Toscana Virus Research Articles

Toscana virus - an emerging Mediterranean arbovirus transmitted by sand flies.

Toscana virus (TOSV) is an emerging arthropod-borne virus (arbovirus) of medical importance that is increasing its range across much of the Mediterranean Basin, Europe and the Middle East. Transmitted by Phlebotomus spp. sand flies, it is the most clinically relevant sand fly-borne phlebovirus. Initially isolated in the Tuscany region of Central Italy, it has now been detected in multiple countries that surround this geographical area. Infection of the vertebrate host can cause fever and neurological disease, following the dissemination of the virus to the brain. The prevalence is high in some regions, with a notable percentage of individuals showing seroconversion. TOSV can be a leading cause of acute meningitis and encephalitis (AME) during the summer months. In this comprehensive review, we will focus on several key topics. We discuss how TOSV has spread to establish outbreaks of infection in both humans and animals around the Mediterranean and the wider region. Clinical aspects of TOSV infection in humans are described, along with the best standards in diagnosis. Finally, we focus our discussion on the role of the sand fly vector, describing their biology, vector competency, implications for putative vertebrate reservoirs, the effect of the climate emergency on sand fly distribution and the putative role that sand fly-derived salivary factors may have on modulating host susceptibility to TOSV infection.

Toscana Virus in Wild-Caught Sand Flies in Portugal, Findings from the National Vector Surveillance Network, 2023.

Phlebotomine sand flies play a crucial role in both human and veterinary medicine, acting as vectors for Leishmania parasites and most known phleboviruses. In Portugal, the REVIVE program, a comprehensive national surveillance network under the Ministry of Health, has included sand fly surveys since 2016. REVIVE aims to identify existing sand fly species in the country, determine which pathogens are circulating among them, and provide actionable insights for prevention and control measures when necessary. In this way, annually, from May to October, health technicians collect sand flies across mainland Portugal with CDC light traps. The collected sand flies are sent to the Centre for Vectors and Infectious Diseases Research for species identification and molecular screening of pathogens. On 21 September 2023, Toscana virus (TOSV), a well-known phlebovirus in the Mediterranean region due to its capacity to cause neurological disease, was detected in a pool of 30 sand flies collected in Algarve, the southernmost region of Portugal. A 668 bp partial sequence of the nucleoprotein gene shows similarity with TOSV strains from Spain. To our knowledge, this is the first detection of TOSV in its vector in this country, having previously only been reported in vertebrate hosts. These findings highlight the important role of ongoing surveillance efforts in monitoring and understanding the dynamics of sand fly-borne diseases in Portugal.

Prevalence of Toscana Virus Antibodies in Residents of Bulgaria: A Nationwide Study Following the Pandemic of COVID-19.

Introduction: Toscana virus (TOSV) is an arthropod-borne virus transmitted by sandflies and is mainly found in countries around the Mediterranean basin. In this article, we present the findings of a seroprevalence study on TOSV in Bulgaria. We aim to assess the current epidemiological situation regarding TOSV in the country and raise clinical awareness. Methods: Serum samples (n = 1892) were collected in December 2023. Serological tests were performed using a commercial anti-TOSV ELISA kit. Results: Specific immunoglobulin G (IgG) antibodies were detected in 6.4% (121/1892) of the participants. A significantly higher seropositivity rate was found in the age group over 65 years compared with the age groups 18-29 and 40-64 (11.8% vs. 3.9% vs. 3.4%), as well as in males compared with females (8.0% vs. 5.3%). The seroprevalence rates in districts ranged from 0% to 18.5%. Higher seropositivity was found in the southern and northern regions. Conclusions: The seroprevalence rate of TOSV in Bulgaria, found in this study, is a significant decrease compared with the seropositivity rate of 24.5% reported in the country in 2018. The reasons for this are unknown and could possibly be related to the COVID-19 pandemic and the constantly changing environmental conditions. There is also a possibility that the higher seropositivity detected in 2018 together with the rise in clinical cases reported from endemic countries around that time might have been due to an unrecognized TOSV outbreak taking place in this period. Continued clinical awareness and surveillance are necessary for recognition and management of potential cases of TOSV neuroinfection, especially during summer.

