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Sandfly-borne phleboviruses are endemic in the Mediterranean basin. However, levels of exposure of human and animal populations are inadequately researched. Toscana virus (TOSV) is present in Portugal where it causes human infection and disease; in contrast there are few data for sandfly fever Sicilian virus (SFSV) which has neither been isolated nor detected by molecular tests and for which there are only limited serological data. The sera collected from 1160 dogs and 189 cats in southern Portugal were tested for the presence of neutralizing antibodies against TOSV and SFSV, two viruses recognized as distinct serocomplexes in the Mediterranean region. Our data showed (i) seropositivity to TOSV and SFSV in dogs at a rate of 6.8 and 50.8 %, respectively, and (ii) that 3.7 % of cats were seropositive for TOSV. TOSV findings are in line with previous results obtained with less stringent serological assays. Our results for SFSV in dogs clearly indicate that the virus is circulating widely and that humans may be exposed to infection via the dogs. Although the presence of SFSV was suggested by haemagglutination inhibition in 4/1690 human sera in 1974, this is the first time, as far as we know, that SFSV has been shown to circulate so widely in dogs in Portugal. Future studies should be directed at isolating strains of SFSV in Portugal from dogs, humans and sandflies collected in high prevalence regions. As dogs appear to be good sentinels for SFSV, their role as a possible reservoir in the natural cycle should also be considered. Grant FP7-261504

Background: Massilia virus (MASV) is a phlebovirus isolated from Phlebotomus perniciosus in various regions of southwestern Europe. It is closely related to human pathogens such as Toscana virus and sandfly fever Naples virus. The natural cycle of phleboviruses is poorly understood. Indeed, experimental studies demonstrate that transovarial and sexual transmission are not efficient enough for the maintenance of the virus in nature and to date there is no convincing evidence that a species of vertebrates is the reservoir of the virus. Here, we studied various transmission routes of MASV taking advantage of experimental colonies representing different species of sand flies. Methodology/Principal findings: In P. perniciosus, four sources of infection were compared: (i) Virus-seeded larval food to the first instar larvae (L1), or (ii) to the fourth instar larvae (L4), (iii) virus-seeded blood meal to adult females, and (iv) virus-seeded sugar meal to adults of both sexes. From 875 adults emerged from infected L1 and L4, only three were positive. In females infected by bloodmeal the infection rate was high before defecation, then it decreased drastically, MASV RNA was detected in only 5 out of 27 post-defecation. Surprisingly, the most efficient route of infection was observed after intake of virus-seeded sugar meal: 72% of females (79/110) and 52% of males (51/99) were found to be MASV RNA-positive. In addition, MASV-infected sandflies regurgitated virus particules into the sugar drop and MASV RNA was detectable in this drop for at least 24 h after regurgitation. MASV RNA was detected in about one third of the P. perniciosus exposed to this sugar drop contaminated by regurgitation. Sugar meal infection was also tested with six other species of sand flies. In males, there were no significant differences in infection rates when compared to P. perniciosus. In females, most species tested showed high infection rate at the beginning but then significant gradual decrease in infection rate during the experiment. Conclusions/Significance: We present the first description of arboviral infection of a dipteran vector using sugar meal. In all seven sand fly species tested, MASV was detected for two weeks post-infection. Our results showed that MASV can be transmitted between P. perniciosus either through co-feeding or via an infected sugar source such as plant sap. These newly described routes of horizontal transmission may play an important role in the circulation of phleboviruses in nature.

International audience; An entomological investigation performed in 2013 covering different bio-geographical areas varying from humid in the north to the arid in the center showed that sand flies of the subgenus Larroussius including Phlebotomus perniciosus, Phlebotomus perfiliewi, and Phlebotomus longicuspis are abundant and widely distributed in Tunisia A total of 3992 collected and pooled with up to 30 specimens per pool based on sex, trapping location and collection date were tested for the presence of phleboviruses by nested reverse transcriptase polymerase chain reaction and sequencing Of a total of 135 pools, 23 were positive, yielding and minimum infection rate of 0.6%. Phylogenetic analysis performed using partial amino acid sequence in the polymerase gene showed that all these phleboviruses were grouped in one cluster clearly distinct from but closely related to Massilia virus and Granada virus This putative novel virus, tentatively called Saddaguia virus (SADV), is widely distributed in Tunisia Together with Toscana, Punique, and Utique viruses, SADV is the fourth recognized phlebovirus to be transmitted by sand flies in Tunisia The medical and public health interest of SADV remains to be investigated.

Leishmaniasis is considered a rural disease in Europe. However, circumstantial evidence has indicated urban transmission of leishmaniasis and phleboviruses in the urban area of Marseille, France. To investigate this urban transmission, sandflies were trapped in 33 locations in the urban area (horse farms, public gardens and a residential area). Sandflies were always captured: 87.8% were Phlebotomus perniciosus, a vector of Leishmania infantum and Toscana and Massilia viruses. RT-PCR and cell culture inoculation identified the Massilia virus in 2/99 pools of sandflies, and PCR identified Leishmania in 5/99. No dual infection was observed, but both pathogens were detected in samples from the same trapping site.

