Naples Virus Research Articles

Serological investigation of phlebovirus exposure in blood donors from the Mediterranean Province of Mersin, Turkey

Phleboviruses are enveloped segmented RNA viruses, capable of inducing febrile disease and/or meningoencephalitis in exposed individuals, according to the infecting strain, following transmission via arthropods. Prototype medically-important phlebovirus strains responsible for sandfly fever are sandfly fever Sicilian virus (SFSV) and sandfly fever Naples virus (SFNV), where the SFSV variant sandfly fever Cyprus virus (SFCV) is also detected in individuals with febrile disease. Toscana virus (TOSV) is unique among phleboviruses as the cause of infections involving central nervous system. In this seroepidemiological study, human exposure to selected medically-important phleboviruses was investigated in healthy adult residents of the Mersin province, Mediterranean Anatolia, Turkey, where the current data on phlebovirus epidemiology is scarce. A total of 1784 healthy individuals (mean age: 34.7±9.6 years; 97.3% were male), accepted as blood donors at the Mersin University Center for Health Research and Application Blood Bank were included in the study after informed consent during a seventeen month period between July 2011 to November 2012. All participants were requested to fill out a questionnaire to reveal risk factors for vector exposure. SFSV, SFNV, SFCV and TOSV IgG antibodies in serum were investigated via a commercial indirect immunofluorescence test (IIFT) (Sandfly Fever Virus IgG Mosaic I; Euroimmun, Germany). Sera interpreted as positive or strong positive for TOSV or SFNV+TOSV in IIFT were evaluated via TOSV virus neutralization test (VNT) for specificity confirmation. IIFT seroreactivity for at least one of the tested phleboviruses was present in 66.8% (1192/1784) of the samples. The most frequently-detected phlebovirus strain was SFSV (51.6%; 920/1784), followed by SFNV (46.4%; 827/1784), TOSV (43.7%; 779/1784) and SFCV (47.3%; 843/1784). Among the reactive sera, 6.6% (79/1192) were positive for a single virus serotype, whereas in 39.8% (475/1192) antibodies reacting with all tested virus serotypes were revealed. A total of 187 sera was included in the TOSV VNT and neutralizing antibodies were detected in 13.9%. According to the IIFT reactivity, residing in rural areas was observed as a statistically significant risk factor for exposure in all phleboviruses tested (p values for SFSV, SFNV, TOSV and SFCV were 0.002, 0.001, <0.001 and 0.003, respectively). TOSV exposure is more frequently detected via IIFT in individuals having pets or domestic farm animals around the living quarters (p=0.005). As a result, frequent exposure to SFSV/SFCV or antigenically similar phlebovirus strains and viruses of the SFNV species were determined in healthy blood donors in Mersin province, located in the Mediterranean region of Turkey. Furthermore, TOSV neutralizing antibodies were detected in selected samples with IIFT reactivity, confirming previous reports suggesting TOSV activity in the region. TOSV and other phleboviruses must be included in the diagnostic work-up in cases with febrile diseases and viral central nervous system infections during the sandfly-active months.

Phlebovirus infections in Greece.

Sandfly fever viruses are endemic in the Mediterranean region causing to humans asymptomatic or mild infections to severe neurological syndromes. In order to investigate the epidemiology of phlebovirus infections in Greece, samples from 499 patients with acute febrile illness (50.2% accompanied by neurological symptoms) were tested during 2010-2014 by serological and molecular methods. Phlebovirus infection was detected in 40 (8%) patients, 25 of them presenting acute neurological infection. Most cases were observed in the summer, with a peak in August. Increased number of cases was observed in 2013, and three of them were observed in Athens. Toscana virus lineage C RNA was detected in one encephalitis case, while the serological results showed that most cases were caused by phleboviruses belonging to the sandfly fever Naples virus serocomplex. This study provided the first insight into the epidemiology of phleboviral disease in Greece.

Toscana virus meningitis case in Switzerland: an example of the ezVIR bioinformatics pipeline utility for the identification of emerging viruses

Toscana virus (TOSV) represents a frequent cause of viral meningitis in the Mediterranean Basin that remains neglected in neighbouring countries. We report a documented TOSV meningitis case in a traveller returning from Tuscany to Switzerland. While routine serological and PCR assays could not discriminate between TOSV and Sandfly fever Naples virus infection, a high-throughput sequencing performed directly on the cerebrospinal fluid specimen and analysed with the ezVIR pipeline provided an unequivocal viral diagnostic. TOSV could be unequivocally considered as the aetiological agent, proving the potential of ezVIR to improve standard diagnostics in cases of infection with uncommon or emerging viruses.

