# External quality assessment for molecular detection of sand fly-borne phleboviruses circulating in the Mediterranean Basin

Nazli Ayhan, Cecile Baronti, Laurence Thrion, Gioia Bongiorno, Carla Maia, Remi N Charrel, Ozge Erisoz Kasap, Ceylan Polat, Gizem Oguz Kaskan, Bulent Alten, et al.

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## To cite this version:

Nazli Ayhan, Cecile Baronti, Laurence Thrion, Gioia Bongiorno, Carla Maia, et al. External quality assessment for molecular detection of sand fly-borne phleboviruses circulating in the Mediterranean Basin. Parasites & Vectors, 2025, 18 (1), pp.173. 10.1186/s13071-025-06785-0. hal-05344680

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# External quality assessment for molecular detection of sand fly-borne phleboviruses circulating in the Mediterranean Basin

Nazli Ayhan¹,²*, Cecile Baronti¹, Laurence Thrion¹, Gioia Bongiorno³, Carla Maia⁴, Remi N. Charrel¹,⁵ and CLIMOS WP2 Phlebovirus Working Group

**Abstract**

**Background** Sand fly-borne phleboviruses (SbPV) are globally distributed and pose potential public health risks. Despite increased detection in recent decades, detailed knowledge of their ecology, characteristics and clinical relevance remains limited. Many cases of SbPV infection likely go unreported or misdiagnosed due to limited awareness and the lack of standardized screening. The External Quality Assessment (EQA) reported herein was organized within the framework of the European Union CLIMOS (EU Climate Monitoring and Decision Support Framework for Sand Fly-borne Diseases Detection and Mitigation) project. The aim of this EQA was to standardize the detection of phleboviruses in order to provide comparable data to feed mathematical models for the surveillance of the impact of climate changes and environmental parameters on the kinetics and diversity of sand fly species and on sand fly-borne microorganisms.

**Methods** Nine laboratories from seven countries participated in the EQA. Each laboratory was provided with eight vials, each containing an anonymous sample; two vials of lyophilized primers and probes to be used for the detection of Toscana virus (TOSV) and several Sandfly fever Sicilian virus (SFSV) species with a reverse-transcriptase PCR (RT-PCR) assay; and one vial of lyophilized primers for the detection of generic phleboviruses with a RT-PCR assay along with the standard operating procedure. The laboratories were instructed to submit their results together with details on the techniques employed.

**Results** All nine laboratories successfully detected the two TOSV- and the one SFSV-positive samples. Only one laboratory, using a generic phlebovirus assay, detected all of the targeted phleboviruses.

**Conclusions** All participating laboratories successfully identified the two TOSV and one SFSV using the proposed RT-qPCR assays, albeit with some variations in cycle threshold values across laboratories. The detection rate of SbPV was lower with the generic Phlebovirus assay than with the specific real-time RT-qPCR assays. This EQA aimed to assess the SbPV detection capabilities of molecular tools and strengthen their use, thereby supporting the involvement of laboratories in virus discovery and surveillance beyond their core expertise.

**Keywords** Phenuiviridae, Phlebovirus, Toscana virus, Sandfly fever Sicilian virus, External Quality Assessment, Phlebovirus toscanaense, Phlebovirus siciliaense, PCR, Detection

<footnote>*Correspondence:
Nazli Ayhan
nazli.ayhan@univ-amu.fr
Full list of author information is available at the end of the article</footnote>
**Background**

Sand fly-borne phleboviruses (SbPV, *Phenuiviridae* fam-
ily) have a worldwide geographical distribution and rep-
resent potential public health concerns [1, 2]. Although
the number of recognized SbPV has increased signifi-
cantly over the two last decades, reflecting heightened
surveillance efforts, comprehensive data on the char-
acteristics, ecological cycles and clinical significance
of most SbPV remain scarce. Many SbPV continue to
be underreported or wrongly identified due to a lack
of widespread awareness and systematic screening
protocols.

Toscana virus (TOSV; *Phlebovirus toscanaense*) and
Sandfly fever Sicilian virus (SFSV; *Phlebovirus sicili-
aense*), both human pathogens, are among the most
well-characterized SbPV. TOSV is of particular impor-
tance due to its ability to cause a spectrum of febrile ill-
nesses, ranging from mild flu-like symptoms, such as
fever, headache, fatigue and retro-orbital pain, to more
severe neuroinvasive diseases, including meningitis and
encephalitis. SFSV, on the other hand, is primarily associ-
ated with sandfly fever, commonly referred to as "three-
day fever," characterized by a sudden onset of high fever,
severe lethargy and general malaise lasting approximately
3–4 days [1–3]. Despite their clinical significance and
well-documented history, both of these viruses remain
neglected in routine diagnostic testing in many regions,
leading to underestimation of their true prevalence and
public health impact.

One of the major challenges in SbPV research and
diagnosis is the lack of standardized and widely accessi-
ble diagnostic tools. While specific assays for TOSV have
been developed, they are not routinely implemented in
clinical or laboratory settings in many endemic coun-
tries, further contributing to their underdiagnosis. Addi-
tionally, there is currently no commercially available
pan-generic assay capable of detecting all SbPV, despite
several individual and comparative diagnostic approaches
having been described in recent studies [4]. The absence
of such broad-spectrum diagnostic tools hinders compre-
hensive surveillance efforts, limiting the ability of such
efforts to assess the full epidemiological impact of SbPV
and their potential to cause emerging infectious diseases.