Toscana virus (TOSV) is a neglected sandfly-borne pathogen in Mediterranean countries. Although discovered four decades ago, articles that describe the clinical aspects are scarce and consist mostly of case reports, with few series of cases. We studied retrospectively symptomatic TOSV infections in patients hospitalized in Marseille (France) from 2004 to 2011. Seventeen patients were classified as probable or confirmed cases. Fourteen cases (82%) occurred between June and September, and 3 cases in March, April and November. Two cases were potentially imported from Croatia and Tuscany. All patients presented with fever and neurological signs were observed such as aseptic meningitis (n = 6), muscular symptoms (n = 3), or encephalitis (n = 4). The outcome was always favorable. At the acute stage, anti TOSV IgM were observed in 14/17 patients, neutralization tests were positive for 3/8 patients, and RT-PCR confirmed TOSV infections in 5/8 CSF specimens.

Sandfly-borne phleboviruses may cause a transient febrile illness (sandfly fever) or more severe neuroinvasive disease. In the Old World, they are vectored by phlebotomine flies, which are widely distributed in the Mediterranean basin, North Africa, the Indian subcontinent, the Middle East and central Asia. High seroprevalence rates have been recorded in humans and domestic animals in areas where sandflies are present. Most published studies have focused on phlebovirus infections of travelers and of soldiers stationed in endemic areas, but the health impact on local populations should not be underestimated, as seroprevalence studies indicate massive circulation of these viruses, even if disease is seldom documented. Except for Toscana virus, which shows a marked neurotropism and is a leading cause of aseptic meningitis in endemic regions, phlebovirus infections are inadequately considered by physicians and are generally underestimated. However, several properties of these viruses suggest that they will extend their geographic range. First, changes in the areas occupied by sandflies as a result of climate change have a direct impact on the epidemiology of associated human and animal diseases. Second, phleboviruses exhibit a high mutation rate, and their tri-segmented genome is prone to reassortment and recombination. Third, distinct virus strains can be transmitted by the same arthropod species. Recent studies have documented the distribution of sandfly-borne phleboviruses in Western Europe, but data for Eastern Europe, the Middle East and Africa are very limited. With the goal of filling knowledge gaps and planning new research programs, we have examined available information and present it as a comprehensive review, with a specific focus on understudied regions. We also discuss the need to conduct studies aimed at developing new antiviral drugs and vaccines.

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.

During summer 2007, a total of 785 phlebotomine flies were trapped in northern Algeria, identified morphologically, organised as monospecific pools and tested for the presence of phlebovirus RNA using degenerate primers. Three pools were positive, and the corresponding PCR products were cloned and sequenced. Viral sequences corresponding to two phleboviruses distinct from each other were detected in sandflies circulating in two close locations (140 km apart) in Northern Algeria. The 3 sequences were aligned with homologous polymerase sequences retrieved from the Genbank database, in order to examine their phylogenetic relationships. One viral sequence (from Phlebotomus papatasi) was closely related to but distinct from a sequence obtained from Phlebotomus ariasi sandflies trapped in Algeria in 2006. The two other viral sequences (from Phlebotomus longicuspis) were genetically distantly related to sequences corresponding to virus members of the Sandfly fever Naples virus species and although falling within the same group, this clearly represents a second distinct novel lineage. These results are indicative of a high genetic heterogeneity within sandflies trapped in a relatively small geographic area. Seroprevalence studies conducted on sera from populations living in the same areas indicated that humans can be infected by these viruses.

A new flavivirus, Ecuador Paraiso Escondido virus (EPEV), named after the village where it was discovered, was isolated from sand flies ( Psathyromyia abonnenci , formerly Lutzomyia abonnenci ) that are unique to the New World. This represents the first sand fly-borne flavivirus identified in the New World. EPEV exhibited a typical flavivirus genome organization. Nevertheless, the maximum pairwise amino acid sequence identity with currently recognized flaviviruses was 52.8%. Phylogenetic analysis of the complete coding sequence showed that EPEV represents a distinct clade which diverged from a lineage that was ancestral to the nonvectored flaviviruses Entebbe bat virus, Yokose virus, and Sokoluk virus and also the Aedes -associated mosquito-borne flaviviruses, which include yellow fever virus, Sepik virus, Saboya virus, and others. EPEV replicated in C6/36 mosquito cells, yielding high infectious titers, but failed to reproduce either in vertebrate cell lines (Vero, BHK, SW13, and XTC cells) or in suckling mouse brains. This surprising result, which appears to eliminate an association with vertebrate hosts in the life cycle of EPEV, is discussed in the context of the evolutionary origins of EPEV in the New World. IMPORTANCE The flaviviruses are rarely (if ever) vectored by sand fly species, at least in the Old World. We have identified the first representative of a sand fly-associated flavivirus, Ecuador Paraiso Escondido virus (EPEV), in the New World. EPEV constitutes a novel clade according to current knowledge of the flaviviruses. Phylogenetic analysis of the virus genome showed that EPEV roots the Aedes -associated mosquito-borne flaviviruses, including yellow fever virus. In light of this new discovery, the New World origin of EPEV is discussed together with that of the other flaviviruses.

