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Toscana Virus Infection
Chapter 8
Toscana Virus Infection Remi N. Charrel Aix Marseille Universite´, IRD French Institute of Research for Development, EHESP French School of Public Health, EPV UMR_D 190 “Emergence des Pathologies Virales”, & IHU Me´diterrane´e Infection, APHM Public Hospitals of Marseille 13385, Marseille, France
CASE PRESENTATION At the beginning of summer, a 17-year-old male, originating from and living in France, without noticeable medical background, visited his medical practitioner for brutal and recent onset characterized by fever, severe headache, and vomiting. After examination, his GP referred the patient to the emergency ward of the public hospital for suspected aseptic meningitis. Upon admission, he presented with the same manifestations in a febrile context (39 C) in spite of self-medication with paracetamol. Examination revealed stiff neck in the absence of photophobia or signs suggesting encephalitis. Laboratory tests showed an increased white blood cell count (WBC) at 11,700/mm3 (80% neutrophils) and a normal C-reactive protein. The cerebrospinal fluid (CSF) contained 840 leukocytes/mm3 (70% lymphocytes) with normal glucose and protein levels. All molecular tests performed on CSF that targeted enteroviruses, herpes simplex virus types 1 and 2, Neisseria meningitidis, and Streptococcus pneumoniae were negative. The patient reported no recent history of travel. Treatment was started with amoxicillin and acyclovir, and the patient was transferred into the Infectious Diseases department. The day after, he was no longer febrile, but still complained of severe headache and vomiting. Routine analysis of the CSF including direct microscopic examination, bacterial culture, and conventional PCR assays for 16 S and 18 S were negative. Real-time RT-PCR for enterovirus, West Nile virus, and Toscana virus were performed. The real-time RT-PCR for Toscana virus was positive (cycle threshold value: 38). Despite low viral load in the CSF, the virus strain was isolated in Vero cells and further characterized by complete genome sequencing.1 Serology using indirect immunofluorescence showed the presence of IgM and IgG in the acute serum. Clinical manifestations rapidly resolved and the patient was discharged the next day. Three days after the diagnosis was established, a field campaign was organized to collect sandflies in the Emerging Infectious Diseases. DOI: http://dx.doi.org/10.1016/B978-0-12-416975-3.00008-X © 2014 Elsevier Inc. All rights reserved.
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vicinity close to the patient’s home. Four CDC miniature light traps were placed in the garden around the house, where the patient used to spend time at nightfall. A total of 17 Phlebotomus perniciosus were trapped but were negative for Toscana virus RNA through real-time RT-PCR. However, the trapping of sandflies in the vicinity close to the patient’s home confirmed that the patient may have been exposed at home to sandflies, known as the vector of Toscana virus. (This is a published case report.1)
1. WHY THIS CASE WAS SIGNIFICANTLY IMPORTANT AS AN EMERGING INFECTION Neurological infections caused by Toscana virus, a sandfly-borne phlebovirus, are common during the summer in regions bordering the Mediterranean Sea (Figure 8.1). Studies collating series of Toscana virus cases demonstrate that Toscana virus is the third cause of aseptic meningitis and meningoencephalitis in the regions of Spain, France, and Italy where the virus circulates in sandflies. Seroprevalence studies performed in southern Europe indicate that a significant proportion of the exposed population (5 to .50%, depending on the studies and on the regions) possess antibodies that react with Toscana virus. According to these data, Toscana virus is the most prevalent arthropod-borne virus in Europe far ahead of tick-borne encephalitis virus, West Nile virus, or dengue virus. As an indicator of medical and scientific attention, a search using the keyword “Toscana virus” retrieved 204 related
FIGURE 8.1 Toscana virus in the Mediterranean Sea.
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articles in PubMed, which correspond to fewer than five articles per year since virus discovery. There is a great need to draw attention to Toscana virus. Because it was discovered in the early 1970s, Toscana virus should be considered as a neglected pathogen rather than an emerging pathogen. As a countermeasure, availability of affordable commercial kits for molecular diagnosis and serology should be aimed for in order to enable dissemination of the diagnostic capacities outside of the few reference centers.
