CoronaVirus Patent

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United States Patent 7,220,852
Rota , et al. May 22, 2007
Coronavirus isolated from humans

Abstract
Disclosed herein is a newly isolated human coronavirus (SARS-CoV), the causative agent of severe acute respiratory syndrome (SARS). Also provided are the nucleic acid sequence of the SARS-CoV genome and the amino acid sequences of the SARS-CoV open reading frames, as well as methods of using these molecules to detect a SARS-CoV and detect infections therewith. Immune stimulatory compositions are also provided, along with methods of their use.

Inventors: Rota; Paul A. (Decatur, GA), Anderson; Larry J. (Atlanta, GA), Bellini; William J. (Lilburn, GA), Burns; Cara Carthel (Avondale Estates, GA), Campagnoli; Raymond (Decatur, GA), Chen; Qi (Marietta, GA), Comer; James A. (Decatur, GA), Emery; Shannon L. (Lusaka, ZM), Erdman; Dean D. (Decatur, GA), Goldsmith; Cynthia S. (Lilburn, GA), Humphrey; Charles D. (Lilburn, GA), Icenogle; Joseph P. (Atlanta, GA), Ksiazek; Thomas G. (Lilburn, GA), Monroe; Stephan S. (Decatur, GA), Nix; William Allan (Bethlehem, GA), Oberste; M. Steven (Lilburn, GA), Peret; Teresa C. T. (Atlanta, GA), Rollin; Pierre E. (Lilburn, GA), Pallansch; Mark A. (Lilburn, GA), Sanchez; Anthony (Lilburn, GA), Tong; Suxiang (Alpharetta, GA), Zaki; Sherif R. (Atlanta, GA)
Assignee: The United States of America as represented by the Secretary of the Department of Health and Human Services, Centers for Disease Control and Prevention (Washington, DC)
N/A (N/A)
Family ID: 38049542
Appl. No.: 10/822,904
Filed: April 12, 2004
Related U.S. Patent Documents
Application Number Filing Date Patent Number Issue Date
60465927 Apr 25, 2003
Current U.S. Class: 536/23.72; 435/235.1
Current CPC Class: A61K 39/215 (20130101); C07K 14/005 (20130101); C12N 7/00 (20130101); A61K 39/12 (20130101); C12N 2770/20021 (20130101); C12N 2770/20022 (20130101); C12N 2770/20034 (20130101)
Current International Class: C12N 15/50 (20060101); C12N 7/00 (20060101)
Field of Search: ;536/23.72 ;514/44 ;435/235.1,320.1
References Cited [Referenced By]
U.S. Patent Documents
2005/0181357 August 2005 Peiris et al.
Foreign Patent Documents
WO 2004/085633 Oct 2004 WO
WO 2004/092360 Oct 2004 WO

Other References

GenBank Accession No. AY274119, “SARS Coronavirus Tor2, complete genome,” version AY274119.1, Apr. 14, 2003. cited by examiner .
GenBank Accession No. AY278487, “SARS coronavirus BJ02, partial genome,” version AY278487.1, Apr. 21, 2003. cited by examiner .
Genbank Accession No. AY278554, “SARS coronavirus CUHK-W1, complete genome.,” version AY278554.1, Apr. 18, 2003. cited by examiner .
GenBank Accession No. AY278491, “SARS Coronavirus HKU-39849, complete genome,” version AY278491.1, Apr. 18, 2003. cited by examiner .
SARS-associated Coronavirus. Genomic Sequence Availability. [online] [retreived on Aug. 8, 2005]. Retreived from the Internet . cited by examiner .
GenBank Accession No. AY274119, Apr. 14, 2003. cited by other .
GenBank Accession No. AY278741, Apr. 21, 2003. cited by other .
“Update: Outbreak of Severe Acute Respiratory Syndrome–Worldwide, 2003,” MMWR Weekly 52:241-248 (2003). cited by other .
Emery et al., “Real-Time Reverse Transcription-Polymerase Chain Reaction Assay for SARS-Associated Coronavirus,” Emerg. Infect. Diseases 10:311-316 (2004). cited by other .
Goldsmith et al., “Ultrastructural Characterization of SARS Coronavirus,” Emerg. Infect. Diseases 10:320-326 (2004). cited by other .
Ksiazek et al., “A Novel Coronavirus Associated with Severe Acute Respiratory Syndrome,” N. Eng. J. Med. 348:1953-1966 (2003). cited by other .
Luo and Luo, “Initial SARS Coronavirus Genome Sequence Analysis Using a Bioinformatics Platform,” 2.sup.nd Asia-Pacific Bioinformatics Conference (APBC2004), Dunedin, New Zealand (2004). cited by other .
Marra et al., “The Genome Sequence of the SARS-Associated Coronavirus,” Science 300:1393-1404 (2003). cited by other .
Supplementary Online Material for Marra et al. <<www.sciencemag.org/cgi/content/full/1085953/DC1>>. cited by other .
Rota et al., “Characterization of a Novel Coronavirus Associated with Severe Acute Respiratory Syndrome,” Science 300:1394-1399 (2003). cited by other .
Supplementary Online Material for Rota et al. <<www.sciencemag.org/cgi/content/full/1085952/DC1>>. cited by other.

Primary Examiner: Mosher; Mary E.
Attorney, Agent or Firm: Klarquist Sparkman LLP
Parent Case Text

PRIORITY CLAIM

This application claims the benefit of U.S. Provisional Patent Application No. 60/465,927 filed Apr. 25, 2003, which is incorporated herein by reference in its entirety.
Claims

We claim:

1. An isolated nucleic acid molecule consisting of the nucleotide sequence as set forth in SEQ ID NO: 1.
Description

STATEMENT OF GOVERNMENT SUPPORT

This invention was made by the Centers for Disease Control and Prevention, an agency of the United States Government. Therefore, the U.S. Government has certain rights in this invention.

FIELD OF THE DISCLOSURE

This invention relates to a newly isolated human coronavirus. More particularly, it relates to an isolated coronavirus genome, isolated coronavirus proteins, and isolated nucleic acid molecules encoding the same. The disclosure further relates to methods of detecting a severe acute respiratory syndrome-associated coronavirus and compositions comprising immunogenic coronavirus compounds.

BACKGROUND

The coronaviruses (order Nidovirales, family Coronaviridae, genus Coronavirus) are a diverse group of large, enveloped, positive-stranded RNA viruses that cause respiratory and enteric diseases in humans and other animals. At approximately 30,000 nucleotides (nt), their genome is the largest found in any of the RNA viruses. Coronaviruses are spherical, 100 160 nm in diameter with 20 40 nm complex club shaped surface projections surrounding the periphery. Coronaviruses share common structural proteins including a spike protein (S), membrane protein (M), envelope protein (E), and, in a subset of coronaviruses, a hemagglutinin-esterase protein (HE). The S protein, a glycoprotein which protrudes from the virus membrane, is involved in host cell receptor binding and is a target for neutralizing antibodies. The E and M proteins are involved in virion formation and release from the host cell. Coronavirus particles are found within the cisternae of the rough endoplasmic reticulum and in vesicles of infected host cells where virions are assembled. The coronavirus genome consists of two open reading frames (ORF1a and ORF1b) yielding an RNA polymerase and a nested set of subgenomic mRNAs encoding structural and nonstructural proteins, including the S, E, M, and nucleocapsid (N) proteins. The genus Coronavirus includes at least 13 species which have been subdivided into at least three groups (groups I, II, and III) on the basis of serological and genetic properties (deVries et al., Sem. Virol. 8:33 47, 1997; Fields et al. eds. Fields Virology, 3rd edition, Raven Press, Philadelphia, 1323 1341, 1996; Mahey and Collier eds. Microbiology and Microbial Infections, Volume 1 Virology, 9.sup.th edition, Oxford University Press, 463 479, 1998).

The three known groups of coronavirus are associated with a variety of diseases of humans and domestic animals (for example, cattle, pigs, cats, dogs, rodents, and birds), including gastroenteritis and upper and lower respiratory tract disease. Known coronaviruses include human Coronavirus 229E (HCoV-229E), canine coronavirus (CCoV), feline infectious peritonitis virus (FIPV), porcine transmissible gastroenteritis virus (TGEV), porcine epidemic diarrhea virus (PEDV), human coronavirus OC43 (HcoV-OC43), bovine coronavirus (BCoV), porcine hemagglutinating encephalomyelitis virus (HEV), rat sialodacryoadenitis virus (SDAV), mouse hepatitis virus (MHV), turkey coronavirus (TCoV), and avian infectious bronchitis virus (IBV-Avian) (Fields et al. eds. Fields Virology, 3rd edition, Raven Press, Philadelphia, 1323 1341, 1996; Mahey and Collier eds. Microbiology and Microbial Infections, Volume 1 Virology, 9.sup.th edition, Oxford University Press, 463 479, 1998).

Coronavirus infections are generally host specific with respect to infectivity and clinical symptoms. Coronaviruses further exhibit marked tissue tropism; infection in the incorrect host species or tissue type may result in an abortive infection, mutant virus production and altered virulence. Coronaviruses generally do not grow well in cell culture, and animal models for human coronavirus infection are lacking. Therefore, little is known about them (Fields et al. eds. Fields Virology, 3rd edition, Raven Press, Philadelphia, 1323 1341, 1996). The known human coronaviruses are notably fastidious in cell culture, preferring select cell lines, organ culture, or suckling mice for propagation. Coronaviruses grown in cell culture exhibit varying degrees of virulence and/or cytopathic effect (CPE) depending on the host cell type and culture conditions. The only human or animal coronavirus which has been shown to grow in Vero E6 cells is PEDV, and it requires the addition of trypsin to culture medium for growth in Vero E6 cells. Moreover, PEDV adapted to Vero E6 cell culture results in a strikingly different CPE, with cytoplasmic vacuoles and the formation of large syncytia (Hofmann and Wyler, J. Clin. Micro. 26:2235 39, 1988; Kusanagi et el., J. Vet. Med. Sci. 554:313 18, 1991).

Coronavirus have not previously been known to cause severe disease in humans, but have been identified as a major cause of upper respiratory tract illness, including the common cold. Repeat infections in humans are common within and across serotype, suggesting that immune response to coronavirus infection in humans is either incomplete or short lived. Coronavirus infection in animals can cause severe enteric or respiratory disease. Vaccination has been used successfully to prevent and control some coronavirus infections in animals. The ability of animal-specific coronaviruses to cause severe disease raises the possibility that coronavirus could also cause more severe disease in humans (Fields et al. eds. Fields Virology, 3rd edition, Raven Press, Philadelphia, 1323 1341, 1996; Mahey and Collier eds. Microbiology and Microbial Infections, Volume 1 Virology, 9.sup.th edition, Oxford University Press, 463 479, 1998).

In late 2002, cases of life-threatening respiratory disease with no identifiable etiology were reported from Guangdong Province, China, followed by reports from Vietnam, Canada, and Hong Kong of severe febrile respiratory illness that spread to household members and health care workers. The syndrome was designated “severe acute respiratory syndrome” (SARS) in February 2003 by the Centers for Disease Control and Prevention (MMWR, 52:241 48, 2003).

Past efforts to develop rapid diagnostics and vaccines for coronavirus infection in humans have been hampered by a lack of appropriate research models and the moderate course of disease in humans. Therefore, a need for rapid diagnostic tests and vaccines exists.

SUMMARY OF THE DISCLOSURE

A newly isolated human coronavirus has been identified as the causative agent of SARS, and is termed SARS-CoV. The nucleic acid sequence of the SARS-CoV genome and the amino acid sequences of the SARS-CoV open reading frames are provided herein.

This disclosure provides methods and compositions useful in detecting the presence of a SARS-CoV nucleic acid in a sample and/or diagnosing a SARS-CoV infection in a subject. Also provided are methods and compositions useful in detecting the presence of a SARS-CoV antigen or antibody in a sample and/or diagnosing a SARS-CoV infection in a subject.

The foregoing and other features and advantages will become more apparent from the following detailed description of several embodiments, which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A B are photomicrographs illustrating typical early cytopathic effects seen with coronavirus isolates and serum from SARS patients. FIG. 1A is a photomicrograph of Vero E6 cells inoculated with an oropharyngeal specimen from a SARS patient (.times.40). FIG. 1B is a photomicrograph of infected Vero E6 cells reacting with the serum of a convalescent SARS patient in an indirect fluorescent antibody (IFA) assay (.times.400).

FIGS. 2A B are electronmicrographs illustrating ultrastructural characteristics of the SARS-associated coronavirus (SARS-CoV). FIG. 2A is a thin-section electron-microscopical view of viral nucleocapsids aligned along the membrane of the rough endoplasmic reticulum (arrow) as particles bud into the cisternae. Enveloped virions have surface projections (arrowhead) and an electron-lucent center. Directly under the viral envelope lies a characteristic ring formed by the helical nucleocapsid, often seen in cross-section. FIG. 2B is a negative stain (methylamine tungstate) electronmicrograph showing stain-penetrated coronavirus particle with the typical internal helical nucleocapsid-like structure and club-shaped surface projections surrounding the periphery of the particle. Bars: 100 nm.

FIG. 3 is an estimated maximum parsimony tree illustrating putative phylogenetic relationships between SARS-CoV and other human and animal coronaviruses. Phylogenetic relationships are based on sequence alignment of 405 nucleotides of the coronavirus polymerase gene ORF1b (nucleic acid 15,173 to 15,578 of SEQ ID NO: 1). The three major coronavirus antigenic groups (I, II and III), represented by HcoV-229E, CCoV, FIPV, TGEV, PEDV, HcoV-OC43, BCoV, HEV, SDAV, MHV, TCoV, and IBV-Avian, are shown shaded. Bootstrap values (100 replicates) obtained from a 50% majority rule consensus tree are plotted at the main internal branches of the phylogram. Branch lengths are proportionate to nucleotide differences.

FIG. 4 is a pictorial representation of neighbor joining trees illustrating putative phylogenetic relationships between SARS-CoV and other human and animal coronaviruses. Amino acid sequences of the indicated SARS-CoV proteins were compared with those from reference viruses representing each species in the three groups of coronaviruses for which complete genomic sequence information was available [group 1: HCoV-229E (AF304460); PEDV (AF353511); TGEV (AJ271965); group 2: BCoV (AF220295); MHV (AF201929); group 3: infectious bronchitis virus (M95169)]. Sequences for representative strains of other coronavirus species, for which partial sequence information was available, were included for some of the structural protein comparisons [group 1: CCoV (D13096); FCoV (AY204704); porcine respiratory coronavirus (Z24675); group 2: HCoV-OC43 (M76373, L14643, M93390); HEV (AY078417); rat coronavirus (AF207551)]. Sequence alignments and neighbor-joining trees were generated by using Clustalx 1.83 with the Gonnet protein comparison matrix. The resulting trees were adjusted for final output using treetool 2.0.1.

FIGS. 5A C are photomicrographs illustrating diffuse alveolar damage in a patient with SARS (FIGS. 5A B), and immunohistochemical staining of SARS-CoV-infected Vero E6 cells (FIG. 5C). FIG. 5A is a photomicrograph of lung tissue from a SARS patient (.times.50). Diffuse alveolar damage, abundant foamy macrophages and multinucleated syncytial cells are present; hematoxylin and eosin stain was used. FIG. 5B is a higher magnification photomicrograph of lung tissue from the same SARS patient (.times.250). Syncytial cells show no conspicuous viral inclusions. FIG. 5C is a photomicrograph of immunohistochemically stained SARS-CoV-infected cells (.times.250). Membranous and cytoplasmic immunostaining of individual and syncytial Vero E6 cells was achieved using feline anti-FIPV-1 ascitic fluid. Immunoalkaline phosphatase with naphthol-fast red substrate and hematoxylin counter stain was used.

FIG. 6A B are electronmicrographs illustrating ultrastructural characteristics of a coronavirus-infected cell in bronchoalveolar lavage (BAL) from a SARS patient. FIG. 6A is an electronmicrograph of a coronavirus-infected cell. Numerous intracellular and extracellular particles are present; virions are indicated by the arrowheads. FIG. 6B is a higher magnification electronmicrograph of the area seen at the arrow in FIG. 6A (rotated clockwise approximately 90.degree.). Bars: FIG. 6A, 1 .mu.m; FIG. 6B, 100 nm.

FIGS. 7A C illustrate the organization of the SARS-CoV genome. FIG. 7A is a diagram of the overall organization of the 29,727-nt SARS-CoV genomic RNA. The 72-nt leader sequence is represented as a small rectangle at the left-most end. ORFs1a and 1b, encoding the nonstructural polyproteins, and those ORFs encoding the S, E, M, and N structural proteins, are indicated. Vertical position of the boxes indicates the phase of the reading frame (phase 1 for proteins above the line, phase two for proteins on the line and phase three for proteins below the line). FIG. 7B is an expanded view of the structural protein encoding region and predicted mRNA transcripts. Known structural protein encoding regions (dark grey boxes) and regions and reading frames for potential products X1 X5 (light gray boxes) are indicated. Lengths and map locations of the 3′-coterminal mRNAs expressed by the SARS-CoV are indicated, as predicted by identification of conserved transcriptional regulatory sequences. FIG. 7C is a digitized image of a nylon membrane showing Northern blot analysis of SARS-CoV mRNAs. Poly(A)+ RNA from infected Vero E6 cells was separated on a formaldehyde-agarose gel, transferred to a nylon membrane, and hybridized with a digoxigenin-labeled riboprobe overlapping the 3′-untranslated region. Signals were visualized by chemiluminescence. Sizes of the SARS-CoV mRNAs were calculated by extrapolation from a log-linear fit of the molecular mass marker. Lane 1, SARS-CoV mRNA; lane 2, Vero E6 cell mRNA; lane 3, molecular mass marker, sizes in kB.

SEQUENCE LISTING

The nucleic and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and three letter code for amino acids, as defined in 37 C.F.R. 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand. In the accompanying sequence listing:

SEQ ID NO: 1 shows the nucleic acid sequence of the SARS-CoV genome.

SEQ ID NO: 2 shows the amino acid sequence of the SARS-CoV polyprotein 1a (encoded by nucleic acid 265 to nucleic acid 13,398 of SEQ ID NO: 1).

SEQ ID NO: 3 shows the amino acid sequence of the SARS-CoV polyprotein 1b (encoded by nucleic acid 13,398 to 21,482 of SEQ ID NO: 1).

SEQ ID NO: 4 shows the amino acid sequence of the SARS-CoV S protein (encoded by nucleic acid 21,492 to 25,256 of SEQ ID NO: 1).

SEQ ID NO: 5 shows the amino acid sequence of the SARS-CoV X1 protein (encoded by nucleic acid 25,268 to 26,089 of SEQ ID NO: 1).

SEQ ID NO: 6 shows the amino acid sequence of the SARS-CoV X2 protein (encoded by nucleic acid 25,689 to 26,150 of SEQ ID NO: 1).

SEQ ID NO: 7 shows the amino acid sequence of the SARS-CoV E protein (encoded by nucleic acid 26,117 to 26,344 of SEQ ID NO: 1).

SEQ ID NO: 8 shows the amino acid sequence of the SARS-CoV M protein (encoded by nucleic acid 26,398 to 27,060 of SEQ ID NO: 1).

SEQ ID NO: 9 shows the amino acid sequence of the SARS-CoV X3 protein (encoded by nucleic acid 27,074 to 27,262 of SEQ ID NO: 1).

SEQ ID NO: 10 shows the amino acid sequence of the SARS-CoV X4 protein (encoded by nucleic acid 27,273 to 27,638 of SEQ ID NO: 1).

SEQ ID NO: 11 shows the amino acid sequence of the SARS-CoV X5 protein (encoded by nucleic acid 27,864 to 28,115 of SEQ ID NO: 1).

SEQ ID NO: 12 shows the amino acid sequence of the SARS-CoV N protein (encoded by nucleic acid 28,120 to 29,385 of SEQ ID NO: 1).

SEQ ID NOs: 13 15 show the nucleic acid sequence of several SARS-CoV-specific oligonucleotide primers.

SEQ ID NOs: 16 33 show the nucleic acid sequence of several oligonucleotide primers/probes used for real-time reverse transcription-polymerase chain reaction (RT-PCR) SARS-CoV assays.

SEQ ID NOs: 34 35 show the nucleic acid sequence of two degenerate primers designed to anneal to sites encoding conserved coronavirus amino acid motifs.

SEQ ID NOs: 36 38 show the nucleic acid sequence of several oligonucleotide primers/probe used as controls in real-time RT-PCR assays.

DETAILED DESCRIPTION OF SEVERAL EMBODIMENTS

I. Abbreviations

TABLE-US-00001 M: coronavirus membrane protein N: coronavirus nucleoprotein ORF: open reading frame PCR polymerase chain reaction RACE: 5′ rapid amplification of cDNA ends RT-PCR: reverse transcription-polymerase chain reaction S: coronavirus spike protein SARS: severe acute respiratory syndrome SARS-CoV: severe acute respiratory syndrome-associated coronavirus TRS: transcriptional regulatory sequence

II. Terms

Unless otherwise noted, technical terms are used according to conventional usage. Definitions of common terms in molecular biology may be found in Benjamin Lewin, Genes VII, published by Oxford University Press, 2000 (ISBN 019879276X); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Publishers, 1994 (ISBN 0632021829); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by Wiley, John & Sons, Inc., 1995 (ISBN 0471186341); and other similar references.

As used herein, the singular terms “a,” “an,” and “the” include plural referents unless context clearly indicates otherwise. Similarly, the word “or” is intended to include “and” unless the context clearly indicates otherwise. Also, as used herein, the term “comprises” means “includes.” Hence “comprising A or B” means including A, B, or A and B. It is further to be understood that all nucleotide sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for description. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

In order to facilitate review of the various embodiments of this disclosure, the following explanations of specific terms are provided:

Adjuvant: A substance that non-specifically enhances the immune response to an antigen. Development of vaccine adjuvants for use in humans is reviewed in Singh et al. (Nat. Biotechnol. 17:1075 1081, 1999), which discloses that, at the time of its publication, aluminum salts and the MF59 microemulsion are the only vaccine adjuvants approved for human use.

Amplification: Amplification of a nucleic acid molecule (e.g., a DNA or RNA molecule) refers to use of a laboratory technique that increases the number of copies of a nucleic acid molecule in a sample. An example of amplification is the polymerase chain reaction (PCR), in which a sample is contacted with a pair of oligonucleotide primers under conditions that allow for the hybridization of the primers to a nucleic acid template in the sample. The primers are extended under suitable conditions, dissociated from the template, re-annealed, extended, and dissociated to amplify the number of copies of the nucleic acid. The product of amplification can be characterized by such techniques as electrophoresis, restriction endonuclease cleavage patterns, oligonucleotide hybridization or ligation, and/or nucleic acid sequencing.

Other examples of amplification methods include strand displacement amplification, as disclosed in U.S. Pat. No. 5,744,311; transcription-free isothermal amplification, as disclosed in U.S. Pat. No. 6,033,881; repair chain reaction amplification, as disclosed in WO 90/01069; ligase chain reaction amplification, as disclosed in EP-A-320,308; gap filling ligase chain reaction amplification, as disclosed in U.S. Pat. No. 5,427,930; and NASBA.TM. RNA transcription-free amplification, as disclosed in U.S. Pat. No. 6,025,134. An amplification method can be modified, including for example by additional steps or coupling the amplification with another protocol.

Animal: Living multi-cellular vertebrate organisms, a category that includes, for example, mammals and birds. The term mammal includes both human and non-human mammals. Similarly, the term “subject” includes both human and veterinary subjects, for example, humans, non-human primates, dogs, cats, horses, and cows.

Antibody: A protein (or protein complex) that includes one or more polypeptides substantially encoded by immunoglobulin genes or fragments of immunoglobulin genes. The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as the myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.

The basic immunoglobulin (antibody) structural unit is generally a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one “light” (about 25 kDa) and one “heavy” (about 50 70 kDa) chain. The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms “variable light chain” (V.sub.L) and “variable heavy chain” (V.sub.H) refer, respectively, to these light and heavy chains.

As used herein, the term “antibodies” includes intact immunoglobulins as well as a number of well-characterized fragments. For instance, Fabs, Fvs, and single-chain Fvs (SCFvs) that bind to target protein (or epitope within a protein or fusion protein) would also be specific binding agents for that protein (or epitope). These antibody fragments are defined as follows: (1) Fab, the fragment which contains a monovalent antigen-binding fragment of an antibody molecule produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain; (2) Fab’, the fragment of an antibody molecule obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain; two Fab’ fragments are obtained per antibody molecule; (3) (Fab’).sub.2, the fragment of the antibody obtained by treating whole antibody with the enzyme pepsin without subsequent reduction; (4) F(ab’).sub.2, a dimer of two Fab’ fragments held together by two disulfide bonds; (5) Fv, a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains; and (6) single chain antibody, a genetically engineered molecule containing the variable region of the light chain, the variable region of the heavy chain, linked by a suitable polypeptide linker as a genetically fused single chain molecule. Methods of making these fragments are routine (see, for example, Harlow and Lane, Using Antibodies: A Laboratory Manual, CSHL, New York, 1999).

Antibodies for use in the methods and devices of this disclosure can be monoclonal or polyclonal. Merely by way of example, monoclonal antibodies can be prepared from murine hybridomas according to the classical method of Kohler and Milstein (Nature 256:495 97, 1975) or derivative methods thereof. Detailed procedures for monoclonal antibody production are described in Harlow and Lane, Using Antibodies: A Laboratory Manual, CSHL, New York, 1999.

Antigen: A compound, composition, or substance that can stimulate the production of antibodies or a T-cell response in an animal, including compositions that are injected or absorbed into an animal. An antigen reacts with the products of specific humoral or cellular immunity, including those induced by heterologous immunogens. In one embodiment, an antigen is a coronavirus antigen.

Binding or Stable Binding: An oligonucleotide binds or stably binds to a target nucleic acid if a sufficient amount of the oligonucleotide forms base pairs or is hybridized to its target nucleic acid, to permit detection of that binding. Binding can be detected by either physical or functional properties of the target:oligonucleotide complex. Binding between a target and an oligonucleotide can be detected by any procedure known to one skilled in the art, including functional or physical binding assays. Binding can be detected functionally by determining whether binding has an observable effect upon a biosynthetic process such as expression of a gene, DNA replication, transcription, translation, and the like.

Physical methods of detecting the binding of complementary strands of DNA or RNA are well known in the art, and include such methods as DNase I or chemical footprinting, gel shift and affinity cleavage assays, Northern blotting, Southern blotting, dot blotting, and light absorption detection procedures. For example, a method which is widely used, because it is so simple and reliable, involves observing a change in light absorption of a solution containing an oligonucleotide (or an analog) and a target nucleic acid at 220 to 300 nm as the temperature is slowly increased. If the oligonucleotide or analog has bound to its target, there is a sudden increase in absorption at a characteristic temperature as the oligonucleotide (or analog) and target dissociate or melt.

The binding between an oligomer and its target nucleic acid is frequently characterized by the temperature (T.sub.m) at which 50% of the oligomer is melted from its target. A higher T.sub.m means a stronger or more stable complex relative to a complex with a lower T.sub.m.

cDNA (complementary DNA): A piece of DNA lacking internal, non-coding segments (introns) and regulatory sequences that determine transcription. cDNA is synthesized in the laboratory by reverse transcription from messenger RNA extracted from cells.

Electrophoresis: Electrophoresis refers to the migration of charged solutes or particles in a liquid medium under the influence of an electric field. Electrophoretic separations are widely used for analysis of macromolecules. Of particular importance is the identification of proteins and nucleic acid sequences. Such separations can be based on differences in size and/or charge. Nucleotide sequences have a uniform charge and are therefore separated based on differences in size. Electrophoresis can be performed in an unsupported liquid medium (for example, capillary electrophoresis), but more commonly the liquid medium travels through a solid supporting medium. The most widely used supporting media are gels, for example, polyacrylamide and agarose gels.

Sieving gels (for example, agarose) impede the flow of molecules. The pore size of the gel determines the size of a molecule that can flow freely through the gel. The amount of time to travel through the gel increases as the size of the molecule increases. As a result, small molecules travel through the gel more quickly than large molecules and thus progress further from the sample application area than larger molecules, in a given time period. Such gels are used for size-based separations of nucleotide sequences.

Fragments of linear DNA migrate through agarose gels with a mobility that is inversely proportional to the log.sub.10 of their molecular weight. By using gels with different concentrations of agarose, different sizes of DNA fragments can be resolved. Higher concentrations of agarose facilitate separation of small DNAs, while low agarose concentrations allow resolution of larger DNAs.

Hybridization: Oligonucleotides and their analogs hybridize by hydrogen bonding, which includes Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary bases. Generally, nucleic acid consists of nitrogenous bases that are either pyrimidines (cytosine (C), uracil (U), and thymine (T)) or purines (adenine (A) and guanine (G)). These nitrogenous bases form hydrogen bonds between a pyrimidine and a purine, and the bonding of the pyrimidine to the purine is referred to as “base pairing.” More specifically, A will hydrogen bond to T or U, and G will bond to C. “Complementary” refers to the base pairing that occurs between to distinct nucleic acid sequences or two distinct regions of the same nucleic acid sequence.

“Specifically hybridizable” and “specifically complementary” are terms that indicate a sufficient degree of complementarity such that stable and specific binding occurs between the oligonucleotide (or its analog) and the DNA or RNA target. The oligonucleotide or oligonucleotide analog need not be 100% complementary to its target sequence to be specifically hybridizable. An oligonucleotide or analog is specifically hybridizable when binding of the oligonucleotide or analog to the target DNA or RNA molecule interferes with the normal function of the target DNA or RNA, and there is a sufficient degree of complementarity to avoid non-specific binding of the oligonucleotide or analog to non-target sequences under conditions where specific binding is desired, for example under physiological conditions in the case of in vivo assays or systems. Such binding is referred to as specific hybridization.

Hybridization conditions resulting in particular degrees of stringency will vary depending upon the nature of the hybridization method of choice and the composition and length of the hybridizing nucleic acid sequences. Generally, the temperature of hybridization and the ionic strength (especially the Na.sup.+ and/or Mg.sup.++ concentration) of the hybridization buffer will determine the stringency of hybridization, though wash times also influence stringency. Calculations regarding hybridization conditions required for attaining particular degrees of stringency are discussed by Sambrook et al. (ed.), Molecular Cloning: A Laboratory Manual, 2.sup.nd ed., vol. 1 3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, chapters 9 and 11; and Ausubel et al. Short Protocols in Molecular Biology, 4.sup.th ed., John Wiley & Sons, Inc., 1999.

For purposes of the present disclosure, “stringent conditions” encompass conditions under which hybridization will only occur if there is less than 25% mismatch between the hybridization molecule and the target sequence. “Stringent conditions” may be broken down into particular levels of stringency for more precise definition. Thus, as used herein, “moderate stringency” conditions are those under which molecules with more than 25% sequence mismatch will not hybridize; conditions of “medium stringency” are those under which molecules with more than 15% mismatch will not hybridize, and conditions of “high stringency” are those under which sequences with more than 10% mismatch will not hybridize. Conditions of “very high stringency” are those under which sequences with more than 6% mismatch will not hybridize.

Immune Stimulatory Composition: A term used herein to mean a composition useful for stimulating or eliciting a specific immune response (or immunogenic response) in a vertebrate. In some embodiments, the immunogenic response is protective or provides protective immunity, in that it enables the vertebrate animal to better resist infection with or disease progression from the organism against which the vaccine is directed.

Without wishing to be bound by a specific theory, it is believed that an immunogenic response may arise from the generation of an antibody specific to one or more of the epitopes provided in the immune stimulatory composition. Alternatively, the response may comprise a T-helper or cytotoxic cell-based response to one or more of the epitopes provided in the immune stimulatory composition. All three of these responses may originate from naive or memory cells. One specific example of a type of immune stimulatory composition is a vaccine.

In some embodiments, an “effective amount” or “immune-stimulatory amount” of an immune stimulatory composition is an amount which, when administered to a subject, is sufficient to engender a detectable immune response. Such a response may comprise, for instance, generation of an antibody specific to one or more of the epitopes provided in the immune stimulatory composition. Alternatively, the response may comprise a T-helper or CTL-based response to one or more of the epitopes provided in the immune stimulatory composition. All three of these responses may originate from naive or memory cells. In other embodiments, a “protective effective amount” of an immune stimulatory composition is an amount which, when administered to a subject, is sufficient to confer protective immunity upon the subject.

Inhibiting or Treating a Disease: Inhibiting the full development of a disease or condition, for example, in a subject who is at risk for a disease such as SARS. “Treatment” refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition after it has begun to develop. As used herein, the term “ameliorating,” with reference to a disease, pathological condition or symptom, refers to any observable beneficial effect of the treatment. The beneficial effect can be evidenced, for example, by a delayed onset of clinical symptoms of the disease in a susceptible subject, a reduction in severity of some or all clinical symptoms of the disease, a slower progression of the disease, a reduction in the number of relapses of the disease, an improvement in the overall health or well-being of the subject, or by other parameters well known in the art that are specific to the particular disease.

