Extensive Diversity of Viruses in Millipedes Collected in the Dong Nai Biosphere Reserve (Vietnam)

Abstract

Advances in sequencing technologies and bioinformatics have led to breakthroughs in the study of virus biodiversity. Millipedes (Diplopoda, Myriapoda, Arthropoda) include more than 12,000 extant species, yet data on virus diversity in Diplopoda are scarce. This study aimed to explore the virome of the millipedes collected in the Dong Nai Biosphere Reserve in Vietnam. We studied 14 species of millipedes and managed to assemble and annotate the complete coding genomes of 16 novel viruses, the partial coding genomes of 10 more viruses, and several fragmented viral sequences, which may indicate the presence of about 54 more viruses in the studied samples. Among the complete and partial genomes, 27% were putative members of the order Picornavirales. Most of the discovered viruses were very distant from the viruses currently present in the relevant databases. At least eight viruses meet the criteria to be recognized as a new species by the International Committee on Taxonomy of Viruses, and, for two of them, a higher taxonomic status (genus and even family) can be suggested.

Keywords: Alternaviridae; Cat Tien National Park; Mitoviridae; Nairoviridae; Picornavirales; Qinviridae; Secoviridae; Xinmoviridae; tropical forest; zhaoviruses.

Figure 1

Total number of viruses (top) and abundance of virus-containing reads (bottom) in each studied pool. Distinct virus groups are marked with colors. The section is marked by crosses if only fragments of the virus genome were obtained.

Figure 2

Genomic structure and phylogenetic relationships of discovered picorna-like viruses. (A) Unrooted phylogenetic tree of the classical members of Picornavirales, discovered picorna-like viruses and related viruses. The tree was constructed using the amino acid sequences of the RdRp-encoding polyprotein, with 1000 bootstrap replicates. Nodes with ≥85% bootstrap support are indicated. The scale bar represents the number of amino acid substitutions per site. Discovered viruses are shown in red. Phylogenetic groups that have ICTV-recognized members are color coded. ICTV-unrecognized groups are shown in black. (B) Subtree of the phylogenetic tree pictured in (A), depicting members of Secoviridae and related viruses. (D) Subtree of the phylogenetic tree pictured in (A), depicting members of Dicistroviridae and related viruses. (F) Subtree of the phylogenetic tree in (A), depicting members of Nora-, Solinvi-, Caliciviridae and related viruses. (C,E,G) Genome scheme of the discovered picorna-like viruses. ORFs are shown in purple. RdRp-encoding ORFs are shown in green. An asterisk (*) above ORF signifies a putative read-through of the UGA codon. Gray blocks indicate estimated gaps. Gray arrowheads indicate the absence of the stop codon within the sequenced region.

Figure 3

Genomic structure and phylogenetic relationships of the newly discovered toli- and noda-like viruses. (A) Unrooted phylogenetic tree of the classical members of Tolivirales, Nodamuvirales, discovered viruses, and related viruses. The tree was constructed using the amino acid sequences of the RdRp-encoding polyprotein, with 1000 bootstrap replicates. Nodes with ≥75% bootstrap support are indicated. The scale bar represents the number of amino acid substitutions per site. Discovered viruses are shown in red. Phylogenetic groups that have ICTV-recognized members are color coded. ICTV-unrecognized groups are shown in black. (B) Genome scheme of the discovered viruses. ORFs are shown in purple. RdRp-encoding ORFs are shown in green. Gray line indicates estimated gaps. (C) Subtree of phylogenetic tree pictured in Figure 2A, depicting members of Nodaviridae and related viruses. (D) Subtree of phylogenetic tree pictured in Figure 2A, depicting members of Carmotetraviridae and related viruses.

Figure 4

Genome and phylogenetic relationships of the Cat Tien Thyropygus nairo-like virus. (A) Phylogenetic tree of Nairoviridae, rooted on the families Mypoviridae and Wupedeviridae, used as outgroups. The tree was constructed using the amino acid sequences of the RdRp (1000 bootstrap replicates; nodes with ≥75% bootstrap support are indicated). The scale bar represents the number of amino acid substitutions per site. The discovered virus is shown in red. (B) Genome scheme of the Cat Tien Thyropygus nairo-like virus. ORFs are shown in purple. The RdRp-encoding ORF is shown in green.

