title: Phleboviruses detection in Phlebotomus perniciosus from a human leishmaniasis focus in South-West Madrid region, Spain.
abstract: BACKGROUND
  Phlebotomus-borne (PhB-) viruses are distributed in large areas of the Old World and are widespread throughout the Mediterranean basin, where recent investigations have indicated that virus diversity is higher than initially suspected. Some of these viruses are causes of meningitis, encephalitis and febrile illnesses. In order to monitor the viral presence and the infection rate of PhB-viruses in a recently identified and well characterized human zoonotic leishmaniasis focus in southwestern Madrid, Spain, a sand fly collection was carried out.
  
  
  METHODS
  Sand fly insects were collected in four stations using CDC light traps during 2012-2013 summer seasons. Screening for Phlebovirus presence both via isolation on Vero cells and via polymerase chain reaction (PCR), using degenerated primers targeting a portion of the L segment, was performed. The serological identity and phylogenetic relationships on the three genomic segments of the viral isolates were carried out.
  
  
  RESULTS
  Six viral isolates belonging to different serological complexes of the genus Phlebovirus were obtained from fifty pools on a total of 963 P. perniciosus (202 females). Phylogenetic analysis and serological assays allowed the identification of two isolates of Toscana virus (TOSV) B genotype, three isolates strongly related to Italian Arbia virus (ARBV), and one isolate of a novel putative Phlebovirus related to the recently characterized Arrabida virus in South Portugal, tentatively named Arrabida-like virus. Positive male sand fly pools suggested that transovarial or venereal transmission could occur under natural conditions.
  
  
  CONCLUSIONS
  Our findings highlighted the presence of different Phlebovirus species in the South-West area of the Madrid Autonomous Community where an outbreak of cutaneous and visceral human leishmaniasis has been recently described. The evidence of viral species never identified before in Spain, as ARBV and Arrabida-like virus, and TOSV B genotype focus stability was demonstrated. Environmental aspects such as climate change, growing urbanization, socio-economic development could have contributed to the genesis of this wide ecological niche of PhB-viruses and Leishmania spp. The potential role of vertebrates as reservoir for the phleboviruses identified and the possibility of Phleboviruses-Leishmania co-infection in the same sand fly should be assessed. Furthermore the PhB-viruses impact on human health should be implemented.
authors.affiliation: National Reference Laboratory for Arboviruses, Department of Infectious, Parasitic and Immune-Mediated Diseases, Istituto Superiore di Sanità, 00161, Rome, Italy. mariaelena.remoli@iss.it.
authors.forename: Maria Elena
authors.initials: ME
authors.lastname: Remoli
authors.affiliation: Unidad de EntomologíaMédica, Servicio de Parasitología, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Ctra. Majadahonda-Pozuelo s/n, 28220, Majadahonda, Madrid, Spain.
authors.forename: Maribel
authors.initials: M
authors.lastname: Jiménez
authors.affiliation: National Reference Laboratory for Arboviruses, Department of Infectious, Parasitic and Immune-Mediated Diseases, Istituto Superiore di Sanità, 00161, Rome, Italy.
authors.forename: Claudia
authors.initials: C
authors.lastname: Fortuna
authors.affiliation: National Reference Laboratory for Arboviruses, Department of Infectious, Parasitic and Immune-Mediated Diseases, Istituto Superiore di Sanità, 00161, Rome, Italy.
authors.forename: Eleonora
authors.initials: E
authors.lastname: Benedetti
authors.affiliation: National Reference Laboratory for Arboviruses, Department of Infectious, Parasitic and Immune-Mediated Diseases, Istituto Superiore di Sanità, 00161, Rome, Italy.
authors.forename: Antonella
authors.initials: A
authors.lastname: Marchi
authors.affiliation: Unit of Global Health, Department of Therapeutic Research and Medicines Evaluation, Istituto Superiore di Sanità, 00161, Rome, Italy.
authors.forename: Domenico
authors.initials: D
authors.lastname: Genovese
authors.affiliation: Unit of Vector-borne Diseases and International Health, Department of Infectious, Parasitic and Immune-Mediated Diseases, Istituto Superiore di Sanità, 00161, Rome, Italy.
authors.forename: Marina
authors.initials: M
authors.lastname: Gramiccia
authors.affiliation: Unidad de EntomologíaMédica, Servicio de Parasitología, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Ctra. Majadahonda-Pozuelo s/n, 28220, Majadahonda, Madrid, Spain.
authors.forename: Ricardo
authors.initials: R
authors.lastname: Molina
authors.affiliation: National Reference Laboratory for Arboviruses, Department of Infectious, Parasitic and Immune-Mediated Diseases, Istituto Superiore di Sanità, 00161, Rome, Italy. mariagrazia.ciufolini@iss.it.
authors.forename: Maria Grazia
authors.initials: MG
authors.lastname: Ciufolini
mesh_terms: D000818:Animals; D005260:Female; D006801:Humans; D007303:Insect Vectors / Q000821:virology*; D007896:Leishmaniasis / Q000453:epidemiology; D008297:Male; D010691:Phlebotomus / Q000821:virology*; D016856:Phlebovirus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification* / Q000502:physiology; D010802:Phylogeny; D000091402:Prohibitins; D013030:Spain / Q000453:epidemiology
keywords: Arbia virus; Human leishmaniasis; Madrid; Novel virus; Phlebotomus perniciosus; Phlebovirus; Spain; Toscana virus