An entomological investigation was carried out in 2014 at two sites located in Central Tunisia, one irrigated and another non-irrigated situated in arid bio-geographical areas. Sand flies of the subgenus Larroussius namely Phlebotomus perfiliewi, Phlebotomus perniciosus, and Phlebotomus longicuspis are the most abundant sand fly species in the irrigated site. However, in the non-irrigated site, Phlebotomus papatasi of the Phlebotomus genus is the most abundant species. A total of 3191 sand flies were collected and pooled with up to 30 specimens per pool based on sex, trapping location and collection date, were tested for the presence of phleboviruses by nested reverse transcriptase polymerase chain reaction in the polymerase gene and sequenced. Of a total of 117 pools, 4 were positive, yielding a minimum infection rate of sand flies with phleboviruses of 0.12%. Phylogenetic analysis performed using partial nucleotide and amino acid sequence in the polymerase gene showed that these phleboviruses belonged to four different clusters corresponding to Toscana virus (TOSV), Saddaguia virus (SADV), Sandfly Fever Sicilian Virus (SFSV) and Utique virus (UTIV). This study provides more evidence that the abundance of P. perfiliewi is associated with the development of irrigation in arid bio-geographical areas of Central Tunisia which may have led to the emergence of phleboviruses. We report the first detection of TOSV from sand flies collected from Central Tunisia.

WOS: 000352218400006, PubMed ID: 25653443, A new phlebovirus, Adana virus, was isolated from a pool of Phlebotomus spp. (Diptera; Psychodidae) in the province of Adana, in the Mediterranean region of Turkey. Genetic analysis based on complete coding of genomic sequences indicated that Adana virus belongs to the Salehabad virus species of the genus Phlebovirus in the family Bunyaviridae. Adana virus is the third virus of the Salehabad virus species for which the complete sequence has been determined. To understand the epidemiology of Adana virus, a seroprevalence study using microneutralization assay was performed to detect the presence of specific antibodies in human and domestic animal sera collected in Adana as well as Mersin province, located 147 km west of Adana. The results demonstrate that the virus is present in both provinces. High seroprevalence rates in goats, sheep, and dogs support intensive exposure to Adana virus in the region, which has not been previously reported for any virus included in the Salehabad serocomplex; however, low seroprevalence rates in humans suggest that Adana virus is not likely to constitute an important public health problem in exposed human populations, but this deserves further studies. IMPORTANCE Until recently, in the genus Phlebovirus, the Salehabad virus species consisted of two viruses: Salehabad virus, isolated from sand flies in Iran, and Arbia virus, isolated from sand flies in Italy. Here we present the isolation and complete genome characterization of the Adana virus, which we propose to be included in the Salehabad virus species. To our knowledge, this is the first report of the isolation and complete genome characterization, from sand flies in Turkey, of a Salehabad virus-related phlebovirus with supporting seropositivity in the Mediterranean, Aegean, and Central Anatolia regions, where phleboviruses have been circulating and causing outbreaks. Salehabad species viruses have generally been considered to be a group of viruses with little medical or veterinary interest. This view deserves to be revisited according to our results, which indicate a high animal infection rate of Adana virus and recent evidence of human infection with Adria virus in Greece., European Union's Seventh Framework ProgrammeEuropean Union (EU) [261504-EDENext-FP7]; FP7 Capacities project GA, The research leading to these results has received funding from the European Union's Seventh Framework Programme for research, technological development and demonstration under Grant Agreement numbers 261504-EDENext-FP7 (http://www.edenext.eu) and FP7 Capacities project GA no228292-EVA (European Virus Archive, http: //www.european-virus-archive.com/). The work of R.N.C. was done under the framework of EurNegVec Cost Action TD1303.

International audience; BACKGROUND: Sandfly-borne phleboviruses are present in North Africa where they can infect humans in regions where Leishmania infantum, the causative agent of zoonotic visceral leishmaniasis in the Western Mediterranean basin is present affecting both humans and dogs. We investigated the capacity of dogs to be used as sentinels for sandfly-borne phleboviruses as previously shown for leishmaniasis.FINDINGS: A total of 312 sera were collected from guard dogs in two different bioclimatic regions (governorates of Kairouan and Bizerte) of Tunisia where zoonotic visceral leishmaniasis has been reported. These sera were tested for the presence of neutralising antibodies against 3 phleboviruses: Toscana virus, Punique virus and Sicilian virus. In the governorate of Kairouan, seroprevalence rates of 7.5%, 43.5%, and 38.1% were observed for Toscana, Punique and Sicilian virus, respectively. A high proportion of sera from the governorate of Bizerte were hemolyzed and showed high cytotoxicity for the cells and subsequently precluded detailed interpretation of this batch. However, validated results for 27 sera were in agreement with data observed in the governorate of Kairouan.CONCLUSIONS: Toscana virus is present in the governorate of Kairouan but at a lower rate compared to Punique and Sicilian viruses. These three sandfly-borne phleboviruses can infect dogs. Direct detection and isolation of the viruses are now to be attempted in animals as well as in humans. Our findings showed that guard dogs are good sentinels for virus transmitted by sandflies and strongly suggested that the high seroprevalence rates observed in dogs merit further attention.

