We describe endogenous viral elements (EVEs) derived from parvoviruses (family

&lt;i&gt;Parvoviridae&lt;/i&gt;) in the long-tailed chinchilla (&lt;i&gt;Chinchilla lanigera&lt;/i&gt;) and degu (&lt;i&gt;Octodon degus&lt;/i&gt;)

genomes. The novel EVEs include Dependovirus-related elements, and representatives of a

clearly distinct parvovirus lineage that also has endogenous representatives in marsupial

 genomes. In the degu, one dependovirus-derived EVEs was found to encode an intact reading

frame, and was differentially expressed &lt;i&gt;in vivo&lt;/i&gt;, with increased expression in the liver

Abascal

Antoine

Ardiles

Arnaud

Belyi

Bezier

Dewannieux

G. Arriagada

Gould

Guindon

Kapoor

Katzourakis

Kumar

Naccache

Okanoya

Opazo

Phan

R. J. Gifford

Rowe

Smuts

English

PubMed

Indexación Scopus.We describe endogenous viral elements (EVEs) derived from parvoviruses (family Parvoviridae) in the genomes of the long tailed chinchilla (Chinchilla lanigera) and the degu (Octodon degus). The novel EVEs include dependovirus-related elements
and representatives of a clearly distinct parvovirus lineage that also has endogenous representatives in marsupial genomes. In
the degu, one dependovirus-derived EVE was found to carry an intact reading frame and was differentially expressed in vivo,
with increased expression in the liverhttps://journals.asm.org/doi/10.1128/jvi.01173-1

Arriagada, Gloria

Gifford, Robert J.

Repositorio Institucional Académico (RIA) de la Universidad Andrés Bello

Journal of Virology

Parvovirus-derived endogenous viral elements in two South American rodent genomes

