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The Samuel Roberts Noble Foundation, Ardmore, Oklahoma, USA

ABSTRACT

Only a few RNA viruses have been discovered from archaeological

sam-ples, the oldest dating from about 750 years ago. Using ancient maize cobs from

An-telope 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

Chrysoviri-dae

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

chryso-virus, putative species in the

Chrysovirus

genus, and nearly complete genomes were

found in three ancient maize samples. We named this new virus

Zea mays

chrysovi-rus 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%

diver-gence.

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

M

aize (

Zea mays

subsp.

mays

) is farmed on every continent except Antarctica.

Civilization owes much to this plant and to the people who first cultivated it. The

wild progenitor of maize remained a mystery for many decades but is now believed to

be the wild Mexican grass, teosinte (

Z. mays

subsp.

parviglumis

), that has the same

number of chromosomes and a remarkably similar arrangement of genes (1–4). More

than 50 symptomatic acute viruses from different families with positive or negative

sense stranded ssRNA (ssRNA), double-stranded RNA (dsRNA), and

single-stranded DNA genomes, have been identified in maize crops (5), but no persistent

viruses have been reported.

Plant persistent viruses are mostly asymptomatic; they transmit only vertically and

do not move from cell-to-cell but are found in all plant cells, moving by cell division (6).

Most plant persistent viruses have dsRNA genomes and are related to viruses that infect

fungi (7). Based on virus biodiversity studies, persistent viruses are the most common

viruses in wild plants (8). The

Chrysoviridae

family includes dsRNA viruses that

persis-tently infect fungi or plants. Chrysoviruses are composed of three to five monocistronic

CitationPeyambari M, Warner S, Stoler N, Rainer D, Roossinck MJ. 2019. A 1,000-year-old RNA virus. J Virol 93:e01188-18.https://doi.org/ 10.1128/JVI.01188-18.

EditorJulie K. Pfeiffer, University of Texas Southwestern Medical Center

Copyright© 2018 American Society for Microbiology.All Rights Reserved.

Address correspondence to Marilyn J. Roossinck, [email protected].

*Present address: Sylvia Warner, The Noble Research Institute, Ardmore, Oklahoma, USA; Nicholas Stoler, Biochemistry and Molecular Biology, Wartik Laboratory Pennsylvania State University, University Park, Pennsylvania, USA; Drew Rainer, Women's Health Medical Group, Fort Worth, Texas, USA.

Received6 July 2018 Accepted26 September 2018

Accepted manuscript posted online10 October 2018

Published10 December 2018

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dsRNA segments, ranging in size from 2.4 to 3.6 kbp, separately encapsidated by

nonenveloped isometric particles 35 to 40 nm in diameter (9–12). Chrysoviruses encode

an RNA-dependent RNA polymerase (RdRp), a coat protein (CP), and p98, a protein with

unknown function.

Raphanus sativus

chrysovirus 1 (RasCV1) was the first chrysovirus

reported to infect plants (9). Most RNA viruses, with short generation times and

error-prone replication, have rapid rates of evolution (13). However, the evolutionary

rate of persistent plant viruses may differ compared to acute viruses (14). RNA

mole-cules are generally considered unstable and readily degraded, but there have been

reports of RNA viruses in plant tissues that are 100 to 750 years old (15–17). In addition,

dsRNA is much more stable than ssRNA since it is not readily subjected to enzymatic or

chemical degradation.

The Antelope House excavation was conducted by Don P. Morris of the National

Park Service from 1970 to 1974. The Antelope House is an Ancestral Puebloan (Anasazi)

ruin, located in a cave in the bottom of Canyon del Muerto, the major tributary of

Canyon de Chelly, in Apache County, Arizona (18), in the area known today as Four

Corners, where the states of New Mexico, Arizona, Colorado, and Utah meet. The ruins

are named after nearby pictographs of antelope and other animals made by the Navajo

in the 1830s. Ancestral Puebloan artifacts have been divided into periods of occupation.

