¿Quién cachopo PATENTA un bichito? Sólo quien tiene intención de utilizarlo
- Autor del tema Raulisimo
- Fecha de inicio
Raulisimo
Será en Octubre
- Desde
- 24 Oct 2009
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- 43.081
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- 84.419
El bichito está PATENTADO por Estados Unidos:
cobi19 isolated from humans
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.
This invention relates to a newly isolated human cobi19. More particularly, it relates to an isolated cobi19 genome, isolated cobi19 proteins, and isolated nucleic acid molecules encoding the same. The disclosure further relates to methods of detecting a severe acute respiratory syndrome-associated cobi19 and compositions comprising immunogenic cobi19 compounds.
The coronaviruses (order Nidovirales, family Coronaviridae, genus cobi19) 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 bichito 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. cobi19 particles are found within the cisternae of the rough endoplasmic reticulum and in vesicles of infected host cells where virions are assembled. The cobi19 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 cobi19 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, 9th edition, Oxford University Press, 463–479, 1998).
The three known groups of cobi19 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 cobi19 229E (HCoV-229E), canine cobi19 (CCoV), feline infectious peritonitis bichito (FIPV), porcine transmissible gastroenteritis bichito (TGEV), porcine epidemic diarrhea bichito (PEDV), human cobi19 OC43 (HcoV-OC43), bovine cobi19 (BCoV), porcine hemagglutinating encephalomyelitis bichito (HEV), rat sialodacryoadenitis bichito (SDAV), mouse hepatitis bichito (MHV), turkey cobi19 (TCoV), and avian infectious bronchitis bichito (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, 9th edition, Oxford University Press, 463–479, 1998).
cobi19 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 bichito production and altered virulence. Coronaviruses generally do not grow well in cell culture, and animal models for human cobi19 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 cobi19 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).
cobi19 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 cobi19 infection in humans is either incomplete or short lived. cobi19 infection in animals can cause severe enteric or respiratory disease. Vaccination has been used successfully to prevent and control some cobi19 infections in animals. The ability of animal-specific coronaviruses to cause severe disease raises the possibility that cobi19 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, 9th 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, amowed 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 cobi19 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 cobi19 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 antiestéticatures and advantages will become more apparent from the amowing 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 cobi19 isolates and serum from SARS patients. FIG. 1A is a photomicrograph of Vero E6 cells inoculated with an oropharyngeal specimen from a SARS patient (×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 (×400).
FIGS. 2A–B are electronmicrographs illustrating ultrastructural characteristics of the SARS-associated cobi19 (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 cobi19 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 frutative phylogenetic relationships between SARS-CoV and other human and animal coronaviruses. Phylogenetic relationships are based on sequence alignment of 405 nucleotides of the cobi19 polymerase gene ORF1b (nucleic acid 15,173 to 15,578 of SEQ ID NO: 1). The three major cobi19 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 frutative 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 bichito (M95169)]. Sequences for representative strains of other cobi19 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 cobi19 (Z24675); group 2: HCoV-OC43 (M76373, L14643, M93390); HEV (AY078417); rat cobi19 (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 (×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 (×250). Syncytial cells show no conspicuous viral inclusions. FIG. 5C is a photomicrograph of immunohistochemically stained SARS-CoV-infected cells (×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 cobi19-infected cell in bronchoalveolar lavage (BAL) from a SARS patient. FIG. 6A is an electronmicrograph of a cobi19-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°). Bars: FIG. 6A, 1 μm; 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 cobi19 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
M: cobi19 membrane protein N: cobi19 nucleoprotein ORF: open reading frame PCR polymerase chain reaction RACE: 5′ rapid amplification of cDNA ends RT-PCR: reverse transcription-polymerase chain reaction S: cobi19 spike protein SARS: severe acute respiratory syndrome SARS-CoV: severe acute respiratory syndrome-associated cobi19 TRS: transcriptional regulatory sequence
https://patents.google.com/patent/US7220852B1/en?oq=US7220852B1
cobi19 isolated from humans
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.
This invention relates to a newly isolated human cobi19. More particularly, it relates to an isolated cobi19 genome, isolated cobi19 proteins, and isolated nucleic acid molecules encoding the same. The disclosure further relates to methods of detecting a severe acute respiratory syndrome-associated cobi19 and compositions comprising immunogenic cobi19 compounds.
The coronaviruses (order Nidovirales, family Coronaviridae, genus cobi19) 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 bichito 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. cobi19 particles are found within the cisternae of the rough endoplasmic reticulum and in vesicles of infected host cells where virions are assembled. The cobi19 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 cobi19 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, 9th edition, Oxford University Press, 463–479, 1998).
