(81) Designated States (unless otherwise indicated, for every
`kind of national protection available): AE, AG, AL, AM,
`AO, AT, AU, AZ, BA, BB, BG, BH, BR, BW, BY, BZ,
`CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, DO,
`DZ, EC, EE, EG, ES, Fl, GB, GD, GE, GH, GM,GT,
`HN, HR, HU, ID,IL,IN,IS, JP, KE, KG, KM,KN,KP,
`KR, KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD,
`ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI,
`NO, NZ, OM,PE, PG, PH, PL, PT, RO, RS, RU, SC, SD,
`SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, TR,
`TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW.
`(84) Designated States (unless otherwise indicated, for every
`kind of regional protection available): ARIPO (BW, GH,
`GM, KE, LR, LS, MW, MZ, NA, SD,SL, SZ, TZ, UG,
`ZM,ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU,TJ,
`TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK,
`(71) Applicant (for all designated States except US): THE
`EE, ES, FI, FR, GB, GR, HR, HU,IE,IS, IT, LT, LU,
`REGENTS OF THE UNIVERSITY OF CALIFOR-
`LV, MC, MK, MT, NL, NO, PL, PT, RO, SE, SL, SK,
`NIA [US/US]; 1111 Franklin Street, 5th Floor, Oakland,
`SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ,
`CA 94607-5200 (US).
`GW, ML, MR,NE, SN, TD, TG).
`(72) Inventors; and
`(75) Inventors/Applicants (or US only): DING, Shou-Wei Published:
`[US/US]; 8797 Barnwood Lane, Riverside, CA 92508
`—__without international search report and to be republished
`(US). WU, Qingfa [CN/US]; 1177 Linden Street, Apt.
`upon receipt of that report (Rule 48. 2(g))
`19, Riverside, CA 92507 (US).
`—___with sequencelisting part ofdescription (Rule 5.2(a))
`(74) Agents: EINHORN, Gregory, P. et al.; Gavrilovich,
`Dodd & Lindsey LLP, 4660 La Jolla Village Drive, Suite
`750, San Diego, CA 92122 (US).
`
`(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`OUT AUIAAOMA AAA
`
`(19) World Intellectual Property Organization
`International Bureau
`
`9 December 2010 (09.12.2010)
`
`(43) International Publication Date
`
`(10) International Publication Number
`WO 2010/141433 A2
`
`(51) International Patent Classification:
`C12Q 1/68 (2006.01)
`C40B 40/06 (2006.01)
`C12@Q 1/70 (2006.01)
`ge
`.
`+
`,
`.
`(21) International Application Number:
`PCT/US2010/036849
`
`(22) International Filing Date:
`
`(25) Filing Language:
`(26) Publication Language:
`(30) Priority Data:
`61/183,377
`61/286,742
`
`1 June 2010 (01.06.2010)
`:
`English
`English
`
`2 June 2009 (02.06.2009)
`15 December 2009 (15.12.2009)
`
`US
`US
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`(54) Title: VIRUS DISCOVERY BY SEQUENCING AND ASSEMBLY OF VIRUS-DERIVED SIRNAS, MIRNAS, PIRNAS
`
`(57) Abstract: In one embodiment, the disclosure provides methods and systems for identifying viral nucleic acids in a sample. In
`another embodiment the invention provides methods for viral genome assembly and viral discovery using small inhibitory RNAs,
`or "small silencing," RNAs (siRNAS), micro-RNAs (miRNAs) and/or PIWI-interacting RNAs (piRNAs), including siRNAS, miR-
`NAsand/or piRNAsisolated or sequenced from invertebrate organisms such as insects (Anthropoda), nematodes (Nemapoda),
`Mollusca, Porifera, and other invertebrates, and/or plants, fungi or algae, Cyanobacteria andthe like.
`
`
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`VIRUS DISCOVERY BY SEQUENCING AND ASSEMBLY
`OF VIRUS-DERIVEDsiRNAs, miRNAs, piRNAs
`
`REFERENCE TO SEQUENCELISTING
`
`This application contains a txt. File containing the sequencelisting, which is
`
`incorporated by reference herein.