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        Arriagada, G., and Gifford, R. J. (2014) Parvovirus-derived endogenous viral elements in two South American rodent genomes. Journal of Virology, 89(20), pp. 12158-12162.  Copyright © 2014  John Wiley & Sons Ltd.  A copy can be downloaded for personal non-commercial research or study, without prior permission or charge   The content must not be changed in any way or reproduced in any format or medium without the formal permission of the copyright holder(s)   When referring to this work, full bibliographic details must be given    http://eprints.gla.ac.uk/95418/       Deposited on:  11 May 2015          Enlighten – Research publications by members of the University of Glasgow http://eprints.gla.ac.uk  1Parvovirus-derived endogenous viral elements in two South American rodent genomes. 1  2 Gloria Arriagada1* and Robert J. Gifford2* 3  4 1Universidad Andres Bello, Departamento de Ciencias Biologicas, Facultad de Ciencias 5 Biologicas, Quillota 980, Viña del Mar, Chile 6  7 2MRC-University of Glasgow Centre for Virus Research, Glasgow, United Kingdom 8  9 *Corresponding authors 10  11 Abstract 12 We describe endogenous viral elements (EVEs) derived from parvoviruses (family 13 Parvoviridae) in the long-tailed chinchilla (Chinchilla lanigera) and degu (Octodon degus) 14 genomes. The novel EVEs include Dependovirus-related elements, and representatives of a 15 clearly distinct parvovirus lineage that also has endogenous representatives in marsupial 16 genomes. In the degu, one dependovirus-derived EVEs was found to encode an intact reading 17 frame, and was differentially expressed in vivo, with increased expression in the liver. 18  19 Main text 20 Parvoviruses are small, non-enveloped viruses containing a single-stranded DNA 21 (ssDNA) genome ~5 kilobases (kb) in length. In recent years, several reports have described 22 endogenous viral elements (EVEs) derived from parvoviruses in animal genomes (1-4). We 23 performed an in silico screen of recently published low-coverage genome sequence 24 assemblies using the database-integrated genome screening (DIGS) tool (5). Screening 25 identified novel parvovirus-related EVEs in two caviomorph rodents - the long-tailed 26 chinchilla (Chinchilla lanigera), and the degu (Octodon degus). A total of twelve novel EVEs 27 were identified in these two species (Table 1). 28  2Chinchillas are medium-sized, crepuscular rodents that live at high altitude in the 29 Andes mountains. Degus are small rodents endemic to the Chilean Matorral ecoregion. 30 Although both species are indigenous to South America, they are relatively distantly related, 31 having diverged ~37 million years ago (MYA) (6).  32 Parvovirus genomes generally comprise two major gene cassettes that separately 33 encode nonstructural (NS) and structural (VP) proteins. Genetic structures of previously 34 described parvovirus-EVEs have included complete viral genomes, intact individual genes, 35 isolated genome fragments, and rearranged complete genomes (2, 3). A similar range of 36 genetic structures were observed here, with five of the novel EVEs representing complete or 37 nearly complete viral genomes spanning both major gene cassettes, while the remainder 38 represented single gene cassettes, or fragments of genes (Figure 1). 39 Phylogenetic analysis of the newly identified EVEs revealed that half of them 40 grouped robustly within the diversity of avian and mammalian Dependoviruses, whereas the 41 others clustered in a single, well-defined clade comprised exclusively of EVEs obtained from 42 the genomes of South American and Australian mammals (Figure 2). In phylogenies, this 43 lineage of EVEs formed a sister clade with exogenous viruses in the Parvovirus and 44 ‘Bufavirus’ (7) genera.  45 None of the novel EVEs were orthologous in the species examined, thus we could not 46 infer minimum dates of integration based on orthology. However, we did identify a pair of 47 endogenous Dependovirus elements (C.lanigera 5a and 5b (see Table 1)) that had apparently 48 been duplicated after integrating into the chinchilla genome. This pair of nearly identical 49 elements shared at least two nonsense mutations (Figure S1), indicating that the coding 50 sequence had degenerated prior to its duplication, and can thus be assumed to have been 51 evolving neutrally in the subsequent period. These elements are at least as old as the 52 duplication event that generated them, which we estimated to have arisen in a duplication 53 event that occurred between 2.8 and 5.6 MYA (Figure S1). 