WO2005000234A2

WO2005000234A2 - Novel coronavirus inhibitors

Info

Publication number: WO2005000234A2
Application number: PCT/US2004/018858
Authority: WO
Inventors: Derhsing Lai; Say Fone Phounsavan
Original assignee: Sars Scientific Corp.
Priority date: 2003-06-12
Filing date: 2004-06-10
Publication date: 2005-01-06
Also published as: WO2005000234A3

Abstract

An agent that inhibits interaction of an intergenic sequence of coronavirus with the coronavirus polymerase complex or component thereof as well as the use of said agent for inhibiting expression of said coronavirus and detecting coronavirus in a subject, as well as kits and compositions comprising such agents.

Description

NOVEL CORONAVIRUS INHIBITORS

FIELD OF THE INVENTION The invention is directed to an agent that inhibits interaction of an intergenic sequence of coronavirus with the coronavirus polymerase complex or component thereof as well as the use of said agent for inhibiting expression of said coronavirus and detecting coronavirus in a subject, as well as kits and compositions comprising such agents.

BACKGROUND OF THE INVENTION Coronaviruses are single-stranded positive stranded RNA viruses having a relatively large genome (27-31 kb) and a halo or crown-like (corona) appearance when viewed under an electron microscope. These viruses are a common cause of mild to moderate upper- respiratory illness in humans and are associated with respiratory, gastrointestinal, liver and neurologic disease in animals. Recently, it has been shown that a novel human coronavirus is the causative agent of Severe Acute Respiratory Syndrome (for review, see Nijayanand et al., 2004, Clin. Med. 4: 152-160) . SARS is a respiratory illness that has recently been reported in Asia, North America, and Europe. The earliest cases occurred in the Guangdong province of China in late 2002. SARS became known to the world at large in Feburary 2003 when a case was reported in Hong Kong. The SARS virus is believed to be spread by droplets produced by coughing and sneezing, but other routes of infection may also be involved, such as fecal contamination. The most commonly reported symptom is fever, with over half of patients reporting general influenza-like symptoms, chills, malaise, loss of appetite, and myalgia. Gastrointestinal symptoms are less common at presentation, including diarrhea, vomiting, and abdominal pain. The mean incubation period of SARS is estimated to be 6.4 days. The estimated case fatality rate is 13% for patients younger than 60 years and 43% for patients aged 60 years or older. (Donnelly CA, et al. "Epidemiological determinants of spread of causal agent of severe acute respiratory syndrome in Hong Kong" Lancet Vol. 361, 03 May 2003). Several strains of SARS-associated coronavirus have been identified to date (http://www.ncbi.nlm.nih.gov/genomes/SARS/SARS.html). These strains have a number of features in common. One strain has been characterized in detail (Rota et al., 2003, Science 300: 1394-9). Upon infection into the cell, which occurs via endocytosis and membrane fusion, the 5 '-most open frame of the viral genome is translated to produce large polyprotein which is cleaved to produce several non- structural proteins which forms the basis of the polymerase complex. This is used as a template to produce negative strand RNAs that in turn act as templates for a "nested" set of mRNAs. Specifically, they all contain an identical 5 'non-translated leader sequence of 72 nucleotides. These structures are thought to be produced by discontinuous transcription of the subgenomic mRNAs and transcription regulatory regions thought to contain the intergenic region, UCUAAA Potential targets for antiviral drugs and vaccines have been proposed (see, for example, Vijayanand et al, supra; Holmes, 2003, J. Clin. Invest. I l l : 1605-1609 and Anand et al., www.scienceexpress.org//10.1126/science.1085658. published May 13, 2003). These include agents that block entry and membrane fusion, assembly, replication and transcription. Additionally, various siRNAs to coronavirus genes have been proposed (see, for example, http://wwwl.qiagen.com/literature/handbooks/PDF/siRNAForGeneSilencing/CoronavirussiR NA/HB siRNA Coronavirus 052003.pdf; http://www.ingenovis.com/siRNA/; http://www.intradigm.com/news_2.htm)

SUMMARY OF THE INVENTION The invention is directed to an agent that inhibits interaction of an intergenic sequence of coronavirus, said intergenic sequence comprising UCUAAA with the polymerase complex of said coronaviurs, or component of said polymerase complex that interacts with said intergenic sequence. In a specific embodiment, the agent blocks the action of said intergenic sequence in facilitating attachment of a leader sequence to the 3 '-ends of negative strand RNA produced. The agent could therefore act to inhibit transcription, translation and replication of the coronavirus. In a specific embodiment, the agent acts to inhibit coronavirus associated with SARS (also referred to as SARS-associated coronavirus). Furthermore, the invention is directed to compositions and kits comprising these agents. The agent may be a ribozyme, nucleic acid molecule, or siRNA. The invention is further directed to a nucleic acid construct, vector and host cell comprising said agent. These agents have various uses. In a specific embodiment, the invention is directed to a method of modulating replication of coronavirus in a host cell infected with said coronavirus or treating a viral infection (e.g., SARS) in a subject resulting from coronavirus infection comprising administering to said host cell or said subject the agent or compositions of the present invention. As used herein, "treating" includes all medically acceptable types of therapeutic intervention including palliation and prophylaxis (prevention) of disease. "Treating" also encompasses any improvement of a disease, including minor improvements. The agent may also be used in the manufacture of a medicament or in a pharmaceutical composition for treating a viral infection. A "subject" refers to mammals and in particular human beings. In a specific embodiment, the agent is an isolated nucleic acid molecule, which (a) comprises a sequence depicted in nnbnnnbnntctaaannnnnnnnn (SEQ ID NO:l) or nnbnnnbnnucuaaannnnnnnnn (SEQ ID NO:2); (b) Comprises a sequence that hybridizes to nnbnnnbnntctaaannnnnnnnn (SEQ ID NO:l) or nnbnnnbnnucuaaannnnnnnnn (SEQ ID NO:2) or (c ) comprises a sequence that is a reverse complement of (a)-(b). The nucleic acid molecule(s) of the present invention may be attached to a solid support. In a specific embodiment, the solid support is a microarray. Furthermore, the invention is directed to computer readable means for storing the nucleic acid sequences of the nucleic acid molecules of the present invention. These nucleic acid molecules can be used to detect the presence or absence of a coronavirus. In one embodiment, the method comprises (a) contacting a nucleic acid molecule present in a sample from a subject with at least one probe or primer derived from said nucleic acid molecules and (b) detecting the presence or absence of coronavirus nucleic acid molecule to detect the presence or absence of said coronavirus. In one specific embodiment, the presence or absence of a coronavirus in a sample from a subject comprising can be detected by a method comprising: (a) contacting a nucleic acid molecule present in the sample with said probe under conditions in which the probe selectively hybridizes to said coronavirus nucleic acid sequence and (b) detecting hybridization of a coronavirus nucleic acid molecule in said sample, wherein the detection of the hybridization indicates the presence of coronavirus in the sample. In another specific embodiment, the presence or absence of coronavirus in a sample from a subject can be detected in a method comprising: (a) contacting the sample with at least one primer derived from said nucleic acid molecule to provide an amplification mixture wherein said first and second primers are sufficient in length to selectively hybridize to said coronavirus nucleic acid sequence and wherein said primer is capable of amplifying a detectable part of a coronavirus nucleic acid if said coronavirus is present in the sample; (b) subjecting the amplification mixture to nucleic acid amplification and (c) detecting whether a part of a coronavirus nucleic acid molecule has been amplified wherein detection of a part indicates the presence of coronavirus in the sample. These nucleic acid molecules may also be used in monitoring treatment of a disease or disorder resulting of coronavirus in a subject comprising (a) contact-rig a sample from a subject periodically with the nucleic acid molecules of the present invention and (b) periodically determining the amount of coronavirus present in said sαbject. Furthermore, the invention is directed to a method for identifying an agent that modulates replication of coronavirus comprising determining if said agent interferes with interaction of said intergenic sequence with said polymerase complex: or portion thereof. The agents of the present invention may be used to identify other agents that interfere with the interaction of the intergenic sequence comprising UCUAAA with the polymerase complex or portion thereof by comparing the effect of said agents of the present invention and candidate agent on interference of interaction of the intergenic sequence depicted in with the polymerase complex or portion thereof of the coronavirus or alternatively in a competitive inhibition assay.