`
`STATEMENT AS TO FEDERALLY SPONSORED RESEARCH
`
`This invention was made with government support under Grant No. AI052447
`
`awarded by the National Institutes of Health (NIH) and Grant No. 2007-353 19-18325
`
`awarded by the USDA. The governmenthascertain rights in the invention.
`
`TECHNICAL FIELD
`
`In one embodiment, the disclosure provides methods and systems for
`
`identifying viral nucleic acids in a sample. In another embodimentthe invention
`
`provides methods for viral genome assembly andviral discovery using small
`
`inhibitory RNAs, or “small silencing,” RNAs (siRNAS), micro-RNAs (miRNAs)
`
`and/or PIWI]-interacting RNAs (piRNAs), including siRNAS, miRNAsand/or
`
`piRNAsisolated or sequenced from invertebrate organisms such as insects
`
`(Anthropoda), nematodes (Nemapoda), Mollusca, Porifera, and other invertebrates,
`
`and/or plants, fungi or algae, Cyanobacteria and the like.
`
`BACKGROUND
`
`Discovery of new virusesis often hindered by difficultics in their
`
`amplification in cell culture and/or lack of their cross-reactivity in serological and
`
`nucleic acid hybridization assays to known viruses. Many new viruses have been
`
`recently identified in environmental and clinical samples using metagenomic
`
`approaches,in whichviral particles are first purified and viral nucleic acid sequences
`
`are then randomly amplified prior to subcloning and sequencing (Delwart, 2007).
`
`The Dicer family of host immune receptors mediates antiviral immunity in
`
`fungi, plants and invertebrate animals by RNA interference (RNAi) or RNA silencing
`
`(1-3). In this immunity, a viral double-stranded RNA (dsRNA) is recognized by Dicer
`
`
`
`and diced into small interfering RNAs (siRNAs). These virus-derived siRNAsare
`
`then loaded into an RNAsilencing complex to act as specificity determinants and to
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`guide slicing of the target viral RNAs by an Argonaute protein (AGO)present in the
`
`complex. Dicer proteins typically contain an RNA helicase domain, a PAZ domain
`
`shared with AGOs, and two tandem type III endoribonuclease (RNaseIII) domains.
`
`Dicer cleaves dsRNA with a simple preference toward a terminus of dsRNA,
`
`producing duplex small RNA fragments of discrete sizes progressively from the
`
`terminus (4).
`
`In addition to siRNAs, microRNAs (miRNAs) and PIWI-interacting RNAs
`
`(piRNAs) also guide RNAsilencing in similar complexes but with distinct AGOs (4-
`
`6). In Drosophila melanogaster, miRNAs and siRNAsare predominantly 22 and 21
`
`nucleotides in length, dependent on Dicer-1 (DCR1) and DCR2 for their biogenesis,
`
`and act in silencing complexes containing AGO1 and AGO2 in the AGO subfamily,
`
`respectively (4-6). In contrast, ~24-30-nt piRNAsare Dicer-independentand require
`
`AGO3, Aubergine (AUB) and PIWI in the PIWI subfamily for their biogenesis (4-6).
`
`Genetic analyses (7-10) have clearly demonstrated a role for D. melanogaster DCR2
`
`in the immunity and biogenesis of viral siRNAstargeting diverse positive-strand (+)
`
`RNAviruses, including Flock house virus (FHV), cricket paralysis virus, Drosophila
`
`C virus (DCY), and Sindbis virus (SINV). Cloning and sequencing of small RNAs
`
`from FHV-infected Drosophila cells further indicate that the viral dsRNA replicative
`
`intermediates (vRI-dsRNA)are the substrate of DCR2 and the precursorofviral
`
`siRNAs(11-12). Drosophila susceptibility to Drosophila X virus (DXV), which
`
`contains a dsRNA genome,is influenced by components from both the siRNA(e.g.,
`
`AGO? & R2D2) and piRNA (e.g., AUB & PIWI) pathways (13). However, detection
`
`of small RNAsderived from any dsRNAvirus has not been reported yet (1, 13).
`
`Virus-derived small RNAswerefirst detected in plants infected with a +RNA
`
`virus (14). The Dicer proteins involved in the production of siRNAstargeting both
`
`+RNA viruses and DNA viruses have been identified in Arabidopsis thaliana (2-3),
`
`which encode AGOsin the AGO subfamily but not in the PIWI subfamily (15).