54 Although most of the novel EVEs contained frameshifts and/or stop codons, 55 insertions encoding apparently intact NS1 proteins were identified in both the chinchilla and 56  3degu genomes. To exclude the possibility that it was somehow derived from contaminating 57 viral DNA (8, 9), one intact element (O.degus-4) was independently amplified from genomic 58 DNA by polymerase chain reaction (PCR). Tissue was obtained from a fresh male headless 59 O.degus cadaver (kindly donated by Dr. Adrian Palacios from Universidad de Valparaiso, 60 Chile). Genomic DNA was extracted from liver tissue, and PCR using primers targeting the 61 5’ and 3’ flanking regions of O.degus-4 (Figure 3a), confirmed the presence of this EVE 62 locus in a second, outbred degu individual, demonstrating that O.degus-4 is a genuinely 63 endogenous sequence. 64 RT-PCR was used to investigate expression of O.degus-4 in distinct degu tissues. 65 RNA was extracted from pancreas, liver, testicle, kidney, suprarenal, spleen and lung tissues 66 (Figure 3b). This analysis revealed that the O.degus-4 replicase is differentially expressed in 67 vivo, with markedly elevated expression of mRNA in the liver, and little or no expression in 68 other tissues. 69 Scientifically, EVEs can be approached from two overlapping but distinct 70 perspectives. Firstly, they can be viewed a kind of genomic ‘fossil record’ from which the 71 long-term, coevolutionary relationships of viruses and hosts can be inferred. In this respect, 72 the presence of a specific, monophyletic lineage of EVEs in both South American and 73 Australian marsupial genomes -and the apparent absence of this lineage from the genomes of 74 Old World rodents - suggests the existence of an ancient parvovirus lineage that evolved in 75 the indigenous mammal populations of biogeographically isolated Southern hemisphere 76 continents (marsupials, xenarthrans), and was acquired by caviomorph rodents subsequent to 77 their colonization of the South American continent (estimated to have occurred ~40 MYA 78 (10)). 79 The second way in which EVEs can be viewed is as host genes. While most EVE 80 sequences are highly degenerated, it is clear that at least a proportion of these elements have 81 been co-opted or ‘exapted’ (i.e. adapted for a function distinct from that for which they 82 originally evolved (11)) to perform physiological functions in their host species (12-15). 83 Dependovirus-derived EVEs encoding intact replicase proteins have previously been 84  4identified in mammalian genomes, including the African elephant (Loxodonta africana) and 85 Hamadryas baboon (Papio hamadryas). However, this is the first study to show that a 86 parvovirus-derived EVE is expressed in vivo in a mammal. While no physiological function 87 has yet been demonstrated for the numerous Parvovirus-related EVEs in mammalian 88 genomes, the identification of an intact element with differential expression across tissues 89 provides further indication that such functions exist. Since degus are experimental organisms 90 that are used currently to research mammalian pathologies and behaviours (16, 17), the 91 identification of an intact, expressed parvovirus-derived EVE in this species suggests a 92 possible path forward for research in this area. 93  94   95  5Figure legends 96  97 Figure 1.  98 (a) Genetic structures of parvovirus-derived EVEs in C.lanigera and O.degus genomes. ORFs 99 were inferred by manual comparison of putative peptide sequences to those of closely related 100 exogenous parvoviruses.  101 (b) Alignment of a pair of duplicated EVEs in the Chinchilla lanigera genome against an 102 adeno-associated virus type 2 (AAV2) reference sequence (GenBank accession #: 103 AF043303.1).  Blocks of six or more amino acids that are conserved between all three 104 sequences are highlighted in grey. Nonsense mutations shared between the two duplicated 105 EVEs are highlighted in black. The two EVEs differed at 8 of 323 nucleotide positions (i.e. an 106 average of 4 nucleotide substitutions has occurred in each insertion), indicating they arose in a 107 duplication event that occurred between 2.8 and 5.6 MYA (assuming a range of mammalian 108 neutral substitution rates from 2.2 to 4.5 x 10-9 per site per year (18)). 109  110  111 Figure 2.  112 Maximum likelihood phylogenies showing the relationships of (a) parvovirus-related, and (b) 113 dependovirus-related EVEs in the C.lanigera and O.degus genomes to exogenous 114 parvoviruses and previously described EVEs. Phylogenies are based on alignments of NS1 115 proteins and putative NS1 proteins encoded by EVE pseudogenes, and were constructed using 116 PHYML (19) and the JTT+ protein substitution model as selected by ProtTest (20). EVEs 117 identified in this study are underlined. The scale bar indicates evolutionary distance in 118 substitutions per amino acid site. Asterisks indicate nodes with maximum likelihood bootstrap 119 support above 75% for 100 bootstrap replicates. Abbreviations: EVE=endogenous viral 120 element; AAV=adeno-associated virus; MV= minute virus; PV=Parvovirus. NS=non-121 structural protein gene cassette; VP=viral capsid protein gene cassette. 