Development of a Reverse Genetics System for Toscana Virus (Lineage A).

Toscana virus (TOSV) is a Phlebovirus in the Phenuiviridae family, order Bunyavirales, found in the countries surrounding the Mediterranean. TOSV is an important cause of seasonal acute meningitis and encephalitis within its range. Here, we determined the full sequence of the TOSV strain 1500590, a lineage A virus obtained from an infected patient (Marseille, 2007) and used this in combination with other sequence information to construct functional cDNA plasmids encoding the viral L, M, and S antigenomic sequences under the control of the T7 RNA promoter to recover recombinant viruses. Importantly, resequencing identified two single nucleotide changes to a TOSV reference genome, which, when corrected, restored functionality to the polymerase L and made it possible to recover infectious recombinant TOSV (rTOSV) from cDNA, as well as establish a minigenome system. Using reverse genetics, we produced an NSs-deletant rTOSV and also obtained viruses expressing reporter genes instead of NSs. The availability of such a system assists investigating questions that require genetic manipulation of the viral genome, such as investigations into replication and tropism, and beyond these fundamental aspects, also the development of novel vaccine design strategies.

An update on Toscana virus distribution, genetics, medical and diagnostic aspects.

BACKGROUND: 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. OBJECTIVE: 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. SOURCES: 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. CONTENT: 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. IMPLICATION: 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.

Virus isolation, genetic characterization and seroprevalence of Toscana virus in Algeria.

Toscana virus (TOSV; Bunyaviridae, Phlebovirus) is transmitted by sandflies of the genus Phlebotomus in the Mediterranean area. One strain of TOSV was isolated from a total of almost 23 000 sandflies collected in Kabylia, Algeria. The complete genome was sequenced, and phylogenetic studies indicated that it was most closely related with TOSV strain from Tunisia within lineage A, which also includes Italian, French and Turkish strains. A seroprevalence study performed on 370 sera collected from people living in the same area showed that almost 50% possessed neutralizing antibodies against TOSV, a rate much higher than that observed in Southern Europe. Sandfly species distribution in the study area suggests that the vector of TOSV in this region belongs to the subgenus Larroussius. These data support the rapid implementation of the diagnosis of TOSV in clinical microbiology laboratories to estimate the burden in patients presenting with neuroinvasive infections and febrile illness.

Urinary detection of toscana virus nucleic acids in neuroinvasive infections.

BACKGROUND: Toscana virus (TOSV) is a sandfly-borne pathogen causing febrile diseases and neuroinvasive infections in humans. Definitive diagnosis of TOSV infections frequently requires the detection of viral RNA in cerebrospinal fluid (CSF) or in circulation, which can be achieved prior to seroconversion. OBJECTIVES: To evaluate TOSV excretion in urine and impact of urine as a diagnostic specimen. STUDY DESIGN: A total of 82 plasma, CSF and urine samples were collected from 24 individuals with a preliminary diagnosis of atypical viral encephalitis, where frequent bacterial fungal and viral causes were ruled out. Phlebovirus and WNV nucleic acids were investigated via real-time and nested polymerase chain reaction (PCR) assays. Commercial immunofluorescence assays were employed for viral IgM detection. Amplicons were characterized via cloning and sequencing. RESULTS: Phlebovirus PCR yielded positive results in 7 out of 14 samples that comprise 4 plasma and 3 urine specimens from 3 individuals. Amplicons were characterized as TOSV genotype A. Investigation of the follow-up samples suggested that virus shedding in urine coincides or follows viremia. Despite conserved sequences observed in paired or sequential plasma-urine specimens, L693S substitution in the viral polymerase was characterized in a urine sample. CONCLUSIONS: These preliminary findings indicate that urine can be employed as a additional clinical sample for TOSV RNA detection in suspected cases, especially in individuals where specimens for viral diagnostics during the early stages of the infection are not available.

Toscana virus meningitis case in Switzerland: an example of the ezVIR bioinformatics pipeline utility for the identification of emerging viruses.

Toscana virus (TOSV) represents a frequent cause of viral meningitis in the Mediterranean Basin that remains neglected in neighbouring countries. We report a documented TOSV meningitis case in a traveller returning from Tuscany to Switzerland. While routine serological and PCR assays could not discriminate between TOSV and Sandfly fever Naples virus infection, a high-throughput sequencing performed directly on the cerebrospinal fluid specimen and analysed with the ezVIR pipeline provided an unequivocal viral diagnostic. TOSV could be unequivocally considered as the aetiological agent, proving the potential of ezVIR to improve standard diagnostics in cases of infection with uncommon or emerging viruses.