Isolated: An “isolated” microorganism (such as a virus, bacterium, fungus, or protozoan) has been substantially separated or purified away from microorganisms of different types, strains, or species. Microorganisms can be isolated by a variety of techniques, including serial dilution and culturing.

An “isolated” biological component (such as a nucleic acid molecule, protein or organelle) has been substantially separated or purified away from other biological components in the cell of the organism in which the component naturally occurs, such as other chromosomal and extra-chromosomal DNA and RNA, proteins, and organelles. Nucleic acids and proteins that have been “isolated” include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids and proteins prepared by recombinant expression in a host cell, as well as chemically synthesized nucleic acids or proteins, or fragments thereof.

Label: A detectable compound or composition that is conjugated directly or indirectly to another molecule to facilitate detection of that molecule. Specific, non-limiting examples of labels include fluorescent tags, enzymatic linkages, and radioactive isotopes.

Nucleic Acid Molecule: A polymeric form of nucleotides, which may include both sense and anti-sense strands of RNA, cDNA, genomic DNA, and synthetic forms and mixed polymers of the above. A nucleotide refers to a ribonucleotide, deoxynucleotide or a modified form of either type of nucleotide. A “nucleic acid molecule” as used herein is synonymous with “nucleic acid” and “polynucleotide.” A nucleic acid molecule is usually at least 10 bases in length, unless otherwise specified. The term includes single- and double-stranded forms of DNA. A polynucleotide may include either or both naturally occurring and modified nucleotides linked together by naturally occurring and/or non-naturally occurring nucleotide linkages.

Oligonucleotide: A nucleic acid molecule generally comprising a length of 300 bases or fewer. The term often refers to single-stranded deoxyribonucleotides, but it can refer as well to single- or double-stranded ribonucleotides, RNA:DNA hybrids and double-stranded DNAs, among others. The term “oligonucleotide” also includes oligonucleosides (that is, an oligonucleotide minus the phosphate) and any other organic base polymer. In some examples, oligonucleotides are about 10 to about 90 bases in length, for example, 12, 13, 14, 15, 16, 17, 18, 19 or 20 bases in length. Other oligonucleotides are about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60 bases, about 65 bases, about 70 bases, about 75 bases or about 80 bases in length. Oligonucleotides may be single-stranded, for example, for use as probes or primers, or may be double-stranded, for example, for use in the construction of a mutant gene. Oligonucleotides can be either sense or anti-sense oligonucleotides. An oligonucleotide can be modified as discussed above in reference to nucleic acid molecules. Oligonucleotides can be obtained from existing nucleic acid sources (for example, genomic or cDNA), but can also be synthetic (for example, produced by laboratory or in vitro oligonucleotide synthesis).

Open Reading Frame (ORF): A series of nucleotide triplets (codons) coding for amino acids without any internal termination codons. These sequences are usually translatable into a peptide/polypeptide/protein/polyprotein.

Operably Linked: A first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence is the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein coding regions, in the same reading frame. If introns are present, the operably linked DNA sequences may not be contiguous.

Pharmaceutically Acceptable Carriers: The pharmaceutically acceptable carriers useful in this disclosure are conventional. Remington’s Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, Pa., 15th Edition (1975), describes compositions and formulations suitable for pharmaceutical delivery of one or more therapeutic compounds or molecules, such as one or more SARS-CoV nucleic acid molecules, proteins or antibodies that bind these proteins, and additional pharmaceutical agents.

In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. For solid compositions (for example, powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically-neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.

Polypeptide: A polymer in which the monomers are amino acid residues which are joined together through amide bonds. When the amino acids are alpha-amino acids, either the L-optical isomer or the D-optical isomer can be used, the L-isomers being preferred. The terms “polypeptide” or “protein” as used herein are intended to encompass any amino acid sequence and include modified sequences such as glycoproteins. The term “polypeptide” is specifically intended to cover naturally occurring proteins, as well as those which are recombinantly or synthetically produced.

Conservative amino acid substitutions are those substitutions that, when made, least interfere with the properties of the original protein, that is, the structure and especially the function of the protein is conserved and not significantly changed by such substitutions. Examples of conservative substitutions are shown below.

TABLE-US-00002 Original Residue Conservative Substitutions Ala Ser Arg Lys Asn Gln, His Asp Glu Cys Ser Gln Asn Glu Asp His Asn; Gln Ile Leu, Val Leu Ile; Val Lys Arg; Gln; Glu Met Leu; Ile Phe Met; Leu; Tyr Ser Thr Thr Ser Trp Tyr Tyr Trp; Phe Val Ile; Leu

Conservative substitutions generally maintain (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain.

The substitutions which in general are expected to produce the greatest changes in protein properties will be non-conservative, for instance changes in which (a) a hydrophilic residue, for example, seryl or threonyl, is substituted for (or by) a hydrophobic residue, for example, leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine or proline is substituted for (or by) any other residue; (c) a residue having an electropositive side chain, for example, lysyl, arginyl, or histadyl, is substituted for (or by) an electronegative residue, for example, glutamyl or aspartyl; or (d) a residue having a bulky side chain, for example, phenylalanine, is substituted for (or by) one not having a side chain, for example, glycine.

Probes and Primers: A probe comprises an isolated nucleic acid attached to a detectable label or other reporter molecule. Typical labels include radioactive isotopes, enzyme substrates, co-factors, ligands, chemiluminescent or fluorescent agents, haptens, and enzymes. Methods for labeling and guidance in the choice of labels appropriate for various purposes are discussed, for example, in Sambrook et al. (ed.), Molecular Cloning: A Laboratory Manual, 2.sup.nd ed., vol. 1 3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989 and Ausubel et al. Short Protocols in Molecular Biology, 4.sup.th ed., John Wiley & Sons, Inc., 1999.

Primers are short nucleic acid molecules, for instance DNA oligonucleotides 10 nucleotides or more in length, for example that hybridize to contiguous complementary nucleotides or a sequence to be amplified. Longer DNA oligonucleotides may be about 15, 20, 25, 30 or 50 nucleotides or more in length. Primers can be annealed to a complementary target DNA strand by nucleic acid hybridization to form a hybrid between the primer and the target DNA strand, and then the primer extended along the target DNA strand by a DNA polymerase enzyme. Primer pairs can be used for amplification of a nucleic acid sequence, for example, by the PCR or other nucleic-acid amplification methods known in the art, as described above.

Methods for preparing and using nucleic acid probes and primers are described, for example, in Sambrook et al. (ed.), Molecular Cloning: A Laboratory Manual, 2.sup.nd ed., vol. 1 3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989; Ausubel et al. Short Protocols in Molecular Biology, 4.sup.th ed., John Wiley & Sons, Inc., 1999; and Innis et al. PCR Protocols, A Guide to Methods and Applications, Academic Press, Inc., San Diego, Calif., 1990. Amplification primer pairs can be derived from a known sequence, for example, by using computer programs intended for that purpose such as Primer (Version 0.5, .COPYRGT. 1991, Whitehead Institute for Biomedical Research, Cambridge, Mass.). One of ordinary skill in the art will appreciate that the specificity of a particular probe or primer increases with its length. Thus, in order to obtain greater specificity, probes and primers can be selected that comprise at least 20, 25, 30, 35, 40, 45, 50 or more consecutive nucleotides of a target nucleotide sequences.

Protein: A biological molecule, particularly a polypeptide, expressed by a gene and comprised of amino acids. A “polyprotein” is a protein that, after synthesis, is cleaved to produce several functionally distinct polypeptides.

Purified: The term “purified” does not require absolute purity; rather, it is intended as a relative term. Thus, for example, a purified protein preparation is one in which the subject protein is more pure than in its natural environment within a cell. Generally, a protein preparation is purified such that the protein represents at least 50% of the total protein content of the preparation.

Recombinant Nucleic Acid: A sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. This artificial combination is often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, for example, by genetic engineering techniques such as those described in Sambrook et al. (ed.), Molecular Cloning: A Laboratory Manual, 2.sup.nd ed., vol. 1 3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989. The term recombinant includes nucleic acids that have been altered solely by addition, substitution, or deletion of a portion of the nucleic acid.

Sample: A portion, piece, or segment that is representative of a whole. This term encompasses any material, including for instance samples obtained from an animal, a plant, or the environment.

An “environmental sample” includes a sample obtained from inanimate objects or reservoirs within an indoor or outdoor environment. Environmental samples include, but are not limited to: soil, water, dust, and air samples; bulk samples, including building materials, furniture, and landfill contents; and other reservoir samples, such as animal refuse, harvested grains, and foodstuffs.

A “biological sample” is a sample obtained from a plant or animal subject. As used herein, biological samples include all samples useful for detection of viral infection in subjects, including, but not limited to: cells, tissues, and bodily fluids, such as blood; derivatives and fractions of blood (such as serum); extracted galls; biopsied or surgically removed tissue, including tissues that are, for example, unfixed, frozen, fixed in formalin and/or embedded in paraffin; tears; milk; skin scrapes; surface washings; urine; sputum; cerebrospinal fluid; prostate fluid; pus; bone marrow aspirates; BAL; saliva; cervical swabs; vaginal swabs; and oropharyngeal wash.

Sequence Identity: The similarity between two nucleic acid sequences, or two amino acid sequences, is expressed in terms of the similarity between the sequences, otherwise referred to as sequence identity. Sequence identity is frequently measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar the two sequences are.

Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith and Waterman (Adv. Appl. Math., 2:482, 1981); Needleman and Wunsch (J. Mol. Biol., 48:443, 1970); Pearson and Lipman (Proc. Natl. Acad. Sci., 85:2444, 1988); Higgins and Sharp (Gene, 73:237 44, 1988); Higgins and Sharp (CABIOS, 5:151 53, 1989); Corpet et al. (Nuc. Acids Res., 16:10881 90, 1988); Huang et al. (Comp. Appls Biosci., 8:155 65, 1992); and Pearson et al. (Meth. Mol. Biol., 24:307 31, 1994). Altschul et al. (Nature Genet., 6:119 29, 1994) presents a detailed consideration of sequence alignment methods and homology calculations.

The alignment tools ALIGN (Myers and Miller, CABIOS 4:11 17, 1989) or LFASTA (Pearson and Lipman, 1988) may be used to perform sequence comparisons (Internet Program.COPYRGT. 1996, W. R. Pearson and the University of Virginia, “fasta20u63” version 2.0u63, release date December 1996). ALIGN compares entire sequences against one another, while LFASTA compares regions of local similarity. These alignment tools and their respective tutorials are available on the Internet at the NCSA website. Alternatively, for comparisons of amino acid sequences of greater than about 30 amino acids, the “Blast 2 sequences” function can be employed using the default BLOSUM62 matrix set to default parameters, (gap existence cost of 11, and a per residue gap cost of 1). When aligning short peptides (fewer than around 30 amino acids), the alignment should be performed using the “Blast 2 sequences” function, employing the PAM30 matrix set to default parameters (open gap 9, extension gap 1 penalties). The BLAST sequence comparison system is available, for instance, from the NCBI web site; see also Altschul et al., J. Mol. Biol., 215:403 10, 1990; Gish. and States, Nature Genet., 3:266 72, 1993; Madden et al., Meth. Enzymol., 266:131 41, 1996; Altschul et al., Nucleic Acids Res., 25:3389 402, 1997; and Zhang and Madden, Genome Res., 7:649 56, 1997.

Orthologs (equivalent to proteins of other species) of proteins are in some instances characterized by possession of greater than 75% sequence identity counted over the full-length alignment with the amino acid sequence of specific protein using ALIGN set to default parameters. Proteins with even greater similarity to a reference sequence will show increasing percentage identities when assessed by this method, such as at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, or at least 98% sequence identity. In addition, sequence identity can be compared over the full length of one or both binding domains of the disclosed fusion proteins.

When significantly less than the entire sequence is being compared for sequence identity, homologous sequences will typically possess at least 80% sequence identity over short windows of 10 20, and may possess sequence identities of at least 85%, at least 90%, at least 95%, or at least 99% depending on their similarity to the reference sequence. Sequence identity over such short windows can be determined using LFASTA; methods are described at the NCSA website. One of skill in the art will appreciate that these sequence identity ranges are provided for guidance only; it is entirely possible that strongly significant homologs could be obtained that fall outside of the ranges provided. Similar homology concepts apply for nucleic acids as are described for protein.

An alternative indication that two nucleic acid molecules are closely related is that the two molecules hybridize to each other under stringent conditions. Representative hybridization conditions are discussed above.

Nucleic acid sequences that do not show a high degree of identity may nevertheless encode similar amino acid sequences, due to the degeneracy of the genetic code. It is understood that changes in nucleic acid sequence can be made using this degeneracy to produce multiple nucleic acid sequences that each encode substantially the same protein.

Specific Binding Agent: An agent that binds substantially only to a defined target. Thus a protein-specific binding agent binds substantially only the defined protein, or to a specific region within the protein. As used herein, a protein-specific binding agent includes antibodies and other agents that bind substantially to a specified polypeptide. The antibodies may be monoclonal or polyclonal antibodies that are specific for the polypeptide, as well as immunologically effective portions (“fragments”) thereof.

The determination that a particular agent binds substantially only to a specific polypeptide may readily be made by using or adapting routine procedures. One suitable in vitro assay makes use of the Western blotting procedure (described in many standard texts, including Harlow and Lane, Using Antibodies: A Laboratory Manual, CSHL, New York, 1999).

Transformed: A “transformed” cell is a cell into which has been introduced a nucleic acid molecule by molecular biology techniques. The term encompasses all techniques by which a nucleic acid molecule might be introduced into such a cell, including transfection with viral vectors, transformation with plasmid vectors, and introduction of naked DNA by electroporation, lipofection, and particle gun acceleration.

Vector: A nucleic acid molecule as introduced into a host cell, thereby producing a transformed host cell. A vector may include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication. A vector may also include one or more selectable marker genes and other genetic elements known in the art.

Virus: Microscopic infectious organism that reproduces inside living cells. A virus typically consists essentially of a core of a single nucleic acid surrounded by a protein coat, and has the ability to replicate only inside a living cell. “Viral replication” is the production of additional virus by the occurrence of at least one viral life cycle. A virus may subvert the host cells’ normal functions, causing the cell to behave in a manner determined by the virus. For example, a viral infection may result in a cell producing a cytokine, or responding to a cytokine, when the uninfected cell does not normally do so.

“Coronaviruses” are large, enveloped, RNA viruses that cause respiratory and enteric diseases in humans and other animals. Coronavirus genomes are non-segmented, single-stranded, positive-sense RNA, approximately 27 31 kb in length. Genomes have a 5′ methylated cap and 3′ poly-A tail, and function directly as mRNA. Host cell entry occurs via endocytosis and membrane fusion, and replication occurs in the cytoplasm. Initially, the 5′ 20 kb of the positive-sense genome is translated to produce a viral polymerase, which then produces a full-length negative-sense strand used as a template to produce subgenomic mRNA as a “nested set” of transcripts. Assembly occurs by budding into the golgi apparatus, and particles are transported to the surface of the cell and released.

III. Overview of Several Embodiments

A newly isolated human coronavirus (SARS-CoV) is disclosed herein. The entire genomic nucleic acid sequence of this virus is also provided herein. Also disclosed are the nucleic acid sequences of the SARS-CoV ORFs, and the polypeptide sequences encoded by these ORFs. Pharmaceutical and immune stimulatory compositions are also disclosed that include one or more SARS-CoV viral nucleic acids, polypeptides encoded by these viral nucleic acids and antibodies that bind to a SARS-CoV polypeptide or SARS-CoV polypeptide fragment.

In one embodiment, a method is provided for detecting the presence of SARS-CoV in a sample. This method includes contacting the sample with a pair of nucleic acid primers that hybridize to a SARS-CoV nucleic acid, wherein at least one primer is 5′-end labeled with a reporter dye, amplifying the SARS-CoV nucleic acid or a fragment thereof from the sample utilizing the pair of nucleic acid primers, electrophoresing the amplified products, and detecting the 5′-end labeled reporter dye, thereby detecting a SARS-CoV. In a specific, non-limiting example, the amplification utilizes RT-PCR. In a further specific example of the provided method, at least one of the nucleic acid primers that hybridize to a SARS-CoV nucleic acid includes a sequence as set forth in any one of SEQ ID NOs: 13 15.

In another example of the provided method, detecting the presence of SARS-CoV in a sample includes contacting the sample with a pair of nucleic acid primers that hybridize to a SARS-CoV nucleic acid, amplifying the SARS-CoV nucleic acid or a fragment thereof from the sample utilizing the pair of nucleic acid primers, adding to the amplified SARS-CoV nucleic acid or the fragment thereof a TaqMan SARS-CoV probe that hybridizes to the SARS-CoV nucleic acid, wherein the TaqMan SARS-CoV probe is labeled with a 5′-reporter dye and a 3′-quencher dye, performing one or more additional rounds of amplification, and detecting fluorescence of the 5′-reporter dye, thereby detecting a SARS-CoV. In a specific, non-limiting example, the amplification utilizes RT-PCR. In a further specific example of the provided method, at least one of the nucleic acid primers that hybridize to a SARS-CoV nucleic acid and/or the TaqMan SARS-CoV probe that hybridizes to the SARS-CoV nucleic acid includes a sequence as set forth in any one of SEQ ID NOs: 16 33.

In another embodiment, a method is provided for detecting a SARS-CoV in a biological sample that contains antibodies. This method includes contacting the biological sample with a SARS-CoV-specific antigen, wherein the antigen includes a SARS-CoV organism and determining whether a binding reaction occurs between the SARS-CoV-specific antigen and an antibody in the biological sample if such is present, thereby detecting SARS-CoV.

In a further embodiment, a method is provided for detecting a SARS-CoV in a biological sample that contains polypeptides and/or fragments thereof. This method includes contacting the biological sample with a SARS-CoV-specific antibody and determining whether a binding reaction occurs between the SARS-CoV-specific antibody and a SARS-CoV polypeptide or fragment thereof in the biological sample if such is present, thereby detecting SARS-CoV. In a specific, non-limiting example, determining whether a binding reaction occurs between the SARS-CoV-specific antibody and a SARS-CoV polypeptide or fragment thereof is carried out in situ or in a tissue sample. In a further specific example, determining whether a binding reaction occurs between the SARS-CoV-specific antibody and a SARS-CoV polypeptide or fragment thereof includes an immunohistochemical assay.

An additional embodiment includes a kit for detecting a SARS-CoV in a sample, including a pair of nucleic acid primers that hybridize under stringent conditions to a SARS-CoV nucleic acid, wherein one primer is 5′-end labeled with a reporter dye. In a specific, non-limiting example, at least one of the nucleic acid primers that hybridize to a SARS-CoV nucleic acid includes a sequence as set forth in any one of SEQ ID NOs: 13 15.

Another example of the provided kit includes a pair of nucleic acid primers that hybridize under high stringency conditions to a SARS-CoV nucleic acid and a TaqMan SARS-CoV probe that hybridizes to the SARS-CoV nucleic acid, wherein the TaqMan SARS-CoV probe is labeled with a 5′-reporter dye and a 3′-quencher dye. In a specific, non-limiting example, at least one of the nucleic acid primers that hybridize to a SARS-CoV nucleic acid and/or the TaqMan SARS-CoV probe that hybridizes to the SARS-CoV nucleic acid includes a sequence as set forth in any one of SEQ ID NOs: 16 33.

Also disclosed herein is a composition including an isolated SARS-CoV organism. In one embodiment, the isolated SARS-CoV organism is an inactive isolated SARS-CoV organism. In another embodiment, the composition includes at least one component selected from the group consisting of pharmaceutically acceptable carriers, adjuvants and combinations of two or more thereof. In yet another embodiment, the composition is introduced into a subject, thereby eliciting an immune response against a SARS-CoV antigenic epitope in a subject.

IV. SARS-CoV Nucleotide and Amino Acid Sequences

The current disclosure provides an isolated SARS-CoV genome, isolated SARS-CoV polypeptides, and isolated nucleic acid molecules encoding the same. In one embodiment, the isolated SARS-CoV genome has a sequence as shown in SEQ ID NO: 1 or an equivalent thereof. Polynucleotides encoding a SARS-CoV polypeptide (encoded by an ORF from within the genome) are also provided, and are termed SARS-CoV nucleic acid molecules. These nucleic acid molecules include DNA, cDNA and RNA sequences which encode a SARS-CoV polypeptide. Specific, non-limiting examples of a SARS-CoV nucleic acid molecule encoding an ORF are nucleic acid 265 to nucleic acid 13,398 of SEQ ID NO: 1 (encoding SARS-CoV 1a, SEQ ID NO: 2), nucleic acid 13,398 to 21,482 of SEQ ID NO: 1 (encoding SARS-CoV 1b, SEQ ID NO: 3), nucleic acid 21,492 to 25,256 of SEQ ID NO: 1 (encoding SARS-CoV S, SEQ ID NO: 4), nucleic acid 25,268 to 26,089 of SEQ ID NO: 1 (encoding SARS-CoV X1, SEQ ID NO: 5), nucleic acid 25,689 to 26,150 of SEQ ID NO: 1 (encoding SARS-CoV X2, SEQ ID NO: 6), nucleic acid 26,117 to 26,344 of SEQ ID NO: 1 (encoding SARS-CoV E, SEQ ID NO: 7), nucleic acid 26,398 to 27,060 of SEQ ID NO: 1 (encoding SARS-CoV M, SEQ ID NO: 8), nucleic acid 27,074 to 27,262 of SEQ ID NO: 1 (encoding SARS-CoV X3, SEQ ID NO: 9), nucleic acid 27,273 to 27,638 of SEQ ID NO: 1 (encoding SARS-CoV X4, SEQ ID NO: 10), nucleic acid 27,864 to 28,115 of SEQ ID NO: 1 (encoding SARS-CoV X5, SEQ ID NO: 11), and nucleic acid 28,120 to 29,385 of SEQ ID NO: 1 (encoding SARS-CoV N, SEQ ID NO: 12).

Oligonucleotide primers and probes derived from the SARS-CoV genome (SEQ ID NO: 1) are also encompassed within the scope of the present disclosure. Such oligonucleotide primers and probes may comprise a sequence of at least about 15 consecutive nucleotides of the SARS-CoV nucleic acid sequence, such as at least about 20, 25, 30, 35, 40, 45, or 50 or more consecutive nucleotides. These primers and probes may be obtained from any region of the disclosed SARS-CoV genome (SEQ ID NO: 1), including particularly from any of the ORFs disclosed herein. Specific, non-limiting examples of oligonucleotide primers derived from the SARS-CoV genome (SEQ ID NO: 1) include: Cor-p-F2 (SEQ ID NO: 13), Cor-p-F3 (SEQ ID NO: 14), Cor-p-R1 (SEQ ID NO: 15), SARS1-F (SEQ ID NO: 16), SARS1-R (SEQ ID NO: 17), SARS2-F (SEQ ID NO: 19), SARS2-R (SEQ ID NO: 20), SARS3-F (SEQ ID NO: 22), SARS3-R (SEQ ID NO: 23), N3-F (SEQ ID NO: 25), N3-R (SEQ ID NO: 26), 3’NTR-F (SEQ ID NO: 28), 3’NTR-R (SEQ ID NO: 29), M-F (SEQ ID NO: 31), and M-R (SEQ ID NO: 32). Specific, non-limiting examples of oligonucleotide probes derived from the SARS-CoV genome (SEQ ID NO: 1) include: SARS1-P (SEQ ID NO: 18), SARS2-P (SEQ ID NO: 21), SARS3-P (SEQ ID NO: 24), N3-P (SEQ ID NO: 27), 3’NTR-P (SEQ ID NO: 30), and M-P (SEQ ID NO: 33).

Nucleic acid molecules encoding a SARS-CoV polypeptide can be operatively linked to regulatory sequences or elements. Regulatory sequences or elements include, but are not limited to promoters, enhancers, transcription terminators, a start codon (for example, ATG), stop codons, and the like.

Additionally, nucleic acid molecules encoding a SARS-CoV polypeptide can be inserted into an expression vector. Specific, non-limiting examples of vectors include, plasmids, bacteriophages, cosmids, animal viruses and yeast artificial chromosomes (YACs) (Burke et al., Science 236:806 12, 1987). Such vectors may then be introduced into a variety of hosts including somatic cells, and simple or complex organisms, such as bacteria, fungi (Timberlake and Marshall, Science 244:1313 17, 1989), invertebrates, plants (Gasser et al., Plant Cell 1:15 24, 1989), and animals (Pursel et al., Science 244:1281 88, 1989).

Transformation of a host cell with an expression vector carrying a nucleic acid molecule encoding a SARS-CoV polypeptide may be carried out by conventional techniques, as are well known to those skilled in the art. By way of example, where the host is prokaryotic, such as E. coli, competent cells that are capable of DNA uptake can be prepared from cells harvested after exponential growth phase and subsequently treated by the CaCl.sub.2 method using procedures well known in the art. Alternatively, MgCl.sub.2 or RbCl can be used. Transformation can also be performed after forming a protoplast of the host cell if desired, or by electroporation.

When the host is a eukaryote, methods of transfection of DNA, such as calcium phosphate coprecipitates, conventional mechanical procedures such as microinjection, electroporation, insertion of a plasmid encased in liposomes, or virus vectors, may be used. Eukaryotic cells can also be cotransformed with SARS-CoV nucleic acid molecules, and a second foreign DNA molecule encoding a selectable phenotype, such as the herpes simplex thymidine kinase gene. Another method is to use a eukaryotic viral vector, such as simian virus 40 or bovine papilloma virus, to transiently infect or transform eukaryotic cells and express the protein (see, for example, Eukaryotic Viral Vectors, Cold Spring Harbor Laboratory, Gluzman ed., 1982).

The SARS-CoV polypeptides of this disclosure include proteins encoded by any of the ORFs disclosed herein, and equivalents thereof. Specific, non-limiting examples of SARS-CoV proteins are provided in SEQ ID NOs: 2 12. Fusion proteins are also contemplated that include a heterologous amino acid sequence chemically linked to a SARS-CoV polypeptide. Exemplary heterologous sequences include short amino acid sequence tags (such as six histidine residues), as well a fusion of other proteins (such as c-myc or green fluorescent protein fusions). Epitopes of the SARS-CoV proteins, that are recognized by an antibody or that bind the major histocompatibility complex, and can be used to induce a SARS-CoV-specific immune response, are also encompassed by this disclosure.

Methods for expressing large amounts of protein from a cloned gene introduced into E. coli may be utilized for the purification and functional analysis of proteins. For example, fusion proteins consisting of amino terminal peptides encoded by a portion of the E. coli lacZ or trpE gene linked to SARS-CoV proteins may be used to prepare polyclonal and monoclonal antibodies against these proteins.

Intact native protein may also be produced in E. coli in large amounts for functional studies. Methods and plasmid vectors for producing fusion proteins and intact native proteins in bacteria are described by Sambrook et al. (ed.), Molecular Cloning: A Laboratory Manual, 2.sup.nd ed., vol. 1 3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989. Such fusion proteins may be made in large amounts, are easy to purify, and can be used to elicit antibody response. Native proteins can be produced in bacteria by placing a strong, regulated promoter and an efficient ribosome-binding site upstream of the cloned gene. If low levels of protein are produced, additional steps may be taken to increase protein production; if high levels of protein are produced, purification is relatively easy. Suitable methods are presented by Sambrook et al. (ed.), Molecular Cloning: A Laboratory Manual, 2.sup.nd ed., vol. 1 3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, and are well known in the art. Often, proteins expressed at high levels are found in insoluble inclusion bodies. Methods for extracting proteins from these aggregates are described by Sambrook et al. (ed.), Molecular Cloning: A Laboratory Manual, 2.sup.nd ed., vol. 1 3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.

Isolation and purification of recombinantly expressed proteins may be carried out by conventional means including preparative chromatography and immunological separations. Additionally, the proteins can be chemically synthesized by any of a number of manual or automated methods of synthesis known in the art.

V. Specific Binding Agents

The disclosure provides specific binding agents that bind to SARS-CoV polypeptides disclosed herein. The binding agent may be useful for purifying and detecting the polypeptides, as well as for detection and diagnosis of SARS-CoV. Examples of the binding agents are a polyclonal or monoclonal antibody, and fragments thereof, that bind to any of the SARS-CoV polypeptides disclosed herein.

Monoclonal or polyclonal antibodies may be raised to recognize a SARS-CoV polypeptide described herein, or a fragment or variant thereof. Optimally, antibodies raised against these polypeptides would specifically detect the polypeptide with which the antibodies are generated. That is, antibodies raised against a specific SARS-CoV polypeptide will recognize and bind that polypeptide, and will not substantially recognize or bind to other polypeptides or antigens. The determination that an antibody specifically binds to a target polypeptide is made by any one of a number of standard immunoassay methods; for instance, the Western blotting technique (Sambrook et al. (ed.), Molecular Cloning: A Laboratory Manual, 2.sup.nd ed., vol. 1 3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).

Substantially pure SARS-CoV recombinant polypeptide antigens suitable for use as immunogen may be isolated from the transformed cells described above, using methods well known in the art. Monoclonal or polyclonal antibodies to the antigens may then be prepared.

Monoclonal antibodies to the polypeptides can be prepared from murine hybridomas according to the classic method of Kohler & Milstein (Nature 256:495 97, 1975), or a derivative method thereof. Briefly, a mouse is repetitively inoculated with a few micrograms of the selected protein immunogen (for example, a polypeptide comprising at least one SARS-CoV-specific epitope, a portion of a polypeptide comprising at least one SARS-CoV-specific epitope, or a synthetic peptide comprising at least one SARS-CoV-specific epitope) over a period of a few weeks. The mouse is then sacrificed, and the antibody-producing cells of the spleen isolated. The spleen cells are fused by means of polyethylene glycol with mouse myeloma cells, and the excess unfused cells destroyed by growth of the system on selective media comprising aminopterin (HAT media). The successfully fused cells are diluted and aliquots of the dilution placed in wells of a microtiter plate where growth of the culture is continued. Antibody-producing clones are identified by detection of antibody in the supernatant fluid of the wells by immunoassay procedures, such as ELISA, as originally described by Engvall (Meth. Enzymol., 70:419 39, 1980), or a derivative method thereof. Selected positive clones can be expanded and their monoclonal antibody product harvested for use. Detailed procedures for monoclonal antibody production are described in Harlow and Lane, Using Antibodies: A Laboratory Manual, CSHL, New York, 1999.

Polyclonal antiserum containing antibodies can be prepared by immunizing suitable animals with a polypeptide comprising at least one SARS-CoV-specific epitope, a portion of a polypeptide comprising at least one SARS-CoV-specific epitope, or a synthetic peptide comprising at least one SARS-CoV-specific epitope, which can be unmodified or modified, to enhance immunogenicity.

Effective antibody production (whether monoclonal or polyclonal) is affected by many factors related both to the antigen and the host species. For example, small molecules tend to be less immunogenic than others and may require the use of carriers and adjuvant. Also, host animals vary in response to site of inoculations and dose, with either inadequate or excessive doses of antigen resulting in low titer antisera. Small doses (ng level) of antigen administered at multiple intradermal sites appear to be most reliable. An effective immunization protocol for rabbits can be found in Vaitukaitis et al. (J. Clin. Endocrinol. Metab., 33:988 91, 1971).

Booster injections can be given at regular intervals, and antiserum harvested when the antibody titer thereof, as determined semi-quantitatively, for example, by double immunodiffusion in agar against known concentrations of the antigen, begins to fall. See, for example, Ouchterlony et al., Handbook of Experimental Immunology, Wier, D. (ed.), Chapter 19, Blackwell, 1973. A plateau concentration of antibody is usually in the range of 0.1 to 0.2 mg/ml of serum (about 12 .mu.M). Affinity of the antisera for the antigen is determined by preparing competitive binding curves, as described, for example, by Fisher (Manual of Clinical Immunology, Ch. 42, 1980).

Antibody fragments may be used in place of whole antibodies and may be readily expressed in prokaryotic host cells. Methods of making and using immunologically effective portions of monoclonal antibodies, also referred to as “antibody fragments,” are well known and include those described in Better & Horowitz, Methods Enzymol. 178:476 96, 1989; Glockshuber et al., Biochemistry 29:1362 67, 1990; and U.S. Pat. No. 5,648,237 (Expression of Functional Antibody Fragments); U.S. Pat. No. 4,946,778 (Single Polypeptide Chain Binding Molecules); and U.S. Pat. No. 5,455,030 (Immunotherapy Using Single Chain Polypeptide Binding Molecules), and references cited therein. Conditions whereby a polypeptide/binding agent complex can form, as well as assays for the detection of the formation of a polypeptide/binding agent complex and quantitation of binding affinities of the binding agent and polypeptide, are standard in the art. Such assays can include, but are not limited to, Western blotting, immunoprecipitation, immunofluorescence, immunocytochemistry, immunohistochemistry, fluorescence activated cell sorting (FACS), fluorescence in situ hybridization (FISH), immunomagnetic assays, ELISA, ELISPOT (Coligan et al., Current Protocols in Immunology, Wiley, NY, 1995), agglutination assays, flocculation assays, cell panning, and the like, as are well known to one of skill in the art.

