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    US7220852B1 Original Document 20200321042732

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    franklin gaona

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    © All Rights Reserved
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    (2) United States Patent Rota et a US007220852B1 US 7,220,852 BL May 22, 2007 (a0) Patent No.: 4s) Date of Patent: (58) 0s) wm @ @ (oo) on) (2) (58) 650) 20050181357 CORONAVIR ISOLATED FROM HUMANS Inventors: Paul A. Rota, Decatur, GA (US): Larry J. Anderson, Atlanta, GA (US); William J. Bellini, [ilbura, GA (US) Cara Carthel Burns, Avondale Esta GA(US); Raymond Campagnoli Decatur, GA (US); Qi Chen, Marietta, smery, Lusaka (ZN); Dean D. Erdman, Decatur, GA (US); Cynthia S. Goldsmith, Litburn GA (US); Charles D. Humphrey Lilburn, GA (US); Joseph P. Teenogle Aalanta, GA (US): Thomas G. Ksiazek, Lithurn, GA (US); Stephan S. Monroe Decatur, GA (US); William Allan Nix, elehem, GA (US); M. Steven Oberste, Lilburn, GA (US); Teresa C, T. Peret, Atlanta, Ga (US); Pierre Rollin, Lilburn, GA (US); Mark A. Pallansch, Lilburn, GA (US); Anthony Sanchez, Lilburn, GA (US); Susan Tong, Alpharetta, GA (US); Sherif R. Zaki, Atlanta, GA (US) 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 (US) Notice: Subject to any disclaimer, the term of this patent is extended or adjusted under 35 USC. 154(b) by 0 days. Appl. No. 10/822,904 Filed: Apr. 12, 2004 Related U.S. Application Data Provisional application No. 604465.9 25, 2008 27 filed on Ape: Int. Cl CRN 15/50 CRN 700 us. cl, (2006.01) (2006.01) ‘§36/28.72; 435/235.1 Pleld of Classification Seareh 536/28.72 S14/48; 435/235.1, 320.1 See application file For complete search history. References Cited US. PATENT DOCUMENTS aus 2005 Pisa ass FOREIGN PATENT DOCUMENTS + 102004 + 102004 wo wo Wo 2004088603 Wo 20081002360, (OTHER PUBLICATIONS, GenBank Accession No. AYITALID, “SARS Coronavinws To? somplete genome,” version AY2741I9.1, Ape M2003 GenBank Accession No. AYITSIN7, “SARS corontvis BID2 patil gnome,” version AV278487, Ape. 21 2003. Genbank Accession No. AY2788S4, “SARS coronasinus CUHK: WI, complete genome.” version AV2TS5S4.1, Ape. 18,2003" GenBank Accession No, AYZTS491, “SARS Coronavins HKU 49, compte genome,” vrsion AVZ7E491.1 Ap. 18,2003 * SARScastociated Coromavinis Genomic Sequence Availability, [online freeived on Aug. 8, 2005) Retival from the Tiere URL: hp nw begs bioinfoSARS> * GenBank Accession No. AY2TAII9, Apr. 14, 2003, GGenliank Accession No. AY2TS741, Apr 21, 2003 inate Outreak of Severe Acute Respiratory Synome—Workvie, 2003 MAIR Weoty52-241-248 (2003, Emery et al, "Real-Time Reverse Transrption-Polymerase Chain Reaction Assay for SARS-Assocatod Coronavinus" Emer, Inet. Diseases 10311316 (2008) Goldsmith ot al, "Ukrasuctural Characterization of SARS CCononvinas” Emer Inet Diseases 10320-926 (2004), Kainz etal, “A Novel Coronvins Associated with Severe Acute Respiratory Syndrome.” N Brg J Med. 348:1983-1966 2003), [Luo and Lo, “Intl SARS Coronavinus Genome Sequence Avaly sis Usinga Biinfomatios Pltforn,” 7 Ase-Pacie Biinermat ‘ex Confrence (APBC2003), Dunetin, New Zealand (200) Marra etal, “The Genome Sequence of the SARS-Assoiated Corsini” Sconce 300-139-1404 (203). Supplementary Online Material for Mara ct al sciencemag ori conta ill 108S953/DCI>>, Rota tal, “Characterization of a Novel Coronavnis Associated wth Severe Acute Respiratory Syadrome," Science 300 13341399 (2009), Supplementary Online Material for Rota etal, >, * cited by examiner Primary Examiner Mary E. Mosher (74) Attorney, Agent, or Firm—Klaeguist Sparkman LLP on ABSTRACT Disclosed herein is a newly isolated human coronavirus (SARS-CoV), the causative agent of severe acute respiratory syncrome (SARS). Also provided are the nvcleic acid sequence of the SARS-CoV genome and the amino acid sequences of the SARS-CoV open reading frames, as well as ricthods of using these molecules to detect a SARS-CoV ‘and detet infections therewith. Immune stimulstory com: positions are also provided, along with methods of their use 1 