Genetic variability of the S segment of Toscana virus.

Toscana virus (TOSV) was originally isolated in 1971 from a pool of Phlebotomus perniciosus sandflies collected in Grosseto province (Central Italy). Since its first isolation, several studies have been conducted in Italy and other Mediterranean countries in order to identify its possible animal reservoirs, spread of infection and genetic variability. Phylogenetic analysis conducted on TOSV genome demonstrated the co-circulation of two major lineages in the Mediterranean areas, TOSV A and TOSV B. This study reports the results of the genetic analysis of 32 viral strains isolated in Italy in the last 30 years from patients hospitalized with neurological disease, from sandflies and from the brain of a bat. The genetic diversity of TOSV was investigated by determining the sequences of the whole S segment. Phylogenetic analysis showed that TOSV A lineage represents the lineage circulating in Italy. Moreover, the current variability of lineage A is similar to that of lineage B.

Toscana virus genome stability: data from a meningoencephalitis case in Mantua, Italy.

In July of 2013, samples from a patient with a neurological syndrome were collected from Mantua hospital and sent to the National Reference Laboratory for Arboviruses (National Institute of Health, Rome). On the basis of the symptoms, serological and molecular assays were performed to diagnose either West Nile virus (WNV) or Toscana virus (TOSV) infection. Molecular and serological tests confirmed TOSV infection. Virus isolation was obtained from cerebrospinal fluid. A full genome sequence was determined from this TOSV strain with next-generation sequencing using Ion Torrent technology. Nucleotide and amino acidic sequences grouped phylogenetically with lineage TOSV A and showed a low genome variability.

Human and entomological surveillance of Toscana virus in the Emilia-Romagna region, Italy, 2010 to 2012.

Toscana virus (TOSV), transmitted by phlebotomine sandflies, is recognised as one of the most important causes of viral meningitis in summer in Mediterranean countries. A surveillance plan based on both human and entomological surveys was started in 2010 in the Emilia-Romagna region, Italy. Clinical samples from patients with neurological manifestations were collected during 2010 to 2012. The surveillance protocol was improved during these years, allowing the detection of 65 human infections. Most of these infections were recorded in hilly areas, where sandflies reach the highest density. Entomological sampling around the homes of the patients resulted in a low number of captured sandflies, while later sampling in a hilly area with high number of human cases (n=21) resulted in a larger number of captured sandflies. Using this approach, 25,653 sandflies were sampled, of which there were 21,157 females, which were sorted into 287 pools. TOSV RNA was detected by real-time PCR in 33 of the pools. The results highlighted the role of Phlebotomus perfiliewi as the main vector of TOSV and a potential link between vector density and virus circulation. This integrated system shows that an interdisciplinary approach improves the sensitiveness and effectiveness of health surveillance.

First detection of Toscana virus in Corsica, France.

Toscana virus (TOSV) was detected for the first time from Phlebotomus perniciosus sandflies in Corsica, a French Mediterranean island. Genetic analysis showed that Corsican TOSV belongs to lineage A, together with Italian, Tunisian, Turkish and other French strains. The demonstration of TOSV in Corsica indicates that autochthonous and tourist populations are at risk of infection. Hence, physicians must consider TOSV as a possible cause of aseptic meningitis and unidentified febrile illness during the warm season.

Isolation of Toscana virus from the cerebrospinal fluid of a man with meningitis in Marseille, France, 2010.

Toscana virus (TOSV; Bunyaviridae, Phlebovirus) is an emerging arthropod-borne virus transmitted by phlebotomine sandflies. TOSV is a frequent cause of central nervous system infection during the warm season in several countries bordering the Mediterranean Sea. Here, we report a case of TOSV aseptic meningitis diagnosed in 2012 in Marseille, France. The virus strain was recovered in cell culture from the cerebrospinal fluid. New-generation sequencing based on Ion Torrent technology was used to determine its complete genome sequence. Phylogenetic analysis based on the partial L segment revealed that this isolate belongs to the lineage B together with other French, Spanish, and Moroccan strains. Although several cases of TOSV meningitis are reported in the literature, few of them are diagnosed by RT-PCR combined with virus isolation and further sequence characterization. This case report supports that virus isolation should be attempted whenever possible because this remains the gold standard technique for diagnosis of arthropod-borne viral infections.