The discovery and development of methods for isolation, characterisation and taxonomy of viruses represents an important milestone in the study, treatment and control of virus diseases during the 20th century. Indeed, by the late-1950s, it was becoming common belief that most human and veterinary pathogenic viruses had been discovered. However, at that time, knowledge of the impact of improved commercial transportation, urbanisation and deforestation, on disease emergence, was in its infancy. From the late 1960s onwards viruses, such as hepatitis virus (A, B and C) hantavirus, HIV, Marburg virus, Ebola virus and many others began to emerge and it became apparent that the world was changing, at least in terms of virus epidemiology, largely due to the influence of anthropological activities. Subsequently, with the improvement of molecular biotechnologies, for amplification of viral RNA, genome sequencing and proteomic analysis the arsenal of available tools for virus discovery and genetic characterization opened up new and exciting possibilities for virological discovery. Many recently identified but “unclassified” viruses are now being allocated to existing genera or families based on whole genome sequencing, bioinformatic and phylogenetic analysis. New species, genera and families are also being created following the guidelines of the International Committee for the Taxonomy of Viruses. Many of these newly discovered viruses are vectored by arthropods (arboviruses) and possess an RNA genome. This brief review will focus largely on the discovery of new arthropod-borne viruses.

As a member of the European Virus Archive (EVA) consortium, our laboratory is developing and maintaining a large collection of viruses. This collection implies the use of a panel of cell lines originating from various animal species. In order to make easier the handling of such a large panel of cell lines, wide spectrum real-time PCR and RT-PCR assays were developed to allow the detection and the quantification of DNA and mRNA of β-actin, one of the most commonly used eukaryotic housekeeping genes. By using two degenerated primers and a unique probe, these two assays were shown to detect nucleic acids of a panel of vertebrate and invertebrate cell lines commonly used in animal virology. This panel included human, monkey, rodent, dog, pig, fish, batrachian, mosquito and tick cell lines. Additionally, the two assays amplified successfully β-actin-encoding sequences of sandflies. Sensitivity evaluation performed on synthetic DNA and RNA sequences showed that the two assays were very sensitive and suitable for accurate quantification. The two assays constitute together a convenient method suitable for multiple purposes. They can be used for instance to estimate the amount of contaminating cellular genetic material prior to sequence-independent amplification of viral genomes achieved before high-throughput sequencing, to evaluate the efficiency of DNase and/or RNase treatments performed on cellular extract and to check nucleic acid extraction by using β-actin-encoding sequences as endogenous control. This assay will constitute a precious tool for virologists working with multiple cell lines or animal models.

