• virology
• genomics

• Whole Genome Sequencing/methods
• Sandfly fever Naples virus/genetics
• Genome, Viral
• Humans
• Animals
• RNA, Viral/genetics
• Phylogeny

• Animals
• Psychodidae/virology
• Psychodidae/classification
• Sandfly fever Naples virus/physiology
• Insect Vectors/virology
• Female
• Humans
• Phlebotomus/virology
• Male

• Horizon 2020 Framework Programme
• Next Generation EU
• HORIZON EUROPE Health
• UK Research and Innovation
• École Doctorale de l'École Pratique des Hautes Études
• Medical Research Council

• viral evolution
• virus-host interactions

• MC_UU_12014/8 Medical Research Council

• Distribution
• France
• PRISMA
• Sand flies
• Systematic review

• France
• Animals
• Insect Vectors/parasitology
• Phlebotomus/classification
• Phlebotomus/parasitology
• Psychodidae/parasitology
• Psychodidae/classification
• Animal Distribution
• Leishmaniasis/transmission
• Leishmaniasis/epidemiology
• Population Density
• Leishmania
• Mediterranean Region
• Climate Change

• CLIMOS Project

• Toscana virus
• genotype
• wild birds
• zoonosis

• Animals
• Humans
• Sandfly fever Naples virus/genetics
• Turkey
• Phlebovirus
• Birds
• Antibodies, Viral

• RT-PCR assay
• arbovirus
• in silico
• phlebovirus
• sand fly

• Animals
• Phlebovirus/genetics
• Psychodidae
• Nucleic Acid Amplification Techniques
• Nucleic Acids
• Phylogeny

• 101057690 European Commission (EC)
• 10038150, 10039289 UK Research and Innovation (UKRI)
• 871029 EC | Horizon 2020 Framework Programme (H2020)

• Laboratory diagnosis
• Neuro-invasive disease
• Phlebovirus
• Phylogenetic analysis
• Toscana virus
• Virus isolation

A novel phlebovirus, Punique virus (PUNV), was discovered and isolated in 2008 from sandflies from Northern Tunisia. PUNV is now classified as a unique member of the Punique phlebovirus species within the Phlebovirus genus in the Phenuiviridae family (order bunyavirales). In this study, we aimed to investigate the transmission dynamics of PUNV in Tunisia. Sandflies were collected during two consecutive years, 2009 and 2010, by CDC light traps. In 2009, a total of 873 sandflies were collected and identified to the species level. Phlebotomus perniciosus was the most abundant species. One pool of P. perniciosus females collected in autumn contained PUNV RNA, yielding an infection rate of 0.11%. The population densities of circulating sandfly species were assessed during May–November 2010 in Northern Tunisia by using sticky traps. Phlebotomus (Larroussius) perniciosus (71.74%) was the most abundant species, followed by Phlebotumus (Larroussius) longicuspis (17.47%), and Phlebotumus (Larroussius) perfiliewi (8.82%). The densities of dominant sandfly species were found to peak in early spring and again in the autumn. In 2010, species identification was not performed, and sandflies were only discriminated on the basis of sex and collection date. Out of 249 pools, three contained PUNV RNA. Each positive pool allowed virus isolation. The three pools of female sandflies containing PUNV RNA were collected in autumn with an infection rate of 0.05%. These findings provide further evidence that P. perniciosus is the main vector of PUNV in Tunisia, and this phlebovirus is endemic in Tunisia. Our findings provided strong evidence of intensive circulation of PUNV in sandflies and hosts through a viral infection buildup process between sandfly vectors and hosts starting at the beginning of the activity of sandflies in spring to reach a maximum during the second main peak in autumn.

• Animals
• DNA Viruses/genetics
• Female
• Phlebotomus/genetics
• Phlebovirus/genetics
• Psychodidae
• RNA, Viral/genetics
• Tunisia/epidemiology
• Viruses, Unclassified/genetics

• Phlebovirus
• Salehabad virus
• Sicilian virus
• Toscana virus
• sand fly borne viruses