2. WHAT IS THE CAUSATIVE AGENT? Toscana virus (Toscana virus; Bunyaviridae; Phlebovirus) is an enveloped, single-strand, negative-sense RNA virus. First isolated in Italy in 1971, Toscana virus is an arthropod-borne virus transmitted by phlebotomine sandflies (Phlebotomus sp. of the subgenus Laroussius). According to the International Classification on the Taxonomy of Viruses, Toscana virus belongs to the species Sandfly fever Naples virus, which includes also several other viruses such as Naples virus and Theran virus; in addition, there are several recently discovered phleboviruses that should be included in this species together with Toscana virus; this is not the role of ICTV since this organism classifies virus down to the level of species, not below the species. Obviously, these newly discovered viruses (Massilia, Granada, Punique) have to be considered as variant genotypes of the species Sandfly fever Naples virus. There is a very limited knowledge on the capacity of these variants’ genotypes to infect humans and to cause disease in humans. Previous phylogenetic analysis of Toscana virus first identified two geographically associated lineages: the lineage A endemic in Italy (including the Sardinia and the Sicily) and the lineage B endemic in Spain.2 New sequences of Toscana virus isolates originating from other countries confirmed this dichotomy: Turkish and Tunisian Toscana virus are more closely related to Italian strains than to the Spanish ones. In contrast, Moroccan Toscana virus is more closely related to Spanish strains than to the Italian ones.3 This dichotomy could be linked to the existence of different vectors (P. perniciosus, P. perfiliewi, other species of Phlebotomus) and/or to the existence of two geographically restricted races of P. perniciosus, the main vector of Toscana virus.2 Interestingly, Toscana virus strains that circulate in France were reported to belong to either of these two lineages.4 Recently, a new lineage of Toscana virus was described in Croatia suggesting a larger genetic diversity than initially believed, and underlining the need to increase the number of Toscana virus sequences and their geographic diversity.5
3. WHAT IS THE FREQUENCY OF THE DISEASE? Because of the arthropod-borne nature of Toscana virus, at-risk areas depend on the distribution of the sandfly species that are capable of transmitting the virus.
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Sandfly distribution is considerably influenced by climatic changes and environmental modifications,6 and recent data suggest that there is tendency to northbound expansion into Austria, Germany, and Switzerland.7 Human cases have been laboratory documented using direct diagnosis (RT-PCR or virus isolation) in Italy, France, Spain, Cyprus, Greece, Portugal, and Croatia.5,8,9 During the last decade, direct and indirect evidence was increasingly reported from the aforementioned regions, but also from regions where Toscana virus was unrecognized such as Morocco and Tunisia in northern Africa, Turkey, and the Mediterranean islands (Corsica, Malta, Elba, Sardinia, and Sicily).10 Recent studies also indicate that Toscana virus is also widely distributed in northern Africa and in Turkey. Since seroprevalence rates frequently reach 5% and can raise up to .50% depending on the region, Toscana virus is one of the most prevalent arboviruses in Europe. Interestingly, there are no data from south-eastern Asian countries such as Taiwan, Hong Kong, and Malaysia, and no reports from Australia and New Zealand. Whether or not this accurately reflects the absence of Toscana virus in these regions remains to be investigated, since this could be falsely reassuring due to the lack of specific studies conducted in these regions. To summarize the extent of recent knowledge about Toscana virus, direct detection was reported in Portugal, Spain, France, Italy, Croatia, Turkey, Morocco, and Tunisia; and indirect (serology-based) evidence was published in Portugal, Spain, France, Italy, Croatia, Greece, Bosnia-Herzegovina, Kosovo, Cyprus, Turkey, and Tunisia.
4. HOW IS THE VIRUS TRANSMITTED? Toscana virus is transmitted to humans and other vertebrates when infected female sandflies take a blood meal. At present, there are no data to assess that either humans or other vertebrates are acting as reservoir for Toscana virus; it is commonly accepted that they are dead-end hosts, and thus do not play a significant role in nature in the virus life cycle. Considering the hypothesis, it seems reasonable to assume that the primary reservoir host is the sandfly in which the virus replicates. Detection of Toscana virus from female sandflies but also from males indicates that the virus is not exclusively acquired through blood meal, and that alternative modes of transmission between sandflies are to be considered. Transovarial and venereal transmission of Toscana virus were demonstrated at the vector level through experiments conducted in insectariums with colonized sandflies.11 16 Currently, maintenance and transmission of Toscana virus in nature appears to depend on the presence of appropriate vector species and their abundance in a local environment. Studies aiming at a better characterization of the determinants driving the ecology of Toscana virus together with environmental parameters within a defined geographic area must be encouraged.
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This knowledge is pivotal for virus control and could be beneficial both for public health and for predicting their potential for emergence in new regions.
5. WHICH FACTORS ARE INVOLVED IN DISEASE PATHOGENESIS? WHAT ARE THE PATHOGENIC MECHANISMS? Various clinical forms of Toscana virus infection are described. They vary from mild febrile illness to peripheral or central neurological manifestations that can be very severe (encephalitis) although fatalities are exceptional. Non-symptomatic infections are suspected but there is no laboratorydocumented case, and if they exist their frequency is unknown. The reasons for such a wide array of clinical features are unexplored.