Vector surveillance programs also play a crucial role
in virus screening. Compared to mosquito vectors, sand
flies remain largely neglected despite their significance
in virus transmission. Studying SbPV in vectors provides
valuable insights into vector species specificity and natu-
ral transmission cycles, which are essential for under-
standing virus ecology, evolution and characterization.
*Phlebotomus perniciosus* and *Ph. perfiliewi* are recog-
nized as the primary vectors of TOSV and *Ph. papataci*
is the primary vector of SFSV. Additionally, recent vector

surveillance and virus screening efforts have led to the
identification of suggested larger varieties of sand fly vec-
tor species [5].

Given the increasing recognition of these viruses and
their potential for causing human diseases, it is impera-
tive to enhance surveillance, to improve diagnostic
capabilities, to understand the natural cycle and to raise
awareness among healthcare professionals and research-
ers. As a part of the European Union CLIMOS (EU Cli-
mate Monitoring and Decision Support Framework
for Sand Fly-borne Diseases Detection and Mitigation)
project, which aims to mitigate climate change impacts
on vector-borne and zoonotic diseases by applying Eco-
health and One Health approaches, with a focus on how
environmental factors influence sand flies and the spread
of sand fly-borne diseases in Europe, we conducted an
External Quality Assessment (EQA) with nine laborato-
ries to evaluate and improve detection capacities. This
initiative aims to address existing gaps in SbPV research,
thereby enabling a more comprehensive understanding
of the transmission dynamics and public health implica-
tions of these viruses, ultimately contributing to more
effective prevention and control strategies. Furthermore,
within the framework of the EU CLIMOS project, the
generation of comparable data is crucial for feeding the
mathematical models that assess the impact of climate
change and environmental factors on the dynamics and
diversity of sand fly species, as well as on the microorgan-
isms they carry.

The EQA study was designed to address the specific
molecular detection of TOSV and several SFSV species,
and for generic SbPV identification, using published
reverse transcription polymerase chain reaction (RT-
PCR) assays. Accordingly, ad-hoc lyophilized primers
and probes (Lyo-P&P) were distributed to nine labora-
tories together with the standard operating procedures
(SOP) and inactivated viruses and mock material. Here
we report the results and analyze the impact of the RNA
purification methods and of the molecular generic kits
used for SbPV detection.

**Methods**

**External quality assessment**

The detection capabilities of nine laboratories from seven
countries participating in the EQA, including five labo-
ratories from European/European Economic Area coun-
tries (Austria, France, Italy, Portugal and Spain), three
laboratories from Türkiye and one laboratory from Israel,
were assessed and evaluated by the French reference
laboratory (Aix Marseille University, France) between
March and September 2023. Each laboratory received a
questionnaire for describing the nucleic acid purification
method, PCR apparatus and PCR master mix used. After
<figure><img src="image_4.png" /><figcaption>**Fig. 1** Geographical representation of the nine laboratories from seven countries that participated in the External Quality Assessment. The numbers indicate the number of laboratories in each country</figcaption></figure>

<table><caption>**Table 1** List of selected phleboviruses included in the sand fly-borne phleboviruses External Quality Assessment panel</caption><thead><tr><th>Identification number</th><th>Virus species</th><th>Virus name/abbreviation</th><th>Virus strain</th><th>EVA number<sup>a</sup></th><th>GenBank number</th></tr></thead><tbody><tr><td>1</td><td>Phlebovirus salehabadense</td><td>Arbia virus/ARBV</td><td>UVE/ARBV/UNK/IT/Phl.35 M6</td><td>001v-EVA102</td><td>EU266620</td></tr><tr><td>2</td><td>Phlebovirus massiliaense</td><td>Massilia virus/MASV</td><td>UVE/MASV/2009/FR/M43</td><td>001 V-02369</td><td>NA</td></tr><tr><td>3</td><td>Phlebovirus puniquense</td><td>Punique virus/PUNV</td><td>Tunisie2009 T101</td><td>001 V-02383</td><td>OM362898</td></tr><tr><td>4</td><td>Phlebovirus siciliaense</td><td>Sandfly fever Sicilian virus/ SFSV</td><td>UVE/SFSV/1943/IT/Sabin</td><td>001v-EVA7</td><td>EF095551</td></tr><tr><td>5</td><td>Phlebovirus toscanaense</td><td>Toscana virus/TOSV</td><td>UVE/TOSV/2010/TN/ T152, Lineage A</td><td>001 V-02119</td><td>JX867534, JX867535, JX867536</td></tr><tr><td>6</td><td>Phlebovirus toscanaense</td><td>Toscana virus/TOSV</td><td>UVE/TOSV/2010/FR/4319, Lineage B</td><td>001 V-02442</td><td>KC776214 KC776215 KC776216</td></tr></tbody></table>

NA: Not available

questionnaire completion, each laboratory received eight vials of inactivated virus or mock samples as well as three vials of lyophilized primers and probes with a SOP (Additional file 1: Data 1) for sample manipulation and PCR methodology (Fig. 1).

## Panel composition

### Sample preparation

The EQA panel consisted of eight anonymized samples including six inactivated virus mentioned in Table 1 and two mock materials (registered non-therapeutic human plasma) (Table 1).

