Ecuador Paraiso Escondido Virus, a New Flavivirus Isolated from New World Sand Flies in Ecuador, Is the First Representative of a Novel Clade in the Genus Flavivirus

Abstract

A new flavivirus, Ecuador Paraiso Escondido virus (EPEV), named after the village where it was discovered, was isolated from sand flies (Psathyromyia abonnenci, formerly Lutzomyia abonnenci) that are unique to the New World. This represents the first sand fly-borne flavivirus identified in the New World. EPEV exhibited a typical flavivirus genome organization. Nevertheless, the maximum pairwise amino acid sequence identity with currently recognized flaviviruses was 52.8%. Phylogenetic analysis of the complete coding sequence showed that EPEV represents a distinct clade which diverged from a lineage that was ancestral to the nonvectored flaviviruses Entebbe bat virus, Yokose virus, and Sokoluk virus and also the Aedes-associated mosquito-borne flaviviruses, which include yellow fever virus, Sepik virus, Saboya virus, and others. EPEV replicated in C6/36 mosquito cells, yielding high infectious titers, but failed to reproduce either in vertebrate cell lines (Vero, BHK, SW13, and XTC cells) or in suckling mouse brains. This surprising result, which appears to eliminate an association with vertebrate hosts in the life cycle of EPEV, is discussed in the context of the evolutionary origins of EPEV in the New World.

Importance: The flaviviruses are rarely (if ever) vectored by sand fly species, at least in the Old World. We have identified the first representative of a sand fly-associated flavivirus, Ecuador Paraiso Escondido virus (EPEV), in the New World. EPEV constitutes a novel clade according to current knowledge of the flaviviruses. Phylogenetic analysis of the virus genome showed that EPEV roots the Aedes-associated mosquito-borne flaviviruses, including yellow fever virus. In light of this new discovery, the New World origin of EPEV is discussed together with that of the other flaviviruses.

FIG 1

C6/36 cell cultures at the 6th passage after 1 day. (Left) Noninfected cells; (right) cells infected with EPEV.

FIG 2

Conserved motifs in the polyprotein of EPEV. The numbers 1 to 4 indicate the conserved enzymatic motifs in the proteins encoded by the NS3 and NS5 genes. Slashes, gaps; residues in yellow, nonconserved positions.

FIG 3

Bayesian phylogeny of the ORFs of the members of the genus Flavivirus. Only posterior probabilities are included. The tree is rooted at the midpoint. Strain names and GenBank accession numbers are given after the names of the viruses, which are abbreviated as follows: KUNV, Kunjin virus; KOUV, Koutango virus; YAOV, Yaounde virus; USUV, Usutu virus; ALFV, Alfuy virus; CPCV, Cacipacore virus; ITV, Israel turkey meningoencephalitis virus; BAGV, Bagaza virus; NTAV, Ntaya virus; TMUV, Tembusu virus; STWV, Sitiawan virus; DEDSV, duck egg drop syndrome virus; ROCV, Rocio virus; ILHV, Ilheus virus; IGUV, Iguape virus; AROAV, Aroa virus; BSQV, Bussuquara virus; NJLV, Naranjal virus; KOKV, Kokobera virus; STRV, Stratford virus; NMV, New Mapoon virus; DENV_1 to DENV_4, dengue virus serotypes 1 to 4, respectively; ZIKV, Zika virus; SPOV, Spondweni virus; KEDV, Kedougou virus; DGV, Donggang virus; UGSV, Uganda S virus; LIV, Louping ill virus; SSEV, Spanish sheep encephalitis virus; TSEV, Turkish sheep encephalitis virus; GGEV, Greek goat encephalitis virus; OHFV, Omsk hemorrhagic fever virus; LGTV, Langat virus; AHFV, Alkhurma virus; KFDV, Kyasanur Forest disease virus; RFV, Royal Farm virus; KSIV, Karshi virus; GGYV, Gadgets Gully virus; KADV, Kadam virus; MEAV, Meaban virus; SREV, Saumarez Reef virus; TYUV, Tyuleniy virus; PPBV, Phnom Penh bat virus; BCV, Batu Cave virus; MMLV, Montana myotis leukoencephalitis virus; RBV, Rio Bravo virus; JUTV, Jutiapa virus; MODV, Modoc virus; MosFV, mosquito flavivirus virus; QBV, Quang Ninh virus; Cv_theileri, Culex theileri flavivirus; NAKV, Nakiwogo virus; PCV, Palm Creek virus; KRV, Kamiti River virus; HANKV, Hanko virus. The definitions of the other abbreviations for viruses are provided in the text.