Molecular Characterization of Hovenia Dulcis-Associated Virus 1 (HDaV1) and 2 (HDaV2): New Tentative Species within the Order Picornavirales

Abstract

In a systematic field survey for plant-infecting viruses, leaf tissues were collected from trees showing virus-like symptoms in Brazil. After viral enrichment, total RNA was extracted and sequenced using the MiSeq platform (Illumina). Two nearly full-length picorna-like genomes of 9534 and 8158 nucleotides were found associated with Hovenia dulcis (Rhamnaceae family). Based upon their genomic information, specific primers were synthetized and used in RT-PCR assays to identify plants hosting the viral sequences. The larger contig was tentatively named as Hovenia dulcis-associated virus 1 (HDaV1), and it exhibited low nucleotide and amino acid identities with Picornavirales species. The smaller contig was related to insect-associated members of the Dicistroviridae family but exhibited a distinct genome organization with three non-overlapping open reading frames (ORFs), and it was tentatively named as Hovenia dulcis-associated virus 2 (HDaV2). Phylogenetic analysis using the amino acid sequence of RNA-dependent RNA polymerase (RdRp) revealed that HDaV1 and HDaV2 clustered in distinct groups, and both viruses were tentatively assigned as new members of the order Picornavirales. HDaV2 was assigned as a novel species in the Dicistroviridae family. The 5' ends of both viruses are incomplete. In addition, a nucleotide composition analysis (NCA) revealed that HDaV1 and HDaV2 have similarities with invertebrate-infecting viruses, suggesting that the primary host(s) of these novel virus species remains to be discovered.

Keywords: HDaV1; HDaV2; Hovenia dulcis; Picornavirales; metagenomics; virome.

Figure 1

Hovenia dulcis symptoms and viral genomic organization. (a) Virus-like symptoms observed in leaves of Hovenia dulcis: interveinal chlorosis. (b) Schematic representation of Hovenia dulcis-associated virus 1 (HDaV1) and Hovenia dulcis-associated virus 2 (HDaV2) genomic organization and sequencing coverage. All open reading frames (ORFs) are represented as arrows pointing from the 5′ to the 3′ end and are colored in grey. Nucleotide positions indicate the start and end of ORFs. ORF 1 and 1b encodes non-structural polyproteins, including putative functional domains (RNA Helicase; 3C peptidase protease; and RdRp: RNA-dependent RNA polymerase). ORF 2 encodes a structural polyprotein with capsid protein domains (Rhv: picornavirus (Rhinovirus) capsid protein-like, CrPV capsid: cricket paralysis virus capsid protein-like). Regions with low coverage are highlighted in red, and the numbered regions were confirmed by RT-PCR and Sanger sequencing.

Figure 2

Phylogeny and genomic organization of representative members of the order Picornavirales. Phylogenetic analysis based on the amino acid sequence of RdRp (RNA-dependent RNA polymerase) of members from six families within the order Picornavirales. Sequences were aligned with ClustalW [30] and the maximum likelihood tree inferred with FastTree [31]. The black circles represent nodes with aLRT ≥0.9. Hovenia dulcis-associated virus 1 (HDaV1) and Hovenia dulcis-associated virus 2 (HDaV2) are highlighted in red. The genome organization was plotted with the Evolview v3 program [34].

Figure 3

Linear discriminant analysis (LDA) used to classify viral sequences into host groups. Linear discriminant analysis comparing nucleotide composition from members of order Picornavirales with known hosts. Invertebrates-infecting viruses are shown in open red circles, plant-infecting viruses are in open green circles, and vertebrate-infecting viruses in open purple circles; Hovenia dulcis-associated virus 1 (HDaV1) is indicated by an open blue triangle, and Hovenia dulcis-associated virus 2 (HDaV2) is indicated by a solid blue triangle. Other picornaviruses (algae, protozoa, and environmental samples) are shown in open black circles.