Binding agents of this disclosure can be bound to a substrate (for example, beads, tubes, slides, plates, nitrocellulose sheets, and the like) or conjugated with a detectable moiety, or both bound and conjugated. The detectable moieties contemplated for the present disclosure can include, but are not limited to, an immunofluorescent moiety (for example, fluorescein, rhodamine), a radioactive moiety (for example, .sup.32P, .sup.125I, .sup.35S), an enzyme moiety (for example, horseradish peroxidase, alkaline phosphatase), a colloidal gold moiety, and a biotin moiety. Such conjugation techniques are standard in the art (for example, see Harlow and Lane, Using Antibodies: A Laboratory Manual, CSHL, New York, 1999; Yang et al., Nature, 382:319 24, 1996).

VI. Detection and Diagnosis of SARS-CoV

A. Nucleic Acid Based Methods of Detection and Diagnosis

A major application of the SARS-CoV sequence information presented herein is in the area of detection and diagnostic testing for SARS-CoV infection. Methods for screening a subject to determine if the subject has been or is currently infected with SARS-CoV are disclosed herein.

One such method includes providing a sample, which sample includes a nucleic acid such as DNA or RNA, and providing an assay for detecting in the sample the presence of a SARS-CoV nucleic acid molecule. Suitable samples include all biological samples useful for detection of viral infection in subjects, including, but not limited to, cells, tissues (for example, lung and kidney), bodily fluids (for example, blood, serum, urine, saliva, sputum, and cerebrospinal fluid), bone marrow aspirates, BAL, and oropharyngeal wash. Additional suitable samples include all environmental samples useful for detection of viral presence in the environment, including, but not limited to, a sample obtained from inanimate objects or reservoirs within an indoor or outdoor environment. The detection in the sample of a SARS-CoV nucleic acid molecule may be performed by a number of methodologies, non-limiting examples of which are outlined below.

In one embodiment, detecting in the sample the presence of a SARS-CoV nucleic acid molecule includes the amplification of a SARS-CoV nucleic acid sequence (or a fragment thereof). Any nucleic acid amplification method can be used. In one specific, non-limiting example, PCR is used to amplify the SARS-CoV nucleic acid sequence(s). In another non-limiting example, RT-PCR can be used to amplify the SARS-CoV nucleic acid sequences. In an additional non-limiting example, transcription-mediated amplification can be used to amplify the SARS-CoV nucleic acid sequences.

In some embodiments, a pair of SARS-CoV-specific primers are utilized in the amplification reaction. One or both of the primers can be end-labeled (for example, radiolabeled, fluoresceinated, or biotinylated). In one specific, non-limiting example, at least one of the primers is 5′-end labeled with the reporter dye 6-carboxyfluorescein (6-FAM). The pair of primers includes an upstream primer (which binds 5′ to the downstream primer) and a downstream primer (which binds 3′ to the upstream primer). In one embodiment, either the upstream primer or the downstream primer is labeled. Specific, non-limiting examples of SARS-CoV-specific primers include, but are not limited to: Cor-p-F2 (SEQ ID NO: 13), Cor-p-F3 (SEQ ID NO: 14), Cor-p-R1 (SEQ ID NO: 15), SARS1-F (SEQ ID NO: 16), SARS1-R (SEQ ID NO: 17), SARS2-F (SEQ ID NO: 19), SARS2-R (SEQ ID NO: 20), SARS3-F (SEQ ID NO: 22), SARS3-R (SEQ ID NO: 23), N3-F (SEQ ID NO: 25), N3-R (SEQ ID NO: 26), 3’NTR-F (SEQ ID NO: 28), 3’NTR-R (SEQ ID NO: 29), M-F (SEQ ID NO: 31), and M-R (SEQ ID NO: 32). Additional primer pairs can be generated, for instance, to amplify any of the specific ORFs described herein, using well known primer design principles and methods.

In one specific, non-limiting example, electrophoresis is used to detect amplified SARS-CoV-specific sequences. Electrophoresis can be automated using many methods well know in the art. In one embodiment, a genetic analyzer is used, such as an ABI 3100 Prism Genetic Analyzer (PE Applied Biosystems, Foster City, Calif.), wherein the bands are analyzed using GeneScan software (PE Applied Biosystems, Foster City, Calif.).

In another specific, non-limiting example, hybridization assays are used to detect amplified SARS-CoV-specific sequences using distinguishing oligonucleotide probes. Such probes include “TaqMan” probes. TaqMan probes consist of an oligonucleotide with a reporter at the 5′-end and a quencher at the 3′-end. In one specific, non-limiting example, the reporter is 6-FAM and the quencher is Blackhole Quencher (Biosearch Tech., Inc., Novato, Calif.). When the probe is intact, the proximity of the reporter to the quencher results in suppression of reporter fluorescence, primarily by fluorescence resonance energy transfer. If the target of interest is present, the TaqMan probe specifically hybridizes between the forward and reverse primer sites during the PCR annealing step. In the process of PCR elongation, the 5′-3′ nucleolytic activity of the Taq DNA polymerase cleaves the hybridized probe between the reporter and the quencher. The probe fragments are then displaced from the target, and polymerization of the strand continues. Taq DNA polymerase does not cleave non-hybridized probe, and cleaves the hybridized probe only during polymerization. The 3′-end of the probe is blocked to prevent extension of the probe during PCR. The 5′-3′ nuclease cleavage of the hybridized probe occurs in every cycle and does not interfere with the exponential accumulation of PCR product. Accumulation of PCR products is detected directly by monitoring the increase in fluorescence of the released reporter. The increase in fluorescence signal is detected only if the target sequence is complementary to the probe and is amplified during PCR. Therefore, non-specific amplification is not detected. SARS-CoV-specific TaqMan probes of the present disclosure include, but are not limited to: SARS1-P (SEQ ID NO: 18), SARS2-P (SEQ ID NO: 21), SARS3-P (SEQ ID NO: 24), N3-P (SEQ ID NO: 27), 3’NTR-P (SEQ ID NO: 30), and M-P (SEQ ID NO: 33), and hybridization assays include, but are not limited to, a real-time RT-PCR assay.

B. Protein Based Methods of Detection and Diagnosis

The present disclosure further provides methods of detecting a SARS-CoV antigen in a sample, and/or diagnosing SARS-CoV infection in a subject by detecting a SARS-CoV antigen. Examples of such methods comprise contacting the sample with a SARS-CoV-specific binding agent under conditions whereby an antigen/binding agent complex can form; and detecting formation of the complex, thereby detecting SARS-CoV antigen in a sample and/or diagnosing SARS-CoV infection in a subject. It is contemplated that at least certain antigens will be on an intact SARS-CoV virion, will be a SARS-CoV-encoded protein displayed on the surface of a SARS-CoV-infected cell expressing the antigen, or will be a fragment of the antigen. Contemplated samples subject to analysis by these methods can comprise any sample, such as a clinical sample, useful for detection of viral infection in a subject.

Methods for detecting antigens in a sample are discussed, for example, in Ausubel et al. Short Protocols in Molecular Biology, 4.sup.th ed., John Wiley & Sons, Inc., 1999. Enzyme immunoassays such as IFA, ELISA and immunoblotting can be readily adapted to accomplish the detection of SARS-CoV antigens according to the methods of this disclosure. An ELISA method effective for the detection of soluble SARS-CoV antigens is the direct competitive ELISA. This method is most useful when a specific SARS-CoV antibody and purified SARS-CoV antigen are available. Briefly: 1) coat a substrate (for example, a microtiter plate) with a sample suspected of containing a SARS-CoV antigen; 2) contact the bound SARS-CoV antigen with a SARS-CoV-specific antibody bound to a detectable moiety (for example, horseradish peroxidase enzyme or alkaline phosphatase enzyme); 3) add purified inhibitor SARS-CoV antigen; 4) contact the above with the substrate for the enzyme; and 5) observe/measure inhibition of color change or fluorescence and quantitate antigen concentration (for example, using a microtiter plate reader).

An additional ELISA method effective for the detection of soluble SARS-CoV antigens is the antibody-sandwich ELISA. This method is frequently more sensitive in detecting antigen than the direct competitive ELISA method. Briefly: 1) coat a substrate (for example, a microtiter plate) with a SARS-CoV-specific antibody; 2) contact the bound SARS-CoV antibody with a sample suspected of containing a SARS-CoV antigen; 3) contact the above with SARS-CoV-specific antibody bound to a detectable moiety (for example, horseradish peroxidase enzyme or alkaline phosphatase enzyme); 4) contact the above with the substrate for the enzyme; and 5) observe/measure color change or fluorescence and quantitate antigen concentration (for example, using a microtiter plate reader).

An ELISA method effective for the detection of cell-surface SARS-CoV antigens is the direct cellular ELISA. Briefly, cells suspected of exhibiting a cell-surface SARS-CoV antigen are fixed (for example, using glutaraldehyde) and incubated with a SARS-CoV-specific antibody bound to a detectable moiety (for example, horseradish peroxidase enzyme or alkaline phosphatase enzyme). Following a wash to remove unbound antibody, substrate for the enzyme is added and color change or fluorescence is observed/measured.

The present disclosure further provides methods of detecting a SARS-CoV-reactive antibody in a sample, and/or diagnosing SARS-CoV infection in a subject by detecting a SARS-CoV-reactive antibody. Examples of such methods comprise contacting the sample with a SARS-CoV polypeptide of this disclosure under conditions whereby a polypeptide/antibody complex can form; and detecting formation of the complex, thereby detecting SARS-CoV antibody in a sample and/or diagnosing SARS-CoV infection in a subject. Contemplated samples subject to analysis by these methods can comprise any sample, such as a clinical sample, as described herein as being useful for detection of viral infection in a subject.

Methods for detecting antibodies in a sample are discussed, for example, in Ausubel et al. Short Protocols in Molecular Biology, 4.sup.th ed., John Wiley & Sons, Inc., 1999. Enzyme immunoassays such as IFA, ELISA and immunoblotting can be readily adapted to accomplish the detection of SARS-CoV antibodies according to the methods of this disclosure. An ELISA method effective for the detection of specific SARS-CoV antibodies is the indirect ELISA method. Briefly: 1) bind a SARS-CoV polypeptide to a substrate (for example, a microtiter plate; 2) contact the bound polypeptide with a sample suspected of containing SARS-CoV antibody; 3) contact the above with a secondary antibody bound to a detectable moiety which is reactive with the bound antibody (for example, horseradish peroxidase enzyme or alkaline phosphatase enzyme); 4) contact the above with the substrate for the enzyme; and 5) observe/measure color change or fluorescence.

Another immunologic technique that can be useful in the detection of SARS-CoV antibodies uses monoclonal antibodies for detection of antibodies specifically reactive with SARS-CoV polypeptides in a competitive inhibition assay. Briefly, a sample suspected of containing SARS-CoV antibodies is contacted with a SARS-CoV polypeptide of this disclosure which is bound to a substrate (for example, a microtiter plate). Excess sample is thoroughly washed away. A labeled (for example, enzyme-linked, fluorescent, radioactive, and the like) monoclonal antibody specific for the SARS-CoV polypeptide is then contacted with any previously formed polypeptide-antibody complexes and the amount of monoclonal antibody binding is measured. The amount of inhibition of monoclonal antibody binding is measured relative to a control (no monoclonal antibody), allowing for detection and measurement of antibody in the sample. The degree of monoclonal antibody inhibition can be a very specific assay for detecting SARS-CoV. Monoclonal antibodies can also be used for direct detection of SARS-CoV in cells or tissue samples by, for example, IFA analysis according to standard methods.

As a further example, a micro-agglutination test can be used to detect the presence of SARS-CoV antibodies in a sample. Briefly, latex beads, red blood cells or other agglutinable particles are coated with a SARS-CoV polypeptide of this disclosure and mixed with a sample, such that antibodies in the sample that are specifically reactive with the antigen crosslink with the antigen, causing agglutination. The agglutinated polypeptide-antibody complexes form a precipitate, visible with the naked eye or measurable by spectrophotometer. In a modification of the above test, SARS-CoV-specific antibodies of this disclosure can be bound to the agglutinable particles and SARS-CoV antigen in the sample thereby detected.

VII. Pharmaceutical and Immune Stimulatory Compositions and Uses Thereof

Pharmaceutical compositions including SARS-CoV nucleic acid sequences, SARS-CoV polypeptides, or antibodies that bind these polypeptides, are also encompassed by the present disclosure. These pharmaceutical compositions include a therapeutically effective amount of one or more SARS-CoV polypeptides, one or more nucleic acid molecules encoding a SARS-CoV polypeptide, or an antibody that binds a SARS-CoV polypeptide, in conjunction with a pharmaceutically acceptable carrier.

Disclosed herein are substances suitable for use as immune stimulatory compositions for the inhibition or treatment of SARS. Particular immune stimulatory compositions are directed against SARS-CoV, and include antigens obtained from SARS-CoV. In one embodiment, an immune stimulatory composition contains attenuated SARS-CoV. Methods of viral attenuation are well know in the art, and include, but are not limited to, high serial passage (for example, in susceptible host cells under specific environmental conditions to select for attenuated virions), exposure to a mutagenic agent (for example, a chemical mutagen or radiation), genetic engineering using recombinant DNA technology (for example, using gene replacement or gene knockout to disable one or more viral genes), or some combination thereof.

In another embodiment, the immune stimulatory composition contains inactivated SARS-CoV. Methods of viral inactivation are well known in the art, and include, but are not limited to, heat and chemicals (for example, formalin, .beta.-propiolactone, and ehtylenimines).

In yet another embodiment, the immune stimulatory composition contains a nucleic acid vector that includes SARS-CoV nucleic acid molecules described herein, or that includes a nucleic acid sequence encoding an immunogenic polypeptide or polypeptide fragment of SARS-CoV or derived from SARS-CoV, such as a polypeptide that encodes a surface protein of SARS-CoV.

In a further embodiment, the immune stimulatory composition contains a SARS-CoV subunit, such as glycoprotein, major capsid protein, or other gene products found to elicit humoral and/or cell mediated immune responses.

The provided immune stimulatory SARS-CoV polypeptides, constructs or vectors encoding such polypeptides, are combined with a pharmaceutically acceptable carrier or vehicle for administration as an immune stimulatory composition to human or animal subjects. In some embodiments, more than one immune stimulatory SARS-CoV polypeptide may be combined to form a single preparation.

The immunogenic formulations may be conveniently presented in unit dosage form and prepared using conventional pharmaceutical techniques. Such techniques include the step of bringing into association the active ingredient and the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers. Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of a sterile liquid carrier, for example, water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets commonly used by one of ordinary skill in the art.

In certain embodiments, unit dosage formulations are those containing a dose or unit, or an appropriate fraction thereof, of the administered ingredient. It should be understood that in addition to the ingredients particularly mentioned above, formulations encompassed herein may include other agents commonly used by one of ordinary skill in the art.

The compositions provided herein, including those for use as immune stimulatory compositions, may be administered through different routes, such as oral, including buccal and sublingual, rectal, parenteral, aerosol, nasal, intramuscular, subcutaneous, intradermal, and topical. They may be administered in different forms, including but not limited to solutions, emulsions and suspensions, microspheres, particles, microparticles, nanoparticles, and liposomes.

The volume of administration will vary depending on the route of administration. By way of example, intramuscular injections may range from about 0.1 ml to about 1.0 ml. Those of ordinary skill in the art will know appropriate volumes for different routes of administration.

A relatively recent development in the field of immune stimulatory compounds (for example, vaccines) is the direct injection of nucleic acid molecules encoding peptide antigens (broadly described in Janeway & Travers, Immunobiology: The Immune System In Health and Disease, page 13.25, Garland Publishing, Inc., New York, 1997; and McDonnell & Askari, N. Engl. J. Med. 334:42 45, 1996). Vectors that include nucleic acid molecules described herein, or that include a nucleic acid sequence encoding an immunogenic SARS-CoV polypeptide may be utilized in such DNA vaccination methods.

Thus, the term “immune stimulatory composition” as used herein also includes nucleic acid vaccines in which a nucleic acid molecule encoding a SARS-CoV polypeptide is administered to a subject in a pharmaceutical composition. For genetic immunization, suitable delivery methods known to those skilled in the art include direct injection of plasmid DNA into muscles (Wolff et al., Hum. Mol. Genet. 1:363, 1992), delivery of DNA complexed with specific protein carriers (Wu et al., J. Biol. Chem. 264:16985, 1989), co-precipitation of DNA with calcium phosphate (Benvenisty and Reshef, Proc. Natl. Acad. Sci. 83:9551, 1986), encapsulation of DNA in liposomes (Kaneda et al., Science 243:375, 1989), particle bombardment (Tang et al., Nature 356:152, 1992; Eisenbraun et al., DNA Cell Biol. 12:791, 1993), and in vivo infection using cloned retroviral vectors (Seeger et al., Proc. Natl. Acad. Sci. 81:5849, 1984). Similarly, nucleic acid vaccine preparations can be administered via viral carrier.

The amount of immunostimulatory compound in each dose of an immune stimulatory composition is selected as an amount that induces an immunostimulatory or immunoprotective response without significant, adverse side effects. Such amount will vary depending upon which specific immunogen is employed and how it is presented. Initial injections may range from about 1 .mu.g to about 1 mg, with some embodiments having a range of about 10 .mu.g to about 800 .mu.g, and still other embodiments a range of from about 25 .mu.g to about 500 .mu.g. Following an initial administration of the immune stimulatory composition, subjects may receive one or several booster administrations, adequately spaced. Booster administrations may range from about 1 .mu.g to about 1 mg, with other embodiments having a range of about 10 .mu.g to about 750 .mu.g, and still others a range of about 50 .mu.g to about 500 .mu.g. Periodic boosters at intervals of 1 5 years, for instance three years, may be desirable to maintain the desired levels of protective immunity.

It is also contemplated that the provided immunostimulatory molecules and compositions can be administered to a subject indirectly, by first stimulating a cell in vitro, which stimulated cell is thereafter administered to the subject to elicit an immune response. Additionally, the pharmaceutical or immune stimulatory compositions or methods of treatment may be administered in combination with other therapeutic treatments.

VIII. Kits

Also provided herein are kits useful in the detection and/or diagnosis of SARS-CoV. This includes kits for use with nucleic acid and protein detection methods, such as those disclosed herein.

The SARS-CoV-specific oligonucleotide primers and probes described herein can be supplied in the form of a kit for use in detection of SARS-CoV. In such a kit, an appropriate amount of one or more of the oligonucleotides is provided in one or more containers, or held on a substrate. An oligonucleotide primer or probe can be provided in an aqueous solution or as a freeze-dried or lyophilized powder, for instance. The container(s) in which the oligonucleotide(s) are supplied can be any conventional container that is capable of holding the supplied form, for instance, microfuge tubes, ampoules, or bottles. In some applications, pairs of primers are provided in pre-measured single use amounts in individual (typically disposable) tubes or equivalent containers. With such an arrangement, the sample to be tested for the presence of a SARS-CoV nucleic acid can be added to the individual tubes and amplification carried out directly.

The amount of each oligonucleotide supplied in the kit can be any appropriate amount, and can depend on the market to which the product is directed. For instance, if the kit is adapted for research or clinical use, the amount of each oligonucleotide primer provided would likely be an amount sufficient to prime several PCR amplification reactions. General guidelines for determining appropriate amounts can be found, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2001; Ausubel et al. (eds.), Short Protocols in Molecular Biology, John Wiley and Sons, New York, N.Y., 1999; and Innis et al., PCR Applications, Protocols for Functional Genomics, Academic Press, Inc., San Diego, Calif., 1999. A kit can include more than two primers, in order to facilitate the amplification of a larger number of SARS-CoV nucleotide sequences.

In some embodiments, kits also include one or more reagents necessary to carry out in vitro amplification reactions, including DNA sample preparation reagents, appropriate buffers (for example, polymerase buffer), salts (for example, magnesium chloride), and deoxyribonucleotides (dNTPs).

Kits can include either labeled or unlabeled oligonucleotide primers and/or probes for use in detection of SARS-CoV nucleotide sequences. The appropriate sequences for such a probe will be any sequence that falls between the annealing sites of the two provided oligonucleotide primers, such that the sequence that the probe is complementary to is amplified during the amplification reaction.

One or more control sequences for use in the amplification reactions also can be supplied in the kit. In other particular embodiments, the kit includes equipment, reagents, and instructions for extracting and/or purifying nucleotides from a sample.

Kits for the detection of SARS-CoV antigen include for instance at least one SARS-CoV antigen-specific binding agent (for example, a polyclonal or monoclonal antibody or antibody fragment). The kits may also include means for detecting antigen:specific binding agent complexes, for instance the specific binding agent may be detectably labeled. If the specific binding agent is not labeled, it may be detected by second antibodies or protein A, for example, which may also be provided in some kits in one or more separate containers. Such techniques are well known.

Another example of an assay kit provided herein is a recombinant SARS-CoV-specific polypeptide (or fragment thereof) as an antigen and an enzyme-conjugated anti-human antibody as a second antibody. Examples of such kits also can include one or more enzymatic substrates. Such kits can be used to test if a sample from a subject contains antibodies against a SARS-CoV-specific protein.

The subject matter of the present disclosure is further illustrated by the following non-limiting Examples.

EXAMPLES

Example 1

Isolation and Characterization of SARS-CoV

Virus Isolation and Ultrastructural Characterization

This example describes the original isolation and characterization of a new human coronavirus from patients with SARS.

A variety of clinical specimens (blood, serum, material from oropharyngeal swabs or washings, material from nasopharyngeal swabs, and tissues of major organs collected at autopsy) from patients meeting the case definition of SARS were sent to the Centers for Disease Control and Prevention (CDC) as part of the etiologic investigation of SARS. These samples were inoculated onto a number of continuous cell lines, including Vero E6, NCI-H292, MDCK, LLC-MK2, and B95-8 cells, and into suckling ICR mice by the intracranial and intraperitoneal routes. All cultures were observed daily for CPE. Maintenance medium was replenished at day seven, and cultures were terminated fourteen days after inoculation. Any cultures exhibiting identifiable CPE were subjected to several procedures to identify the cause of the effect. Suckling mice were observed daily for fourteen days, and any sick or dead mice were further tested by preparing a brain suspension that was filtered and subcultured. Mice that remained well after fourteen days were killed, and their test results were recorded as negative.

Two cell lines, Vero E6 cells and NCI-H292 cells, inoculated with oropharyngeal specimens from Patient 16 (a 46 year old male physician with an epidemiologic link to a hospital with multiple SARS patients) initially showed CPE (Table 1)

TABLE-US-00003 TABLE 1 Specimens from patients with SARS that were positive for SARS-CoV by one or more methods*. Exposure Findings Patient and on Chest Hospital- Serologic No. Setting Age/Sex Radiograph ization Results Specimen Isolation RT-PCR 1 Singapore, 53 yr/F Pneumonia Yes + Nasal, – Not done hospital oropharyngeal swabs 2.dagger. Hong 36 yr/F Pneumonia Yes + Nasal, swab – Not done Kong, hotel 3 Hong 22 yr/M Pneumonia Yes + Swab – – Kong, hotel 4.dagger. Hong 39 yr/M Pneumonia Yes + Nasal, – – Kong, pharyngeal hotel swab 5 Hong 49 Yr/M Pneumonia Yes Not done Sputum + + Kong, hotel 6.dagger-dbl. Hong 46 yr/M Pneumonia Yes + Kidney, lung, +.sctn. + Kong, bronchoalveolar hotel lavage 7 Vietnam, Adult/ Pneumonia Yes – Oropharyngeal + + hospital unknown wash 8 Vietnam, Adult/ Pneumonia Yes – Oropharyngeal – + hospital unknown wash 9 Vietnam, Adult/ Pneumonia Yes – Oropharyngeal – + hospital unknown wash 10 Vietnam, Adult/ Pneumonia Yes – Oropharyngeal – + hospital unknown wash 11 Vietnam, Adult/ Pneumonia Yes – Oropharyngeal – + hospital unknown wash 12 Vietnam, Adult/ Pneumonia Yes – Oropharyngeal – + hospital unknown wash 13 Vietnam, Adult/ Pneumonia Yes – Oropharyngeal + + hospital unknown wash 14 Vietnam, Adult/ Pneumonia Yes – Oropharyngeal – + hospital unknown wash 15 Vietnam, Adult/ Pneumonia Yes – Oropharyngeal – + hospital unknown wash 16 Vietnam, 46 yr/M Pneumonia Yes + Nasal, + + hospital oropharyngeal swabs 17 Canada, 43 yr/M Pneumonia Yes Not done Lung, bone – family marrow 18 Taiwan, 51 yr/F Pneumonia Yes – Sputum – + family 19 Hong Adult/F Pneumonia Yes + Oropharyngeal – + Kong, wash hotel *Plus signs denote positive results, and minus signs negative results. The serologic and RT-PCR assays were not necessarily performed on samples obtained at the same time. .dagger.This was a late specimen, antibody positive at first sample. .dagger-dbl.Travel included China, Hong Kong (hotel), and Hanoi (the patient was the index patient in the French Hospital). .sctn.Isolation was from the kidney only. Isolation was from the oropharyngeal only.

The CPE in the Vero E6 cells was first noted on the fifth day post-inoculation; it was focal, with cell rounding and a refractive appearance in the affected cells that was soon followed by cell detachment (FIG. 1A). The CPE spread quickly to involve the entire cell monolayer within 24 to 48 hours. Subculture of material after preparation of a master seed stock (used for subsequent antigen production) resulted in the rapid appearance of CPE, as noted above, and in complete destruction of the monolayer in the inoculated flasks within 48 hours. Similar CPE was also noted in four additional cultures: three cultures of respiratory specimens (two oropharyngeal washes and one sputum specimen) and one culture of a suspension of kidney tissue obtained at autopsy. In these specimens, the initial CPE was observed between day two and day four and, as noted above, the CPE rapidly progressed to involve the entire cell monolayer.

Tissue culture samples showing CPE were prepared for electron-microscopical examination. Negative-stain electron-microscopical specimens were prepared by drying culture supernatant, mixed 1:1 with 2.5% paraformaldehyde, onto Formvarcarbon-coated grids and staining with 2% methylamine tungstate. Thin-section electron-microscopical specimens were prepared by fixing a washed cell pellet with 2.5% glutaraldehyde and embedding the cell pellet in epoxy resin. In addition, a master seed stock was prepared from the remaining culture supernatant and cells by freeze-thawing the culture flask, clarifying the thawed contents by centrifugation at 1000.times.g, and dispensing the supernatant into aliquots stored in gas phase over liquid nitrogen. The master seed stock was subcultured into 850-cm.sup.2 roller bottles of Vero E6 cells for the preparation of formalin-fixed positive control cells for immunohistochemical analysis, mixed with normal Vero E6 cells, and gamma-irradiated for preparation of spot slides for IFA tests or extracted with detergent and gamma-irradiated for use as an ELISA antigen for antibody tests.

Examination of CPE-positive Vero E6 cells by thin-section electron microscopy revealed characteristic coronavirus particles within the cisternae of the rough endoplasmic reticulum and in vesicles (FIG. 2A) (Becker et al., J. Virol. 1:1019 27, 1967; Oshiro et al. J. Gen. Virol. 12:161 8, 1971). Extracellular particles were found in large clusters and adhering to the surface of the plasma membrane. Negative-stain electron microscopy identified coronavirus particles, 80 to 140 nm in diameter, with 20- to 40-nm complex surface projections surrounding the periphery (FIG. 2B). Hemagglutinin esterase-type glycoprotein projections were not seen.

The isolation and growth of a human-derived coronavirus in Vero E6 cells were unexpected. The previously known human coronaviruses are notably fastidious, preferring select cell lines, organ culture, or suckling mice for propagation. The only human or animal coronavirus which has been shown to grow in Vero E6 cells is PEDV, and it requires the addition of trypsin to culture medium for growth in the cells. Moreover, PEDV adapted to growth in Vero E6 cells results in a strikingly different CPE, with cytoplasmic vacuoles and the formation of large syncytia. Syncytial cells were only observed occasionally in monolayers of Vero E6 cells infected with the SARS-CoV; they clearly do not represent the dominant CPE.

Reverse Transcription-Polymerase Chain Reaction and Sequencing

For RT-PCR assays, cell-culture supernatants were placed in lysis buffer. RNA extracts were prepared from 100 .mu.l of each specimen (or culture supernatant) with the automated NucliSens extraction system (bioMerieux, Durham, N.C.). Initially, degenerate, inosine-containing primers IN-2 (+) 5’GGGTTGGGACTA TCCTAAGTGTGA3′ (SEQ ID NO: 34) and IN-4 (-) 5’TAACACACAACICCATCA TCA3′ (SEQ ID NO: 35) were designed to anneal to sites encoding conserved amino acid motifs that were identified on the basis of alignments of available coronavirus ORF1a, ORF1b, S, HE, M, and N gene sequences. Additional, SARS-specific, primers Cor-p-F2 (+) 5’CTAACATGCTTAGGATAATGG3′ (SEQ ID NO: 13), Cor-p-F3 (+) 5’GCCTCTCTTGTTCTTGCTCGC3′ (SEQ ID NO: 14), and Cor-p-R1 (-) 5’CAGGTAAGCGTAAAACTCATC3 (SEQ ID NO: 15) were designed as sequences were generated from RT-PCR products amplified with the degenerate primers. These SARS-specific primers were used to test patient specimens for SARS (see below). Primers used for specific amplification of human metapneumovirus have been described by Falsey et al. (J. Infect. Dis. 87:785 90, 2003).

For RT-PCR products of less than 3 kb, cDNA was synthesized in a 20 .mu.l reaction mixture containing 500 ng of RNA, 200 U of Superscript.TM. II reverse transcriptase (Invitrogen Life Technologies, Carlsbad, Calif.), 40 U of RNasin (Promega Corp., Madison, Wis.), 100 mM each dNTP (Roche Molecular Biochemicals, Indianapolis, Ind.), 4 .mu.l of 5.times. reaction buffer (Invitrogen Life Technologies, Carlsbad, Calif.), and 200 pmol of the reverse primer. The reaction mixture, except for the reverse transcriptase, was heated to 70.degree. C. for 2 minutes, cooled to 4.degree. C. for 5 minutes and then heated to 42.degree. C. in a thermocycler. The mixture was held at 42.degree. C. for 4 minutes, and then the reverse transcriptase was added, and the reactions were incubated at 42.degree. C. for 45 minutes. Two microliters of the cDNA reaction was used in a 50 .mu.l PCR reaction containing 67 mM Tris-HCl (pH 8.8), 1 mM each primer, 17 mM ammonium sulfate, 6 mM EDTA, 2 mM MgCl.sub.2, 200 mM each dNTP, and 2.5 U of Taq DNA polymerase (Roche Molecular Biochemicals, Indianapolis, Ind.). The thermocycler program for the PCR consisted of 40 cycles of denaturation at 95.degree. C. for 30 seconds, annealing at 42.degree. C. for 30 seconds, and extension at 65.degree. C. for 30 seconds. For SARS-CoV-specific primers, the annealing temperature was increased to 55.degree. C.

For amplification of fragments longer than 3 kb, regions of the genome between sections of known sequence were amplified by means of a long RT-PCR protocol and SARS-CoV-specific primers. First-strand cDNA synthesis was performed at 42.degree. C. or 50.degree. C. using Superscript.TM. II RNase H reverse transcriptase (Invitrogen Life Technologies, Carlsbad, Calif.) according to the manufacturer’s instructions with minor modifications. Coronavirus-specific primers (500 ng) and SARS-CoV RNA (350 ng) were combined with the PCR Nucleotide Mix (Roche Molecular Biochemicals, Indianapolis, Ind.), heated for 1 minute at 94.degree. C., and cooled to 4.degree. C. in a thermocycler. The 5.times. first-strand buffer, dithiothreitol (Invitrogen Life Technologies, Carlsbad, Calif.), and Protector RNase Inhibitor (Roche Molecular Biochemicals, Indianapolis, Ind.) were added, and the samples were incubated at 42.degree. C. or 50.degree. C. for 2 minutes. After reverse transcriptase (200 U) was added, the samples were incubated at 42.degree. C. or 50.degree. C. for 1.5 to 2 hours. Samples were inactivated at 70.degree. C. for 15 minutes and subsequently treated with 2 U of RNase H (Roche Molecular Biochemicals, Indianapolis, Ind.) at 37.degree. C. for 30 minutes. Long RT-PCR amplification of 5- to 8-kb fragments was performed using Taq Plus Precision (Stratagene, La Jolla, Calif.) and AmpliWax PCR Gem 100 beads (Applied Biosystems; Foster City, Calif.) for “hot start” PCR with the following thermocycling parameters: denaturation at 94.degree. C. for 1 minute followed by 35 cycles of 94.degree. C. for 30 seconds, 55.degree. C. for 30 seconds, an increase of 0.4 degrees per second up to 72.degree. C., and 72.degree. C. for 7 to 10 minutes, with a final extension at 72.degree. C. for 10 minutes. RT-PCR products were separated by electrophoresis on 0.9% agarose TAE gels and purified by use of a QIAquick Gel Extraction Kit (Qiagen, Inc., Santa Clarita, Calif.).

In all cases, the RT-PCR products were gel-isolated and purified for sequencing by means of a QIAquick Gel Extraction kit (Qiagen, Inc., Santa Clarita, Calif.). Both strands were sequenced by automated methods, using fluorescent dideoxy-chain terminators (Applied Biosystems; Foster City, Calif.).