`
`Cloning and sequencing ofplant viral siRNAs suggest that they may be processed
`
`either from vRI-dsRNAorhairpin regions of single-stranded RNA precursors (16-
`
`20). Production of viral siRNAshas also been demonstrated in fungi, silkworms,
`
`mosquitoes, and nematodesin response to infection with +RNA viruses and viral
`
`small silencing RNAsproduced in fungi and mosquitoes have recently been cloned
`
`and sequenced (21-25).
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`The available data thusillustrate that accumulation of virus-derived small
`
`silencing RNAsis a commonfeature of an active immuneresponseto viral infection
`
`in diverse eukaryotic host species.
`
`SUMMARY
`
`The disclosure provides a method for viral discovery that is independent of
`
`either amplification or purification of viral particles. Many humandiseases such as
`
`approximately half of all analyzed cases of human encephalitis and gastroenteritis,
`
`have no identified etiology. Thus, discovery of new viruses should facilitate
`
`identification of human pathogenic viruses, improve the understandingof their
`
`transmission and provide diagnostic tools and targets for the developmentofanti-
`
`virals.
`
`The disclosure provides methodsofidentifying viral nucleic acid, assembling
`
`viral genomesand discovering viruses based upon the mechanism ofinvertebrate,
`
`plant, algae, fungal etc. processing of viral small inhibitory RNAs, or “small
`
`silencing,” RNAs (siRNAS), micro-RNAs (miRNAs) and/or PIW]-interacting RNAs
`
`(piRNAs), including miRNA-, piIRNA-, siRNA and/or RNAi-mediated viral
`
`immunity in plants and invertebrates, including insects (Drosophila melanogaster and
`
`mosquitoes) and nematodes (Caenorhabditis elegans), and algae, fungus,
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`Cyanobacteria and thelike.
`
`In alternative embodiments, the invention provides methods comprising:
`
`(a) (i) obtaining a plurality of naturally occurring 18-28 nucleotide RNA
`
`fragments, or siRNAs, or miRNAsand/or piRNAs, to generate an RNAlibrary, or,
`
`obtaining a plurality of 18-28 nucleotide RNA fragments, or siRNAs, or miRNAs
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`25
`
`and/or piRNAsfrom an organism or organisms, or a plant or plants; and
`
`(11)
`
`determining the sequence of the RNA fragments, or siRNAs, or
`
`
`
`miRNAsand/or piRNAs, and using those sequences to assemble the RNA fragments,
`
`or siRNAs, or miRNAs and/or piRNAs into at least one contiguous unit (‘a contig’’)
`
`comprising a plurality of the nucleotide RNA fragments siRNAs, miRNAsand/or
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`piRNAs; or
`
`(b) the method of (a), wherein the contigs are assembled using the help of a
`
`computer program, wherein optionally the computer program is VELVET.
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`In alternative embodiments, the methods further comprise determining the
`
`sequence of the assembled contiguous unit, or the contig; or further comprise:
`
`(a) searching a databaseof viral or microorganism sequencesusingthe at least
`
`one contiguous sequenceto identify a viral or microorganism genome,nucleic acid or
`
`protein-encoding sequence, or subsequencethereof, having significant homology to
`
`the assembled contiguous unit; or
`
`(b) the method of (a), wherein the database comprises non-redundant
`
`nucleotide sequences; or
`
`(c) the method of (a), wherein the database comprises in silico translation
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`sequences.
`
`wherein optionally the assembled contig sequence hassignificant homology to
`
`a knownviral genus or genome.
`
`In alternative embodiments, the methods further comprise searching a
`
`database of viral or microorganism sequences using the at least one contiguous
`
`sequenceto identify a viral or microorganism genome,nucleic acid or protein-
`
`encoding sequence, or subsequence thereof, having at least about 50% to about 100%
`
`percent homologyto all or part of the assembled contiguous sequence.
`
`In alternative embodiments, the methods further comprise making a
`
`phylogenetic analysis of the identified viral or microorganism genome,nucleic acid or
`
`protein-encoding sequence with the contiguous sequence.
