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Preservation of Viral Genomes in 700-y-old Caribou Feces From a Subarctic Ice Patch

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Preservation of Viral Genomes in 700-y-old Caribou Feces From a Subarctic Ice Patch

Terry Fei Fan Ng et al. Proc Natl Acad Sci U S A.

Abstract

Viruses preserved in ancient materials provide snapshots of past viral diversity and a means to trace viral evolution through time. Here, we use a metagenomics approach to identify filterable and nuclease-resistant nucleic acids preserved in 700-y-old caribou feces frozen in a permanent ice patch. We were able to recover and characterize two viruses in replicated experiments performed in two different laboratories: a small circular DNA viral genome (ancient caribou feces associated virus, or aCFV) and a partial RNA viral genome (Ancient Northwest Territories cripavirus, or aNCV). Phylogenetic analysis identifies aCFV as distantly related to the plant-infecting geminiviruses and the fungi-infecting Sclerotinia sclerotiorum hypovirulence-associated DNA virus 1 and aNCV as within the insect-infecting Cripavirus genus. We hypothesize that these viruses originate from plant material ingested by caribou or from flying insects and that their preservation can be attributed to protection within viral capsids maintained at cold temperatures. To investigate the tropism of aCFV, we used the geminiviral reverse genetic system and introduced a multimeric clone into the laboratory model plant Nicotiana benthamiana. Evidence for infectivity came from the detection of viral DNA in newly emerged leaves and the precise excision of the viral genome from the multimeric clones in inoculated leaves. Our findings indicate that viral genomes may in some circumstances be protected from degradation for centuries.

Keywords: aDNA; ancient virus; metagenomics; paleopathology; reverse genetics.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Metagenomic identification and PCR screening of ancient viruses cryogenically preserved in frozen caribou feces and in alpine ice cores. (A) Schematic of KfTe-1-C1-5 ice patch core used in this study, describing the carbon dating of Caribou feces, depth of the samples, and the metagenomic and PCR result for aCFV and aNCV using three different extraction methods. The carbon dating and depth were described in a previous report (10). (B) Comparison of the viruses detected in the ancient caribou fecal virome with the present-day caribou fecal virome.
Fig. 2.
Fig. 2.
Genome organization and phylogenetic analyses of caribou feces-derived viruses. (A) Phylogenetic analysis of Rep proteins in plant-infecting geminiviruses, fungi-infecting SsHADV-1, gemycircularviruses including the extant gemycircularvirus FaGmV-13 in modern caribou feces, and other circular DNA genomes including aCFV. (B) Phylogenetic analysis of the partial RNA-dependent RNA polymerase of aNCV with other invertebrate-infecting cripaviruses. CP, capsid protein; NS, nonstructural protein; Rep, replication-associated protein.
Fig. 3.
Fig. 3.
Infectivity of the reconstituted aCFV in N. benthamiana plants. (A) Plasmid map of a 1.2-mer multimeric aCFV clone in pSL1180 vector for particle bombardment. Restriction site positions are shown relative to the original genome. (B) PCR result using the preinoculation plasmid, in which the outward-facing VB primers direct the amplification of a 4,355-bp fragment containing the vector. The recombinant plasmid used for agroinoculation was constructed similarly and is not shown here. (C) Schematic representation of the replicational release of a 1.0-mer circular genome from a 1.2-mer vector construct, adapted from the well-established geminiviral model (25). Replication is initiated at a nick in the stem loop indicated by the first yellow triangle and ended at the later stem loop at one genome length. This replicational released circular genome was then detected by PCR in Fig. 3 D and E. (D) Schematic representation of the recovered aCFV genomic DNA postinoculation of N. benthamiana plants. (E) PCR result of the particle bombarded and agroinoculated plants using VB and VC primers. The VB primers direct the amplification of a 625-bp fragment. The VB and VC PCR reactions demonstrated the precise excision of aCFV from the plasmid vector during replication and the circular nature of the excised genome. (F) N. benthamiana plants at 21 d postbombardment of multimeric aCFV clones. White arrow shows the location of the young newly emerged leaves taken for PCR testing; the orange arrow shows one of the leaves that was inoculated.

Comment in

  • Freezing viruses in time.
    Holmes EC. Holmes EC. Proc Natl Acad Sci U S A. 2014 Nov 25;111(47):16643-4. doi: 10.1073/pnas.1419827111. Epub 2014 Nov 17. Proc Natl Acad Sci U S A. 2014. PMID: 25404311 Free PMC article. No abstract available.

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