The Basketmaker III and Pueblo I, II, and III occupations were involved in agricultural

development. During Basketmaker III the Ancestral Puebloans become farmers living in

small villages. The Ancestral Puebloans found the canyons an ideal place to plant crops

like maize, beans, and squash. More than two tons of vegetal refuse, in highly

recognizable form, were recovered at Antelope House. It is clear from the vegetal

remains that maize was a major food (over 39% of the gross vegetal weight was

contributed by maize). The remains of maize recovered at Antelope House consisted of

cobs, ears with kernels, kernels alone, husks, leaves, shanks, stem portions, and tassels

and archeologically dated from

700 to 1,300 CE (Basketmaker III, Pueblo I, Pueblo II,

Early Pueblo III, Middle Pueblo III, and Late Pueblo III) (18).

In this study, by screening archeological and modern maize cobs, we discovered a

persistent plant RNA virus with three dsRNAs of 3.3 to 4.2 kbp related to known

chrysoviruses. Evidence of this virus was found in a total of 39 samples dating to about

1,000 years ago, making this the oldest plant virus described to date.

RESULTS

Ancient virus sequences.

For this study, we obtained 312 maize cobs from

Ante-lope house through the Western Archeological and Conservation Center (National Parks

Service). Cobs were selected based on provenance, and to obtain a variety of ages

(Table S1). To search for viruses, total nucleic acids were extracted, enriched for dsRNA,

and converted to cDNA in a clean lab, followed by multiplexing and sequence analysis

using an Illumina HiSeq 2000. Sequence data were assembled and compared to the

GenBank database. The most common virus-like sequence was related to

Anthurium

mosaic-associated virus, a virus from an ornamental plant in Hawaii. Partial sequences

of this virus were found in a total of 39 samples, and nearly complete genomes were

found in three samples (Table S1). We designated this putative novel virus

Zea mays

chrysovirus 1 (ZMCV1). The ZMCV1 genome has three RNA segments (Fig. 1A).

Se-quence analysis predicted one open reading frame (ORF) in each dsRNA encoding a

putative CP, RdRp, and p98. The N-terminal

650-amino-acid (aa) residues of the CP

shared significant sequence similarities among the ancient ZMCV1 and other related

chrysoviruses. The RdRp, in addition to eight conserved motifs characteristic of RdRps

(Fig. 1B), contains a consensus sequence known as phytoreovirus S7 domain at the

N-terminal region. This domain is widely distributed in the

Chrysoviridae

,

Totiviridae

,

Reoviridae

, and

Endornaviridae

families (9, 19–21). RNA 3 codes p98 with an unknown

function. This protein has the motif PGDGXCXXHX, which was previously described for

the chrysovirus Amasya cherry disease-associated virus (22). This motif, along with

three other motifs, constitute the conserved core of ovarian tumor gene-like

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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.

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family of predicted proteases (23–25). The function of this putative protease is yet to be

determined.

The ages of the three cobs with nearly complete genomes, as well as two cobs with

partial sequences of the virus, were confirmed by radiocarbon (

14

C) dating (Fig. 2). All

conventional ages were calibrated to calendar years using OxCal 4.3 (26) and the

IntCal13 calibration curve (27). These samples ranged in age from

1030 to 1290 CE.

Contigs from ancient samples more than 500 bp in length were aligned and

compared to a consensus sequence to provide a picture of the genetic diversity in each

genomic RNA of ancient ZMCV1. Genetic diversity was calculated by counting

differ-ences between the individual contigs and the consensus sequence and was

signifi-cantly lower in RNA 2 that encodes the RdRp than in RNAs 1 and 3 that encode the CP

and p98, respectively; RNA 1 and RNA 3 showed similar genetic diversities (Table 1). This

analysis does not take reverse transcription-PCR (RT-PCR) or sequencing errors into

account and hence is artificially high, it but provides a general comparison of diversity

among the three RNAs.

Three dsRNA segments in modern maize.

Uninfected plants do not contain

detectable amounts of large dsRNAs, so the presence of dsRNAs is a sign of viral

infection (28). We enriched for dsRNAs in modern maize cultivars and teosinte

acces-sions and found a pattern of dsRNAs that was consistent with chrysoviruses with three

segments ranging in size from 3.3 to 4.2 kb, in addition to other dsRNA segments that

were not characterized (Fig. 3A; Table 2). Resistance to DNase I and RNase A in a high

salt concentration confirmed the dsRNA nature of the nucleic acids.