DEFINITIONS AND TERMS In the nucleic acid sequences set forth in the application and claims, below is a table listing the nucleotides symbols depicted in each of the nucleic acids set forth and their meaning

The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims. Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Whiere the stated range includes one or both of the limits, ranges excluding either both of those included limits are also included in the invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described. It must be noted that as used herein and in the appended claims, the singular forms "a," "and" and "the" include plural references unless the context clearly dictates otherwise.

DETAILED DESCRIPTION OF THE INVENTION The agent of the present invention inhibits interaction of an intergenic sequence of coronavirus with polymerase complex or component of said polymerase complex that interacts with said intergenic sequence. The polymerase complex contains several non- structural proteins, one of which is an RNA-dependent RNA polymerase and another is an adenosine triphosphatase helicase. In a specific embodiment, the coronavinxs is a SARS- associated coronavirus. The SARS-associated coronavirus includes but is not limited to SARS coronavirus BJ04 (ID No. AY279354); SARS coronavirus BJ03 (ID No. AY278490); SARS coronavirus BJ02 (ID No. AY278487); SARS coronavirus ZJ01(AY^"297028); SARS coronavirus (ID No. NC004718); SARS coronavirus CUHK-W1 (AY278554), SARS coronavirus BJ01 (ID No. AY278488); SARS coronavirus TOR2 (AY2741 19); SARS coronavirus TWl (ID no. AY291451); SARS coronavirus isolate Sin2774 QD No. AY283798); SARS coronavirus isolate SIN2748 (AY283797); SARS coronavirus isolate SIN2679 (ID No. 283796); SARS coronavirus isolate SIN2677 (ID No. 283795); SARS coronavirus isolate SIN2500 (ID No. AY283794); SARS coronavirus CUHK-SulO (ID No. AY282752); SARS coronavirus GZ50 (ID No. AY304495); SARS coronavirus HSR1 (AY323977); complete genome, SARS coronavirus Frankfurt 1 (AY291315); SARS coronavirus Taiwan TCI (AY338174); SARS coronavirus Taiwan TC2 (A T 338175); SARS coronavirus Taiwan TC3 (AY348314); SARS coronavirus TWC (ID No. AΥ321118); SARS coronavirus TW9 (ID No. AY502932) SARS coronavirus TW8 (ID No. AY502931) SARS coronavirus TW7 (ID No. AY502930) SARS coronavirus TW6 (ID No. AY502929) SARS coronavirus TW5 (ID No. AY502928) SARS coronavirus TW4 (ID No. AY502927) SARS coronavirus TW3 (ID No. AY502926) SARS coronavirus TW2 (ID No. AY502925) SARS coronavirus TWl (ID No. AY502924) SARS coronavirus TW10 (ID No. NY502923); SARS coronavirus ShanghaiQXCl (ID No. AY502959); SARS coronavirus ShanghaiQXC2 (ID No. AY502960); SARS coronavirus GD69 (ID No. AY313906); SARS coronavirus FRA (ID No. AY310120); SARS coronavirus SoD(ID No. AY461660); SARS coronavirus Sino3- 11 (ID No. AY485278) SARS coronavirus Sinol-11 (ID No. AY485277) SARS coronavirus CUHK-AG03 (ID No. AY345988); SARS coronavirus CUHK-AG02 (ID No. AY345987); SARS coronavirus CUHK-AG01 (ID No. AY345986); SARS coronavirus PUMC03 (ID No. AY357076); SARS coronavirus PUMC02 (ID No. AY357075); SARS coronavirus PUMC01 (ID No. AY350750); SARS coronavirus SZ16 (ID No. AY304488); SARS coronavirus SZ3 (ID No. AY304486); SARS coronavirus AS (ID No. AY427439); SARS coronavirus TWC3 (ID No. AY362699); SARS coronavirus TWC2 (ID No. AY362698); SARS coronavirus TWY (ID No. AP006561); SARS coronavirus TWS (ID No. AP006560); SARS coronavirus TWK (ID No. AP006559); SARS coronavirus TWJ (ID No. AP006558); SARS coronavirus TWH (ID No. AP006557); Embodiments are described below. Generally, nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art. The methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. See, e.g., Sarnbrook et al, Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press (1989) and Sarnbrook et al., Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Press (2001); Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992, and Supplements to 2000); Ausubel et al., Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology~4th Ed., Wiley & Sons (1999). Enzymatic reactions and purification techniques are performed according to manufacturer's specifications, as commonly accomplished in the art or as described herein. The nomenclatures used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery.

Agents The agents of the present invention may be an isolated nucleic acid molecule or nucleic acid molecule. As defined herein, "isolated nucleic acid molecule" is a nucleic acid molecule which is substantially separated from other cellular components that naturally are associated with a native nucleic acid molecule in its natural host cell, e.g., ribosomes, polymerase and is removed from its original environment and is thus altered "by the hand of man" from its natural state.. The terms "nucleic acid molecule" and "polynucleotide" are synonymous and will be used interchangeably. As defined herein, " nucleic acid molecule" or "polynucleotide" refers to a polymeric form of nucleotides and includes RNA, cDNA, genomic DNA and synthetic forms and mixed polymers of the above. The term "nucleic acid molecule" refers to a molecule of at least 10 bases in length. A polynucleotide may contain naturally occurring and/or modified nucleotides linked together by naturally occurring and/or non-naturally occurring nucleotide linkages. Modifications of nucleotides include but are not limited to labels, methylation and substitution with an analog. The polynucleotides may also contain modified oligonucleotide backbones. These backbones include, without limitation, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. Representative United States patents that teach the preparation of the above phosphorus-containing linkages include but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; and 5,625,050. Other modified oligonucleotide backbones do not include a phosphorus atom, but have backbones that are formed by short chain alkyl or cycloalkyl intemucleoside linkages, mixed heteroatom and alkyl or cycloalkyl intemucleoside linkages, or one or more short chain heteroatomic or heterocyclic intemucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulf oxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH₂ component parts. Representative U.S. patents that teach the preparation of the above backbones include, but are not limited to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437 and 5,677,439. The nucleic acid molecules of the present invention include oligonucleotide mimetics, where both the sugar and the intemucleoside linkage are replaced with novel groups, such as peptide nucleic acids (PNA). In PNA compounds, the phosphodiester backbone of the nucleic acid is replaced with an amide-containing backbone, in particular by repeating N-(2- aminoethyl) glycine units linked by amide bonds. Nucleobases are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone, typically by methylene carbonyl linkages. PNA can be synthesized using a modified peptide synthesis protocol. PNA oligomers can be synthesized by both Fmoc and tboc methods. Representative U.S. patents that teach the preparation of PNA compounds include but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by reference.