• Guillain–Barré syndrome
• Toscana virus
• West Nile virus

• Leishmania
• Toscana virus
• phenuiviridae
• phlebovirus
• sandfly

• Animals
• Bunyaviridae Infections/transmission
• Humans
• Insect Vectors/parasitology
• Insect Vectors/virology
• Leishmania tropica/isolation & purification
• Leishmaniasis, Cutaneous/transmission
• Phlebotomus/classification
• Phlebotomus/parasitology
• Phlebotomus/virology
• Phylogeny
• Psychodidae/classification
• Psychodidae/parasitology
• Psychodidae/virology
• RNA, Viral
• Sandfly fever Naples virus/genetics
• Sandfly fever Naples virus/isolation & purification
• Turkey/epidemiology
• Vector Borne Diseases/parasitology
• Vector Borne Diseases/transmission
• Vector Borne Diseases/virology

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

• Bunyavirales
• Phlebovirus
• Toscana virus
• reverse genetics

Toscana virus (TOSV) is endemic in the Mediterranean basin, where it is transmitted by sand flies. TOSV can infect humans and cause febrile illness as well as neuroinvasive infections affecting the central and peripheral nervous systems. Although TOSV is a significant human pathogen, it remains neglected and there are consequently many gaps of knowledge. Recent seroepidemiology studies and case reports showed that TOSV’s geographic distribution is much wider than was assumed a decade ago. The apparent extension of the TOSV circulation area raises the question of the sandfly species that are able to transmit the virus in natural conditions. Phlebotomus (Ph.)perniciosus and Ph. perfiliewi were historically identified as competent species. Recent results suggest that other species of sand flies could be competent for TOSV maintenance and transmission. Here we organize current knowledge in entomology, epidemiology, and virology supporting the possible existence of additional phlebotomine species such as Ph. longicuspis, Ph. sergenti, Ph. tobbi, Ph. neglectus, and Sergentomyia minuta in TOSV maintenance. We also highlight some of the knowledge gaps to be addressed in future studies.

• Bunyavirales
• Mediterranean area
• Phenuiviridae
• Phlebotomus
• Sandfly fever Naples phlebovirus
• Sergentomyia
• Toscana virus
• arbovirus
• arthropod-borne
• phlebotomine
• sand fly
• sandfly

We describe a novel virus, designated Ntepes virus (NPV), isolated from sand flies in Kenya. NPV has the characteristic phlebovirus trisegmented genome architecture and is related to, but distinct from, Gabek Forest phlebovirus. Diverse cell cultures derived from wildlife, livestock, and humans were susceptible to NPV, with pronounced permissiveness in swine and rodent cells. NPV infection of newborn mice caused rapid and fatal illness. Permissiveness for NPV replication in sand fly cells, but not mosquito cells, suggests a vector-specific adaptation. Specific neutralizing antibodies were found in 13.9% (26/187) of human serum samples taken at the site of isolation of NPV as well as a disparate site in northeastern Kenya, suggesting a wide distribution. We identify a novel human-infecting arbovirus and highlight the importance of rural areas in tropical Africa for arbovirus surveillance as well as extending arbovirus surveillance to include hematophagous arthropods other than mosquitoes.

• Gabek Forest phlebovirus
• Kenya
• Ntepes virus
• arbovirus
• neutralizing antibodies
• phlebovirus
• sand flies
• virus discovery
• viruses

• Adolescent
• Adult
• Animals
• Antibodies, Neutralizing/immunology
• Antibodies, Viral/immunology
• Bunyaviridae Infections/epidemiology
• Bunyaviridae Infections/immunology
• Bunyaviridae Infections/transmission
• Bunyaviridae Infections/virology
• Cell Line
• Child
• Female
• Genome, Insect
• Genome, Viral
• Genomics/methods
• Geography, Medical
• Humans
• Insect Vectors/virology
• Kenya/epidemiology
• Male
• Mice
• Phlebovirus/classification
• Phlebovirus/genetics
• Phlebovirus/immunology
• Phlebovirus/isolation & purification
• Phylogeny
• Psychodidae/classification
• Psychodidae/genetics
• Psychodidae/virology
• Public Health Surveillance
• Young Adult

We report a rare case of Toscana virus infection imported into Switzerland in a 23-year old man who travelled to Imperia (Italy) 10 days before onset of symptoms. Symptoms included both meningitis and as well epididymitis. This is only the fourth case of Toscana virus reported in Switzerland.

The patient presented with lymphocytic meningitis and scrotal pain due to epididymitis. Meningitis was initially treated with ceftriaxone. Herpes simplex, tick-borne encephalitis, enterovirus, measles, mumps, rubella and Treponema pallidum were excluded with specific polymerase chain reaction (PCR) or serology. In support of routine diagnostic PCR and serology assays, unbiased viral metagenomic sequencing was performed of cerebrospinal fluid and serum. Toscana virus infection was identified in cerebrospinal fluid and the full coding sequence could be obtained. Specific PCR in cerebrospinal fluid and blood and serology with Immunoglobulin (Ig) M and IgG against Toscana virus confirmed our diagnosis. Neurological symptoms recovered spontaneously after 5 days.