The sequence of the leader was obtained from the subgenomic mRNA coding for the N gene and from the 5′ terminus of genomic RNA. The 5′ rapid amplification of cDNA ends (RACE) technique (Harcourt et al., Virology 271:334 49, 2000) was used with reverse primers specific for the N gene or for the 5′ untranslated region. RACE products were either sequenced directly or were cloned into a plasmid vector before sequencing. A primer that was specific for the leader of SARS-CoV was used to amplify the region between the 5′-terminus of the genome and known sequences in the rep gene. The 3′-terminus of the genome was amplified for sequencing by use of an oligo-(dT) primer and primers specific for the N gene.

Once the complete SARS-CoV genomic sequence had been determined, it was confirmed by sequencing a series of independently amplified RT-PCR products spanning the entire genome. Positive- and negative-sense sequencing primers, at intervals of approximately 300 nt, were used to generate a confirmatory sequence with an average redundancy of 9.1. The confirmatory sequence was identical to the original sequence. The genomic sequence (SEQ ID NO: 1) was published in the GenBank sequence database (Accession No. AY278741) on Apr. 21, 2003.

Sequence Analysis

Predicted amino acid sequences were compared with those from reference viruses representing each species for which complete genomic sequence information was available: group 1 representatives included human coronavirus 229E (GenBank Accession No. AF304460), porcine epidemic diarrhea virus (GenBank Accession No. AF353511), and transmissible gastroenteritis virus (GenBank Accession No. AF271965); group 2 representatives included bovine coronavirus (GenBank Accession No. AF220295) and mouse hepatitis virus (GenBank Accession No. AF201929); group 3 was represented by infectious bronchitis virus (GenBank Accession No. M95169). Sequences for representative strains of other coronavirus species for which partial sequence information was available were included for some of the structural protein comparisons: group 1 representative strains included canine coronavirus (GenBank Accession No. D13096), feline coronavirus (GenBank Accession No. AY204704), and porcine respiratory coronavirus (GenBank Accession No. Z24675); and group 2 representatives included three strains of human coronavirus OC43 (GenBank Accession Nos. M76373, L14643 and M93390), porcine hemagglutinating encephalomyelitis virus (GenBank Accession No. AY078417), and rat coronavirus (GenBank Accession No. AF207551).

Partial nucleotide sequences of the polymerase gene were aligned with published coronavirus sequences, using CLUSTAL W for Unix (version 1.7; Thompson et al., Nucleic Acids Res. 22:4673 80, 1994). Phylogenetic trees were computed by maximum parsimony, distance, and maximum likelihood-based criteria analysis with PAUP (version 4.0.d10; Swofford ed., Phylogenetic Analysis using Parsimony and other Methods, Sinauer Associates, Sunderland, Mass.). When compared with other human and animal coronaviruses, the nucleotide and deduced amino acid sequence from this region had similarity scores ranging from 0.56 to 0.63 and from 0.57 to 0.74, respectively. The highest sequence similarity was obtained with group II coronaviruses. The maximum-parsimony tree obtained from the nucleotide-sequence alignment is shown in FIG. 3. Bootstrap analyses of the internal nodes at the internal branches of the tree provided strong evidence that the SARS-CoV is genetically distinct from other known coronaviruses.

Microarray analyses (using a long oligonucleotide DNA microarray with array elements derived from highly conserved regions within viral families) of samples from infected and uninfected cell cultures gave a positive signal for a group of eight oligonucleotides derived from two virus families: Coronaviridae and Astroviridae (Wang et al., PNAS 99:15687 92, 2002). All of the astroviruses and two of the coronavirus oligonucleotides share a consensus sequence motif that maps to the extreme 3′-end of astroviruses and two members of the coronavirus family: avian infectious bronchitis and turkey coronavirus (Jonassen et al., J. Gen. Virol. 79:715 8, 1998). Results were consistent with the identity of the isolate as a coronavirus.

Additional sequence alignments and neighbor-joining trees were generated by using ClustalX (Thompson et al., Nucleic Acids Res. 25:4876 82, 1997), version 1.83, with the Gonnet protein comparison matrix. The resulting trees were adjusted for final output by using treetool version 2.0.1. Uncorrected pairwise distances were calculated from the aligned sequences by using the Distances program from the Wisconsin Sequence Analysis Package, version 10.2 (Accelrys, Burlington, Mass.). Distances were converted to percent identity by subtracting from 100. The amino acid sequences for three well-defined enzymatic proteins encoded by the rep gene and the four major structural proteins of SARS-CoV were compared with those from representative viruses for each of the species of coronavirus for which complete genomic sequence information was available (FIG. 4, Table 2). The topologies of the resulting phylograms are remarkably similar (FIG. 4). For each protein analyzed, the species formed monophyletic clusters consistent with the established taxonomic groups. In all cases, SARS-CoV sequences segregated into a fourth, well-resolved branch. These clusters were supported by bootstrap values above 90% (1000 replicates). Consistent with pairwise comparisons between the previously characterized coronavirus species (Table 2), there was greater sequence conservation in the enzymatic proteins (3CL.sup.pro, polymerase (POL), and helicase (HEL)) than among the structural proteins (S, E, M, and N). These results indicate that SARS-CoV is not closely related to any of the previously characterized coronaviruses and forms a distinct group within the genus Coronavirus.

TABLE-US-00004 TABLE 2 Pairwise amino acid identities of coronavirus proteins. Group Virus 3CLPRO POL HEL S E M N Pairwise Amino Acid Identity (Percent) G1 HCoV-229E 40.1 58.8 59.7 23.9 22.7 28.8 23.0 PEDV 44.4 59.5 61.7 21.7 17.6 31.8 22.6 TGEV 44.0 59.4 61.2 20.6 22.4 30.0 25.6 G2 BCoV 48.8 66.3 68.3 27.1 20.0 39.7 31.9 MHV 49.2 66.5 67.3 26.5 21.1 39.0 33.0 G3 IBV 41.3 62.5 58.6 21.8 18.4 27.2 24.0 Predicted Protein Length (aa) SARS-CoV 306 932 601 1255 76 221 422 CoV Range 302 307 923 940 506 600 1173 1452 76 108 225 262 377 454

Example 2

Detection of SARS-CoV in a Subject

This example demonstrates the detection of SARS-CoV in patient specimens using SARS-CoV-specific primers.

The SARS-specific primers Cor-p-F2 (SEQ ID NO: 13), Cor-p-F3 (SEQ ID NO: 14) and Cor-p-R1 (SEQ ID NO: 15) were used to test patient specimens for SARS. One primer for each set was 5′-end-labeled with 6-FAM to facilitate GeneScan analysis. One-step amplification reactions were performed with the Access RT-PCR System (Promega, Madison, Wis.) as described by Falsey et al., J. Infect. Dis. 87:785 90, 2003. Positive and negative RT-PCR controls, containing standardized viral RNA extracts, and nuclease-free water were included in each run. Amplified 6-FAM-labeled products were analyzed by capillary electrophoresis on an ABI 3100 Prism Genetic Analyzer with GeneScan software (version 3.1.2; Applied Biosystems; Foster City, Calif.). Specimens were considered positive for SARS-CoV if the amplification products were within one nucleotide of the expected product size (368 nucleotides for Cor-p-F2 or Cor-p-R1 and 348 nucleotides for Cor-p-F3 or Cor-p-R1) for both specific primer sets, as confirmed by a second PCR reaction from another aliquot of RNA extract in a separate laboratory. Where DNA yield was sufficient, the amplified products were also sequenced. Additionally, as described above, microarray-based detection of SARS-CoV in patient specimens was carried out (Wang et al., PNAS 99:15687 92, 2002 and Bohlander et al., Genomics 13:1322 24, 1992).

Example 3

Immunohistochemical and Histopathological Analysis, and Electron-Microscopical Analysis of Bronchoalveolar Lavage Fluid

This example illustrates immunohistochemical, histopathological and electron-microscopical analysis of Vero E6 cells infected with the SARS-CoV and tissue samples from SARS patients.

Formalin-fixed, paraffin-embedded Vero E6 cells infected with the SARS-CoV and tissues obtained from patients with SARS were stained with hematoxylin and eosin and various immunohistochemical stains. Immunohistochemical assays were based on a method described previously for hantavirus (Zaki et al., Amer. J. Pathol. 146:552 79, 1995). Briefly, 4-.mu.m sections were deparaffinized, rehydrated, and digested in Proteinase K for 15 minutes. Slides were then incubated for 60 minutes at room temperature with monoclonal antibodies, polyclonal antiserum or ascitic fluids derived from animal species with reactivities to various known coronaviruses, and with a convalescent-phase serum specimen from a patient with SARS.

Optimal dilutions of the primary antibodies were determined by titration experiments with coronavirus-infected cells from patients with SARS and with noninfected cells or, when available, with concentrations recommended by the manufacturers. After sequential application of the appropriate biotinylated link antibody, avidin-alkaline phosphatase complex, and naphthol-fast red substrate, sections were counterstained in Mayer’s hematoxylin and mounted with aqueous mounting medium. The following antibody and tissue controls were used: serum specimens from noninfected animals, various coronavirus-infected cell cultures and animal tissues, noninfected cell cultures, and normal human and animal tissues. Tissues from patients were also tested by immunohistochemical assays for various other viral and bacterial pulmonary pathogens. In addition, a BAL specimen was available from one patient for thin-section electron-microscopical evaluation.

Lung tissues were obtained from the autopsy of three patients and by open lung biopsy of one patient, 14 19 days following onset of SARS symptoms. Confirmatory laboratory evidence of infection with coronavirus was available for two patients (patients 6 and 17) and included PCR amplification of coronavirus nucleic acids from tissues, viral isolation from BAL fluid or detection of serum antibodies reactive with coronavirus (Table 1). For two patients, no samples were available for molecular, cell culture, or serological analysis; however, both patients met the CDC definition for probable SARS cases and had strong epidemiologic links with laboratory-confirmed SARS cases. Histopathologic evaluation of lung tissues of the four patients showed diffuse alveolar damage at various levels of progression and severity. Changes included hyaline membrane formation, interstitial mononuclear inflammatory infiltrates, and desquamation of pneumocytes in alveolar spaces (FIG. 5A). Other findings identified in some patients included focal intraalveolar hemorrhage, necrotic inflammatory debris in small airways, and organizing pneumonia. Multinucleated syncytial cells were identified in the intraalveolar spaces of two patients who died 14 and 17 days, respectively, after onset of illness. These cells contained abundant vacuolated cytoplasm with cleaved and convoluted nuclei. No obvious intranuclear or intracytoplasmic viral inclusions were identified (FIG. 5B), and electron-microscopical examination of a limited number of these syncytial cells revealed no coronavirus particles. No definitive immunostaining was identified in tissues from SARS patients with the use of a battery of immunohistochemical stains reactive with coronaviruses from antigenic groups I, II, and III. In addition, no staining of patient tissues was identified with the use of immunohistochemical stains for influenzaviruses A and B, adenoviruses, Hendra and Nipah viruses, human metapneumovirus, respiratory syncytial virus, measles virus, Mycoplasma pneumoniae, and Chlamydia pneumoniae.

Evaluation of Vero E6 cells infected with coronavirus isolated from a patient with SARS revealed viral CPE that included occasional multinucleated syncytial cells but no obvious viral inclusions (FIG. 5C). Immunohistochemical assays with various antibodies reactive with coronaviruses from antigenic group I, including HCoV-229E, FIPV and TGEV, and with an immune serum specimen from a patient with SARS, demonstrated strong cytoplasmic and membranous staining of infected cells (FIG. 5C and Table 3); however, cross-reactivity with the same immune human serum sample and FIPV antigen was not observed. No staining was identified with any of several monoclonal or polyclonal antibodies reactive with coronaviruses in antigenic group II (HCoV-OC43, BCoV and MHV) or group III (TCoV and IBV-Avian). Electron microscopical examination of a BAL fluid from one patient revealed many coronavirus-infected cells (FIGS. 6A B).

TABLE-US-00005 TABLE 3 Immunohistochemical reactivities of various polyclonal group I anti- coronavirus reference antiserum samples with a coronavirus isolated from a patient with SARS and with selected antigenic group I coronaviruses. Immunohistochemical reactivity of antiserum with coronavirus-infected culture cells HCoV-229E SARS-CoV (mouse 3T3- FIPV-1 Antiserum (Vero E6) hAPN) (BHK-fAPN) Convalescent- + + – phase SARS (patient 3) Guinea pig anti- + + – HCoV-229E Rabbit anti- + + + HCoV-229E Feline anti- + + + FIPV-1 Porcine anti- + – + TGEV

Example 4

SARS-CoV Serologic Analysis

This example illustrates representative methods of performing serological analysis of SARS-CoV.

Spot slides were prepared by applying 15 .mu.l of the suspension of gamma-irradiated mixed infected and noninfected cells onto 12-well Teflon-coated slides. Slides were allowed to air dry before being fixed in acetone. Slides were then stored at -70.degree. C. until used for IFA tests (Wulff and Lange, Bull. WHO 52:429 36, 1975). An ELISA antigen was prepared by detergent extraction and subsequent gamma irradiation of infected Vero E6 cells (Ksiazek et al., J. Infect. Dis. 179 (suppl. 1):S191 8, 1999). The optimal dilution (1:1000) for the use of this antigen was determined by checkerboard titration against SARS patient serum from the convalescent phase; a control antigen, similarly prepared from uninfected Vero E6 cells, was used to control for specific reactivity of tested sera. The conjugates used were goat antihuman IgG, IgA, and IgM conjugated to fluorescein isothiocyanate and horseradish peroxidase (Kirkegaard and Perry, Gaithersburg, Md.), for the IFA test and ELISA, respectively. Specificity and cross-reactivity of a variety of serum samples to the newly identified virus were evaluated by using the tests described herein. For this evaluation, serum from SARS patients in Singapore, Bangkok and Hong Kong was used, along with serum from healthy blood donors from the CDC serum bank and from persons infected with known human coronavirus (human coronaviruses OC43 and 229E) (samples provided by E. Walsh and A. Falsey, University of Rochester School of Medicine and Dentistry, Rochester, N.Y.).

Spot slides with infected cells reacted with serum from patients with probable SARS in the convalescent phase (FIG. 1B). Screening of a panel of serum from patients with suspected SARS from Hong Kong, Bangkok, Singapore as well as the United States showed a high level of specific reaction with infected cells, and conversion from negative to positive reactivity or diagnostic rises in the IFA test by a factor of four. Similarly, tests of these same serum samples with the ELISA antigen showed high specific signal in the convalescent-phase samples and conversion from negative to positive antibody reactivity or diagnostic increases in titer (Table 4).

TABLE-US-00006 TABLE 4 Results of serological testing with both IFA assay and ELISA in SARS patients tested against the newly isolated human coronavirus. Source Serum No. Days After Onset ELISA Titer* IFA Titer* Hong Kong 1.1 4 <100 <25 Hong Kong 1.2 13 .gtoreq.6400 1600 Hong Kong 2.1 11 400 100 Hong Kong 2.2 16 1600 200 Hong Kong 3.1 7 <100 <25 Hong Kong 3.2 17 .gtoreq.6400 800 Hong Kong 4.1 8 <100 <25 Hong Kong 4.2 13 1600 50 Hong Kong 5.1 10 100 <25 Hong Kong 5.2 17 .gtoreq.6400 1600 Hong Kong 6.1 12 1600 200 Hong Kong 6.2 20 .gtoreq.6400 6400 Hong Kong 7.1 17 400 50 Hong Kong 7.2 24 .gtoreq.6400 3200 Hong Kong 8.1 3 <100 <25 Hong Kong 8.2 15 .gtoreq.6400 200 Hong Kong 9.1 5 <100 <25 (Hanoi) Hong Kong 9.2 11 .gtoreq.6400 1600 Bangkok 1.1 2 <100 <25 Bangkok 1.2 4 <100 <25 Bangkok 1.3 7 <100 <25 Bangkok 1.4 15 1600 200 United States 1.1 2 <100 <25 United States 1.2 6 400 50 United States 1.3 13 .gtoreq.6400 800 Singapore 1.1 2 100 <25 Singapore 1.2 11 .gtoreq.6400 800 Singapore 2.1 6 100 <25 Singapore 2.2 25 .gtoreq.6400 400 Singapore 3.1 6 100 <25 Singapore 3.2 14 .gtoreq.6400 400 Singapore 4.1 5 100 <25 Singapore 4.2 16 1600 400 *Reciprocal of dilution

Information from the limited numbers of samples tested suggests that antibody is first detectable by IFA assay and ELISA between one and two weeks after the onset of symptoms in the patient. IFA testing and ELISA of a panel of 384 randomly selected serum samples (from U.S. blood donors) were negative for antibodies to the new coronavirus, with the exception of one specimen that had minimal reactivity on ELISA. A panel of paired human serum samples with diagnostic increases (by a factor of four or more) in antibody (with very high titers to the homologous viral antigen in the convalescent-phase serum) to the two known human coronaviruses, OC43 (13 pairs) and 229E (14 pairs), showed no reactivity in either acute- or convalescent-phase serum with the newly isolated coronavirus by either the IFA test or the ELISA.

Example 5

Poly(A).sup.+ RNA Isolation and Northern Hybridization

This example illustrates a representative method of Northern hybrididization to detect SARS-CoV messages in Vero E6 cells.

Total RNA from infected or uninfected Vero E6 cells was isolated with Trizol reagent (Invitrogen Life Technologies, Carlsbad, Calif.) used according to the manufacturer’s recommendations. Poly(A).sup.+ RNA was isolated from total RNA by use of the Oligotex Direct mRNA Kit (Qiagen, Inc., Santa Clarita, Calif.), following the instructions for the batch protocol, followed by ethanol precipitation. RNA isolated from 1 cm.sup.2 of cells was separated by electrophoresis on a 0.9% agarose gel containing 3.7% formaldehyde, followed by partial alkaline hydrolysis (Ausubel et al. eds. Current Protocols in Molecular Biology, vol. 1, John Wiley & Sons, Inc., NY, N.Y., Ch. 4.9, 1996). RNA was transferred to a nylon membrane (Roche Molecular Biochemicals, Indianapolis, Ind.) by vacuum blotting (Bio-Rad, Hercules, Calif.) and fixed by UV cross-linking. The DNA template for probe synthesis was generated by RT-PCR amplification of SARS-CoV nt 29,083 to 29,608 (SEQ ID NO: 1), by using a reverse primer containing a T7 RNA polymerase promoter to facilitate generation of a negative-sense riboprobe. In vitro transcription of the digoxigenin-labeled riboprobe, hybridization, and detection of the bands were carried out with the digoxigenin system by using manufacturer’s recommended procedures (Roche Molecular Biochemicals, Indianapolis, Ind.). Signals were visualized by chemiluminescence and detected with x-ray film.

Example 6

SARS-CoV Genome Organization

This example illustrates the genomic organization of the SARS-CoV genome, including the location of SARS-CoV ORFs.

The genome of SARS-CoV is a 29,727-nucleotide, polyadenylated RNA, and 41% of the residues are G or C (range for published coronavirus complete genome sequences, 37% to 42%). The genomic organization is typical of coronaviruses, having the characteristic gene order [5′-replicase (rep), spike (S), envelope (E), membrane (M), nucleocapsid (N)-3′] and short untranslated regions at both termini (FIG. 7A, Table 5). The SARS-CoV rep gene, which comprises approximately two-thirds of the genome, encodes two polyproteins (encoded by ORF1a and ORF1b) that undergo co-translational proteolytic processing. There are four ORFs downstream of rep that encode the structural proteins, S, E, M, and N, which are common to all known coronaviruses. The hemagglutinin-esterase gene, which is present between ORF1b and S in group 2 and some group 3 coronaviruses (Lai and Holmes, in Fields Virology, eds. Knipe and Howley, Lippincott Williams and Wilkins, New York, 4.sup.th edition, 2001, Ch. 35), was not found in SARS-CoV.

Coronaviruses also encode a number of non-structural proteins that are located between S and E, between M and N, or downstream of N. These non-structural proteins, which vary widely among the different coronavirus species, are of unknown function and are dispensable for virus replication (Lai and Holmes, in Fields Virology, eds. Knipe and Howley, Lippincott Williams and Wilkins, New York, 4.sup.th edition, 2001, Ch. 35). The genome of SARS-CoV contains ORFs for five non-structural proteins of greater than 50 amino acids (FIG. 7B, Table 5). Two overlapping ORFs encoding proteins of 274 and 154 amino acids (termed X1 (SEQ ID NO: 5) and X2 (SEQ ID NO: 6), respectively) are located between S (SEQ ID NO: 4) and E (SEQ ID NO: 7). Three additional non-structural genes, X3 (SEQ ID NO: 9), X4 (SEQ ID NO: 10), and X5 (SEQ ID NO: 11) (encoding proteins of 63, 122, and 84 amino acids, respectively), are located between M (SEQ ID NO: 8) and N (SEQ ID NO: 12). In addition to the five ORFs encoding the non-structural proteins described above, there are also two smaller ORFs between M and N, encoding proteins of less than 50 amino acids. Searches of the GenBank database (BLAST and FastA) indicated that there is no significant sequence similarity between these non-structural proteins of SARS-CoV and any other proteins.

The coronavirus rep gene products are translated from genomic RNA, but the remaining viral proteins are translated from subgenomic mRNAs that form a 3′-coterminal nested set, each with a 5′-end derived from the genomic 5′-leader sequence. The coronavirus subgenomic mRNAs are synthesized through a discontinuous transcription process, the mechanism of which has not been unequivocally established (Lai and Holmes, in Fields Virology, eds. Knipe and Howley, Lippincott Williams and Wilkins, New York, 4.sup.th edition, 2001, Ch. 35; Sawicki and Sawicki, Adv. Exp. Med. Biol. 440:215 19, 1998). The SARS-CoV leader sequence was mapped by comparing the sequence of 5′-RACE products synthesized from the N gene mRNA with those synthesized from genomic RNA. A sequence, AAACGAAC (nucleotides 65 72 of SEQ ID NO: 1), was identified immediately upstream of the site where the N gene mRNA and genomic sequences diverged. This sequence was also present upstream of ORF1a and immediately upstream of five other ORFs (Table 5), suggesting that it functions as the conserved core of the transcriptional regulatory sequence (TRS).

In addition to the site at the 5′-terminus of the genome, the TRS conserved core sequence appears six times in the remainder of the genome. The positions of the TRS in the genome of SARS-CoV predict that subgenomic mRNAs of 8.3, 4.5, 3.4, 2.5, 2.0, and 1.7 kb, not including the poly(A) tail, should be produced (FIGS. 7A B, Table 5). At least five subgenomic mRNAs were detected by Northern hybridization of RNA from SARS-CoV-infected cells, using a probe derived from the 3′-untranslated region (FIG. 7C). The calculated sizes of the five predominant bands correspond to the sizes of five of the predicted subgenomic mRNAs of SARS-CoV; the possibility that other, low-abundance mRNAs are present cannot be excluded. By analogy with other coronaviruses (Lai and Holmes, in Fields Virology, eds. Knipe and Howley, Lippincott Williams and Wilkins, New York, 4.sup.th edition, 2001, Ch. 35), the 8.3-kb and 1.7-kb subgenomic mRNAs are monocistronic, directing translation of S and N, respectively, whereas multiple proteins are translated from the 4.5-kb (X1, X2, and E), 3.4-kb (M and X3), and 2.5-kb (X4 and X5) mRNAs. A consensus TRS is not found directly upstream of the ORF encoding the predicted E protein, and a monocistronic mRNA that would be predicted to code for E could not be clearly identified by Northern blot analysis. It is possible that the 3.6-kb band contains more than one mRNA species or that the monocistronic mRNA for E is a low-abundance message.

TABLE-US-00007 TABLE 5 Locations of SARS-CoV ORFs and sizes of proteins and mRNAs Genome Location Predicted Size ORF TRS.sup.a ORF Start ORF End Protein (aa) mRNA (nt).sup.b 1a 72 265 13,398 4,378 29,727 1b 13,398 21,482 2,695 S 21,491 21,492 25,256 1,255 8,308.sup.c X1 25,265 25,268 26,089 274 4,534.sup.c X2 25,689 26,150 154 E 26,117 26,344 76 M 26,353 26,398 27,060 221 3,446.sup.c X3 27,074 27,262 63 X4 27,272 27,273 27,638 122 2,527.sup.c X5 27,778 27,864 28,115 84 2,021.sup.d N 28,111 28,120 29,385 422 1,688.sup.c .sup.aThe location is the 3′-most nucleotide in the consensus TRS, AAACGAAC. .sup.bNot including poly(A). Predicted size is based on the position of the conserved TRS. .sup.cCorresponding mRNA detected by Northern blot analysis (FIG. 7C) .sup.dNo mRNA corresponding to utilization of this consensus TRS was detected by Northern blot analysis (FIG. 7C)

Example 7

Real-Time RT-PCR Assay for SARS-CoV Detection

This example demonstrates the use of SARS-CoV-specific primers and probes in a real-time RT-PCR assay to detect SARS-CoV in patient specimens.

A variant of the real-time format, based on TaqMan probe hydrolysis technology (Applied Biosystems, Foster City, Calif.), was used to analyze a total of 340 clinical specimens collected from 246 persons with confirmed or suspected SARS-CoV infection. Specimens included oro- and nasopharyngeal swabs (dry and in viral transport media), sputa, nasal aspirates and washes, BAL, and lung tissue specimens collected at autopsy.

Nucleic Acid Extraction

SARS-CoV nucleic acids were recovered from clinical specimens using the automated NucliSens extraction system (bioMerieux, Durham, N.C.). Following manufacturer’s instructions, specimens received in NucliSens lysis buffer were incubated at 37.degree. C. for 30 min with intermittent mixing, and 50 .mu.L of silica suspension, provided in the extraction kit, was added and mixed. The contents of the tube were then transferred to a nucleic acid extraction cartridge and processed on an extractor workstation. Approximately 40 50 .mu.L of total nucleic acid eluate was recovered into nuclease-free vials and either tested immediately or stored at -70.degree. C.

Primers and Probes

Multiple primer and probe sets were designed from the SARS-CoV polymerase 1b (nucleic acid 13,398 to 21,482 of SEQ ID NO: 1) and nucleocapsid gene (nucleic acid 28,120 to 29,385 of SEQ ID NO: 1) sequences by using Primer Express software version 1.5 or 2.0.0 (Applied Biosystems, Foster City, Calif.) with the following default settings: primer melting temperature (T.sub.M) set at 60.degree. C.; probe T.sub.M set at 10.degree. C. greater than the primers at approximately 70.degree. C.; and no guanidine residues permitted at the 5′ probe termini. All primers and probes were synthesized by standard phosphoramidite chemistry techniques. TaqMan probes were labeled at the 5′-end with the reporter 6-FAM and at the 3′-end with the quencher Blackhole Quencher 1 (Biosearch Technologies, Inc., Novato, Calif.). Optimal primer and probe concentrations were determined by cross-titration of serial twofold dilutions of each primer against a constant amount of purified SARS-CoV RNA. Primer and probe concentrations that gave the highest amplification efficiencies were selected for further study (Table 6).

TABLE-US-00008 TABLE 6 Primers and probes used for real-time RT-PCR assays.sup.a Genomic Assay ID Primer/probe Sequence Region Primary diagnostic assay SARS1 F CATGTGTGGCGGCTCACTATAT (SEQ ID NO: 16) RNA Pol R GACACTATTAGCATAAGCAGTTGTAGCA (SEQ ID NO: 17) P TTAAACCAGGTGGAACATCATCCGGTG (SEQ ID NO: 18) SARS2 F GGAGCCTTGAATACACCCAAAG (SEQ ID NO: 19) Nucleocapsid R GCACGGTGGCAGCATTG (SEQ ID NO: 20) P CCACATTGGCACCCGCAATCC (SEQ ID NO: 21) SARS3 F CAAACATTGGCCGCAAATT (SEQ ID NO: 22) Nucleocapsid R CAATGCGTGACATTCCAAAGA (SEQ ID NO: 23) P CACAATTTGCTCCAAGTGCCTCTGCA (SEQ ID NO: 24) To confirm positive results N3 F GAAGTACCATCTGGGGCTGAG (SEQ ID NO: 25) Nucleocapsid R CCGAAGAGCTACCCGACG (SEQ ID NO: 26) P CTCTTTCATTTTGCCGTCACCACCAC (SEQ ID NO: 27) 3′-NTR F AGCTCTCCCTAGCATTATTCACTG (SEQ ID NO: 28) 3′-NTR R CACCACATTTTCATCGAGGC (SEQ ID NO: 29) P TACCCTCGATCGTACTCCGCGT (SEQ ID NO: 30) M F TGTAGGCACTGATTCAGGTTTTG (SEQ ID NO: 31) M protein R CGGCGTGGTCTGTATTTAATTTA (SEQ ID NO: 32) P CTGCATACAACCGCTACCGTATTGGAA (SEQ ID NO: 33) .sup.aRT-PCR, reverse transcription-polymerase chain reaction; F, forward primer; R, reverse primer; P, probe; NTR, nontranslated region.

Real-Time RT-PCR Assay

The real-time RT-PCR assay was performed by using the Real-Time One-Step RT-PCR Master Mix (Applied Biosystems, Foster City, Calif.). Each 25-.mu.L reaction mixture contained 12.5 .mu.L of 2.times. Master Mix, 0.625 .mu.L of the 40.times. MultiScribe and RNase Inhibitor mix, 0.25 .mu.L of 10 .mu.M probe, 0.25 .mu.L each of 50 .mu.M forward and reverse primers, 6.125 .mu.L of nuclease-free water, and 5 .mu.L of nucleic acid extract. Amplification was carried out in 96-well plates on an iCycler iQ Real-Time Detection System (Bio-Rad, Hercules, Calif.). Thermocycling conditions consisted of 30 minutes at 48.degree. C. for reverse transcription, 10 minutes at 95.degree. C. for activation of the AmpliTaq Gold DNA polymerase, and 45 cycles of 15 seconds at 95.degree. C. and 1 minute at 60.degree. C. Each run included one SARS-CoV genomic template control and at least two no-template controls for the extraction (to check for contamination during sample processing) and one no-template control for the PCR-amplification step. As a control for PCR inhibitors, and to monitor nucleic acid extraction efficiency, each sample was tested by real-time RT-PCR for the presence of the human ribonuclease (RNase) P gene (GenBank Accession No. NM.sub.–006413) by using the following primers and probe: forward primer 5′-AGATTTGGACCTGCGAGCG-3′ (SEQ ID NO: 36); reverse primer 5′-GAGCGGCTGTCTCCACAAGT-3′ (SEQ ID NO: 37); probe 5′-TTCTGACC TGAAGGCTCTGCGCG-3′ (SEQ ID NO: 38). The assay reaction was performed identically to that described above except that primer concentrations used were 30 .mu.M each. Fluorescence measurements were taken and the threshold cycle (C.sub.T) value for each sample was calculated by determining the point at which fluorescence exceeded a threshold limit set at the mean plus 10 standard deviations above the baseline. A test result was considered positive if two or more of the SARS genomic targets showed positive results (C.sub.T.ltoreq.45 cycles) and all positive and negative control reactions gave expected values.

While this disclosure has been described with an emphasis upon preferred embodiments, it will be obvious to those of ordinary skill in the art that variations and equivalents of the preferred embodiments may be used and it is intended that the disclosure may be practiced otherwise than as specifically described herein. Accordingly, this disclosure includes all modifications encompassed within the spirit and scope of the disclosure as defined by the claims below.

