Evaluation of a Trio Toscana Virus Real-Time RT-PCR Assay Targeting Three Genomic Regions Within Nucleoprotein Gene
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pathogens Article Evaluation of a Trio Toscana Virus Real-Time RT-PCR Assay Targeting Three Genomic Regions within Nucleoprotein Gene Laurence Thirion 1 , Laura Pezzi 1,2 , Irene Pedrosa-Corral 3 , Sara Sanbonmatsu-Gamez 3, Xavier De Lamballerie 1, Alessandra Falchi 2, Mercedes Perez-Ruiz 3 and Remi N. Charrel 1,* 1 Unité des Virus Émergents (UVE: Aix-Marseille Univ-IRD 190-Inserm 1207-IHU Méditerranée Infection), 13005 Marseille, France; [email protected] (L.T.); [email protected] (L.P.); [email protected] (X.D.L.) 2 UR7310, Laboratoire de Virologie, Université de Corse-Inserm, 20250 Corte, France; [email protected] 3 Servicio de Microbiología, Hospital Universitario Virgen de las Nieves, Instituto de Investigación Biosanitaria ibs.Granada, 18014 Granada, Spain; [email protected] (I.P.-C.); [email protected] (S.S.-G.); [email protected] (M.P.-R.) * Correspondence: [email protected] Abstract: Toscana virus (TOSV) can cause central nervous system infections in both residents of and travelers to Mediterranean countries. Data mining identified three real-time RT-qPCR assays for detecting TOSV RNA targeting non-overlapping regions in the nucleoprotein gene. Here, they were combined to create a multi-region assay named Trio TOSV RT-qPCR consisting of six primers Citation: Thirion, L.; Pezzi, L.; and three probes. In this study, (i) we evaluated in silico the three RT-qPCR assays available in the Pedrosa-Corral, I.; literature for TOSV detection, (ii) we combined the three systems to create the Trio TOSV RT-qPCR, Sanbonmatsu-Gamez, S.; (iii) we assessed the specificity and sensitivity of the three monoplex assays versus the Trio TOSV Lamballerie, X.D.; Falchi, A.; RT-qPCR assay, and (iv) we compared the performance of the Trio TOSV RT-qPCR assay with one Perez-Ruiz, M.; Charrel, R.N. of the reference monoplex assays on clinical samples. In conclusion, the Trio TOSV RT-qPCR assay Evaluation of a Trio Toscana Virus performs equally or better than the three monoplex assays; therefore, it provides a robust assay that Real-Time RT-PCR Assay Targeting can be used for both research and diagnostic purposes. Three Genomic Regions within Nucleoprotein Gene. Pathogens 2021, Keywords: Phlebovirus; Phenuiviridae; meningitis; sand fly; Phlebotomus; arbovirus; Mediterranean; 10, 254. https://doi.org/10.3390/ diagnostics pathogens10030254 Academic Editor: Anna Honko Received: 26 October 2020 1. Introduction Accepted: 16 February 2021 Toscana virus (TOSV) (species Sandfly fever Naples phlebovirus, genus Phlebovirus, family Published: 24 February 2021 Phenuiviridae) is a negative-stranded, tri-segmented enveloped RNA virus transmitted by sandflies in the Mediterranean Basin [1]. Three genetic lineages have been identified to Publisher’s Note: MDPI stays neutral date, known as A, B, and C [2]. Lineage A has been reported so far in Italy, France, Turkey, with regard to jurisdictional claims in Tunisia, and Algeria from both human cases and sandfly vectors [2,3]. Lineage B strains published maps and institutional affil- have been described in Portugal, Spain, France, Morocco, Croatia, and Turkey [2,3]. TOSV iations. strains belonging to lineage C have been reported in Croatia and in Greece; although, the existence of this new lineage has been recognized based on partial sequences only, since the virus has not been isolated so far, nor characterized through complete genome sequencing [4–6]. Copyright: © 2021 by the authors. TOSV is the etiological agent of central nervous system infections in both residents Licensee MDPI, Basel, Switzerland. of and travelers to Mediterranean countries [3,7]. TOSV is also among the three most This article is an open access article prevalent causes of acute aseptic meningitis in TOSV endemic areas [7,8]. The disease can distributed under the terms and be severe, with almost 50% of neurological forms including encephalitis manifestations; conditions of the Creative Commons furthermore, permanent sequelae and fatal cases are rare but have been reported [2,9–11]. Attribution (CC BY) license (https:// Despite the evidence of TOSV circulation in an expanding number of countries and its creativecommons.org/licenses/by/ implication in neurological infections, there is little interest for this virus in the scientific 4.0/). Pathogens 2021, 10, 254. https://doi.org/10.3390/pathogens10030254 https://www.mdpi.com/journal/pathogens Pathogens 2021, 10, 254 2 of 11 community, and scarce knowledge among physicians on its symptoms spectrum [2]. Due to the lack of specificity of the clinical manifestations, laboratory diagnosis is necessary to differentiate TOSV infections from those caused by other etiological agents, and real-time RT-PCR is now considered the test of reference for diagnostic purpose at the acute stage of the infection [2,3]. The aim of this study was