[Sandfly virus meningitis in a Danish traveller returning from Tuscany]. / Sandfluevirusmeningitis hos en dansk turist efter ophold i Toscana.

We report the first case of Sandfly virus meningitis in a Danish traveller returning from Tuscany. A 52 year-old man was admitted with headache, fever and photophobia. Spinal fluid showed evidence of aseptic meningitis. Indirect immuno-fluorescence assays showed presence of immunoglobulin (Ig)M and IgG antibodies reactive against Toscana virus, and Phlebovirus RNA was detected in blood by real-time polymerase chain reaction. The patient recovered spontaneously. Since Sandfly virus is a very common cause of meningitis in the Mediterranean countries, it is important to be aware of this disease in travellers returning from these areas.

Granada virus: a natural phlebovirus reassortant of the sandfly fever Naples serocomplex with low seroprevalence in humans.

A new member of the phlebovirus genus, tentatively named Granada virus, was detected in sandflies collected in Spain. By showing the presence of specific neutralizing antibodies in human serum collected in Granada, we show that Granada virus infects humans. The analysis of the complete genome of Granada virus revealed that this agent is likely to be a natural reassortant of the recently described Massilia virus (donor of the long and short segments) with a yet unidentified phlebovirus (donor of the medium segment).

Genetic diversity of Toscana virus.

Distribution of Toscana virus (TOSV) is evolving with climate change, and pathogenicity may be higher in nonexposed populations outside areas of current prevalence (Mediterranean Basin). To characterize genetic diversity of TOSV, we determined the coding sequences of isolates from Spain and France. TOSV is more diverse than other well-studied phleboviruses (e.g.,Rift Valley fever virus).

Reverse transcription, real-time PCR assay for detection of Toscana virus.

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 reverse transcription, 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 TICD(50) 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.

Cocirculation of 2 genotypes of Toscana virus, southeastern France.

Toscana virus (TOSV), an arthropodborne phlebovirus transmitted by sandflies, can cause febrile illness and meningitis. The vector of TOSV in France was unknown. We detected TOSV RNA in 2 (female Phlebotomus perniciosus) of 61 pools of sandflies captured in southeastern France. Two genotypes of TOSV were identified.

Genetic variability of the M genome segment of clinical and environmental Toscana virus strains.

Twenty-seven strains of Toscana virus, collected over a period of 23 years and isolated from several localities and from different hosts (humans, arthropods and a bat), were investigated by sequencing of a portion of the M genomic segment comprising the G(N) glycoprotein coding region. Sequence data indicated that the divergence among isolates ranged from 0 to 5.7 % at the nucleotide level and from 0 to 3.4 % at the amino acid level. Phylogenetic analysis revealed four main clusters. A close correspondence between viral strains and area/year of isolation could not be demonstrated, whilst co-circulation of different viral strains in the same area and in the same time period was observed for both patients and environmental viral isolates. Alignment of the deduced amino acid sequences and evolutionary analysis indicated that most of the sites along the gene may be invariable because of purifying and/or neutral selection.

Toscana virus central nervous system infections in southern Italy.

Toscana virus was detected by reverse transcription-nested PCR in 5.6% of cerebrospinal fluid (CSF) samples from patients with meningitis and encephalitis during the summer in southern Italy. The central nervous system infections were associated with young adults and with a substantially benign clinical course. Presenting features and CSF findings are also discussed in the present report.

Emergence of Toscana virus in Europe.

Toscana virus (TOSV) is an arthropod-borne virus first identified in 1971 from the sandfly Phlebotomus perniciosus in central Italy. Many case reports in travelers and clinical research and epidemiologic studies conducted around the Mediterranean region have shown that TOSV has a tropism for the central nervous system (CNS) and is a major cause of meningitis and encephalitis in countries in which it circulates. In central Italy, TOSV is the most frequent cause of meningitis from May to October, far exceeding enteroviruses. In other northern Mediterranean countries, TOSV is among the 3 most prevalent viruses associated with meningitis during the warm seasons. Therefore, TOSV must be considered an emerging pathogen. Here, we review the epidemiology of TOSV in Europe and determine questions that should be addressed in future studies. Despite increasing evidence of its major role in medicine as an emerging cause of CNS infections, TOSV remains an unstudied pathogen, and few physicians are aware of its potential to cause CNS infections.