Non-infectious virus samples were prepared by using virus culture supernatant that was heat-inactivated at 60 °C for 1 h. Inactivation was demonstrated by the absence of cytopathic effect after two passages in Vero cells (ATCC CCL-81; American Type Culture Collection, Manassas, VA, USA) (CVCL_0059) and the lack of increased viral RNA titer in the supernatant 7 days after inoculation as measured by virus-specific RT-PCR assays. Each 2-ml glass vial was filled with 0.4 ml of inactivated material before lyophilization.

<footnote><sup>a</sup> European Virus Archive (EVA); accessible at https://www.european-virus-archive.com/evag-portal</footnote>
<table><caption>**Table 2** References, primer sequences, target segments and genes, positions, and amplicon size details of assays used in the External Quality Assessment</caption><thead><tr><th>Assay</th><th>Reference</th><th>Primer/Probe</th><th>5' → 3' Sequence</th><th>Target</th><th>Position</th><th>Amplicon size (in nucleotides)</th></tr></thead><tbody><tr><td rowspan="12">Toscana virus (TOSV) trio</td><td rowspan="3">[6]</td><td>STOS-F</td><td>TGCTTTTCTTGATGAGTCTGCAG</td><td rowspan="3">S RNA, N gene</td><td rowspan="3">1718–1807</td><td rowspan="3">89</td></tr><tr><td>STOS-R</td><td>CAATGCGCTTYGGRTCAAA</td></tr><tr><td>STOS-P</td><td>FAM-ATCAATGCATGGGTRAAT GAGTTTGCTTACC-TAMRA</td></tr><tr><td rowspan="3">[7]</td><td>TOS F</td><td>GGGTGCATCATGGCTCTT</td><td rowspan="3">S RNA, N gene</td><td rowspan="3">1381–1531</td><td rowspan="3">150</td></tr><tr><td>TOS R</td><td>GCAGRGACACCATCACTCTGTC</td></tr><tr><td>TOS P</td><td>FAM-CAATGGCATCCATAGTGG TCCCAGA-TAMRA</td></tr><tr><td rowspan="3">[8]</td><td>TOS-IMT-F</td><td>TCTCCCAGGAAATGACATCC</td><td rowspan="3">S RNA, N gene</td><td rowspan="3">621–725</td><td rowspan="3">104</td></tr><tr><td>TOS-IMT-R</td><td>AGATGGGWGTCTCTGGTCAT</td></tr><tr><td>TOS-IMT-P</td><td>FAM-TGTGGTYCAAGCAGCACG GGTG-TAMRA</td></tr><tr><td rowspan="12">Pan-SFSV (Sandfly fever Sicilian virus)</td><td rowspan="12">[9]</td><td>SFSV-AII-F</td><td>ATGGASGASTACCAGAARATYGC</td><td rowspan="12">S RNA, N gene</td><td rowspan="12">1655–1761</td><td rowspan="12">106</td></tr><tr><td>Corfou-Toros-F</td><td>ATGGAGGACTACCAGAAG ATCGC</td></tr><tr><td>Corfou-Toros-R</td><td>CTAGCATCAAAACCYTGGTAA GCAAA</td></tr><tr><td>SFSV-DAHV-R</td><td>CTGGCATCAAAAYCCYTGA TAIGCAAA</td></tr><tr><td>SFSV-DAHV-F</td><td>ATGGACGAGTACCAGAAA ATTGC</td></tr><tr><td>Corfou-Toros-P</td><td>FAM-TTCGGTGAGCAGGCTATA GATGA-TAMRA</td></tr><tr><td>SFSV-P1</td><td>FAM-TTTGGAGAACAGGCCATT GATGAG-TAMRA</td></tr><tr><td>SFSV-P2</td><td>FAM-TTTGGAGAGCAGGCTATT GATGAG-TAMRA</td></tr><tr><td>TBPVL2759F</td><td>CAGCATGGIGGICTIAGAGAGAT</td><td>L RNA</td><td>2786–3300</td><td>514</td></tr><tr><td>TBPVL3267R</td><td>TGIAGIATSCCYTGCATCAT</td><td>RdRp gene</td><td>2786–3309</td><td>523</td></tr><tr><td>HRT-GL2759F</td><td>CAGCATGGIGGIYTIAGRGAAATY TATGT</td><td></td><td></td><td></td></tr><tr><td>HRT-GL3276R</td><td>GAWGTRWARTGCAGGATICCY TGCATCAT</td><td></td><td></td><td></td></tr></tbody></table>

F Forward primer, L large, N viral nucleocapsid protein, P probe, R reverse primer, S small,

## Lyophilization

**Preparation and aliquoting** Sucrose was added to the inactivated virus samples to achieve a final concentration of 0.2 M. Tubes were placed on aluminum plates and pre-cooled at – 80 °C. Aliquots of 400 µL per tube were prepared, sealed with caps, and transferred to a pre-frozen lyophilizer (Cryotec—Bench-Top Pilot Lyophilizer).

**Lyophilization process** The lyophilization cycle was initiated using the Cryotec system. Upon completion, the vacuum was broken by adjusting the vacuum break knob. The chamber was filled with nitrogen by opening the nitrogen valve, and the tubes were sealed under nitrogen conditions to ensure sample integrity.