This case of Toscana virus infection highlights the benefits of unbiased metagenomic sequencing to support routine diagnostics in rare or unexpected viral infections. With increasing travel histories of patients, physicians should be aware of imported Toscana virus as the agent for viral meningitis and meningoencephalitis.

The online version of this article (10.1186/s12879-019-4231-9) contains supplementary material, which is available to authorized users.

• Clinical metagenomics
• Epididymitis
• Meningitis
• Switzerland
• Toscana virus

• Arbovirus
• Toscana virus
• arthropods
• fleas
• lice
• phlebovirus
• sand fly
• ticks
• virus

Sandflies are well known vectors for Leishmania but also transmit a number of arthropod-borne viruses (arboviruses). Few studies have addressed the interaction between sandflies and arboviruses. RNA interference (RNAi) mechanisms utilize small non-coding RNAs to regulate different aspects of host-pathogen interactions. The small interfering RNA (siRNA) pathway is a broad antiviral mechanism in insects. In addition, at least in mosquitoes, another RNAi mechanism mediated by PIWI interacting RNAs (piRNAs) is activated by viral infection. Finally, endogenous microRNAs (miRNA) may also regulate host immune responses. Here, we analyzed the small non-coding RNA response to Vesicular stomatitis virus (VSV) infection in the sandfly Lutzoymia longipalpis. We detected abundant production of virus-derived siRNAs after VSV infection in adult sandflies. However, there was no production of virus-derived piRNAs and only mild changes in the expression of vector miRNAs in response to infection. We also observed abundant production of virus-derived siRNAs against two other viruses in Lutzomyia Lulo cells. Together, our results suggest that the siRNA but not the piRNA pathway mediates an antiviral response in sandflies. In agreement with this hypothesis, pre-treatment of cells with dsRNA against VSV was able to inhibit viral replication while knock-down of the central siRNA component, Argonaute-2, led to increased virus levels. Our work begins to elucidate the role of RNAi mechanisms in the interaction between L. longipalpis and viruses and should also open the way for studies with other sandfly-borne pathogens.

Sandflies are important insect vectors that transmit many species of Leishmania, bacteria and viruses. We know very little about how this insect vector responds to viral infection. RNA interference (RNAi) utilizes small non-coding RNAs to regulate different aspects of animal physiology, including immune responses. Small interfering RNAs (siRNAs) mediate a major antiviral response in insects. Virus-derived PIWI-interacting RNAs (piRNAs) can also be generated during infection, at least in some insects. Finally, endogenous microRNAs (miRNA) can regulate the host response to infection. Here we show that virus infection triggers activation of the siRNA pathway but not production of piRNAs in the sandfly Lutzomyia longipalpis. Furthermore, activation or inhibition of the siRNA pathway had a direct effect on viral replication. We also show that virus infection caused mild changes to the expression of endogenous miRNAs. Our work describes for the first time a model to study virus infection in sandflies and highlights the importance of the siRNA pathway for the control of virus infection in L. longipalpis. The framework described here can be used to explore other aspects of the vector-pathogen interactions.

• Animals
• Host-Pathogen Interactions
• Insect Vectors/parasitology
• Insect Vectors/virology
• Leishmania/physiology
• MicroRNAs/genetics
• Psychodidae/genetics
• Psychodidae/immunology
• Psychodidae/physiology
• Psychodidae/virology
• RNA Interference
• RNA, Small Interfering/genetics
• RNA, Untranslated
• RNA, Viral/genetics
• Vesicular stomatitis Indiana virus/genetics
• Vesicular stomatitis Indiana virus/physiology
• Virus Replication

• Conselho Nacional de Desenvolvimento Científico e Tecnológico
• Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
• Fundação de Amparo à Pesquisa do Estado de Minas Gerais
• Agence Nationale de la Recherche

• bedbugs
• fleas
• lice
• mosquitoes
• ticks

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.

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.