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.

BackgroundSandfly-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.FindingsA 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.ConclusionsToscana 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.

Serosurvey for zoonotic viral and bacterial pathogens among slaughtered livestock in Egypt.

Zoonotic diseases are an important cause of human morbidity and mortality. Animal populations at locations with high risk of transmission of zoonotic pathogens offer an opportunity to study viral and bacterial pathogens of veterinary and public health concern. Blood samples were collected from domestic and imported livestock slaughtered at the Muneeb abattoir in central Egypt in 2009. Samples were collected from cattle (n=161), buffalo (n=153), sheep (n=174), and camels (n=10). Samples were tested for antibodies against Leptospira spp. by a microscopy agglutination test, Coxiella burnetii by enzyme immunoassay, Brucella spp. by standard tube agglutination, and Rift Valley Fever virus (RVFV), Crimean-Congo hemorrhagic fever virus (CCHFV), sandfly fever Sicilian virus (SFSV), and sandfly fever Naples virus (SFNV) by enzyme-linked immunosorbent assay. Antibodies against Leptospira spp. were identified in 64 (40%) cattle, 45 (29%) buffalo, 71 (41%) sheep, and five (50%) camels; antibodies against C. burnetii in six (4%) buffalo, 14 (8%) sheep, and seven (70%) camels; and antibodies against Brucella spp. in 12 (8%) cattle, one (1%) buffalo, seven (4%) sheep, and one (10%) camel. Antibodies against RVFV were detected in two (1%) cattle and five (3%) buffalo, and antibodies against CCHFV in one (1%) cow. No antibodies against SFSV or SFNV were detected in any species. RESULTS indicate that livestock have been exposed to a number of pathogens, although care must be taken with interpretation. It is not possible to determine whether antibodies against Leptospira spp. and RVFV in cattle and buffalo are due to prior vaccination or natural exposure. Similarly, antibodies identified in animals less than 6 months of age may be maternal antibodies transferred through colostrum rather than evidence of prior exposure. RESULTS provide baseline evidence to indicate that surveillance within animal populations may be a useful tool to monitor the circulation of pathogens of veterinary and public health concern in Egypt.

Viral Isolates of a Novel Putative Phlebovirus in the Marche Region of Italy

Thirty pools from 900 (540 females and 360 males) Phlebotomus perfiliewi sandflies collected during the summer of 2012 in the Fermo area (Marche Region, central Italy) were tested for the presence of Phleboviruses. A nested polymerase chain reaction was performed using degenerated primers amplifying a fragment of the polymerase gene (large segment) and a fragment of the nucleoprotein gene (small segment) of the genus Phlebovirus. One pool was positive for Toscana virus, as expected from results of studies in the area, and six pools were positive for a putative novel Phlebovirus. Virus isolation in Vero cells was performed. Minimum field infection rates/1,000 insects processed for the novel and Toscana viruses were 6.7 and 1.0, respectively. Phylogenetic analysis of the novel Phlebovirus, tentatively named Fermo virus, placed it in the Sandfly Fever Naples virus serocomplex.

Characterization of the Sandfly fever Naples species complex and description of a new Karimabad species complex (genus Phlebovirus, family Bunyaviridae).

Genomic and antigenic characterization of members of the Sandfly fever Naples virus (SFNV) complex reveals the presence of five clades that differ in their geographical distribution. Saint Floris and Gordil viruses, both found in Africa, form one clade; Punique, Granada and Massilia viruses, all isolated in the western Mediterranean, constitute a second; Toscana virus, a third; SFNV isolates from Italy, Cyprus, Egypt and India form a fourth; while Tehran virus and a Serbian isolate Yu 8/76, represent a fifth. Interestingly, this last clade appears not to express the second non-structural protein ORF. Karimabad virus, previously classified as a member of the SFNV complex, and Gabek Forest virus are distinct and form a new species complex (named Karimabad) in the Phlebovirus genus. In contrast with the high reassortment frequency observed in some South American phleboviruses, the only virus of the SFNV complex with evidence of reassortment was Granada virus.