Automated PNA synthesis is readily achievable on commercial synthesizers (see, e.g., "PNA

User's Guide," Rev. 2, February 1998, Perseptive Biosystems Part No. 60138, Applied

Biosystems, Inc., Foster City, Calif). The nucleic acid molecule may interact with the negative strand of the coronavirus RNA in the cytoplasm to interfere with its transcription to messenger RNA. The nucleic acid molecule may in one embodiment include but is not limited to: (a) a nucleic acid molecule comprising a sequence depicted in SEQ ID NO:l or SEQ ID NO:2; (b) one or more nucleic acid molecules that hybridizes to SEQ ID NO: 1 or SEQ ID NO: 2 and (c) a reverse complement of (a)-(b). In a specific embodiment, the nucleic acid molecule includes but is not limited to (a) a nucleic acid molecule comprising sequence depicted in SEQ ID NO: 3

(wsnstctaaacgmacwwrm) or SEQ ID NO:4 (wsnsucuaaacgmacwwrm); (b) a nucleic acid molecule that hybridizes to SEQ ID NO: 2 or SEQ ID NO: 3 and (c ) a reverse complement of

(a)-(b). In a more specific embodiment, the nucleic acid molecule is depicted in ag gtctaaacgaactaac (SEQ ID NO:5) or agngucuaaacgaacuaac (SEQ ID NO:6). In even more specific embodiments, the nucleic acid molecule has the sequence selected from the group consisting of:

5'-gatctgttctctaaacgaacttta-3'(SEQ ID NO:7) 5'-tggcatcgatctaaagtcttatga-3'(SEQ ID NO:8)

5'-gctgaactctctaaatgagccgct-3'(SEQ ID NO:9)

5'-cccaaattttctaaattgttcaca-3'(SEQ ID NO: 10)

5'-actgtacagtctaaaatgtctgac-3'(SEQ ID NO:l 1)

5'-tttgctgtgtctaaaggtttcttt-3'(SEQ ID NO: 12) 5'-gagaatgactctaaagaagggttt-3'(SEQ ID NO:13)

5'-tttgctgtttctaaacccatgggt-3'(SEQ ID NO: 14)

5'-acctgaccctctaaagccaactaa-3'(SEQ ID NO: 15)

5'-atcttctggtctaaacgaactaac-3'(SEQ ID NO: 16)

5'-gctgaggcatctaaaaagcctcgc-3'(SEQ ID NO:17) 5'-gaucuguucucuaaacgaacuuua-3'(SEQ ID NO: 18)

5'-uggcaucgaucuaaagucuuauga-3'(SEQ ID NO: 19)

5'-gcugaacucucuaaaugagccgcu-3'(SEQ ID NO:20)

5'-cccaaauuuucuaaauuguucaca-3'(SEQ ID NO:21) 5'-acuguacagucuaaaaugucugac-3'(SEQ ID NO: 22) 5'-uuugcugugucuaaagguuucuuu-3'(SEQ ID NO:23) 5'-gagaaugacucuaaagaaggguuu-3'(SEQ ID NO: 24) 5'-uuugcuguuucuaaacccaugggu-3'(SEQ ID NO:25) 5'-accugacccucuaaagccaacuaa-3'(SEQ ID NO:26) 5'-aucuucuggucuaaacgaacuaac-3'(SEQ ID NO: 27) 5'-gcugaggcaucuaaaaagccucgc-3'(SEQ ID NO: 28)

In a more specific embodiment, the nucleic acid molecule has the sequence: 5'- gatctgttctctaaacgaacttta-3'(SEQ ID NO:7) or 5'-tggcatcgatctaaagtcttatga-3'(SEQ ID NO:8), 5'- gaucuguucucuaaacgaacuuua-3'(SEQ ID NO:18), 5'-uggcaucgaucuaaagucuuauga-3'(SEQ ID NO: 19). A polynucleotide "hybridizes" to another polynαcleotide, when a single-stranded form of the polynucleotide can anneal to the other polynucleotide under the appropriate conditions of temperature and solution ionic strength (see Sarnbrook et al., supra). The conditions of temperature and ionic strength determine the "stringency" of the hybridization. For preliminary screening for homologous nucleic acids, low stringency hybridization conditions, corresponding to a temperature of 42°C, can be used, e.g., 5X SSC, 0.1% SDS, 0.25% milk, and no formamide; or 40% formamide, 5X SSC, 0.5%SDS). Moderate stringency hybridization conditions correspond to a higher temperature of 55°C. The polynucleotides may also, e.g., 40% formamide, with 5X or 6X SCC. High stringency hybridization conditions correspond to the highest temperature of 65°C, e.g., 50 % formamide, 5X or 6X

SSC. Hybridization requires that the two nucleic acids contain complementary sequences, although depending on the stringency of the hybridization, mismatches between bases are possible. The appropriate stringency for hybridizing nucleic acids depends on the length of the nucleic acids and the degree of complementation, variables well known in the art. The greater the degree of similarity or homology between two nucleotide sequences, the greater the value of T_m for hybrids of nucleic acids having those sequences. The relative stability (corresponding to higher Tm) of nucleic acid hybridizations decreases in the following order: RNA:RNA, DNA:RNA, DNA:DNA. The reverse complement of the polynucleotide depicted in SEQ ID NO: 1 or SEQ ID NO:2 is depicted in SEQ ID NO:29 (nnnnnnnnntttaganαdnnndnn) or SEQ ID NO:30 (nnnnnnnnnuuuaganndnnndnn). In a more specific embodiment, the reverse complement of the polynucleotide depicted in SEQ ID NO:3 and 4 is depicted in SEQ ID NO:31 (kywwgtbcgtttagasnsw) and SEQ ID NO:32 (kywwgubcguuuagasnsw). In an even more specific embodiment, it has the sequence depicted in SEQ ID NO: 33 (gttagttcgtttagacnct) or SEQ ID NO: 34 (guuaguucguuuagacncu). In yet a more specific embodiment, it has the sequence selected from the group consisting of

5 '-taaagttcgtttagagaacagatc-3' (SEQ ID NO:35)

5'-tcataagactttagatcgatgcca-3' (SEQ ID NO:36) 5'-agcggctcatttagagagttcagc-3' (SEQ ID NO:37)

5'-tgtgaacaatttagaaaatttggg-3' (SEQ ID NO: 38)

5'-gtcagacattttagactgtacagt-3' (SEQ ID NO:39)

5'-aaagaaacctttagacacagcaaa-3' (SEQ ID NO:40)

5'-aaacccttctttagagtcattctc-3' (SEQ ID NO:41) 5'-acccatgggtttagaaacagcaaa-3'(SEQ ID NO:42)

5'-ttagttggctttagagggtcaggt-3'(SEQ ID NO:43)

5'-gttagttcgtttagaccagaagat-3'(SEQ ID NO:44)

5'-gcgaggctttttagatgcctcagc-3'(SEQ ID NO:45)

5'-uaaaguucguuuagagaacagauc-3' (SEQ ID NO:46) 5'-ucauaagacuuuagaucgaugcca-3' (SEQ ID NO:47)

5'-agcggcucauuuagagaguucagc-3' (SEQ ID NO:48)

5'-ugugaacaauuuagaaaauuuggg-3' (SEQ ID NO:49)

5'-gucagacauuuuagacuguacagu-3' (SEQ ID NO:50)

