Complete Genome of a Novel Endornavirus Assembled from Next-Generation Sequence Data

Abstract

Endornaviruses have large double-stranded RNA (dsRNA) genomes that carry a single open reading frame (ORF). Here we report the complete genome of a novel endornavirus, assembled from next-generation sequence data generated from Vitis vinifera-extracted dsRNA. Two different fungal hosts have been identified for this virus, suggesting that horizontal transmission of the virus is possible.

GENOME ANNOUNCEMENT

The family Endornaviridae is a new virus family (1) that contains viruses infecting plants, fungi, and oomycetes. Endornaviruses have double-stranded RNA (dsRNA) genomes which carry a single open reading frame (ORF) (2). An RNA-dependent RNA polymerase (RdRp) domain is the only conserved domain among all members, while other domains, like methyl-transferase, helicase, and glycosyltransferase, are present in some, but not all members (5). Endornaviruses are not encapsidated, they cause persistent infections, and horizontal transmissions are believed not to occur in plants or fungi (2, 5).

Here we report the complete genome of a novel dsRNA virus, assembled from next-generation sequence data of dsRNA purified from grapevine. We propose that this novel virus is a member of the family Endornaviridae and suggest the name grapevine endophyte endornavirus (GEEV).

Double-stranded RNA was extracted using cellulose affinity chromatography from 10 g of phloem tissue from a diseased Vitis vinifera cv. Shiraz vine (6). A sequencing library was prepared from the dsRNA using a ScriptSeq v2 RNA-Seq Library preparation kit (Epicentre) and sequenced as short reads using an Illumina HiScanSQ system. The short reads were trimmed and filtered for quality, and 6.4 million reads with a length of 57 nucleotides (nt) each were used in a de novo assembly using CLC Genomics Workbench v. 4.9 (CLC bio). The resulting scaffolds were subjected to homology searches against the NCBI nonredundant nucleotide database (blastn and tblastx) and a 12,201-bp contig, aligning to Chalara elegans endornavirus 1 (CeEV1) (GenBank accession no. GQ494150), was identified (4). The similarity, however, was low, suggesting a novel endornavirus. Yeast (Saccharomyces cerevisiae) poly(A) polymerase (Affymetrix) was used, together with two genome-specific primers and an oligo(dT) primer, in a poly(A)-tailing reaction on dsRNA to confirm the 5′ and 3′ ends. The complete genome is 12,154 bp long and contains a single ORF whose product has a predicted molecular mass of 452.7 kDa. Predicted protein domains include a viral helicase domain near the middle of the polyprotein and an RdRp near the C terminus. Phylogenetic analysis was performed between the RdRps of the new endornavirus (GEEV) and 10 other endornaviruses for which nucleotide sequences are available. As expected, GEEV is most closely related to CeEV1, with 47.64% identity in the RdRp on the amino acid level. Read mapping was performed against GEEV using MAQ v. 0.7.1 (3). A total of 146,438 reads, representing 2.28% of the data, were mapped with an average depth of 687 nt and a genome coverage of 99.9%. Diagnostic primers were designed to screen grapevine dsRNA as well as grapevine endophytic fungi with reverse transcription-PCR (RT-PCR). The virus was detected in grapevine in which a fungal host could not be identified. It was also detected in fungal isolates from two different vines, a Stemphylium isolate and an Aureobasidium isolate. The viral hosts are therefore still unclear, but there is strong evidence to suggest that horizontal transmission of the virus is a possibility.

Nucleotide sequence accession number.

The genome sequence of GEEV has been deposited in NCBI GenBank under accession no. JX678977.