**Lyophilized primers and probes (Lyo-P&P)** Primers and probes for two real-time, reverse-transcriptase PCR (RT-qPCR) specific assays for TOSV and Pan-SFSV and primers for one conventional end-point Pan-Phlebovirus RT-PCR assay were lyophilized as described [11] (Table 2). The TOSV trio RT-qPCR assay included, in the same reaction, three monoplex assays [6-8] as previously described [12]. The Pan-SFSV RT-qPCR assay was not specific for SFSV but could also detect other SFSV-like viruses such as Corfou (CFUV), Dashli (DASV) and Toros (TORV) viruses. The Pan-Phlebovirus RT-PCR assay, is one of the most comprehensive assay for detecting phleboviruses [4].
**Submission, evaluation and EQA scoring of EQA results**

Participants received a Microsoft Excel (Microsoft Corp., Redmond, WA, USA) form to submit EQA results (Additional file 2: Data 2) on which they reported positive/ negative results for the three molecular assays and the amplification threshold cycle (Ct) values for the RT-qPCR assays (TOSV trio and Pan-SFSV). Data were collected and analyzed using Microsoft Excel 2011 to calculate the percentage of correct results for each assay and overall.

**Results**

**EQA participation**

The results of the nine laboratories from seven countries were analyzed, and the results are shown in Table 3.

**Nucleic acid extraction**

The techniques used for nucleic acid extraction are presented in Table 3. Five laboratories used nucleic acid extraction kits manufactured by Qiagen (Hilden, Germany), of which four were manual methods. The other laboratories used the extraction kits manufactured by Resnova s.r.l. (Brescia, Italy) (n=1), Promega (Madison, WI, USA) (n=1), Thermo Fisher Scientific (Waltham, MA, USA) (n=1) and Precision System Science Co., Ltd (MagLead system; Matsudo, Japan) (n = 1). For nucleic acid purification, the starting volume varied between laboratories (Table 3).

**Real-time RT-qPCR molecular detection methodology**

The real-time RT-qPCR kits used by the laboratories are presented in Table 3. Five laboratories used the RT-qPCR one-step SuperScript III Platinum kit manufactured by Thermo Fisher Scientific, and two laboratories used the RT-qPCR kits from Meridian Bioscience (Cincinnati, OH, USA). The TaqMan RT-qPCR kit from Thermo Fischer Scientific and RT-qPCR kits from Promega and NZYtech (Lisbon, Portugal) were used by two laboratories of which laboratory #5 used both the Thermo Fisher Scientific TaqMan and NZYtech kits.

**Conventional RT-PCR molecular detection methodology**

Table 3 presents the techniques used for conventional RT-PCR molecular detection methods. Six laboratories used three different Thermo Fisher Scientific kits. Kits from Meridian Bioscience (n=1), New England Biolabs (Ipswich, MA, USA) (n=1) and Qiagen (n=1) were also used.

**Real-time RT-qPCR and conventional RT-PCR results**

**TOSV samples using Toscana virus trio assay**

All nine laboratories successfully detected the two TOSV-positive samples. The Ct values varied for both TOSV lineage A (25.9–32.8) and for TOSV lineage B

(23.3–33.5) (Table 4). None of the laboratories reported results showing problems of carry-over contamination.

**SFSV sample using the Pan-SFSV assay**

All nine laboratories successfully detected the SFSV-positive sample, with Ct values ranging from 26.0 to 31.0 (Table 4). None of the laboratories reported results showing problems of carry-over contamination.

**All samples using the Pan-Phlebovirus assay**

Only laboratory #1 successfully detected all six samples of SbPV (Table 4). Four laboratories detected five out of six positive samples, three laboratories detected three positive samples and one laboratory detected two positive samples (Table 4). Unique virus (PUNV) and Arbia (ARBV) virus were detected by all nine laboratories, Massilia virus (MASV) was detected by eight laboratories, SFSV and TOSV lineage A was detected by 5 laboratories and TOSV lineage B was detected by only one laboratory.

**Evaluation of detection success rate**

For the TOSV trio assay and Pan-SFSV assay, all laboratories had 100% success of detection. In contrast, sample detection ranged between 33.3% and 100% for the Pan-Phlebovirus assay, with one laboratory detecting all six (100%) positive samples, and four, three and one laboratories detecting five (83.3%), three (50%) and one (33.3%) samples, respectively (Table 4). The overall SbPV detection success of the EQA was between 77.7% and 100% (Table 4).

**Discussion**

This EQA was organized within the framework of the EU CLIMOS Project to standardize detection of SbPV in order to provide comparable data to feed mathematical models for the surveillance of the impact of climate changes and environmental parameters on the kinetics and diversity of sand fly species and on the sand fly-borne microorganisms. Accordingly, the nine laboratories participating in this EQA were those participating in the CLIMOS project.