5'-aaagaaaccuuuagacacagcaaa-3' (SEQ ID NO:51) 5'-aaacccuucuuuagagucauucuc-3' (SEQ ID NO:52)

5'-acccauggguuuagaaacagcaaa-3'(SEQ ID NO:53)

5'-uuaguuggcuuuagagggucaggu-3'(SEQ ID NO: 54)

5'-guuaguucguuuagaccagaagau-3'(SEQ ID NO:55)

5'-gcgaggcuuuuuagaugccucagc-3'(SEQ ID NO:56) In a most specific embodiment, the nucleic acid molecule has the sequence: 5'- taaagttcgtttagagaacagatc-3' (SEQ ID NO:35), 5'-tcataagactttagatcgatgcca-3' (SEQ ID NO:36),

5'-uaaaguucguuuagagaacagauc-3' (SEQ ID NO:46) or 5'-ucauaagacuuuagaucgaugcca-3'

(SEQ ID NO:47). The nucleic acid molecules of the present invention are at least 24 nucleotides in length and may be 25 nucleotides in length or 30, 40 or 50 nucleotides in length. The agent may also be an antisense nucleic acid to the intergenic sequence comprising

UCUAAA. As defined herein, an "antisense" nucleic acid molecule is a nucleic acid molecule that interacts with the positive strand of said coronavirus. The antisense nucleic acid molecule may be 10 to 60 bases in length. Preferably, the antisense nucleic acid is 19 or 20 bases in length but may also be 25, 30, 35, 40, 45 50 55 or 50 bases in length. The agent may also be a ribozyme. Specifically, the ribozyme may be specific to the intergenic sequence comprising UCUAAA. Ribozymes are RNA-protein complexes that cleave nucleic acids in the site-specific fashion. A ribozyme targets the RNA genome and RNA transcripts and copies thereof. Each ribozyme molecule contains a catalytically active segment capable of cleaving the plus or minus strand of the coronavirus RNA, and further comprises flanking sequences having a nucleotide sequence complementary to portions of the target RNA. The flanking sequences serve to anneal the ribozyme to the RNA in a site- specific manner. Absolute complementarity of the flanking sequences to the target sequence is not necessary, however, as only an amount of complementarity sufficient to form a duplex with the target RNA and to allow the catalytically active segment of the ribozyme to cleave at the target sites is necessary. Thus, only sufficient complementarity to permit the ribozyme to be hybridizable with the target RNA is required. In preferred embodiments of the present invention the enzymatic RNA molecule is formed in a hammerhead motif but the ribozyme may also be formed in the motif of a hairpin, hepatitis delta virus, group I intron or RNAse P RNA (in association with an RNA guide sequence). Examples of hammerhead motifs are described by Rossi et al., AIDS Res. Hum. Retrovir. 8:183 (1992), hairpin motifs are described by Hampel et al., Biochem. 28:4929 (1989) and Hampel et al., Nucl. Acids Res. 18:299 (1990), the hepatitis delta vims motif is exemplified in Perrotta and Been, Biochem. 31:16 (1992), an RNAseP motif is described in Gueerier-Ta ada et al., Cell 35:849 (1983), and examples of the group I intron motif are described in Cech et al., U.S. Pat. No. 4,987,071, each of the foregoing disclosures being incorporated herein by reference. Furthermore, the agent of the present invention may be a small or short interfering RNA (siRNA) for RNA interference (RNAi) strategy (PNAS USA, 2003, 100, 2014-2018). The term siRNA means short interfering RNA which is double-stranded RNA that is less than 30 bases and preferably 21-25 bases in length and inhibits expression of a target nucleic acid molecule. In a specific embodiment it is substantially identical to the intergenic region depicted UCUAAA. As defined herein, "substantially identical" means it has at least 80% identity to said region and/or sequence, It may also have 85%, 90%, 95%, 97%, 98%, and 99% identity. The length of sequence identity comparison may be over a stretch of at least about nine nucleotides, usually at least about 20 nucleotides, more usually at least about 24 nucleotides. As a practical matter, whether any particular nucleic acid molecule or polypeptide is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a nucleotide sequence of the presence invention can be determined conventionally using known computer programs. Examples include but are not limited to programs in Wisconsin Package Version 10.0, Genetics Computer Group (GCG), Madison, Wis. FASTA, which includes, e.g., the programs FASTA2 and FASTA3, provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences (Pearson, Methods Enzymol. 183: 63-98 (1990); Pearson, Methods Mol. Biol. 132: 185-219 (2000); Pearson, Methods Enzymol 266: 227-258 (1996); Pearson, J Mol. Biol. 276: 71-84 (1998)). Unless otherwise specified, default parameters for a particular program or algorithm are used. For instance, percent sequence identity between nucleic acid sequences can be determined using FASTA with its default parameters (a word size of 6 and the NOP AM factor for the scoring matrix) or using Gap with its default parameters as provided in GCG Version 6.1. The agents of the present invention (polynucleotides, antisense nucleic acid molecules, siRNA, ribozymes) can be prepared using methods known in the art. For example, they can be chemically synthesized by standard ethods known in the art, e.g., by use of an automated DNA synthesizer which is commercially available (e.g., Applied Biosystems, Biosearch). Furthermore, the agents of the present invention may also be prepared αsing other methods known in the art, e.g., polymerase chain reaction using primers having an appropriate sequence or restriction digestion of cloned nucleic acid fragments (see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Press (2001); Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992, and Supplements to 2000); Ausubel et al., Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology--4th Ed., Wiley & Sons (1999)). In a specific embodiment, ribozymes, polynucleotides or siRNA may be prepared by chemical synthesis or produced by recombinant vectors according to methods established for the synthesis of nucleic acid molecules. See, e.g., Sambrook et al., Molecular Cloning, A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001). The polynucleotide of the present invention may be expressed by inserting the nucleic acid sequence or a nucleic acid construct comprising the sequence into an appropriate vector for expression. In creating the expression vector, the polynucleotide is located in the vector so that it is operably linked with the appropriate control sequences for expression. The ribozyme sequence may be synthesized, for example, using RNA polymerases such as T7 or

SP6.

Substrate In a specific embodiment, the nucleic acid molecules of the present invention, may be attached to a substrate. A substrate may be solid including, without limitation, glass, amorphous silicon, crystalline silicon, paper or plastics. Examples of plastics include, without limitation, polymethylacrylic, polyethylene, polypropylene, polyacrylate, polymethylmethacrylate, polyvinylchloride, polytetrafluoroethylene, polystyrene, polycarbonate, polyacetal, poly sulf one, celluloseacetate, cellulosenitrate, nitrocellulose, or mixtures thereofor porous, a membrane, typically comprising nitrocellulose, nylon, or positively-charged derivati-zed nylon planar or non-planar, unitary or distributed. The nucleic acid molecule may be attached covalently or applied to a derivatized surface in a chaotropic agent that facilitates denaturation and adherence by presumed noncovalent interactions, or some combinations thereof. In a more specific embodiment, the substrate is a microarray. "Micro-array" as defined herein is a substrate-bound collection of a plurality nucleic acids, hybridization to each of the plurality of bound nucleic acids being separately detectable. The microarray may comprise a plurality of nucleic acid molecules depicted in SEQ ID NO: 1,2, 29 and/or 30, more specifically in SEQ ID NOS: 3, 4, 31 and/or 32), even more specifically in (SEQ ID NOS: 5, 6, 33 and/or 34), and most specifically in SEQ ID NOS: 7-28 and 35-56. Alternatively the microarray may further comprise other coronavirus nucleic acid molecules.

