Negeviruses isolated from mosquitoes in the Brazilian Amazon

Abstract

Background: There are several groups of viruses including Insect Specific Viruses (ISV) such as the taxon Negevirus, a group of viruses phylogenetically related to plant viruses. Negeviruses replicate in mosquito cells, but not in vertebrate cells.

Methods: Pools of hematophagous arthropods were inoculated in Vero and C6/36 cells. The cells were observed to detect possible cytopathic effect. Then, indirect immunofluorescence, RT-PCR, and nucleotide sequencing were performed.

Results: Seven samples which presented negative results for flaviviruses, alphaviruses and bunyaviruses, but showed cytopathic effect in C6/36 cells were sequenced. We identified the occurrence of a variety of ISVs, most of them belonging to the taxon Negevirus: The Brejeira, Negev, Cordoba and Wallerfield viruses, including a new virus for science, tentatively named Feitosa virus.

Conclusions: We detected negeviruses in the Amazon region, including two viruses that were isolated for the first time in Brazil: Cordoba virus and the Negev virus and, a new virus for science: the Feitosa virus.

Keywords: Arboviruses; Insect-specific viruses; Negevirus.

Fig. 1

Cytopathic effect (CPE) observed in infected C6/36 cells (red arrow). a Cells inoculated with the sample BE AR 805503 (CPE at 6th day post-infection), showing destruction of cell’s monolayer; b cells inoculated with the sample BE AR 805529 (CPE at 4th day post-infection), showing destruction of cell’s monolayer and formation of clumps; c cells inoculated with the sample BE AR 805511 (CPE at 6th day post-infection), showing destruction of cell’s monolayer and large cells (larger than normal cells); d–f cells inoculated with the sample BE AR 805514 (CPE at 6th day post-infection), BE AR 820396 (CPE at 6th dpi) and BE AR 805520 (CPE at 6th day post-infection), respectively, showing destruction of the cell’s monolayer; g cells inoculated with the sample BE AR 805525 (CPE at 6th day post-infection), highlighting destruction of the cell’s monolayer and large cells (larger than normal cells); h negative control (C6/36 cells) showing no CPE (100X)

Fig. 2

Phylogenetic tree based on the amino acid sequences of viral polymerase (RdRp) from ORF 1 of insect-specific viruses belonging to the taxon Negevirus. Phylogeny generated with the Maximum Likelihood method and LG model. The virus strains obtained in the study are in blue font and arranged in a cartoon format, while the virus groups related to the isolated viruses are collapsed. Viruses with all three ORFs are highlighted in yellow; those with two ORFs are highlighted in blue; and those with only one ORF are highlighted in green. The host that originated each isolate is identified with an image of the host (mosquito, bee, plant), identified in the legend. The bottom bar represents the rate of amino acid replacement

Fig. 3

Phylogenetic tree of the concatenated nucleotide sequences of ORF1, ORF2 and ORF3 of the isolated strains (in red font) and other strains available in the Genbank, using the Maximum Likelihood method and GTR model. The bottom bar represents the rate of nucleotide replacement. a BRJV; b NEGV; c CDBV; d WALV; e FEITV