A novel and diverse family of filamentous DNA viruses associated with parasitic wasps

Abstract

Large dsDNA viruses from the Naldaviricetes class are currently composed of four viral families infecting insects and/or crustaceans. Since the 1970s, particles described as filamentous viruses (FVs) have been observed by electronic microscopy in several species of Hymenoptera parasitoids but until recently, no genomic data was available. This study provides the first comparative morphological and genomic analysis of these FVs. We analyzed the genomes of seven FVs, six of which were newly obtained, to gain a better understanding of their evolutionary history. We show that these FVs share all genomic features of the Naldaviricetes while encoding five specific core genes that distinguish them from their closest relatives, the Hytrosaviruses. By mining public databases, we show that FVs preferentially infect Hymenoptera with parasitoid lifestyle and that these viruses have been repeatedly integrated into the genome of many insects, particularly Hymenoptera parasitoids, overall suggesting a long-standing specialization of these viruses to parasitic wasps. Finally, we propose a taxonomical revision of the class Naldaviricetes in which FVs related to the Leptopilina boulardi FV constitute a fifth family. We propose to name this new family, Filamentoviridae.

Keywords: EVE; Lefavirales; Naldaviricetes; dsDNA virus; filamentous virus; horizontal gene transfer; parasitoid wasp.

Figure 1.

Diagrams of the four circularized FV genomes. CcFV1 = Cotesia congregata FV 1 from Cotesia congregata (laboratory line, MsT), CcFV2 = Cotesia congregata catalpae FV 2 from Cotesia congregata catalpae (line recently obtained from a wild population, CcC), EfFV = Encarsia formosa FV and the previously published LbFV. Predicted ORFs are displayed as blue rectangles, where dark blue and light blue represent positive and negative strands, respectively. The GC content along the genome is displayed with bars, where black bars represent GC > mean GC content and grey bars represent GC < mean GC content. Coverage corresponding to long read technologies (Nanopore, 454 or PacBio) is plotted in green, while coverage corresponding to short reads (Illumina) is displayed in orange. Repeated elements of interest are plotted in the center with the homologous repeat sequences (hrs) in red, the best motifs elements in grey (with light grey = sense strand and dark grey = antisense strand) and the direct repeats in yellow. First ORF was set as the DNApol for all three new genomes, as it is usually done for related viruses, except for the previously published LbFV where the DNApol corresponds to orf58.

Figure 2.

Heatmap representing the core gene content of FVs related to LbFV compared to virus representatives of Naldaviricetes. A cladogram phylogeny is reported on the left. Taxonomic affiliation of considered viruses is reported on the right. The rows represent the viral species, and the columns represent the genes grouped according to their potential functions. Colored cells indicate the presence of the gene in viral genomes. Of note, the homolog of Baculoviruses helicase is the helicase 2 in Nudiviruses, Hytrosaviruses, and FVs.

Figure 3.

Phylogenetic tree of large dsDNA viruses from the Naldaviricetes class. Relationships were inferred using ML analysis in IQ-TREE v2 from twenty-five virus species with 17,114 sites with 16,650 distinct patterns at the amino-acid level. Bootstrap values are shown at each node (SH-aLRT support (%)/ultrafast bootstrap (%)). The scale bar indicates the average number of amino acid substitutions per site. Viral families are represented by the following colors: brown = Nudiviridae, yellow = Baculoviridae, purple = Hytrosaviridae, light blue = proposed Filamentoviridae and green = AmFV-like (Supplementary Tables S1 and S2). After each virus name a drawing represents the order of arthropod to which the infected host belongs. Hymenoptera have two lifestyle categories indicated: (1) endoparasitoids or (2) free-living species. DmFV and LhFV are likely the same virus infecting the Drosophila parasitoid L. heterotoma, although DmFV has been initially detected from a pool of wild-caught Drosophila (Section 2.5).

Figure 4.