The distribution of sand flies in the Mediterranean region is well documented [13, 14]. Recent studies suggest that the relationship between SbPV and their phlebotomine vector species is not as restrictive as once thought [5]. It is likely that different SbPV can be transmitted by the same sand fly species and that a single sand fly species may carry different SbPV. In addition, sand flies are rarely tested individually but rather pooled in groups of 30 or 50 individual flies, so that the chance that two viruses are present in the same pool is not negligible. To better understand SbPV circulation, robust detection
<table><caption>**Table 3** Details on RNA extraction, PCR methods and kits and PCR volumes used by laboratories participating in the External Quality Assessment</caption><thead><tr><th>Laboratory identification number</th><th>Volume used for RNA purification (µl)</th><th>Elution volume (µl)</th><th>Volume of RNA per RT-PCR (µl)</th><th>Kit/apparatus used for extraction</th><th>Kit for real-time RT-qPCR</th><th>Kit for conventional RT-PCR</th></tr></thead><tbody><tr><td>Reference Laboratory</td><td>200</td><td>60</td><td>5</td><td>Qiagen: EZ1 Advanced XL with virus minikit v2.0 (9018708) (automated)</td><td>Thermo Fisher Scientific: RT-qPCRone-step SuperScript III Platinum (11732088)</td><td>Thermo Fisher Scientific: SuperScript™ III One-Step RT-PCR System with Platinum (10928042)</td></tr><tr><td>1</td><td>200</td><td>60</td><td>5</td><td>Resnova: MAGPURIX VIRAL/Pathogen Nucleic Acids Extraction Kit A (ZP02011) (automated)</td><td>Meridian Bioscience: 2 x SensiFAST Probe No-ROX Mix Bioline (BIO-76001)</td><td>Meridian Bioscience: MyTaqMix One Step RT-PCR Bioline (MDX062)</td></tr><tr><td>2</td><td>300</td><td>50</td><td>5</td><td>Promega: Maxwell® 16 Viral Total Nucleic Acid Purification Kit (AS1150) (automated)</td><td>Promega: GoTaq® Probe 1-Step RT-qPCR System (A6120)</td><td>Promega: Access RT-PCR System (A1260)</td></tr><tr><td>3</td><td>200</td><td>60</td><td>5</td><td>Thermo Fisher Scientific: MagMAX™ Viral/Pathogen Ultra Nucleic Acid extraction kit (A42356) (manual)</td><td>Thermo Fisher Scientific: RT-qPCRone-step SuperScript III Platinum (11732088)</td><td>Thermo Fisher Scientific: SuperScript™ III One-Step RT-PCR System with Platinum (12574018)</td></tr><tr><td>4</td><td>400</td><td>100</td><td>5</td><td>Precision System Science Co.: MagLEAD 12gC (MagDEA Dx SV 400) (automated)</td><td>Meridian Bioscience: Inhibitor-Tolerant RT-qPCR Mix (MDX016)</td><td>Qiagen: OneStep RT-PCR Kit (210212)</td></tr><tr><td>5</td><td>140</td><td>60</td><td>5</td><td>Qiagen: QiaAmp Viral RNA Mini Lit (52904) (manual)</td><td>NZY Tech: NZYSpeedy One-step RT-qPCR Probe Master Mix (MB35201)</td><td>Thermo Fisher Scientific: SuperScript™ IV First-Strand Synthesis System (18091050)</td></tr><tr><td>6</td><td>200</td><td>30</td><td>5 in RT-PCR, 4 in qPCR</td><td>Qiagen: RNeasy Mini Kit (74104) (manual)</td><td>Thermo Fisher Scientific: Taqman master mix (4444434)</td><td>Thermo Fisher Scientific: DreamTaq PCR Master Mix (2X) (K1071)</td></tr><tr><td>7</td><td>200</td><td>30</td><td>5</td><td>Qiagen: RNeasy Mini Kit (74104) (manual)</td><td>New England Biolabs: Luna Universal Probe One-Step RT-qPCR Kit (E30065)</td><td>Thermo Fisher Scientific: SuperScript™ III One-Step RT-PCR System with Platinum (10928042)</td></tr><tr><td>8</td><td>200</td><td>60</td><td>5</td><td>Qiagen: EZ1 Advanced XL with virus minikit v2.0 (9018708) (automated)</td><td>Thermo Fisher Scientific: RT-qPCRone-step SuperScript III Platinum (11732088)</td><td>Thermo Fisher Scientific: SuperScript™ III One-Step RT-PCR (11732020)</td></tr><tr><td>9</td><td>200</td><td>50</td><td>5</td><td>Qiagen: RNeasy Mini Kit (74104) (manual)</td><td>Thermo Fisher Scientific: RT-qPCRone-step SuperScript III Platinum (11732088)</td><td>Thermo Fisher Scientific: SuperScript™ III One-Step RT-PCR System with Platinum (12574018)</td></tr></tbody></table>