SEQUENCE LISTINGS

1

38129727DNACoronavirusmisc_feature(265)..(13398)ORF 1a 1ttattaggtt tttacctacc caggaaaagc caaccaacct cgatctcttg tagatctgtt 60ctctaaacga actttaaaat ctgtgtagct gtcgctcggc tgcatgccta gtgcacctac 120gcagtataaa caataataaa ttttactgtc gttgacaaga aacgagtaac tcgtccctct 180tctgcagact gcttacggtt tcgtccgtgt tgcagtcgat catcagcata cctaggtttc 240gtccgggtgt gaccgaaagg taagatggag agccttgttc ttggtgtcaa cgagaaaaca 300cacgtccaac tcagtttgcc tgtccttcag gttagagacg tgctagtgcg tggcttcggg 360gactctgtgg aagaggccct atcggaggca cgtgaacacc tcaaaaatgg cacttgtggt 420ctagtagagc tggaaaaagg cgtactgccc cagcttgaac agccctatgt gttcattaaa 480cgttctgatg ccttaagcac caatcacggc cacaaggtcg ttgagctggt tgcagaaatg 540gacggcattc agtacggtcg tagcggtata acactgggag tactcgtgcc acatgtgggc 600gaaaccccaa ttgcataccg caatgttctt cttcgtaaga acggtaataa gggagccggt 660ggtcatagct atggcatcga tctaaagtct tatgacttag gtgacgagct tggcactgat 720cccattgaag attatgaaca aaactggaac actaagcatg gcagtggtgc actccgtgaa 780ctcactcgtg agctcaatgg aggtgcagtc actcgctatg tcgacaacaa tttctgtggc 840ccagatgggt accctcttga ttgcatcaaa gattttctcg cacgcgcggg caagtcaatg 900tgcactcttt ccgaacaact tgattacatc gagtcgaaga gaggtgtcta ctgctgccgt 960gaccatgagc atgaaattgc ctggttcact gagcgctctg ataagagcta cgagcaccag 1020acacccttcg aaattaagag tgccaagaaa tttgacactt tcaaagggga atgcccaaag 1080tttgtgtttc ctcttaactc aaaagtcaaa gtcattcaac cacgtgttga aaagaaaaag 1140actgagggtt tcatggggcg tatacgctct gtgtaccctg ttgcatctcc acaggagtgt 1200aacaatatgc acttgtctac cttgatgaaa tgtaatcatt gcgatgaagt ttcatggcag 1260acgtgcgact ttctgaaagc cacttgtgaa cattgtggca ctgaaaattt agttattgaa 1320ggacctacta catgtgggta cctacctact aatgctgtag tgaaaatgcc atgtcctgcc 1380tgtcaagacc cagagattgg acctgagcat agtgttgcag attatcacaa ccactcaaac 1440attgaaactc gactccgcaa gggaggtagg actagatgtt ttggaggctg tgtgtttgcc 1500tatgttggct gctataataa gcgtgcctac tgggttcctc gtgctagtgc tgatattggc 1560tcaggccata ctggcattac tggtgacaat gtggagacct tgaatgagga tctccttgag 1620atactgagtc gtgaacgtgt taacattaac attgttggcg attttcattt gaatgaagag 1680gttgccatca ttttggcatc tttctctgct tctacaagtg cctttattga cactataaag 1740agtcttgatt acaagtcttt caaaaccatt gttgagtcct gcggtaacta taaagttacc 1800aagggaaagc ccgtaaaagg tgcttggaac attggacaac agagatcagt tttaacacca 1860ctgtgtggtt ttccctcaca ggctgctggt gttatcagat caatttttgc gcgcacactt 1920gatgcagcaa accactcaat tcctgatttg caaagagcag ctgtcaccat acttgatggt 1980atttctgaac agtcattacg tcttgtcgac gccatggttt atacttcaga cctgctcacc 2040aacagtgtca ttattatggc atatgtaact ggtggtcttg tacaacagac ttctcagtgg 2100ttgtctaatc ttttgggcac tactgttgaa aaactcaggc ctatctttga atggattgag 2160gcgaaactta gtgcaggagt tgaatttctc aaggatgctt gggagattct caaatttctc 2220attacaggtg tttttgacat cgtcaagggt caaatacagg ttgcttcaga taacatcaag 2280gattgtgtaa aatgcttcat tgatgttgtt aacaaggcac tcgaaatgtg cattgatcaa 2340gtcactatcg ctggcgcaaa gttgcgatca ctcaacttag gtgaagtctt catcgctcaa 2400agcaagggac tttaccgtca gtgtatacgt ggcaaggagc agctgcaact actcatgcct 2460cttaaggcac caaaagaagt aacctttctt gaaggtgatt cacatgacac agtacttacc 2520tctgaggagg ttgttctcaa gaacggtgaa ctcgaagcac tcgagacgcc cgttgatagc 2580ttcacaaatg gagctatcgt tggcacacca gtctgtgtaa atggcctcat gctcttagag 2640attaaggaca aagaacaata ctgcgcattg tctcctggtt tactggctac aaacaatgtc 2700tttcgcttaa aagggggtgc accaattaaa ggtgtaacct ttggagaaga tactgtttgg 2760gaagttcaag gttacaagaa tgtgagaatc acatttgagc ttgatgaacg tgttgacaaa 2820gtgcttaatg aaaagtgctc tgtctacact gttgaatccg gtaccgaagt tactgagttt 2880gcatgtgttg tagcagaggc tgttgtgaag actttacaac cagtttctga tctccttacc 2940aacatgggta ttgatcttga tgagtggagt gtagctacat tctacttatt tgatgatgct 3000ggtgaagaaa acttttcatc acgtatgtat tgttcctttt accctccaga tgaggaagaa 3060gaggacgatg cagagtgtga ggaagaagaa attgatgaaa cctgtgaaca tgagtacggt 3120acagaggatg attatcaagg tctccctctg gaatttggtg cctcagctga aacagttcga 3180gttgaggaag aagaagagga agactggctg gatgatacta ctgagcaatc agagattgag 3240ccagaaccag aacctacacc tgaagaacca gttaatcagt ttactggtta tttaaaactt 3300actgacaatg ttgccattaa atgtgttgac atcgttaagg aggcacaaag tgctaatcct 3360atggtgattg taaatgctgc taacatacac ctgaaacatg gtggtggtgt agcaggtgca 3420ctcaacaagg caaccaatgg tgccatgcaa aaggagagtg atgattacat taagctaaat 3480ggccctctta cagtaggagg gtcttgtttg ctttctggac ataatcttgc taagaagtgt 3540ctgcatgttg ttggacctaa cctaaatgca ggtgaggaca tccagcttct taaggcagca 3600tatgaaaatt tcaattcaca ggacatctta cttgcaccat tgttgtcagc aggcatattt 3660ggtgctaaac cacttcagtc tttacaagtg tgcgtgcaga cggttcgtac acaggtttat 3720attgcagtca atgacaaagc tctttatgag caggttgtca tggattatct tgataacctg 3780aagcctagag tggaagcacc taaacaagag gagccaccaa acacagaaga ttccaaaact 3840gaggagaaat ctgtcgtaca gaagcctgtc gatgtgaagc caaaaattaa ggcctgcatt 3900gatgaggtta ccacaacact ggaagaaact aagtttctta ccaataagtt actcttgttt 3960gctgatatca atggtaagct ttaccatgat tctcagaaca tgcttagagg tgaagatatg 4020tctttccttg agaaggatgc accttacatg gtaggtgatg ttatcactag tggtgatatc 4080acttgtgttg taataccctc caaaaaggct ggtggcacta ctgagatgct ctcaagagct 4140ttgaagaaag tgccagttga tgagtatata accacgtacc ctggacaagg atgtgctggt 4200tatacacttg aggaagctaa gactgctctt aagaaatgca aatctgcatt ttatgtacta 4260ccttcagaag cacctaatgc taaggaagag attctaggaa ctgtatcctg gaatttgaga 4320gaaatgcttg ctcatgctga agagacaaga aaattaatgc ctatatgcat ggatgttaga 4380gccataatgg caaccatcca acgtaagtat aaaggaatta aaattcaaga gggcatcgtt 4440gactatggtg tccgattctt cttttatact agtaaagagc ctgtagcttc tattattacg 4500aagctgaact ctctaaatga gccgcttgtc acaatgccaa ttggttatgt gacacatggt 4560tttaatcttg aagaggctgc gcgctgtatg cgttctctta aagctcctgc cgtagtgtca 4620gtatcatcac cagatgctgt tactacatat aatggatacc tcacttcgtc atcaaagaca 4680tctgaggagc actttgtaga aacagtttct ttggctggct cttacagaga ttggtcctat 4740tcaggacagc gtacagagtt aggtgttgaa tttcttaagc gtggtgacaa aattgtgtac 4800cacactctgg agagccccgt cgagtttcat cttgacggtg aggttctttc acttgacaaa 4860ctaaagagtc tcttatccct gcgggaggtt aagactataa aagtgttcac aactgtggac 4920aacactaatc tccacacaca gcttgtggat atgtctatga catatggaca gcagtttggt 4980ccaacatact tggatggtgc tgatgttaca aaaattaaac ctcatgtaaa tcatgagggt 5040aagactttct ttgtactacc tagtgatgac acactacgta gtgaagcttt cgagtactac 5100catactcttg atgagagttt tcttggtagg tacatgtctg ctttaaacca cacaaagaaa 5160tggaaatttc ctcaagttgg tggtttaact tcaattaaat gggctgataa caattgttat 5220ttgtctagtg ttttattagc acttcaacag cttgaagtca aattcaatgc accagcactt 5280caagaggctt attatagagc ccgtgctggt gatgctgcta acttttgtgc actcatactc 5340gcttacagta ataaaactgt tggcgagctt ggtgatgtca gagaaactat gacccatctt 5400ctacagcatg ctaatttgga atctgcaaag cgagttctta atgtggtgtg taaacattgt 5460ggtcagaaaa ctactacctt aacgggtgta gaagctgtga tgtatatggg tactctatct 5520tatgataatc ttaagacagg tgtttccatt ccatgtgtgt gtggtcgtga tgctacacaa 5580tatctagtac aacaagagtc ttcttttgtt atgatgtctg caccacctgc tgagtataaa 5640ttacagcaag gtacattctt atgtgcgaat gagtacactg gtaactatca gtgtggtcat 5700tacactcata taactgctaa ggagaccctc tatcgtattg acggagctca ccttacaaag 5760atgtcagagt acaaaggacc agtgactgat gttttctaca aggaaacatc ttacactaca 5820accatcaagc ctgtgtcgta taaactcgat ggagttactt acacagagat tgaaccaaaa 5880ttggatgggt attataaaaa ggataatgct tactatacag agcagcctat agaccttgta 5940ccaactcaac cattaccaaa tgcgagtttt gataatttca aactcacatg ttctaacaca 6000aaatttgctg atgatttaaa tcaaatgaca ggcttcacaa agccagcttc acgagagcta 6060tctgtcacat tcttcccaga cttgaatggc gatgtagtgg ctattgacta tagacactat 6120tcagcgagtt tcaagaaagg tgctaaatta ctgcataagc caattgtttg gcacattaac 6180caggctacaa ccaagacaac gttcaaacca aacacttggt gtttacgttg tctttggagt 6240acaaagccag tagatacttc aaattcattt gaagttctgg cagtagaaga cacacaagga 6300atggacaatc ttgcttgtga aagtcaacaa cccacctctg aagaagtagt ggaaaatcct 6360accatacaga aggaagtcat agagtgtgac gtgaaaacta ccgaagttgt aggcaatgtc 6420atacttaaac catcagatga aggtgttaaa gtaacacaag agttaggtca tgaggatctt 6480atggctgctt atgtggaaaa cacaagcatt accattaaga aacctaatga gctttcacta 6540gccttaggtt taaaaacaat tgccactcat ggtattgctg caattaatag tgttccttgg 6600agtaaaattt tggcttatgt caaaccattc ttaggacaag cagcaattac aacatcaaat 6660tgcgctaaga gattagcaca acgtgtgttt aacaattata tgccttatgt gtttacatta 6720ttgttccaat tgtgtacttt tactaaaagt accaattcta gaattagagc ttcactacct 6780acaactattg ctaaaaatag tgttaagagt gttgctaaat tatgtttgga tgccggcatt 6840aattatgtga agtcacccaa attttctaaa ttgttcacaa tcgctatgtg gctattgttg 6900ttaagtattt gcttaggttc tctaatctgt gtaactgctg cttttggtgt actcttatct 6960aattttggtg ctccttctta ttgtaatggc gttagagaat tgtatcttaa ttcgtctaac 7020gttactacta tggatttctg tgaaggttct tttccttgca gcatttgttt aagtggatta 7080gactcccttg attcttatcc agctcttgaa accattcagg tgacgatttc atcgtacaag 7140ctagacttga caattttagg tctggccgct gagtgggttt tggcatatat gttgttcaca 7200aaattctttt atttattagg tctttcagct ataatgcagg tgttctttgg ctattttgct 7260agtcatttca tcagcaattc ttggctcatg tggtttatca ttagtattgt acaaatggca 7320cccgtttctg caatggttag gatgtacatc ttctttgctt ctttctacta catatggaag 7380agctatgttc atatcatgga tggttgcacc tcttcgactt gcatgatgtg ctataagcgc 7440aatcgtgcca cacgcgttga gtgtacaact attgttaatg gcatgaagag atctttctat 7500gtctatgcaa atggaggccg tggcttctgc aagactcaca attggaattg tctcaattgt 7560gacacatttt gcactggtag tacattcatt agtgatgaag ttgctcgtga tttgtcactc 7620cagtttaaaa gaccaatcaa ccctactgac cagtcatcgt atattgttga tagtgttgct 7680gtgaaaaatg gcgcgcttca cctctacttt gacaaggctg gtcaaaagac ctatgagaga 7740catccgctct cccattttgt caatttagac aatttgagag ctaacaacac taaaggttca 7800ctgcctatta atgtcatagt ttttgatggc aagtccaaat gcgacgagtc tgcttctaag 7860tctgcttctg tgtactacag tcagctgatg tgccaaccta ttctgttgct tgaccaagtt 7920cttgtatcag acgttggaga tagtactgaa gtttccgtta agatgtttga tgcttatgtc 7980gacacctttt cagcaacttt tagtgttcct atggaaaaac ttaaggcact tgttgctaca 8040gctcacagcg agttagcaaa gggtgtagct ttagatggtg tcctttctac attcgtgtca 8100gctgcccgac aaggtgttgt tgataccgat gttgacacaa aggatgttat tgaatgtctc 8160aaactttcac atcactctga cttagaagtg acaggtgaca gttgtaacaa tttcatgctc 8220acctataata aggttgaaaa catgacgccc agagatcttg gcgcatgtat tgactgtaat 8280gcaaggcata tcaatgccca agtagcaaaa agtcacaatg tttcactcat ctggaatgta 8340aaagactaca tgtctttatc tgaacagctg cgtaaacaaa ttcgtagtgc tgccaagaag 8400aacaacatac cttttagact aacttgtgct acaactagac aggttgtcaa tgtcataact 8460actaaaatct cactcaaggg tggtaagatt gttagtactt gttttaaact tatgcttaag 8520gccacattat tgtgcgttct tgctgcattg gtttgttata tcgttatgcc agtacataca 8580ttgtcaatcc atgatggtta cacaaatgaa atcattggtt acaaagccat tcaggatggt 8640gtcactcgtg acatcatttc tactgatgat tgttttgcaa ataaacatgc tggttttgac 8700gcatggttta gccagcgtgg tggttcatac aaaaatgaca aaagctgccc tgtagtagct 8760gctatcatta caagagagat tggtttcata gtgcctggct taccgggtac tgtgctgaga 8820gcaatcaatg gtgacttctt gcattttcta cctcgtgttt ttagtgctgt tggcaacatt 8880tgctacacac cttccaaact cattgagtat agtgattttg ctacctctgc ttgcgttctt 8940gctgctgagt gtacaatttt taaggatgct atgggcaaac ctgtgccata ttgttatgac 9000actaatttgc tagagggttc tatttcttat agtgagcttc gtccagacac tcgttatgtg 9060cttatggatg gttccatcat acagtttcct aacacttacc tggagggttc tgttagagta 9120gtaacaactt ttgatgctga gtactgtaga catggtacat gcgaaaggtc agaagtaggt 9180atttgcctat ctaccagtgg tagatgggtt cttaataatg agcattacag agctctatca 9240ggagttttct gtggtgttga tgcgatgaat ctcatagcta acatctttac tcctcttgtg 9300caacctgtgg gtgctttaga tgtgtctgct tcagtagtgg ctggtggtat tattgccata 9360ttggtgactt gtgctgccta ctactttatg aaattcagac gtgtttttgg tgagtacaac 9420catgttgttg ctgctaatgc acttttgttt ttgatgtctt tcactatact ctgtctggta 9480ccagcttaca gctttctgcc gggagtctac tcagtctttt acttgtactt gacattctat 9540ttcaccaatg atgtttcatt cttggctcac cttcaatggt ttgccatgtt ttctcctatt 9600gtgccttttt ggataacagc aatctatgta ttctgtattt ctctgaagca ctgccattgg 9660ttctttaaca actatcttag gaaaagagtc atgtttaatg gagttacatt tagtaccttc 9720gaggaggctg ctttgtgtac ctttttgctc aacaaggaaa tgtacctaaa attgcgtagc 9780gagacactgt tgccacttac acagtataac aggtatcttg ctctatataa caagtacaag 9840tatttcagtg gagccttaga tactaccagc tatcgtgaag cagcttgctg ccacttagca 9900aaggctctaa atgactttag caactcaggt gctgatgttc tctaccaacc accacagaca 9960tcaatcactt ctgctgttct gcagagtggt tttaggaaaa tggcattccc gtcaggcaaa 10020gttgaagggt gcatggtaca agtaacctgt ggaactacaa ctcttaatgg attgtggttg 10080gatgacacag tatactgtcc aagacatgtc atttgcacag cagaagacat gcttaatcct 10140aactatgaag atctgctcat tcgcaaatcc aaccatagct ttcttgttca ggctggcaat 10200gttcaacttc gtgttattgg ccattctatg caaaattgtc tgcttaggct taaagttgat 10260acttctaacc ctaagacacc caagtataaa tttgtccgta tccaacctgg tcaaacattt 10320tcagttctag catgctacaa tggttcacca tctggtgttt atcagtgtgc catgagacct 10380aatcatacca ttaaaggttc tttccttaat ggatcatgtg gtagtgttgg ttttaacatt 10440gattatgatt gcgtgtcttt ctgctatatg catcatatgg agcttccaac aggagtacac 10500gctggtactg acttagaagg taaattctat ggtccatttg ttgacagaca aactgcacag 10560gctgcaggta cagacacaac cataacatta aatgttttgg catggctgta tgctgctgtt 10620atcaatggtg ataggtggtt tcttaataga ttcaccacta ctttgaatga ctttaacctt 10680gtggcaatga agtacaacta tgaacctttg acacaagatc atgttgacat attgggacct 10740ctttctgctc aaacaggaat tgccgtctta gatatgtgtg ctgctttgaa agagctgctg 10800cagaatggta tgaatggtcg tactatcctt ggtagcacta ttttagaaga tgagtttaca 10860ccatttgatg ttgttagaca atgctctggt gttaccttcc aaggtaagtt caagaaaatt 10920gttaagggca ctcatcattg gatgctttta actttcttga catcactatt gattcttgtt 10980caaagtacac agtggtcact gtttttcttt gtttacgaga atgctttctt gccatttact 11040cttggtatta tggcaattgc tgcatgtgct atgctgcttg ttaagcataa gcacgcattc 11100ttgtgcttgt ttctgttacc ttctcttgca acagttgctt actttaatat ggtctacatg 11160cctgctagct gggtgatgcg tatcatgaca tggcttgaat tggctgacac tagcttgtct 11220ggttataggc ttaaggattg tgttatgtat gcttcagctt tagttttgct tattctcatg 11280acagctcgca ctgtttatga tgatgctgct agacgtgttt ggacactgat gaatgtcatt 11340acacttgttt acaaagtcta ctatggtaat gctttagatc aagctatttc catgtgggcc 11400ttagttattt ctgtaacctc taactattct ggtgtcgtta cgactatcat gtttttagct 11460agagctatag tgtttgtgtg tgttgagtat tacccattgt tatttattac tggcaacacc 11520ttacagtgta tcatgcttgt ttattgtttc ttaggctatt gttgctgctg ctactttggc 11580cttttctgtt tactcaaccg ttacttcagg cttactcttg gtgtttatga ctacttggtc 11640tctacacaag aatttaggta tatgaactcc caggggcttt tgcctcctaa gagtagtatt 11700gatgctttca agcttaacat taagttgttg ggtattggag gtaaaccatg tatcaaggtt 11760gctactgtac agtctaaaat gtctgacgta aagtgcacat ctgtggtact gctctcggtt 11820cttcaacaac ttagagtaga gtcatcttct aaattgtggg cacaatgtgt acaactccac 11880aatgatattc ttcttgcaaa agacacaact gaagctttcg agaagatggt ttctcttttg 11940tctgttttgc tatccatgca gggtgctgta gacattaata ggttgtgcga ggaaatgctc 12000gataaccgtg ctactcttca ggctattgct tcagaattta gttctttacc atcatatgcc 12060gcttatgcca ctgcccagga ggcctatgag caggctgtag ctaatggtga ttctgaagtc 12120gttctcaaaa agttaaagaa atctttgaat gtggctaaat ctgagtttga ccgtgatgct 12180gccatgcaac gcaagttgga aaagatggca gatcaggcta tgacccaaat gtacaaacag 12240gcaagatctg aggacaagag ggcaaaagta actagtgcta tgcaaacaat gctcttcact 12300atgcttagga agcttgataa tgatgcactt aacaacatta tcaacaatgc gcgtgatggt 12360tgtgttccac tcaacatcat accattgact acagcagcca aactcatggt tgttgtccct 12420gattatggta cctacaagaa cacttgtgat ggtaacacct ttacatatgc atctgcactc 12480tgggaaatcc agcaagttgt tgatgcggat agcaagattg ttcaacttag tgaaattaac 12540atggacaatt caccaaattt ggcttggcct cttattgtta cagctctaag agccaactca 12600gctgttaaac tacagaataa tgaactgagt ccagtagcac tacgacagat gtcctgtgcg 12660gctggtacca cacaaacagc ttgtactgat gacaatgcac ttgcctacta taacaattcg 12720aagggaggta ggtttgtgct ggcattacta tcagaccacc aagatctcaa atgggctaga 12780ttccctaaga gtgatggtac aggtacaatt tacacagaac tggaaccacc ttgtaggttt 12840gttacagaca caccaaaagg gcctaaagtg aaatacttgt acttcatcaa aggcttaaac 12900aacctaaata gaggtatggt gctgggcagt ttagctgcta cagtacgtct tcaggctgga 12960aatgctacag aagtacctgc caattcaact gtgctttcct tctgtgcttt tgcagtagac 13020cctgctaaag catataagga ttacctagca agtggaggac aaccaatcac caactgtgtg 13080aagatgttgt gtacacacac tggtacagga caggcaatta ctgtaacacc agaagctaac 13140atggaccaag agtcctttgg tggtgcttca tgttgtctgt attgtagatg ccacattgac 13200catccaaatc ctaaaggatt ctgtgacttg aaaggtaagt acgtccaaat acctaccact 13260tgtgctaatg acccagtggg ttttacactt agaaacacag tctgtaccgt ctgcggaatg 13320tggaaaggtt atggctgtag ttgtgaccaa ctccgcgaac ccttgatgca gtctgcggat 13380gcatcaacgt ttttaaacgg gtttgcggtg taagtgcagc ccgtcttaca ccgtgcggca 13440caggcactag tactgatgtc gtctacaggg cttttgatat ttacaacgaa aaagttgctg 13500gttttgcaaa gttcctaaaa actaattgct gtcgcttcca ggagaaggat gaggaaggca 13560atttattaga ctcttacttt gtagttaaga ggcatactat gtctaactac caacatgaag 13620agactattta taacttggtt aaagattgtc cagcggttgc tgtccatgac tttttcaagt 13680ttagagtaga tggtgacatg gtaccacata tatcacgtca gcgtctaact aaatacacaa 13740tggctgattt agtctatgct ctacgtcatt ttgatgaggg taattgtgat acattaaaag 13800aaatactcgt cacatacaat tgctgtgatg atgattattt caataagaag gattggtatg 13860acttcgtaga gaatcctgac atcttacgcg tatatgctaa cttaggtgag cgtgtacgcc 13920aatcattatt aaagactgta caattctgcg atgctatgcg tgatgcaggc attgtaggcg 13980tactgacatt agataatcag gatcttaatg ggaactggta cgatttcggt gatttcgtac 14040aagtagcacc aggctgcgga gttcctattg tggattcata ttactcattg ctgatgccca 14100tcctcacttt gactagggca ttggctgctg agtcccatat ggatgctgat ctcgcaaaac 14160cacttattaa gtgggatttg ctgaaatatg attttacgga agagagactt tgtctcttcg 14220accgttattt taaatattgg gaccagacat accatcccaa ttgtattaac tgtttggatg 14280ataggtgtat ccttcattgt gcaaacttta atgtgttatt ttctactgtg tttccaccta 14340caagttttgg accactagta agaaaaatat ttgtagatgg tgttcctttt gttgtttcaa 14400ctggatacca ttttcgtgag ttaggagtcg tacataatca ggatgtaaac ttacatagct 14460cgcgtctcag tttcaaggaa cttttagtgt atgctgctga tccagctatg catgcagctt 14520ctggcaattt attgctagat aaacgcacta catgcttttc agtagctgca ctaacaaaca 14580atgttgcttt tcaaactgtc aaacccggta attttaataa agacttttat gactttgctg 14640tgtctaaagg tttctttaag gaaggaagtt ctgttgaact aaaacacttc ttctttgctc 14700aggatggcaa cgctgctatc agtgattatg actattatcg ttataatctg ccaacaatgt 14760gtgatatcag acaactccta ttcgtagttg aagttgttga taaatacttt gattgttacg 14820atggtggctg tattaatgcc aaccaagtaa tcgttaacaa tctggataaa tcagctggtt 14880tcccatttaa taaatggggt aaggctagac tttattatga ctcaatgagt tatgaggatc 14940aagatgcact tttcgcgtat actaagcgta atgtcatccc tactataact caaatgaatc 15000ttaagtatgc