[image:4.594.56.354.71.308.2]

FIG 2Morphological 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.

TABLE 1Comparative genetic diversity in CP, RdRp, and p98 of ancient ZMCV1

Virus segment Mutation frequency (10–2)a

RdRp 0.7A

CP 1.3B

p98 1.4B

aSubstitutions, insertions, and deletions were counted equally in determining mutation frequencies.

Statistical differences in genetic variations were determined using ANOVA (P⬍0.05). Mutation frequencies with the same superscript capital letter are not statistically different.

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The modern maize and teosinte dsRNAs were recalcitrant to cDNA synthesis using

standard random priming methods (29). Hence, we generated three sets of specific

primers based on the ancient ZMCV1 sequences for the RdRp, CP, and p98. We were

able to amplify the appropriate-sized fragments from dsRNA from modern maize

(cultivar Japonica Striped) (Fig. 3B), but not teosinte. The Japonica Striped maize

cultivar was selected for further investigation. Using primer walking, we obtained the

nearly complete genome sequences from RNAs 1, 2, and 3 from ZMCV1 in modern

maize, but we were never able to amplify anything from teosinte, even using

degen-erate primers. Sequence comparison of the genomes of modern and ancient ZMCV1

showed 96.7, 98, and 97.4% identities in RNAs 1, 2 and 3, respectively, suggesting that

ZMCV1 has changed about 3% in the 1,000 years that it has been evolving through

vertical passage in maize, although we cannot be certain that the ZMCV1 in the

Japonica Strip cultivar is a direct descendant of the virus found in the Ancient Puebloan

maize.

Using published methods for chrysovirus purification from plant tissues (9), we

attempted to isolate virus particles from modern maize leaves. We obtained a pellet

[image:5.594.43.400.70.205.2]

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.

TABLE 2Maize cultivars and teosinte accession numbers tested for ZMCV1-like dsRNAs

Cultivar/speciesa Accession no.b Presence of dsRNAc Positive by RT-PCRd

Maize

Calico Popcorn NA ⫹ ⫺

Japonica Striped NA ⫹ ⫹

Jade Blue Dwarf NA ⫹ NT

Yellow Field Dent NA ⫹ ⫺

Mini Indian NA ⫹ NT

Tom Thumb NA ⫹ NT

Goldan Bantum NA ⫹ NT

Japanese Hulless NA ⫹ NT

Country Gentleman NA ⫹ NT

Blue Jade NA ⫹ NT

Ashworth Sweet NA ⫹ ⫺

Teosinte

Z. mayssubsp.parviglumis PI 384066 ⫹ ⫺

Z. mayssubsp.parviglumis PI 566687 ⫹ ⫺

Z. mayssubsp.mexicana Ames 8,083 ⫹ ⫺

Z. mayssubsp.mexicana PI 566674 ⫹ ⫺

aCultivar names for maize (Zea mayssubsp.mays) and species names for Teosinte.

bMaize seeds were purchased from commercial seed companies, and do not have accession numbers;

Teosinte seeds were obtained from the USDA. NA, not applicable.

cThat is, the presence () or absence (–) of dsRNAs, as shown in Fig. 3A.

dNot all maize cultivars were tested by RT-PCR using ZMCV1-specific primers. NT, not tested.

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after centrifugation through a 10% sucrose cushion, which normally contains only virus

particles. This pellet contained the viral dsRNAs, but we were unable to visualize any

particles by transmission electron microscopy (TEM), most likely due to the very low

concentration of this virus.

Northern blot analysis.

The dsRNAs from modern maize and teosinte were

separated on an agarose gel, transferred to a nylon membrane, and probed with

chemiluminescence-labeled cDNA clones of RdRp-1, CP-1, and p98-3 (Fig. 3C). The

dsRNAs from maize annealed with the appropriate probes but not those from

teosinte, confirming that ZMCV1 is not found in teosinte, so if the dsRNAs represent

a chrysovirus it is a different virus.