Numb-associated kinases regulate sandfly-borne Toscana virus entry

ABSTRACT Sandfly-borne Toscana virus (TOSV) is an enveloped tri-segmented negative single-strand RNA Phlebovirus. It is an emerging virus predominantly endemic in southwestern Europe and Northern Africa. Although TOSV infection is typically asymptomatic or results in mild febrile disease, it is neurovirulent and ranks among the three most common causes of summer meningitis in certain regions. Despite this clinical significance, our understanding of the molecular aspects and host factors regulating phlebovirus infection is limited. This study characterized the early steps of TOSV infection. Our findings reveal that two members of the Numb-associated kinases family of Ser/Thr kinases, namely adaptor-associated kinase 1 (AAK1) and cyclin G-associated kinase (GAK), play a role in regulating the early stages of TOSV entry. FDA-approved inhibitors targeting these kinases demonstrated significant inhibition of TOSV infection. This study suggests that AAK1 and GAK represent druggable targets for inhibiting TOSV infection and, potentially, related Phleboviruses.

Fifty years after the first identification of Toscana virus in Italy: Genomic characterization of viral isolates within lineage A and aminoacidic markers of evolution

Toscana Virus (TosV) was firstly isolated from phlebotomine in our Institute about fifty years ago. Later, in 1984–1985, TosV infection, although asymptomatic in most cases, was shown to cause disease in humans, mainly fever and meningitis. By means of genetic analysis of part of M segment, we describe 3 new viral isolates obtained directly from cerebrospinal fluid or sera samples of patients diagnosed with TosV infection in July 2020 in Tuscany region. Phylogenesis was used to propose the clustering of TosV lineage A strains in 3 main groups, whereas deep mutational analysis based on 12 amino acid positions, allowed the identification of 9 putative strains. We discuss deep mutational analysis as a method to identify molecular signature of host adaptation and/or pathogenesis.

Seroprevalence and Risk Factors Associated with Phleboviruses and Crimean-Congo Hemorrhagic Fever Virus among Blood Donors in Central Tunisia.

The aim of this study was to determine the prevalence of six viruses, from two families of the order Bunyavirales, in the general population of central Tunisia. Sera collected from 377 asymptomatic blood donors were serologically assayed for Rift Valley fever virus (RVFV), Crimean-Congo hemorrhagic fever virus (CCHFV), and four sandfly-borne phleboviruses: Toscana virus (TOSV), sandfly fever Naples virus (SFNV), sandfly fever Sicilian virus (SFSV), and sandfly fever Cyprus virus (SFCV). Of the 377 subjects enrolled in this study, 17.3% were IgG positive for at least one of the viruses tested. The most frequently detected antibodies were against TOSV (13.3%), followed by SFCV (2.9%), RVFV (1.9%), SFSV (1.3%), and SFNV (1.1%). Only one sample was IgG positive for CCHFV. Dual reactivity was observed in nine cases: SFSV + SFCV in three cases (0.8%) and TOSV + SFNV, TOSV + SFCV, and TOSV + RVFV in two cases (0.5%) each. 15.9% of donors were IgG positive against sandfly-borne phleboviruses. Among the 65 donors IgG positive for phleboviruses, 50.8% were from rural areas compared to 12.3% from urban areas (p < 0.001); 92.3% had animals in their living quarters (p = 0.009); and 70.8% lived in the vicinity of stagnant water (p = 0.062). Seroprevalence was significantly higher among donors living with chronic diseases (p = 0.039). Furthermore, the seroprevalence of phleboviruses was higher in Kairouan, the central governorate, than in the two coastal governorates: Monastir and Sousse, with 33.4%, 24.2%, and 14.9%, respectively. The presence of antibodies in the general population needs further investigation to better assess the extent of these viruses. Only TOSV was known to have an extensive circulation in Tunisia and in North Africa. Continued surveillance and interventions are necessary to detect the emergence of all arboviruses and to prevent further transmission.

Urine: A Pitfall for Molecular Detection of Toscana Virus? An Analytical Proof-of-Concept Study.