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

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. 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. 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.

Prevalence of antibodies to phleboviruses within the sand fly fever Naples virus species in humans, northern Greece

Phleboviruses are commonly detected in the Mediterranean region. In order to estimate the seroprevalence to Toscana virus (TOSV) and TOSV-like viruses in the human population in northern Greece, we tested serum samples from 595 apparently healthy individuals aged 1–87 years (median: 45 years) for the presence of TOSV IgG antibodies. A seroprevalence of 11.26% was observed, ranging from 0% to 23.5%. Seropositivity was significantly lower in children and adults <30 years of age. The high seroprevalence (<10%) observed in several prefectures of northern Greece suggests that an important proportion of infections caused by TOSV or TOSV-like viruses may be asymptomatic or mild, and therefore underestimated. Increased awareness of physicians is needed during the summer months, when sand flies are active and have the potential to transmit phleboviruses to humans.

Toscana Virus Isolated from Sandflies, Tunisia

Toscana Virus Isolated from Sandflies, Tunisia

A Review of Zoonotic Disease Surveillance Supported by the Armed Forces Health Surveillance Center

The Armed Forces Health Surveillance Center (AFHSC), Division of Global Emerging Infections Surveillance and Response System conducts disease surveillance through a global network of US Department of Defense research laboratories and partnerships with foreign ministries of agriculture, health and livestock development in over 90 countries worldwide. In 2010, AFHSC supported zoonosis survey efforts were organized into four main categories: (i) development of field assays for animal disease surveillance during deployments and in resource limited environments, (ii) determining zoonotic disease prevalence in high-contact species which may serve as important reservoirs of diseases and sources of transmission, (iii) surveillance in high-risk human populations which are more likely to become exposed and subsequently infected with zoonotic pathogens and (iv) surveillance at the human-animal interface examining zoonotic disease prevalence and transmission within and between human and animal populations. These efforts have aided in the detection, identification and quantification of the burden of zoonotic diseases such as anthrax, brucellosis, Crimean Congo haemorrhagic fever, dengue fever, Hantaan virus, influenza, Lassa fever, leptospirosis, melioidosis, Q fever, Rift Valley fever, sandfly fever Sicilian virus, sandfly fever Naples virus, tuberculosis and West Nile virus, which are of military and public health importance. Future zoonotic surveillance efforts will seek to develop local capacity for zoonotic surveillance focusing on high risk populations at the human-animal interface.