To the Editor: Toscana virus (TOSV; genus Phlebovirus, family Bunyaviridae) is transmitted by sandflies, mostly the species Phlebotomus perniciosus and P. perfiliewi (1). Initially discovered in central Italy, TOSV was recently identified in other European countries (i.e., Portugal, Spain, France, Croatia, and Turkey) (2). TOSV is a primary cause of aseptic meningitis during warm months (2). A seroprevalence study suggested that TOSV is present in Tunisia and may cause neuroinvasive infections, but definitive evidence of TOSV circulation has not been possible because it is difficult to distinguish from the antigenically related phlebovirus Punique virus (3,4); both viruses are members of the species Sandfly fever Naples virus. We investigated the prevalence of TOSV among sandflies in northern Tunisia. A total of 5,288 sandflies (3,547 females, 1,740 males) were collected during June–October 2010 by using CDC light traps (John W. Hock Company, Gainesville, FL, USA) at Utique (37°08′N, 7°74′E), a focus for visceral leishmaniasis in northern Tunisia. Sandflies were separated by sex and trapping nights and pooled with >30 specimens by pool. Pools were processed as described (4) and subjected to PCR detection of phlebovirus RNA targeting 2 genes independently (4–6) and virus isolation onto Vero cells. Of 249 pools processed, 8 strains of phleboviruses were isolated: 2 TOSV, 3 Punique virus, and 3 other phleboviruses currently being characterized. TOSV strains were obtained from 2 pools of sandflies trapped in September 2010, T152 and T166, consisting of 30 males and 30 females, respectively. These pools were positive for TOSV RNA by sequencing of 2 PCR products (201-nt and 280-nt sections in the large [L] and small [S] gene segments, respectively). Supernatant of the third passage was prepared for electron microscopy, which showed spherical and pleomorphic structures, 80–120 nm in diameter, compatible with viruses of the family Bunyaviridae. Complete genome sequencing was then done by using the Ion PGM Sequencer (Life Technologies SAS, Saint Aubin, France) (7); a total of 165,307 reads were obtained, of which 135,700 matched with the sequence of TOSV Iss.PhL3 used as reference. The viral genome of TOSV Tunisia-2010-T152 (GenBank accession nos. {"type":"entrez-nucleotide-range","attrs":{"text":"JX867534-JX867536","start_term":"JX867534","end_term":"JX867536","start_term_id":"469785662","end_term_id":"469785666"}}JX867534-JX867536) was composed of 12,488 nt; the complete sequence consisted of 1,869 nt, 4,215 nt, and 6,404 nt for the S, medium [M], and L RNA segments, respectively. The partial S sequence of the TOSV Tunisia-2010-T166 strain (GenBank accession nos. {"type":"entrez-nucleotide-range","attrs":{"text":"JX867537-JX867539","start_term":"JX867537","end_term":"JX867539","start_term_id":"469785669","end_term_id":"469785677"}}JX867537-JX867539) was identical to that of T152, but 1 synonymous mutation was observed in the partial L sequence and 1 nonsynonymous mutation in the partial M sequence (I906V). The TOSV Tunisia-2010-T152 strain was aligned with homologous sequences retrieved from the GenBank database. Genetic distances were calculated at the amino acid and nucleotide levels by using the p-distance algorithm (Technical Appendix Tables 1–3). Phylogenetic studies were performed by using the neighbor-joining method in MEGA5 (8) (Figure). The robustness of the nodes was tested by 1,000 bootstrap replications. We found that TOSV Tunisia-2010-T152 was most closely related to the prototype strain from Italy, Iss.PhL3, with nucleotide/amino acid distances of 0.031/0.052, 0.032/0.073, and 0.039/0.012 for the S, M, and L RNA sequences, respectively. Together, these genetic distances and phylogram topologies indicate that TOSV Tunisia-2010-T152 is most closely related to strains within the Italian lineage, although it may represent a distinct sublineage, more distantly related to strains belonging to the Spanish lineage (9). Figure Phylogenetic analysis of 3 segments of Toscana virus (TOSV) isolates from pools of sandflies collected in Tunisia and homologous sequences of other selected phleboviruses. A) Large segments; B) medium segments; C) small segments. Sequences are identified ... Concomitantly with virus isolation, the phenology of sandfly species was studied during May–November 2010. Sandflies were identified, and the density was calculated as described (10). Most of the sandflies belonged to the subgenus Larroussius (98.3%). P. perniciosus sandflies were the most abundant species (71.74%), followed by P. longicuspis (17.47%) and P. perfiliewi (8.82%). Other sandfly species, such as Phlebotomus (Phlebotomus) papatasi, Phlebotomus (Paraphlebotomus) sergenti, Sergentomyia minuta parotti, S. christophersi, and S. antennata were found, but these were much less abundant. The phenology of 3 main sandfly species showed 2 main peaks: 1 small peak in June and a second, larger peak during September–October (Technical Appendix Figure). In summary, of a total of 5,288 sandflies collected, 2 pools were positive for TOSV, yielding an infection rate of 0.03%. A similar infection rate was observed in Spain (0.05%) (9); however, the infection rates in Italy (0.22%) and in France (0.29%) are substantially higher (1,6). The isolation of TOSV from male and female sandflies suggests transovarial transmission in nature, as reported in Italy and Spain (1,9). In southern European countries, TOSV is mostly transmitted by P. perniciosus and P. perfiliewi sandflies (1,6,9), whereas P. perniciosus, P. longicuspis, and P. perfiliewi are the most abundant sandfly species in northern Tunisia. It is therefore probable that TOSV is transmitted by sandfly species of the subgenus Larroussius. We found that 2 phleboviruses belonging to the Sandfly fever Naples virus species, TOSV and Punique virus, are cocirculating in northern Tunisia. This finding calls for further investigation of these viruses’ potential effect on human health in this area. Technical Appendix: Genetic pairwise distances between Toscana virus strains and selected phleboviruses and phenology of sandflies collected in Utique, Governorate of Bizerte, Tunisia. Click here to view.(401K, pdf)

Background In northern Tunisia, the co-circulation of two related sand fly-borne phleboviruses, Toscana virus (TOSV) and Punique virus (PUNV) was previously demonstrated. In contrast to TOSV, a prominent human pathogen, there is no data supporting that PUNV is capable to infect and cause disease to humans. We studied the respective involvement of TOSV and PUNV in human infections in northern Tunisia through a seroprevalence study. Methods The presence of TOSV and PUNV neutralising antibodies (NT-Ab) was tested in human sera collected from 5 districts of the governorate of Bizerte, and the titres of NT-Ab were estimated by microneutralisation (MN) assay. Principal Findings A total of 1,273 sera were processed. TOSV and PUNV NT-Ab were detected in 522 (41%) and 111 sera (8.72%) respectively. TOSV seroprevalence varied from 17.2% to 59.4% depending on the district. Analysis of TOSV geometric mean titre values demonstrated a constant increase according to the age. The vast majority of sera containing NT-Ab were found to be more reactive toward TOSV than PUNV. Indeed, past infections with PUNV and TOSV were undisputable for 5 and 414 sera, respectively. Conclusions PUNV may be capable to infect humans but at a low rate. TOSV is responsible for the vast majority of human infections by sand fly-borne phleboviruses in northern Tunisia. TOSV must be considered by physician and tested in diagnostic laboratories for patients with meningitis and unexplained fever in northern Tunisia., Author Summary In northern Tunisia, two different pheboviruses are known to circulate in sand fly population, Toscana virus (TOSV) and Punique virus (PUNV). In contrast to TOSV, a prominent human pathogen, there is no data supporting that PUNV is capable to infect humans and to cause a disease. We studied the respective involvement of TOSV and PUNV in human infections in northern Tunisia through a seroprevalence study. Because TOSV and PUNV are antigenically and genetically closely related, it is difficult to distinguish between them by using broadly reactive serological tests, such as enzyme-linked immunosorbent assay (ELISA). Thus, we developed a method of microneutralisation assay using the two viruses in a comparative manner. A total of 1,273 sera were processed. We provide first evidence to support (i) that Punique virus may be capable to infect humans but at a low rate, (ii) that TOSV, the most prevalent arbovirus in Southern Europe, is responsible for the vast majority of human infections by sand fly-borne phleboviruses in northern Tunisia. Therefore, it is important to consider TOSV as an important pathogen that needs to be included in all virological diagnostic concerning patients with meningitis and unexplained febrile illness originated from Northern Tunisia.