DISCUSSION

We have determined the nearly complete genomic sequence and genome

organi-zation of ZMCV1, a tentative member of the

Chrysoviridae

family, obtained from ancient

maize cobs and modern maize leaf tissue. ZMCV1 is the first dsRNA plant virus obtained

from ancient samples dating from ca. 1030 to 1210 CE and is the oldest known plant

virus. By resolving the dsRNA extracted from modern maize into three bands by

agarose gel electrophoresis, followed by molecular cloning, cDNA sequencing, and

Northern hybridization analysis of ZMCV1 dsRNAs, the presence of three distinct dsRNA

segments was confirmed. We analyzed dsRNA sequences using specific primers for

each segment, designed based on ancient ZMCV1 sequences. dsRNA 1 encodes the

putative CP, and dsRNA 2 codes for the putative RdRp, while dsRNA 3 encodes a protein

with unknown function that has some similarities to known proteases. RNA

ligase-mediated RACE (30), poly(A) tailing 3

=

-RACE (3

=

-rapid amplification of cDNA ends), and

poly(G/C) 5

=

-RACE were unsuccessful, and hence the complete sequences of the 5

=

and

3

=

untranslated regions (UTRs) are unknown. This may be due to modified nucleotides

or cross-linking in portions of the dsRNA genome that make it recalcitrant to normal

cDNA synthesis. This is not unique to ZMCV1 but has been found in a variety of dsRNA

viral genomes from both plants and fungi (unpublished data). The partial sequences

included most of the 5

=

and 3

=

UTRs of three dsRNAs, based on related chrysovirus

sequences. Phylogenetic analysis of the putative RdRp indicated that this is a novel

tentative member of

Chrysoviridae

family, most closely related to

Anthurium

mosaic-associated virus and

Persea americana

chrysovirus (Fig. 4). Because of the apparently

low concentration of ZMCV1 in maize tissue, we were unable to examine virus particles

by TEM, but the presence of viral genomic dsRNA in pellets from 10% sucrose cushions,

which normally contain only virus particles, implies that the virus does package its

genome into virions. In screening several accessions of teosinte, we found a profile of

dsRNAs similar to those from maize cultivars, but we were unable to find any evidence

of ZMCV1 in teosinte by using specific primers or Northern blot analysis. Either the

dsRNAs of teosinte cultivars are not ZMCV1 and this virus was introduced to maize after

divergence from teosinte, or the virus has diverged too much in teosinte to be

identified even by Northern blot analysis that can detect more divergent nucleic acids.

In the ancient ZMCV1 sequences, the mutation frequency of the RdRp-encoding

segment is significantly lower than that of the CP (

P

0.005) and p98 (

P

0.03)

encoding segments, but there is no significant difference between CP and p98

(

P

0.82). Thus, the RdRp gene shows different patterns of evolution compared to CP

and p98. This is not surprising, since RdRp is an essential enzyme for all RNA viruses and

is usually highly conserved. These data indicate that the RdRp, CP, and p98 probably

have different evolutionary trajectories and are under different selection pressures.

In RNA viruses, mutation rates are determined by the presence or absence of

proofreading mechanisms, the mode of replication, and host factors (31). The classical

viewpoint that RNA viruses are replicated with low fidelity and evolve rapidly does not

appear to hold true for ZMCV1 or other persistent plant viruses (6, 14). Previous

estimates have been based on presumed ages of divergence of persistent viruses based

on the divergence of their plant hosts. In this study, we provide direct evidence that the

evolution of ZMCV1 in maize is indeed slower than that calculated for other viruses (32),

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although population studies on dsRNA viruses are rare, and there is some evidence that

long-term evolution of RNA viruses is not a precise extrapolation of what has been

found in experimental studies (33, 34).