6. WHAT ARE THE CLINICAL MANIFESTATIONS? After a short incubation period (2 7 days), the onset is brutal with non-specific signs of viral febrile illness associated or not with central nervous system manifestations (aseptic meningitis or meningoencephalitis).17,18 Neuroinvasive infections usually begin with headache, fever, nausea, vomiting, and myalgia. Physical examination may show neck rigidity, Kernig sign, and in some cases consciousness, tremors, paresis, and nystagmus. In most cases, the cerebrospinal fluid contains more than 5 10 cells/mL with normal levels of glucose and proteins. Leucopenia or leucocytosis can be observed. The outcome is usually favorable without reported sequelae. Although the vast majority of cases have a favorable outcome, some severe cases of Toscana virus infections were described.8,9,18 20 Recent studies have reported a larger extent for neurological symptoms than initially identified. Other neurological manifestations such as deafness,21,22 persistent personality alterations,23 fasciitis and myositis,24 and speech disorders and paresis25 were reported.
7. HOW DO YOU DIAGNOSE? Toscana virus infection can be diagnosed using virus isolation, serological tests, and molecular tests (RT-PCR). These methods are often combined in clinical microbiology laboratories. In the absence of direct evidence of Toscana virus (RT-PCR or virus isolation), demonstration of a seroconversion or presence of Toscana virus IgM in the CSF is needed to assess a confirmed case. To avoid misclassification of suspect Toscana virus cases, the case definition of the National Notifiable Diseases Surveillance System of the US Center for Disease Control and Prevention should be used.26 As demonstrated from this case, virus isolation should be attempted whenever biological material is available since it is the gold standard technique. It is important to underline that commonly used serological tests to detect IgG
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and IgM such as immunofluorescence (IFA) and ELISA have a poor capacity to discriminate between Toscana virus and other related virus belonging to the same serocomplex (Naples, Massilia, Granada, Punique, Theran). Because of antigenic closeliness, cross-reactivity must be anticipated and ELISA/IFA results must be confirmed by neutralization tests. The increasing number of recently discovered sandfly-borne phleboviruses will render confirmation using neutralization assays more and more indispensable. There are several molecular tests that can be used for direct detection of the genome of Toscana virus in CSF, in serums (and also in sandflies).4,27 It is important to underline that there is a striking need for standardized diagnostic kits (commercially available at affordable prices) to include the detection of Toscana virus in syndromes such as “fever of unknown origin” and “CNS infections,” and to organize large-scale epidemiologic studies in at-risk regions.
8. HOW DO YOU DIFFERENTIATE THIS DISEASE FROM SIMILAR ENTITIES? The few studies reporting series of cases are in agreement for denying the existence of specific characters of infection due to Toscana virus.20,28,29 For instance, clinical and paraclinical signs are similar for aseptic meningitis due to enteroviruses and to Toscana virus. Only the seasonal aspect may be differential since Toscana virus cases can occur only when sandflies are circulating, which may drastically vary from one region to another, but can be roughly estimated between March and November with a clear peak in July and August. Other than neuroinvasive infections, summer fevers are generally not laboratory documented since these patients are not hospitalized and thus escape from subsequent specific virological investigations. Research projects focused on general medicine practices might be organized to study the incidence of this virus in febrile illness.
9. WHAT IS THE THERAPEUTIC APPROACH? There are no approved drugs for treatment of Toscana virus infections. The majority of in vitro studies were done using Sicilian virus that is very distantly related to Toscana virus.30,31 None of the volunteers who were treated with ribavirin were infected when they were experimentally infected with Sicilian virus.32 The pyrazine derivatives T-705 and T-1106 showed in vitro activity against Naples virus with a lower toxicity than ribavirin.33,34
10. WHAT ARE THE PREVENTIVE AND INFECTION CONTROL MEASURES? There is no licensed vaccine for Toscana virus. Repellents, insecticides, and impregnated bed nets may be efficient at the individual level, but rarely used
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because sandflies are commonly unnoticed.35 38 Insecticide spraying significantly decreases the incidence of sandfly-borne diseases only if the spraying is continuous, whereas sporadic campaigns are considered to be ineffective. Alternatives such as insecticide-impregnated curtains,39 insecticideimpregnated dog collars,40 insecticide-treated sugar baits, pheromone dispenser baits,41,42 and cultivation of noxious plants against sandflies43 have been recently developed and are being studied.