Toscana virus (TOSV), a sandfly-borne virus, is an important etiological agent in human acute meningitis and meningoencephalitis in the Mediterranean area during the summer. However, the actual number of TOSV infections is underestimated. Laboratory confirmation is necessary because TOSV infection has overlapping clinical features with other neuro-invasive viral infections. Nowadays, the reference test for direct diagnosis in the acute phase of TOSV infection is the PCR based method for detecting TOSV in cerebrospinal fluid and/or plasma, serum, or blood. Although poorly employed, urine is another helpful biological matrix for TOSV detection. Urine is a matrix rich in PCR inhibitors that affect PCR efficiency; consequently, false negatives could be generated. To investigate the potential effect of urine PCR inhibitors on TOSV detection, we compared undiluted and diluted urine using 10-fold series of spiked TOSV. The results showed a significant improvement in TOSV detection performance in diluted urine (1 TCID50 vs. 1 × 104 TCID50 limit of detection and 101.35% vs. 129.62% efficiency, respectively, in diluted and undiluted urine). In conclusion, our data provide preliminary important insights into the use of diluted urine to limit the impact of the inhibitory effects of urine on the detection of TOSV in RT-PCR-based approaches.

Analysis of Negative-Strand RNA Viruses by RT-qPCR: Rift Valley Fever Virus and Toscana Virus.

RT-qPCR allows the detection of viruses and the monitoring of viral replication. This technique was extensively employed during the SARS-CoV-2 pandemic, where it demonstrated its efficiency and robustness. Here we describe the analysis of Rift Valley fever and Toscana virus infections over time, achieved through the RT-qPCR quantification of the viral genome. We further elaborate on the method to discriminate between genomic and antigenomic viral RNAs by using primers specific for each strand during the reverse transcription step.

Making Rift Valley Fever Viral Particles Fluorescent.

Rift Valley fever virus (RVFV) is a mosquito-borne pathogen that represents a significant threat to both human and veterinary public health. Since its discovery in the Great Rift Valley of Kenya in the 1930s, the virus has spread across Africa and beyond, now posing a risk of introduction into Southern Europe and Asia. Despite recent progresses, early RVFV-host cell interactions remain largely uncharacterized. In this method chapter, we delineate the procedure for labeling RVFV particles with fluorescent organic dyes. This approach makes it feasible to visualize single viral particles in both fixed and living cells and study RVFV entry into host cells. We provide additional examples with two viruses closely related to RVFV, namely, Toscana virus and Uukuniemi virus. Furthermore, we illustrate how to utilize fluorescent viral particles to examine and quantify each step of the cell entry program of RVFV, which includes state-of-the-art fluorescence-based detection techniques such as fluorescence microscopy, flow cytometry, and fluorimetry.

Co-Circulation of Leishmania Parasites and Phleboviruses in a Population of Sand Flies Collected in the South of Portugal.

In the Old World, phlebotomine sand flies from the genus Phlebotomus are implicated in the transmission of Leishmania spp. parasites (Kinetoplastida: Trypanosomatidae) and viruses belonging to the genus Phlebovirus (Bunyavirales: Phenuiviridae). Two of the five sand fly species known to occur in Portugal, Phlebotomus perniciosus and Ph. ariasi, the former being the most ubiquitous, are recognized vectors of Leishmania infantum, which causes visceral leishmaniasis, the most prevalent form of leishmaniasis in the country. Phlebotomus perniciosus is also the vector of the neurotropic Toscana virus, which can cause aseptic meningitis. Entomological surveillance is essential to provide fundamental data about the presence of vectors and the pathogens they can carry. As such, and given the lack of data in Portugal, an entomological survey took place in the Algarve, the southernmost region of the country, from May to October 2018. Polymerase chain reaction assays were performed in order to detect the presence of the above-mentioned pathogens in sand fly pools. Not only were both Leishmania parasites and phleboviruses detected during this study, but more importantly, it was the first time their co-circulation was verified in the same sand fly population collected in Portugal.

Do birds play a role in the transmission of Toscana virus? Initial isolation results from birds in northernmost Türkiye.