There are a number of possible scenarios that could explain low variation in

persistent plant viruses, as seen when we compare the ancient and modern ZMCV1. It

is likely that there is no benefit for a persistent plant virus that is perfectly adapted to

its host and transmitted only through vertical passage to offspring of the same host to

change. So, in this constant environment, all mutations are probably deleterious and

removed through purifying selection. Another possibility is a higher fidelity of the

RdRps of dsRNA viruses, although this has not been studied. In addition, dsRNA viruses

do not undergo exponential replication but rather use a stamping machine mode of

replication, which would limit variation (14). Finally, RNA molecules are much less prone

to deamination and depurination than DNA molecules, and all double-stranded

mol-ecules are protected from other posttranscriptional modifications such as hydrolysis

and enzymatic degradation that affect single-stranded molecules (both RNA and DNA)

(35). In a study of Barley stripe mosaic virus (BSMV) discovered in an

750-year-old

archaeological sample of barley seeds, the levels of variation in comparison to modern

BSMV were considerably lower than would be expected from experimental studies on

RNA virus evolution (34). In that study the authors also used C-U changes as a signal of

deamination to confirm the ancient nature of the RNA (34). In the present study, we

could not verify the ancient nature of the RNA by this method since we do not have

significant modern sequence data for comparison, and the dsRNA nature of the

genome makes deamination much less likely than for an ssRNA genome such as BSMV.

However, all of the ancient-sample work was done under strict clean-lab conditions,

FIG 4Phylogenetic analysis based on amino acid sequences of RdRps of ancient and modern ZMCV1 and selected members ofChrysoviridae. 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 sativuschrysovirus 1 was used as an outgroup.

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and no modern corn was worked on in the lab until after the work on ancient materials

was completed, making contamination highly unlikely.

MATERIALS AND METHODS

Plant materials.The maize cultivars and teosinte accessions used in this study are commercial seeds that were provided by the Hudson Valley Seed Company or germplasm provided by the U.S. Department of Agriculture (USDA) (Table 2). Most of the maize varieties are local cultivars. Maize and teosinte plants were grown in an insect-proof growth chamber to protect them from acute plant viruses, which are predominantly transferred by vectors. Seeds were planted at one seed per 10-cm plastic pot and placed in a climate-controlled growth chamber set at 25°C with a photoperiod of 16 h of light and 8 h of darkness. Plant leaves were used for dsRNA extraction.

dsRNA extraction and molecular characterization.Extraction of dsRNA from ancient tissues was performed in a HEPA filter-equipped clean lab, using protective clothing, to prevent contamination of the samples. Approximately 4 g of ancient tissue was chopped using a razor blade into pieces of about 3 mm3and placed into 2 Bio 101 tubes containing 750l of extraction buffer (28) and 750l of Tris-EDTA

(TE)-saturated phenol-chloroform (1:1). Tubes were placed in a Savant 120 cell disruptor and pulverized twice for 45 s at a speed setting of 6.5. The resulting paste was removed and placed into a 50-ml centrifuge tube containing 3.5 ml of extraction buffer and 3.5 ml of TE-saturated phenol-chloroform. The remainder of the dsRNA extraction protocol was as described previously (28). The extracted dsRNAs were used to synthesize cDNA using tagged random priming, as previously described (29), followed by pooling of up to 96 samples for sequence analysis on Illumina HiSeq 2000. Sequence data were assembled using PRICE (36) for 10 cycles, seeding with 25 randomly chosen reads. Assembled contigs were compared to GenBank using BLASTX and tBLASTX.

The dsRNAs were extracted from modern maize cultivars and teosinte accessions by CF11 (Whatman, UK) cellulose chromatography, as described previously for fungal samples (28), from 5 g of fresh leaf tissue. The extracted dsRNAs were tested for the presence of ZMCV1 by RT-PCR using specific primers of CP, RdRp, and p98. Several primers were designed based on the RdRp, CP, and p98 sequences of ancient maize samples and used for RT-PCR, followed by sequence analysis of the PCR products at the Genomic Core Facility of Pennsylvania State University, University Park, PA. Sequence data were assembled, and nucleotide sequence and putative protein sequences of ZMCV1 RNA 1, RNA 2, and RNA 3 were analyzed for ORFs using the ORF finding operation in Geneious version 10.1.3 (37). Sequence similarity searches using each dsRNA segment of ZMCV1 to screen for virus-related sequences in eukaryotic genomes were conducted using the BLAST N program available online from National Center for Biotechnology Infor-mation (NCBI).