• Animals
• Base Sequence
• Female
• Genome, Viral/genetics
• Humans
• Insect Vectors/virology
• Iran
• Male
• Phlebotomus Fever/transmission
• Phlebotomus Fever/virology
• Phlebovirus/classification
• Phlebovirus/genetics
• Phlebovirus/isolation & purification
• Phylogeny
• Psychodidae/virology
• RNA, Viral/genetics
• Sequence Analysis, RNA

• Europe 7th PCRDT
• H2020 European Research Council
• H2020 European Research Council (BE)
• European Cooperation in Science and Technology

• Sandfly Fever Naples Virus
• Toscana virus
• arbovirus
• phlebovirus
• sandfly fever

Recently, Balkan virus (BALKV, family Phenuiviridae, genus Phlebovirus) was discovered in sand flies collected in Albania and genetically characterised as a member of the Sandfly fever Naples species complex. To gain knowledge concerning the geographical area where exposure to BALKV exists, entomological surveys were conducted in 2014 and 2015, in Croatia, Bosnia and Herzegovina (BH), Kosovo, Republic of Macedonia and Serbia.

A total of 2830 sand flies were trapped during 2014 and 2015 campaigns, and organised as 263 pools. BALKV RNA was detected in four pools from Croatia and in one pool from BH. Phylogenetic relationships were examined using sequences in the S and L RNA segments. Study of the diversity between BALKV sequences from Albania, Croatia and BH showed that Albanian sequences were the most divergent (9–11% [NP]) from the others and that Croatian and BH sequences were grouped (0.9–5.4% [NP]; 0.7–5% [L]). The sand fly infection rate of BALKV was 0.26% in BH and 0.27% in Croatia. Identification of the species content of pools using cox1 and cytb partial regions showed that the five BALKV positive pools contained Phlebotomus neglectus DNA; in four pools, P neglectus was the unique species, whereas P. tobbi DNA was also detected in one pool.

We report here (i) the first direct evidence that the Balkan virus initially described in coastal Albania has a much wider dissemination area than originally believed, (ii) two real-time RT-PCR assays that may be useful for further screening of patients presenting with fever of unknown origin that may be caused by Balkan virus infection, (iii) entomological results suggesting that Balkan virus is likely transmitted by Phlebotomus neglectus, and possibly other sand fly species of the subgenus Larroussius. So far, BALKV has been detected only in sand flies. Whether BALKV can cause disease in humans is unknown and remains to be investigated.

• Arbovirus
• Bunyaviridae
• Emergence
• Fever
• Meningitis
• Phlebotomus
• Phlebovirus
• Phylogeny
• Sand fly
• Toscana virus

• Toscana virus
• Turkey
• West Nile virus
• avian
• bird
• phlebovirus

• Animals
• Animals, Domestic/virology
• Humans
• Insect Vectors/virology
• Phlebotomus Fever/transmission
• Phlebotomus Fever/veterinary
• Phlebovirus/classification
• Phlebovirus/genetics
• Psychodidae/virology
• Seroepidemiologic Studies

• SAXS
• Toscana virus
• negative-sense single-stranded RNA virus
• negative-stain TEM
• nucleoprotein oligomers
• nucleoproteins
• oligomers
• phlebovirus
• ribonucleoprotein

• Bunyaviridae
• Toscana virus
• arbovirus(es)
• field studies
• sand fly (flies)
• vector-borne

• Bunyaviridae
• Phlebotomus
• Toscana virus
• arbovirus
• phlebovirus

• Anatolia
• Asia minor
• Phlebovirus
• Turkey
• bunyavirus
• sand fly

• Animals
• Asia/epidemiology
• Humans
• Phlebotomus Fever/epidemiology
• Phlebotomus Fever/veterinary
• Phlebovirus/classification
• Phlebovirus/isolation & purification
• Psychodidae/growth & development
• Psychodidae/virology
• Vertebrates

• Arbovirus(es)
• Leishmania
• Phebovirus
• Toscana virus
• Turkey
• bunyaviruses
• canine
• dogs
• sandfly (flies)

• Emerging
• Mediterranean basin
• Phlebotomus
• Phlebovirus
• Saddaguia virus
• Sand flies
• Sandfly fever sicilian virus
• Toscana virus
• Utique virus

Toscana virus (TOSV) is an arthropod-borne phlebovirus belonging to the Sandfly fever Naples virus species (genus Phlebovirus, family Bunyaviridae). Here, we report the complete coding sequences of six TOSV strains isolated from human patients having acquired the infection in southeastern France during a 12-year period.

