Co-circulation of a novel phlebovirus and Massilia virus in sandflies, Portugal

Abstract

Background: 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.

Findings: 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.

Conclusions: 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.

Fig. 1

Map depicting the geographic location of the trapping sites from where the sandflies have been collected and the positive sites where the Alcube and Massilia viruses were detected

Fig. 2

Phylogenetic analyses based on complete genomes of the selected members of the Phlebovirus genus, including the putative novel Alcube virus species and two new Massilia viruses. The phylogenetic trees were inferred on the basis of complete L, M and S (N and NS) protein sequences with the use of the Bayesian Markov chain Monte Carlo method in MrBayes v3.2.5 (http://mrbayes.sourceforge.net/index.php) and, in parallel, the maximum-likelihood methods using PhyML v3.0 (http://www.atgc-montpellier.fr/phyml/). Statistical support of grouping from Bayesian posterior probabilities (clade credibilities ≥90 %) and maximum-likelihood bootstrap replicates (≥70 %) is indicated with an asterisk. The taxon information includes the GenBank accession number and virus abbreviation. The recognized and/or proposed species or species complexes of phleboviruses are indicated to the right of the trees. The Alcube virus and Massilia viruses generated during this study are bolded. The scale bar represents amino acid substitutions per site