Novel Phlebovirus in Febrile Child, Greece

To the Editor: Phleboviruses (family Bunyaviridae, genus Phlebovirus) are arthropod-borne, single-stranded, RNA viruses. Their genome consists of 3 segments—small, medium, and large—which encode the nucleoprotein and nonstructural proteins, the envelope glycoproteins, and the viral polymerase, respectively (1). The genus Phlebovirus consists at least 60 antigenically distinct serotypes, including the sandfly fever viruses transmitted to humans by phlebotomine sandflies. In the Mediterranean region, 3 phleboviruses are known to circulate: Toscana virus, sandfly fever Naples virus, and sandfly fever Sicilian virus. Sandfly fever Naples virus and sandfly fever Sicilian virus cause a transient febrile illness, whereas Toscana virus is sometimes neurovirulent, leading to aseptic meningitis and meningoencephalitis (2,3). Phleboviruses have been detected in Greece in clinically ill persons and in sandflies; seroprevalance in humans is high, especially in the Ionian islands (3–7). In addition, in 2002, a Sicilian-like virus (Cyprus virus) was responsible for a major outbreak of febrile illness among Greek Army forces in Cyprus (8). We report genetic detection and sequencing of an Adria virus from a boy who was hospitalized because of simple febrile seizure; the identical sequence was initially detected in sandflies collected in a coastal area in Albania. On September 23, 2009, a 2.5-year-old boy was admitted to the Hippokration Hospital of Thessaloniki, Greece, after a single episode of simple febrile seizure. The patient was febrile (38.2°C) and had vomited 1 time while in nursery school. He had sudden onset of eye gaze, perioral cyanosis, masseter muscle spasm, generalized tonic convulsions of the body and extremities, and involuntary loss of urine. The episode lasted ≈3 minutes, after which the child became irritable and sleepy. At the time of hospital admission (30 minutes later), he was afebrile and conscious. The boy’s history contained no previous neurologic or developmental disabilities and no family history of epilepsy or febrile seizures. Clinical examination, which included a thorough general and detailed neurologic evaluation, revealed no abnormalities except mild rhinitis. Laboratory tests showed leukocytosis (22,600 cells/μL) with 85.7% neutrophils. Blood levels of electrolytes, urea nitrogen, creatinine, glucose, albumin, bilirubin, alkaline phosphatase, and aminotransferases; prothrombin time; and urinalysis results were within reference limits. Electroencephalogram showed no brain abnormalities. Lumbar puncture and neuroimaging were not considered necessary. After 2 days of hospitalization, the child recovered, was free of signs and symptoms, and was discharged from the hospital with a diagnosis of simple febrile seizure and mild upper respiratory infection. According to a report from his mother 1 year later, the child remains well without any recurrence of febrile or other type of seizures. Viral RNA was extracted from the patient’s blood sample taken at the time of hospital admission. Nested reverse transcription–PCR using degenerate primers was applied to amplify a 222-bp fragment of the large RNA segment of phleboviruses (9). The retrieved sequence was identical to sequences detected in sandflies collected in 2005 in the Adriatic coastal region of Albania; that strain was provisionally named Adria virus (10). Adria virus is distinct from other recognized members of the genus Phlebovirus and clusters with phleboviruses of the Salehabad serocomplex, such as Salehabad virus and Arbia virus, differing by 21.6% and 29.6% with Salehabad virus and Arbia virus at the nucleotide level and by 3% and 17.7% at the amino acid level, respectively (Table). Table Percentage nucleotide and amino acid sequence divergence among phleboviruses* Detection of the Adria virus sequence in the patient’s blood suggests that this virus is pathogenic to humans. As expected, serologic testing of the sample taken at the time of admission produced negative results for phleboviruses; a convalescent-phase blood sample was not available. Although the course of the disease in the child was mild, further studies will show the role of this strain in public health. Because the duration of viremia in persons with phlebovirus infections is short, use of molecular methods for the laboratory diagnosis of phleboviral infections is limited; and even when a phleboviral infection is confirmed by serologic testing, the exact strain is difficult to determine. Physicians in Greece, as in other Mediterranean countries, should be aware of the circulation of phleboviruses and potential risk for phlebovirus-associated infections during the summer. Such infections, especially with neurologic signs, should be included in the differential diagnosis of summer febrile syndromes.

Sandfly fever virus activity in central/northern Anatolia, Turkey: first report of Toscana virus infections

AbstractSandfly fever viruses (SFVs) cause febrile diseases as well as aseptic meningitis/encephalitis and include serotypes sandfly fever Sicilian virus (SFSV), sandfly fever Naples virus (SFNV) and Toscana virus (TOSV). Infections are endemic in the Mediterranean basin and data on SFV activity in Turkey are limited. In this study, sera from 1533 blood donors from the Ankara, Konya, Eskişehir and Zonguldak provinces of Turkey were evaluated for SFV exposure by indirect immunofluorescence test (IIFT) and confirmed by virus neutralization test (VNT). One hundred and two patients with central nervous system (CNS) infections of unknown aetiology were also tested via IIFT and real-time reverse-transcription PCR for SFV/TOSV. Rate of overall IgG reactivity in IIFT was 32.9% (505/1533) among blood donors. TOSV exposure was confirmed by VNT in all study regions. Exposure to the recently-identified serotype sandfly fever Turkish virus, as evaluated by VNT, was revealed in Konya and Ankara. SFNV exposure was identified in Konya and SFSV was observed to be present in all regions except Zonguldak. TOSV RNA was detected in 15.7% (16/102) and was accompanied by TOSV IgM in 25% (4/16) of the patients. Partial L and S sequences suggested that TOSV circulating in Turkey can be grouped into TOSV genotype A strains. Exposure to TOSV and other SFV serotypes was revealed in blood donors and CNS infections by TOSV were identified for the first time in Turkey. Infections are observed to be endemic in central Anatolia and should be considered as aetiologic agents in cases/outbreaks of fever and meningoencephalitis.