Recent research has prioritized emerging and re-emerging diseases that affect human and animal health, particularly to describe how these diseases enter countries and determine their transmission cycles. Given that migratory birds play a significant role in spreading infections, the present study analysed their migration paths and specimens to investigate Orthoflavivirus, Orthonairovirus, Alphavirus and Phlebovirus in birds in Samsun province, Türkiye. For these viruses, 312 samples from 56 birds were analysed using RT-PCR and qRT-PCR. Toscana virus (TOSV) was identified in 14 birds (four mallards, five partridges, four quails and one pigeon), representing 25% of the birds sampled. Genotype B was reported in all 14 birds. After inoculating the positive tissues in cell cultures, TOSV was isolated from the organs of pigeons, mallards and partridges. This is the first time TOSV has been isolated in cell culture from birds and indicates that they may play a role in spreading TOSV in Türkiye. The results also suggest that TOSV might be carried between countries by migratory birds.

Neutralizing based seroprevalence study of Toscana virus in livestock from Algeria

Toscana virus is a sandfly-borne human pathogen belonging to Phlebovirus genus into Phenuiviridae family. It is emerging in north Africa posing a complex threat to public health. TOSV is heavily affecting sandfly-exposed people in northern Algeria. A larger distribution has recently been stated in Algeria by using dog sera. Dog exposure to TOSV was repeatedly identified in north Algeria, with 4.56% lately detected to possess respective neutralizing antibodies. However, evidence for TOSV has only been observed in dogs among various species of domestic animals. Therefore, we attempted to assess sera from 221 livestock comprising cattle, sheep, goats, rabbits and horses, to identify the presence of TOSV neutralizing antibodies. The study was conducted during 2017, in 11 areas from the governorates of Blida, Medea, Algiers, Tipaza, Ain Defla, Tissemsilt in the north center, and Setif, Mila, Tizi Ouzou, Jijel in the northeast of Algeria. Positive results were obtained in 14.6% (12/82) cattle, 17.18% (11/64) sheep, 15% (3/20) horses and 3.33% (1/30) goats, whereas rabbits remained negative. Positive samples originated mainly from the north centre, with new areas being first-ever detected. The seroprevalence was noticed to be very strongly related to sample origin (p < 0.01). Females (OR=4.09) were observed to be more likely infected. Our findings represent a further proof of TOSV circulation in Algeria. Moreover, they revealed a potential role of livestock (p = 0.00731) in its natural cycle. This fact emphasize how important is to elucidate the exact contribution of livestock to the epidemiology of sandfly-borne phleboviruses, and their impact on public health.

Melittin-Related Peptides Interfere with Sandfly Fever Naples Virus Infection by Interacting with Heparan Sulphate.

Emerging viruses pose an important global public health challenge, and early action is needed to control their spread. The Bunyaviridae family contains a great number of arboviruses which are potentially pathogenic for humans. For example, phleboviruses affect a large range of hosts, including humans and animals. Some infections usually have an asymptomatic course, but others lead to severe complications, such as Toscana virus, which is able to cause meningitis and encephalitis. Unfortunately, to date, no vaccines or antiviral treatments have been found. In the present study, we evaluated the effect of melittin-related peptides, namely the frog-derived RV-23 and AR-23, on sandfly fever Naples virus infection in vitro. Both peptides exhibited a strong antiviral activity by targeting the viral particles and blocking the virus-cell interaction. Their action was directed to an early phase of SFNV infection, in particular at viral adsorption on host cells, by interfering with the binding of common glycosaminoglycan receptors. Given the better antimicrobial behavior of AR-23 and RV-23 compared to melittin in terms of selectivity, our studies expand our understanding of the potential of these peptides as antimicrobials and stimulate further investigations in the direction of novel antiviral strategies against phlebovirus infection.

Comprehensive Cross-Sectional Evaluation of Human Sandfly-Borne Phlebovirus Exposure in an Endemic Region.