Computer Readable Means A further aspect of the invention is a compixter readable means for storing the nucleic acid sequences of the instant invention. In a preferred embodiment, the invention provides a computer readable means for storing, for example sequences encompassing SEQ ID NOS:2- 49 as described herein, as the complete set of sequences or in any combination. The records of the computer readable means can be accessed for reading and display and for interface with a computer system for the application of programs allowing for the location of data upon a query for data meeting certain criteria, the comparison of sequences, the alignment or ordering of sequences meeting a set of criteria, and the like. The nucleic acid sequences of the invention are particularly useful as components in databases useful for search analyses as well as in sequence analysis algorithms. As used herein, the terms "nucleic acid sequences of the invention" mean any detectable chemical or physical characteristic of a nucleic acid molecule of the invention that is or may be reduced to or stored in a computer readable form. These include, without limitation, chromatographic scan data or peak data, photographic data or scan data therefrom, and mass spectrographic data. This invention provides computer readable media having stored thereon sequences of the invention. A computer readable medium may comprise one or more of the following: a nucleic acid sequence comprising a sequence of a nucleic acid sequence of the invention; a set of nucleic acid sequences wherein at least one of said sequences comprises the sequence of a nucleic acid sequence of the invention; a data set representing a nucleic acid sequence comprising the sequence of one or more nucleic acid sequences of the invention; a set of nucleic acid sequences wherein at least one of said sequences comprises the sequence of a nucleic acid sequence of the invention; a data set representing a nucleic acid sequence comprising the sequence of a nucleic acid sequence of the invention. The computer readable medium can be any composition of matter used to store information or data, including, for example, commercially available floppy disks, tapes, hard drives, compact disks, and video disks. Also provided by the invention are methods for the analysis of character sequences, particularly genetic sequences. Preferred methods of sequence analysis include, for example, methods of sequence homology analysis, such as identity and similarity analysis, RNA structure analysis, sequence assembly, cladistic analysis, sequence motif analysis, open reading frame determination, nucleic acid base calling, and sequencing chromatogram peak analysis. A computer-based method is provided for performing nucleic acid sequence identity or similarity identification, particularly a method for identifying potential agents that interferes with interaction of a coronavirus intergenic sequence comprising UCUAAA with a coronaviras polymerase complex. This method comprises the steps of providing a nucleic acid sequence comprising the sequence of a nucleic acid of the invention in a computer readable medium; providing a library of nucleic acid sequences and comparing said nucleic acid sequence to at least one nucleic acid sequenceto identify sequence identity or similarity.

Uses Diagnostics The present invention also relates to qualitative and quantitative diagnostic assays, kits and methods for detecting, diagnosing and monitoring coronaviras infection (particularly SARS) in a sample from a subject by detecting the presence or absence of coronaviras in said sample. The sample may be obtained from cells, tissue, feces or bodily fluids such as blood, saliva, respiratory secretions and urine from said subject. In a specific embodiment, the presence or absence a coronavirus nucleic acid molecule is detected using probes or primers derived from the nucleic acid molecules of the present invention. The presence of the coronaviras nucleic acid molecule or alternatively increased amount of said coronavirus nucleic acid molecule compared to an earlier time point or compared to levels of coronavirus nucleic acid present from a sample from an uninfected subject indicates that the subject has been infected. A probe or primer is at least 10 nucleotides in length, more preferably at least 12, more preferably at least 14 and even more preferably at least 16 or 17 nucleotides in length. In an even more preferred embodiment, the probe or primer is at least 18 nucleotides in length, even more preferably at least 19 nucleotides. Primers and probes may also be longer in length. For instance, a probe or primer may be 20, 25 nucleotides in length, or may be 30, 40 or 50 nucleotides in length.

Methods The expression of coronavirus is measured by determining the amount of coronavirus nucleic acid expressed using methods known in the art including but not limited to Northern blot, microarray, dot or slot blots, in situ hybridization and qualitative or quantitative reverse transcriptase PCR. In a specific embodiment, the nucleic acid molecule(s) of the present invention or probes derived from the nucleic acid molecules of the present invention are hybridized to a nucleic acid obtained from a sample or in situ, and the presence or absence of the coronaviras nucleic acid is detected. The probe may be part of a microarray on a solid support. In a specific embodiment, the nucleic acid molecule is labeled with a any detectable reporter or signal moiety including, but not limited to radioisotopes, enzymes, antigens, antibodies, spectrophotometiric reagents, chemiluminescent reagents, fluorescent and another light producing chemicals. Additionally, these probes may be modified without changing the substance of their purpose by terminal addition of nucleotides designed to incorporate restriction sites or other useful sequences, proteins, signal generating ligands such as acridinium esters, and/or paramagnetic particles. These probes may also be modified by the addition of a capture moiety (including, but not limited to paramagnetic particles, biotin, fluorescein, dioxigenin, antigens, antibodies) or attached to the walls of microtiter trays to assist in the solid phase capture and purification of these probes and any DNA or RNA hybridized to these probes. Fluorescein may be used as a signal moiety as well as a capture moiety, the latter by interacting with an anti-fluorescein antibody. In another specific embodiment, an RNA extracted from a sample is transcribed in a reaction mixture containing one or more primers derived from the nucleic acid molecules of the present invention. Methods of performing primer-directed amplification are also well- known in the art; see, for example, McPherson, PCR Basics: From Background to Bench, Springer Verlag (2000); Innis et al. (eds.), PCR Applications: Protocols for Functional Genomics, Academic Press (1999); Gelfand et al. (eds.), PCR Strategies, Academic Press (1998); Newton et al, PCR, Springer- Verlag New York (1997); Burke (ed.), PCR: Essential Techniques, John Wiley & Son Ltd (1996); White (ed.), PCR Cloning Protocols: From Molecular Cloning to Genetic Engineering, Vol. 67, Humana Press (1996); McPherson et al. (eds.), PCR 2: A Practical Approach, Oxford University Press, Inc. (1995). Methods for performing RT-PCR are collected, e.g., in Siebert et al. (eds.), GeneCloning and Analysis by RT-PCR, Eaton Publishing Company/Bio. The TechniquesBooks Division, 1998; Siebert (ed.), PCR Technique:RT-PCR, Eaton Publishing Company/BioTechniques Books (1995). Amplified nucleic acid may be detected using methods known in the art, by, for example detecting the labeled amplified nucleic acid or alternatively by mass spectrometry. The agents, particularly nucleic acid molecules of the present invention, may also be used to monitor populations of subjects having susceptibility to diseases resulting from coronaviras infection, eg., SARS. Specifically, samples are obtained at various times. In the case of SARS, it would be over a 7-21 day period. Probes or primers derived from the nucleic acid molecules of the present invention may be used to detect and quantitate the amount of coronaviras present in said subject. Alternatively, the probes or primers derived from the nucleic acid molecules of the present invention may be used to monitor the progress of treatment. Again nucleic acid molecules from a sample from a subject are contacted with probes or primers derived from the nucleic acid molecules of the present invention are contacted with samples from a subject obtained at various times. A decrease in the amount of coronaviras nucleic acid detected is indicative of effectiveness of treatment. The agents of the present invention may also be used to identify an agent effective for modulating replication of coronavirus by comparing the ability of said candidate agent to inhibit interaction of the intergenic region with the coronavirus polymerase complex or portion thereof with the agent of the present invention. Alternatively the effectiveness of the candidate agent may be determined by measuring the effectiveness of said candidate agent in inhibiting the action of the agent of the present invention in modulating the replication of the coronavirus.