cattagtgca aagaatagag ctcgcaccgt agctggtgtc tctatctgta 15060gtactatgac aaatagacag tttcatcaga aattattgaa gtcaatagcc gccactagag 15120gagctactgt ggtaattgga acaagcaagt tttacggtgg ctggcataat atgttaaaaa 15180ctgtttacag tgatgtagaa actccacacc ttatgggttg ggattatcca aaatgtgaca 15240gagccatgcc taacatgctt aggataatgg cctctcttgt tcttgctcgc aaacataaca 15300cttgctgtaa cttatcacac cgtttctaca ggttagctaa cgagtgtgcg caagtattaa 15360gtgagatggt catgtgtggc ggctcactat atgttaaacc aggtggaaca tcatccggtg 15420atgctacaac tgcttatgct aatagtgtct ttaacatttg tcaagctgtt acagccaatg 15480taaatgcact tctttcaact gatggtaata agatagctga caagtatgtc cgcaatctac 15540aacacaggct ctatgagtgt ctctatagaa atagggatgt tgatcatgaa ttcgtggatg 15600agttttacgc ttacctgcgt aaacatttct ccatgatgat tctttctgat gatgccgttg 15660tgtgctataa cagtaactat gcggctcaag gtttagtagc tagcattaag aactttaagg 15720cagttcttta ttatcaaaat aatgtgttca tgtctgaggc aaaatgttgg actgagactg 15780accttactaa aggacctcac gaattttgct cacagcatac aatgctagtt aaacaaggag 15840atgattacgt gtacctgcct tacccagatc catcaagaat attaggcgca ggctgttttg 15900tcgatgatat tgtcaaaaca gatggtacac ttatgattga aaggttcgtg tcactggcta 15960ttgatgctta cccacttaca aaacatccta atcaggagta tgctgatgtc tttcacttgt 16020atttacaata cattagaaag ttacatgatg agcttactgg ccacatgttg gacatgtatt 16080ccgtaatgct aactaatgat aacacctcac ggtactggga acctgagttt tatgaggcta 16140tgtacacacc acatacagtc ttgcaggctg taggtgcttg tgtattgtgc aattcacaga 16200cttcacttcg ttgcggtgcc tgtattagga gaccattcct atgttgcaag tgctgctatg 16260accatgtcat ttcaacatca cacaaattag tgttgtctgt taatccctat gtttgcaatg 16320ccccaggttg tgatgtcact gatgtgacac aactgtatct aggaggtatg agctattatt 16380gcaagtcaca taagcctccc attagttttc cattatgtgc taatggtcag gtttttggtt 16440tatacaaaaa cacatgtgta ggcagtgaca atgtcactga cttcaatgcg atagcaacat 16500gtgattggac taatgctggc gattacatac ttgccaacac ttgtactgag agactcaagc 16560ttttcgcagc agaaacgctc aaagccactg aggaaacatt taagctgtca tatggtattg 16620ctactgtacg cgaagtactc tctgacagag aattgcatct ttcatgggag gttggaaaac 16680ctagaccacc attgaacaga aactatgtct ttactggtta ccgtgtaact aaaaatagta 16740aagtacagat tggagagtac acctttgaaa aaggtgacta tggtgatgct gttgtgtaca 16800gaggtactac gacatacaag ttgaatgttg gtgattactt tgtgttgaca tctcacactg 16860taatgccact tagtgcacct actctagtgc cacaagagca ctatgtgaga attactggct 16920tgtacccaac actcaacatc tcagatgagt tttctagcaa tgttgcaaat tatcaaaagg 16980tcggcatgca aaagtactct acactccaag gaccacctgg tactggtaag agtcattttg 17040ccatcggact tgctctctat tacccatctg ctcgcatagt gtatacggca tgctctcatg 17100cagctgttga tgccctatgt gaaaaggcat taaaatattt gcccatagat aaatgtagta 17160gaatcatacc tgcgcgtgcg cgcgtagagt gttttgataa attcaaagtg aattcaacac 17220tagaacagta tgttttctgc actgtaaatg cattgccaga aacaactgct gacattgtag 17280tctttgatga aatctctatg gctactaatt atgacttgag tgttgtcaat gctagacttc 17340gtgcaaaaca ctacgtctat attggcgatc ctgctcaatt accagccccc cgcacattgc 17400tgactaaagg cacactagaa ccagaatatt ttaattcagt gtgcagactt atgaaaacaa 17460taggtccaga catgttcctt ggaacttgtc gccgttgtcc tgctgaaatt gttgacactg 17520tgagtgcttt agtttatgac aataagctaa aagcacacaa ggataagtca gctcaatgct 17580tcaaaatgtt ctacaaaggt gttattacac atgatgtttc atctgcaatc aacagacctc 17640aaataggcgt tgtaagagaa tttcttacac gcaatcctgc ttggagaaaa gctgttttta 17700tctcacctta taattcacag aacgctgtag cttcaaaaat cttaggattg cctacgcaga 17760ctgttgattc atcacagggt tctgaatatg actatgtcat attcacacaa actactgaaa 17820cagcacactc ttgtaatgtc aaccgcttca atgtggctat cacaagggca aaaattggca 17880ttttgtgcat aatgtctgat agagatcttt atgacaaact gcaatttaca agtctagaaa 17940taccacgtcg caatgtggct acattacaag cagaaaatgt aactggactt tttaaggact 18000gtagtaagat cattactggt cttcatccta cacaggcacc tacacacctc agcgttgata 18060taaagttcaa gactgaagga ttatgtgttg acataccagg cataccaaag gacatgacct 18120accgtagact catctctatg atgggtttca aaatgaatta ccaagtcaat ggttacccta 18180atatgtttat cacccgcgaa gaagctattc gtcacgttcg tgcgtggatt ggctttgatg 18240tagagggctg tcatgcaact agagatgctg tgggtactaa cctacctctc cagctaggat 18300tttctacagg tgttaactta gtagctgtac cgactggtta tgttgacact gaaaataaca 18360cagaattcac cagagttaat gcaaaacctc caccaggtga ccagtttaaa catcttatac 18420cactcatgta taaaggcttg ccctggaatg tagtgcgtat taagatagta caaatgctca 18480gtgatacact gaaaggattg tcagacagag tcgtgttcgt cctttgggcg catggctttg 18540agcttacatc aatgaagtac tttgtcaaga ttggacctga aagaacgtgt tgtctgtgtg 18600acaaacgtgc aacttgcttt tctacttcat cagatactta tgcctgctgg aatcattctg 18660tgggttttga ctatgtctat aacccattta tgattgatgt tcagcagtgg ggctttacgg 18720gtaaccttca gagtaaccat gaccaacatt gccaggtaca tggaaatgca catgtggcta 18780gttgtgatgc tatcatgact agatgtttag cagtccatga gtgctttgtt aagcgcgttg 18840attggtctgt tgaataccct attataggag atgaactgag ggttaattct gcttgcagaa 18900aagtacaaca catggttgtg aagtctgcat tgcttgctga taagtttcca gttcttcatg 18960acattggaaa tccaaaggct atcaagtgtg tgcctcaggc tgaagtagaa tggaagttct 19020acgatgctca gccatgtagt gacaaagctt acaaaataga ggagctcttc tattcttatg 19080ctacacatca cgataaattc actgatggtg tttgtttgtt ttggaattgt aacgttgatc 19140gttacccagc caatgcaatt gtgtgtaggt ttgacacaag agccttgtca aacttgaact 19200taccaggctg tgatggtggt agtttgtatg tgaataagca tgcattccac actccagctt 19260tcgataaaag tgcatttact aatttaaagc aattgccttt cttttactat tctgatagtc 19320cttgtgagtc tcatggcaaa caagtagtgt cggatattga ttatgttcca ctcaaatctg 19380ctacgtgtat tacacgatgc aatttaggtg gtgctgtttg cagacaccat gcaaatgagt 19440accgacagta cttggatgca tataatatga tgatttctgc tggatttagc ctatggattt 19500acaaacaatt tgatacttat aacctgtgga atacatttac caggttacag agtttagaaa 19560atgtggctta taatgttgtt aataaaggac actttgatgg acacgccggc gaagcacctg 19620tttccatcat taataatgct gtttacacaa aggtagatgg tattgatgtg gagatctttg 19680aaaataagac aacacttcct gttaatgttg catttgagct ttgggctaag cgtaacatta 19740aaccagtgcc agagattaag atactcaata atttgggtgt tgatatcgct gctaatactg 19800taatctggga ctacaaaaga gaagccccag cacatgtatc tacaataggt gtctgcacaa 19860tgactgacat tgccaagaaa cctactgaga gtgcttgttc ttcacttact gtcttgtttg 19920atggtagagt ggaaggacag gtagaccttt ttagaaacgc ccgtaatggt gttttaataa 19980cagaaggttc agtcaaaggt ctaacacctt caaagggacc agcacaagct agcgtcaatg 20040gagtcacatt aattggagaa tcagtaaaaa cacagtttaa ctactttaag aaagtagacg 20100gcattattca acagttgcct gaaacctact ttactcagag cagagactta gaggatttta 20160agcccagatc acaaatggaa actgactttc tcgagctcgc tatggatgaa ttcatacagc 20220gatataagct cgagggctat gccttcgaac acatcgttta tggagatttc agtcatggac 20280aacttggcgg tcttcattta atgataggct tagccaagcg ctcacaagat tcaccactta 20340aattagagga ttttatccct atggacagca cagtgaaaaa ttacttcata acagatgcgc 20400aaacaggttc atcaaaatgt gtgtgttctg tgattgatct tttacttgat gactttgtcg 20460agataataaa gtcacaagat ttgtcagtga tttcaaaagt ggtcaaggtt acaattgact 20520atgctgaaat ttcattcatg ctttggtgta aggatggaca tgttgaaacc ttctacccaa 20580aactacaagc aagtcaagcg tggcaaccag gtgttgcgat gcctaacttg tacaagatgc 20640aaagaatgct tcttgaaaag tgtgaccttc agaattatgg tgaaaatgct gttataccaa 20700aaggaataat gatgaatgtc gcaaagtata ctcaactgtg tcaatactta aatacactta 20760ctttagctgt accctacaac atgagagtta ttcactttgg tgctggctct gataaaggag 20820ttgcaccagg tacagctgtg ctcagacaat ggttgccaac tggcacacta cttgtcgatt 20880cagatcttaa tgacttcgtc tccgacgcag attctacttt aattggagac tgtgcaacag 20940tacatacggc taataaatgg gaccttatta ttagcgatat gtatgaccct aggaccaaac 21000atgtgacaaa agagaatgac tctaaagaag ggtttttcac ttatctgtgt ggatttataa 21060agcaaaaact agccctgggt ggttctatag ctgtaaagat aacagagcat tcttggaatg 21120ctgaccttta caagcttatg ggccatttct catggtggac agcttttgtt acaaatgtaa 21180atgcatcatc atcggaagca tttttaattg gggctaacta tcttggcaag ccgaaggaac 21240aaattgatgg ctataccatg catgctaact acattttctg gaggaacaca aatcctatcc 21300agttgtcttc ctattcactc tttgacatga gcaaatttcc tcttaaatta agaggaactg 21360ctgtaatgtc tcttaaggag aatcaaatca atgatatgat ttattctctt ctggaaaaag 21420gtaggcttat cattagagaa aacaacagag ttgtggtttc aagtgatatt cttgttaaca 21480actaaacgaa catgtttatt ttcttattat ttcttactct cactagtggt agtgaccttg 21540accggtgcac cacttttgat gatgttcaag ctcctaatta cactcaacat acttcatcta 21600tgaggggggt ttactatcct gatgaaattt ttagatcaga cactctttat ttaactcagg 21660atttatttct tccattttat tctaatgtta cagggtttca tactattaat catacgtttg 21720gcaaccctgt catacctttt aaggatggta tttattttgc tgccacagag aaatcaaatg 21780ttgtccgtgg ttgggttttt ggttctacca tgaacaacaa gtcacagtcg gtgattatta 21840ttaacaattc tactaatgtt gttatacgag catgtaactt tgaattgtgt gacaaccctt 21900tctttgctgt ttctaaaccc atgggtacac agacacatac tatgatattc gataatgcat 21960ttaattgcac tttcgagtac atatctgatg ccttttcgct tgatgtttca gaaaagtcag 22020gtaattttaa acacttacga gagtttgtgt ttaaaaataa agatgggttt ctctatgttt 22080ataagggcta tcaacctata gatgtagttc gtgatctacc ttctggtttt aacactttga 22140aacctatttt taagttgcct cttggtatta acattacaaa ttttagagcc attcttacag 22200ccttttcacc tgctcaagac atttggggca cgtcagctgc agcctatttt gttggctatt 22260taaagccaac tacatttatg ctcaagtatg atgaaaatgg tacaatcaca gatgctgttg 22320attgttctca aaatccactt gctgaactca aatgctctgt taagagcttt gagattgaca 22380aaggaattta ccagacctct aatttcaggg ttgttccctc aggagatgtt gtgagattcc 22440ctaatattac aaacttgtgt ccttttggag aggtttttaa tgctactaaa ttcccttctg 22500tctatgcatg ggagagaaaa aaaatttcta attgtgttgc tgattactct gtgctctaca 22560actcaacatt tttttcaacc tttaagtgct atggcgtttc tgccactaag ttgaatgatc 22620tttgcttctc caatgtctat gcagattctt ttgtagtcaa gggagatgat gtaagacaaa 22680tagcgccagg acaaactggt gttattgctg attataatta taaattgcca gatgatttca 22740tgggttgtgt ccttgcttgg aatactagga acattgatgc tacttcaact ggtaattata 22800attataaata taggtatctt agacatggca agcttaggcc ctttgagaga gacatatcta 22860atgtgccttt ctcccctgat ggcaaacctt gcaccccacc tgctcttaat tgttattggc 22920cattaaatga ttatggtttt tacaccacta ctggcattgg ctaccaacct tacagagttg 22980tagtactttc ttttgaactt ttaaatgcac cggccacggt ttgtggacca aaattatcca 23040ctgaccttat taagaaccag tgtgtcaatt ttaattttaa tggactcact ggtactggtg 23100tgttaactcc ttcttcaaag agatttcaac catttcaaca atttggccgt gatgtttctg 23160atttcactga ttccgttcga gatcctaaaa catctgaaat attagacatt tcaccttgct 23220cttttggggg tgtaagtgta attacacctg gaacaaatgc ttcatctgaa gttgctgttc 23280tatatcaaga tgttaactgc actgatgttt ctacagcaat tcatgcagat caactcacac 23340cagcttggcg catatattct actggaaaca atgtattcca gactcaagca ggctgtctta 23400taggagctga gcatgtcgac acttcttatg agtgcgacat tcctattgga gctggcattt 23460gtgctagtta ccatacagtt tctttattac gtagtactag ccaaaaatct attgtggctt 23520atactatgtc tttaggtgct gatagttcaa ttgcttactc taataacacc attgctatac 23580ctactaactt ttcaattagc attactacag aagtaatgcc tgtttctatg gctaaaacct 23640ccgtagattg taatatgtac atctgcggag attctactga atgtgctaat ttgcttctcc 23700aatatggtag cttttgcaca caactaaatc gtgcactctc aggtattgct gctgaacagg 23760atcgcaacac acgtgaagtg ttcgctcaag tcaaacaaat gtacaaaacc ccaactttga 23820aatattttgg tggttttaat ttttcacaaa tattacctga ccctctaaag ccaactaaga 23880ggtcttttat tgaggacttg ctctttaata aggtgacact cgctgatgct ggcttcatga 23940agcaatatgg cgaatgccta ggtgatatta atgctagaga tctcatttgt gcgcagaagt 24000tcaatggact tacagtgttg ccacctctgc tcactgatga tatgattgct gcctacactg 24060ctgctctagt tagtggtact gccactgctg gatggacatt tggtgctggc gctgctcttc 24120aaataccttt tgctatgcaa atggcatata ggttcaatgg cattggagtt acccaaaatg 24180ttctctatga gaaccaaaaa caaatcgcca accaatttaa caaggcgatt agtcaaattc 24240aagaatcact tacaacaaca tcaactgcat tgggcaagct gcaagacgtt gttaaccaga 24300atgctcaagc attaaacaca cttgttaaac aacttagctc taattttggt gcaatttcaa 24360gtgtgctaaa tgatatcctt tcgcgacttg ataaagtcga ggcggaggta caaattgaca 24420ggttaattac aggcagactt caaagccttc aaacctatgt aacacaacaa ctaatcaggg 24480ctgctgaaat cagggcttct gctaatcttg ctgctactaa aatgtctgag tgtgttcttg 24540gacaatcaaa aagagttgac ttttgtggaa agggctacca ccttatgtcc ttcccacaag 24600cagccccgca tggtgttgtc ttcctacatg tcacgtatgt gccatcccag gagaggaact 24660tcaccacagc gccagcaatt tgtcatgaag gcaaagcata cttccctcgt gaaggtgttt 24720ttgtgtttaa tggcacttct tggtttatta cacagaggaa cttcttttct ccacaaataa 24780ttactacaga caatacattt gtctcaggaa attgtgatgt cgttattggc atcattaaca 24840acacagttta tgatcctctg caacctgagc tcgactcatt caaagaagag ctggacaagt 24900acttcaaaaa tcatacatca ccagatgttg atcttggcga catttcaggc attaacgctt 24960ctgtcgtcaa cattcaaaaa gaaattgacc gcctcaatga ggtcgctaaa aatttaaatg 25020aatcactcat tgaccttcaa gaattgggaa aatatgagca atatattaaa tggccttggt 25080atgtttggct cggcttcatt gctggactaa ttgccatcgt catggttaca atcttgcttt 25140gttgcatgac tagttgttgc agttgcctca agggtgcatg ctcttgtggt tcttgctgca 25200agtttgatga ggatgactct gagccagttc tcaagggtgt caaattacat tacacataaa 25260cgaacttatg gatttgttta tgagattttt tactcttgga tcaattactg cacagccagt 25320aaaaattgac aatgcttctc ctgcaagtac tgttcatgct acagcaacga taccgctaca 25380agcctcactc cctttcggat ggcttgttat tggcgttgca tttcttgctg tttttcagag 25440cgctaccaaa ataattgcgc tcaataaaag atggcagcta gccctttata agggcttcca 25500gttcatttgc aatttactgc tgctatttgt taccatctat tcacatcttt tgcttgtcgc 25560tgcaggtatg gaggcgcaat ttttgtacct ctatgccttg atatattttc tacaatgcat 25620caacgcatgt agaattatta tgagatgttg gctttgttgg aagtgcaaat ccaagaaccc 25680attactttat gatgccaact actttgtttg ctggcacaca cataactatg actactgtat 25740accatataac agtgtcacag atacaattgt cgttactgaa ggtgacggca tttcaacacc 25800aaaactcaaa gaagactacc aaattggtgg ttattctgag gataggcact caggtgttaa 25860agactatgtc gttgtacatg gctatttcac cgaagtttac taccagcttg agtctacaca 25920aattactaca gacactggta ttgaaaatgc tacattcttc atctttaaca agcttgttaa 25980agacccaccg aatgtgcaaa tacacacaat cgacggctct tcaggagttg ctaatccagc 26040aatggatcca atttatgatg agccgacgac gactactagc gtgcctttgt aagcacaaga 26100aagtgagtac gaacttatgt actcattcgt ttcggaagaa acaggtacgt taatagttaa 26160tagcgtactt ctttttcttg ctttcgtggt attcttgcta gtcacactag ccatccttac 26220tgcgcttcga ttgtgtgcgt actgctgcaa tattgttaac gtgagtttag taaaaccaac 26280ggtttacgtc tactcgcgtg ttaaaaatct gaactcttct gaaggagttc ctgatcttct 26340ggtctaaacg aactaactat tattattatt ctgtttggaa ctttaacatt gcttatcatg 26400gcagacaacg gtactattac cgttgaggag cttaaacaac tcctggaaca atggaaccta 26460gtaataggtt tcctattcct agcctggatt atgttactac aatttgccta ttctaatcgg 26520aacaggtttt tgtacataat aaagcttgtt ttcctctggc tcttgtggcc agtaacactt 26580gcttgttttg tgcttgctgc tgtctacaga attaattggg tgactggcgg gattgcgatt 26640gcaatggctt gtattgtagg cttgatgtgg cttagctact tcgttgcttc cttcaggctg 26700tttgctcgta cccgctcaat gtggtcattc aacccagaaa caaacattct tctcaatgtg 26760cctctccggg ggacaattgt gaccagaccg ctcatggaaa gtgaacttgt cattggtgct 26820gtgatcattc gtggtcactt gcgaatggcc ggacaccccc tagggcgctg tgacattaag 26880gacctgccaa aagagatcac tgtggctaca tcacgaacgc tttcttatta caaattagga 26940gcgtcgcagc gtgtaggcac tgattcaggt tttgctgcat acaaccgcta ccgtattgga 27000aactataaat taaatacaga ccacgccggt agcaacgaca atattgcttt gctagtacag 27060taagtgacaa cagatgtttc atcttgttga cttccaggtt acaatagcag agatattgat 27120tatcattatg aggactttca ggattgctat ttggaatctt gacgttataa taagttcaat 27180agtgagacaa ttatttaagc ctctaactaa gaagaattat tcggagttag atgatgaaga 27240acctatggag ttagattatc cataaaacga acatgaaaat tattctcttc ctgacattga 27300ttgtatttac atcttgcgag ctatatcact atcaggagtg tgttagaggt acgactgtac 27360tactaaaaga accttgccca tcaggaacat acgagggcaa ttcaccattt caccctcttg 27420ctgacaataa atttgcacta acttgcacta gcacacactt tgcttttgct tgtgctgacg 27480gtactcgaca tacctatcag ctgcgtgcaa gatcagtttc accaaaactt ttcatcagac 27540aagaggaggt tcaacaagag ctctactcgc cactttttct cattgttgct gctctagtat 27600ttttaatact ttgcttcacc attaagagaa agacagaatg aatgagctca ctttaattga 27660cttctatttg tgctttttag cctttctgct attccttgtt ttaataatgc ttattatatt 27720ttggttttca ctcgaaatcc aggatctaga agaaccttgt accaaagtct aaacgaacat 27780gaaacttctc attgttttga cttgtatttc tctatgcagt tgcatatgca ctgtagtaca 27840gcgctgtgca tctaataaac ctcatgtgct tgaagatcct tgtaaggtac aacactaggg 27900gtaatactta tagcactgct tggctttgtg ctctaggaaa ggttttacct tttcatagat 27960ggcacactat ggttcaaaca tgcacaccta atgttactat caactgtcaa gatccagctg 28020gtggtgcgct tatagctagg tgttggtacc ttcatgaagg tcaccaaact gctgcattta 28080gagacgtact tgttgtttta aataaacgaa caaattaaaa tgtctgataa tggaccccaa 28140tcaaaccaac gtagtgcccc ccgcattaca tttggtggac ccacagattc aactgacaat 28200aaccagaatg gaggacgcaa tggggcaagg ccaaaacagc gccgacccca aggtttaccc 28260aataatactg cgtcttggtt cacagctctc actcagcatg gcaaggagga acttagattc 28320cctcgaggcc agggcgttcc aatcaacacc aatagtggtc cagatgacca aattggctac 28380taccgaagag ctacccgacg agttcgtggt ggtgacggca aaatgaaaga gctcagcccc 28440agatggtact tctattacct aggaactggc ccagaagctt cacttcccta cggcgctaac 28500aaagaaggca tcgtatgggt tgcaactgag ggagccttga atacacccaa agaccacatt 28560ggcacccgca atcctaataa caatgctgcc accgtgctac aacttcctca aggaacaaca 28620ttgccaaaag gcttctacgc agagggaagc agaggcggca gtcaagcctc ttctcgctcc 28680tcatcacgta gtcgcggtaa ttcaagaaat tcaactcctg gcagcagtag gggaaattct 28740cctgctcgaa tggctagcgg aggtggtgaa actgccctcg cgctattgct gctagacaga 28800ttgaaccagc ttgagagcaa agtttctggt aaaggccaac aacaacaagg ccaaactgtc 28860actaagaaat ctgctgctga ggcatctaaa aagcctcgcc aaaaacgtac tgccacaaaa 28920cagtacaacg tcactcaagc atttgggaga cgtggtccag aacaaaccca aggaaatttc 28980ggggaccaag acctaatcag acaaggaact gattacaaac attggccgca aattgcacaa 29040tttgctccaa gtgcctctgc attctttgga atgtcacgca ttggcatgga agtcacacct 29100tcgggaacat ggctgactta tcatggagcc attaaattgg atgacaaaga tccacaattc 29160aaagacaacg tcatactgct gaacaagcac attgacgcat acaaaacatt cccaccaaca 29220gagcctaaaa aggacaaaaa gaaaaagact gatgaagctc agcctttgcc gcagagacaa 29280aagaagcagc ccactgtgac tcttcttcct gcggctgaca tggatgattt ctccagacaa 29340cttcaaaatt ccatgagtgg agcttctgct gattcaactc aggcataaac actcatgatg 29400accacacaag gcagatgggc tatgtaaacg ttttcgcaat tccgtttacg atacatagtc 29460tactcttgtg cagaatgaat tctcgtaact aaacagcaca agtaggttta gttaacttta 29520atctcacata gcaatcttta atcaatgtgt aacattaggg aggacttgaa agagccacca 29580cattttcatc gaggccacgc ggagtacgat cgagggtaca gtgaataatg ctagggagag 29640ctgcctatat ggaagagccc taatgtgtaa aattaatttt agtagtgcta tccccatgtg 29700attttaatag cttcttagga gaatgac 2972724382PRTCoronavirus 2Met Glu Ser Leu Val Leu Gly Val Asn Glu Lys Thr His Val Gln Leu1 5 10 15Ser Leu Pro Val Leu Gln Val Arg Asp Val Leu Val Arg Gly Phe Gly 20 25 30Asp Ser Val Glu Glu Ala Leu Ser Glu Ala Arg Glu His Leu Lys Asn 35 40

45Gly Thr Cys Gly Leu Val Glu Leu Glu Lys Gly Val Leu Pro Gln Leu 50 55 60Glu Gln Pro Tyr Val Phe Ile Lys Arg Ser Asp Ala Leu Ser Thr Asn65 70 75 80His Gly His Lys Val Val Glu Leu Val Ala Glu Met Asp Gly Ile Gln 85 90 95Tyr Gly Arg Ser Gly Ile Thr Leu Gly Val Leu Val Pro His Val Gly 100 105 110Glu Thr Pro Ile Ala Tyr Arg Asn Val Leu Leu Arg Lys Asn Gly Asn 115 120 125Lys Gly Ala Gly Gly His Ser Tyr Gly Ile Asp Leu Lys Ser Tyr Asp 130 135 140Leu Gly Asp Glu Leu Gly Thr Asp Pro Ile Glu Asp Tyr Glu Gln Asn145 150 155 160Trp Asn Thr Lys His Gly Ser Gly Ala Leu Arg Glu Leu Thr Arg Glu 165 170 175Leu Asn Gly Gly Ala Val Thr Arg Tyr Val Asp Asn Asn Phe Cys Gly 180 185 190Pro Asp Gly Tyr Pro Leu Asp Cys Ile Lys Asp Phe Leu Ala Arg Ala 195 200 205Gly Lys Ser Met Cys Thr Leu Ser Glu Gln Leu Asp Tyr Ile Glu Ser 210 215 220Lys Arg Gly Val Tyr Cys Cys Arg Asp His Glu His Glu Ile Ala Trp225 230 235 240Phe Thr Glu Arg Ser Asp Lys Ser Tyr Glu His Gln Thr Pro Phe Glu 245 250 255Ile Lys Ser Ala Lys Lys Phe Asp Thr Phe Lys Gly Glu Cys Pro Lys 260 265 270Phe Val Phe Pro Leu Asn Ser Lys Val Lys Val Ile Gln Pro Arg Val 275 280 285Glu Lys Lys Lys Thr Glu Gly Phe Met Gly Arg Ile Arg Ser Val Tyr 290 295 300Pro Val Ala Ser Pro Gln Glu Cys Asn Asn Met His Leu Ser Thr Leu305 310 315 320Met Lys Cys Asn His Cys Asp Glu Val Ser Trp Gln Thr Cys Asp Phe 325 330 335Leu Lys Ala Thr Cys Glu His Cys Gly Thr Glu Asn Leu Val Ile Glu 340 345 350Gly Pro Thr Thr Cys Gly Tyr Leu Pro Thr Asn Ala Val Val Lys Met 355 360 365Pro Cys Pro Ala Cys Gln Asp Pro Glu Ile Gly Pro Glu His Ser Val 370 375 380Ala Asp Tyr His Asn His Ser Asn Ile Glu Thr Arg Leu Arg Lys Gly385 390 395 400Gly Arg Thr Arg Cys Phe Gly Gly Cys Val Phe Ala Tyr Val Gly Cys 405 410 415Tyr Asn Lys Arg Ala Tyr Trp Val Pro Arg Ala Ser Ala Asp Ile Gly 420 425 430Ser Gly His Thr Gly Ile Thr Gly Asp Asn Val Glu Thr Leu Asn Glu 435 440 445Asp Leu Leu Glu Ile Leu Ser Arg Glu Arg Val Asn Ile Asn Ile Val 450 455 460Gly Asp Phe His Leu Asn Glu Glu Val Ala Ile Ile Leu Ala Ser Phe465 470 475 480Ser Ala Ser Thr Ser Ala Phe Ile Asp Thr Ile Lys Ser Leu Asp Tyr 485 490 495Lys Ser Phe Lys Thr Ile Val Glu Ser Cys Gly Asn Tyr Lys Val Thr 500 505 510Lys Gly Lys Pro Val Lys Gly Ala Trp Asn Ile Gly Gln Gln Arg Ser 515 520 525Val Leu Thr Pro Leu Cys Gly Phe Pro Ser Gln Ala Ala Gly Val Ile 530 535 540Arg Ser Ile Phe Ala Arg Thr Leu Asp Ala Ala Asn His Ser Ile Pro545 550 555 560Asp Leu Gln Arg Ala Ala Val Thr Ile Leu Asp Gly Ile Ser Glu Gln 565 570 575Ser Leu Arg Leu Val Asp Ala Met Val Tyr Thr Ser Asp Leu Leu Thr 580 585 590Asn Ser Val Ile Ile Met Ala Tyr Val Thr Gly Gly Leu Val Gln Gln 595 600 605Thr Ser Gln Trp Leu Ser Asn Leu Leu Gly Thr Thr Val Glu Lys Leu 610 615 620Arg Pro Ile Phe Glu Trp Ile Glu Ala Lys Leu Ser Ala Gly Val Glu625 630 635 640Phe Leu Lys Asp Ala Trp Glu Ile Leu Lys Phe Leu Ile Thr Gly Val 645 650 655Phe Asp Ile Val Lys Gly Gln Ile Gln Val Ala Ser Asp Asn Ile Lys 660 665 670Asp Cys Val Lys Cys Phe Ile Asp Val Val Asn Lys Ala Leu Glu Met 675 680 685Cys Ile Asp Gln Val Thr Ile Ala Gly Ala Lys Leu Arg Ser Leu Asn 690 695 700Leu Gly Glu Val Phe Ile Ala Gln Ser Lys Gly Leu Tyr Arg Gln Cys705 710 715 720Ile Arg Gly Lys Glu Gln Leu Gln Leu Leu Met Pro Leu Lys Ala Pro 725 730 735Lys Glu Val Thr Phe Leu Glu Gly Asp Ser His Asp Thr Val Leu Thr 740 745 750Ser Glu Glu Val Val Leu Lys Asn Gly Glu Leu Glu Ala Leu Glu Thr 755 760 765Pro Val Asp Ser Phe Thr Asn Gly Ala Ile Val Gly Thr Pro Val Cys 770 775 780Val Asn Gly Leu Met Leu Leu Glu Ile Lys Asp Lys Glu Gln Tyr Cys785 790 795 800Ala Leu Ser Pro Gly Leu Leu Ala Thr Asn Asn Val Phe Arg Leu Lys 805 810 815Gly Gly Ala Pro Ile Lys Gly Val Thr Phe Gly Glu Asp Thr Val Trp 820 825 830Glu Val Gln Gly Tyr Lys Asn Val Arg Ile Thr Phe Glu Leu Asp Glu 835 840 845Arg Val Asp Lys Val Leu Asn Glu Lys Cys Ser Val Tyr Thr Val Glu 850 855 860Ser Gly Thr Glu Val Thr Glu Phe Ala Cys Val Val Ala Glu Ala Val865 870 875 880Val Lys Thr Leu Gln Pro Val Ser Asp Leu Leu Thr Asn Met Gly Ile 885 890 895Asp Leu Asp Glu Trp Ser Val Ala Thr Phe Tyr Leu Phe Asp Asp Ala 900 905 910Gly Glu Glu Asn Phe Ser Ser Arg Met Tyr Cys Ser Phe Tyr Pro Pro 915 920 925Asp Glu Glu Glu Glu Asp Asp Ala Glu Cys Glu Glu Glu Glu Ile Asp 930 935 940Glu Thr Cys Glu His Glu Tyr Gly Thr Glu Asp Asp Tyr Gln Gly Leu945 950 955 960Pro Leu Glu Phe Gly Ala Ser Ala Glu Thr Val Arg Val Glu Glu Glu 965 970 975Glu Glu Glu Asp Trp Leu Asp Asp Thr Thr Glu Gln Ser Glu Ile Glu 980 985 990Pro Glu Pro Glu Pro Thr Pro Glu Glu Pro Val Asn Gln Phe Thr Gly 995 1000 1005Tyr Leu Lys Leu Thr Asp Asn Val Ala Ile Lys Cys Val Asp Ile 1010 1015 1020Val Lys Glu Ala Gln Ser Ala Asn Pro Met Val Ile Val Asn Ala 1025 1030 1035Ala Asn Ile His Leu Lys His Gly Gly Gly Val Ala Gly Ala Leu 1040 1045 1050Asn Lys Ala Thr Asn Gly Ala Met Gln Lys Glu Ser Asp Asp Tyr 1055 1060 1065Ile Lys Leu Asn Gly Pro Leu Thr Val Gly Gly Ser Cys Leu Leu 1070 1075 1080Ser Gly His Asn Leu Ala Lys Lys Cys Leu His Val Val Gly Pro 1085 1090 1095Asn Leu Asn Ala Gly Glu Asp Ile Gln Leu Leu Lys Ala Ala Tyr 1100 1105 1110Glu Asn Phe Asn Ser Gln Asp Ile Leu Leu Ala Pro Leu Leu Ser 1115 1120 1125Ala Gly Ile Phe Gly Ala Lys Pro Leu Gln Ser Leu Gln Val Cys 1130 1135 1140Val Gln Thr Val Arg Thr Gln Val Tyr Ile Ala Val Asn Asp Lys 1145 1150 1155Ala Leu Tyr Glu Gln Val Val Met Asp Tyr Leu Asp Asn Leu Lys 1160 1165 1170Pro Arg Val Glu Ala Pro Lys Gln Glu Glu Pro Pro Asn Thr Glu 1175 1180 1185Asp Ser Lys Thr Glu Glu Lys Ser Val Val Gln Lys Pro Val Asp 1190 1195 1200Val Lys Pro Lys Ile Lys Ala Cys Ile Asp Glu Val Thr Thr Thr 1205 1210 1215Leu Glu Glu Thr Lys Phe Leu Thr Asn Lys Leu Leu Leu Phe Ala 1220 1225 1230Asp Ile Asn Gly Lys Leu Tyr His Asp Ser Gln Asn Met Leu Arg 1235 1240 1245Gly Glu Asp Met Ser Phe Leu Glu Lys Asp Ala Pro Tyr Met Val 1250 1255 1260Gly Asp Val Ile Thr Ser Gly Asp Ile Thr Cys Val Val Ile Pro 1265 1270 1275Ser Lys Lys Ala Gly Gly Thr Thr Glu Met Leu Ser Arg Ala Leu 1280 1285 1290Lys Lys Val Pro Val Asp Glu Tyr Ile Thr Thr Tyr Pro Gly Gln 1295 1300 1305Gly Cys Ala Gly Tyr Thr Leu Glu Glu Ala Lys Thr Ala Leu Lys 1310 1315 1320Lys Cys Lys Ser Ala Phe Tyr Val Leu Pro Ser Glu Ala Pro Asn 1325 1330 1335Ala Lys Glu Glu Ile Leu Gly Thr Val Ser Trp Asn Leu Arg Glu 1340 1345 1350Met Leu Ala His Ala Glu Glu Thr Arg Lys Leu Met Pro Ile Cys 1355 1360 1365Met Asp Val Arg Ala Ile Met Ala Thr Ile Gln Arg Lys Tyr Lys 1370 1375 1380Gly Ile Lys Ile Gln Glu Gly Ile Val Asp Tyr Gly Val Arg Phe 1385 1390 1395Phe Phe Tyr Thr Ser Lys Glu Pro Val Ala Ser Ile Ile Thr Lys 1400 1405 1410Leu Asn Ser Leu Asn Glu Pro Leu Val Thr Met Pro Ile Gly Tyr 1415 1420 1425Val Thr His Gly Phe Asn Leu Glu Glu Ala Ala Arg Cys Met Arg 1430 1435 1440Ser Leu Lys Ala Pro Ala Val Val Ser Val Ser Ser Pro Asp Ala 1445 1450 1455Val Thr Thr Tyr Asn Gly Tyr Leu Thr Ser Ser Ser Lys Thr Ser 1460 1465 1470Glu Glu His Phe Val Glu Thr Val Ser Leu Ala Gly Ser Tyr Arg 1475 1480 1485Asp Trp Ser Tyr Ser Gly Gln Arg Thr Glu Leu Gly Val Glu Phe 1490 1495 1500Leu Lys Arg Gly Asp Lys Ile Val Tyr His Thr Leu Glu Ser Pro 1505 1510 1515Val Glu Phe His Leu Asp Gly Glu Val Leu Ser Leu Asp Lys Leu 1520 1525 1530Lys Ser Leu Leu Ser Leu Arg Glu Val Lys Thr Ile Lys Val Phe 1535 1540 1545Thr Thr Val Asp Asn Thr Asn Leu His Thr Gln Leu Val Asp Met 1550 1555 1560Ser Met Thr Tyr Gly Gln Gln Phe Gly Pro Thr Tyr Leu Asp Gly 1565 1570 1575Ala Asp Val Thr Lys Ile Lys Pro His Val Asn His Glu Gly Lys 1580 1585 1590Thr Phe Phe Val Leu Pro Ser Asp Asp Thr Leu Arg Ser Glu Ala 1595 1600 1605Phe Glu Tyr Tyr His Thr Leu Asp Glu Ser Phe Leu Gly Arg Tyr 1610 1615 1620Met Ser Ala Leu Asn His Thr Lys Lys Trp Lys Phe Pro Gln Val 1625 1630 1635Gly Gly Leu Thr Ser Ile Lys Trp Ala Asp Asn Asn Cys Tyr Leu 1640 1645 1650Ser Ser Val Leu Leu Ala Leu Gln Gln Leu Glu Val Lys Phe Asn 1655 1660 1665Ala Pro Ala Leu Gln Glu Ala Tyr Tyr Arg Ala Arg Ala Gly Asp 1670 1675 1680Ala Ala Asn Phe Cys Ala Leu Ile Leu Ala Tyr Ser Asn Lys Thr 1685 1690 1695Val Gly Glu Leu Gly Asp Val Arg Glu Thr Met Thr His Leu Leu 1700 1705 1710Gln His Ala Asn Leu Glu Ser Ala Lys Arg Val Leu Asn Val Val 1715 1720 1725Cys Lys His Cys Gly Gln Lys Thr Thr Thr Leu Thr Gly Val Glu 1730 1735 1740Ala Val Met Tyr Met Gly Thr Leu Ser Tyr Asp Asn Leu Lys Thr 1745 1750 1755Gly Val Ser Ile Pro Cys Val Cys Gly Arg Asp Ala Thr Gln Tyr 1760 1765 1770Leu Val Gln Gln Glu Ser Ser Phe Val Met Met Ser Ala Pro Pro 1775 1780 1785Ala Glu Tyr Lys Leu Gln Gln Gly Thr Phe Leu Cys Ala Asn Glu 1790 1795 1800Tyr Thr Gly Asn Tyr Gln Cys Gly His Tyr Thr His Ile Thr Ala 1805 1810 1815Lys Glu Thr Leu Tyr Arg Ile Asp Gly Ala His Leu Thr Lys Met 1820 1825 1830Ser Glu Tyr Lys Gly Pro Val Thr Asp Val Phe Tyr Lys Glu Thr 1835 1840 1845Ser Tyr Thr Thr Thr Ile Lys Pro Val Ser Tyr Lys Leu Asp Gly 1850 1855 1860Val Thr Tyr Thr Glu Ile Glu Pro Lys Leu Asp Gly Tyr Tyr Lys 1865 1870 1875Lys Asp Asn Ala Tyr Tyr Thr Glu Gln Pro Ile Asp Leu Val Pro 1880 1885 1890Thr Gln Pro Leu Pro Asn Ala Ser Phe Asp Asn Phe Lys Leu Thr 1895 1900 1905Cys Ser Asn Thr Lys Phe Ala Asp Asp Leu Asn Gln Met Thr Gly 1910 1915 1920Phe Thr Lys Pro Ala Ser Arg Glu Leu Ser Val Thr Phe Phe Pro 1925 1930 1935Asp Leu Asn Gly Asp Val Val Ala Ile Asp Tyr Arg His Tyr Ser 1940 1945 1950Ala Ser Phe Lys Lys Gly Ala Lys Leu Leu His Lys Pro Ile Val 1955 1960 1965Trp His Ile Asn Gln Ala Thr Thr Lys Thr Thr Phe Lys Pro Asn 1970 1975 1980Thr Trp Cys Leu Arg Cys Leu Trp Ser Thr Lys Pro Val Asp Thr 1985 1990 1995Ser Asn Ser Phe Glu Val Leu Ala Val Glu Asp Thr Gln Gly Met 2000 2005 2010Asp Asn Leu Ala Cys Glu Ser Gln Gln Pro Thr Ser Glu Glu Val 2015 2020 2025Val Glu Asn Pro Thr Ile Gln Lys Glu Val Ile Glu Cys Asp Val 2030 2035 2040Lys Thr Thr Glu Val Val Gly Asn Val Ile Leu Lys Pro Ser Asp 2045 2050 2055Glu Gly Val Lys Val Thr Gln Glu Leu Gly His Glu Asp Leu Met 2060 2065 2070Ala Ala Tyr Val Glu Asn Thr Ser Ile Thr Ile Lys Lys Pro Asn 2075 2080 2085Glu Leu Ser Leu Ala Leu Gly Leu Lys Thr Ile Ala Thr His Gly 2090 2095 2100Ile Ala Ala Ile Asn Ser Val Pro Trp Ser Lys Ile Leu Ala Tyr 2105 2110 2115Val Lys Pro Phe Leu Gly Gln Ala Ala Ile Thr Thr Ser Asn Cys 2120 2125 2130Ala Lys Arg Leu Ala Gln Arg Val Phe Asn Asn Tyr Met Pro Tyr 2135 2140 2145Val Phe Thr Leu Leu Phe Gln Leu Cys Thr Phe Thr Lys Ser Thr 2150 2155 2160Asn Ser Arg Ile Arg Ala Ser Leu Pro Thr Thr Ile Ala Lys Asn 2165 2170 2175Ser Val Lys Ser Val Ala Lys Leu Cys Leu Asp Ala Gly Ile Asn 2180 2185 2190Tyr Val Lys Ser Pro Lys Phe Ser Lys Leu Phe Thr Ile Ala Met 2195 2200 2205Trp Leu Leu Leu Leu Ser Ile Cys Leu Gly Ser Leu Ile Cys Val 2210 2215 2220Thr Ala Ala Phe Gly Val Leu Leu Ser Asn Phe Gly Ala Pro Ser 2225 2230 2235Tyr Cys Asn Gly Val Arg Glu Leu Tyr Leu Asn Ser Ser Asn Val 2240 2245 2250Thr Thr Met Asp Phe Cys Glu Gly Ser Phe Pro Cys Ser Ile Cys 2255 2260 2265Leu Ser Gly Leu Asp Ser Leu Asp Ser Tyr Pro Ala Leu Glu Thr 2270 2275 2280Ile Gln Val Thr Ile Ser Ser Tyr Lys Leu Asp Leu Thr Ile Leu 2285 2290 2295Gly Leu Ala Ala Glu Trp Val Leu Ala Tyr Met Leu Phe Thr Lys 2300 2305 2310Phe Phe Tyr Leu Leu Gly Leu Ser Ala Ile Met Gln Val Phe Phe 2315 2320 2325Gly Tyr Phe Ala Ser His Phe Ile Ser Asn Ser Trp Leu Met Trp 2330 2335 2340Phe Ile Ile Ser Ile Val Gln Met Ala Pro Val Ser Ala Met Val 2345 2350 2355Arg Met Tyr Ile Phe Phe Ala Ser Phe Tyr Tyr Ile Trp Lys Ser 2360 2365 2370Tyr Val His Ile Met Asp Gly Cys Thr Ser Ser Thr Cys Met Met 2375 2380 2385Cys Tyr Lys Arg Asn Arg Ala Thr Arg Val Glu Cys Thr Thr Ile 2390 2395 2400Val Asn Gly Met Lys Arg Ser Phe Tyr Val Tyr Ala Asn Gly Gly 2405 2410 2415Arg Gly Phe Cys Lys Thr His Asn Trp Asn Cys Leu Asn Cys Asp 2420 2425 2430Thr Phe Cys Thr Gly Ser Thr Phe Ile Ser Asp Glu Val Ala Arg 2435 2440 2445Asp Leu Ser Leu Gln Phe Lys Arg Pro Ile Asn Pro Thr Asp Gln 2450 2455 2460Ser Ser Tyr Ile Val Asp Ser Val Ala Val Lys Asn Gly Ala Leu 2465 2470 2475His Leu Tyr Phe Asp Lys Ala Gly Gln Lys Thr Tyr Glu Arg His 2480 2485 2490Pro Leu Ser His Phe Val