The putative amino acid RdRp sequences of ancient and modern ZMCV1 and those of related chrysoviruses were aligned using the MUSCLE default settings in the Geneious program. After manual editing of the alignments, they were used for phylogenetic analyses and tree construction using MrBayes (38) via the Geneious plugin. The RdRp sequence ofRaphanus sativuschrysovirus 1 (RasCV1) was used as the outgroup. The ZMCV1 sequence was the consensus sequence of the three nearly complete RdRp sequences from ancient corn. The rate matrix was set to a Poisson distribution with a gamma rate variation. The total chain length was 100,000, and branch lengths were unconstrained. Amino acid signatures and protein motifs were identified by searching the Conserved Domain Database in the NCBI proteomics tools.

Numerous attempts at 5=and 3=RACE using various methods were unsuccessful (data not shown) due to common problems with dsRNA viruses of plants and fungi.

Primers used in this study.The following primers were used in this study: RdRp-1F, GGCATGGTA CCTGATG; RdRp-1R, GGCTTCAACGGTATCC; RdRp-2F, GGTCCACGATTTGGTACG; RdRp-2R, GTGTACAGGA CGTACG; RdRp-3F, GCCAAAGTTTGAATCCGCC; RdRp-3R, CCTTATTCGAGGCCAAGCCC; RdRp-4F, CGATGC GCAAGTACGGG; RdRp-4R, CCAGTCACGGCTCATAGCC; RdRp-5F, CCAGATGGCTGTGGCAGG; RdRp-5R, CC TGTGCATACGCATTGC; RdRp-6F, GCGCTATGGTGTAAAG; RdRp-6R, GGTTACTTACCTCACG; RdRp-7F, GGG AGGCAAGTGCTACCC; RdRp-7R, CGACAAGTGGTTCTGCTCC; RdRp-8F, GGTACACCATGGTGAATG; RdRp-8R, CCCTGCCTCATAGACCC; RdRp-5=RACE, CTTGAGATGGCCGCAC; RdRp-3=RACE, GGTAGATAGCTGACTG; RdRp-degenerate-F, ACCGTCGTGCAYGARGGNGA; RdRp-degenerate-R, GCGACATACATRTANGCRTG; CP-1F, CCATATCATCCAAGTCATC; CP-1R, GCTAAAGACCTCAGTAAGCC; CP-2F, GATGCTAGAGCGACGGCCCG; 2R, CACCCGAATGGGCACATCAAG; 3F, CCGGGATTTGGTGTGAG; 3R, GTCACGTTCTTCTCGGC; CP-4F, GCATGGGGCTTTGTGTG; CP-4R, CCATCATCCTTCTTACTGGC; CP-5F, CGGGTGGGGATGATTC; CP-5R, GTATGGCCAGAGCTAACC; CP-6F, GGTCAGGACAAGCCTGATG; CP-6R, CTTCTTGTCACTCAGC; CP-7F, GGGC ATGGAACTGTCG; CP-7R, CTCCTGTAGGTTGGTACG; CP-8F, GCGGGTGAACCAAGTC; CP-8R, GGCAGCCTTG GCTATC; CP-9F, GGCATCTGTGTTAAGTCGG; CP-9R, CCAATGAAGGACTCAGC; CP-10F, GCACGACTGCTGGA ATCAC; CP-10R, GCACGTACGCCTTATCAAGG; CP-11F, GGACGCACAGGACATAAG; CP-11R, GGTGGTGTAC GTGCTGC; CP-12F, GCGTCGAGTAGAAGTAG; CP-12R, CTCAGCGTGCGCCTGCTC; CP-13F, GCCCATGAAGAC AAAC; CP-13R, GCTTTCCTAGCCTTATGCC; CP-14F, CCCAAAGGATGGCAGAAGC; CP-14R, CGCACACCTCAT CACAACG; CP-15F, GGCTGCGATGTATGAT; CP-15R, CATATGCCCATAGCGGC; CP-16F, GGATGACTGGTCTA GAAG; CP-16R, CTACACAATATACAAGC; CP-5=RACE, CCTGGAGACTTGGTTCACC; CP-3=RACE, CCAACCTA CAGGAGGAG; CP-degenerate-F, GAGGCTGATAAGATHGCNAARGC; CP-degenerate-R, GACGTGGTTNACRT ACCARTAYT; p98-1F, GGCAAGTGCCAGGAATCATATGG; p98-1R, CAGTTCAGCATTTCTTGACC; p98-2F, GGT CAAGAAATGCTGAACTG; p98-2R, GTTATTCATCACCTTGGGCACC; p98-3F, GCCTCAGAGGCGTGACAAG; p98-3R CGTACCTTCATTGTCTACCTCC; p98-4F, GGCATTGAGAGTTTGCCC; p98-4R, CTTGCCATAGTCCAC ACC; p98-5F, GGTCTACGACCTAAGG; p98-5R, CCTCGTAACTCCGAAGG; p98-6F, CGGGCCCATGTGTAAG; p98-6R, CGATAAGTACCGTTGGC; p98-7F, CGGATGCTCCTGTTGTG; p98-7R, GTGCCAAACCAATGGAGG;

