Reversetranscription, real-time PCR assay for detection of Toscana virus

doi:10.1016/j.jcv.2007.05.003

Journal of Clinical Virology

Volume 39, Issue 4, August 2007, Pages 276-281

https://doi.org/10.1016/j.jcv.2007.05.003 Get rights and content

Abstract

Background

The arthropod-borne Toscana virus is a common cause of acute neurological infection in the Mediterranean basin. Recently, a new lineage, highly divergent from the Italian prototype, has been reported in Spain.

Objective

We describe a reversetranscription, real-time PCR assay for detection of both Toscana virus genotypes. The real-time PCR uses a TaqMan^® probe and an internal control to identify false negative results.

Study design

A conserved region of the two known lineages of Toscana virus, located at the 3′ end of the small segment of their genomes, was chosen to design both the primers and the probe.

Results

The sensitivity of the assay was 0.0158 TICD₅₀ per reaction of Toscana virus, equivalent to seven copies of cDNA. No other phleboviruses or RNA viruses were amplified by this specific real-time PCR.

Conclusions

The assay seems to be sensitive, reliable and easy to be applied in the diagnosis of autochthonous and/or imported suspected cases of Toscana virus infection.

Introduction

Toscana virus (TOSV, Phlebovirus, Bunyaviridae) is an important etiological agent of acute meningitis and meningoencephalitis in Mediterranean countries (Charrel et al., 2005, Verani et al., 1984, Ehrnst et al., 1985, Calisher et al., 1987, Nicoletti et al., 1991, Eitrem et al., 1991, Schwarz et al., 1995a, Mendoza-Montero et al., 1998). Seroprevalence studies show that more than 20% of the general population has been infected in regions of Spain (Sanbonmatsu-Gámez et al., 2005) and Italy (Valassina et al., 2003).

Virological diagnosis has been done by serological studies using ELISA (Echevarría et al., 2003) and/or neutralization tests (Mendoza-Montero et al., 1998). However, in the acute phase of the infection, sensitive etiological diagnosis relies on direct detection of TOSV in the CSF, either by cell culture or by nucleic acid amplification techniques (Schwarz et al., 1995b, Valassina et al., 1996, Sánchez-Seco et al., 2003). The advantage of molecular techniques is that a result can be obtained in 24–48 h, and cell culture must be maintained at least 14 days to discard negative results. Real-time PCR is substituting classical PCR methods on virological diagnosis, because it is less time-consuming and reduces the risk of contamination (Espy et al., 2006).

Recently, Sanbonmatsu-Gámez et al. (2005) have suggested the existence of at least two lineages of TOSV, Italian and Spanish, based on nucleotide sequence differences found among strains from both areas within a fragment of the L gene and the complete N gene.

The aim of this work was to develop a reversetranscriptase (RT)-real-time PCR for the diagnosis of CNS infection caused by all the known variants of TOSV.

Section snippets

Viral strains used for sensitivity and specificity studies

The STI-62100 strain of the Spanish lineage of TOSV was chosen for optimizing the real-time PCR method. The strain derived from the Spanish TOSV Isolate (STI)-62100, recovered in 2003 from a CSF sample. Aliquots of 100 μl of 10-fold dilutions of STI-62100 were used in parallel to calculate the TCID₅₀ in Vero cell lines (Hsiung, 1994) and the equivalent sensitivity of the real-time PCR assay.

To determine the specificity of the assay, the following several RNA viruses were tested by the real-time

Development of the real-time PCR

Serial 10-fold dilutions down to 10 copies/μl and 2-fold dilutions from 10 to 1 copies/μl of plasmid DNA from pTOS and pIC were obtained. A 5 μl aliquot of each dilution was separately amplified by the real-time PCR assay. The limit of detection, at which 100% of five replicates amplified with a crossing point value under 40, was estimated as 7 copies/reaction of pTOS and 1 copy/reaction of pIC plasmid DNA. Fluorescent signals in channel F1 and F2 were only seen in those capillaries with pTOS and

Discussion

Viral meningitis is usually a mild and self-limited disease. Thus, aetiological diagnosis is desirable to ease the management of the patient and to avoid unnecessary antibiotic treatment. Cell culture has been the gold standard for enterovirus detection from CSF specimens. Nucleic acid amplification assays have probably converted in a better alternative due to their great sensitivity, and they are now the new gold standard for the diagnosis of enterovirus infection (Debiasi and Tyler, 2004).

Acknowledgements

We thank Francisca García, María Angeles Rivera and María José Ruiz, for their technical assistance during the performance of this work. We are indebted to Ma. Carmen Peláez and Stephen Colussi for proofreading the English text.