Sandfly-borne phleboviruses are endemic in countries around the Mediterranean Basin and pose a significant health threat for populations, with symptoms spanning from febrile diseases to central nervous system involvement. We carried out a comprehensive cross-sectional screening via microneutralization (MN) assays for a quantitative assessment of neutralizing antibodies (NAs) to seven phleboviruses representing three distinct serocomplexes, using samples previously screened via immunofluorescence assays (IFAs) in Turkey, an endemic region with various phleboviruses in circulation. We detected NAs to three phleboviruses: Toscana virus (TOSV), sandfly fever Naples virus (SFNV), and sandfly fever Sicilian virus (SFSV), while assays utilizing Adana virus, Punique virus, Massilia virus, and Zerdali virus remained negative. The most frequently observed virus exposure was due to TOSV, with a total prevalence of 22.6%, followed by SFNV (15.3%) and SFSV (12.1%). For each virus, IFA reactivity was significantly associated with NA detection, and further correlated with NA titers. TOSV and SFSV seroreactivities were co-detected, suggesting exposure to multiple pathogenic viruses presumably due to shared sandfly vectors. In 9.6% of the samples, multiple virus exposure was documented. In conclusion, our findings demonstrate widespread exposure to distinct pathogenic phleboviruses, for which diagnostic testing and serological screening efforts should be directed.

Current Status of Vector-Borne Diseases in Croatia: Challenges and Future Prospects.

Different vector-borne pathogens are present or have (re-)emerged in Croatia. Flaviviruses tick-borne encephalitis (TBEV), West Nile (WNV), and Usutu (USUV) are widely distributed in continental regions, while Toscana virus (TOSV) and sandfly fever viruses are detected at the Croatian littoral. Recently, sporadic clinical cases of Tahyna orthobunyavirus (TAHV) and Bhanja bandavirus infection and seropositive individuals have been reported in continental Croatia. Acute infections and serologic evidence of WNV, TBEV, USUV, and TAHV were also confirmed in sentinel animals and vectors. Autochthonous dengue was reported in 2010 at the Croatian littoral. Lyme borreliosis is the most widely distributed vector-borne bacterial infection. The incidence is very high in northwestern and eastern regions, which correlates with numerous records of Ixodes ricinus ticks. Acute human Anaplasma phagocytophilum infections are reported sporadically, but there are many records of serologic evidence of anaplasmosis in animals. Mediterranean spotted fever (Rickettsia conorii) and murine typhus (Rickettsia typhi) are the main rickettsial infections in Croatia. Human leishmaniasis is notified sporadically, while serologic evidence of leishmaniasis was found in 11.4% of the Croatian population. After the official eradication of malaria in 1964, only imported cases were reported in Croatia. Since vector-borne diseases show a growing trend, continuous monitoring of vectors is required to protect the population from these infections.

The phenuivirus Toscana virus makes an atypical use of vacuolar acidity to enter host cells.

Toscana virus is a major cause of arboviral disease in humans in the Mediterranean basin during summer. However, early virus-host cell interactions and entry mechanisms remain poorly characterized. Investigating iPSC-derived human neurons and cell lines, we found that virus binding to the cell surface was specific, and 50% of bound virions were endocytosed within 10 min. Virions entered Rab5a+ early endosomes and, subsequently, Rab7a+ and LAMP-1+ late endosomal compartments. Penetration required intact late endosomes and occurred within 30 min following internalization. Virus entry relied on vacuolar acidification, with an optimal pH for viral membrane fusion at pH 5.5. The pH threshold increased to 5.8 with longer pre-exposure of virions to the slightly acidic pH in early endosomes. Strikingly, the particles remained infectious after entering late endosomes with a pH below the fusion threshold. Overall, our study establishes Toscana virus as a late-penetrating virus and reveals an atypical use of vacuolar acidity by this virus to enter host cells.

Phlebovirus detection on phlebotomine sandflies in Lampedusa Island (Italy).

Phleboviruses are common human pathogens diffused on the Mediterranean area whose infection can cause the typical prodromal symptom of a mild three‑days fever. In particular, Toscana Virus (TOSV) has a great concern since its capacity to provoke central nervous system disorders like meningoencephalitis. Furthermore, as the phlebotomine arthropod vectors represent the main carrier for pathogens of the genus Leishmania as well, the purpose of the study was to investigate the presence of TOSV in Lampedusa, Italy previously reported for leishmaniosis infection cases. The survey was carried out through an initial sampling phase of sand flies, by means of CDC light traps, and a second step of molecular analyses. The genomic S‑segment of TOSV was targeted. The positive samples were sequenced and compared with those available in GenBank™ using Basic Local Alignments Tool (BLAST) analyses. The study revealed for the first time the presence of TOSV in Lampedusa, Italy. The entomological studies directed on vectors are currently widely used in sand fly surveillance, and new data on TOSV are of public health concern.