qPCR Quantitative PCR, RT-PCR reverse-transcriptase PCR, RT-qPCR reverse-transcriptase quantitative PCR
<table><caption>**Table 4** Results from the Toscana virus- and Sandfly fever Sicilian virus-specific reverse-transcriptase PCR assays and from the Pan-generic real-time PCR assays, from the different participating laboratories.</caption><thead><tr><th>Laboratory identity number</th><th>TOSV (positive n=2)</th><th>TOSV (Lineage A and B), Ct</th><th>%<sup>b</sup></th><th>Negative (n=6)</th><th>Pan-SFSV (positive n=1)</th><th>SFSV Ct<sup>a</sup></th><th>%<sup>b</sup></th><th>Negative (n=7)</th><th>Pan-Phlebovirus (positive n=6)</th><th>Detected phleboviruses<sup>c</sup></th><th>%<sup>b</sup></th><th>Negative (n=2)</th><th>Total<sup>b</sup></th></tr></thead><tbody><tr><td>Reference Laboratory</td><td>TOSV A 26.2<br>TOSV B 26.4</td><td>100</td><td>6</td><td>1</td><td>26.0</td><td>100</td><td>7</td><td>6</td><td>6</td><td>PUNV/ARBV,<br>MASV, SFSV,<br>TOSV A and B</td><td>100</td><td>2</td><td>100%</td></tr><tr><td>1</td><td>TOSV A 26.6 TOSV B 27.1</td><td>100</td><td>6</td><td>1</td><td>26.0</td><td>100</td><td>7</td><td>6</td><td>5</td><td>PUNV/ARBV,<br>MASV, SFSV,<br>TOSV A and B</td><td>100.0</td><td>2</td><td>100.0%</td></tr><tr><td>2</td><td>TOSV A 28.9 TOSV B 28.1</td><td>100</td><td>6</td><td>1</td><td>28.6</td><td>100</td><td>7</td><td>5</td><td>2</td><td>PUNV/ARBV,<br>MASV, SFSV, TOSV A</td><td>83.3</td><td>3</td><td>83.3%</td></tr><tr><td>3</td><td>TOSV A 32.8<br>TOSV B 32.1</td><td>100</td><td>6</td><td>1</td><td>29.2</td><td>100</td><td>7</td><td>2</td><td>3</td><td>PUNV/ARBV,<br>MASV</td><td>33.3</td><td>6</td><td>77.7%</td></tr><tr><td>4</td><td>TOSV A 25.9 TOSV B 26.1</td><td>100</td><td>6</td><td>1</td><td>23.3</td><td>100</td><td>7</td><td>3</td><td>3</td><td>PUNV/ARBV,<br>MASV</td><td>50.0</td><td>5</td><td>83.3%</td></tr><tr><td>5</td><td>TOSV A 30.0<br>TOSV B 30.0</td><td>100</td><td>6</td><td>1</td><td>31.0</td><td>100</td><td>7</td><td>5</td><td>5</td><td>PUNV/ARBV,<br>MASV, SFSV, TOSV A</td><td>83.3</td><td>3</td><td>94.4%</td></tr><tr><td>6</td><td>TOSV A 31.8<br>TOSV B 33.4</td><td>100</td><td>6</td><td>1</td><td>28.8</td><td>100</td><td>7</td><td>5</td><td>3</td><td>PUNV/ARBV,<br>MASV, SFSV, TOSV A</td><td>83.3</td><td>3</td><td>94.4%</td></tr><tr><td>7</td><td>TOSV A 31.2 TOSV B 33.5</td><td>100</td><td>6</td><td>1</td><td>29.7</td><td>100</td><td>7</td><td>3</td><td>3</td><td>PUNV/ARBV,<br>MASV</td><td>50.0</td><td>5</td><td>83.3%</td></tr><tr><td>8</td><td>TOSV A 28.9 TOSV B 29.5</td><td>100</td><td>6</td><td>1</td><td>27.1</td><td>100</td><td>7</td><td>5</td><td>5</td><td>PUNV/ARBV,<br>MASV, SFSV, TOSV A</td><td>83.3</td><td>3</td><td>94.4%</td></tr><tr><td>9</td><td>TOSV A 31.0<br>TOSV B 32.0</td><td>100</td><td>6</td><td>1</td><td>30.0</td><td>100</td><td>7</td><td>3</td><td>3</td><td>PUNV/ARBV,<br>MASV</td><td>50.0</td><td>5</td><td>83.3%</td></tr></tbody></table>

SFSV Sandfly fever Sicilian virus. For definitions of ARBV, MASV, PUNV and TOSV, see Table 1

<sup>a</sup> The specific RT-qPCR assay outcomes are included as cycle threshold (Ct) values

<sup>b</sup> The accuracy percentage was calculated for each individual assay and for the combined results (Total column)
assays are essential, particularly those capable of being
deployed across wide geographic regions.

Over the last few decades, numerous studies have
sought to screen vector-borne SbPV, leading to the dis-
covery of novel SbPV and the detection of known ones in
previously unrecognized areas [1–3, 15–17]. The ability
to detect and to identify circulating SbPV is also crucial
for recognizing potential pathogens and enhancing pre-
vention methods and strategies.

The three assays selected for testing in this study are
intended for use in epidemiological studies and virus dis-
covery/detection studies. They are not meant for diag-
nostic purposes, with the exception of the TOSV trio
assay [12].

We targeted two known pathogens in this EQA, TOSV and SFSV. For TOSV, a specific trio RT-qPCR assay was selected, comprising three monoplex systems. This assay was chosen because it had been previously validated also for the clinical diagnostics of TOSV infection [12]. For SFSV, we selected a real-time RT-qPCR assay that allows detection of not only SFSV but also the genetically closely related CFUV, DASV and TORV identified in Greece, Iran and Türkiye, respectively [16]. Interestingly, CFUV, DASV and TORV are now considered to be unique members of the *Phlebovirus corfouense*, *Phlebovirus dashliense* and *Phlebovirus toroense* species respectively. Whether these related viruses are pathogenic to humans remains unknown. It should also be noted that SFSV is also the unique member of the *Phlebovirus siciliaense*.

Additionally, the single Pan-Phlebovirus RT-PCR assay was able to detect the novel and already recognized SbPV (such as ARBV, MASV and PUNV viruses) incorporated into the EQA. For selecting the pan-generic assay we relied on a study that compared four assays [4]; then, based on these findings, we selected the system developed by Matsuno et al. [10] as it demonstrated a good balance between sensitivity and detection spectrum.