Figure 5

Genomic structure and phylogenetic relationships of the Cat Tien Plusioglyphiulus xinmo-like virus. (A) Midpoint-rooted phylogenetic tree of the Cat Tien Plusioglyphiulus xinmo-like virus. The tree was constructed using the amino acid sequences of the RdRp (1000 bootstrap replicates; nodes with ≥80% bootstrap support are marked). The scale bar represents the number of amino acid substitutions per site. The discovered virus is shown in red. Clades representing Xinmoviridae and Lispiviridae families are highlighted. (B) Genome scheme of the Cat Tien Plusioglyphiulus xinmo-like virus. ORFs are shown in purple. RdRp-encoding ORF is marked in green.

Figure 6

Genomic structure and phylogenetic relationships of the Cat Tien Hyleoglomeris arti-like virus. (A) Phylogenetic tree of the family Artiviridae, rooted on the families Giardiviridae and Inseviridae, used as outgroups. The tree was constructed using the amino acid sequences of the RdRp (1000 bootstrap replicates; nodes with ≥85% bootstrap support are marked). The scale bar represents the number of amino acid substitutions per site. The discovered virus is marked in red. (B) Genome scheme of the Cat Tien Hyleoglomeris arti-like virus. ORFs are shown in purple. The RdRp-encoding ORF is marked in green.

Figure 7

Genomic structure and phylogenetic relationships of the Cat Tien alterna-like virus. (A) Phylogenetic tree of the family Alternaviridae. The tree was constructed using the amino acid sequences of the RdRp (1000 bootstrap replicates; nodes with ≥85% bootstrap support are indicated), and rooted on the family Artiviridae, used as an outgroup. The scale bar represents the number of amino acid substitutions per site. The discovered virus is marked in red. (B) Genome scheme of the Cat Tien alterna-like virus. ORFs are shown in purple. The RdRp-encoding ORF is marked in green.

Figure 8

Genomic structure and phylogenetic relationships of discovered unuamitoviruses (A) Phylogenetic tree of the genus Unuamitovirus, rooted on the genus Duamitovirus, used as an outgroup. The tree was constructed using the amino acid sequences of the RdRp (1000 bootstrap replicates; nodes with ≥85% bootstrap support are indicated). The scale bar represents the number of amino acid substitutions per site. Discovered viruses are marked in red. (B) Scheme of the discovered unuamitoviruses genomes. The RdRp-encoding ORF is marked in green. Each * above an ORF signifies a UGA codon that encodes Trp. Gray blocks indicate estimated gaps. Gray arrowheads indicate the absence of the stop codon within the sequenced region.

Figure 9

Genomic structure and phylogenetic relationships of the Cat Tien Plusioglyphiulus chu-like virus (A) Midpoint-rooted phylogenetic tree of the family Chuviridae. The tree was constructed using the amino acid sequences of the RdRp (1000 bootstrap replicates; nodes with ≥85% bootstrap support are indicated). The scale bar represents the number of amino acid substitutions per site. The Cat Tien Plusioglyphiulus chu-like virus is marked in red. (B) Scheme of the Cat Tien Plusioglyphiulus chu-like virus genome. ORFs are shown in purple. The RdRp-encoding ORF is marked in green. The gray block indicates the estimated gap.

Figure 10

Genomic structure and phylogenetic relationship of zhao-like viruses. (A) Phylogenetic tree of zhaoviruses, rooted in the Cilio–Brinovirus clade. The tree was constructed using the amino acid sequences of the RdRp (1000 bootstrap replicates; nodes with ≥85% bootstrap support are marked). The scale bar represents the number of amino acid substitutions per site. The discovered virus is marked in red. Viruses using the ciliate translation table are italicized. (B) Scheme of the Cat Tien Thyropygus zhao-like virus genome. ORFs are shown in purple. The RdRp-encoding ORF is marked in green.

Figure 11

Genomic structure and phylogenetic relationships of the Cat Tien Plusioglyphiulus quin-like virus. (A) Phylogenetic tree of the family Qinviridae, rooted on members of the families Yueviridae, Myriaviridae, and Aspiviridae, used as outgroups. The tree was constructed using the amino acid sequences of the RdRp (1000 bootstrap replicates; nodes with ≥85% bootstrap support are indicated). The scale bar represents the number of amino acid substitutions per site. The discovered virus is marked in red. (B) Scheme of the Cat Tien Plusioglyphiulus quin-like virus genome. ORFs are shown in purple. The RdRp-encoding ORF is marked in green.

Figure 12

Schemes of the virus-like elements discovered in the Hyleoglomeris cattienensis. ORFs are shown in purple. RdRp-encoding ORFs are marked in green.