REFERENCES 1. Nougairede A, Bichaud L, Thiberville SD, et al. Isolation of Toscana virus from the cerebrospinal fluid of a man with meningitis in Marseille, France, 2010. Vector Borne Zoonotic Dis 2013;13:685 8. 2. Sanbonmatsu-Ga´mez S, Pe´rez-Ruiz M, Collao X, et al. Toscana virus in Spain. Emerg Infect Dis 2005;11:1701 7. 3. Ergu¨nay K, Saygan MB, Aydo˘gan S, et al. Sandfly fever virus activity in central/northern anatolia, Turkey: first report of Toscana virus infections. Clin Microbiol Infect 2011;17:575 81. 4. Charrel RN, Izri A, Temmam S, et al. Cocirculation of 2 genotypes of Toscana virus, southeastern France. Emerg Infect Dis 2007;13:465 8. 5. Punda-Poli´c V, Mohar B, Duh D, et al. Evidence of an autochthonous Toscana virus strain in Croatia. J Clin Virol 2012;55:4 7. 6. Weaver SC, Reisen WK. Present and future arboviral threats. Antiviral Res 2010;85:328 45. 7. Fischer D, Moeller P, Thomas SM, Naucke TJ, Beierkuhnlein C. Combining climatic projections and dispersal ability: a method for estimating the responses of sandfly vector species to climate change. PLoS Negl Trop Dis 2011;5:e1407. 8. Depaquit J, Grandadam M, Fouque F, Andry PE, Peyrefitte C. Arthropod-borne viruses transmitted by Phlebotomine sandflies in Europe: a review. Euro Surveill 2010;15:19507. 9. Charrel RN, Gallian P, Navarro-Mari JM, et al. Emergence of Toscana virus in Europe. Emerg Infect Dis 2005;11:1657 63. 10. Alkan C, Bichaud L, de Lamballerie X, Alten B, Gould EA, Charrel RN. Sandfly-borne phleboviruses of Eurasia and Africa: epidemiology, genetic diversity, geographic range, control measures. Antiviral Res 2013;100:54 74. 11. Ciufolini MG, Maroli M, Guandalini E, Marchi A, Verani P. Experimental studies on the maintenance of Toscana and Arbia viruses (Bunyaviridae: Phlebovirus). Am J Trop Med Hyg 1989;40:669 75. 12. Ciufolini MG, Maroli M, Verani P. Laboratory reared sandflies (Diptera: Psychodidae) and studies on phleboviruses. Parassitologia 1991;33(Suppl):137 42. 13. Ciufolini MG, Maroli M, Verani P. Growth of two phleboviruses after experimental infection of their suspected sand fly vector, Phlebotomus perniciosus (Diptera: Psychodidae). Am J Trop Med Hyg 1985;34:174 9. 14. Maroli M, Ciufolini MG, Verani P. Vertical transmission of Toscana virus in the sandfly, Phlebotomus perniciosus, via the second gonotrophic cycle. Med Vet Entomol 1993;7:283 6. 15. Tesh RB, Modi GB. Maintenance of Toscana virus in Phlebotomus perniciosus by vertical transmission. Am J Trop Med Hyg 1987;36:189 93.
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16. Tesh RB, Lubroth J, Guzman H. Simulation of arbovirus overwintering: survival of Toscana virus (Bunyaviridae: Phlebovirus) in its natural sand fly vector Phlebotomus perniciosus. Am J Trop Med Hyg 1992;47:574 81. 17. Dionisio D, Valassina M, Ciufolini MG, et al. Encephalitis without meningitis due to sandfly fever virus serotype toscana. Clin Infect Dis 2001;32:1241 3. 18. Baldelli F, Ciufolini MG, Francisci D, et al. Unusual presentation of life-threatening Toscana virus meningoencephalitis. Clin Infect Dis 2004;38:515 20. 19. Cusi MG, Savellini GG, Zanelli G. Toscana virus epidemiology: from Italy to beyond. Open Virol J 2010;4:22 8. 20. Vocale C, Bartoletti M, Rossini G, et al. Toscana virus infections in northern Italy: laboratory and clinical evaluation. Vector Borne Zoonotic Dis 2012;12:526 9. 21. Martinez-Garcia FA, Moreno-Docon A, Segovia-Hernandez M, Fernandez-Barreiro A. Deafness as a sequela of Toscana virus meningitis. Med Clin (Barc) 2008;130:639. 22 Paul C, Schwarz TF, Meyer CG, Jager G. Neurological symptoms after an infection by the sandfly fever virus. Dtsch Med Wochenschr 1995;120:1468 72. 