Journal of Clinical Virology 66 (2015) 107–111
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A rapid and specific real time RT-PCR assay for diagnosis of Toscana virus infection Nadège Brisbarre a,∗ , Sébastien Plumet b,1 , Christelle Cotteaux-Lautard c,2 , Sébastien F. Emonet b,3 , Frédéric Pages c,4 , Isabelle Leparc-Goffart b,5 a
UMR 7268 ADES, Aix-Marseille Université/EFS/CNRS, 27 Boulevard Jean Moulin, 13385 Marseille Cedex 05, France CNR Arbovirus IRBA (Institut de Recherche Biomédicale des Armées), 111 Avenue de la Corse BP40026, 13568 Marseille Cedex 02, France c Unité d’entomologie Médicale, IRBA (Institut de recherche Biomédicale des Armées), 111 Avenue de la Corse BP40026, 13568 Marseille Cedex 02, France b
a r t i c l e
i n f o
Article history: Received 8 August 2014 Received in revised form 26 February 2015 Accepted 6 March 2015 Keywords: Toscana virus Bunyaviridae Arbovirus Diagnosis Real-time RT-PCR
a b s t r a c t Background: To scan a virus (TOSV) belongs to the Phlebovirus genus within the Bunyaviridae family. TOSV is an arbovirus transmitted by sandflies. In Mediterranean countries, TOSV is one of the major viral pathogens involved in aseptic meningitis and meningoencephalitis. Objectives: Development and assessment of a new sensitive and specific real-time RT-PCR assay for TOSV diagnosis. Study design: TOSV-specific primers and probe targeting the S-segment of the genome were designed, based on recent TOSV sequences available in public databases. Sensitivity was assessed using 10-fold serial dilutions of a RNA transcript and serial dilutions of TOSV strains isolated from infected human beings. Specificity was determined by testing RNA extracts from closely related Phleboviruses. The assay was then used for TOSV infection diagnosis in 971 clinical samples and for TOSV detection in 2000 sandflies. Results: The real-time RT-PCR assay exhibited a sensitivity of under 257 copies per reaction for the RNA transcripts and 0.0056 and 0.014 TCID50 of Italian and Spanish TOSV genotypes per reaction, respectively. No other close Phleboviruses were detected. TOSV was identified in 17 clinical samples and in 3 sandflies. Conclusions: The assay described is a rapid, robust and reliable real-time RT-PCR test for accurate diagnosis of human TOSV infection as well as for the surveillance of TOSV in vector populations. © 2015 Elsevier B.V. All rights reserved.
1. Background
∗ Corresponding author. Tel.: +33 491324553. E-mail addresses:
[email protected] (N. Brisbarre),
[email protected] (S. Plumet),
[email protected] (C. Cotteaux-Lautard),
[email protected] (S.F. Emonet),
[email protected] (F. Pages),
[email protected] (I. Leparc-Goffart). 1 Permanent address: DGA MNRBC, Site du Bouchet, 5 rue Lavoisier, 91710 Vert le Petit, France. 2 Permanent address: UMR-MD1, Faculté de Médecine, 27 Boulevard Jean Moulin, 13385 Marseille Cedex 05, France. 3 Permanent address: Unité de Virologie, Institut de Recherche Biomédicale des Armées, 21 Avenue Tony Garnier, 69007 Lyon, France. 4 Permanent address: Cire Océan Indien, Institut de Veille Sanitaire (InVS), Saint Denis, la Réunion Ou Cire OI, ARS Océan Indien, 2 bis Avenue Georges Brassens, CS 61002–97743 Saint Denis Cedex 9, La Reunion, France. 5 Permanent address: HIA Laveran, 34 Boulevard Laveran, 13013 Marseille, France. http://dx.doi.org/10.1016/j.jcv.2015.03.007 1386-6532/© 2015 Elsevier B.V. All rights reserved.