Performance of Various Commercial Assays for the Detection of Toscana Virus Antibodies

Sandfly fever virus (SFV) serotypes sandfly fever Naples virus, sandfly fever Sicilian virus, and sandfly fever Cyprus virus cause febrile diseases, whereas Toscana virus (TOSV) is responsible for aseptic meningoencephalitis. Diagnosis and surveillance of TOSV depend heavily on virus serology, and various commercial assays utilizing various antigen sources and formats have been available. The aim of this study was to perform comparative evaluation of commercially available serological assays for anti-TOSV immunoglobulins. A collection of 120 sera from healthy blood donors from an endemic region, previously identified to be reactive for antibodies against various SFV serotypes by indirect immunofluorescence test (IIFT), was reevaluated for IgG/IgM via IIFT, enzyme-linked immunosorbent assay, and an immunoblot assay manufactured by Euroimmun, Diesse, and Mikrogen, respectively. Virus neutralization test (VNT) was performed for 99 sera using standard TOSV, sandfly fever Sicilian virus, and sandfly fever Naples virus strains. A total of 89 samples (74.2%) were reactive for TOSV IgG in at least one of the commercial assays, and 31 samples (31.3%) were reactive in VNT for various SFV serotypes. Average percentage agreements among commercial assays and between VNT and the commercial assays were noted as 57.8% and 62.6%, respectively. No significant correlation between assay results and VNT titers was observed. SFV IgM antibodies were detected in a total of eight samples (6.7%) via IIFT, which were nonreactive in enzyme-linked immunosorbent assay and VNT. Commercial diagnostic immunoassays displayed slight to fair agreement for TOSV IgG as assessed via kappa and percentage agreement values. The results could only be confirmed via virus neutralization in a portion of the samples, and overall agreement between the commercial assays and VNT was slight. Commercial assays such as immunoblot can be used in addition to VNT for confirmation of TOSV exposure.

Development of a Flow-Trough Microarray based Reverse Transcriptase Multiplex Ligation-Dependent Probe Amplification Assay for the Detection of European Bunyaviruses

It is suspected that apart from tick-borne encephalitis virus several additional European Arboviruses such as the sandfly borne Toscana virus, sandfly fever Sicilian virus and sandfly fever Naples virus, mosquito-borne Tahyna virus, Inkoo virus, Batai virus and tick-borne Uukuniemi virus cause aseptic meningo-encephalitis or febrile disease in Europe. Currently, the microarray technology is developing rapidly and there are many efforts to apply it to infectious diseases diagnostics. In order to arrive at an assay system useful for high throughput analysis of samples from aseptic meningo-encephalitis cases the authors developed a combined multiplex ligation-dependent probe amplification and flow-through microarray assay for the detection of European Bunyaviruses. These results show that this combined assay indeed is highly sensitive, and specific for the accurate detection of multiple viruses.