Toscana virus (TOSV) is an arthropod-borne virus, identified in 1971, from Phlebotomus perniciosus and Phlebotomus perfiliewi in central Italy. TOSV belongs to the Phlebovirus genus within the Bunyaviridae family. As other bunyaviruses, the genome of TOSV consists of 3 segments (S for small, M for Medium, and L for Large) respectively encoding non structural and capsid proteins, envelope structural proteins, and the viral RNA-dependant RNA-polymerase. It is transmitted by sand flies. Therefore its distribution is dictated by that of the arthropod vectors, and virus circulation peaks during summertime when sandfly populations are active. Here, we reviewed the epidemiology of TOSV in the old world. First evidence of its pathogenicity for humans, specifically its propensity to cause central nervous system (CNS) infections such as meningitis and encephalitis, was reported in central Italy. After 2000, it was recognized that TOSV had a much larger geographic distribution than initially believed, and was present in most of the Western European countries located on the northern border of the Mediterranean Sea (Portugal, Spain, France, Greece, Croatia) as well as eastern countries such as Cyprus and Turkey. In the countries where TOSV is present, it is among the three most prevalent viruses in meningitis during the warm seasons, together with enteroviruses and herpesviruses. Up to now, epidemiological data concerning Northern Africa and other countries located south of the Mediterranean are scarce. TOSV must be considered an emerging pathogen. Despite the important role played by TOSV in CNS infections, it remains a neglected agent and is rarely considered by physicians in diagnostic algorithms of CNS infections and febrile illness during the warm season, probably because of the lack of information.

This article presents methods to analyze global spatial relationships between two variables in two different sets of fixed points. Analysis of spatial relationships between two phenomena is of great interest in health geography and epidemiology, especially to highlight competing interest between phenomena or evidence of a common environmental factor. Our general approach extends the Moran and Pearson indices to the bivariate case in two different sets of points. The case where the variables are Boolean is treated separately through methods using nearest neighbors distances. All tests use Monte-Carlo simulations to estimate their probability distributions, with options to distinguish spatial and no spatial correlation in the special case of identical sets analysis. Implementation in a Geographic Information System (SavGIS) and real examples are used to illustrate these spatial indices and methods in epidemiology.

International audience; Sandflies are widely distributed around the Mediterranean Basin. Therefore, human populations in this area are potentially exposed to sandfly-transmitted diseases, including those caused by phleboviruses. Whilst there are substantial data in countries located in the northern part of the Mediterranean basin, few data are available for North Africa. In this study, a total of 1489 sandflies were collected in 2008 in Tunisia from two sites, bioclimatically distinct, located 235 km apart, and identified morphologically. Sandfly species comprised Phlebotomus perniciosus (52.2%), Phlebotomus longicuspis (30.1%), Phlebotomus papatasi (12.0%), Phlebotomus perfiliewi (4.6%), Phlebotomus langeroni (0.4%) and Sergentomyia minuta (0.5%). PCR screening, using generic primers for the genus Phlebovirus, resulted in the detection of ten positive pools. Sequence analysis revealed that two pools contained viral RNA corresponding to a novel virus closely related to sandfly fever Naples virus. Virus isolation in Vero cells was achieved from one pool. Genetic and phylogenetic characterization based on sequences in the three genomic segments showed that it was a novel virus distinct from other recognized members of the species. This novel virus was provisionally named Punique virus. Viral sequences in the polymerase gene corresponding to another phlebovirus closely related to but distinct from sandfly fever Sicilian virus were obtained from the eight remaining positive pools.

A seroprevalence study was carried out in the region of Marseille (south-eastern France) to address the public health importance of sandfly fever Sicilian virus (SFSV) and SFSV-like viruses, as recently recognized vectors of those viruses are present in this area. The low seroprevalence rate observed in this study suggests that SFSV is not likely to be of major medical importance in the Marseille area.