Toscana virus (TOSV) is a member of the sandfly fever Naples group, genus Phlebovirus, family Bunyaviridae. TOSV is an enveloped virus, with a genome composed of three single-stranded RNA segments. The large segment (L) encodes the RNA-dependent RNA-polymerase and the medium segment (M), the most variable, encodes viral glycoproteins (GN , GC ) and a non-structural protein (NSM ); the small segment (S), that utilizes an ambisense coding strategy, encodes the nucleoprotein (N) and a non-structural protein (NSS ) [1–3]. TOSV is an arbovirus transmitted by sandflies from the Phlebotomus perniciosus and Phlebotomus perfiliewi species, which are present in northern Africa and Western Europe (Spain, Italy and France) and along the coast in North Africa and from Italy to Eastern Turkey [4]. The virus was first isolated in Italy from a phlebotomine sandfly in 1971. As its infection is generally asymptomatic, TOSV was only associated with human disease in 1983 when the virus was isolated
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Table 1 Primers and probes in the S-segment for Toscana virus detection. Name
Sequence (5 −4 )
Nucleotide position
TOS-IMT-F TOS-IMT-R TOS-IMT-P TOS-S-F Tos-ST7-F Tos-S-R
TCTCCCAGGAAATGACATCC AGATGGGWGTCTCTGGTCAT FAM-TGTGGTYCAAGCAGCACGGGTG-TAMRA TAGGGAGATGCAATCCAGAGCTGTCATTCT TAATACGACTCACTATAGGGAGATGCAATCCAGAGCTGTCATTCT TCATAGGGGTGGGTAGTGGGGGGGA
594–613 678–698 627–648 25–56 25–56 960–984
Nucleotide positions were determined on sequence FJ153286.
from a patient with acute meningitis [5,6]. Subsequent cases have been reported repeatedly in Italy, Spain, France, Portugal, Greece, Cyprus, Turkey, Croatia, Bosnia-Herzegovina, Morocco and Tunisia [7]. TOSV is now known to be a major cause of aseptic meningitis and meningoencephalitis in the Mediterranean Basin during the summer season (June–October) with a peak in August when vector activity is high. TOSV is endemic in the French Mediterranean Basin and its surveillance is associated to the surveillance of the WestNile virus [8,9]. Only limited epidemiological data are available in France. A study conducted from 2000 to 2002 showed the involvement of TOSV in 1.5% of encephalitis [10]. In the city of Marseille (South of France) TOSV immunoglobulin G (IgG) was detected in 18.9% of patients hospitalized with central nervous system (CNS) infection [11]. Among blood donors from the south of France, two studies indicated seroprevalence (IgG) ranging from 6.25 to 13.3% [12]. TOSV is currently the only Phlebovirus of medical importance identified in France. Phylogenetic studies have shown that TOSV can be divided in two different genotypes (genotypes A and B), which cocirculate in France [13,14]. Besides TOSV, two other Phleboviruses transmitted by sandflies can be encountered in the Mediterranean Basin: sandfly fever Naples virus (SFNV) and sandfly fever Sicilian virus (SFSV), which also lead to a self-limited flu-like syndrome in humans [4]. It appears important for diagnosis and global surveillance to use a specific real-time RT-PCR system, that allows detection of the 2 TOSV genotypes and that discriminate it from other viruses of the same genus (particularly from SFNV and SFSV).
Glutamax Dulbecco’s Modified Eagle Medium (DMEM, Gibco: Life Technologies, Saint Aubin, France) supplemented with 5% heatinactivated fetal calf serum (FCS, Eurobio, Courtaboeuf, France). Each viral strain was checked by sequence analysis in the S-segment (data not shown). 3.2. Virus titration Virus titers were measured using the 50% Tissue Culture Infective Dose assay (TCID50 ). Briefly, tenfold dilutions were used to infect confluent Vero cells in a 96-well plate in DMEM (2% FCS) at 37 ◦ C. Wells were classified as positive (cytopathic effect) versus negative (no cytopathic effect) after 4 days of culture. Titers (TCID50 /ml) were calculated using the Reed–Muench method [15]. TOSV, SFNV and SFSV titers were all close to 106 –107 TCID50 /ml. 3.3. Field-caught sandfly samples Sandflies (n = 2000) were trapped in the South–East of France overnight from July to September 2009, using CDC miniature light traps (John W. Hock, Gainesville, FL, USA). Insects were transported alive to the laboratory, stored at −20 ◦ C and identified. Abdomens and thoraxes were individually ground with glass beads in phosphate buffered saline (PBS; Lonza, Amboise, France) 3% milk. For real-time RT-PCR TOSV detection, pools of 10–25 flies were grouped according to sex and species.
2. Objectives
3.4. Clinical samples
Objectives of this study were to develop a robust and specific real-time RT-PCR, TOSV-IMT assay, that (i) was validated for detection of both TOSV genotypes, (ii) could discriminate between TOSV and close viral neighbors, (iii) was efficient for the diagnosis of TOSV infection in humans and in sandflies. Molecular detection is widely used in laboratories since this approach is rapid and sensitive. Specificity of the assay was assessed using three different Phleboviruses (SFNV, SFSV, TOSV strains). Sensitivity was determined according to the number of RNA transcript copies but also according to infectious titer. This real-time RT-PCR assay was used for TOSV diagnosis in clinical samples, such as cerebrospinal fluid (CSF), serum and plasma as well as in insects.
