Viola phlebovirus is a novel Phlebotomus fever serogroup member identified in Lutzomyia (Lutzomyia) longipalpis from Brazilian Pantanal

Abstract

Background: High throughput sequencing (HTS) boosted the discovery of novel viruses and new variants of known viruses. Here we investigated the presence of viruses in 12 pools of sand flies captured in three climatic periods in RAPELD grids at Rio Claro, Chapada dos Guimarães and at Pirizal, North Pantanal, Mato Grosso State, Midwestern Brazil by HTS, viral isolation of a putative Phlebovirus positive pool in Vero cells, RT-PCR and transmission electron microscopy (TEM).

Results: One pool containing three Lutzomyia (Lutzomyia) longipalpis sand flies captured in the transitional climatic period in North Pantanal showed a tripartite genomic sequence of a putative novel Phlebovirus belonging to the phlebotomus fever serogroup. Phylogenetic analysis revealed this virus is closely related and share a common ancestor with phleboviruses included in the same clade: Chagres, Urucuri and Uriurana virus. RNA-dependent RNA polymerase (RdRP) presented 60%, 59% and 58% of amino-acid (aa) similarity with these phleboviruses, respectively. Similarity of Nucleoprotein and NSs protein codified by ambissense strategy of segment S was of 49% and 37%, respectively, with the proteins of the closest phlebovirus, Uriurana virus. Glycoproteins (G1, G2) and NSm protein presented 49% and 48% aa similarity with Chagres and Uriurana virus, respectively. Uriurana virus was isolated from sand flies in Brazilian Amazon and Urucuri from rodents in Utinga forest, Pará State. Chagres virus is an arbovirus responsible for outbreaks of febrile illness in Panama. This phlebovirus was isolated in Vero cells, confirmed by TEM and RT-PCR for the L segment of the virus, and named Viola phlebovirus.

Conclusions: HTS, viral isolation, RT-PCR and TEM showed the presence of one virus in sand flies from North Pantanal with identity to a putative novel Phlebovirus from phlebotomus fever serogroup, named Viola phlebovirus.

Keywords: Bunyavirales; High throughput sequencing; Phenuiviridae; Phylogeny; RNA virus; Viola phlebovirus; Viral isolation.

Fig. 1

Location of RAPELD systems used in the study for sand flies collections in three climatic periods in North Pantanal (1) and Chapada dos Guimarães National Park (2), Mato Grosso State, Midwestern Brazil. Collection plots are marked with blue dots in each RAPELD system grade

Fig. 2

RT-PCR for a region of L segment (460 bp) at passage 4 (a) and transmission electron microscopy of Viola phlebovirus in Vero cells at passage 5 (b)

Fig. 3

a Genomic organization of Viola phlebovirus L segment; and b phylogenetic tree based on amino acid sequences of phleboviruses by maximum likelihood method Jones-Taylor-Thornton model. Phlebotomus fever and Uukuniemi serogroups are marked in orange and purple, respectively. Bars indicate serocomplexes

Fig. 4

a Genomic organization of Viola phlebovirus M segment; and b phylogenetic tree based on amino acid sequences of phleboviruses by maximum likelihood method Jones-Taylor-Thornton model. Phlebotomus fever and Uukuniemi serogroups are marked in orange and purple, respectively. Bars indicate serocomplexes

Fig. 5

a Genomic organization of Viola phlebovirus S segment; and b phylogenetic tree based on amino acid sequences of phleboviruses by maximum likelihood method Jones-Taylor-Thornton model. Phlebotomus fever and Uukuniemi serogroup are marked in orange and purple, respectively. Bars indicate serocomplexes