Claim, 7 Drawing Sheets U.S. Patent May 22,2007 Sheet 1 of 7 US 7,220,852 BI U.S. Patent May 22,2007 Sheet 2 of 7 US 7,220,852 BI U.S. Patent May 22,2007 Sheet 3 of 7 US 7,220,852 BI SARS Cov SN BO } 10 nt US. Patent May 22,2007 Sheet 4 of 7 US 7,220,852 B1 REDV ce HOoV.220 SARS-CoV G2 aE HeveCov FIG. 4 US 7,220,852 B1 Sheet 5 of 7 May 22, 2007 U.S. Patent U.S. Patent May 22,2007 Sheet 6 of 7 US 7,220,852 BI FIG. 7A FIG. 7B 20001 25,000 30,0 .3kb quayed “S' L007 77 Ae LJ 1 yayg Ta 7S8'077°L SA US 7,220,852 BI 1 ‘CORONAVIRUS ISOLATED FROM HUMANS PRIORITY CLAIM, ‘This application claims the benefit of U.S. Provisional Patent Application No. 601465,927 filed Apr. 25, 2008, ‘which is incorporated herein by reference in its entirety. 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 US. Government has certain rights inthis invention FIELD OF THE DISCLOSURE ‘This invention relates to @ newly isolated human coro- navinus, More paniculary, it relates to an isolated coranavi- ris genome, isolated coronavirus proteins, and isolated 2 rueleie acid molecules encoding the same, The disclosure farther relates to methods of detecting a severe acute respi- ratory syadrome-sssociated coronavirus and compositions ‘comprising immunogenic coronsvires compounds. BACKGROUND, ‘The coronaviruses (onder Nidovirales, family Coronavir- dae, genus Coronavirus) area diverse group of lage, evel- ‘oped, postive-stranded RNA viruses that cause respiratory and enteric diseases in. humans and other animals. At ‘approximately 30,000 aucletides (at), their genome isthe largest found in any ofthe RNA viruses. Coronaviruses are spherical, 100-160 nz in diameter with 20-40 am complex club shaped surface projections surounding the periphery. ‘Cononaviruses share common structural proteins ineluding 8 spike protein (S), membrane protein (M), envelope protein (Bj, and, in a subset of coronaviruses, a hemagautinin- ‘esterase protein (HE), The S protein, a glycoprotein which protrudes from the virus membrane, is involved in host cell receptor binding and isa target for neutralizing antibodies. ‘The E and M proteins ae involved in virion formation and release from the host cell. Coronavirus particles are found ‘within the cisternae ofthe rough endoplasmic reticulum and in vesicles of infected host cells where virions are assembled, The coronavirus genome consists of two open reading frames (ORFIa and ORFIb) yielding an RNA. polymerase anda nested set of subgenomic mRNAs encod- ing srctural and nonstructural proteins, including the 8, E, M, and nucleocapsid (N) proteins. The genus Coronavirus inchudes at least 13 species which have been subdivided into ‘atleast three groups (groups I, I, and I} on the basis of seeological and genetic properties (deVries etal, Sem, Fro 8:33-47, 1997; Fieks etal. eds. Fields Frology, 3d edition, Raven Press, Philadelphia, 1323-1341, 1996: Mabey and Collier eds. Microbiology and Microbial Infections, Volume 1 Virology, 9 eaition, 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 fmaman Coro havinus 229E, (HCOV-229E), canine coronavirus (CCoV), feline infectious peritonitis vir (FIPV), porcine trans sible gastroenteritis virus (TGEV), porcine epidemic diat- thea vis (PEDV), human cormavinis OCA3. (HeoV- ” © 2 0C43), bovine coronavirus (BCoV), porcine hemagglutinating encephlomyelitis vius (HEV), rat sialo- dacryoadenits ira (SDAV), mouse hepatitis virus (MEV), turkey comnavirus (TCoV), and avian infetious bronchitis virus (IBV-Avian) (Fields etal, eds. Fields Virology, 3rd edition, Raven Press, Philadelphia, 1323-1341, 1996; Mahey and Collier eds. Microbiology and Microbial Infec- tions, Volume 1 Virology, 9 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 tropisn; infection inthe ineor- rect host species or tissue type may result in an abortive infection, mutant vitus production and altered virulence. Coronaviruses generally do not grow well in call culture, ‘nd animal models for human cononavinis infection are Jacking. Therefore ite is known about them (Fields et al cis. Fields Virology, 3rd eition, Raven Press, Philadelphia, 1323-1341, 