Experimental Infection of Sand Flies by Massilia Virus and Viral Transmission by Co-Feeding on Sugar Meal.

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.

Isolation and sequencing of Dashli virus, a novel Sicilian-like virus in sandflies from Iran; genetic and phylogenetic evidence for the creation of one novel species within the Phlebovirus genus in the Phenuiviridae family.

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.

Seroprevalence of Sandfly-Borne Phleboviruses Belonging to Three Serocomplexes (Sandfly fever Naples, Sandfly fever Sicilian and Salehabad) in Dogs from Greece and Cyprus Using Neutralization Test.

Phleboviruses transmitted by sandflies are endemic in the Mediterranean area. The last decade has witnessed the description of an accumulating number of novel viruses. Although, the risk of exposure of vertebrates is globally assessed, detailed geographic knowledge is poor even in Greece and Cyprus where sandfly fever has been recognized for a long time and repeatedly. A total of 1,250 dogs from mainland Greece and Greek archipelago on one hand and 422 dogs from Cyprus on the other hand have been sampled and tested for neutralising antibodies against Toscana virus (TOSV), Sandfly fever Sicilian virus (SFSV), Arbia virus, and Adana virus i.e. four viruses belonging to the 3 sandfly-borne serocomplexes known to circulate actively in the Mediterranean area. Our results showed that (i) SFSV is highly prevalent with 71.9% (50.7-84.9% depending on the region) in Greece and 60.2% (40.0-72.6%) in Cyprus; (ii) TOSV ranked second with 4.4% (0-15.4%) in Greece and 8.4% (0-11.4%) in Cyprus; (iii) Salehabad viruses (Arbia and Adana) displayed also substantial prevalence rates in both countries with values ranging from 0-22.6% depending on the region and on the virus strain used in the test. These results demonstrate that circulation of viruses transmitted by sand flies can be estimated qualitatively using dog sera. As reported in other regions of the Mediterranean, these results indicate that it is time to shift these viruses from the "neglected" status to the "priority" status in order to stimulate studies aiming at defining and quantifying their medical and veterinary importance and possible public health impact. Specifically, viruses belonging to the Sandfly fever Sicilian complex should be given careful consideration. This calls for implementation of direct and indirect diagnosis in National reference centers and in hospital microbiology laboratories and systematic testing of unelucidated febrile illness and central and peripheral nervous system febrile manifestations.

Neutralization-based seroprevalence of Toscana virus and sandfly fever Sicilian virus in dogs and cats from Portugal.

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.

Isolation, full genomic characterization and neutralization-based human seroprevalence of Medjerda Valley virus, a novel sandfly-borne phlebovirus belonging to the Salehabad virus complex in northern Tunisia.

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.

Ecuador Paraiso Escondido Virus, a New Flavivirus Isolated from New World Sand Flies in Ecuador, Is the First Representative of a Novel Clade in the Genus Flavivirus.

UNLABELLED: 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.

Isolation, genetic characterization, and seroprevalence of Adana virus, a novel phlebovirus belonging to the Salehabad virus complex, in Turkey.

UNLABELLED: 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.

Presence of sandfly-borne phleboviruses of two antigenic complexes (Sandfly fever Naples virus and Sandfly fever Sicilian virus) in two different bio-geographical regions of Tunisia demonstrated by a microneutralisation-based seroprevalence study in dogs.

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.

Phlebotomus sergenti a common vector of Leishmania tropica and Toscana virus in Morocco.

BACKGROUND & OBJECTIVES: An entomological study using CDC miniature light-traps was performed in El Hanchane locality, where cutaneous leishmaniasis (CL) was emerging during the summer of 2011. The aim of this study is to identify the vectors of Leishmania and of phleboviruses. METHODS: In the field, a total of 643 sandfly specimens were collected, identified by morphological keys and categorized by sex and species. A total of nine distinct species were morphologically identified where seven belonged to the Phlebotomus genus and two species to the Sergentomyia genus. Phlebotomus sergenti was the most abundant species (76%). Phleboviruses were detected by nested RT-PCR using 30 pooled sandflies while P. sergenti females were tested individually for infections of Leishmania species. RESULTS: By using ITS1-PCR-RFLP approach, Leishmania tropica DNA was detected in 10 females, caught in this emerging focus, and provide additional evidence in favour of the role of P. sergenti as vector of L. tropica in Morocco. Real-time PCR screening for phlebovirus RNA, using an assay targeting the polymerase gene, showed positive result in one pool of male P. sergenti. INTERPRETATION & CONCLUSION: In this study, P. sergenti were infected by L. tropica and Toscana virus. To our knowledge, actually this is the first time that Toscana virus has been detected in P. sergenti.

Development of generic Taqman PCR and RT-PCR assays for the detection of DNA and mRNA of ß-actin-encoding sequences in a wide range of animal species.

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 ß-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 ß-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 ß-actin-encoding sequences as endogenous control. This assay will constitute a precious tool for virologists working with multiple cell lines or animal models.

Co-circulation of Toscana virus and Punique virus in northern Tunisia: a microneutralisation-based seroprevalence study.

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.

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

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

Sandfly-borne phleboviruses of Eurasia and Africa: epidemiology, genetic diversity, geographic range, control measures.

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.

Emergence of Toscana virus in the mediterranean area.

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.

Epidemiologic relationship between Toscana virus infection and Leishmania infantum due to common exposure to Phlebotomus perniciosus sandfly vector.

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).

Statistical methods for bivariate spatial analysis in marked points. Examples in spatial epidemiology.

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.

Molecular and serological evidence for the presence of novel phleboviruses in sandflies from northern algeria.

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.

Punique virus, a novel phlebovirus, related to sandfly fever Naples virus, isolated from sandflies collected in Tunisia.

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.