23. Serata D, Rapinesi C, Del Casale A, et al. Personality changes after Toscana virus encephalitis in a 49-year-old man: a case report. Int J Neurosci 2011;121:165 9. 24. Doudier B, Ninove L, Million M, de Lamballerie X, Charrel RN, Brouqui P. Unusual Toscana virus encephalitis in southern France. Med Mal Infect 2011;41:50 1. 25. Sanbonmatsu-Gamez S, Perez-Ruiz M, Palop-Borras B, Navarro-Mari JM. Unusual manifestation of Toscana virus infection, Spain. Emerg Infect Dis 2009;15:347 8. 26. Anonymous. Case definitions: nationally notifiable conditions infectious and noninfectious case. Atlanta, GA: Centers for Disease Control and Prevention; 2012. 27. Cusi MG, Savellini GG. Diagnostic tools for Toscana virus infection. Expert Rev Anti Infect Ther 2011;9:799 805. 28. Jaijakul S, Arias CA, Hossain M, Arduino RC, Wootton SH, Hasbun R. Toscana meningoencephalitis: a comparison to other viral central nervous system infections. J Clin Virol 2012;55:204 8. 29. de Ory F, Avello´n A, Echevarrı´a JE, et al. Viral infections of the central nervous system in Spain: a prospective study. J Med Virol 2013;85:554 62. 30. Kirsi JJ, North JA, McKernan PA, et al. Broad-spectrum antiviral activity of 2-betaD-ribofuranosylselenazole-4-carboxamide, a new antiviral agent. Antimicrob Agents Chemother 1983;24:353 61. 31. Crance JM, Gratier D, Guimet J, Jouan A. Inhibition of sandfly fever Sicilian virus (Phlebovirus) replication in vitro by antiviral compounds. Res Virol 1997;148:353 65. 32. Huggins JW. Prospects for treatment of viral hemorrhagic fevers with ribavirin, a broadspectrum antiviral drug. Rev Infect Dis 1989;11(Suppl. 4):S750 61. 33. Gowen BB, Wong MH, Jung KH, et al. In vitro and in vivo activities of T-705 against arenavirus and bunyavirus infections. Antimicrob Agents Chemother 2007;51:3168 76. 34. Gowen BB, Wong MH, Jung KH, Smee DF, Morrey JD, Furuta Y. Efficacy of favipiravir (T-705) and T-1106 pyrazine derivatives in phlebovirus disease models. Antiviral Res 2010;86:121 7. 35. Alten B, Caglar SS, Kaynas S, Simsekand FM. Evaluation of protective efficacy of K-OTAB impregnated bednets for cutaneous leishmaniasis control in Southeast AnatoliaTurkey. J Vector Ecol 2003;28:53 64. 36. Elnaiem DA, Elnahas AM, Aboud MA. Protective efficacy of lambdacyhalothrinimpregnated bednets against Phlebotomus orientalis, the vector of visceral leishmaniasis in Sudan. Med Vet Entomol 1999;13:310 4.
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37. Jalouk L, Al Ahmed M, Gradoni L, Maroli M. Insecticide-treated bednets to prevent anthroponotic cutaneous leishmaniasis in aleppo governate, Syria: results from two trials. Trans R Soc Trop Med Hyg 2007;101:360 7. 38. Faiman R, Cun˜o R, Warburg A. Control of phlebotomine sand flies with vertical fine-mesh nets. J Med Entomol 2009;46:820 31. 39. Maroli M, Majori G. Permethrin-impregnated curtains against phlebotomine sand flies (Diptera: Psychodidae): laboratory and field studies. Parassitologia 1991;33:399 404. 40. Killick-Kendrick R, Killick-Kendrick M, Focheux C. Protection of dogs from the bites of phlebotomine sand flies by deltamethrin collars for the control of canine leishmaniasis. Med Vet Entomol 1997;11:105 11. 41. Bray DP, Alves GB, Dorval ME, Brazil RP, Hamilton JG. Synthetic sex pheromone attracts the leishmaniasis vector Lutzomyia longipalpis to experimental chicken sheds treated with insecticide. Parasit Vectors 2010;3:16. 42. Bray DP, Bandi KK, Brazil RP, Oliveira AG, Hamilton JG. Synthetic sex pheromone attracts the leishmaniasis vector Lutzomyia longipalpis (Diptera: Psychodidae) to traps in the field. J Med Entomol 2009;46:428 34. 43. Schlein Y, Jacobson RL, Mu¨ller GC. Sand fly feeding on noxious plants: a potential method for the control of leishmaniasis. Am J Trop Med Hyg 2001;65:300 3.