• Agmeroul
• Ait Makhlouf
• Azilal
• El Hanchane
• Essaouira
• Guimi
• Louata
• Maghreb
• Meningitis
• Morocco
• Northern Africa
• Ouarzazate
• Phlebovirus
• Sandflies
• Sefrou
• Toscana virus
• Zaouit Sidi Othmane

• Animals
• Bunyaviridae Infections/epidemiology
• Bunyaviridae Infections/virology
• Chlorocebus aethiops
• DNA, Viral/analysis
• Disease Reservoirs/statistics & numerical data
• Disease Reservoirs/virology
• Genotype
• Humans
• Insect Vectors/virology
• Molecular Epidemiology
• Morocco/epidemiology
• Phlebotomus/virology
• Phlebovirus/classification
• Phlebovirus/genetics
• Phylogeny
• Polymerase Chain Reaction/methods
• Vero Cells

• Arbia virus
• Human leishmaniasis
• Madrid
• Novel virus
• Phlebotomus perniciosus
• Phlebovirus
• Spain
• Toscana virus

Southern Anatolia in Turkey at the border with Syria, where many refugee camps are settled, is endemic for sandfly-borne leishmaniasis. Sandfly-borne phleboviruses are also known to circulate in this region, although their relevance in terms of medical implications is virtually unknown. Therefore, the specific objectives of our study were firstly to identify isolate and characterise potentially pathogenic phleboviruses in sandflies; secondly to determine the complete genomic sequence of any viruses that we were able to isolate; and thirdly, to further our understanding of the potential medical importance and epidemiological significance of these viruses. To achieve these objectives, we organised field campaigns in 2012 and 2013. Two new phleboviruses (Toros and Zerdali viruses) were isolated and characterized by complete genome sequencing and phylogenetic analyses. Toros virus was genetically most closely related to Corfou virus within the Sandfly fever Sicilian group. Zerdali virus was most closely related to Tehran virus within the Sandfly fever Naples species. Although these new viruses belong to genetic groups that include several human pathogens, it is not yet clear if Toros and Zerdali viruses can infect humans and cause disease such as sandfly fever. Consequently, the availability of these genetically characterized infectious viruses will enable seroprevalence studies to establish their medical importance in this region and to assist the health agencies to develop appropriate and effective disease control strategies.

We provide evidence that sandfly-borne phleboviruses belonging to 3 distinct genetic and phylogenetic groups (Sandfly fever Naples virus [SFNV], Sandfly fever Sicilian virus [SFSV], and Salehabad virus [SALV]) co-circulate in Adana city, in Mediterranean Turkey. While Adana virus was recently described as a new member of the SALV species, Zerdali and Toros viruses are described here as new phleboviruses genetically closely related to SFNV and SFSV, respectively. In this study, isolated and characterised these two new viruses by determining their complete genome sequence and by phylogenetic analysis. This study demonstrates that 3 distinct viruses can co-circulate in the same geographic area and based on their phylogenetic relationships and association with sandflies are likely to be transmitted by these arthropod vectors. Our molecular and phylogenetic data are important for establishing group-specific molecular detection assays in order to further understand of the possible impact of these viruses in animal and human health in this region of Turkey.

• Animals
• Female
• Genome, Viral
• Genotype
• Insect Vectors/virology
• Male
• Phlebovirus/genetics
• Phylogeny
• Psychodidae/virology
• Sandfly fever Naples virus/genetics
• Turkey/epidemiology

• Phlebovirus
• RT-PCR assay
• Sandfly/mosquito-borne
• Tick-borne

• Bunyaviridae Infections/diagnosis
• Bunyaviridae Infections/virology
• Humans
• Molecular Diagnostic Techniques/methods
• Phlebovirus/classification
• Phlebovirus/genetics
• Phlebovirus/isolation & purification
• Reverse Transcriptase Polymerase Chain Reaction/methods
• Sensitivity and Specificity
• Virology/methods

• Europe
• Phlebotomus
• sampling
• sand flies

• Animals
• DNA Barcoding, Taxonomic
• Europe
• Insect Vectors/microbiology
• Insect Vectors/parasitology
• Insect Vectors/physiology
• Phlebotomus/microbiology
• Phlebotomus/parasitology
• Phlebotomus/physiology
• Population Density
• Population Dynamics
• Population Surveillance/methods

In Portugal, entomological surveys to detect phleboviruses in their natural vectors have not been performed so far. Thus, the aims of the present study were to detect, isolate and characterize phleboviruses in sandfly populations of Portugal.