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according to the method recommended by the manufacturer (Roche Diagnostics). The ZMCV1 CP-1, RdRp-1, and p98-3 primers were used to amplify templates for probes from cDNA clones that were representative of ZMCV1 RNAs 1, 2, and 3, respectively. The labeling was done during fragment amplifications using DIG-11-dUTP and a deoxynucleoside triphosphate mix (DIG-11-dUTP: dTTP, 1:3; with equimolar amounts of dATP, dCTP, and dGTP) in an Idaho Technologies Rapid Cycler. These PCR products (540, 670, and 570 bp, respectively) were purified by a Cycle Pure kit (Omega) and used as probes to detect ZMCV1 (RNAs 1, 2, and 3) in dsRNA from modern maize cultivars and teosinte accessions.

The dsRNAs were isolated from leaf tissues, separated by 1.2% agarose gel electrophoresis in Tris-borate-EDTA, and denatured by soaking the gel in 50 mM NaOH for 30 min. The gel was soaked in 50 mM sodium borate with three changes, for 5 min each time, and the denatured RNA was transferred to Hybond N⫹nylon membrane (Amersham/GE Healthcare) via capillary action in 20⫻SSC (1⫻SSC is 0.15 M NaCl plus 0.015 M sodium citrate) buffer overnight. The RNA was fixed on the membrane by UV cross-linking in a Stratalinker at 200 J. Prehybridization, hybridization, and stringency washes were performed as previously described (39), except that prehybridization and hybridization were carried out at 52°C. For chemiluminescence detection, the blot was incubated in antibody solution, anti-DIG AP conjugate (Roche), and CDP-STAR (Roche) according to the manufacturer’s instructions.

Carbon dating.Five maize cob samples positive for ZMCV1 (i.e., 48, 74, 154, 175, and 201) were processed for radiocarbon measurement at the Human Paleoecology and Isotope Geochemistry Lab at Pennsylvania State University. Cobs were sonicated in 18.2M⍀water to removed adhering sediment and then subjected to acid/base/acid pretreatment of sequential baths in 1 N HCl and 0.1 N NaOH at 70°C for 20 min on a heater block. The initial acid wash dissolved using exogenous carbonate, and repeated base washes extracted organic contaminants such as humic acids. The final acid wash removed any secondary carbonates formed during the base treatment, and then the samples were rinsed in 18.2 M⍀water at 70°C to remove chlorides. Sample CO2was produced by combustion at 900°C for 3 h in evacuated sealed

quartz tubes using a CuO oxygen source and Ag wire to remove chlorides. Primary (OXII) and secondary (FIRI-H) standards, as well as a⬎50k BP wood background, were processed along with the unknowns. Sample CO2was reduced to graphite at 550°C by hydrogen reduction onto a Fe catalyst with reaction

water drawn off with Mg(ClO4)2.

Radiocarbon measurements were made on a National Electrostatics Corporation 500-kV 1.5SDH-1 compact accelerator mass spectrometer at the PSU AMS14C Laboratory. Ratios were corrected with

background subtractions and normalized to the OXII standard. Conventional 14C ages were ␦13C

corrected for mass-dependent fractionation with ␦13C values measured on the AMS (40). Because

fractionation during sample graphitization or AMS measurement can cause these values to differ from the␦13C of the original material, these values are not reported.