1996). The known human coronaviruses are notably fastidious in cell elture, preferring select cell ines, ‘omgan culture, or suckling mice for propagation, Coronavi- rises grown. in cell culture exhibit varying degrees of virulence andor cytopathic eect (CPE) depending on the host eell type and culture conditions. The only human oF animal coronavimos which has been shown to grow in Vero 6 cells is PEDY, and it requires the ation of trypsin to culture medium for growth in Vero E6 cells. Moreover, PEDY adapted to Vero B6 cell culture results ina strikingly different CPE, with eytoplasmic vacuoles andthe formation of large syneytia (Hofmann and. Wyler, J. Clin, Micro 26.2235-39, 1988; Kusanagi et el, J. er. Med. Se. $54 313-18, 1991). Coronavirus have not previously been known 10 cause severe disease in humans, but have been identified as a major cause of upper respiratory tract ilness, including the common cold. Repeat infections in humans are common within and across serotype, suggesting that immune response to coronavins infection in humans is either income plete or short lived, Coronavirs infection in animals can cause severe enteric or respiratory disease. Vaccination has been used succesfilly to prevent and control some caro- navins infections in animals. The ability of animal-specitic coronavimises to cause severe disease rises the possibilty that conmavinis could also cause more severe disease in hhumans Fields etal. es. Fields Vrofogy: 3 edition, Raven Press, Philadelphia, 1323-1341, 1996; Mahey and Collier cds. Microbiology and Microbial Infections, Volume 1 Viol cay, 9 edition, Oxford University Press, 463-479, 1998) In late 2002, cases of life-threatening respiratory disease ‘ith no identifiable etiology were reported from Guangsong Province, China, followed by reports from Vietnam, Canada, and Hong Kong of severe febrile respiratory illness that spread to houschold members and healthcare workers. The symdrome was designated “severe acute respiratory syn- drome” (SARS) in February 2003 by the Centers for Disease Control and Prevention (MMIVR, 52:241-48, 2003), Past efforts to develop rapid diagnostics and vaccines for coronavimis infection in hamans have been hampered by @ lack of appropriate research modes and the moderate course of disease in humans. Therefore, a need for rapid diagnos tests and vaovines exits SUMMARY OF THE DISCLOSURE A newly isolated human coronavirus has been identified as the causative agent of SARS, and is termed SARS-CoV. US 7,220,852 BI 7 ‘The mucleie acid sequence of the SARS-CoV genome and the amino aeid sequences of the SARS-CoV open reading frames are provided herein ‘This disclosure provides methods and compositions use- ful in detecting the presence of a SARS-CoV’ mucleie aid in ‘2 sample andlor 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 ant body ina sample andor diagnosing a SARS-CoV infection ina subject. ‘The foregoing and other features and advantages will become more apparent from the following deailed deserip- tion of several embodiments, which proceeds with relerence to the accompanying figures. BRIEF DESCRIPTION OF THE FIGURES FIGS. 14-B are photomicrographs illustrating typical carly cytopathic effects seen with coronavirus isolates und Serum om SARS patents FIG. 1A a photomicrograph of Vero £6 cells inoculated with an oropharyngeal specimen fom a SARS patient (40). FIG. 1B i a photomicrograph of infected Vero 6 cells reacting with the serum of a ‘convalescent SARS patient in an indirect fuorescentanti- body (IPA) assay (4400), FIGS. 24-1 are electroamicrographs illustrating whim structural characteristics of the SARS-associated coranavi- ns (SARS-CoV), FIG. 2A isa thin-section elecron-micro- scopical view of viral nucleocapsids aligned along the membrane of the rough endoplasmic reticulum (arrow) as particles bud into the cistemse. Enveloped virions have surface projections (arrowhead) and an electron-ucent cen- ter, Dircelly under the viral eavelope lies a characteristic ring fommed hy the helical nucleocapsid, often seen in cross-section. FIG, 2B is a negative stain (methylamine ‘ungstate) electronmierograph showing. stain-penetrated ‘coronavirus particle with the typical internal helical nucleo- ‘capsic-like strvcture and club-shaped surfice projections surrounding the periphery ofthe particle. Bars: 100 nm. FIG. 3 isan estimated maximum parsimony toe illustrat. ing putative phylogenetic relationships between SARS-CoV ‘and other human and animal coronaviruses. Phylogenetic relationships are based on sequence alignment of 405 nucle- ‘tides of the coronavirus polymerase gene ORF (aucleie acid 15,173 to 13,578 of SEQ ID NO: 1. The three major ‘coronavirus antigenic groups (I, Il and I}, represented by HeoV-229E, CCoV, FIPV, TRV, PEDV, HeoV-0C43, BCoV, HEV, SDAV, MEV, TCoV, and IBV-Avian, are shown shaded. Bootstrap values (100 replicates) obtained fiom a 50% majority rule consensus tre are plotted at the ‘main internal branches of the phylogram, Branch lengths are proportionate to nucleotide differences, FIG. 4 isa pictorial representation of neighbor joining trees illustating putative phylogenetic relationships berwoen SARS-CoV’ and other human and animal corona ruses, Amino acid sequences of the indicated SARS-CoV proteins were compared with thse from reference viruses representing each species in the three groups of coronavi- ruses for which complete genomic sequence information was available [group 1: HCoV-229E (AF304860}; PEDV (AF3S3511); TGEV (43271965); group 2: BCoV (AF220295); MH (AF201929); group 3: infectious bron- hits 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: CoV (D13096), FCoV_ (AY204704); porcine respiratory coronavirus (224675), group 2: HCoV-OCHS (M76373,_ 114648, 1M93390); HEV (AYO78417); rat coronavirus (AF207551)) Sequence alignments and neighbor joining trees were gen- x0 ” 4s so © 4 erated by using Clasalx 1.83 with the Gonnet protsin comparison matrix. The resting trees were adjusted for final opt using teetol 2.04 FIGS, 84-C are photomicrographs illuseaing diuse alveolar dasage ina patient with SARS (FIGS, §4-B) and ‘umunohistochemical sini of SARS-CoV dnfectd Vero 6 cells (FIG. 8C), FIG. 8A i 2 photomicrograph of hung tissue fom a SARS patent e80), Difse alveolar damsge, sundant foamy macrophages and molinucleated syncytial cells are present: hematoxylin and eosin stain was used. 1G. $58 is higher magnification photomicrograph of lng tissue fromthe same SARS patient (<250)- Syncytial cells show no conspicuous viral inclusions. FIG. 8C is a photomicrograph of immunohistochemically. stained SARS-CoV%infected cell 250), Membranows an eytoplasmic immunostaining of individual and syncytial Ver E6cels was achieved using feline anticFIPV-I ascitic Mid. Immunoalkaline phos- intase with naphthol-fast rd substrate and hematoxylin ouster stain Was use. FIG. 64-B ae elecronmierogrphs illustrating ultra stroctural characteristics of a coronaviws-infcted cell in bronchoalveolar lavage (BAL) fom a SARS pation FIG. Ais an eleetronmicrograph of a coronavirus-nfcted cell. Numerous intcellular and extracellular particles are present Visions ae indicated by the arwheads. FIG. 63 is ‘higher magnification electronmicrograph ofthe area seen a the aon in FIG. 6A (routed clockse approximately 90°), Bars: FIG. 6A, 1 um; FIG, 8, 100 nm. FIGS, 7A. C illustrate the organization ofthe SARS-CoV’ genome FIG. 7Aisa diagram ofthe overall organization of the 29,727-n1 SARS-COV genomie RNA. The T-nt leader sequence is represented asa small tangle at the let most end. ORFsIa and 1, encoding the nnstrctural polypro- teins, and those ORF encoding eS, E, Mand structural proiens, ar indicated. Veical position of the boxes indi Cates the phase of the reading frame (phase 1 for proteins above the fine, phase two fr proteins onthe Tine and phase tive for proteins below the line). FIG. 7B isan expanded ‘view ofthe strctural protein encoding region and predicted ‘ARNA traserpts. Known structural protein encoding regions (dark prey boxes) and regions and reading frames for potential produts X1-XS (ht gray boxes) are indi cated. Lengths and map locations of the 3

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