https://doi.org/10.1128/JVI.73.1.251-259.1999 PMID: 9847328

39. Charrel RN, Izri A, Temmam S, Delaunay P, Toga I, Dumon H, et al. Cocirculation of 2 genotypes of Toscana virus, southeastern France. Emerg Infect Dis. 2007;13(3):465-8. https://doi.org/10.3201/eid1303.061086 PMID: 17552102

40. Nougairede A, Bichaud L, Thiberville S-D, Ninove L, Zandotti C, de Lamballerie X, et al. Isolation of Toscana virus from the cerebrospinal fluid of a man with meningitis in Marseille, France, 2010. Vector Borne Zoonotic Dis. 2013;13(9):685-8. https://doi.org/10.1089/vbz.2013.1316 PMID: 23808972

41. Lindenbach BD. Measuring HCV infectivity produced in cell culture and in vivo. Tang H, editor. Hepatitis C. Methods in molecular biology™. Humana Press; 2009; vol 510.

42. Pérez-Ruiz M, Collao X, Navarro-Mari JM, Tenorio A. Reverse transcription, real-time PCR assay for detection of Toscana virus. J Clin Virol. 2007;39(4):276-81.

43. R Core Team. R: A language and environment for statistical computing. Vienna, Austria: R Foundation for Statistical Computing; 2018. Available from: https://www.R-project.org/

44. Franz AWE, Kantor AM, Passarelli AL, Clem RJ. Tissue Barriers to Arbovirus Infection in Mosquitoes. Viruses. 2015;7(7):3741-67. https://doi.org/10.3390/v7072795 PMID: 26184281

45. Kramer LD, Hardy JL, Presser SB, Houk EJ. Dissemination barriers for western equine encephalomyelitis virus in *Culex tarsalis* infected after ingestion of low viral doses. Am J Trop Med Hyg. 1981;30(1):190-7. https://doi.org/10.4269/ajtmh.1981.30.190 PMID: 7212166

46. Hardy JL, Houk EJ, Kramer LD, Reeves WC. Intrinsic factors affecting vector competence of mosquitoes for arboviruses. Annu Rev Entomol. 1983;28:229-62. https://doi.org/10.1146/annurev.en.28.010183.001305 PMID: 6131642

47. Carpenter A, Clem RJ. Factors Affecting Arbovirus Midgut Escape in Mosquitoes. Pathogens. 2023;12(2):220.

48. Behura SK, Gomez-Machorro C, deBruyn B, Lovin DD, Harker BW, Romero-Severson J, et al. Influence of mosquito genotype on transcriptional response to dengue virus infection. Funct Integr Genomics. 2014;14(3):581-9. https://doi.org/10.1007/s10142-014-0376-1 PMID: 24798794

49. Ciota AT, Chin PA, Ehrbar DJ, Micieli MV, Fonseca DM, Kramer LD. Differential effects of temperature and mosquito genetics determine transmissibility of arboviruses by *Aedes aegypti* in Argentina. Am J Trop Med Hyg. 2018;99(2):417.

50. Pesko K, Westbrook CJ, Mores CN, Lounibos LP, Reiskind MH. Effects of infectious virus dose and bloodmeal delivery method on susceptibility of *Aedes aegypti* and *Aedes albopictus* to Chikungunya virus. J Med Entomol. 2009;46(2):395-9.

51. Pongsiri A, Ponlawat A, Thaisomboonsuk B, Jarman RG, Scott TW, Lambrechts L. Differential susceptibility of two field *Aedes aegypti* populations to a low infectious dose of dengue virus. PLoS One. 2014;9(3):e92971. https://doi.org/10.1371/journal.pone.0092971 PMID: 24664142

52. Roundy CM, Azar SR, Rossi SL, Huang JH, Leal G, Yun R, et al. Variation in *Aedes aegypti* Mosquito Competence for Zika Virus Transmission. Emerg Infect Dis. 2017;23(4):625-32. https://doi.org/10.3201/eid2304.161484 PMID: 28287375
53. Xi Z, Ramirez JL, Dimopoulos G. The Aedes aegypti toll pathway controls dengue virus infection. PLoS Pathog. 2008;4(7):e1000098. https://doi.org/10.1371/journal.ppat.1000098 PMID: 18604274

54. Saraiva RG, Kang S, Simoes ML, Anglero-Rodriguez YI, Dimopoulos G. Mosquito gut antiparasitic and antiviral immunity. Dev Comp Immunol. 2016;64:53-64.

55. Tikhe CV, Dimopoulos G. Mosquito antiviral immune pathways. Dev Comp Immunol. 2021;116:103964.

56. Jupatanakul N, Sim S, Dimopoulos G. The insect microbiome modulates vector competence for arboviruses. Viruses. 2014;6(11):4294-313. https://doi.org/10.3390/v6i114294 PMID: 25393895

57. Angleró-Rodríguez YI, Talyuli OA, Blumberg BJ, Kang S, Demby C, Shields A, et al. An *Aedes aegypti*-associated fungus increases susceptibility to dengue virus by modulating gut trypsin activity. Elife. 2017;6:e28844. https://doi.org/10.7554/eLife.28844 PMID: 29205153

58. Wu P, Sun P, Nie K, Zhu Y, Shi M, Xiao C, et al. A Gut Commensal Bacterium Promotes Mosquito Permissiveness to Arboviruses. Cell Host Microbe. 2019;25(1):101-12.e5. https://doi.org/10.1016/j.chom.2018.11.004 PMID: 30595552