Kits The invention is further directed to diagnostic kits. These comprise a nucleic acid molecule(s) of the present invention and/or probe or primer derived from said nucleic acid molecule. The kits may further comprise other coronaviras sequences, instractions for use and at least one reagent necessary for perfomu-ng the assay, such as a buffer and/or reverse transcriptase. In another embodiment, the kits of the present invention comprise microa-rrays of probes to detect coronaviras in a sample.

Therapeutic The agents of the present invention may be used to modulate replication of coronaviras in a host cell infected with said coronaviras and thus treat a viral infection resulting from a coronaviras in a subject. In one embodiment, on nucleic acid molecule of the present invention is administered. In another embodiment, a plurality of nucleic acid molecules of the present invention are administered. The agents and particularly the nucleic acid molecules of the present invention may be expressed from a viral vector, often a vector based upon a replication incompetent retro virus, an adenoviras, or an adeno-associated virus (AAV) — for the purpose of gene therapy. The agents of the present invention (e.g., nucleic acid molecules) may be introduced directly to the subject using methods known in the art (see, for example, Rolland, 1998, Crit. Rev. Therap. Drug Carrier Systems 15:143-198 and references cited therein and Ulmer, 1993, Science 259: 1745-1749). The uptake of naked nucleic acid molecule may be increased by coating the nucleic acid molecule onto biodegradable beads, which are efficiently transported into the cells. Other methods to directly introduce the agents of the present invention into cells or exploit receptors on the surface of cell^'s include but are not limited to the use of liposomes and lipids, ligands for specific cell surface receptors, cell receptors, and calcium phosphate and other chemical mediators, microinjections directly to single cells, electroporation and homologous recombination. Liposomes are commercially available from Gibco BRL, for example, as LIPOFECTIN^" and LIPOFECTACE^", which are formed of cationic lipids such as N-[l-(2,3 dioleyloxy)-propyl]-n,n,n-trimethylammonium chloride (DOTMA) and dimethyl dioctadecylammonium bromide (DDAB). Numerous methods are also published for making liposomes, known to those skilled in the art. For example, nucleic acid- lipid complexes— lipid carriers can be associated with naked nucleic acids (e.g., plasmid DNA) to facilitate passage through cellular membranes. Cationic, anionic, or neutral lipids can be used for this purpose. However, cationic lipids are preferred because they have been shown to associate better with DNA which, generally, h-as a negative charge. Cationic lipids have also been shown to mediate intracellular delivery of plasmid DNA (Feigner and Ringold, Nature 337:387 (1989)). Intravenous injection of cationic lipid- plasmid complexes into mice has been shown to result in expression of the DNA. in lung (Brigham et al., Am. J. Med. Sci.298:278 (1989)). See also, Osaka et al., J. Ptiarm. Sci. 85(6):612-618 (1996); San et al., Human Gene Therapy 4:781-788 (1993); Senior et al., Biochemica et Biophysica Acta 1070:173-179 (1991); Kabanov and Kabanov, Bioconjugate Chem. 6:7-20 (1995); Remy et al., Bioconjugate Chem. 5:647-654 (1994); Behr, J-P., Bioconjugate Chem 5:382-389 (1994); Behr et al., Proc. Natl. Acad. Sci., USA. 86:6982- 6986 (1989); and Wyman et al., Biochem. 36:3008-3017 (1997). Cationic lipids are known to those of ordinary skill in the art. Representative cationic lipids include those disclosed, for example, in U.S. Pat. No. 5,283,185; and e.g., U.S. Pat. No. 5,767,099. In a preferred embodiment, the cationic lipid is N.sup.4 -spernxine cholesteryl carbamate (GL-67) disclosed in U.S. Pat. No. 5,767,099. Additional preferred lipids include N4 _spermidine cholestryl carbamate (GL-53) and 1-(N4 -spermi-nd) -2,3- dilaurylglycerol carbamate (GL-89). In another embodiment of the therapeutic methods of the present invention, a therapeutically effective amount of a pharmaceutical composition comprising the agent of the present invention is administered. Such a composition typically contains from about 0.1 to 90% by weight of a therapeutic agent of the invention formulated in and/or with a pharmaceutically acceptable carrier or excipient. Pharmaceutical formulation is a well- established art, and is further described in Gennaro (ed.), Remington: The Science and Practice of Pharmacy, 20th ed., Lippincott, Williams & Wilkins (2000); Ansel et al.,

Pharmaceutical Dosage Forms and Drag Delivery Systems, 7th ed., Lippincott Williams & Wilkins (1999); and Kibbe (ed.), Handbook of Pharmaceutical Excipients American Pharmaceutical Association, 3rd ed. (2000). Briefly, formulation of the pharmaceutical compositions of the present invention will depend upon the route chosen for administration. The pharmaceutical compositions utilized in this invention can be administered by various routes including both enteral and parenteral routes, including oral, intravenous, intramuscular, subcutaneous, inhalation, intrathecal, intraventricular, transmucosal, transdermal, intranasal, intraperitoneal, and intrapulmonary. The pharmaceutical composition may comprise one or more agents of the present invention. Oral dosage forms can be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for ingestion by the patient. Solid formulations of the compositions for oral administration can contain suitable carriers or excipients, such as carbohydrate or protein fillers, such as sugars, including lactose, sucrose, mannitol, or sorbitol; starch from com, wheat, rice, potato, or other plants; cellulose, such as meth l cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, or microcrystalline cellulose; gums including arabic and tragacanth; proteins such as gelatin and collagen; inorganics, such as kaolin, calcium carbonate, dicalcium phosphate, sodium chloride; and other agents such as acacia and alginic acid. Agents that facilitate disintegration and/or solubilization can be added, such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof, such as sodium alginate, microcrystalline cellulose, corn starch, sodium starch glycolate, and alginic acid. Tablet binders that can be used include acacia, methylcellulose, carboxymethylcellulose, polyvinylpyrrolidone (Povidon™), hydroxypropyl methylcellulose, sucrose, starch and ethylcellulose. Lubricants that can be used include magnesium stearates, stearic acid, silicone fluid, talc, waxes, oils, and colloidal silica. Fillers, agents that facilitate disintegration and/or solubilization, tablet binders and lubricants, including the aforementioned, can be used singly or in combination. Solid oral dosage forms need not be uniform throughout. For example, dragee cores can be used in conjunction with suitable coatings, such as concentrated sugar solutions, which can also contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Oral dosage forms of the present invention include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a coating, such as glycerol or sorbitol. Push-fit capsules can contain active ingredients mixed with a filler or binders, such as lactose or starches, lubricants, such as talc or magnesium stearate, and, optionally, stabilizers. In soft capsules, the active compounds can be dissolved or suspended in suitable liquids, such as fatty oils, liquid, or liquid polyethylene glycol with or without stabilizers. Additionally, dyestuffs or pigments can be added to the tablets or dragee coatings for product identification or to characterize the quantity of active compound, i.e., dosage. Liquid formulations of the pharmaceutical compositions for oral (enteral) administration are prepared in water or other aqueous vehicles and can contain various suspending agents such as methylcellulose, alginates, tragacanth, pectin, kelgin, carrageenan, acacia,polyvinylpyrrolidone, and polyvinyl alcohol. The liquid formulations can also include solutions, emulsions, syrups and elixirs containing, together with the active compound(s), wetting agents, sweeteners, and coloring and flavoring agents. The pharmaceutical compositions of the present invention can also be formulated for parenteral administration. Formulations for parenteral administration can be in the form of aqueous or non-aqueous isotonic sterile injection solutions or suspensions. For intravenous injection, water soluble versions of the compounds of the present invention are formulated in, or if provided as a lyophilate, mixed with, a physiologically acceptable fluid vehicle, such as 5% dextrose ("D5"), physiologically buffered saline, 0.9% saline, Hanks' solution, or Ringer's solution. Intravenous formulations may include carriers, excipients or stabilizers including, without limitation, calcium, human serum albumin, citrate, acetate, calcium chloride, carbonate, and other salts. Intramuscular preparations, e.g. a sterile formulation of a suitable soluble salt form of the compounds of the present invention, can be dissolved and administered in a pharmaceutical excipient such as Water-for-Injection, 0.9% saline, or 5% glucose solution. Alternatively, a suitable insoluble form of the compound can be prepared and administered as a suspension in an aqueous base or a pharmaceutically acceptable oil base, such as an ester of a long chain fatty acid (e.g., ethyl oleate), fatty oils such as sesame oil, triglycerides, or liposomes. Parenteral formulations of the compositions can contain various carriers such as vegetable oils, dimethylacetamide, dimethylformamide, ethyl lactate, ethyl carbonate, isopropyl myristate, ethanol, polyols (glycerol, propylene glycol, liquid polyethylene glycol, and the like). Aqueous injection suspensions can also contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Non-lipid polycationic amino polymers can also be used for delivery. Optionally, the suspension can also contain suitable stabilizers or agents that increase the solubility of the compounds to allow for the preparation of highly concentrated solutions. Pharmaceutical compositions of the present invention can also be formulated to permit injectable, long-term, deposition. Injectable depot forms may be made by formir g microencapsulated matrices of the compound in biodegradable polymers such as polylactide- polyglycolide. Depending upon the ratio of drug to polymer and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) andpoly (anhydrides). Depot injectable formulations are also prepared by entrapping the drag in microemulsions that are compatible with body tissues. Inhalation formulations can also readily be formulated. For inhalation, various powder and liquid formulations can be prepared. For aerosol preparations, a sterile formulation of the compound or salt form of the compound may be used in inhalers, such as metered dose inhalers, and nebulizers. Aerosolized forms may be especially useful for treating respiratory disorders. Alternatively, the compounds of the present invention can be in powder form for reconstitution in the appropriate pharmaceutically acceptable carrier at the time of delivery. The pharmaceutically active compound in the pharmaceutical compositions of the present invention can be provided as the salt of a variety of acids, including but not limited to hydrochloric, sulfuric, acetic, lactic, tartaric, malic, and succinic acid. Salts tend to be more soluble in aqueous or other protonic solvents than are the corresponding free base forms.