`
`In alternative embodiments, the methods further comprise identifying and
`
`annotating the phylogenetic analysis of the identified viral sequence with the
`
`contiguous sequence.
`
`In alternative embodiments, the obtained RNA or nucleotide sequences are
`
`substantially purified or isolated from an organism ofinterest.
`
`In alternative embodiments, the methods further comprise substantially
`
`purifying small RNA fragments, or siRNAs, or miRNAs and/or piRNAs, from an
`
`organism of interest and sequencing the RNA fragments to obtain an RNAlibrary.
`
`In alternative embodiments, the methods further comprise removing
`
`sequenced segments from the library that overlap with the genomic sequence of the
`
`organism of interest from which the RNA wasderived.
`
`In alternative embodiments, the methods further comprise filling in gaps
`
`between the contiguous sequences. In one embodiment,filling in the gaps between
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`the contiguous sequences comprises use of RT-PCR and/or sequencing to fill in gaps
`
`between the contiguous sequences.
`
`In alternative embodiments, the methods further comprise completing a
`
`genomic sequenceof a virus or a microorganism comprising the contiguous sequence
`
`using 5’-RACEand 3’-RACE.
`
`In one embodiment, the organism or organismsis/are an invertebrate, an insect
`
`(Anthropoda), a nematode (Nemapoda), a Mollusca, a Porifera, a plant, a fungi, an
`
`algae, a Cyanobacteria; or the organism or organismsare identified or unidentified
`
`and are derived from an environmental sample. In one embodiment, the
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`environmental sample is a soil sample, a water sample or an air sample.
`
`In one embodiment, the invention provides methods for identifying a virus,
`
`comprising:
`
`constructing a small RNAlibrary from an organism or organisms;
`
`deep sequencing the small RNAlibrary;
`
`assembling the sequenced small RNAsusing(a) all of the sequenced small
`
`RNAsof 18-28 nucleotides in length, or siRNAs, or miRNAsand/or piRNAs; or (b)
`
`small RNAs, or siRNAs, or miRNAs and/or piRNAs,of a defined length into at
`
`plurality of contigs;
`
`identifying and removing those assembled sequences mapped onto the genome
`
`of the organism to provide an enriched set of contigs;
`
`performing a homology search of contigs against knownviruses at both the
`
`nucleotide and protein levels;
`
`optionally using RT-PCR and sequencingto fill the gaps between the contigs
`
`that show limited similarities with a knownvirus;
`
`completing the full-length genomic sequenceof the identified virus with 5’-
`
`RACEand 3’-RACE;and
`
`annotating the identified virus.
`
`In one embodiment, the organism or organismsis/are an invertebrate, an insect
`
`(Anthropoda), a nematode (Nemapoda), a Mollusca, a Porifera, a plant, a fungi, an
`
`algae, a Cyanobacteria; or the organism or organismsare identified or unidentified
`
`and are derived from an environmental sample. In one embodiment, the
`
`environmental sample is a soil sample, a water sample or an air sample.
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`The details of one or more embodimentsof the disclosure are set forth in the
`
`accompanying drawingsandthe description below. Other features, objects, and
`
`advantages of the disclosure will be apparent from the description and drawings, and
`
`from the claims.
`
`All publications mentioned herein are incorporated herein by reference in full
`
`for the purpose of describing anddisclosing the methodologies, which are described
`
`in the publications, which might be used in connection with the description herein.
`
`The publications discussed above and throughout the text are providedsolely for their
`
`disclosure priorto the filing date of the present application. Nothing herein is to be
`
`construed as an admission that the inventors are not entitled to antedate such
`
`disclosure by virtue of prior disclosure.
`
`DESCRIPTION OF DRAWINGS
`
`Figure 1A and Figure B showsdistribution of assembled viral siRNA contigs
`
`on the tripartite and bipartite RNA genomeof (a) CMV and (b) FHV; as discussed in
`
`detail in Example 1, below.
`
`Figure 2 showsthe discovery of three new viruses by assembly ofviral
`
`siRNAs. Total of 54, 34 and 19 contigs assembled from sequenced siRNAs were
`
`mapped to DmTV, DmBV and DmTRYV,respectively. The genome organization of
`
`EEV was shownas a reference for DmTRV. % protein sequence identities of
`
`assembled contigs (red bars) of the three viruses to related viruses were shown on the
`
`top or below;as discussed in detail in Example 1, below.