Among the six vials containing phleboviruses, the two
TOSV and one SFSV samples were detectable not only
using the RT-qPCR primers and probes but also by the
conventional and RT-PCR reagents. The contents of
three vials were detectable by conventional RT-PCR rea-
gents only, namely MASV, PUNV and ARBV. The two
latter viruses were detected by all nine participating
laboratories whereas MASV was detected by eight of the
nine laboratories. It should be emphasized here that spe-
cies identification was not part of the EQA trial since it
requires additional sequencing techniques.

Although all laboratories successfully identified the
two TOSV and the one SFSV using the proposed RT-
qPCR assays, there was some variations in the Ct values
across laboratories, possibly due to differences in RNA

extraction and/or RT-qPCR sensitivity. Four laboratories
using manual extraction kits reported higher Ct values,
potentially indicating RNA loss during extraction, which
is consistent with previous findings that manual extrac-
tion can result in lower viral RNA yield compared to
automated protocols [18]. For the Pan-Phlebovirus RT-
PCR assay, the SbPV detection rate was lower than that
of the specific real-time RT-qPCR assays. As the results
of the specific real-time RT-qPCR assays showed, the lab-
oratories who had high Ct values for the TOSV and SFSV
samples were unable to detect these samples with the
Pan-Phlebovirus RT-PCR assay. This outcome reflects the
known trade-off between the broad detection spectrum
and reduced sensitivity of pan-generic PCR assays. As
expected, the RT-qPCR assays showed a better sensitiv-
ity for detecting TOSV and SFSV compared to the Pan-
Phlebovirus generic RT-PCR assay, as previously shown
for TOSV [19]. The objectives of the two types of tests
were not the same: (i) the specific real-time TOSV test is
part of a detection/diagnosis logic, whereas (ii) the pan-
generic test approach (Pan-SFSV and Pan-Phlebovirus)
corresponds to a more open approach aimed at discover-
ing new viruses and assessing the presence of recognized
viruses in a given geographic area. The overall success
rate for SbPV detection ranged from 77.7% to 100%,
demonstrating the high detection capability among par-
ticipating laboratories.

It can be argued that strictly speaking, the study
reported here is not an EQA since the molecular detec-
tion tools were distributed to the participating labo-
ratories. Laboratories provided their own nucleic acid
extraction techniques, reverse transcriptase and Mas-
termix kits and PCR equipment. Moreover, most of the
participating laboratories did not routinely carry out
molecular detection of SbPV prior to this project, except
for the Italian and Israeli laboratories. The observed, sat-
isfactory results show that providing freeze-dried rea-
gents for diagnosis allows accurate detection within a
timeframe compatible with the completion of a scientific
project. Indeed, these laboratories are now implementing
phlebovirus detection locally on trapped sand flies with-
out the need to ship material to a reference laboratory.
Our results also lay the groundwork for the rapid deploy-
ment of a detection or diagnostic capability on a site that
has the technical tools, in the absence of specific exper-
tise in the field.

The aim of this EQA was not only to demonstrate the
laboratories' abilities to detect SbPV, but also to improve
the use of molecular tools that will enable research insti-
tutions to set up or take part in research and surveil-
lance programs in the fields of "virus discovery" or "virus
detection" in addition to their primary areas of expertise.
By strengthening infrastructure and expertise, capacity-building initiatives improve the ability of institutions to conduct accurate and timely molecular diagnostics, to contribute to research and development and to respond effectively to emerging public health challenges. Collaboration and knowledge-sharing among international laboratories are crucial components of these efforts. Ultimately, molecular capacity-building empowers countries to address local health issues more effectively and contribute to global health security.

## Conclusions

This EQA, conducted within the framework of the EU CLIMOS Project, successfully harmonized the molecular detection of SbPV by nine laboratories, enabling comparable data to be obtained for climate and environmental impact modeling. The study confirmed the high sensitivity of specific RT-qPCR assays for TOSV and SFSV, while the Pan-Phlebovirus RT-PCR assay proved valuable for broader virus detection despite its lower sensitivity.

Variation in Ct values, particularly higher Ct values from laboratories with using manual RNA extraction, highlights probable RNA loss during extraction. Importantly, this EQA facilitated the implementation of molecular detection tools in previously inexperienced laboratories, demonstrating the feasibility of decentralized viral screening.

Beyond evaluating detection capabilities, this EQA aimed to strengthen molecular capacity, supporting virus surveillance and discovery efforts. By fostering international collaboration, it enhances preparedness for emerging infectious diseases and contributes to global health security.

## Abbreviations

<table><tr><td>ARBV</td><td>Arbia virus</td></tr><tr><td>CFUV</td><td>Corfou virus</td></tr><tr><td>DASV</td><td>Dashli virus</td></tr><tr><td>EQA</td><td>External Quality Assessment</td></tr><tr><td>EU</td><td>European Union</td></tr><tr><td>F</td><td>Forward</td></tr><tr><td>L</td><td>Large segment</td></tr><tr><td>Lyo-P&amp;P</td><td>Lyophilized primers and probes</td></tr><tr><td>MASV</td><td>Massilia virus</td></tr><tr><td>N</td><td>Viral nucleocapsid protein</td></tr><tr><td>Nts</td><td>Nucleotides</td></tr><tr><td>P</td><td>Probe</td></tr><tr><td>PUNV</td><td>Punique virus</td></tr><tr><td>PCR</td><td>Polymerase chain reaction</td></tr><tr><td>R</td><td>Reverse</td></tr><tr><td>RT-qPCR</td><td>Reverse-transcriptase PCR</td></tr><tr><td>S</td><td>Small segment</td></tr><tr><td>SbPV</td><td>Sand fly-borne phleboviruses</td></tr><tr><td>SFSV</td><td>Sandfly fever Sicilian virus</td></tr><tr><td>SOP</td><td>Standard operating procedure</td></tr><tr><td>TORV</td><td>Toros virus</td></tr><tr><td>TOSV</td><td>Toscana virus</td></tr></table>

# Supplementary Information

The online version contains supplementary material available at https://doi.org/10.1186/s13071-025-06785-0.

