A 1,000-Year-Old RNA Virus

Abstract

Only a few RNA viruses have been discovered from archaeological samples, the oldest dating from about 750 years ago. Using ancient maize cobs from Antelope house, Arizona, dating from ca. 1,000 CE, we discovered a novel plant virus with a double-stranded RNA genome. The virus is a member of the family Chrysoviridae that infect plants and fungi in a persistent manner. The extracted double-stranded RNA from 312 maize cobs was converted to cDNA, and sequences were determined using an Illumina HiSeq 2000. Assembled contigs from many samples showed similarity to Anthurium mosaic-associated virus and Persea americana chrysovirus, putative species in the Chrysovirus genus, and nearly complete genomes were found in three ancient maize samples. We named this new virus Zea mays chrysovirus 1. Using specific primers, we were able to recover sequences of a closely related virus from modern maize and obtained the nearly complete sequences of the three genomic RNAs. Comparing the nucleotide sequences of the three genomic RNAs of the modern and ancient viruses showed 98, 96.7, and 97.4% identities, respectively. Hence, in 1,000 years of maize cultivation, this virus has undergone about 3% divergence.IMPORTANCE A virus related to plant chrysoviruses was found in numerous ancient samples of maize, with nearly complete genomes in three samples. The age of the ancient samples (i.e., about 1,000 years old) was confirmed by carbon dating. Chrysoviruses are persistent plant viruses. They infect their hosts from generation to generation by transmission through seeds and can remain in their hosts for very long time periods. When modern corn samples were analyzed, a closely related chrysovirus was found with only about 3% divergence from the ancient sequences. This virus represents the oldest known plant virus.

Keywords: ancient tissue; maize; virus evolution.

FIG 1

(A) Genome organization of ancient ZMCV1. ZMCV1 genomic dsRNA 1 with a single ORF (nucleotides [nt] 165 to 4133) coding for a putative CP, ZMCV1 genomic dsRNA 2 with a single ORF (nt 159 to 3431) coding for a putative RdRp, and ZMCV1 genomic dsRNA 3 with a single ORF (nt 113 to 3313) coding for p98, a protein with unknown function, are depicted. (B) Multiple alignment of putative RdRp encoded by the ancient and modern ZMCV1 RNA 2 with the RdRps of additional chrysoviruses. Numbers I to VIII refer to the eight motifs conserved in the RdRps of dsRNA viruses of lower eukaryotes (41, 42). Accession numbers are provided in Materials and Methods.

FIG 2

Morphological variability of ancient maize cobs collected from Antelope House. Samples 48, 74, and 154 have nearly complete genomes of ZMCV1. Samples 201 and 175 have partial sequences of ZMCV1. The estimated ages based on carbon dating are shown for each sample in the table below.

FIG 3

(A) Agarose gel electrophoresis of dsRNAs extracted from modern maize cultivars. Lanes: 1, Japonica Striped; 2, Calico; 3, Jade Blue Dwarf; M, DNA marker (lambda DNA digested with EcoRI and HindIII). (B) Agarose gel electrophoresis profile of RT-PCR products of modern ZMCV1 genomic dsRNA segments isolated from modern maize leaf tissue. Lanes: 1, CP; 2, RdRp; 3, p98; M, DNA marker (lambda DNA digested with PstI). (C) Northern blot analysis of modern maize and teosinte total RNA probed for ZMCV1 CP, RdRp, and p98.

FIG 4

Phylogenetic analysis based on amino acid sequences of RdRps of ancient and modern ZMCV1 and selected members of Chrysoviridae. Bayesian analysis was done using the MrBayes plugin in Geneious software. Posterior probabilities are indicated by numbers at the nodes. The viral siglas and accession numbers of sequences used in the analyses are given in Materials and Methods. Species indicated in boldface are isolated from plants. Raphanus sativus chrysovirus 1 was used as an outgroup.