`
`Figure 3A, Figure 3B, Figure 3C illustrate a phylogenetic analysis of newly
`
`identified viruses (indicated by a red arrow) according to the similarities of viral
`
`RdRPs with Clustal W method; as discussed in detail in Example 1, below.
`
`Figure 4A and Figure 4Billustrate the distribution of assembled viral siRNA
`
`contigs on the monopartite genome and bipartite RNA genome of SINV and a new
`
`nodavirus respectively; as discussed in detail in Example 1, below.
`
`Figure 5 illustrates position and distribution of FHV and SINV siRNA contigs
`
`assembled from small RNAs sequenced from (Figure 5A) Drosophila S2 cells
`
`infected with the B2-deletion mutant of FHV (11), (Figure 5B) a transgenic C.
`
`elegans strain in the RNAi-defective 1 (rde-1) mutant background carrying an FHV
`
`RNAI replicon in which the coding sequence of B2 wasreplaced by that of GFP (29),
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`and (Figure 5C) adult mosquitoes infected with SINV (22); as discussed in detail in
`
`Example 2, below.
`
`Figure 6 illustrates discovery of dsRNA viruses DTV (Figure 6A) and DBV
`
`(Figure 6B), and +RNA viruses DTrV (Figure 6C) and MNV (Figure 6D) from S2-
`
`GMRcells by vsSAR; as discussed in detail in Example 2, below.
`
`Figure 7 illustrates the S2-GMRcells contained four infectious RNA viruses:
`
`Figure 7A illustrates DTV, DBV, DXV and ANV wereall detected by RT-PCRin
`
`non-contaminated S2 cells 4 days after inoculation with the supernatant of the S2-
`
`GMRcells; Figure 7B illustrates detection of a DI-RNA derived from ANV RNA2 in
`
`S2-GMRcells (right lane) and in S2 after inoculation with the supernatant of S2-
`
`GMRcells (left lane) by Northern blot hybridizations using a probe recognizing the
`
`3’-terminal 120 nt of RNA2; Figure 7C illustrates structure of the cloned DI-RNA of
`
`ANV(top) and mapping of the perfect-matched 21-nt siRNAs sequenced from S2-
`
`GMRcells to the positive (blue) and negative (red) strands of ANV RNA2 (20-nt
`
`windows) (bottom) ; as discussed in detail in Example 2, below.
`
`Figure 8 illustrates size distribution (Figure 8A) and aggregate nucleotide
`
`composition (Figure 8B) of virus-derived small RNAs in Drosophila OSS cells; as
`
`discussed in detail in Example 2, below.
`
`Like reference symbols in the various drawingsindicate like elements.
`
`DETAILED DESCRIPTION
`
`In alternative embodiments the invention provides methods for viral genome
`
`assembly andviral discovery using small inhibitory RNAs,or “small silencing,”
`
`RNAs(siRNAS), micro-RNAs (miRNAs) and/or PIW]-interacting RNAs (piRNAs),
`
`including siRNAS, miRNAsand/or piRNAsisolated or sequenced from invertebrate
`
`organismssuchas insects (Anthropoda), nematodes (Nemapoda), Mollusca, Porifera,
`
`and other invertebrates, and/or plants, fungi or algae, Cyanobacteria and the like.
`
`As described in Example 2, we found that viral small silencing RNAs
`
`produced by invertebrate animals are overlapping in sequence and can assemble into
`
`long contiguous fragments of the invading viral genome from small RNAlibraries
`
`sequenced by next generation platforms. Based on this finding, we developed an
`
`approach of virus discovery in invertebrates by deep sequencing and assembly oftotal
`
`small RNAs (vdSAR)isolated from a host organism ofinterest.
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`As described in Example 2, alternative embodiments of the invention revealed
`
`mix infection of Drosophila cell lines and adult mosquitoes by multiple RNA viruses,
`
`five of which were new. Analysis of small RNAs from mix infected Drosophila cells
`
`showed that infection ofall three distinct dsRNA virusestriggered production of viral
`
`siRNAswith features similar to siRNAs derived from +RNA viruses. Ourstudy also
`
`revealed production and assembly of virus-derived piRNAsin Drosophila cells,
`
`suggesting a novel function of piRNAsin viral immunity. Thus, unique features of
`
`the invention’s vdSAR can discover new invertebrate and arthropod-borne animal and
`
`human viral pathogens.