From May to October 2007–2008, 896 female sandflies were trapped in Arrábida region, located on the southwest coast of Portugal. Phlebovirus RNA was detected by using a pan-phlebovirus RT-PCR in 4 out of 34 Phlebotomus perniciosus pools. Direct sequencing of the amplicons showed that 2 samples exhibited 72 % nucleotide identity with Arbia virus, and two showed 96 % nucleotide identity with Massilia virus. The Arbia-like virus (named Alcube virus) was isolated in cell culture and complete genomic sequences of one Alcube and two Massila viruses were determined using next-generation sequencing technology. Phylogenetic analysis demonstrated that Alcube virus clustered with members of the Salehabad virus species complex. Within this clade, Alcube virus forms a monophyletic lineage with the Arbia, Salehabad and Adana viruses sharing a common ancestor. Arbia virus has been identified as the most closely related virus with 20-28 % nucleotide and 10-27 % amino acid divergences depending on the analysed segment.

We have provided genetic evidence for the circulation of a novel phlebovirus species named Alcube virus in Ph. perniciosus and co-circulation of Massilia virus, in Arrábida region, southwest of Portugal. Further epidemiological investigations and surveillance for sandfly-borne phleboviruses in Portugal are needed to elucidate their medical importance.

• Arbovirus
• Bunyaviridae
• Diagnosis
• Real-time RT-PCR
• Toscana virus

• Emerging
• Mediterranean basin
• Phlebotomus
• Phlebovirus
• Saddaguia virus
• Sand flies

In this chapter, we describe 73 zoonotic viruses that were isolated in Northern Eurasia and that belong to the different families of viruses with a single-stranded RNA (ssRNA) genome. The family includes viruses with a segmented negative-sense ssRNA genome (families Bunyaviridae and Orthomyxoviridae) and viruses with a positive-sense ssRNA genome (families Togaviridae and Flaviviridae). Among them are viruses associated with sporadic cases or outbreaks of human disease, such as hemorrhagic fever with renal syndrome (viruses of the genus Hantavirus), Crimean–Congo hemorrhagic fever (CCHFV, Nairovirus), California encephalitis (INKV, TAHV, and KHATV; Orthobunyavirus), sandfly fever (SFCV and SFNV, Phlebovirus), Tick-borne encephalitis (TBEV, Flavivirus), Omsk hemorrhagic fever (OHFV, Flavivirus), West Nile fever (WNV, Flavivirus), Sindbis fever (SINV, Alphavirus) Chikungunya fever (CHIKV, Alphavirus) and others. Other viruses described in the chapter can cause epizootics in wild or domestic animals: Geta virus (GETV, Alphavirus), Influenza A virus (Influenzavirus A), Bhanja virus (BHAV, Phlebovirus) and more. The chapter also discusses both ecological peculiarities that promote the circulation of these viruses in natural foci and factors influencing the occurrence of epidemic and epizootic outbreaks

Our aim is to detect the infection by Toscana virus (TOSV) and other Phleboviruses in the sera and cerebro-spinal fluid (CSF) of patients with meningitis in Tunisia. We examined various species of phlebotomus present in Tunisia to determine whether or not a direct relationship exists between cases of meningitis and the viruses circulating in the insect vectors.

Patients with the meningeal syndrome were tested for anti-TOSV IgM and IgG using an indirect Enzyme-Linked Immunosorbent Assay (ELISA) and for the presence of TOSV and other Phleboviruses using a RT-PCR test.

An entomological study was carried out using CDC light traps to trap sandflies in different bioclimatic zones of Tunisia. Collected sandflies were tested by RT-PCR for the presence of TOSV and other Phleboviruses and subsequently by viral isolation on Vero cells.

Of 263 patients were tested using ELISA of which 12.16% (n = 32/263) were IgM positive for anti TOSV. Of these 32 patients, 78% (n = 25/32) were IgG positive. 12.86% (n = 18/140) of the CSF samples tested by RT-PCR were positive for the Toscana virus.

One CSF sample tested by RT-PCR revealed the presence of Sandfly Fever Sicilian Virus (SFSV). The Punique virus was identified in one sandfly pool.

This study confirms, for the first time, that TOSV is involved in a neurological disorder in North Africa. The incidence of this involvement in Tunisia conforms with observations made in other Mediterranean countries. Moreover, for the first time, a molecular approach was used to detect SFSV in a Tunisian patient displaying neurological symptoms.

The online version of this article (doi:10.1186/s12879-014-0598-9) contains supplementary material, which is available to authorized users.