After pharmaceutical compositions have been prepared, they are packaged in an appropriate container and labeled for treatment of an indicated condition. The active compound will be present in an amount effective to achieve the intended purpose. The determination of an effective dose is well within the capability of those skilled in the art. The therapeutically effective dose of the pharmaceutical agents of the present invention can be estimated initially by in vitro tests, such as cell culture assays, followed by assay in model animals, usually mice, rats, rabbits, dogs, or pigs. The animal model can also be used to determine an initial preferred concentration range and route of administration. For example, the ED₅₀ (the dose therapeutically effective in 50% of the population) and LD₅₀ (the dose lethal to 50% of the population) can be determined in one or more cell culture of animal model systems. The dose ratio of toxic to therapeutic effects is the therapeutic index, which can be expressed as LE>_5C/ED₅₀. Pharmaceutical compositions that exhibit large therapeutic indices are preferred. The data obtained from cell culture assays and animal studies are used in formulating an initial dosage range for human use, and preferably provide a range of circulating concentrations that includes the ED₅₀ with little or no toxicity. After administration, or between successive administrations, the circulating concentration of active agent varies within this range depending upon pharmacokinetic factors well known in the art, such as the dosage form employed, sensitivity of the patient, and the route of administration. The exact dosage will be determined by the practitioner, in light of factors specific to the subject requiring treatment. Factors that can be taken into account by the practitioner include the severity of the disease state, general -health of the subject, age, weight, gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy. Long-acting pharmaceutical compositions can be administered every 3 to 4 days, every week, or once every two weeks depending on half- life and clearance rate of the particular formulation. Normal dosage amounts may vary from 0.1 to 100,000 micrograms, up to a total dose of about 1 g, depending upon the route of administration. Where the therapeutic agent is a protein or antibody of the present invention, the therapeutic protein or antibody agent typically is administered at a daily dosage of 0.01 mg to 30 mg/kg of body weight of the patient (e.g., 1 mg/kg to 5 mg/kg). The pharmaceutical formulation can be administered in multiple doses per day, if desired, to achieve the total desired daily dose. Conventional methods, known to those of ordinary skill in the art of medicine, can be used to administer the phar aceutical formulation(s) of the present invention to the patient. The pharmaceutical compositions of the present invention can be administered alone, or in combination with other therapeutic agents or interventions. Specifically, the compositions of the present invention may further comprise a plurality of agents of the present invention. Furthermore, the composition may comprise another agent that inhibits the replication of coronaviras. Examples include but are not limited to agents inhibiting entry of coronavirus into a host cell and replication inhibitors. The invention described and claimed herein is not to be limited in scope by the specific embodiments herein disclosed, since these embodiments are intended as illustrations of several aspects of the invention. Any equivalent embodiments are intended to be within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. Various references are cited herein, the disclosure of which are incorporated by reference in their entireties.

Claims

WHAT IS CLAIMED IS: 1. An agent that inhibits interaction of an intergenic sequence of coronavirus, said intergenic sequence depicted in UCLT AAA with the polymerase complex of said polymerase complex or component thereof of said polymerase complex interacting with said intergenic sequence.

2. The agent according to claim 1 wherein said coronaviras is a causative agent of

Severe Acute Respiratory Syndrome or associated with SARS.

3. The agent according to claim 1, wherein said agent is selected from the group consisting of a polynucleotide, a ribozyme and a small or short interfering RNA (siRNA).

4. The agent of claim 1 , wherein said coronaviras is selected from the selected from the group consisting of SARS coronavirus BJ04 (ID No. AY279354); SARS coronavirus BJ03 (ID No. AY278490); SARS coronaviras BJ02 (ID No. AY278487); SARS coronaviras ZJ01(AY297028); SA-R-S coronaviras (ID No. NC004718); SARS coronavirus CUHK-Wl (AY278554), SARS coronavirus BJOl (ID No. AY278488); SARS coronavirus TOR2 (AY274119); SARS coronavirus TWl (ID no. AY291451); SARS coronaviras isolate Sin2774 (ID No. AY283798); SARS coronavirus isolate SIN2748 (AY283797); SARS coronavirus isolate SIN2679 (ID No. 283796); SARS coronaviras isolate SIN2677 (ID No. 283795); SARS coronavirus isolate SIN2500 (ID No. AY283794); SARS coronavirus CUHK-SulO (ID No. AY282752); SARS coronavirus GZ50 (ID No. AY304495); SARS coronavirus HSR1 (AY323977); complete genome, SARS coronaviras Frankfurt 1 (AY291315); SARS coronaviras Taiwan TCI (AY338174); SARS coronaviras Taiwan TC2 (AY338175); SARS coronaviras Taiwan TC3 (AY348314); SARS coronavirus TWC (ID No. AY321118); SARS coronavirus TW9 (ID No. AY502932) SARS coronavirus TW8 (ID No. AY502931) SARS coronaviras TW7 (ID No. AY502930) SARS coronaviras TW6 (ID No. AY502929) SARS coronavirus TW5 (ID No. AY502928) SARS coronaviras TW4 (ID No. AY502927) SARS coronavirus TW3 (ID No. AY502926) SARS coronaviras TW2 (ID No. AY502925) SARS coronavirus TWl (ID No. AY502924) SARS coronavirus TW10 (ID No. AY502923); SARS coronaviras ShanghaiQXCl (ID No. AY502959); SARS coronaviras ShanghaiQXC2 (ID No . AY502960); SARS coronavirus GD69 (ID No.