`
`As used herein and in the appended claims, the singular forms "a,” "and,” and
`
`"the" include plural referents unless the context clearly dictates otherwise. Thus, for
`
`example, reference to "an siRNA"includes a plurality of such siRNAsand reference
`
`to "the virus" includes reference to one or moreviruses, and so forth.
`
`Unless defined otherwise, all technical and scientific terms used herein have
`
`the same meaning as commonly understood to one of ordinary skill in the art to which
`
`this disclosure belongs. Although any methods and reagents similar or equivalent to
`
`those described herein can be usedin the practice of the disclosed methods and
`
`compositions, the exemplary methods and materials are now described.
`
`Also, the use of“or means “and/or” unless stated otherwise. Similarly,
`99 66
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`“include,”
`“includes,” and “including” are
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`“comprise,”
`
`“comprises,
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`comprising”
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`interchangeable and not intendedto be limiting.
`
`It is to be further understood that where descriptions of various embodiments
`
`use the term “comprising,” those skilled in the art would understand that in some
`
`specific instances, an embodiment can bealternatively described using language
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`“consisting essentially of’ or “consisting of.”
`
`The disclosure of U.S. Patent No. 7,211,390, describing techniques associated
`
`with “deep sequencing” is incorporated herein by reference.
`
`In immunity, viral infection induces production of virus-derived small
`
`interfering RNAs (siRNAs), pi-RNAs and miRNAsthat subsequently guide specific
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`30
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`viral RNA clearance by the RNAinterference (RNAi), pi-RNA and miRNA
`
`mechanism. In D. melanogaster, for example, siRNAs of 21 nucleotides long
`
`targeting several positive-strand (+) RNAviruses are produced by Dicer-2 from
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`processing dsRNAreplicative intermediates synthesized during viral RNA
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`replication. Assisted by the dsRNA-binding protein R2D2, these viral siRNAs are
`
`then loaded in Argonaute-2 to direct viral RNA clearance (Galiana-Arnouxetal.,
`
`2006; Wanget al., 2006; Zambonetal., 2006). As a counter defense, viruses encode
`
`essential pathogenesis proteins that are viral suppressors of RNAi (VSRs) (Li and
`
`Ding, 2006; Mlotshwaet al., 2008). VSRs mayinhibit either the production or
`
`activity of viral siRNAsby targeting the dsRNA precursors, siRNAs or Argonaute
`
`proteins. Several nuclear-replicating DNA viruses produce virus-derived microRNAs
`
`following infection of their mammalian host cells and many proteins encoded by
`
`mammalian RNA and DNAviruses exhibit VSR activity. However, the current
`
`consensusis that in vertebrates viral dsRNAtriggers PKR and interferon responses
`
`instead of the RNAi response.
`
`The disclosure provides a method for virus discovery that is independent of
`
`either amplification or purification of viral particles. Many of the human diseases
`
`such as approximately half of all analyzed cases of human encephalitis and
`
`gastroenteritis, have no identified etiology. Thus, discovery of new virusesor the
`
`identification of the presence of viral infection can facilitate identification of human
`
`pathogenic viruses, improve our understanding of their transmission and provide
`
`diagnostic tools and targets for the developmentofanti-virals.
