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

Abstract

BackgroundHigh 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).ResultsOne 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.ConclusionsHTS, 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.Electronic supplementary materialThe online version of this article (10.1186/s13071-018-2985-3) contains supplementary material, which is available to authorized users.

Aedes aegypti (Aag2)-derived clonal mosquito cell lines reveal the effects of pre-existing persistent infection with the insect-specific bunyavirus Phasi Charoen-like virus on arbovirus replication

“…PCLV levels remained consistently low over multiple passages, and in the case of Aag2-AF12 cells were so low as to be only intermittently detectable. In the wild, insect-specific viruses primarily cause persistent infection of mosquitoes, and comparison of the PCLV-low clones Aag2-AF10 and Aag2-AF12 to parental Aag2 cells allowed us to test the impact of persistent insect-specific virus infection on arbovirus replication, in comparison to previous studies that tested the impact of acute insect-specific virus infection on arbovirus replication [31,34–37,41–43]. We observed no consistent impact of PCLV on the replication of representative flaviviruses (DENV-2, ZIKV), alphaviruses (SINV) or rhabdoviruses (VSV), representing both +ssRNA and -ssRNA arboviruses.…”

“…There is no consensus on how insect-specific viruses affect arbovirus replication in tissue culture or in vivo , with the experimental outcome varying depending on the combination of arbovirus, insect-specific virus and mosquito species (and potentially the specific mosquito line or cell line used), as well as other variations in the experimental set up [15]. Thus, previous studies have found insect-specific viruses to either increase [29,30], decrease [19,31–41] or have no effect [31,35,40,42,43] on the replication of various arboviruses across different mosquito species and cell lines. The majority of these studies were performed in the context of acute insect-specific virus infection, which may not accurately recapitulate the effects of the persistent insect-specific virus infections more commonly encountered in nature.…”

Novel Viruses in Mosquitoes from Brazilian Pantanal

“…Female mosquitoes were identified alive after immobilization (4 °C by 4 min) using dichotomy keys [22]; their dissected salivary glands [23] were pooled together ( n = 3 to 117) according to date, place of collection, species, and gender; then homogenized in 0.4 mL of RNAse free phosphate saline buffer (pH 7.2) and centrifuged (5000× g for 4 min at 4 °C). RNA was extracted from the supernatant (0.2 mL) with a High Pure Viral RNA Kit (Roche) without RNA carrier, quantified (quantifluor RNA system; Quantus fluorometer, Promega, Madison, WI, USA), reverse transcribed (GoScript, Promega, Madison, WI, USA), and amplified in quintuplicates with a viral random PCR after double-strand cDNA synthesis (Klenow DNA polymerase I, New Englands BioLabs, Ipswich, MA, EUA) as previously described [19,21,24]. PCR products were purified with 20% polyethylene glycol, quantified with a quantifluor one dsDNA system (Quantus Fluorometer, Promega, Madison, WI, USA) and sequenced after genomic library preparation with the Truseq DNA PCR-free library kit (Illumina, San Diego, CA, USA) using 2 × 100 paired-end reads in two lanes with 60 GB on a Hiseq 2500 platform (Illumina, San Diego, CA, USA).…”

“…Previous studies from our group already described new ISVs from Chuviridae, Rhabdoviridae, Partitiviridae, and Totiviridae families infecting the salivary glands of mosquitoes [19,20]. A novel phlebotomus fever serogroup member from Phlebovirus genus, Phenuiviridae family, named Viola virus was identified in Lutzomiya longipalpis from the High Pantanal region [21]. Therefore, this study aimed to identify the sialovirome of culicinae mosquitoes captured in High Pantanal, Mato Grosso State, Central-Western Brazil.…”

Primera aproximación a la fauna flebotomínea (Psychodidae: Phlebotominae) del departamento de Atlántico

“…El género Lutzomyia (Lu) o "moscas de la arena", está representado por cerca de 530 especies presentes en el Nuevo Mundo (Shimabukuro et al, 2017;Adams y Shimabukuro, 2018). Las hembras tienen la necesidad de ingerir sangre de diversas fuentes de vertebrados y esta condición hace que su participación sea relevante en la transmisión de varias enfermedades zoonóticas como las leishmaniasis (Killick-Kendrick, 1990) y la bartonelosis (Birtles, 2001), así como agentes virales como Flavivirus, Orvivirus, Phlebovirus y Vesiculovirus (Comer y Tesh, 1991;Ashford, 2001;de Carvalho et al, 2018).…”