Clinical samples (n = 971) came from patients with fever, meningitis or meningoencephalitis without traveling history and living in the south of France, suspected to have been infected with West Nile virus (WNV) and hospitalized between May and October 2009–2013. These samples had previously been analyzed by specific real-time RT-qPCR assays for SFNV, SFSV, WNV (data not shown) and for TOSV (assay of this study) [16,17].
3. Study design 3.1. Virus and cells Viruses used were: SFNV (patient isolate NAMRU 840055/R3like), SFSV (Sabin strain) and 3 isolates of TOSV: H/IMTSSA isolate (FJ153286) and 2 isolates of Spanish (1.107 TCID50 /ml) and Italian (4.107 TCID50 /ml) lineage (Timone Hospital, Marseille, France). Propagation of TOSV, SFNV, SFSV was performed in subconfluent VERO cells using a multiplicity of infection of 0.1 in
3.5. Primers and probe design Primers and the probe used in this study (Table 1) were designed to target a conserved region of the S-segment (Fig. 1), using alignments of selected sequences from the NCBI GenBank database (FJ153285, FJ153286, EF120631, EU327772, X53794, EF201828, EF201833). Our system amplifies a short sequence (85 bp) of the N gene and uses a dual-labeled probe (5 FAM/3 TAMRA). 3.6. RNA extraction RNA was extracted from 140 l of viral (TOSV, SFNV or SFSV) supernatants, serum, CSF or insect pool samples using a QIAamp viral RNA mini kit (Qiagen, Hilden, Germany), according to manufacturer’s recommendations.
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Fig. 1. Partial nucleotide sequence alignment of Phleboviruses of the S-segment. Sequences of forward (F) and reverse (R) primers and probe (P) targeting this region are indicated in bold. Partial sequences of Toscana virus (TOSV): TOSV ELB (EF201833), H/IMTSSA (FJ153286), ESH 62100 (FJ153285), GR40 (EF120631), SI-1812 (EU327772), ISS Phl. 3 (X53794) and Sandfly Naples fever virus (SFNV): SFNV NAMRU 840,055 (EF201828), SFNV NAMRU 840,055 (EF201829), SFNV R-3 (EF201832), SFNV YU 8-76 (EF201831), SFNV P7101795 (EF201830) are shown. Viruses isolated from human samples are in classical format and those from Phlebotomus are in Italic. Dots represent identical nucleotides when compared with the aligned primers/probe sequences or reference sequences. Black star represents homology due to mixed base inclusion.
3.7. Preparation of RNA standard An in vitro transcribed RNA from a portion of the N gene of TOSV H/IMTSSA isolate was used to validate our real-time RT-PCR assay. Primers TOS-S-F/R were used to amplify part of the N gene (Table 1). The PCR product was purified using a QIAquick gel extraction kit (Qiagen, Hilden, Germany) and ligated into the pCR2.1 vector using a TA cloning kit (Invitrogen: Life Technologies, Saint Aubin, France). The recombinant plasmid was used to transform competent One Shot INV␣F´ı Chemically Competent Escherichia coli 20 (Invitrogen: Life Technologies, Saint Aubin, France). The sequence of selected clones was checked by sequencing. The insert was amplified using the primers TOS-S-T7-F/TOSV-R (Table 1) and then purified using a QIAquick gel purification kit (Qiagen, Hilden, Germany). In vitro transcription was performed from 1 g of purified DNA by using the T7 RNA polymerase (Roche Diagnostics, Meylan, France) via the T7 promoter introduced in the primer TOS-S-T7-F. In order to remove the DNA template, the reaction mixture was incubated with 20 U of DNase I (Roche Diagnostics, Meylan, France) and resulting RNA was then purified with an RNeasy Mini Kit (Qiagen, Hilden, Germany). The number of RNA transcript copies (molecules/l) was calculated as follows: C × A/L where C represents the concentration of RNA (g/ml) assessed by OD measurement, A is Avogadro’s number (6.023 × 1023 ) and L is the estimated molecular weight of the RNA transcript.