Asn Leu Asp Asn Leu Arg Ala Asn Asn 2495 2500 2505Thr Lys Gly Ser Leu Pro Ile Asn Val Ile Val Phe Asp Gly Lys 2510 2515 2520Ser Lys Cys Asp Glu Ser Ala Ser Lys Ser Ala Ser Val Tyr Tyr 2525 2530 2535Ser Gln Leu Met Cys Gln Pro Ile Leu Leu Leu Asp Gln Val Leu 2540 2545 2550Val Ser Asp Val Gly Asp Ser Thr Glu Val Ser Val Lys Met Phe 2555 2560 2565Asp Ala Tyr Val Asp Thr Phe Ser Ala Thr Phe Ser Val Pro Met 2570 2575 2580Glu Lys Leu Lys Ala Leu Val Ala Thr Ala His Ser Glu Leu Ala 2585 2590 2595Lys Gly Val Ala Leu Asp Gly Val Leu Ser Thr Phe Val Ser Ala 2600 2605 2610Ala Arg Gln Gly Val Val Asp Thr Asp Val Asp Thr Lys Asp Val 2615 2620 2625Ile Glu Cys Leu Lys Leu Ser His His Ser Asp Leu Glu Val Thr 2630 2635 2640Gly Asp Ser Cys Asn Asn Phe Met Leu Thr Tyr Asn Lys Val Glu 2645 2650 2655Asn Met Thr Pro Arg Asp Leu Gly Ala Cys Ile Asp Cys Asn Ala 2660 2665 2670Arg His Ile Asn Ala Gln Val Ala Lys Ser His Asn Val Ser Leu 2675 2680 2685Ile Trp Asn Val Lys Asp Tyr Met Ser Leu Ser Glu Gln Leu Arg 2690 2695 2700Lys Gln Ile Arg Ser Ala Ala Lys Lys Asn Asn Ile Pro Phe Arg 2705 2710 2715Leu Thr Cys Ala Thr Thr Arg Gln Val Val Asn Val Ile Thr Thr 2720 2725 2730Lys Ile Ser Leu Lys Gly Gly Lys Ile Val Ser Thr Cys Phe Lys 2735 2740 2745Leu Met Leu Lys Ala Thr Leu Leu Cys Val Leu Ala Ala Leu Val 2750 2755 2760Cys Tyr Ile Val Met Pro Val His Thr Leu Ser Ile His Asp Gly 2765 2770 2775Tyr Thr Asn Glu Ile Ile Gly Tyr Lys Ala Ile Gln Asp Gly Val 2780 2785 2790Thr Arg Asp Ile Ile Ser Thr Asp Asp Cys Phe Ala Asn Lys His 2795 2800 2805Ala Gly Phe Asp Ala Trp Phe Ser Gln Arg Gly Gly Ser Tyr Lys 2810 2815 2820Asn Asp Lys Ser Cys Pro Val Val Ala Ala Ile Ile Thr Arg Glu 2825 2830 2835Ile Gly Phe Ile Val Pro Gly Leu Pro Gly Thr Val Leu Arg Ala 2840 2845 2850Ile Asn Gly Asp Phe Leu His Phe Leu Pro Arg Val Phe Ser Ala 2855 2860 2865Val Gly Asn Ile Cys Tyr Thr Pro Ser Lys Leu Ile Glu Tyr Ser 2870 2875 2880Asp Phe Ala Thr Ser Ala Cys Val Leu Ala Ala Glu Cys Thr Ile 2885 2890 2895Phe Lys Asp Ala Met Gly Lys Pro Val Pro Tyr Cys Tyr Asp Thr 2900 2905 2910Asn Leu Leu Glu Gly Ser Ile Ser Tyr Ser Glu Leu Arg Pro Asp 2915 2920 2925Thr Arg Tyr Val Leu Met Asp Gly Ser Ile Ile Gln Phe Pro Asn 2930 2935 2940Thr Tyr Leu Glu Gly Ser Val Arg Val Val Thr Thr Phe Asp Ala 2945 2950 2955Glu Tyr Cys Arg His Gly Thr Cys Glu Arg Ser Glu Val Gly Ile 2960 2965 2970Cys Leu Ser Thr Ser Gly Arg Trp Val Leu Asn Asn Glu His Tyr 2975 2980 2985Arg Ala Leu Ser Gly Val Phe Cys Gly Val Asp Ala Met Asn Leu 2990 2995 3000Ile Ala Asn Ile Phe Thr Pro Leu Val Gln Pro Val Gly Ala Leu 3005 3010 3015Asp Val Ser Ala Ser Val Val Ala Gly Gly Ile Ile Ala Ile Leu 3020 3025 3030Val Thr Cys Ala Ala Tyr Tyr Phe Met Lys Phe Arg Arg Val Phe 3035 3040 3045Gly Glu Tyr Asn His Val Val Ala Ala Asn Ala Leu Leu Phe Leu 3050 3055 3060Met Ser Phe Thr Ile Leu Cys Leu Val Pro Ala Tyr Ser Phe Leu 3065 3070 3075Pro Gly Val Tyr Ser Val Phe Tyr Leu Tyr Leu Thr Phe Tyr Phe 3080 3085 3090Thr Asn Asp Val Ser Phe Leu Ala His Leu Gln Trp Phe Ala Met 3095 3100 3105Phe Ser Pro Ile Val Pro Phe Trp Ile Thr Ala Ile Tyr Val Phe 3110 3115 3120Cys Ile Ser Leu Lys His Cys His Trp Phe Phe Asn Asn Tyr Leu 3125 3130 3135Arg Lys Arg Val Met Phe Asn Gly Val Thr Phe Ser Thr Phe Glu 3140 3145 3150Glu Ala Ala Leu Cys Thr Phe Leu Leu Asn Lys Glu Met Tyr Leu 3155 3160 3165Lys Leu Arg Ser Glu Thr Leu Leu Pro Leu Thr Gln Tyr Asn Arg 3170 3175 3180Tyr Leu Ala Leu Tyr Asn Lys Tyr Lys Tyr Phe Ser Gly Ala Leu 3185 3190 3195Asp Thr Thr Ser Tyr Arg Glu Ala Ala Cys Cys His Leu Ala Lys 3200 3205 3210Ala Leu Asn Asp Phe Ser Asn Ser Gly Ala Asp Val Leu Tyr Gln 3215 3220 3225Pro Pro Gln Thr Ser Ile Thr Ser Ala Val Leu Gln Ser Gly Phe 3230 3235 3240Arg Lys Met Ala Phe Pro Ser Gly Lys Val Glu Gly Cys Met Val 3245 3250 3255Gln Val Thr Cys Gly Thr Thr Thr Leu Asn Gly Leu Trp Leu Asp 3260 3265 3270Asp Thr Val Tyr Cys Pro Arg His Val Ile Cys Thr Ala Glu Asp 3275 3280 3285Met Leu Asn Pro Asn Tyr Glu Asp Leu Leu Ile Arg Lys Ser Asn 3290 3295 3300His Ser Phe Leu Val Gln Ala Gly Asn Val Gln Leu Arg Val Ile 3305 3310 3315Gly His Ser Met Gln Asn Cys Leu Leu Arg Leu Lys Val Asp Thr 3320 3325 3330Ser Asn Pro Lys Thr Pro Lys Tyr Lys Phe Val Arg Ile Gln Pro 3335 3340 3345Gly Gln Thr Phe Ser Val Leu Ala Cys Tyr Asn Gly Ser Pro Ser 3350 3355 3360Gly Val Tyr Gln Cys Ala Met Arg Pro Asn His Thr Ile Lys Gly 3365 3370 3375Ser Phe Leu Asn Gly Ser Cys Gly Ser Val Gly Phe Asn Ile Asp 3380 3385 3390Tyr Asp Cys Val Ser Phe Cys Tyr Met His His Met Glu Leu Pro 3395 3400 3405Thr Gly Val His Ala Gly Thr Asp Leu Glu Gly Lys Phe Tyr Gly 3410 3415 3420Pro Phe Val Asp Arg Gln Thr Ala Gln Ala Ala Gly Thr Asp Thr 3425 3430 3435Thr Ile Thr Leu Asn Val Leu Ala Trp Leu Tyr Ala Ala Val Ile 3440 3445 3450Asn Gly Asp Arg Trp Phe Leu Asn Arg Phe Thr Thr Thr Leu Asn 3455 3460 3465Asp Phe Asn Leu Val Ala Met Lys Tyr Asn Tyr Glu Pro Leu Thr 3470 3475 3480Gln Asp His Val Asp Ile Leu Gly Pro Leu Ser Ala Gln Thr Gly 3485 3490 3495Ile Ala Val Leu Asp Met Cys Ala Ala Leu Lys Glu Leu Leu Gln 3500 3505 3510Asn Gly Met Asn Gly Arg Thr Ile Leu Gly Ser Thr Ile Leu Glu 3515 3520 3525Asp Glu Phe Thr Pro Phe Asp Val Val Arg Gln Cys Ser Gly Val 3530 3535 3540Thr Phe Gln Gly Lys Phe Lys Lys Ile Val Lys Gly Thr His His 3545 3550 3555Trp Met Leu Leu Thr Phe Leu Thr Ser Leu Leu Ile Leu Val Gln 3560 3565 3570Ser Thr Gln Trp Ser Leu Phe Phe Phe Val Tyr Glu Asn Ala Phe 3575 3580 3585Leu Pro Phe Thr Leu Gly Ile Met Ala Ile Ala Ala Cys Ala Met 3590 3595 3600Leu Leu Val Lys His Lys His Ala Phe Leu Cys Leu Phe Leu Leu 3605 3610 3615Pro Ser Leu Ala Thr Val Ala Tyr Phe Asn Met Val Tyr Met Pro 3620 3625 3630Ala Ser Trp Val Met Arg Ile Met Thr Trp Leu Glu Leu Ala Asp 3635 3640 3645Thr Ser Leu Ser Gly Tyr Arg Leu Lys Asp Cys Val Met Tyr Ala 3650 3655 3660Ser Ala Leu Val Leu Leu Ile Leu Met Thr Ala Arg Thr Val Tyr 3665 3670 3675Asp Asp Ala Ala Arg Arg Val Trp Thr Leu Met Asn Val Ile Thr 3680 3685 3690Leu Val Tyr Lys Val Tyr Tyr Gly Asn Ala Leu Asp Gln Ala Ile 3695 3700 3705Ser Met Trp Ala Leu Val Ile Ser Val Thr Ser Asn Tyr Ser Gly 3710 3715 3720Val Val Thr Thr Ile Met Phe Leu Ala Arg Ala Ile Val Phe Val 3725 3730 3735Cys Val Glu Tyr Tyr Pro Leu Leu Phe Ile Thr Gly Asn Thr Leu 3740 3745 3750Gln Cys Ile Met Leu Val Tyr Cys Phe Leu Gly Tyr Cys Cys Cys 3755 3760 3765Cys Tyr Phe Gly Leu Phe Cys Leu Leu Asn Arg Tyr Phe Arg Leu 3770 3775 3780Thr Leu Gly Val Tyr Asp Tyr Leu Val Ser Thr Gln Glu Phe Arg 3785 3790 3795Tyr Met Asn Ser Gln Gly Leu Leu Pro Pro Lys Ser Ser Ile Asp 3800 3805 3810Ala Phe Lys Leu Asn Ile Lys Leu Leu Gly Ile Gly Gly Lys Pro 3815 3820 3825Cys Ile Lys Val Ala Thr Val Gln Ser Lys Met Ser Asp Val Lys 3830 3835 3840Cys Thr Ser Val Val Leu Leu Ser Val Leu Gln Gln Leu Arg Val 3845 3850 3855Glu Ser Ser Ser Lys Leu Trp Ala Gln Cys Val Gln Leu His Asn 3860 3865 3870Asp Ile Leu Leu Ala Lys Asp Thr Thr Glu Ala Phe Glu Lys Met 3875 3880 3885Val Ser Leu Leu Ser Val Leu Leu Ser Met Gln Gly Ala Val Asp 3890 3895 3900Ile Asn Arg Leu Cys Glu Glu Met Leu Asp Asn Arg Ala Thr Leu 3905 3910 3915Gln Ala Ile Ala Ser Glu Phe Ser Ser Leu Pro Ser Tyr Ala Ala 3920 3925 3930Tyr Ala Thr Ala Gln Glu Ala Tyr Glu Gln Ala Val Ala Asn Gly 3935 3940 3945Asp Ser Glu Val Val Leu Lys Lys Leu Lys Lys Ser Leu Asn Val 3950 3955 3960Ala Lys Ser Glu Phe Asp Arg Asp Ala Ala Met Gln Arg Lys Leu 3965 3970 3975Glu Lys Met Ala Asp Gln Ala Met Thr Gln Met Tyr Lys Gln Ala 3980 3985 3990Arg Ser Glu Asp Lys Arg Ala Lys Val Thr Ser Ala Met Gln Thr 3995 4000 4005Met Leu Phe Thr Met Leu Arg Lys Leu Asp Asn Asp Ala Leu Asn 4010 4015 4020Asn Ile Ile Asn Asn Ala Arg Asp Gly Cys Val Pro Leu Asn Ile 4025 4030 4035Ile Pro Leu Thr Thr Ala Ala Lys Leu Met Val Val Val Pro Asp 4040 4045 4050Tyr Gly Thr Tyr Lys Asn Thr Cys Asp Gly Asn Thr Phe Thr Tyr 4055 4060 4065Ala Ser Ala Leu Trp Glu Ile Gln Gln Val Val Asp Ala Asp Ser 4070 4075 4080Lys Ile Val Gln Leu Ser Glu Ile Asn Met Asp Asn Ser Pro Asn 4085 4090 4095Leu Ala Trp Pro Leu Ile Val Thr Ala Leu Arg Ala Asn Ser Ala 4100 4105 4110Val Lys Leu Gln Asn Asn Glu Leu Ser Pro Val Ala Leu Arg Gln 4115 4120 4125Met Ser Cys Ala Ala Gly Thr Thr Gln Thr Ala Cys Thr Asp Asp 4130 4135 4140Asn Ala Leu Ala Tyr Tyr Asn Asn Ser Lys Gly Gly Arg Phe Val 4145 4150 4155Leu Ala Leu Leu Ser Asp His Gln Asp Leu Lys Trp Ala Arg Phe 4160 4165 4170Pro Lys Ser Asp Gly Thr Gly Thr Ile Tyr Thr Glu Leu Glu Pro 4175 4180 4185Pro Cys Arg Phe Val Thr Asp Thr Pro Lys Gly Pro Lys Val Lys 4190 4195 4200Tyr Leu Tyr Phe Ile Lys Gly Leu Asn Asn Leu Asn Arg Gly Met 4205 4210 4215Val Leu Gly Ser Leu Ala Ala Thr Val Arg Leu Gln Ala Gly Asn 4220 4225 4230Ala Thr Glu Val Pro Ala Asn Ser Thr Val Leu Ser Phe Cys Ala 4235 4240 4245Phe Ala Val Asp Pro Ala Lys Ala Tyr Lys Asp Tyr Leu Ala Ser 4250 4255 4260Gly Gly Gln Pro Ile Thr Asn Cys Val Lys Met Leu Cys Thr His 4265 4270 4275Thr Gly Thr Gly Gln Ala Ile Thr Val Thr Pro Glu Ala Asn Met 4280 4285 4290Asp Gln Glu Ser Phe Gly Gly Ala Ser Cys Cys Leu Tyr Cys Arg 4295 4300 4305Cys His Ile Asp His Pro Asn Pro Lys Gly Phe Cys Asp Leu Lys 4310 4315 4320Gly Lys Tyr Val Gln Ile Pro Thr Thr Cys Ala Asn Asp Pro Val 4325 4330 4335Gly Phe Thr Leu Arg Asn Thr Val Cys Thr Val Cys Gly Met Trp 4340 4345 4350Lys Gly Tyr Gly Cys Ser Cys Asp Gln Leu Arg Glu Pro Leu Met 4355 4360 4365Gln Ser Ala Asp Ala Ser Thr Phe Leu Asn Gly Phe Ala Val 4370 4375 438032695PRTCoronavirus 3Arg Val Cys Gly Val Ser Ala Ala Arg Leu Thr Pro Cys Gly Thr Gly1 5 10 15Thr Ser Thr Asp Val Val Tyr Arg Ala Phe Asp Ile Tyr Asn Glu Lys 20 25 30Val Ala Gly Phe Ala Lys Phe Leu Lys Thr Asn Cys Cys Arg Phe Gln 35 40 45Glu Lys Asp Glu Glu Gly Asn Leu Leu Asp Ser Tyr Phe Val Val Lys 50 55 60Arg His Thr Met Ser Asn Tyr Gln His Glu Glu Thr Ile Tyr Asn Leu65 70 75 80Val Lys Asp Cys Pro Ala Val Ala Val His Asp Phe Phe Lys Phe Arg 85 90 95Val Asp Gly Asp Met Val Pro His Ile Ser Arg Gln Arg Leu Thr Lys 100 105 110Tyr Thr Met Ala Asp Leu Val Tyr Ala Leu Arg His Phe Asp Glu Gly 115 120 125Asn Cys Asp Thr Leu Lys Glu Ile Leu Val Thr Tyr Asn Cys Cys Asp 130 135 140Asp Asp Tyr Phe Asn Lys Lys Asp Trp Tyr Asp Phe Val Glu Asn Pro145 150 155 160Asp Ile Leu Arg Val Tyr Ala Asn Leu Gly Glu Arg Val Arg Gln Ser 165 170 175Leu Leu Lys Thr Val Gln Phe Cys Asp Ala Met Arg Asp Ala Gly Ile 180 185 190Val Gly Val Leu Thr Leu Asp Asn Gln Asp Leu Asn Gly Asn Trp Tyr 195 200 205Asp Phe Gly Asp Phe Val Gln Val Ala Pro Gly Cys Gly Val Pro Ile 210 215 220Val Asp Ser Tyr Tyr Ser Leu Leu Met Pro Ile Leu Thr Leu Thr Arg225 230 235 240Ala Leu Ala Ala Glu Ser His Met Asp Ala Asp Leu Ala Lys Pro Leu 245 250 255Ile Lys Trp Asp Leu Leu Lys Tyr Asp Phe Thr Glu Glu Arg Leu Cys 260 265 270Leu Phe Asp Arg Tyr Phe Lys Tyr Trp Asp Gln Thr Tyr His Pro Asn 275 280 285Cys Ile Asn Cys Leu Asp Asp Arg Cys Ile Leu His Cys Ala Asn Phe 290 295 300Asn Val Leu Phe Ser Thr Val Phe Pro Pro Thr Ser Phe Gly Pro Leu305 310 315 320Val Arg Lys Ile Phe Val Asp Gly Val Pro Phe Val Val Ser Thr Gly 325 330 335Tyr His Phe Arg Glu Leu Gly Val Val His Asn Gln Asp Val Asn Leu 340 345 350His Ser Ser Arg Leu Ser Phe Lys Glu Leu Leu Val Tyr Ala Ala Asp 355 360 365Pro Ala Met His Ala Ala Ser Gly Asn Leu Leu Leu Asp Lys Arg Thr 370 375 380Thr Cys Phe Ser Val Ala Ala Leu Thr Asn Asn Val Ala Phe Gln Thr385 390 395 400Val Lys Pro Gly Asn Phe Asn Lys Asp Phe Tyr Asp Phe Ala Val Ser 405 410 415Lys Gly Phe Phe Lys Glu Gly Ser Ser Val Glu Leu Lys His Phe Phe 420 425 430Phe Ala Gln Asp Gly Asn Ala Ala Ile Ser Asp Tyr Asp Tyr Tyr Arg 435 440 445Tyr Asn Leu Pro Thr Met Cys Asp Ile Arg Gln Leu Leu Phe Val Val 450 455 460Glu Val Val Asp Lys Tyr Phe Asp Cys Tyr Asp Gly Gly Cys Ile Asn465 470 475 480Ala Asn Gln Val Ile Val Asn Asn Leu Asp Lys Ser Ala Gly Phe Pro 485 490 495Phe Asn Lys Trp Gly Lys Ala Arg Leu Tyr Tyr Asp Ser Met Ser Tyr 500 505 510Glu Asp Gln Asp Ala Leu Phe Ala Tyr Thr Lys Arg Asn Val Ile Pro 515 520 525Thr Ile Thr Gln Met Asn Leu Lys Tyr Ala Ile Ser Ala Lys Asn Arg 530 535 540Ala Arg Thr Val Ala Gly Val Ser Ile Cys Ser Thr Met Thr Asn Arg545 550 555

