A Multicomponent Animal Virus Isolated from Mosquitoes

Abstract

RNA viruses exhibit a variety of genome organization strategies, including multicomponent genomes in which each segment is packaged separately. Although multicomponent genomes are common among viruses infecting plants and fungi, their prevalence among those infecting animals remains unclear. We characterize a multicomponent RNA virus isolated from mosquitoes, designated Guaico Culex virus (GCXV). GCXV belongs to a diverse clade of segmented viruses (Jingmenvirus) related to the prototypically unsegmented Flaviviridae. The GCXV genome comprises five segments, each of which appears to be separately packaged. The smallest segment is not required for replication, and its presence is variable in natural infections. We also describe a variant of Jingmen tick virus, another Jingmenvirus, sequenced from a Ugandan red colobus monkey, thus expanding the host range of this segmented and likely multicomponent virus group. Collectively, this study provides evidence for the existence of multicomponent animal viruses and their potential relevance for animal and human health.

Figure 1

The genome of GCXV is enveloped and divided into five single-stranded, positive-sense RNA segments. A) Distinct RNA segments of GCXV on an agarose gel. Segment #s are shown in black, ladder band sizes (nt) are shown in white. B) Selective degradation with RNase I demonstrates that GCXV has a single-stranded RNA genome. U – untreated, D – DNase I treated, R – RNase I treated. Segment 4 amplicons were run on a separate gel. C) Genome schematic with all ORFs ≥400 nt. Dotted lines indicate regions putatively translated through -1 ribosomal frameshifting (arrows indicate slippery heptanucleotides); solid lines indicate the predicted ORFs based on the first conserved AUG. D) Treatment with NP40 resulted in highly diminished RNA copy numbers, lack of growth and absence of CPE in C6/36 cells, indicating that GCXV is enveloped. See also Figures S2–S3 and Tables S2–S3.

Figure 2

Segment 5 of GCXV is not required for replication in C6/36 cells and is variably present in natural isolates. A) Cell culture supernatants resulting from transfection with GCXV segments 1–5 (passage 1) and segments 1–4 (passage 2). Segment #s are shown in black, ladder band sizes (nt) are shown in white. B) Unrooted, nt-level phylogenetic trees including all six isolates of GCXV. ACH27 and TR7094 lack segment 5. Color indicates country of collection: blue for Peru, red for Panama and green for Trinidad. Branch labels represent bootstrap support values. Scale bar indicates number of nt changes per site. See also Table S4.

Figure 3

The dose-response curve for GCXV supports a multicomponent genome organization. Plaque count is shown on the y-axis and the relative dilution of the virus stock is shown on the x-axis. The analysis included 5 replicates per dilution (circles). The black line is the best-fitting line from a Poisson generalized linear model, with 95% confidence intervals (grey band). The grey lines represent expected slopes for multiplicities of infection of 1–5.

Figure 4

Multiple combinations of GCXV genome segments were detected in infected cells. A representative 63x magnification image is shown from the co-hybridization of probes for segments 3–5. Four different infected cell types were observed: closed arrowhead – missing segments 4 and 5; open arrowhead – missing segment 5; full arrow – missing segment 4 (note: segment 5 present in low abundance in indicated cell); and dashed arrow – containing all assayed segments. The scale bar represents 10 µm. See also Figure S1 and Table S1.

Figure 5

GCXV belongs to an insect-specific clade within the segmented Jingmenviruses. Maximum-likelihood (ML) trees are shown with bootstrap support values from both the ML and neighbor-joining (NJ) trees: ML/NJ. Scale indicates number of amino acid changes per site. See also Table S5.