59. Vazeille M, Gaborit P, Mousson L, Girod R, Failloux A-B. Competitive advantage of a dengue 4 virus when co-infecting the mosquito *Aedes aegypti* with a dengue 1 virus. BMC Infect Dis. 2016;16:318. https://doi.org/10.1186/s12879-016-1666-0 PMID: 27390932

60. White AV, Fan M, Mazzara JM, Roper RL, Richards SL. Mosquito-infecting virus Espirito Santo virus inhibits replication and spread of dengue virus. J Med Virol. 2021;93(6):3362-73. https://doi.org/10.1002/jmv.26686 PMID: 33219544

61. Liu Z, Zhang Z, Lai Z, Zhou T, Jia Z, Gu J, et al. Temperature Increase Enhances *Aedes albopictus* Competence to Transmit Dengue Virus. Front Microbiol. 2017;8:2337. https://doi.org/10.3389/fmicb.2017.02337 PMID: 29250045

62. Ciota AT, Keyel AC. The Role of Temperature in Transmission of Zoonotic Arboviruses. Viruses. 2019;11(11):1013. https://doi.org/10.3390/v11111013 PMID: 31683823

63. Alomar AA, Alto BW. Temperature-Mediated Effects on Mayaro Virus Vector Competency of Florida *Aedes aegypti* Mosquito Vectors. Viruses. 2022;14(5):880. https://doi.org/10.3390/v14050880 PMID: 35632622

64. Svobodová M, Alten B, Zídková L, Dvorák V, Hlavacková J, Mysková J, et al. Cutaneous leishmaniasis caused by *Leishmania infantum* transmitted by *Phlebotomus tobbi*. Int J Parasitol. 2009;39(2):251-6. https://doi.org/10.1016/j.ijpara.2008.06.016 PMID: 18761342

65. Gouzelou E, Haralambous C, Amro A, Mentis A, Pratlong F, Dedet J-P, et al. Multilocus microsatellite typing (MLMT) of strains from Turkey and Cyprus reveals a novel monophyletic *L. donovani* sensu lato group. PLoS Negl Trop Dis. 2012;6(2):e1507. https://doi.org/10.1371/journal.pntd.0001507 PMID: 22348162

66. Rogers MB, Downing T, Smith BA, Imamura H, Sanders M, Svobodova M, et al. Genomic confirmation of hybridisation and recent inbreeding in a vector-isolated *Leishmania* population. PLoS Genet. 2014;10(1):e1004092. https://doi.org/10.1371/journal.pgen.1004092 PMID: 24453988

67. Armstrong PM, Ehrlich HY, Magalhaes T, Miller MR, Conway PJ, Bransfield A, et al. Successive blood meals enhance virus dissemination within mosquitoes and increase transmission potential. Nat microbiol. 2020;5(2):239-47.

68. Antoniou M, Gramiccia M, Molina R, Dvorak V, Wolf P. The role of indigenous phlebotomine sandflies and mammals in the spreading of leishmaniasis agents in the Mediterranean region. Euro Surveill. 2013;18(30):20540. https://doi.org/10.2807/1560-7917.es2013.18.30.20540 PMID: 23929183

69. World Health Organization. Arboviruses and human disease: report of a WHO scientific group [meeting held in Geneva from 26 September to 1 October 1966]. World Health Organization; 1967.

70. Fares W, Dachraoui K, Barhoumi W, Cherni S, Chelbi I, Zhioua E. Co-circulation of Toscana virus and *Leishmania infantum* in a focus of zoonotic visceral leishmaniasis from Central Tunisia. Acta Trop. 2020;204:105342. https://doi.org/10.1016/j.actatropica.2020.105342 PMID: 31954137

71. Saadawi WK, Abozaid FD, Almukhtar M, Annajar BB, Shaibi T. Seroprevalence study of Toscana virus in Yafran area, Libya. J Vector Borne Dis. 2022;59(2):186-9. https://doi.org/10.4103/0972-9062.335728 PMID: 36124486

72. Dvorak V, Kasap OE, Ivovic V, Mikov O, Stefanovska J, Martinkovic F, et al. (2020) Sand flies (Diptera: Psychodidae) in eight Balkan countries: historical review and region-wide entomological survey. Parasit Vectors. 2020;13:1-15.

73. Şuleşço T, Erisoz Kasap O, Halada P, Oğuz G, Rusnac D, Gresova M, et al. Phlebotomine sand fly survey in the Republic of Moldova: species composition, distribution and host preferences. Parasit Vectors. 2021;14(1):371. https://doi.org/10.1186/s13071-021-04858-4 PMID: 34289883

74. Calzolari M, Ferrarini G, Bonilauri P, Lelli D, Chiapponi C, Bellini R, et al. Co-circulation of eight different phleboviruses in sand flies collected in the Northern Apennine Mountains (Italy). Infect Genet Evol. 2018;64:131-4. https://doi.org/10.1016/j.meegid.2018.06.014 PMID: 29902582

75. Bichaud L, Izri A, de Lamballerie X, Moureau G, Charrel RN. First detection of Toscana virus in Corsica, France. Clin Microbiol Infect. 2014;20(2):O101-4. https://doi.org/10.1111/1469-0691.12347 PMID: 23992202

76. Alexander AJT, Confort M-P, Desloire S, Dunlop JI, Kuchi S, Sreenu VB, et al. Development of a Reverse Genetics System for Toscana Virus (Lingeage A). Viruses. 2020;12(4):411. https://doi.org/10.3390/v12040411 PMID: 32272808