Gene-order conservation among Naldaviricetes assessed using gene parity plot comparisons. All genes are represented by dots following the order of the genes in the reference genome on the x-axis, and the positions of the homologs in the other genome on the y-axis. (A) Gene parity plot comparison of the circularized FV genomes. CcFV1 genome is set as reference and its gene order is compared to that of CcFV2 in red, LbFV in deep blue and EfFV in green. Orange boxes highlight microsyntenies present in all the FVs presented. (B) GpSGHV-Uga gene organization compared to that of MdSGHV. Genes specific to hytrosaviruses are represented in light blue and genes shared with FVs in orange. (C) Hytrosavirus gene order relative to CcFV1, with MdSGHV in purple and GpSGHV-Uga in yellow. (D) Gene-order comparison between a gammabaculovirus: NeseNPV (set as reference), two alphabaculoviruses: AcMNPV in red and LdMNPV in orange, and a betabaculovirus: CpGV in grey. Virus species abbreviations and number of genes (#) are those given in Supplementary Table S2.

Figure 5.

FV-like core sequences were detected in the genome assemblies of numerous Hymenoptera, Diptera, and Lepidoptera. (A) Table showing presence/absence of putative FV-like core genes in considered insect assemblies. A TBLASTN approach combined with alien index filtering and phylogenetic validation was used (note that a given species may be represented by several assemblies derived from different populations) (see also Supplementary Fig. S6 and S7 and Supplementary Table S8). The colors of the boxes represent the ORFs completeness status: black color represents a most probably complete ORF (that spans at least 70 per cent the size of the best FV BLAST hit), gray color represents a probably incomplete ORF (spanning less than 70 per cent the size of the best FV BLAST hit), red color represents a pseudogene or an ORF with a premature stop codon. More than one color in the same box indicates that the genome assembly of the species contains several copies with different status endogenous/exogenous as summarized in the left column. Several colors in one box indicate the probable presence of both endogenous and exogenous viral elements. The cyan circles along the taxon names indicate parasitoid species. (B) The circular phylogenetic tree shows the evolutionary relationships between the three insect orders: blue = Hymenoptera, light gray = Diptera, dark gray = Lepidoptera. Each colored dash along the leaves of the tree stands for the status of the FV elements (green = endogenous FV, dark blue = exogenous FV). The phylogenetic cladogram was reconstructed based on the taxonomical NCBI level of all genomes surveyed in this analysis using the NCBITaxa ete3 function in Python. The percentage of species with FV-like core sequences is displayed for each insect order in the inset.

Figure 6.

Typical structures observed by TEM in cells from two parasitoid wasps infected by FVs. (A, C, E) Cotesia congregata adult wasp calyx (CcC wasp line) and (B, D, F) Leptopilina boulardi adult wasp oviduct (B, D, F). (A, B) Details obtained using higher magnification of the views in the white frames TEM pictures of a calyx cell producing FVs, the white frames represent magnified sections in panels C and E. (C, D) Magnified TEM images of filamentous particles observed within C. congregata and L. boulardi cells, respectively. (E, F) TEM images of typical structures showing arrays filamentous particles in C. congregata and L. boulardi, respectively. N: nucleus and C: cytoplasm. The arrows designate enveloped particles, arrowheads nucleocapsids (in longitudinal section for C, D and cross-section for E, F), and white circle one of the abundant membrane vesicles observed in the cytoplasm.

Figure 7.

Electron microscope images showing filamentous particles in C. congregata adult wasp. (A) TEM photographs of a calyx cell producing large number of nucleocapsids, the inset at the bottom right represents a magnified section of the cell nucleus. (B) Image of enveloped filamentous particles obtained using high magnification. (C) Images of a calyx cell producing FVs, the inset at the top left represents a magnified section of the cell cytoplasm. (D) Images of wasp calyx lumen, the inset at the top right represents a magnified section of the lumen containing both FV and bracovirus particles. N: nucleus, C: cytoplasm, VS: virogenic stroma and N BV: nucleus containing bracovirus particles. White arrow: enveloped filamentous particle, white arrowheads: mitochondria lined with nucleocapsids, white circle: two bracovirus particles.