Additional file 1: Data file 1.

Additional file 2: Data file 2.

## Acknowledgements

We would like to thank Camille Placidi, Gregory Molle, Sandie Escotte and Quentin di Brasi for technical assistance.

CLIMOS WP2 Phlebovirus Working Group: Ozge Erisoz Kasap¹, Ceylan Polat², Gizem Oguz Kaskan¹, Bulent Alten¹, Eduardo Berriatua³, José Risueño³, Clara Muñoz-Hernández³,⁴, Edwin Kniha⁵, Katharina Platzgummer⁵, Julia Walochnik⁵, Ricardo Parreira⁶, Yasemin Coşgun⁷, Umut Berberoğlu⁷, Oscar David Kirstein⁸, Victoria Indenbaum⁸, Ravit Koren⁸, Osnat Halpern⁸, Yaniv Lustig⁸, Muhammed Nalcaci⁹, Kardelen Yetismis⁹, Seray Toz⁹, Yusuf Ozbel⁹, Jerome Depaquit¹⁰, Nalia Mekarnia¹⁰, Claudia Fortuna¹¹, Giulia Marsili¹¹

¹Department of Biology, Ecology Section, Faculty of Science, VERG Laboratories, Hacettepe University, Ankara, Turkey. ²Department of Medical Microbiology, Faculty of Medicine, Hacettepe University, Ankara, Turkey. ³Departamento de Sanidad Animal, Facultad de Veterinaria, Campus Regional de Excelencia Internacional "Campus Mare Nostrum", Universidad de Murcia, Murcia, Spain.

⁴Health and Biotechnology Research Group (SaBio), Institute for Game and Wildlife Research (IREC), CSIC-UCLM-JCCM, Ciudad Real, Spain. ⁵Institute of Specific Prophylaxis and Tropical Medicine, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria (Kinderspitalgasse 15, 1090 Vienna). ⁶Global Health and Tropical Medicine, GHTM, LA-REAL, Instituto de Higiene e Medicina Tropical, Universidade NOVA de Lisboa, Lisbon, Portugal. ⁷General Directorate of Public Health, Ministry of Health, Microbiology Reference and Biological Materials Laboratory, Ankara, Turkey. ⁸National Zoonotic Reference Laboratory, Central Virology Laboratory, Ministry of Health, Sheba Medical Center, Israel. ⁹Department of Parasitology, Faculty of Medicine, Ege University, Izmir, Turkey. ¹⁰Université de Reims Champagne-Ardenne, Faculté de Pharmacie, ANSES, SFR Cap Santé, EA7510 ESCAPE-USC VECPAR, Reims, France, Laboratoire de Parasitologie-Mycologie, CHU de Reims, Reims, France. ¹¹Istituto Superiore di Sanità, Department of Infectious Diseases, Vector-borne diseases unit, Rome, Italy

## Author contributions

NA worked on data curation, formal analysis, methodology, writing and editing. RNC supervised and worked on data curation, writing and editing, CB, LT, GB and CM worked on the project administration, the methodology and writing the original draft. All authors contributed to the methodology. All authors reviewed and approved the final version of the manuscript.

## Funding

This study was funded by European Commission grant 101057690 and UKRI grants 10038150 and 10039289, and is catalogued by the CLIMOS Scientific Committee as CLIMOS number 022 (http://www.climos-project.eu). 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. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. For the purposes of Open Access, the authors have applied a CC BY [option: CC BY-ND] public copyright license to any Author Accepted Manuscript version arising from this submission. CLIMOS forms part of the Climate Change and Health Cluster comprising six Horizon Europe projects: BlueAdapt, CATALYSE, CLIMOS, HIGH Horizons, IDAlert and TRIGGER.

## Availability of data and materials

Data is provided within the manuscript or supplementary information files.

## Declarations

### Ethics approval and consent to participate

Not applicable.
**Consent for publication**

Not applicable.

**Competing interests**

The authors declare no competing interests.

**Author details**

¹Unite des Virus Emergents (UVE), L Université d Aix-Marseille–Universita di Corsica, IRD 190, Inserm 1207–L Institut de recherche biomédicale des armées (IRBA), 19–21, Bd. Jean Moulin, 13005 Marseille, France. ²Centre National de Reference des Arbovirus, Inserm-IRBA, Marseille, France. ³Department of Infectious Diseases, Vector-borne Diseases Unit, Istituto Superiore di Sanità, Rome, Italy. ⁴Global Health and Tropical Medicine (GHTM), Associate Laboratory in Translation and Innovation Towards Global Health, LA-REAL, Instituto de Higiene e Medicina Tropical (IHMT), Universidade NOVA de Lisboa, Lisboa, Portugal. ⁵Laboratoire des Infections Virales Aigues et Tropicales, AP-HM Hopitaux Universitaires de Marseille, Marseille, France.

Received: 7 January 2025 Accepted: 28 March 2025

Published: 12 May 2025

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