122  123  6Figure 3. 124 (a) Schematic representation of endogenous viral element (EVE) O.degus-4. The replicase 125 ORF is shown as a white box and the 5’ and 3’ genomic sequences as black lines. The relative 126 positions of primers used in this study are shown. Primers F1 and F2, positioned at the 127 extreme ends of the EVE were used to amplify genomic DNA and sequence PCR products. 128 Primers G1 and G2, positioned in the genomic flanking sequence, were used to control for 129 genomic contamination of the RNA preparations. The internal primers I1 and I2 were used to 130 detect O.degus-4 replicase mRNA.  131  132 (b) Tissue-specific expression of a dependovirus-related EVE mRNA in the degu. Total RNA 133 was extracted from O.degus pancreas, lung, testicle, suprarenal gland, spleen, liver and 134 kidney. After cDNA generation, expression of the O.degus-4 EVE was detected using primers 135 designed to amplify the region 519-689 of the predicted mRNA (top panel). OdGAPDH 136 mRNA (XM_004643553.1) was used as a positive control of mRNA presence (middle panel). 137 To discard genomic DNA contamination in the cDNA preparations, primers aligning 35 138 nucleotides (nt) upstream and 85 nt downstream the predicted element in the genomic 139 sequence NW_004524640.1 were used (bottom panel). The different tissues analyzed are 140 indicated above the figure. Genomic DNA was used as a positive amplification control. 141  142 Acknowledgements 143 This work was supported by FONDECYT 1130852 (G.A.), and UK Medical Research 144 Council grant number MC_UU_12014 (R.G.). 145  146 References 147  148  149 1. Belyi VA, Levine AJ, Skalka AM. 2010. Sequences from ancestral single-stranded 150 DNA viruses in vertebrate genomes: the parvoviridae and circoviridae are more than 151 40 to 50 million years old. J Virol 84:12458-12462. 152 2. Kapoor A, Simmonds P, Lipkin WI. 2010. Discovery and characterization of 153 mammalian endogenous parvoviruses. J Virol 84:12628-12635. 154  73. Katzourakis A, Gifford RJ. 2010. Endogenous viral elements in animal genomes. 155 PLoS Genet 6:e1001191. 156 4. Liu H, Fu Y, Xie J, Cheng J, Ghabrial SA, Li G, Peng Y, Yi X, Jiang D. 2011. 157 Widespread endogenization of densoviruses and parvoviruses in animal and human 158 genomes. J Virol 85:9863-9876. 159 5. Gifford RJ. 2014. DIGS v1.0, Available from http://paleovirology.org.uk/. 160 6. Opazo JC. 2005. A molecular timescale for caviomorph rodents (Mammalia, 161 Hystricognathi). Molecular phylogenetics and evolution 37:932-937. 162 7. Phan TG, Vo NP, Bonkoungou IJ, Kapoor A, Barro N, O'Ryan M, Kapusinszky 163 B, Wang C, Delwart E. 2012. Acute diarrhea in West African children: diverse 164 enteric viruses and a novel parvovirus genus. J Virol 86:11024-11030. 165 8. Naccache SN, Greninger AL, Lee D, Coffey LL, Phan T, Rein-Weston A, 166 Aronsohn A, Hackett J, Jr., Delwart EL, Chiu CY. 2013. The perils of pathogen 167 discovery: origin of a novel parvovirus-like hybrid genome traced to nucleic acid 168 extraction spin columns. J Virol 87:11966-11977. 169 9. Smuts H, Kew M, Khan A, Korsman S. 2014. Novel Hybrid Parvovirus-Like 170 Virus, NIH-CQV/PHV, Contaminants in Silica Column-Based Nucleic Acid 171 Extraction Kits. J Virol 88:1398. 172 10. 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Bezier A, Annaheim M, Herbiniere J, Wetterwald C, Gyapay G, Bernard-184 Samain S, Wincker P, Roditi I, Heller M, Belghazi M, Pfister-Wilhem R, 185 Periquet G, Dupuy C, Huguet E, Volkoff AN, Lanzrein B, Drezen JM. 2009. 186 Polydnaviruses of braconid wasps derive from an ancestral nudivirus. Science 187 323:926-930. 188 14. Rowe HM, Trono D. 2011. Dynamic control of endogenous retroviruses during 189 development. Virology 411:273-287. 190 15. Dewannieux M, Heidmann T. 2013. Endogenous retroviruses: acquisition, 191 amplification and taming of genome invaders. Curr Opin Virol 3:646-656. 192 16. Okanoya K, Tokimoto N, Kumazawa N, Hihara S, Iriki A. 2008. Tool-use 193 training in a species of rodent: the emergence of an optimal motor strategy and 194 functional understanding. PloS one 3:e1860. 195 17. Ardiles AO, Tapia-Rojas CC, Mandal M, Alexandre F, Kirkwood A, Inestrosa 196 NC, Palacios AG. 2012. Postsynaptic dysfunction is associated with spatial and 197 object recognition memory loss in a natural model of Alzheimer's disease. 198 Proceedings of the National Academy of Sciences of the United States of America 199 109:13835-13840. 