primers and probe were tested with blast analysis to determine potential hybridization with other nucleotide sequences of close Phleboviruses (SFNV and SFSV). The primers/probe demonstrated better hybridization specificity for TOSV nucleotide sequences than previous published detection systems, particularly concerning the closer SFNV nucleotide sequence that can cause some cross reactivity (Fig. 1). 4.2. Sensitivity and specificity assessment The detection limit of the TOSV-IMT assay was assessed using quantified RNA in vitro transcripts. Transcribed TOSV H/IMTSSA RNA was diluted in the range of 109 –101 molecules/l and then tested by TOSV-IMT RT-qPCR. The limit of detection was estimated to be at least 257 copies of RNA transcript per reaction (Fig. 2A). RNA standard detection was linear over a 7 log10 range from 2.57 × 109 (Ct 9.88) to 2.57 × 103 (Ct 31) (R2 > 0.99). Within this range, efficiency was assessed at 88% on the
3.8. Real-time RT-PCR assay The reaction was performed on a LightCycler instrument (Roche Diagnostics, Meylan, France). The assay was carried out with the SuperScript III Platinum One-Step Quantitative RT-PCR System (Invitrogen: Life Technologies, Saint Aubin, France). Reaction mix composition was: 5 l RNA extract or 2 l RNA transcript dilution, 0.4 M TOS-IMT-F, 0.4 M TOS-IMT-R and 0.2 M TOS-IMT-P in reagent mix (following the manufacturer’s instructions). Reaction conditions were: RT at 45 ◦ C/15 min, polymerase activation at 95 ◦ C/2 min, 40 cycles of PCR at 95 ◦ C/15 s, 57 ◦ C/5 s and 60 ◦ C/30 s. A single fluorescence reading was taken in each cycle at the extension step. Measurements were expressed according to a determined cycle threshold (Ct) value. All assays were validated by positive and negative controls. 4. Results 4.1. Design of TOSV specific primers and probe Primers and the probe target the S-segment of TOSV, which is a well conserved segment largely represented in sequence databases and for which new sequences are submitted regularly [13]. The
Fig. 2. Assessment of TOSV-IMT assay sensitivity and specificity. Sensitivity was assessed with 10-fold serial dilutions of RNA transcript (from 109 to 101 molecules/l). Analysis was performed in triplicate on two different LightCycler automates (A). Sensitivity and specificity were tested with 10-fold serial dilutions ranging from 104 to 10−3 TCID50 /l for Italian strains (B) and from 2.8 × 103 to 2.8 × 10−3 TCID50 /l for Spanish strains (C) (analysis was performed in duplicate for Spanish strains).
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basis of linear regression of triplicate data (slope −3.66, R2 > 0.99). Inter-assay coefficient variation of the mean Ct was 0.09% (range 0.16–0.06%). TOSV-IMT assay sensitivity was also tested on two strains of TOSV (Italian and Spanish, 4.107 and 1.107 TCID50 /ml, respectively) (Fig. 2B and C). Ten-fold serial dilutions were used for each titrated strain. RNA was extracted from infected supernatant and 5 l of each TOSV dilution were tested using the TOSV-IMT system. Results showed that the detection limits of the assay for Italian and Spanish strains were 0.0056 and 0.014 TCID50 /reaction, respectively. Reactions for each strain showed good linearity until ∼10−1 TCID50 /l. Moreover, the assay efficiency in the linear range for Italian and Spain strains was 87.5% (slope −3.66, R2 > 0.99) and 102% (slope −3.26, R2 > 0.98), respectively. Specificity was determined by analyzing RNA from TOSV and from two close Phleboviruses: SFNV and SFSV liable to exhibit some cross reactivity with TOSV. Viral RNA was extracted from infected supernatants of VERO cultures and tested by TOSV-IMT assay. As a result, only TOSV RNA was specifically amplified but neither SFNV nor SFSV RNAs. 4.3. TOSV investigation in clinical samples TOSV-IMT assay was then used for TOSV infection diagnosis on clinical samples. A network has been set up in the south of France for the specific surveillance of WNV infection. Samples from patients with suspected viral meningitis or meningoencephalitis were sent to the French National Reference Center for Arboviruses for WNV, TOSV, SFNV and SFSV diagnosis. Overall a total of 971 samples (CSF and sera) were received between 2009 and 2013. RNA was extracted from each serum or CSF sample and tested using specific realtime assays. All samples were negative for the RNA detection of WNV, SFNV and SFSV. 17 patients (1.75%) were found positive by the TOSV-IMT assay (with 5% alpha error) (Table 2). All of these patients had clinical signs such as fever and CNS diseases. For 12 of the 17 patients molecular diagnosis was confirmed by serological diagnosis. For 5 of the 17 patients, seroconversion (confirmation diagnosis) could not be attested since no subsequent sample was sent to the laboratory (10–15 days after the first sample). Virus isolation on VERO cells was attempted on the 6 positive samples from 2009. TOSV was isolated from 1CSF (strain TOSV/Pt11368/FR/2009). 