Toscana Virus Infection Remi N. Charrel Aix Marseille Universite´, IRD French Institute of Research for Development, EHESP French School of Public Health, EPV UMR_D 190 “Emergence des Pathologies Virales”, & IHU Me´diterrane´e Infection, APHM Public Hospitals of Marseille 13385, Marseille, France
CASE PRESENTATION At the beginning of summer, a 17-year-old male, originating from and living in France, without noticeable medical background, visited his medical practitioner for brutal and recent onset characterized by fever, severe headache, and vomiting. After examination, his GP referred the patient to the emergency ward of the public hospital for suspected aseptic meningitis. Upon admission, he presented with the same manifestations in a febrile context (39 C) in spite of self-medication with paracetamol. Examination revealed stiff neck in the absence of photophobia or signs suggesting encephalitis. Laboratory tests showed an increased white blood cell count (WBC) at 11,700/mm3 (80% neutrophils) and a normal C-reactive protein. The cerebrospinal fluid (CSF) contained 840 leukocytes/mm3 (70% lymphocytes) with normal glucose and protein levels. All molecular tests performed on CSF that targeted enteroviruses, herpes simplex virus types 1 and 2, Neisseria meningitidis, and Streptococcus pneumoniae were negative. The patient reported no recent history of travel. Treatment was started with amoxicillin and acyclovir, and the patient was transferred into the Infectious Diseases department. The day after, he was no longer febrile, but still complained of severe headache and vomiting. Routine analysis of the CSF including direct microscopic examination, bacterial culture, and conventional PCR assays for 16 S and 18 S were negative. Real-time RT-PCR for enterovirus, West Nile virus, and Toscana virus were performed. The real-time RT-PCR for Toscana virus was positive (cycle threshold value: 38). Despite low viral load in the CSF, the virus strain was isolated in Vero cells and further characterized by complete genome sequencing.1 Serology using indirect immunofluorescence showed the presence of IgM and IgG in the acute serum. Clinical manifestations rapidly resolved and the patient was discharged the next day. Three days after the diagnosis was established, a field campaign was organized to collect sandflies in the Emerging Infectious Diseases. DOI: http://dx.doi.org/10.1016/B978-0-12-416975-3.00008-X © 2014 Elsevier Inc. All rights reserved.
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vicinity close to the patient’s home. Four CDC miniature light traps were placed in the garden around the house, where the patient used to spend time at nightfall. A total of 17 Phlebotomus perniciosus were trapped but were negative for Toscana virus RNA through real-time RT-PCR. However, the trapping of sandflies in the vicinity close to the patient’s home confirmed that the patient may have been exposed at home to sandflies, known as the vector of Toscana virus. (This is a published case report.1)
1. WHY THIS CASE WAS SIGNIFICANTLY IMPORTANT AS AN EMERGING INFECTION Neurological infections caused by Toscana virus, a sandfly-borne phlebovirus, are common during the summer in regions bordering the Mediterranean Sea (Figure 8.1). Studies collating series of Toscana virus cases demonstrate that Toscana virus is the third cause of aseptic meningitis and meningoencephalitis in the regions of Spain, France, and Italy where the virus circulates in sandflies. Seroprevalence studies performed in southern Europe indicate that a significant proportion of the exposed population (5 to .50%, depending on the studies and on the regions) possess antibodies that react with Toscana virus. According to these data, Toscana virus is the most prevalent arthropod-borne virus in Europe far ahead of tick-borne encephalitis virus, West Nile virus, or dengue virus. As an indicator of medical and scientific attention, a search using the keyword “Toscana virus” retrieved 204 related
FIGURE 8.1 Toscana virus in the Mediterranean Sea.
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articles in PubMed, which correspond to fewer than five articles per year since virus discovery. There is a great need to draw attention to Toscana virus. Because it was discovered in the early 1970s, Toscana virus should be considered as a neglected pathogen rather than an emerging pathogen. As a countermeasure, availability of affordable commercial kits for molecular diagnosis and serology should be aimed for in order to enable dissemination of the diagnostic capacities outside of the few reference centers.