Toscana Virus Infection in American Traveler Returning from Sicily, 2009

Toscana Virus Infection in American Traveler Returning from Sicily, 2009

Toscana Virus Infection Imported from Elba into Switzerland

To the Editor: Toscana virus (TOSV) is a serotype of Sandfly fever Naples virus (SFNV) within the family Bunyaviridae and the genus Phlebovirus. TOSV is transmitted to humans by sandflies (Phlebotomus spp.) and is a prominent cause of aseptic meningitis in Mediterranean countries (1). In Italy, for populations living in rural areas and persons engaging in outdoor activities, the highest risk for acquiring TOSV is from August through October (1). TOSV infections should therefore be considered in travelers returning from the Mediterranean area who have fever and signs of meningitis. Laboratory diagnosis of TOSV infections is often limited to the detection of immunoglobulin (Ig) M and IgG because of the short period of viremia and the low amount of virus in the cerebrospinal fluid (CSF) during the acute phase (2). We report a reverse transcription–PCR (RT-PCR)–confirmed TOSV infection acquired on the island of Elba that was then imported into Switzerland. A 17-year-old man was referred to Basel University Medical Clinic, Liestal, Switzerland, in August 2009 with headache, recurrent episodes of vomiting, photophobia and phonophobia, and an elevated temperature of 38.1°C. The patient had returned to Switzerland from a vacation on the island of Elba, Italy, 14 days before. He recalled that he had received multiple insect bites on the beach. Cardiopulmonary and neurologic examination showed tachycardia and nuchal rigidity. Results of a complete blood count and liver and kidney function tests showed no abnormalities. CSF analysis showed lymphocytic pleocytosis (47 cells/µL), and aseptic meningitis of viral origin was suspected. Empirical treatment with acyclovir (2.3 g/day) was started for the first 48 hours. The results of a PCR for herpesviruses were negative in the CSF sample, and serologic testing showed no evidence of acute infection with herpesviruses. CSF, urine, and blood cultures showed negative results for fungi and bacteria, including mycobacteria. The patient did not show signs of immune deficiency. Serum and CSF samples were sent to the Bernhard-Nocht-Institute for Tropical Medicine in Germany for SFNV diagnostics. Results of immunofluorescent assays for TOSV and SFNV were positive with IgM titers of 1,280 and 160, respectively (cut-off 20) and IgG titers of 5,120 and 640, respectively (cut-off 20). Real-time RT-PCRs for detection of TOSV and SFNV were performed using the CSF sample according to a recently published protocol (3). A positive result was obtained for TOSV, and the PCR result was confirmed by sequencing the PCR product. Phylogenetic analysis demonstrated that the TOSV from Elba clustered with the TOSV A lineage (Figure). Attempts to isolate TOSV from the CSF sample in cell culture failed. The patient was afebrile on the second day of hospitalization, headache vanished on the third day, and he was discharged on day 5 restitutio ad integrum (fully recovered). Figure Bayesian phylogenetic tree of Toscana virus (TOSV) and Sandfly fever Naples virus (SFNV) strains. For each sequence used, GenBank accession number, strain designation, and strain origin are shown. Phylognetic analysis was performed by using MrBayes 3.0 ... This report demonstrates the presence of TOSV on the island of Elba by molecular detection and typing. This finding is in agreement with previous serologic reports on imported TOSV infections from this area into central Europe (5,6). However, because of serologic cross-reactivity, serologic tests are usually not able to clearly discriminate between TOSV and other SFNV infections (2). Real-time RT-PCR is the most appropriate tool for the differentiation of TOSV from other SFNV infections and enables molecular typing of amplified sequences. The Bayesian phlyogenetic tree calculated with the short PCR fragment (111 bp, GenBank accession no. {type:entrez-nucleotide,attrs:{text:GU270841,term_id:283971013,term_text:GU270841}}GU270841) of the nucleocapsid coding sequence (Figure) shows the same topology of the main clades when compared with trees obtained with the complete N coding sequence (7,8). The assignment of the TOSV from Elba to lineage A (Figure) is consistent with results of previous studies, demonstrating that this is the dominant genotype of TOSV in mainland Italy and the island of Sardinia (8). The presence of TOSV A on the island of Elba is a major public health issue for the local population and for the >2 million tourists that visit Elba every year (9). Given the incidence of TOSV infections in other surrounding Mediterranean countries, one could assume that the virus is present in other islands of the Mediterranean, posing a public health problem for the resident population and tourists alike. Molecular and serologic surveillance studies in Mediterranean countries could identify potential high risk areas for TOSV infections to help prevent exposure of local residents and tourists to the virus. Moreover, the risk of transfusion-associated transmission of arboviruses in European countries should be addressed.

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.

Arthropod-borne viruses transmitted by Phlebotomine sandflies in Europe: a review

Phlebotomine sandflies are known to transmit leishmaniases, bacteria and viruses that affect humans and animals in many countries worldwide. These sandfly-borne viruses are mainly the Phlebovirus, the Vesiculovirus and the Orbivirus. Some of these viruses are associated with outbreaks or human cases in the Mediterranean Europe. In this paper, the viruses transmitted by Phlebotomine sandflies in Europe (Toscana virus, Sicilian virus, sandfly fever Naples virus) are reviewed and their medical importance, geographical distribution, epidemiology and potential spreading discussed. Data on vertebrate reservoirs is sparse for sandfly fever viruses. The factor currently known to limit the spread of diseases is mainly the distribution areas of potential vectors. The distribution areas of the disease may not be restricted to the areas where they have been recorded but could be as wide as those of their vectors, that is to say Larroussius and P. papatasi mainly but not exclusively. Consequently, field work in form of viral isolation from sandflies and possible reservoirs as well as laboratory work to establish vectorial competence of colonised sandflies need to be encouraged in a near future, and epidemiological surveillance should be undertaken throughout the European Union.

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.