Characterization of a novel insect-specific flavivirus from Brazil: potential for inhibition of infection of arthropod cells with medically important flaviviruses

Abstract

In the past decade, there has been an upsurge in the number of newly described insect-specific flaviviruses isolated pan-globally. We recently described the isolation of a novel flavivirus (tentatively designated 'Nhumirim virus'; NHUV) that represents an example of a unique subset of apparently insect-specific viruses that phylogenetically affiliate with dual-host mosquito-borne flaviviruses despite appearing to be limited to replication in mosquito cells. We characterized the in vitro growth potential and 3' untranslated region (UTR) sequence homology with alternative flaviviruses, and evaluated the virus's capacity to suppress replication of representative Culex spp.-vectored pathogenic flaviviruses in mosquito cells. Only mosquito cell lines were found to support NHUV replication, further reinforcing the insect-specific phenotype of this virus. Analysis of the sequence and predicted RNA secondary structures of the 3' UTR indicated NHUV to be most similar to viruses within the yellow fever serogroup and Japanese encephalitis serogroup, and viruses in the tick-borne flavivirus clade. NHUV was found to share the fewest conserved sequence elements when compared with traditional insect-specific flaviviruses. This suggests that, despite apparently being insect specific, this virus probably diverged from an ancestral mosquito-borne flavivirus. Co-infection experiments indicated that prior or concurrent infection of mosquito cells with NHUV resulted in a significant reduction in virus production of West Nile virus (WNV), St Louis encephalitis virus (SLEV) and Japanese encephalitis virus. The inhibitory effect was most effective against WNV and SLEV with over a 10(6)-fold and 10(4)-fold reduction in peak titres, respectively.

Figure 1

Phase contrast image depicting NHUV cytopathology in C6/36 cells in vitro; A) negative control mock infected, B) NHUV infected cells with syncytia.

Figure 2

Epifluorescent images of IFA tests in the various cell types examined

Figure 3

Phylogenetic analysis based on nucleotide sequences of complete polyprotein coding sequences. Phylogenies were constructed using the maximum likelihood method with labeled bootstrap percentages as support. Labels include taxon name and accession number. NHUV is highlighted in gray and clades are labeled by host association designations on the far right of the figure.

Figure 4

Mfold generated prediction and labels denoting conserved secondary structure and sequence elements for CFAV (shown in alternating display for clarity), TBEV, MODV, WNV, and NHUV. Nucleotides included in conserved MBFV sequences such the pentanucleotide, conserved sequence 1 (CS1), and CS2 are highlighted with grey circles. A) Key structures identified in CFAV include the 3’ LSH with an internal conserved pentanucleotide (CACCG), a Y-shaped element, and a conserved hexanucleotide sequence element. B). TBEV had the 3’LSH, pentanucleotide (CACAG), SL2, and Y-1 with an internal hexanucleotide sequence. C) MODV demonstrated the 3’ LSH, pentanucleotide (CUCAG), and Y-1 with internal hexanucleotide sequence.multiple. D) WNV showed a 3’LSH, the conserved pentanucleotide sequence (CACAG), SL2, conserved sequences CS1, CS2, and CS3. E) NHUV was found to have a 3’ LSH, a conserved pentanucleotide (CACAG), SL2, and only CS1 and CS2.

Figure 5

Histograms demonstrating threonine, arginine, leucine, and proline codon usage frequencies for NHUV, WNV, CxFV, and MODV.

Figure 6

Inhibition of WNV, JEV, and SLEV on C6/36 cells in the presence of NHUV. A) Replication kinetics of WNV compared with WNV dual-infection with NHUV at day 0, day 1, day 3, and day 5 post-NHUV inoculations of C6/36 cells. B) JEV dual-infection with NHUV at day 0, and day 3 post-NHUV infection. C) SLEV dual infection with NHUV at day 0 and day 3 post-NHUV infection. Cells were inoculated with an MOI of 0.1 of the representative MBFV and exposed to NHUV at an MOI of 5. Time points were collected daily for seven days following infection with each MBFV.