4.4. TOSV investigation in insect samples Human cases of Toscana virus infection lead to an entomological study. CDC light traps for sandflies were hung overnight in the areas where patients were suspected to have been infected. During the summer of 2009 some 2000 sandflies (∼90% P. perniciosus, ∼5% Phlebotomus ariasi, ∼5% Sergentomia minuta) were captured. After identification and grinding of insects, RNA was extracted from pools of 10–25 sandflies and submitted to TOSV-IMT assay as described above. 3 pools were found positive. 2 l of the initial ground Table 2 Use of TOSV-IMT real-time RT-PCR assay on human and insect samples. Samples
Positive TOV-IMT real-time RT-PCR
Human samples analysed
971
17
Animal samples analysed P. perniciosus P. ariasi
∼1800 ∼200
3 0
TOSV-IMT system was routinely used as diagnostic tool during research for arbovirus infections in human sera or LCR as well as in crushes from phlebotomines captured. 3 samples found positive by real-time RT-PCR lead to virus isolation by cell culture. All the positive patients had clinical signs such as fever and CNS diseases.
material, corresponding to each individual sandfly from the positive pools, was directly submitted to TOSV-IMT assay: 3 P. perniciosus (1 male and 2 female) were found positive and carried at least 106 copies of TOSV genome (Table 2). Individual culture on VERO cells of the ground material from the three positive sandflies led to isolation of 2 Toscana viruses: strains TOSV/Phl. 51/FR/2009 (site 1, male) and TOSV/Phl. 233/FR/2009 (site 2, female).
5. Discussion TOSV is an important etiological agent of acute meningitis and meningoencephalitis in Mediterranean countries. The development of a specific molecular test for TOSV detection is essential for the early diagnosis of viral CNS infection, particularly in countries where the virus is endemic. The aim of this study was to design and assess a specific and sensitive real-time RT-PCR assay for molecular identification, in the context of TOSV infection surveillance by the French National Reference Center for Arboviruses. The TOSV-IMT assay presented here has the advantage of being updated with the sequence of the latest isolates compared to the previous published system [17,18]. Viral load levels reached during human TOSV infection are unknown. Thus, the availability of a high-sensitivity test is critical to achieve efficient detection for diagnosis. We have designed a robust and sensitive real-time RT-PCR for TOSV that allowed the detection of 257 copies of RNA transcript per reaction, or between 0.00112 and 0.0028 TCID50 per l of TOSV isolates from various locations (Italy and Spain). This test is more sensitive than another published test (0.0158 TCID50 ) [18]. Sensitivity in terms of viral load turns out to be less than 1 TCID50 : this is probably due to noninfectious viruses when TOSV is produced in in vitro cell culture systems. These non-infectious viruses increase the number of available genomes for molecular detection but are not counted as viable particles. Compared to the previously published system, the TOSV-IMT system was designed with a different target region; selected primers/probe show less homology with the sequences of SFNV which is the closest phylogenetically related Phlebovirus and accordingly no cross reaction was found during testing, either with SFNV or with other Phleboviruses [4]. In addition, our assay was capable of detecting the TOSV genome in various samples (clinical and insect samples). The TOSV-IMT test has been used to study TOSV propagation among vector populations. We were able to find male and female sandflies infected with TOSV, this is quite significant as male sandflies do not feed on blood: viral transmission among insects is therefore not restricted to blood-feeding females. It thus confirms that vertical transmission among sandflies exists for TOSV in their natural life cycle [19]. Our results revealed that infected sandflies carried at least 106 copies of TOSV genome. This is significantly higher than previous viral loads observed in experimentally infected sandflies [20]. It may be possible that viral infection is more efficient in natural than experimental infection among sandflies, and contribute to explain why TOSV is so efficiently transmitted by sandflies to human beings. Our real-time one step TOSV-IMT RT-PCR provides a rapid and sensitive tool for TOSV detection. Its high specificity and sensitivity makes it a useful tool for clinical and entomologic fields of investigation into TOSV infection.
Funding None.
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