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The majority of giant algal viruses belong to the family Phycodnaviridae, class Algavirales, phylum Nucleocytoviricota. Among them, the genus Chlorovirus is the most studied, with three recognized groups based on genomics and host range, although many fundamental questions remain to be elucidated, particularly regarding their diversity. In this study, we focus on betachloroviruses, a poorly explored subgroup that infects the alga Micractinium conductrix Pbi. Here, we describe the isolation and genomic analysis of 11 new betachloroviruses from water samples collected in Nebraska, USA. With 25 fully sequenced genomes now available, we assessed the genomic diversity of these viruses. They have double-stranded DNA genomes ranging from 295 to 374 kbp, encoding hundreds of ORFs, of which a large number (~40%) lack known function. Comparative genomics and phylogenetic analyses revealed three species of betachlorovirus, each with high intra-species genomic identity. Notably, some isolates with over 99.5% genomic identity display markedly different plaque phenotypes, which led us to propose the use of the term genomovar among giant algal viruses, a concept potentially applicable to other giant viral groups yet to be explored. Altogether, this work advances our understanding of betachloroviruses and highlights the importance of linking viral genotype to phenotype, opening new avenues for exploring the diversity of giant algal viruses.
402038/2023-1 National Council for Scientific and Technological Development, APQ-01057-23 Fundação de Amparo à Pesquisa do Estado de Minas Gerais, Finance Code 001 Coordenação de Aperfeicoamento de Pessoal de Nível Superior, 1736030 U.S. National Science Foundation
Figure 1. Phylogenetics and genomic characteristics of betachloroviruses. Phylogenetic reconstruction of betachloroviruses. The phylogeny was made based on six concatenated genes (A32, PolB, SFII, TFIIB, Topo, VLTF3) considered the best hallmark genes for the phylum Nucleocytoviricota. New isolates are represented in pink labels, while already known isolates are in black. ATCV-1, a gammachlorovirus, was used as an outgroup. Only bootstrap values above 80 are represented. The tree scale refers to the substitution rate of amino acids. Colored bars indicate the different genomic elements found in the virus isolates.
Figure 2. Genomic content of betachloroviruses. (A) Genes commonly found in betachloroviruses, separated based on functional categories. Only five of the 24 known minor capsid proteins of chloroviruses (P1 to P5) are represented in the graph, although homologs of other genes have been found in different isolates. Three different glycosyltransferase homologs were included to highlight possible distinct glycosylation machinery/pattern expressed by each isolate; (B) tRNA diversity in betachloroviruses. The number of anticodons is indicated in the respective square of each isolate. The scale bar indicates the range of tRNA counts in the isolate.
Figure 3. Genome synteny of betachloroviruses. New and old isolates are included in the analyses, indicating conserved collinearity among the genomes, except the NE-JV-1 and NE-P-6-s. The isolates of each cluster were chosen randomly for this analysis. Inversions in some genomic regions are observed. The color grade represents BLASTn identity percentage.
Figure 4. Betachloroviruses are clustered into three species. Hierarchical cluster analysis reveals three distinctive groups in the ‘Betachlorovirus’ subgenus. The grouping reflects a 94% cutoff value for species designation. We refer to these groups as species I, II and III. The scale bar indicates the range of ANI values.
Figure 5. Pan-genome and COG-sharing pattern in betachloroviruses. (A) Pan-genome evolution of betachloroviruses. Pan-genome and core-genome are represented by circles and triangles, respectively; (B) Upset plot of COG-sharing considering the three betachloroviruses species. The number of COGs found per species is indicated in the horizontal bars. Vertical bars indicate the number of COGs shared between the species; (C) Bipartite network graph representing the COG-sharing pattern of betachloroviruses. Colored nodes represent the isolates, while nodes in the grayscale represent the COGs. White nodes are the core genome, gray nodes represent the satellite genome, and dark gray nodes stand for singletons. In all images, each species is represented by a different color: species I, purple; species II, yellow; species III, blue.
Figure 6. Betachloroviruses genomovars exhibit different plaque phenotypes. (A) ANI matrix heatmap of betachloroviruses sharing ANI >99%, grouped by species and genomovars; (B) Schematic representation of different plaque phenotypes of betachloroviruses. Plaques can be classified by size as small (<2 mm), medium (2–4 mm), and large (>4 mm), with regular (continuous line) or irregular (discontinuous line) edges, and the clarity of plaque morphology, being regular (white plaque) or cloudy (green plaque). Plaques also exhibit varying levels in the clarity of the plaque field, being clear (white plaque) or cloudy (green plaque), as well as differences in the definition at the plaque boundary, having a sharp plaque boundary (white inside border) or a soft plaque boundary (green inside boarder); (C) Plaque phenotypes of different isolates of betachloroviruses. All photos were taken after 12 days of infection. Black arrows indicate soft plaque boundaries in isolate P-NE-11.