560Gln Phe His Gln Lys Leu Leu Lys Ser Ile Ala Ala Thr Arg Gly Ala 565 570 575Thr Val Val Ile Gly Thr Ser Lys Phe Tyr Gly Gly Trp His Asn Met 580 585 590Leu Lys Thr Val Tyr Ser Asp Val Glu Thr Pro His Leu Met Gly Trp 595 600 605Asp Tyr Pro Lys Cys Asp Arg Ala Met Pro Asn Met Leu Arg Ile Met 610 615 620Ala Ser Leu Val Leu Ala Arg Lys His Asn Thr Cys Cys Asn Leu Ser625 630 635 640His Arg Phe Tyr Arg Leu Ala Asn Glu Cys Ala Gln Val Leu Ser Glu 645 650 655Met Val Met Cys Gly Gly Ser Leu Tyr Val Lys Pro Gly Gly Thr Ser 660 665 670Ser Gly Asp Ala Thr Thr Ala Tyr Ala Asn Ser Val Phe Asn Ile Cys 675 680 685Gln Ala Val Thr Ala Asn Val Asn Ala Leu Leu Ser Thr Asp Gly Asn 690 695 700Lys Ile Ala Asp Lys Tyr Val Arg Asn Leu Gln His Arg Leu Tyr Glu705 710 715 720Cys Leu Tyr Arg Asn Arg Asp Val Asp His Glu Phe Val Asp Glu Phe 725 730 735Tyr Ala Tyr Leu Arg Lys His Phe Ser Met Met Ile Leu Ser Asp Asp 740 745 750Ala Val Val Cys Tyr Asn Ser Asn Tyr Ala Ala Gln Gly Leu Val Ala 755 760 765Ser Ile Lys Asn Phe Lys Ala Val Leu Tyr Tyr Gln Asn Asn Val Phe 770 775 780Met Ser Glu Ala Lys Cys Trp Thr Glu Thr Asp Leu Thr Lys Gly Pro785 790 795 800His Glu Phe Cys Ser Gln His Thr Met Leu Val Lys Gln Gly Asp Asp 805 810 815Tyr Val Tyr Leu Pro Tyr Pro Asp Pro Ser Arg Ile Leu Gly Ala Gly 820 825 830Cys Phe Val Asp Asp Ile Val Lys Thr Asp Gly Thr Leu Met Ile Glu 835 840 845Arg Phe Val Ser Leu Ala Ile Asp Ala Tyr Pro Leu Thr Lys His Pro 850 855 860Asn Gln Glu Tyr Ala Asp Val Phe His Leu Tyr Leu Gln Tyr Ile Arg865 870 875 880Lys Leu His Asp Glu Leu Thr Gly His Met Leu Asp Met Tyr Ser Val 885 890 895Met Leu Thr Asn Asp Asn Thr Ser Arg Tyr Trp Glu Pro Glu Phe Tyr 900 905 910Glu Ala Met Tyr Thr Pro His Thr Val Leu Gln Ala Val Gly Ala Cys 915 920 925Val Leu Cys Asn Ser Gln Thr Ser Leu Arg Cys Gly Ala Cys Ile Arg 930 935 940Arg Pro Phe Leu Cys Cys Lys Cys Cys Tyr Asp His Val Ile Ser Thr945 950 955 960Ser His Lys Leu Val Leu Ser Val Asn Pro Tyr Val Cys Asn Ala Pro 965 970 975Gly Cys Asp Val Thr Asp Val Thr Gln Leu Tyr Leu Gly Gly Met Ser 980 985 990Tyr Tyr Cys Lys Ser His Lys Pro Pro Ile Ser Phe Pro Leu Cys Ala 995 1000 1005Asn Gly Gln Val Phe Gly Leu Tyr Lys Asn Thr Cys Val Gly Ser 1010 1015 1020Asp Asn Val Thr Asp Phe Asn Ala Ile Ala Thr Cys Asp Trp Thr 1025 1030 1035Asn Ala Gly Asp Tyr Ile Leu Ala Asn Thr Cys Thr Glu Arg Leu 1040 1045 1050Lys Leu Phe Ala Ala Glu Thr Leu Lys Ala Thr Glu Glu Thr Phe 1055 1060 1065Lys Leu Ser Tyr Gly Ile Ala Thr Val Arg Glu Val Leu Ser Asp 1070 1075 1080Arg Glu Leu His Leu Ser Trp Glu Val Gly Lys Pro Arg Pro Pro 1085 1090 1095Leu Asn Arg Asn Tyr Val Phe Thr Gly Tyr Arg Val Thr Lys Asn 1100 1105 1110Ser Lys Val Gln Ile Gly Glu Tyr Thr Phe Glu Lys Gly Asp Tyr 1115 1120 1125Gly Asp Ala Val Val Tyr Arg Gly Thr Thr Thr Tyr Lys Leu Asn 1130 1135 1140Val Gly Asp Tyr Phe Val Leu Thr Ser His Thr Val Met Pro Leu 1145 1150 1155Ser Ala Pro Thr Leu Val Pro Gln Glu His Tyr Val Arg Ile Thr 1160 1165 1170Gly Leu Tyr Pro Thr Leu Asn Ile Ser Asp Glu Phe Ser Ser Asn 1175 1180 1185Val Ala Asn Tyr Gln Lys Val Gly Met Gln Lys Tyr Ser Thr Leu 1190 1195 1200Gln Gly Pro Pro Gly Thr Gly Lys Ser His Phe Ala Ile Gly Leu 1205 1210 1215Ala Leu Tyr Tyr Pro Ser Ala Arg Ile Val Tyr Thr Ala Cys Ser 1220 1225 1230His Ala Ala Val Asp Ala Leu Cys Glu Lys Ala Leu Lys Tyr Leu 1235 1240 1245Pro Ile Asp Lys Cys Ser Arg Ile Ile Pro Ala Arg Ala Arg Val 1250 1255 1260Glu Cys Phe Asp Lys Phe Lys Val Asn Ser Thr Leu Glu Gln Tyr 1265 1270 1275Val Phe Cys Thr Val Asn Ala Leu Pro Glu Thr Thr Ala Asp Ile 1280 1285 1290Val Val Phe Asp Glu Ile Ser Met Ala Thr Asn Tyr Asp Leu Ser 1295 1300 1305Val Val Asn Ala Arg Leu Arg Ala Lys His Tyr Val Tyr Ile Gly 1310 1315 1320Asp Pro Ala Gln Leu Pro Ala Pro Arg Thr Leu Leu Thr Lys Gly 1325 1330 1335Thr Leu Glu Pro Glu Tyr Phe Asn Ser Val Cys Arg Leu Met Lys 1340 1345 1350Thr Ile Gly Pro Asp Met Phe Leu Gly Thr Cys Arg Arg Cys Pro 1355 1360 1365Ala Glu Ile Val Asp Thr Val Ser Ala Leu Val Tyr Asp Asn Lys 1370 1375 1380Leu Lys Ala His Lys Asp Lys Ser Ala Gln Cys Phe Lys Met Phe 1385 1390 1395Tyr Lys Gly Val Ile Thr His Asp Val Ser Ser Ala Ile Asn Arg 1400 1405 1410Pro Gln Ile Gly Val Val Arg Glu Phe Leu Thr Arg Asn Pro Ala 1415 1420 1425Trp Arg Lys Ala Val Phe Ile Ser Pro Tyr Asn Ser Gln Asn Ala 1430 1435 1440Val Ala Ser Lys Ile Leu Gly Leu Pro Thr Gln Thr Val Asp Ser 1445 1450 1455Ser Gln Gly Ser Glu Tyr Asp Tyr Val Ile Phe Thr Gln Thr Thr 1460 1465 1470Glu Thr Ala His Ser Cys Asn Val Asn Arg Phe Asn Val Ala Ile 1475 1480 1485Thr Arg Ala Lys Ile Gly Ile Leu Cys Ile Met Ser Asp Arg Asp 1490 1495 1500Leu Tyr Asp Lys Leu Gln Phe Thr Ser Leu Glu Ile Pro Arg Arg 1505 1510 1515Asn Val Ala Thr Leu Gln Ala Glu Asn Val Thr Gly Leu Phe Lys 1520 1525 1530Asp Cys Ser Lys Ile Ile Thr Gly Leu His Pro Thr Gln Ala Pro 1535 1540 1545Thr His Leu Ser Val Asp Ile Lys Phe Lys Thr Glu Gly Leu Cys 1550 1555 1560Val Asp Ile Pro Gly Ile Pro Lys Asp Met Thr Tyr Arg Arg Leu 1565 1570 1575Ile Ser Met Met Gly Phe Lys Met Asn Tyr Gln Val Asn Gly Tyr 1580 1585 1590Pro Asn Met Phe Ile Thr Arg Glu Glu Ala Ile Arg His Val Arg 1595 1600 1605Ala Trp Ile Gly Phe Asp Val Glu Gly Cys His Ala Thr Arg Asp 1610 1615 1620Ala Val Gly Thr Asn Leu Pro Leu Gln Leu Gly Phe Ser Thr Gly 1625 1630 1635Val Asn Leu Val Ala Val Pro Thr Gly Tyr Val Asp Thr Glu Asn 1640 1645 1650Asn Thr Glu Phe Thr Arg Val Asn Ala Lys Pro Pro Pro Gly Asp 1655 1660 1665Gln Phe Lys His Leu Ile Pro Leu Met Tyr Lys Gly Leu Pro Trp 1670 1675 1680Asn Val Val Arg Ile Lys Ile Val Gln Met Leu Ser Asp Thr Leu 1685 1690 1695Lys Gly Leu Ser Asp Arg Val Val Phe Val Leu Trp Ala His Gly 1700 1705 1710Phe Glu Leu Thr Ser Met Lys Tyr Phe Val Lys Ile Gly Pro Glu 1715 1720 1725Arg Thr Cys Cys Leu Cys Asp Lys Arg Ala Thr Cys Phe Ser Thr 1730 1735 1740Ser Ser Asp Thr Tyr Ala Cys Trp Asn His Ser Val Gly Phe Asp 1745 1750 1755Tyr Val Tyr Asn Pro Phe Met Ile Asp Val Gln Gln Trp Gly Phe 1760 1765 1770Thr Gly Asn Leu Gln Ser Asn His Asp Gln His Cys Gln Val His 1775 1780 1785Gly Asn Ala His Val Ala Ser Cys Asp Ala Ile Met Thr Arg Cys 1790 1795 1800Leu Ala Val His Glu Cys Phe Val Lys Arg Val Asp Trp Ser Val 1805 1810 1815Glu Tyr Pro Ile Ile Gly Asp Glu Leu Arg Val Asn Ser Ala Cys 1820 1825 1830Arg Lys Val Gln His Met Val Val Lys Ser Ala Leu Leu Ala Asp 1835 1840 1845Lys Phe Pro Val Leu His Asp Ile Gly Asn Pro Lys Ala Ile Lys 1850 1855 1860Cys Val Pro Gln Ala Glu Val Glu Trp Lys Phe Tyr Asp Ala Gln 1865 1870 1875Pro Cys Ser Asp Lys Ala Tyr Lys Ile Glu Glu Leu Phe Tyr Ser 1880 1885 1890Tyr Ala Thr His His Asp Lys Phe Thr Asp Gly Val Cys Leu Phe 1895 1900 1905Trp Asn Cys Asn Val Asp Arg Tyr Pro Ala Asn Ala Ile Val Cys 1910 1915 1920Arg Phe Asp Thr Arg Val Leu Ser Asn Leu Asn Leu Pro Gly Cys 1925 1930 1935Asp Gly Gly Ser Leu Tyr Val Asn Lys His Ala Phe His Thr Pro 1940 1945 1950Ala Phe Asp Lys Ser Ala Phe Thr Asn Leu Lys Gln Leu Pro Phe 1955 1960 1965Phe Tyr Tyr Ser Asp Ser Pro Cys Glu Ser His Gly Lys Gln Val 1970 1975 1980Val Ser Asp Ile Asp Tyr Val Pro Leu Lys Ser Ala Thr Cys Ile 1985 1990 1995Thr Arg Cys Asn Leu Gly Gly Ala Val Cys Arg His His Ala Asn 2000 2005 2010Glu Tyr Arg Gln Tyr Leu Asp Ala Tyr Asn Met Met Ile Ser Ala 2015 2020 2025Gly Phe Ser Leu Trp Ile Tyr Lys Gln Phe Asp Thr Tyr Asn Leu 2030 2035 2040Trp Asn Thr Phe Thr Arg Leu Gln Ser Leu Glu Asn Val Ala Tyr 2045 2050 2055Asn Val Val Asn Lys Gly His Phe Asp Gly His Ala Gly Glu Ala 2060 2065 2070Pro Val Ser Ile Ile Asn Asn Ala Val Tyr Thr Lys Val Asp Gly 2075 2080 2085Ile Asp Val Glu Ile Phe Glu Asn Lys Thr Thr Leu Pro Val Asn 2090 2095 2100Val Ala Phe Glu Leu Trp Ala Lys Arg Asn Ile Lys Pro Val Pro 2105 2110 2115Glu Ile Lys Ile Leu Asn Asn Leu Gly Val Asp Ile Ala Ala Asn 2120 2125 2130Thr Val Ile Trp Asp Tyr Lys Arg Glu Ala Pro Ala His Val Ser 2135 2140 2145Thr Ile Gly Val Cys Thr Met Thr Asp Ile Ala Lys Lys Pro Thr 2150 2155 2160Glu Ser Ala Cys Ser Ser Leu Thr Val Leu Phe Asp Gly Arg Val 2165 2170 2175Glu Gly Gln Val Asp Leu Phe Arg Asn Ala Arg Asn Gly Val Leu 2180 2185 2190Ile Thr Glu Gly Ser Val Lys Gly Leu Thr Pro Ser Lys Gly Pro 2195 2200 2205Ala Gln Ala Ser Val Asn Gly Val Thr Leu Ile Gly Glu Ser Val 2210 2215 2220Lys Thr Gln Phe Asn Tyr Phe Lys Lys Val Asp Gly Ile Ile Gln 2225 2230 2235Gln Leu Pro Glu Thr Tyr Phe Thr Gln Ser Arg Asp Leu Glu Asp 2240 2245 2250Phe Lys Pro Arg Ser Gln Met Glu Thr Asp Phe Leu Glu Leu Ala 2255 2260 2265Met Asp Glu Phe Ile Gln Arg Tyr Lys Leu Glu Gly Tyr Ala Phe 2270 2275 2280Glu His Ile Val Tyr Gly Asp Phe Ser His Gly Gln Leu Gly Gly 2285 2290 2295Leu His Leu Met Ile Gly Leu Ala Lys Arg Ser Gln Asp Ser Pro 2300 2305 2310Leu Lys Leu Glu Asp Phe Ile Pro Met Asp Ser Thr Val Lys Asn 2315 2320 2325Tyr Phe Ile Thr Asp Ala Gln Thr Gly Ser Ser Lys Cys Val Cys 2330 2335 2340Ser Val Ile Asp Leu Leu Leu Asp Asp Phe Val Glu Ile Ile Lys 2345 2350 2355Ser Gln Asp Leu Ser Val Ile Ser Lys Val Val Lys Val Thr Ile 2360 2365 2370Asp Tyr Ala Glu Ile Ser Phe Met Leu Trp Cys Lys Asp Gly His 2375 2380 2385Val Glu Thr Phe Tyr Pro Lys Leu Gln Ala Ser Gln Ala Trp Gln 2390 2395 2400Pro Gly Val Ala Met Pro Asn Leu Tyr Lys Met Gln Arg Met Leu 2405 2410 2415Leu Glu Lys Cys Asp Leu Gln Asn Tyr Gly Glu Asn Ala Val Ile 2420 2425 2430Pro Lys Gly Ile Met Met Asn Val Ala Lys Tyr Thr Gln Leu Cys 2435 2440 2445Gln Tyr Leu Asn Thr Leu Thr Leu Ala Val Pro Tyr Asn Met Arg 2450 2455 2460Val Ile His Phe Gly Ala Gly Ser Asp Lys Gly Val Ala Pro Gly 2465 2470 2475Thr Ala Val Leu Arg Gln Trp Leu Pro Thr Gly Thr Leu Leu Val 2480 2485 2490Asp Ser Asp Leu Asn Asp Phe Val Ser Asp Ala Asp Ser Thr Leu 2495 2500 2505Ile Gly Asp Cys Ala Thr Val His Thr Ala Asn Lys Trp Asp Leu 2510 2515 2520Ile Ile Ser Asp Met Tyr Asp Pro Arg Thr Lys His Val Thr Lys 2525 2530 2535Glu Asn Asp Ser Lys Glu Gly Phe Phe Thr Tyr Leu Cys Gly Phe 2540 2545 2550Ile Lys Gln Lys Leu Ala Leu Gly Gly Ser Ile Ala Val Lys Ile 2555 2560 2565Thr Glu His Ser Trp Asn Ala Asp Leu Tyr Lys Leu Met Gly His 2570 2575 2580Phe Ser Trp Trp Thr Ala Phe Val Thr Asn Val Asn Ala Ser Ser 2585 2590 2595Ser Glu Ala Phe Leu Ile Gly Ala Asn Tyr Leu Gly Lys Pro Lys 2600 2605 2610Glu Gln Ile Asp Gly Tyr Thr Met His Ala Asn Tyr Ile Phe Trp 2615 2620 2625Arg Asn Thr Asn Pro Ile Gln Leu Ser Ser Tyr Ser Leu Phe Asp 2630 2635 2640Met Ser Lys Phe Pro Leu Lys Leu Arg Gly Thr Ala Val Met Ser 2645 2650 2655Leu Lys Glu Asn Gln Ile Asn Asp Met Ile Tyr Ser Leu Leu Glu 2660 2665 2670Lys Gly Arg Leu Ile Ile Arg Glu Asn Asn Arg Val Val Val Ser 2675 2680 2685Ser Asp Ile Leu Val Asn Asn 2690 269541255PRTCoronavirus 4Met Phe Ile Phe Leu Leu Phe Leu Thr Leu Thr Ser Gly Ser Asp Leu1 5 10 15Asp Arg Cys Thr Thr Phe Asp Asp Val Gln Ala Pro Asn Tyr Thr Gln 20 25 30His Thr Ser Ser Met Arg Gly Val Tyr Tyr Pro Asp Glu Ile Phe Arg 35 40 45Ser Asp Thr Leu Tyr Leu Thr Gln Asp Leu Phe Leu Pro Phe Tyr Ser 50 55 60Asn Val Thr Gly Phe His Thr Ile Asn His Thr Phe Gly Asn Pro Val65 70 75 80Ile Pro Phe Lys Asp Gly Ile Tyr Phe Ala Ala Thr Glu Lys Ser Asn 85 90 95Val Val Arg Gly Trp Val Phe Gly Ser Thr Met Asn Asn Lys Ser Gln 100 105 110Ser Val Ile Ile Ile Asn Asn Ser Thr Asn Val Val Ile Arg Ala Cys 115 120 125Asn Phe Glu Leu Cys Asp Asn Pro Phe Phe Ala Val Ser Lys Pro Met 130 135 140Gly Thr Gln Thr His Thr Met Ile Phe Asp Asn Ala Phe Asn Cys Thr145 150 155 160Phe Glu Tyr Ile Ser Asp Ala Phe Ser Leu Asp Val Ser Glu Lys Ser 165 170 175Gly Asn Phe Lys His Leu Arg Glu Phe Val Phe Lys Asn Lys Asp Gly 180 185 190Phe Leu Tyr Val Tyr Lys Gly Tyr Gln Pro Ile Asp Val Val Arg Asp 195 200 205Leu Pro Ser Gly Phe Asn Thr Leu Lys Pro Ile Phe Lys Leu Pro Leu 210 215 220Gly Ile Asn Ile Thr Asn Phe Arg Ala Ile Leu Thr Ala Phe Ser Pro225 230 235 240Ala Gln Asp Ile Trp Gly Thr Ser Ala Ala Ala Tyr Phe Val Gly Tyr 245 250 255Leu Lys Pro Thr Thr Phe Met Leu Lys Tyr Asp Glu Asn Gly Thr Ile 260 265 270Thr Asp Ala Val Asp Cys Ser Gln Asn Pro Leu Ala Glu Leu Lys Cys 275 280 285Ser Val Lys Ser Phe Glu Ile Asp Lys Gly Ile Tyr Gln Thr Ser Asn 290 295 300Phe Arg Val Val Pro Ser Gly Asp

Val Val Arg Phe Pro Asn Ile Thr305 310 315 320Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Lys Phe Pro Ser 325 330 335Val Tyr Ala Trp Glu Arg Lys Lys Ile Ser Asn Cys Val Ala Asp Tyr 340 345 350Ser Val Leu Tyr Asn Ser Thr Phe Phe Ser Thr Phe Lys Cys Tyr Gly 355 360 365Val Ser Ala Thr Lys Leu Asn Asp Leu Cys Phe Ser Asn Val Tyr Ala 370 375 380Asp Ser Phe Val Val Lys Gly Asp Asp Val Arg Gln Ile Ala Pro Gly385 390 395 400Gln Thr Gly Val Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe 405 410 415Met Gly Cys Val Leu Ala Trp Asn Thr Arg Asn Ile Asp Ala Thr Ser 420 425 430Thr Gly Asn Tyr Asn Tyr Lys Tyr Arg Tyr Leu Arg His Gly Lys Leu 435 440 445Arg Pro Phe Glu Arg Asp Ile Ser Asn Val Pro Phe Ser Pro Asp Gly 450 455 460Lys Pro Cys Thr Pro Pro Ala Leu Asn Cys Tyr Trp Pro Leu Asn Asp465 470 475 480Tyr Gly Phe Tyr Thr Thr Thr Gly Ile Gly Tyr Gln Pro Tyr Arg Val 485 490 495Val Val Leu Ser Phe Glu Leu Leu Asn Ala Pro Ala Thr Val Cys Gly 500 505 510Pro Lys Leu Ser Thr Asp Leu Ile Lys Asn Gln Cys Val Asn Phe Asn 515 520 525Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Pro Ser Ser Lys Arg 530 535 540Phe Gln Pro Phe Gln Gln Phe Gly Arg Asp Val Ser Asp Phe Thr Asp545 550 555 560Ser Val Arg Asp Pro Lys Thr Ser Glu Ile Leu Asp Ile Ser Pro Cys 565 570 575Ser Phe Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Ala Ser Ser 580 585 590Glu Val Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Asp Val Ser Thr 595 600 605Ala Ile His Ala Asp Gln Leu Thr Pro Ala Trp Arg Ile Tyr Ser Thr 610 615 620Gly Asn Asn Val Phe Gln Thr Gln Ala Gly Cys Leu Ile Gly Ala Glu625 630 635 640His Val Asp Thr Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile 645 650 655Cys Ala Ser Tyr His Thr Val Ser Leu Leu Arg Ser Thr Ser Gln Lys 660 665 670Ser Ile Val Ala Tyr Thr Met Ser Leu Gly Ala Asp Ser Ser Ile Ala 675 680 685Tyr Ser Asn Asn Thr Ile Ala Ile Pro Thr Asn Phe Ser Ile Ser Ile 690 695 700Thr Thr Glu Val Met Pro Val Ser Met Ala Lys Thr Ser Val Asp Cys705 710 715 720Asn Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys Ala Asn Leu Leu Leu 725 730 735Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg Ala Leu Ser Gly Ile 740 745 750Ala Ala Glu Gln Asp Arg Asn Thr Arg Glu Val Phe Ala Gln Val Lys 755 760 765Gln Met Tyr Lys Thr Pro Thr Leu Lys Tyr Phe Gly Gly Phe Asn Phe 770 775 780Ser Gln Ile Leu Pro Asp Pro Leu Lys Pro Thr Lys Arg Ser Phe Ile785 790 795 800Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Ala Asp Ala Gly Phe Met 805 810 815Lys Gln Tyr Gly Glu Cys Leu Gly Asp Ile Asn Ala Arg Asp Leu Ile 820 825 830Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu Pro Pro Leu Leu Thr 835 840 845Asp Asp Met Ile Ala Ala Tyr Thr Ala Ala Leu Val Ser Gly Thr Ala 850 855 860Thr Ala Gly Trp Thr Phe Gly Ala Gly Ala Ala Leu Gln Ile Pro Phe865 870 875 880Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile Gly Val Thr Gln Asn 885 890 895Val Leu Tyr Glu Asn Gln Lys Gln Ile Ala Asn Gln Phe Asn Lys Ala 900 905 910Ile Ser Gln Ile Gln Glu Ser Leu Thr Thr Thr Ser Thr Ala Leu Gly 915 920 925Lys Leu Gln Asp Val Val Asn Gln Asn Ala Gln Ala Leu Asn Thr Leu 930 935 940Val Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile Ser Ser Val Leu Asn945 950 955 960Asp Ile Leu Ser Arg Leu Asp Lys Val Glu Ala Glu Val Gln Ile Asp 965 970 975Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln Thr Tyr Val Thr Gln 980 985 990Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser Ala Asn Leu Ala Ala 995 1000 1005Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser Lys Arg Val Asp 1010 1015 1020Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro Gln Ala Ala 1025 1030 1035Pro His Gly Val Val Phe Leu His Val Thr Tyr Val Pro Ser Gln 1040 1045 1050Glu Arg Asn Phe Thr Thr Ala Pro Ala Ile Cys His Glu Gly Lys 1055 1060 1065Ala Tyr Phe Pro Arg Glu Gly Val Phe Val Phe Asn Gly Thr Ser 1070 1075 1080Trp Phe Ile Thr Gln Arg Asn Phe Phe Ser Pro Gln Ile Ile Thr 1085 1090 1095Thr Asp Asn Thr Phe Val Ser Gly Asn Cys Asp Val Val Ile Gly 1100 1105 1110Ile Ile Asn Asn Thr Val Tyr Asp Pro Leu Gln Pro Glu Leu Asp 1115 1120 1125Ser Phe Lys Glu Glu Leu Asp Lys Tyr Phe Lys Asn His Thr Ser 1130 1135 1140Pro Asp Val Asp Leu Gly Asp Ile Ser Gly Ile Asn Ala Ser Val 1145 1150 1155Val Asn Ile Gln Lys Glu Ile Asp Arg Leu Asn Glu Val Ala Lys 1160 1165 1170Asn Leu Asn Glu Ser Leu Ile Asp Leu Gln Glu Leu Gly Lys Tyr 1175 1180 1185Glu Gln Tyr Ile Lys Trp Pro Trp Tyr Val Trp Leu Gly Phe Ile 1190 1195 1200Ala Gly Leu Ile Ala Ile Val Met Val Thr Ile Leu Leu Cys Cys 1205 1210 1215Met Thr Ser Cys Cys Ser Cys Leu Lys Gly Ala Cys Ser Cys Gly 1220 1225 1230Ser Cys Cys Lys Phe Asp Glu Asp Asp Ser Glu Pro Val Leu Lys 1235 1240 1245Gly Val Lys Leu His Tyr Thr 1250 12555274PRTCoronavirus 5Met Asp Leu Phe Met Arg Phe Phe Thr Leu Gly Ser Ile Thr Ala Gln1 5 10 15Pro Val Lys Ile Asp Asn Ala Ser Pro Ala Ser Thr Val His Ala Thr 20 25 30Ala Thr Ile Pro Leu Gln Ala Ser Leu Pro Phe Gly Trp Leu Val Ile 35 40 45Gly Val Ala Phe Leu Ala Val Phe Gln Ser Ala Thr Lys Ile Ile Ala 50 55 60Leu Asn Lys Arg Trp Gln Leu Ala Leu Tyr Lys Gly Phe Gln Phe Ile65 70 75 80Cys Asn Leu Leu Leu Leu Phe Val Thr Ile Tyr Ser His Leu Leu Leu 85 90 95Val Ala Ala Gly Met Glu Ala Gln Phe Leu Tyr Leu Tyr Ala Leu Ile 100 105 110Tyr Phe Leu Gln Cys Ile Asn Ala Cys Arg Ile Ile Met Arg Cys Trp 115 120 125Leu Cys Trp Lys Cys Lys Ser Lys Asn Pro Leu Leu Tyr Asp Ala Asn 130 135 140Tyr Phe Val Cys Trp His Thr His Asn Tyr Asp Tyr Cys Ile Pro Tyr145 150 155 160Asn Ser Val Thr Asp Thr Ile Val Val Thr Glu Gly Asp Gly Ile Ser 165 170 175Thr Pro Lys Leu Lys Glu Asp Tyr Gln Ile Gly Gly Tyr Ser Glu Asp 180 185 190Arg His Ser Gly Val Lys Asp Tyr Val Val Val His Gly Tyr Phe Thr 195 200 205Glu Val Tyr Tyr Gln Leu Glu Ser Thr Gln Ile Thr Thr Asp Thr Gly 210 215 220Ile Glu Asn Ala Thr Phe Phe Ile Phe Asn Lys Leu Val Lys Asp Pro225 230 235 240Pro Asn Val Gln Ile His Thr Ile Asp Gly Ser Ser Gly Val Ala Asn 245 250 255Pro Ala Met Asp Pro Ile Tyr Asp Glu Pro Thr Thr Thr Thr Ser Val 260 265 270Pro Leu6154PRTCoronavirus 6Met Met Pro Thr Thr Leu Phe Ala Gly Thr His Ile Thr Met Thr Thr1 5 10 15Val Tyr His Ile Thr Val Ser Gln Ile Gln Leu Ser Leu Leu Lys Val 20 25 30Thr Ala Phe Gln His Gln Asn Ser Lys Lys Thr Thr Lys Leu Val Val 35 40 45Ile Leu Arg Ile Gly Thr Gln Val Leu Lys Thr Met Ser Leu Tyr Met 50 55 60Ala Ile Ser Pro Lys Phe Thr Thr Ser Leu Ser Leu His Lys Leu Leu65 70 75 80Gln Thr Leu Val Leu Lys Met Leu His Ser Ser Ser Leu Thr Ser Leu 85 90 95Leu Lys Thr His Arg Met Cys Lys Tyr Thr Gln Ser Thr Ala Leu Gln 100 105 110Glu Leu Leu Ile Gln Gln Trp Ile Gln Phe Met Met Ser Arg Arg Arg 115 120 125Leu Leu Ala Cys Leu Cys Lys His Lys Lys Val Ser Thr Asn Leu Cys 130 135 140Thr His Ser Phe Arg Lys Lys Gln Val Arg145 150776PRTCoronavirus 7Met Tyr Ser Phe Val Ser Glu Glu Thr Gly Thr Leu Ile Val Asn Ser1 5 10 15Val Leu Leu Phe Leu Ala Phe Val Val Phe Leu Leu Val Thr Leu Ala 20 25 30Ile Leu Thr Ala Leu Arg Leu Cys Ala Tyr Cys Cys Asn Ile Val Asn 35 40 45Val Ser Leu Val Lys Pro Thr Val Tyr Val Tyr Ser Arg Val Lys Asn 50 55 60Leu Asn Ser Ser Glu Gly Val Pro Asp Leu Leu Val65 70 758221PRTCoronavirus 8Met Ala Asp Asn Gly Thr Ile Thr Val Glu Glu Leu Lys Gln Leu Leu1 5 10 15Glu Gln Trp Asn Leu Val Ile Gly Phe Leu Phe Leu Ala Trp Ile Met 20 25 30Leu Leu Gln Phe Ala Tyr Ser Asn Arg Asn Arg Phe Leu Tyr Ile Ile 35 40 45Lys Leu Val Phe Leu Trp Leu Leu Trp Pro Val Thr Leu Ala Cys Phe 50 55 60Val Leu Ala Ala Val Tyr Arg Ile Asn Trp Val Thr Gly Gly Ile Ala65 70 75 80Ile Ala Met Ala Cys Ile Val Gly Leu Met Trp Leu Ser Tyr Phe Val 85 90 95Ala Ser Phe Arg Leu Phe Ala Arg Thr Arg Ser Met Trp Ser Phe Asn 100 105 110Pro Glu Thr Asn Ile Leu Leu Asn Val Pro Leu Arg Gly Thr Ile Val 115 120 125Thr Arg Pro Leu Met Glu Ser Glu Leu Val Ile Gly Ala Val Ile Ile 130 135 140Arg Gly His Leu Arg Met Ala Gly His Pro Leu Gly Arg Cys Asp Ile145 150 155 160Lys Asp Leu Pro Lys Glu Ile Thr Val Ala Thr Ser Arg Thr Leu Ser 165 170 175Tyr Tyr Lys Leu Gly Ala Ser Gln Arg Val Gly Thr Asp Ser Gly Phe 180 185 190Ala Ala Tyr Asn Arg Tyr Arg Ile Gly Asn Tyr Lys Leu Asn Thr Asp 195 200 205His Ala Gly Ser Asn Asp Asn Ile Ala Leu Leu Val Gln 210 215 220963PRTCoronavirus 9Met Phe His Leu Val Asp Phe Gln Val Thr Ile Ala Glu Ile Leu Ile1 5 10 15Ile Ile Met Arg Thr Phe Arg Ile Ala Ile Trp Asn Leu Asp Val Ile 20 25 30Ile Ser Ser Ile Val Arg Gln Leu Phe Lys Pro Leu Thr Lys Lys Asn 35 40 45Tyr Ser Glu Leu Asp Asp Glu Glu Pro Met Glu Leu Asp Tyr Pro 50 55 6010122PRTCoronavirus 10Met Lys Ile Ile Leu Phe Leu Thr Leu Ile Val Phe Thr Ser Cys Glu1 5 10 15Leu Tyr His Tyr Gln Glu Cys Val Arg Gly Thr Thr Val Leu Leu Lys 20 25 30Glu Pro Cys Pro Ser Gly Thr Tyr Glu Gly Asn Ser Pro Phe His Pro 35 40 45Leu Ala Asp Asn Lys Phe Ala Leu Thr Cys Thr Ser Thr His Phe Ala 50 55 60Phe Ala Cys Ala Asp Gly Thr Arg His Thr Tyr Gln Leu Arg Ala Arg65 70 75 80Ser Val Ser Pro Lys Leu Phe Ile Arg Gln Glu Glu Val Gln Gln Glu 85 90 95Leu Tyr Ser Pro Leu Phe Leu Ile Val Ala Ala Leu Val Phe Leu Ile 100 105 110Leu Cys Phe Thr Ile Lys Arg Lys Thr Glu 115 1201184PRTCoronavirus 11Met Cys Leu Lys Ile Leu Val Arg Tyr Asn Thr Arg Gly Asn Thr Tyr1 5 10 15Ser Thr Ala Trp Leu Cys Ala Leu Gly Lys Val Leu Pro Phe His Arg 20 25 30Trp His Thr Met Val Gln Thr Cys Thr Pro Asn Val Thr Ile Asn Cys 35 40 45Gln Asp Pro Ala Gly Gly Ala Leu Ile Ala Arg Cys Trp Tyr Leu His 50 55 60Glu Gly His Gln Thr Ala Ala Phe Arg Asp Val Leu Val Val Leu Asn65 70 75 80Lys Arg Thr Asn12422PRTCoronavirus 12Met Ser Asp Asn Gly Pro Gln Ser Asn Gln Arg Ser Ala Pro Arg Ile1 5 10 15Thr Phe Gly Gly Pro Thr Asp Ser Thr Asp Asn Asn Gln Asn Gly Gly 20 25 30Arg Asn Gly Ala Arg Pro Lys Gln Arg Arg Pro Gln Gly Leu Pro Asn 35 40 45Asn Thr Ala Ser Trp Phe Thr Ala Leu Thr Gln His Gly Lys Glu Glu 50 55 60Leu Arg Phe Pro Arg Gly Gln Gly Val Pro Ile Asn Thr Asn Ser Gly65 70 75 80Pro Asp Asp Gln Ile Gly Tyr Tyr Arg Arg Ala Thr Arg Arg Val Arg 85 90 95Gly Gly Asp Gly Lys Met Lys Glu Leu Ser Pro Arg Trp Tyr Phe Tyr 100 105 110Tyr Leu Gly Thr Gly Pro Glu Ala Ser Leu Pro Tyr Gly Ala Asn Lys 115 120 125Glu Gly Ile Val Trp Val Ala Thr Glu Gly Ala Leu Asn Thr Pro Lys 130 135 140Asp His Ile Gly Thr Arg Asn Pro Asn Asn Asn Ala Ala Thr Val Leu145 150 155 160Gln Leu Pro Gln Gly Thr Thr Leu Pro Lys Gly Phe Tyr Ala Glu Gly 165 170 175Ser Arg Gly Gly Ser Gln Ala Ser Ser Arg Ser Ser Ser Arg Ser Arg 180 185 190Gly Asn Ser Arg Asn Ser Thr Pro Gly Ser Ser Arg Gly Asn Ser Pro 195 200 205Ala Arg Met Ala Ser Gly Gly Gly Glu Thr Ala Leu Ala Leu Leu Leu 210 215 220Leu Asp Arg Leu Asn Gln Leu Glu Ser Lys Val Ser Gly Lys Gly Gln225 230 235 240Gln Gln Gln Gly Gln Thr Val Thr Lys Lys Ser Ala Ala Glu Ala Ser 245 250 255Lys Lys Pro Arg Gln Lys Arg Thr Ala Thr Lys Gln Tyr Asn Val Thr 260 265 270Gln Ala Phe Gly Arg Arg Gly Pro Glu Gln Thr Gln Gly Asn Phe Gly 275 280 285Asp Gln Asp Leu Ile Arg Gln Gly Thr Asp Tyr Lys His Trp Pro Gln 290 295 300Ile Ala Gln Phe Ala Pro Ser Ala Ser Ala Phe Phe Gly Met Ser Arg305 310 315 320Ile Gly Met Glu Val Thr Pro Ser Gly Thr Trp Leu Thr Tyr His Gly 325 330 335Ala Ile Lys Leu Asp Asp Lys Asp Pro Gln Phe Lys Asp Asn Val Ile 340 345 350Leu Leu Asn Lys His Ile Asp Ala Tyr Lys Thr Phe Pro Pro Thr Glu 355 360 365Pro Lys Lys Asp Lys Lys Lys Lys Thr Asp Glu Ala Gln Pro Leu Pro 370 375 380Gln Arg Gln Lys Lys Gln Pro Thr Val Thr Leu Leu Pro Ala Ala Asp385 390 395 400Met Asp Asp Phe Ser Arg Gln Leu Gln Asn Ser Met Ser Gly Ala Ser 405 410 415Ala Asp Ser Thr Gln Ala 4201321DNAArtificial SequenceSynthetic oligonucleotide. 13ctaacatgct taggataatg g 211421DNAArtificial SequenceSynthetic oligonucleotide. 14gcctctcttg ttcttgctcg c 211521DNAArtificial SequenceSynthetic oligonucleotide. 15caggtaagcg taaaactcat c 211622DNAArtificial SequenceSynthetic oligonucleotide. 16catgtgtggc ggctcactat at 221728DNAArtificial SequenceSynthetic oligonucleotide. 17gacactatta gcataagcag ttgtagca

281827DNAArtificial SequenceSynthetic oligonucleotide. 18ttaaaccagg tggaacatca tccggtg 271922DNAArtificial SequenceSynthetic oligonucleotide. 19ggagccttga atacacccaa ag 222017DNAArtificial SequenceSynthetic oligonucleotide. 20gcacggtggc agcattg 172121DNAArtificial SequenceSynthetic oligonucleotide. 21ccacattggc acccgcaatc c 212219DNAArtificial SequenceSynthetic oligonucleotide. 22caaacattgg ccgcaaatt 192321DNAArtificial SequenceSynthetic oligonucleotide. 23caatgcgtga cattccaaag a 212426DNAArtificial SequenceSynthetic oligonucleotide. 24cacaatttgc tccaagtgcc tctgca 262521DNAArtificial SequenceSynthetic oligonucleotide. 25gaagtaccat ctggggctga g 212618DNAArtificial SequenceSynthetic oligonucleotide. 26ccgaagagct acccgacg 182726DNAArtificial SequenceSynthetic oligonucleotide. 27ctctttcatt ttgccgtcac caccac 262824DNAArtificial SequenceSynthetic oligonucleotide. 28agctctccct agcattattc actg 242920DNAArtificial SequenceSynthetic oligonucleotide. 29caccacattt tcatcgaggc 203022DNAArtificial SequenceSynthetic oligonucleotide. 30taccctcgat cgtactccgc gt 223123DNAArtificial SequenceSynthetic oligonucleotide. 31tgtaggcact gattcaggtt ttg 233223DNAArtificial SequenceSynthetic oligonucleotide. 32cggcgtggtc tgtatttaat tta 233327DNAArtificial SequenceSynthetic oligonucleotide. 33ctgcatacaa ccgctaccgt attggaa 273424DNAArtificial SequenceSynthetic oligonucleotide. 34gggttgggac tatcctaagt gtga 243521DNAArtificial SequenceSynthetic oligonucleotide. 35taacacacaa cnccatcatc a 213619DNAArtificial SequenceSynthetic oligonucleotide. 36agatttggac ctgcgagcg 193720DNAArtificial SequenceSynthetic oligonucleotide. 37gagcggctgt ctccacaagt 203823DNAArtificial SequenceSynthetic oligonucleotide. 38ttctgacctg aaggctctgc gcg 23

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