`
`Thedisclosure is based, in part, on the understanding of the mechanism of
`
`RNAi-mediated, including pi-RNA-, miRNA- and siRNA-based, viral immunity. In
`
`this immunity, viral infection induces production of virus-derived small interfering
`
`RNAs(siRNAs), pi-RNAs and miRNAs, that subsequently guide specific viral RNA
`
`clearance by the RNAinterference (RNAi) (pi-RNA-, miRNA- and siRNA-based)
`
`mechanism.In D. melanogaster, for example, siRNAs of 21 nucleotides long
`
`targeting several positive-strand (+) RNAvirusesare produced by Dicer-2 from
`
`processing dsRNA replicative intermediates synthesized during viral RNA
`
`replication. Assisted by the dsRNA-binding protein R2D2, these viral siRNAs are
`
`then loaded in Argonaute-2 to direct viral RNA clearance (Galiana-Arnouxetal.,
`
`2006; Wanget al., 2006; Zambonet al., 2006). As a counter defense, viruses encode
`
`essential pathogenesis proteins that are viral suppressors of RNAi (VSRs)(Li and
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`Ding, 2006; Mlotshwaet al., 2008). VSRs mayinhibit either the production or
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`activity of viral siRNAs, pi-RNAs and miRNAsbytargeting the dsRNA precursors,
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`siRNAs, pi-RNAs and miRNAsor Argonaute proteins. Several nuclear-replicating
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`DNAviruses produce virus-derived microRNAs following infection of their
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`mammalian host cells and many proteins encoded by mammalian RNA and DNA
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`viruses exhibit VSR activity (Ding and Voinnet, 2007). However, the current
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`consensusis that in vertebrates viral dsRNA triggers PKR and interferon responses
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`instead of the RNAi (siRNAs, pi-RNAs and miRNAs) response.
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`The disclosure demonstrates by the next generation sequencing technologies
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`viral siRNAs, pi-RNAs and miRNAsproducedin plants andfruit flies infected with
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`positive-strand RNA viruses, which are closely related to human pathogenic viruses
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`such as poliovirus and West Nile virus. The results show that viral siRNAs produced
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`by the host immune system in responseto viral infection are overlapping in sequence
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`and can be assembled back into long continuous fragments (contigs) of the infecting
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`viral RNA genomeusing assembly programs developed for short read genome
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`sequencing. Unlike individual siRNAs, pi-RNAs and miRNAs, the contigs assembled
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`from viral siRNAs, pi-RNAs and miRNAscanbetranslated into protein sequences in
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`silico for homology searches to identify new viruses that may be only distantly related
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`to known viruses.
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`The disclosure demonstrates that deep sequencing by the next generation
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`technologies and assembly of virus-derived siRNAs, pi-RNAs and miRNAs can be
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`employed as a new approach for virus discovery and identification. Indeed, the
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`examination of a recently sequenced small RNAlibrary (Flyntet al., 2009) made
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`from a Drosophila cell line found that the cell line is infected with at least five distinct
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`RNAviruses. These include two knownviruses and three new viruses belonging to
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`different genera not previously described. Since virus infection of plants and
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`invertebrates inevitably results in the production of virus-derived siRNAs, pi-RNAs
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`and miRNAs,this invention does not depend on the ability to either amplify the virus
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`or purify the viral particle to enrich viral nucleic acids, which is essential for the
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`current technologies. Importantly, any viruses detected by the methodofthe
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`disclosure are live and replication-competent because viral siRNAs, pi-RNAs and
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`miRNAsare products of an active host immune response to viral infection.
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`The observation that individual viral siRNAs, pi-RNAs and miRNAscan be
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`assembled back to longer genome fragments of the invading virus provides an
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`exciting new methodforvirus discovery by deep sequencing and assemblyofviral
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`siRNAs, pi-RNAs and miRNAs. Unlike individual siRNAs, pi-RNAs and miRNAs,
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`the contigs assembled from viral siRNAs, pi-RNAs and miRNAs can betranslated
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`into protein sequencesin silico for homology searches to identify new viruses that
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`may bedistantly related to knownviruses.
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`The disclosure provides a frame of the VDsiR comprised of bioinformatics
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`analysis and experimental verification. Small RNA assembling is a useful component
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`of the system, the numberof input sequences and distinct programs have impact on
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`the output. In a pilot study (described herein), Velvet was foundto be a useful
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`program for the project, which employs the principle of de Bruijn graphsto build up
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`continuous sequence from short reads in short run time (Zerbinoet al, 2008).
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`The disclosure thus provides in one embodiment, a method comprising (1)
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`obtaining nucleotide sequences from a small RNAlibraries comprising a plurality of
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`naturally occurring 18-28 nucleotide RNA fragments to obtain a sequenced small
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`RNAlibrary; (ii) assembling the sequencesin the sequenced small RNAlibrary into
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`at least one contiguous sequence comprising a plurality of nucleotide RNA fragment
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`sequences; optionally filling in gaps in a sequence by RT-PCRtechniques; (iii)
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`searching a database of viral sequence usingthe at least one contiguous sequence to
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`identify a viral sequence having at least 50%-100 percent homology to the contiguous
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`sequence;(iv) identifying and annotating the phylogenetic analysis of the identified
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`viral sequence with the contiguous sequence.
