Co-circulation of a novel phlebovirus and Massilia virus in sandflies, Portugal
© Amaro et al. 2015
Received: 4 August 2015
Accepted: 20 October 2015
Published: 24 October 2015
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.
The world-wide impact of sandflies (Diptera: Psychodidae) as significant public health concern and vectors of several zoonotic diseases affecting humans is well known. The most widely distributed pathogens transmitted by sandflies are protozoan parasites of the genus Leishmania, but they also act as vectors of several phleboviruses (Phlebovirus genus, Bunyaviridae family) that are associated with human illness (from transient febrile illness to severe neuroinvasive diseases) [1–3]. Based on antigenic and genomic relationships, six species complexes of phleboviruses are recognized and/or proposed so far for which transmission by sandflies has been shown [4–10]. In the Mediterranean Basin, phleboviruses are emerging agents of infectious diseases whose real medical importance has not yet been fully addressed. Until very recentely only Toscana virus, a member of the Sandfly fever Naples virus species complex was known to circulate in Portugal after first being reported in 1985 when a tourist was infected in the Algarve region, in the south of the country [11, 12]. However, no entomological surveys to detect phleboviruses in their natural vectors have been performed so far. The aims of the present study were to detect, isolate and characterize phleboviruses in sandfly populations of Portugal.
Genetic distances (nucleotide and amino acid divergences) between sequences of the L, M, and S (nucleocapsid, N; nonstructural, NS) genes and proteins of selected phleboviruses and Alcube virus
Nucleotide/amino acid divergences (%)
To date, Toscana virus [11, 12] from the Sandfly fever Naples virus species complex is the only phlebovirus known to circulate in sandfly populations of Portugal. In this study, a novel phlebovirus species, designated Alcube virus, was discovered in phlebotomine sandflies (Ph. perniciosus) in Arrábida region, located on the southwest coast of Portugal. The virus was isolated and characterized using full-length genome sequence data. Phylogenies demonstrated that Alcube virus clustered with members of the Salehabad virus species complex within the genus Phlebovirus and forms with the Arbia, Salehabad and Adana viruses a distinct monophyletic lineage within this clade. In Turkey, high Adana virus seroprevalence rates in goats, sheep, and dogs were demonstrated . In contrast, low seroprevalence rates in humans suggest that Adana virus is not likely to constitute an important public health problem in Turkey . In the Arrábida region, we also detected the presence and co-circulation of Massilia virus, a member of the Sandfly fever Naples serocomplex which was described only in France so far. This is the first report of Massilia virus outside France. The medical and public health impact of Massila virus in Portugal remains to be investigated. Although the number of sandflies trapped was relatively small, the number of phlebovirus-infected pools were similar to those reported from France and Italy [15, 16]. These findings suggested that a relatively high proportion of sandflies are naturally infected. The results of this study calls for further epidemiological investigations and surveillance for sandfly-borne phleboviruses to elucidate their medical and veterinary importance.
In conclusion, 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, a previously recognized phlebotomine-borne phlebovirus, in Arrábida region, southwest of Portugal.
This work was partially funded by the FCT project “New arboviruses isolated in Portugal. Risk assessment and public health application (PTDC/SAU-SAP/119199/2010)”.
The GenBank accession numbers for the complete genome of Alcube virus are KR363190, KR363191, and KR363192. The GenBank accession numbers for the complete genomes of Massila viruses are KT906098, KT906099, KT906100, KT906101, KT906102, and KT906103.
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