Accession number(s). Viruses used in the phylogenetic analysis were as follows: Grapevine-associated chrysovirus (GaCV1), ADO60926.1; Isaria javanica chrysovirus 1 (IjCV1), YP_009337840.1;

Aspergillus fumigatus chrysovirus (AfuCV), CAX48749.1; Penicillium chrysogenum chrysovirus (PcV), YP_392482.1; Amasya cherry disease-associated chrysovirus (ACDACV),CAH03664.1;Anthurium mosaic-associated virus (AMaV),ACU11563.1;Brassica campestrischrysovirus 1 (BcCV1),AKU48197.1; Colletotri-chum gloeosporioideschrysovirus 1 (CgCV1),ALW95408.1;Cryphonectria nitschkeichrysovirus 1 (CnCV1), ACT79255.1;Fusarium oxysporumchrysovirus 1 (FoCV1),ABQ53134.1;Helminthosporium victoria145 s virus (Hv145SV), YP_052858.1; Macrophomina phaseolina chrysovirus 1 (MpCV1),ALD89090.1;Persea americanachrysovirus (PaCV),AJA37498.1;Raphanus sativuschrysovirus 1 (RasCV1),AFE83590.1; and

Verticillium dahlia chrysovirus 1 (VdCV1),ADG21213.1. For sequences of ZMCV1, which were newly determined in the present study, JS is from modern corn, and the remainder are numbered according to sample number: ZMCV1-JS-CP,MH931186; ZMCV1-JS-RdRp,MH931187; ZMCV1-JS-p98,MH931188; ZMCV1-154-CP,MH931189; ZMCV1-154-RdRp,MH931190; ZMCV1-154-p98,MH931191; ZMCV1-48-CP, MH931192; ZMCV1-48-RdRp,MH931193; ZMCV1-48-p98,MH931194; ZMCV1-74-CP,MH931195; ZMCV1-74-RdRp, MH931196; ZMCV1-74-p98, MH931197; ZMCV1-201-CP, MH936007; ZMCV1-201-RdRp, MH931198; ZMCV1-201-p98 MH931199; ZMCV1-141-CP, MH931200; ZMCV1-141-RdRp, MH931201; ZMCV1-141-p98, MH936006; ZMCV1-63-CP, MH931203; ZMCV1-63-RdRp, MH931202; ZMCV1-80-CP, MH931204; ZMCV1-80-RdRp, MH936014; ZMCV1-241-CP, MH931205; ZMCV1-241-RdRp, MH936017; ZMCV1-305-RdRp,MH931206; ZMCV1-248-RdRp,MH931207; ZMCV1-306-RdRp,MH931208; ZMCV1-304-RdRp,MH936015; and ZMCV1-147-RdRp,MH936016.

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SUPPLEMENTAL MATERIAL

Supplemental material for this article may be found at

https://doi.org/10.1128/JVI

.01188-18

.

SUPPLEMENTAL FILE 1, PDF file, 0.9 MB.

ACKNOWLEDGMENTS

We thank Douglas J. Kennett and Brendan J. Culleton for assistance in carbon dating,

which was performed at the Human Paleoecology and Isotope Geochemistry Lab at

Pennsylvania State University. We thank the Western Archeological Center for allowing

us to perform destructive analysis on the maize samples. We thank Michael Clegg for

supporting the early stages of this work.

This study was supported by the Samuel Roberts Noble Foundation, The

Pennsyl-vania State University College of Agricultural Science, and the Huck Institutes of Life

Sciences.

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Figure

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, ZMCV1genomic dsRNA 2 with a single ORF (nt 159 to 3431) coding for a putative RdRp, and ZMCV1 genomic dsRNA 3 w
TABLE 1 Comparative genetic diversity in CP, RdRp, and p98 of ancient ZMCV1
FIG 3 (A) Agarose gel electrophoresis of dsRNAs extracted from modern maize cultivars
FIG 4 Phylogenetic analysis based on amino acid sequences of RdRps of ancient and modern ZMCV1 andselected members of Chrysoviridae

References

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