AY313906); SARS coronavirus FRA (ID No. AY310120); SARS coronavirus SoD(ID No. AY461660); SARS coronavirus Sino3-ll (ID No. AY485278) SARS coronavirus Sinol-11 (ID No. AY485277) SARS coronavirus CUHK-AG03 (ID No. AY345988); SARS coronavirus CUHK-AG02 (ID No. AY345987); SARS coronavirus CUHK-AG01 (ID No. AY345986); SARS coronaviras PUMC03 (ID No. AY357076); SARS coronaviras PUMC02 (ID No. AY357075); SARS coronaviras PUMC01 (ID No. AY350750); SARS coronavirus SZ16 (ID No. AY304488); SARS coronavirus SZ3 (ID NTo. AY304486); SARS coronaviras AS (ID No. AY427439); SARS coronaviras TWC3 (ID NTo. AY362699); SARS coronaviras TWC2 (ID No. AY362698); SARS coronaviras TWY (ID No. AP006561); SARS coronavirus TWS (ID No. AP006560); SARS coronaviras TWK (ID No. AP006559); SARS coronavirus TWJ (ID No. AP006558); SARS coronavirus TWH (ID No. AP006557).

5. A nucleic acid construct, vector or host cell comprising the agent of claim 3.

6. A composition comprising the agent of claim 1 and a carrier.

7. The composition of claim 6, which comprises a plurality of said agents.

8. The composition according to claim 6 which further comprises an agent which inhibits expression of coronavirus.

9.A method of modulating replication of coronavirus in a host cell infected with said coronavirus comprising administering to said host cell the agent of claim 1 or composition of claim 8 in an amount effective to modulate replication of said virus.

10. The method according to claim 9, wherein the replication is inhibited.

11. A pharmaceutical composition comprising an effective amount of the agent of claim 1 for use in modulating replication of coronavirus.

12. Use of the agent of clam 1 for the manufacture of a medicament for treating SARS.

13. A kit comprising the agent of claim 1.

14. The kit according to claim 13, wherein said agent is labeled with a detectable substance.

15. An isolated nucleic acid molecule comprising (a) a nucleic acid sequence depicted in SEQ ID NO: 1 or SEQ ID NO:2; (b) a nucleic acid sequence that hybridizes to SEQ ID NO:l or SEQ ID NO: 2 or (c) a reverse complement of (a) -(b) and has the sequence depicted in SEQ ID NO: 29 or SEQ ID NO:30.

16. The isolated nucleic acid molecule of claim 15 wherein said nucleic acid molecule is depicted in SEQ ID NO:l or SEQ ID NO:2.

5 17. The isolated nucleic acid molecule of claim 15 wherein said molecule comprises (a) a nucleic acid sequence depicted in SEQ ID NO:3 or SEQ ID NO:4; (b) a nucleic acid sequence that hybridizes to SEQ ID NO:3 or SEQ ID NO:4 or (c) a reverse complement of (a)-(b) and has the sequence depicted in SEQ ID NO:31 or SEQ ID NO:32.

10 18. The isolated nucleic acid molecule of claim 15 wherein said nucleic acid molecule is depicted in SEQ ID NO:5, 6, 33 or 34.

19. The isolated nucleic acid molecule of claim 15, wherein said molecule is selected from the group consisting of SEQ ID NO:7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,

15 20, 21, 22, 23, 24, 25, 26, 27, 28, 32, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, -45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55 or 56.

20. The isolated nucleic acid molecule of claim 15, wherein said nucleic acid sequence is selected from the group consisting of 5'-gatctgttctctaaacgaacttta-3'(SEQ ID 0 NO:7), 5'-tggcatcgatctaaagtcttatga-3'(SEQ ID NO:8), 5'-gaucuguucucuaaacgaacuιuua-3'(SEQ ID NO:18), 5'-uggcaucgaucuaaagucuuauga-3'(SEQ ID NO:19)

21. A method of detecting the presence of absence of a coronavirus in a subject comprising: contacting a nucleic acid molecule from a sample from a subject with at least one 5 primer or probe derived from the nucleic acid molecule of claim 18 and detecting the presence or absence of a coronaviras nucleic acid molecule in said sample to detect the presence or absence of said coronaviras.

22. The method according to claim 21, wherein said method further comprises 0 isolating nucleic acid from said subject.

23. A method for monitoring treatment or progression of a disease or disorder resulting from coronavirus infection in a subject comprising contacting a sample -from a subject periodically with a primer or probe derived from the nucleic acid molecule of claim 15 5 and periodically determining the amount of coronavirus present in said subject.

24. The method according to claim 23, wherein the amount of the nucleic acid molecule present in said sample is determined by PCR or nucleic acid hybridization.

25. A method for identifying an agent effective for modulating replication of coronavirus comprising determining if a candidate agent interferes with interaction of a coronavirus intergenic sequence comprising UCUAA with a coronavirus polymerase complex or component of said polymerase complex that interacts with said intergenic sequence.

26. A method for identifying an agent effective for modulating replication of coronavirus comprising comparing the effect of the agent of claim 1 and candidate agent on interference of interaction of a coronaviras intergenic sequence UCUAA with the polymerase complex of said coronavirus or portion of said polymerase complex interacting with said intergenic sequence.

27. A method for identifying an agent effective for modulating replication of coronavirus comprising measuring the effectiveness of said candidate agent in inhibiting the action of the agent of claim 1 in modulating the replication of the coronavirus

28. A kit comprising at least one primer or probe derived from the nucleic acid molecule of claim 15.

29. The kit of claim 28, comprising a plurality of primers or probes derived from the nucleic acid molecule of claim 18.

30. The kit of claim 28, further comprising one or more nucleic acid molecules derived from a coronavirus nucleic acid molecule and/or enzymes.

31. The kit of claim 28, wherein said coronaviras is a coronavirus associated with SARS.

32. A solid support comprising the nucleic acid molecule of claim 15.

33. The solid support according to claim 32, wherein said solid, support is a microarray.

34. The solid support according to claim 32, wherein said solid support comprises a plurality of said nucleic acid molecules.

35. The solid support of claim 32, wherein said solid support is a microarray further comprising a nucleic acid molecule derived from coronavirus nucleic acid.

36- A computer readable means for storing the nucleic acid sequences of the nucleic acid molecule of claim 15.

37. A computer-based method for identifying potential agents that interferes with interaction of a coronaviras intergenic sequence comprising UCUAAA with a coronaviras polymerase complex comprising (a) providing a nucleic acid from the computer readable medium of claim 36; (b) providing library of nucleic acid sequences and comparing the nucleic acid sequences of (a) and (b) to identify sequence identity or similarity.