2. WHAT IS THE CAUSATIVE AGENT? Toscana virus (Toscana virus; Bunyaviridae; Phlebovirus) is an enveloped, single-strand, negative-sense RNA virus. First isolated in Italy in 1971, Toscana virus is an arthropod-borne virus transmitted by phlebotomine sandflies (Phlebotomus sp. of the subgenus Laroussius). According to the International Classification on the Taxonomy of Viruses, Toscana virus belongs to the species Sandfly fever Naples virus, which includes also several other viruses such as Naples virus and Theran virus; in addition, there are several recently discovered phleboviruses that should be included in this species together with Toscana virus; this is not the role of ICTV since this organism classifies virus down to the level of species, not below the species. Obviously, these newly discovered viruses (Massilia, Granada, Punique) have to be considered as variant genotypes of the species Sandfly fever Naples virus. There is a very limited knowledge on the capacity of these variants’ genotypes to infect humans and to cause disease in humans. Previous phylogenetic analysis of Toscana virus first identified two geographically associated lineages: the lineage A endemic in Italy (including the Sardinia and the Sicily) and the lineage B endemic in Spain.2 New sequences of Toscana virus isolates originating from other countries confirmed this dichotomy: Turkish and Tunisian Toscana virus are more closely related to Italian strains than to the Spanish ones. In contrast, Moroccan Toscana virus is more closely related to Spanish strains than to the Italian ones.3 This dichotomy could be linked to the existence of different vectors (P. perniciosus, P. perfiliewi, other species of Phlebotomus) and/or to the existence of two geographically restricted races of P. perniciosus, the main vector of Toscana virus.2 Interestingly, Toscana virus strains that circulate in France were reported to belong to either of these two lineages.4 Recently, a new lineage of Toscana virus was described in Croatia suggesting a larger genetic diversity than initially believed, and underlining the need to increase the number of Toscana virus sequences and their geographic diversity.5
3. WHAT IS THE FREQUENCY OF THE DISEASE? Because of the arthropod-borne nature of Toscana virus, at-risk areas depend on the distribution of the sandfly species that are capable of transmitting the virus.
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Sandfly distribution is considerably influenced by climatic changes and environmental modifications,6 and recent data suggest that there is tendency to northbound expansion into Austria, Germany, and Switzerland.7 Human cases have been laboratory documented using direct diagnosis (RT-PCR or virus isolation) in Italy, France, Spain, Cyprus, Greece, Portugal, and Croatia.5,8,9 During the last decade, direct and indirect evidence was increasingly reported from the aforementioned regions, but also from regions where Toscana virus was unrecognized such as Morocco and Tunisia in northern Africa, Turkey, and the Mediterranean islands (Corsica, Malta, Elba, Sardinia, and Sicily).10 Recent studies also indicate that Toscana virus is also widely distributed in northern Africa and in Turkey. Since seroprevalence rates frequently reach 5% and can raise up to .50% depending on the region, Toscana virus is one of the most prevalent arboviruses in Europe. Interestingly, there are no data from south-eastern Asian countries such as Taiwan, Hong Kong, and Malaysia, and no reports from Australia and New Zealand. Whether or not this accurately reflects the absence of Toscana virus in these regions remains to be investigated, since this could be falsely reassuring due to the lack of specific studies conducted in these regions. To summarize the extent of recent knowledge about Toscana virus, direct detection was reported in Portugal, Spain, France, Italy, Croatia, Turkey, Morocco, and Tunisia; and indirect (serology-based) evidence was published in Portugal, Spain, France, Italy, Croatia, Greece, Bosnia-Herzegovina, Kosovo, Cyprus, Turkey, and Tunisia.
4. HOW IS THE VIRUS TRANSMITTED? Toscana virus is transmitted to humans and other vertebrates when infected female sandflies take a blood meal. At present, there are no data to assess that either humans or other vertebrates are acting as reservoir for Toscana virus; it is commonly accepted that they are dead-end hosts, and thus do not play a significant role in nature in the virus life cycle. Considering the hypothesis, it seems reasonable to assume that the primary reservoir host is the sandfly in which the virus replicates. Detection of Toscana virus from female sandflies but also from males indicates that the virus is not exclusively acquired through blood meal, and that alternative modes of transmission between sandflies are to be considered. Transovarial and venereal transmission of Toscana virus were demonstrated at the vector level through experiments conducted in insectariums with colonized sandflies.11 16 Currently, maintenance and transmission of Toscana virus in nature appears to depend on the presence of appropriate vector species and their abundance in a local environment. Studies aiming at a better characterization of the determinants driving the ecology of Toscana virus together with environmental parameters within a defined geographic area must be encouraged.
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This knowledge is pivotal for virus control and could be beneficial both for public health and for predicting their potential for emergence in new regions.
5. WHICH FACTORS ARE INVOLVED IN DISEASE PATHOGENESIS? WHAT ARE THE PATHOGENIC MECHANISMS? Various clinical forms of Toscana virus infection are described. They vary from mild febrile illness to peripheral or central neurological manifestations that can be very severe (encephalitis) although fatalities are exceptional. Non-symptomatic infections are suspected but there is no laboratorydocumented case, and if they exist their frequency is unknown. The reasons for such a wide array of clinical features are unexplored.