200 18. Kumar S, Subramanian S. 2002. Mutation rates in mammalian genomes. 201 Proceedings of the National Academy of Sciences of the United States of America 202 99:803-808. 203 19. Guindon S, Dufayard JF, Lefort V, Anisimova M, Hordijk W, Gascuel O. 2010. 204 New algorithms and methods to estimate maximum-likelihood phylogenies: assessing 205 the performance of PhyML 3.0. Systematic biology 59:307-321. 206 20. Abascal F, Zardoya R, Posada D. 2005. ProtTest: selection of best-fit models of 207 protein evolution. Bioinformatics (Oxford, England) 21:2104-2105. 208  209 GenericParvovirinae genomeO.degus  1C.lanigera  2C.lanigera  1O.degus  5NS1 (replicase) VP2 (capsid)~5 kbC.lanigera  4O.degus  6C.lanigera  5C.lanigera  3C.lanigera  6O.degus  4O.degus  2x233DependovirusParvovirus-relatedO.degus  3Chinchilla 5a   NKNFPFS?PLDKMIVWWEEGKMTAKVVESLTATLGSSKVRVDEKGKSSDELDQSPAChinchilla 5b   NKNFPFS?PLDKMIVWWEEGKITAKVVESLTASVGSSKVRVDEKGKSSDELDQSPAAAV2            NENFPFNDCVDKMVIWWEEGKMTAKVVESAKAILGGSKVRVDQKCKSSAQIDPTPV Chinchilla 5a   IVIRNTDMCTLVDGN*TALEHQQCLQDWMSTFQLAYSLELSFGKITKQEVKDChinchilla 5b   IVNSNTDMCTLVDGN*TAWEHQQCL*DWMSTFQLAYSLELSFGKITKQEVKDAAV2            IVTSNTNMCAVIDGNSTTFEHQQPLQDRMFKFELTRRLDHDFGKVTKQEVKDa)b)b) Reptile*Mammalian*AvianDependovirus NS1a)Parvo/Bufavirus-related NS1degenerated EVESouthernhemiisphereParvovirus-related EVEsParvovirus****AmdovirusBufavirusO.degus-1O.degus-2O.degus-3C.lanigera-1C.lanigera-2C.lanigera-4O.degus-4Baboon EVEBovine AAVCaprine AAVElephant EVEMicrobat EVEWallaby EVEGuinea pig EVEDuck parvovirusGoose parvovirusSnake parvovirusAvian AAV-1Avian AAV-1 (VR-865)Gray fox amdovirusMink amdovirusWallaby EVEGuinea pig EVEOpossum EVEPorcine PV TornauPorcine PV Feline PV Mink PV Canine PV Rat PV 1aMouse PV 1aMouse PV 1bPV H1Rat MV 1bRat MV 1aRat MV 1cPV LuIIIHamster PVMouse MVbMouse MVaMouse MV IBufavirus-1Bufavirus-3Bufavirus-2AAV4AAV2EVE with intact ORFEVE with intact, transcribed ORF3OdGAPDH3O.degus 4 replicasepancreaslungtesticlesuprarenalspleenliver kidney3genomic flank primersgenomic DNA O. degus 4 replicase  ORFG1G2I1I2F1F2abTable 1. Parvovirus EVEs in C. lanigera and O.degus genomes   Element   Accession number Orientation Structure Genus1 Match coordinates       Scaffold Viral genome2       start end start end           C.lanigera-1  JH721894.1 -ve NS-VP Parvo 14636996 14641202 1137 4398 C.lanigera-2  JH721993.1 -ve NS-VP Parvo 459340 462818 1065 4428 C.lanigera-3  JH721905.1 -ve NS Dependo 7599480 7600235 717 896 C.lanigera-4*  JH721911.1 -ve NS Dependo 18094486 18095973 330 1684 C.lanigera-5a  JH721873.1 +ve NS Dependo 22669255 22669578 1428 1724 C.lanigera-5b  JH721873.1 +ve NS Dependo 22702412 22702712 1428 1724 C.lanigera-6  JH721896.1 -ve VP Dependo 13652894 13654833 4054 4407 O.degus-1  JH651603.1 +ve NS-VP Parvo 365906 369616 507 4443 O.degus-2   JH651624.1 -ve NS-VP Parvo 3857091 3858404 1089 2652 O.degus-3    JH651827.1 -ve NS-VP Parvo 1343057 1345620 295 3142 O.degus-4*  JH651579.1 +ve NS Dependo 8427679 8429061 321 1784 O.degus-5  JH651577.1 +ve VP Parvo 12296699 12296983 4135 4419 O.degus-6  JH651549.1 -ve VP Dependo 16377593 16377937 4063 4401            Footnote: 1 Genus assignment based on phylogenetic analysis of the NS1 gene (see Figure 2). Dependo- = Dependovirus, Parvo- = parvovirus related EVEs 2 Viral genome coordinates based on pairwise alignment to genus-specific reference sequences; adeno-associated virus 2 (AF043303.1) for Dependoviruses, and mouse minute virus (J02275.1) for Parvovirus-like elements. Asterisks denote elements encoding intact open reading frames.   

ABSTRACT
          
            We describe endogenous viral elements (EVEs) derived from parvoviruses (family
            Parvoviridae
            ) in the genomes of the long-tailed chinchilla (
            Chinchilla lanigera
            ) and the degu (
            Octodon degus
            ). The novel EVEs include dependovirus-related elements and representatives of a clearly distinct parvovirus lineage that also has endogenous representatives in marsupial genomes. In the degu, one dependovirus-derived EVE was found to carry an intact reading frame and was differentially expressed
            in vivo
            , with increased expression in the liver.
          </jats:p

Gloria Arriagada

Robert J. Gifford

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Parvovirus-Derived Endogenous Viral Elements in Two South American Rodent Genomes

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Parvovirus-derived endogenous viral elements in two South American rodent genomes

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Repositorio Institucional Académico (RIA) de la Universidad Andrés Bello

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