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`It will be understood that a sequence library may be providedbya third party
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`or made available to a user by any number of ways(i.e., internet, computer readable
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`medium andthelike) and thus the process described above can be adapted to carry the
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`process andidentify or annotate a virus accordingly. In some embodiment, however,
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`the library may be a sample library comprising substantially purified RNA from an
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`organism of interest. In such instances, deep sequencing techniquesare carried out
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`and a sequence library created. In yet another embodiment, a sample comprising
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`substantially purifying small RNA fragments from an organism ofinterest are
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`provided in which case sequencing the RNA fragments to obtain the small RNA
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`library is performed.
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`In yet another embodiment, if a gross RNA sample from an organism is
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`provided or where increased homology searchingis desired, the method may
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`optionally include removing sequenced segments from the library that overlap with
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`the genomic sequence of the organism of interest from which the RNA wasderived.
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`In yet another embodiment, the method further comprises completing a
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`genomic sequence of a virus comprising the contiguous sequence using 5’-RACE and
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`3’-RACE.
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`For example, the disclosure demonstrates in the specific embodiments and
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`proof of principle that a method includingthe steps of construction of a small RNA
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`library from cell culture or adults insects such as mosquitoesor fruit flies; deep
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`sequencing of the small RNAlibraries with an Hfimina 2G Analyser; assembly of the
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`sequenced small RNAs by Velvet using either all of the sequenced small RNAsof 18-
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`28 nucleotides in length or small RNAsof specific lengths such as 21-nt and 22-nt,
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`which most likely represent the products of Drosophila Dicer-2 and Dicer-1,
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`respectively, to generate a contig(s), contigs of virus-derived siRNAs mayinclude
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`features such as specific enrichment of 21- to 22-nt small RNAs, the presence of
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`small RNAsof both polarities and the high density of siRNAs (number of
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`siRNAs/length of contigs); identification and removal of those assembled sequences
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`mapped onto the host genome when the genome sequence is known, which reduces
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`the numberof the candidates for next steps; homology search of contigs with known
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`virus at both the nucleotide and protein levels; in an optional embodiment, RT-PCR
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`and sequencing can be usedto fill the gaps between the contigs that show limited
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`similarities with a known virus; optionally completing the full-length genomic
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`sequence ofthe identified virus with 5’-RACEand 3’-RACE; and annotation and
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`phylogenetic analysis of the identified virus, resulted in the identification of 2 known
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`viruses and 3 novel viruses from a D. melanogaster sample.
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`Asused herein a sample is any sample that can contain a virus. Thus, the
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`sample can be obtained from the environment, from a specific organism (including
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`plants, insects and mammals). An environmental sample can be obtained from any
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`number of sources (as described above), including, for example, insect feces, hot
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`springs, soil and the like. Any source of nucleic acids in purified or non-purified form
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`can be utilized as starting material. Thus, the nucleic acids may be obtained from any
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`source which is contaminated by an infectious organism (e.g. a virus). The sample can
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`be an extract from any bodily sample such as blood, urine, spinal fluid, tissue, vaginal
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`swab, stool, amniotic fluid or buccal mouthwash from any mammalian organism. For
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`non-mammalian(e.g., invertebrates) organisms the sample can be a tissue sample,
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`salivary sample, fecal material or material in the digestive tract of the organism. For
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`example, in horticulture and agricultural testing the sample can be a plant, soil, liquid
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`or other horticultural or agricultural product; in food testing the sample can be fresh
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`food or processed food (for example infant formula, seafood, fresh produce and
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`packaged food); and in environmental testing the sample can be liquid, soil, sewage
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`treatment, sludge and any other sample in the environment.
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`The sample can be processed using techniques knownin the art for deep
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`sequencing. In some embodiments, the sample is treated with an RNaseinhibitor to
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`prevent degradation of RNA oligonucleotides in t