6. WHAT ARE THE CLINICAL MANIFESTATIONS? After a short incubation period (2 7 days), the onset is brutal with non-specific signs of viral febrile illness associated or not with central nervous system manifestations (aseptic meningitis or meningoencephalitis).17,18 Neuroinvasive infections usually begin with headache, fever, nausea, vomiting, and myalgia. Physical examination may show neck rigidity, Kernig sign, and in some cases consciousness, tremors, paresis, and nystagmus. In most cases, the cerebrospinal fluid contains more than 5 10 cells/mL with normal levels of glucose and proteins. Leucopenia or leucocytosis can be observed. The outcome is usually favorable without reported sequelae. Although the vast majority of cases have a favorable outcome, some severe cases of Toscana virus infections were described.8,9,18 20 Recent studies have reported a larger extent for neurological symptoms than initially identified. Other neurological manifestations such as deafness,21,22 persistent personality alterations,23 fasciitis and myositis,24 and speech disorders and paresis25 were reported.
7. HOW DO YOU DIAGNOSE? Toscana virus infection can be diagnosed using virus isolation, serological tests, and molecular tests (RT-PCR). These methods are often combined in clinical microbiology laboratories. In the absence of direct evidence of Toscana virus (RT-PCR or virus isolation), demonstration of a seroconversion or presence of Toscana virus IgM in the CSF is needed to assess a confirmed case. To avoid misclassification of suspect Toscana virus cases, the case definition of the National Notifiable Diseases Surveillance System of the US Center for Disease Control and Prevention should be used.26 As demonstrated from this case, virus isolation should be attempted whenever biological material is available since it is the gold standard technique. It is important to underline that commonly used serological tests to detect IgG
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and IgM such as immunofluorescence (IFA) and ELISA have a poor capacity to discriminate between Toscana virus and other related virus belonging to the same serocomplex (Naples, Massilia, Granada, Punique, Theran). Because of antigenic closeliness, cross-reactivity must be anticipated and ELISA/IFA results must be confirmed by neutralization tests. The increasing number of recently discovered sandfly-borne phleboviruses will render confirmation using neutralization assays more and more indispensable. There are several molecular tests that can be used for direct detection of the genome of Toscana virus in CSF, in serums (and also in sandflies).4,27 It is important to underline that there is a striking need for standardized diagnostic kits (commercially available at affordable prices) to include the detection of Toscana virus in syndromes such as “fever of unknown origin” and “CNS infections,” and to organize large-scale epidemiologic studies in at-risk regions.
8. HOW DO YOU DIFFERENTIATE THIS DISEASE FROM SIMILAR ENTITIES? The few studies reporting series of cases are in agreement for denying the existence of specific characters of infection due to Toscana virus.20,28,29 For instance, clinical and paraclinical signs are similar for aseptic meningitis due to enteroviruses and to Toscana virus. Only the seasonal aspect may be differential since Toscana virus cases can occur only when sandflies are circulating, which may drastically vary from one region to another, but can be roughly estimated between March and November with a clear peak in July and August. Other than neuroinvasive infections, summer fevers are generally not laboratory documented since these patients are not hospitalized and thus escape from subsequent specific virological investigations. Research projects focused on general medicine practices might be organized to study the incidence of this virus in febrile illness.
9. WHAT IS THE THERAPEUTIC APPROACH? There are no approved drugs for treatment of Toscana virus infections. The majority of in vitro studies were done using Sicilian virus that is very distantly related to Toscana virus.30,31 None of the volunteers who were treated with ribavirin were infected when they were experimentally infected with Sicilian virus.32 The pyrazine derivatives T-705 and T-1106 showed in vitro activity against Naples virus with a lower toxicity than ribavirin.33,34
10. WHAT ARE THE PREVENTIVE AND INFECTION CONTROL MEASURES? There is no licensed vaccine for Toscana virus. Repellents, insecticides, and impregnated bed nets may be efficient at the individual level, but rarely used
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because sandflies are commonly unnoticed.35 38 Insecticide spraying significantly decreases the incidence of sandfly-borne diseases only if the spraying is continuous, whereas sporadic campaigns are considered to be ineffective. Alternatives such as insecticide-impregnated curtains,39 insecticideimpregnated dog collars,40 insecticide-treated sugar baits, pheromone dispenser baits,41,42 and cultivation of noxious plants against sandflies43 have been recently developed and are being studied.
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