Seroprevalence of Toscana and sandfly fever Sicilian viruses in humans and livestock animals from western Saudi Arabia

High seroprevalence rates of several phleboviruses have been reported in domestic animals and humans in sandfly-infested regions. Sandfly Fever Sicilian virus (SFSV) and Toscana virus (TOSV) are two of these viruses commonly transmitted by Phlebotomus sandflies. While SFSV can cause rapidly resolving mild febrile illness, TOSV could involve the central nervous system (CNS), causing diseases ranging from aseptic meningitis to meningoencephalitis. Sandfly-associated phleboviruses have not been investigated before in Saudi Arabia and are potential causes of infection given the prevalence of sandflies in the country. Here, we investigated the seroprevalence of SFSV and TOSV in the western region of Saudi Arabia in samples collected from blood donors, livestock animals, and animal handlers. An overall seroprevalence of 9.4% and 0.8% was found in humans for SFSV and TOSV, respectively. Seropositivity was significantly higher in non-Saudis compared to Saudis and increased significantly with age especially for SFSV. The highest seropositivity rate was among samples collected from animal handlers. Specifically, in blood donors, 6.4% and 0.7% tested positive for SFSV and TOSV nAbs, respectively. Animal handlers showed higher seroprevalence rates of 16% and 1% for anti-SFSV and anti-TOSV nAbs, respectively, suggesting that contact with livestock animals could be a risk factor. Indeed, sera from livestock animals showed seropositivity of 53.3% and 4.4% in cows, 27.5% and 7.8% in sheep, 2.2% and 0.0% in goats, and 10.0% and 2.3% in camels for SFSV and TOSV, respectively. Together, these results suggest that both SFSV and TOSV are circulating in the western region of Saudi Arabia in humans and livestock animals, albeit at different rates, and that age and contact with livestock animals could represent risk factors for infection with these viruses.

The Role of the Leishmania infantum Infected Dogs as a Potential Reservoir Host for Toscana Virus in a Zoonotic Visceral Leishmaniasis Focus of Northern Tunisia.

The role of dogs as reservoir hosts for Toscana virus (TOSV) remains undetermined. This study investigated TOSV and Leishmania infantum infections in one healthy and three infected dogs with Leishmania (A, B, C) following natural exposition to sandfly bites in a focus of zoonotic visceral leishmaniasis (ZVL) located in Northern Tunisia from June to October 2020. At the end of the exposition period, infected and healthy dogs were examined for TOSV and L. infantum infections by xenodiagnosis using a colony of Phlebotomus perniciosus. Pools of freshly engorged P. perniciosus at days 0 and those at days 7 post-feeding were screened for TOSV and L. infantum by nested PCR in the polymerase gene and kinetoplast minicircle DNA, respectively. In the exposure site, P. pernicious is the most abundant sandfly species. The infection rates of sandflies with TOSV and L. infantum were 0.10 and 0.05%, respectively. Leishmania infantum DNA and TOSV RNA were detected in P. perniciosus females fed on dog B and C, respectively. The isolation of TOSV in Vero cells was achieved from two pools containing P. perniciosus fed on dog C. No pathogens were detected in P. perniciosus females fed on dog A and on control dog. We report for the first time the reservoir competence of dog with ZVL in the transmission of TOSV to sandfly vectors in natural settings, in addition to its role as a main reservoir host of L. infantum.

Persistence of Toscana virus in sugar and blood meals of phlebotomine sand flies: epidemiological and experimental consequences

Many virological studies have tested the persistence of enveloped RNA viruses in various environmental and laboratory conditions and shown their short-term persistence. In this article, we analyzed Toscana virus (TOSV) infectivity, a pathogenic sandfly-borne phlebovirus, in two different conditions: in the sugar meal and blood meal of sand flies. Our results showed that TOSV RNA was detectable up to 15 days in sugar solution at 26 °C and up to 6 h in blood at 37 °C. Moreover, TOSV remains infective for 7 days in sugar solution and for minimum 6 h in rabbit blood. TOSV has shown persistent infectivity/viability under different conditions, which may have important epidemiological consequences. These results strengthen new hypotheses about the TOSV natural cycle, such as the possibility of horizontal transmission between sand flies through infected sugar meal.