Phlebotomine sandflies are vectors of a large variety of arboviruses, mostly in the genus Phlebovirus. Many were discovered during the last decade, but were only partially sequenced because of extremely high genetic diversity. Next-Generation sequencing (NGS) is a key tool to achieve rapid sequencing of these viruses. Here we present the results obtained with a custom-made automated platform for high throughput processing (HTP) of field-trapped sandflies. The aim is to present an integrated approach to discover new/existing viruses vectored by sandflies, to characterize these viruses by complete genome sequencing, and to study their potential public health impact for humans and non human vertebrates. Sandflies were collected in France, Tunisia, Algeria, Turkey and Iran. They were processed on a custom-made automated HTP platform. RT-PCR assays targeting conserved regions within the genus Phlebovirus were performed both directly on sandfly extracted RNA and on supernatant of Vero cells, and further sequenced. Sandflies and cell culture that were PCR-positive, as well as supernatant of flasks with cytopathic effect were sequenced using a de novo protocol onto a Ion Torrent PGM sequencer. A total of 29,664 sandflies were collected and processed onto the platform. Origin and number of specimens are as follows: France (n = 3,087), Tunisia (n = 8,206), Algeria (n = 1,283), Turkey (n = 12,000), Iran (n = 5,089). We isolated 9 virus strains in France (2 Toscana, 7 Massilia), 10 strains in Tunisia (2 Toscana, 5 Punique, 3 new viruses still unnamed), 1 strain in Algeria (1 Toscana), 2 strains in Turkey (new phlebovirus, provisionally named Damyeri), 1 strain in Iran (new phlebovirus, provisionally named Dashli). Analysis using complete gene sequences allowed to produce full protein phylograms for isolated virus strains. Seroprevalence studies were performed to define whether these viruses infect humans and non human vertebrates in regions where sandflies were trapped or not. Seroprevalence results will be presented for France and Tunisia. A total of 23 strains were isolated, 6 of which correspond to new sandfly-borne phleboviruses that have been completely sequenced through NGS. Screening field-trapped sandflies is suitable to inventory recognized and new viruses. Combining cell culture for isolation and NGS for sequencing is cost-effective and adapted to process high quantities of insects. Serological tools are a rapid and easy method to address potential public health impact which must be confirmed by molecular diagnostic derived from complete sequence data obtained through NGS.

Sand flies are recognised vectors of parasites in the genus Leishmania and a number of arthropod-borne viruses, in particular viruses within the genus Phlebovirus, family Bunyaviridae. In southern France, Toscana phlebovirus (TOSV) is recognized as a prominent cause of summer meningitis. Since Leishmania and TOSV have a common vector (Phlebotomus perniciosus), an epidemiologic link has been assumed for a long time. However, there is no scientific evidence of such a link between human leishmaniosis and phleboviral infections. To identify a possible link, we investigated the presence and distribution of antibodies against these two microorganisms (i) in individuals and (ii) at a spatial level in the city of Marseille (south-eastern France). Five hundred sera were selected randomly in the biobank of the Department of Parasitology of the Public Hospitals of Marseille. All sera were previously tested for IgG against Leishmania by Western Blotting, and TOSV IgG were detected by indirect immunofluorescence. The seropositivity rates were 21.4% for TOSV and 28% for Leishmania. Statistical analysis demonstrated that seropositivity for one pathogen was significantly associated with seropositivity to the other pathogen. This result provided the first robust evidence for the existence of an epidemiological relationship between Leishmania infantum and TOSV. Addresses of tested patients were geolocalized and integrated into Geographical Information System software, in order to test spatial relationship between the two pathogens. Spatial analysis did not allow to identify (i) specific patterns for the spatial distribution of positive serological results for TOSV or Leishmania, and (ii) a spatial relationship between Leishmania and TOSV positive serological results. This may reflect the fact that the sample studied was not powerful enough to demonstrate either a spatial clustering or co-location, i.e. that the actual risk exposure area is smaller than the mean of distance between patients in our study (245 m)., Author Summary Sand flies are tiny insects widely distributed in peri-Mediterranean countries where they can transmit Leishmania parasite and Toscana virus (TOSV). Since those microorganisms have a common vector, an epidemiologic link has been assumed for a long time. However, there is no scientific evidence of such a link between human leishmaniosis and phleboviral infections. To identify a possible link, we investigated the presence and distribution of antibodies to both Leishmania and TOSV in 500 sera of individuals living in Marseille, France. Antibodies are markers of past infections and reflect the exposition of population to pathogens. Statistical analysis demonstrated that seropositivity to one of these pathogens was significantly associated with seropositivity to the other pathogen. This result provided first robust evidence for an epidemiological relationship between Leishmania infantum and TOSV. Home addresses of tested patients were geolocalized in order to test spatial relationship between the two pathogens. This spatial analysis did not allow identifying either a spatial clustering or co-location, most probably because the sample was not powerful enough. Risk-factors of infection by TOSV (or Leishmania) would be investigated in seropositive patients' neighbourhood, in a range inferior to the mean of distance between patients in our study (245 meters).