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Epidemiology, Isolation, and Genetic Characterization of Toscana Virus in Algerian Patients Displaying Neurological Infection, 2016–2018
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The current study reports the results of the diagnosis of neuro-invasive Toscana virus (TOSV) infection in Algeria between 2016 and 2018 and describes the first isolation of TOSV strain from human samples in North Africa.
Cerebrospinal fluid (CSF) and sera samples were obtained from 720 hospitalized patients displaying neurological infection symptoms of unknown etiology, of which 604 were screened for TOSV. The diagnosis was performed by serological and/or RT-PCR tests. In addition, TOSV was isolated in vivo and in vitro from CSF and genetically characterized.
23 cases of TOSV neurological infections were detected. Cases were located in 11 Wilayas (administrative provinces), mainly in northern Algeria. In addition, we report the isolation of TOSV strain belonging to lineage A from human samples with its complete coding sequence.
Even though the number of infections is probably underestimated, TOSV is endemic in Algeria, with several cases of neuro-invasive diseases in humans recorded each year. Therefore, the diagnosis of TOSV should be included in the differential diagnosis of neurological diseases, especially aseptic meningitis, during the period of activity of the phlebotomine vector. Further studies are required to measure precisely the nationwide prevalence of TOSV in Algeria.
Identification of Toscana virus in natural population of sand flies (Diptera: Psychodidae) from Moroccan leishmaniasis foci
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RNA was extracted from ground female sand fly pools using the One for All – Qiagen (Germany) RNA/DNA extraction kit on a semi-automated extractor system (Bio-Sprint 96 – Qiagen, Germany) according to the manufacturer’s instructions, retro transcribed and subjected to real-time polymerase chain reaction (RT-PCR). TOSV detection in sand fly pools was carried out on CFX 96 Thermal cycler (BIO-RAD) according to Pérez-Ruiz et al. [25] protocol. Where p is the proportion (n/N) of individuals of one particular species found (n) divided by the total number of individuals found (N), ln is the natural logarithm, Σ is the sum of the calculations, and s is the number of species [26].
Phlebotomine sand flies are known as vectors of various pathogens such as Leishmania sp parasite and Toscana virus (TOSV). Leishmaniasis is endemic in Morocco, and TOSV is increasingly reported. Our objective is to analyze the specific composition of the natural population of sand flies in endemic and non endemic area of leishmaniasis in Morocco, thus evaluated their infection by Toscana virus.
Sand flies were collected by CDC miniature light traps from seven different localities with an altitude range from 399 m to 1496 m. Synanthropic index was calculated for each sand fly species. The collected female sand flies were grouped in 73 pools, with a maximum of 50 specimens per pool, and submitted to real time PCR for TOSV detection.
8 sand fly species were identified morphologically: 5 of the Phlebotomus genus and 3 of the Sergentomyia genus. Phlebotomus sergenti was the most abundant species comprising of 43,12% of identified sand flies, followed by P. papatasi (18,89%) and P. longicuspis (13,43%). Estimated synanthropic indices for these species were between + 1.1 and + 12.6 suggesting a high preference to anthropogenic environments. A total of 3558 sand fly females were grouped in 73 pools (up to 50 sand flies per pool) for TOSV detection. TOSV was detected in one pool (out of 6 tested) from Lalla Laaziza locality (Chichaoua Province) where P. sergenti was the most abundant sand fly species.
We reported the TOSV for the first time in a central Morocco, where cutaneous leishmaniasis by L. tropica is endemic. This result has epidemiological importance for both researchers and health authorities to monitor circulation of TOSV and implement a surveillance plan of sand fly-borne phleboviruses in Morocco.
An update on Toscana virus distribution, genetics, medical and diagnostic aspects
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Citation Excerpt :
It is important to underline that there are so far no data supporting the fact that one lineage would be more pathogenic in humans than another. Three molecular assays based on real-time RT-PCR have been published [62–64]. The three assays present similar performances, although the first two are the more commonly used.
Toscana virus is an arbovirus transmitted by sand flies within the Mediterranean area where it can cause febrile illness and neuroinvasive infections during the seasonal circulation period of the vector. Although it is an important cause of meningitis and encephalitis, it remains a neglected virus with limited published data, as demonstrated by <250 peer-reviewed articles since the 1970s.
The last review article on Toscana virus was published in 2012. The aim was to compile peer-reviewed articles to provide an updated review highlighting recent findings to complement previous review articles.
PubMed database was searched using the ‘Toscana virus’ keyword from 2010 to present. A total of 152 articles were retrieved and identified studies were assessed for novel information on virus genetics, and geographic and medical aspects compared with existing knowledge reported in previous review articles.