to perform data mining in order to identify peer-reviewed articles describing monoplex real-time RT-qPCR assays for detecting TOSV RNA, and to combine them to create a multi-region assay. Multi-region assays consist of the combination of two or more monoplex assays in the same reaction. The advantages of targeting different regions within the same gene have been recently described for SARS-CoV-2 [12]. A similar approach, consisting of targeting several regions in different genes, has been implemented for several pathogens such as human immunodeficiency virus (HIV), hepatitis B and C viruses (HBV and HCV), chikungunya virus (CHIKV), and Zaire ebolavirus (EBOV) [13–20]. In this study, (i) we evaluated in silico the three RT-qPCR assays available in the literature for TOSV detection, (ii) we combined the three systems to create the Trio TOSV RT-qPCR assay, (iii) we assessed the specificity and sensitivity of the three monoplex assays versus the Trio TOSV RT-qPCR assay, and (iv) we compared the performance of the Trio TOSV RT-qPCR assay with one of the reference monoplex assays on clinical samples. 2. Materials and Methods 2.1. Inventory of Published TOSV RT-qPCR Assays and Design of the Trio TOSV RT-qPCR Test TOSV RT-qPCR assays were searched for in NCBI PubMED using the search terms “Toscana”, “TOSV”, “polymerase chain reaction”, and “PCR” until April 2020. The three retrieved assays [21–23] were evaluated by in silico analysis using complete and partial se- quences covering the S RNA region representative of the three TOSV lineages (6 sequences of lineage A, 8 of lineage B, and 2 of lineage C). Moreover, strains from phylogenetically close phleboviruses belonging to Sandfly Fever Naples virus, Salehabad virus, and Sandfly Fever Sicilian virus serocomplexes were added to the alignment. The list of sequences is available in Figure1. The aim of this in silico analysis was to evaluate the adequacy of avail- able TOSV assays for covering existing genetic variability among TOSV lineages, without detecting viruses other than TOSV. Sequence alignments were performed by using MEGA 7 software. Primers and probes of the systems were plotted against sequence alignment and evaluated for the number and position of mismatches as previously described [19,20,24]. Figure 1. Cont. Pathogens 2021, 10, 254 3 of 11 Figure 1. In silico analysis of the three RT-qPCR for TOSV detection included in the Trio TOSV RT-qPCR assay. In blue, TOSV strains; in yellow, viruses belonging to Sandfly Fever Naples virus serocomplex; in green, viruses belonging to Salehabad virus serocomplex; in orange, Sandfly Fever Sicilian virus serocomplex. TOSV genetic lineage (A–C) is indicated after the symbol “_” in the strain’s name. 2.2. RT-qPCR Assays RT-qPCR reactions for the three monoplex assays and the “designed in this study” Trio TOSV RT-qPCR assay were performed with a SuperScript® III Platinum® One-Step RT-qPCR Kit with ROX (#11732-088, Invitrogen—Thermo Fisher Scientific, Waltham, MA, USA) on a BioRad CFX96TM thermal cycler, software version 3.1 (Bio-Rad Laboratories, Hercules, CA, USA). A volume of 5 µL of RNA was added to 20 µL of mix containing 12.5 µL of 2× Reaction Mix, 0.5 µL of Superscript III RT/Platinum Taq Mix, and primers and probes at the concentrations described in Table1. Cycling conditions were: 50 ◦C for 15 min; 95 ◦C for 2 min; 45 cycles of 95 ◦C for 15 s; 60 ◦C for 45 s. All probes were labeled with the same dye (FAM). There were no modifications for neither the sequence nor the concentrations of the primers and probes of the three monoplex assays when combined in the same reaction tube. The only difference is that the quencher of the probe described Pathogens 2021, 10, 254 4 of 11 by Pérez-Ruiz et al. has been modified to TAMRA instead of Dabcyl that was used in the original article. The reason for this is the need to have the same quencher for the probes of the three assays included in the Trio test. Table 1. Primers and probes included in the Trio TOSV RT-qPCR assay. Amplicon Reference Primer/Probe 5’!3’ Sequence Target Position a Concentration [nM] Size (nts) STOS-F TGCTTTTCTTGATGAGTCTGCAG 1718–1736 1000 Pérez-Ruiz et al. [23] STOS-R CAATGCGCTTYGGRTCAAAS RNA, N gene 1785–180790 1000 STOS-P FAM-ATCAATGCATGGGTRAATGAGTTTGCTTACC-TAMRA 1742–1772 200 TOS F GGGTGCATCATGGCTCTT 1381–1398 500 Weidmann et al. [22] TOS R GCAGRGACACCATCACTCTGTCS RNA, N gene 1510–1531151 500 TOS P FAM-CAATGGCATCCATAGTGGTCCCAGA-TAMRA 1415–1439 200 TOS-IMT-F TCTCCCAGGAAATGACATCC 621–640 400 Brisbarre et al. [21] TOS-IMT-R AGATGGGWGTCTCTGGTCATS RNA, N gene 706–72585 400 TOS-IMT-P FAM-TGTGGTYCAAGCAGCACGGGTG-TAMRA 654–675 200 a Refers to the sequence of TOSV strain 181135-14 (GenBank accession number KU573066). 2.3. Generation of RNA Synthetic Transcript (standard RNA) An in-house synthetic standard RNA was used for the evaluation process of the Trio TOSV RT-qPCR assay, containing the three regions targeted by the three monoplex RT-qPCR assays included in the Trio assay.