A new phlebovirus, Medjerda Valley virus (MVV), was isolated from one pool of Phlebotomus sp. (Diptera; Psychodidae) sandflies trapped in the vicinity of the Utique site, northern Tunisia. Genetic analysis based on complete coding of genomic sequences of the three RNA segments indicated that MVV is most closely related to members of the Salehabad virus species, where it is the fourth virus for which the complete sequence is available. A seroprevalence study was performed to search for neutralizing antibodies in human sera in the same region. The results demonstrate that in this area, MVV can readily infect humans despite low seroprevalence rates. Salehabad species viruses have generally been considered to be a group of viruses with little medical or veterinary interest. This view deserves to be revisited according to our human seroprevalence results, together with high animal infection rate of Adana virus and recent evidence of human infection with Adria virus in Greece. Further studies are needed to investigate the capacity of each specific member of the Salehabad virus species to cause human or animal diseases.

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.

Phlebotomine sandflies are vectors of phleboviruses that cause sandfly fever or meningitis with significant implications for public health. Although several strains of these viruses had been isolated in Iran in the late 1970's, there was no recent data about the present situation at the outset of this study. Entomological investigations performed in 2009 and 2011 in Iran collected 4,770 sandflies from 10 different regions. Based on morphological identification, they were sorted into 315 pools according to species, sex, trapping station and date of capture. A phlebovirus, provisionally named Dashli virus (DASHV), was isolated from one pool of Sergentomyia spp, and subsequently DASHV RNA was detected in a second pool of Phlebotomus papatasi. Genetic and phylogenetic analyses based on complete coding genomic sequences indicated that (i) DASHV is most closely related to the Iranian isolates of Sandfly fever Sicilian virus [SFSV], (ii) there is a common ancestor to DASHV, Sandfly fever Sicilian- (SFS) and SFS-like viruses isolated in Italy, India, Turkey, and Cyprus (lineage I), (iii) DASHV is more distantly related with Corfou and Toros viruses (lineage II) although common ancestry is supported with 100% bootstrap, (iii) lineage I can be subdivided into sublineage Ia including all SFSV, SFCV and SFTV except those isolated in Iran which forms sublineage Ib (DASHV). Accordingly, we suggest to approve Sandfly fever Sicilian virus species consisting of the all aforementioned viruses. Owing that most of these viruses have been identified in human patients with febrile illness, DASHV should be considered as a potential human pathogen in Iran., Author summary Phlebotomine sandflies are vectors of phleboviruses that cause sandfly fever or meningitis with significant implications for public health. Although several strains of these viruses had been isolated in Iran in the late 1970's, there was no recent data about the present situation at the outset of this study. Entomological investigations performed in 2009 and 2011 in Iran collected 4,770 sandflies from 10 different regions. A phlebovirus, provisionally named Dashli virus (DASHV), was isolated / detected in two pools. DASHV strain was isolated in cell culture and complete genome sequence was determined. Sequence analysis indicated that (i) DASHV is most closely related to the Iranian isolates of Sandfly fever Sicilian virus [SFSV], a virus that is known to cause self-resolutive incapacitating febrile illness in humans, (ii) there is a common ancestor to DASHV and all other variants of SFSV isolated in Italy, India, Turkey, and Cyprus (lineage I), (iii) DASHV is more distantly related with Corfou and Toros viruses (lineage II) although common ancestry is supported with 100% bootstrap, (iii) lineage I can be subdivided into sublineage Ia including all SFSV strains, whereas Iranian viruses are most closely related and should be individualized as DASHV (sublineage Ib). Although discovered first in the 1940's, SFSV is still listed as "tentative species" by the International Committee for Taxonomy of Viruses. Based on the results described in this study, we propose to approve Sandfly fever Sicilian virus species. Owing that most of these viruses have been identified in human patients with febrile illness, DASHV should be considered as a potential human pathogen in Iran.

Background Cutaneous leishmaniasis is endemic to the Pacific coast of Ecuador, and Nyssomyia trapidoi is considered to be its main vector. Dujardin et al. [1] recorded some differences in body pigmentation and isoenzymatic profiles in sympatric populations of Ny. trapidoi from the Pacific coast of Ecuador and suggested the existence of two cryptic species. Methods Entomological collections were performed in November 2008 and March 2011 in the locality of Paraíso Escondido using CDC miniature light traps and human bait. Morphological, isoenzymatical and molecular (sequencing of cytochome b and cytochrome c oxidase 1 of the mitochondrial DNA) analyses, such as detection of Leishmania DNA and phlebovirus RNA in some females, were performed. Results Neighbor-joining trees from mitochondrial sequences grouped all of Ecuadorian Ny. trapidoi (including the two color variants) in one cluster, except for two specimens which clustered separately in both genes. Isoenzymatic characterization confirmed that the color variants belong to the same population. Additionally, 11.5% of females were found by PCR to contain Endotrypanum monterogeii kinetoplastid DNA. All pools of Ny. trapidoi were negative for phlebovirus RNA. Conclusion Analysis of mitochondrial gene sequences and isoenzymes was unable to support the existence of two sibling species within Ny. trapidoi, which is a probable vector of Endotrypanum monterogeii.