Studies addressing medical, veterinary and entomological aspects have provided evidence that Toscana virus is present in North Africa, in the Balkan Peninsula, and in most of the Mediterranean islands. Besides the two previously recognized genetic lineages, a novel evolutionary lineage has been identified in the Balkan Peninsula. Co-circulation of two genetic lineages has been demonstrated in France, in Turkey and in Croatia. In addition to meningitis and meningo-encephalitis, which have been reported for 40 years, various neuroinvasive forms have been recently reported such as Guillain–Barré syndrome, hydrocephalus, myositis, fasciitis, polymyeloradiculopathy, deafness and facial paralysis.
Because it is endemic in countries bordering the Mediterranean, physicians should include Toscana virus in the differential diagnosis of patients presenting with febrile illness and/or neurological manifestations.
Epidemiology of Toscana virus in South Tuscany over the years 2011-2019
2020, Journal of Clinical Virology
Toscana virus (TOSV) is a Phlebovirus transmitted to humans by phlebotomines and represent an etiological agent of acute aseptic meningitis (AAM) in countries where the virus is endemic, including Italy. Incidence of TOSV infections is closely associated with the geographical distribution of the phlebotomine vectors which in turn is affected by climate changes that determine survival and spread. As a result, TOSV infections show a seasonal trend with a peak of incidence in summer months.
To measure the prevalence of TOSV infections in AAM patients in central Italy and evaluate the climate changes in phlebotomine vectors ecology and virus propagation.
One thousand and seventy-three cerebrospinal fluid samples (CSFs), collected from patients with suspected viral meningitis, were collected over nine years (2011-2019) during the May to October period and tested for viruses most commonly associated with AAM. Serum samples addressed to the Microbiology and Virology Unit of “S. Maria delle Scotte” Hospital for confirmation acute TOSV infection (n = 324) were tested for TOSV-specific IgM and IgG.
Among the CSF samples, 1.3% were positive for Enteroviruses; 0.9% for Varicella zoster virus, 1.9% for Herpes simplex virus type-1/2 and 4.6% for TOSV. Serum IgM analyses disclosed TOSV-specific IgM in 27.1% of sera suggesting the predominant involvement of TOSV in neuroinvasive infections.
This data confirms the predominant role of TOSV as causative agent of AAM during the summer time in endemic countries. Moreover, climate changes affecting phlebotomine vectors persistence, reproduction and activity could be involved in the cyclic nature of TOSV infection reported during the last nine years.
Analytical validation of viral CNS Flow Chip kit for detection of acute meningitis and encephalitis
2018, Journal of Virological Methods
A new molecular assay (Viral CNS Flow Chip kit, Master Diagnóstica, Spain) has been developed for the detection of eight viruses causing acute meningitis and encephalitis, i.e. herpes simplex viruses 1–2, varicella zoster virus, human enterovirus, human parechovirus, Toscana virus, human cytomegalovirus and Epstein Barr virus. The new assay is a multiplex one-step RT-PCR followed by automatic flow-through hybridization, colorimetric detection and image analysis.
The limit of detection was 50 copies/reaction, and 10 copies/reaction for human enterovirus and the other seven viruses, respectively. The analytical validation was performed with nucleic acids extracted from 268 cerebrospinal fluid samples and the results were compared with routine molecular assays. An excellent coefficient of agreement was observed between V-CNS and routine assays [kappa index: 0.948 (95%CI: 0.928-0.968)]. The overall sensitivity and specificity was 95.9% (95%CI: 91.2–98.3%) and 99.9% (95%CI: 99.6–100%), respectively.
Viral CNS Flow Chip kit is an efficient multiplex platform for the detection of the main viruses involved in acute meningitis and encephalitis. The inclusion of a TOSV genome target may improve the laboratory diagnosis of viral neurological infections in endemic areas.
A rapid and specific real time RT-PCR assay for diagnosis of Toscana virus infection
2015, Journal of Clinical Virology
Citation Excerpt :
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 μl 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 non-infectious viruses when TOSV is produced in in vitro cell culture systems.
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.
Development and assessment of a new sensitive and specific real-time RT-PCR assay for TOSV diagnosis.
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.
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.
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.

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Reversetranscription, real-time PCR assay for detection of Toscana virus

Abstract

Background

Objective

Study design

Results

Conclusions

Introduction

Section snippets

Viral strains used for sensitivity and specificity studies

Development of the real-time PCR

Discussion

Acknowledgements

Lancet

J Clin Virol

Lancet

Res Virol

Emergence of Toscana virus in Europe

Emerg Infect Dis

Applications and challenges of real-time PCR for the clinical microbiology laboratory

Molecular methods for diagnosis of viral encephalitis

Clin Microbiol Rev

Sandfly fever among Swedish tourists

Scand J Infect Dis

Real-time PCR in clinical microbiology: applications for routine laboratory testing

Clin Microbiol Rev

Rapid detection of enteroviruses in small volumes of natural waters by real-time quantitative reverse transcriptase PCR

Appl Env Microbiol

Virus assay, neutralization test, and antiviral assay