The three known groups of cobi19 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 cobi19 229E (HCoV-229E), canine cobi19 (CCoV), feline infectious peritonitis bichito (FIPV), porcine transmissible gastroenteritis bichito (TGEV), porcine epidemic diarrhea bichito (PEDV), human cobi19 OC43 (HcoV-OC43), bovine cobi19 (BCoV), porcine hemagglutinating encephalomyelitis bichito (HEV), rat sialodacryoadenitis bichito (SDAV), mouse hepatitis bichito (MHV), turkey cobi19 (TCoV), and avian infectious bronchitis bichito (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, 9th edition, Oxford University Press, 463–479, 1998).
cobi19 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 bichito production and altered virulence. Coronaviruses generally do not grow well in cell culture, and animal models for human cobi19 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 cobi19 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).
cobi19 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 cobi19 infection in humans is either incomplete or short lived. cobi19 infection in animals can cause severe enteric or respiratory disease. Vaccination has been used successfully to prevent and control some cobi19 infections in animals. The ability of animal-specific coronaviruses to cause severe disease raises the possibility that cobi19 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, 9th 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, amowed 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 cobi19 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 cobi19 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 antiestéticatures and advantages will become more apparent from the amowing 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 cobi19 isolates and serum from SARS patients. FIG. 1A is a photomicrograph of Vero E6 cells inoculated with an oropharyngeal specimen from a SARS patient (×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 (×400).
FIGS. 2A–B are electronmicrographs illustrating ultrastructural characteristics of the SARS-associated cobi19 (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 cobi19 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 frutative phylogenetic relationships between SARS-CoV and other human and animal coronaviruses. Phylogenetic relationships are based on sequence alignment of 405 nucleotides of the cobi19 polymerase gene ORF1b (nucleic acid 15,173 to 15,578 of SEQ ID NO: 1). The three major cobi19 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 frutative 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 bichito (M95169)]. Sequences for representative strains of other cobi19 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 cobi19 (Z24675); group 2: HCoV-OC43 (M76373, L14643, M93390); HEV (AY078417); rat cobi19 (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 (×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 (×250). Syncytial cells show no conspicuous viral inclusions. FIG. 5C is a photomicrograph of immunohistochemically stained SARS-CoV-infected cells (×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 cobi19-infected cell in bronchoalveolar lavage (BAL) from a SARS patient. FIG. 6A is an electronmicrograph of a cobi19-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°). Bars: FIG. 6A, 1 μm; 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 cobi19 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
M: cobi19 membrane protein N: cobi19 nucleoprotein ORF: open reading frame PCR polymerase chain reaction RACE: 5′ rapid amplification of cDNA ends RT-PCR: reverse transcription-polymerase chain reaction S: cobi19 spike protein SARS: severe acute respiratory syndrome SARS-CoV: severe acute respiratory syndrome-associated cobi19 TRS: transcriptional regulatory sequence
https://patents.google.com/patent/US7220852B1/en?oq=US7220852B1
cerilloprieto
Gitanista
Más barato y efectivo que cualquier otro terrorismo anterior.
Primero doblegaron gobiernos de Europa mediante democracias corruptas financiadas por su banca mundial (Sistema Financiero), que llevaron a dos guerras mundiales ante la oposición de algunos países con un mínimo de dignidad. Una vez vencida la oposición, tras la guerra implantaron bases, armamento, ejército, dividiendo Europa en dos bloques. Posteriormente, en los 60 crearon el terrorismo rojo (ETA, Brigadas Rojas, IRA, Baader-Meinhof, Separatismo Corso, etc...). Los actos militares de estos grupos iban descendiendo según íbamos poniendo el trastero, hasta perder totalmente las pocas riendas de soberanía nacional que quedaban. Pero les parece siempre poco, y crean el terrorismo islámico, probado primero con éxito en Africa, y posteriormente en pleno corazón de occidente. Cuando parece que cesan los actos de los jovenlandeses locos, nos sueltan un bichito de diseño superresistente, a la par que dan orden a sus gobiernos títeres de hacer todo lo posible para que se propague, bajo la excusa aparente de políticos ineficientes que toman pocas medidas y tarde.
Primero doblegaron gobiernos de Europa mediante democracias corruptas financiadas por su banca mundial (Sistema Financiero), que llevaron a dos guerras mundiales ante la oposición de algunos países con un mínimo de dignidad. Una vez vencida la oposición, tras la guerra implantaron bases, armamento, ejército, dividiendo Europa en dos bloques. Posteriormente, en los 60 crearon el terrorismo rojo (ETA, Brigadas Rojas, IRA, Baader-Meinhof, Separatismo Corso, etc...). Los actos militares de estos grupos iban descendiendo según íbamos poniendo el trastero, hasta perder totalmente las pocas riendas de soberanía nacional que quedaban. Pero les parece siempre poco, y crean el terrorismo islámico, probado primero con éxito en Africa, y posteriormente en pleno corazón de occidente. Cuando parece que cesan los actos de los jovenlandeses locos, nos sueltan un bichito de diseño superresistente, a la par que dan orden a sus gobiernos títeres de hacer todo lo posible para que se propague, bajo la excusa aparente de políticos ineficientes que toman pocas medidas y tarde.
Raulisimo
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Tierra Azul
Madmaxista
vaya hombre empezaste bien hasta que soltaste ese video, en serio no habeis pensado que este tio tambien esta metido en la hez y que lo de q es una psyops...? en fin, no nos salvara nadie salvo nosotros mismos
Raulisimo
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Nosotros tampoco podemos salvarnos.
Es obvio que la propaganda de Trump es eso: propaganda.
Pero marca una declaración de intenciones. Nadie suelta semejante campaña de marketing si no tiene las cosas bien atadas.
De la Dictadura no nos libra nadie, nos pongamos como nos pongamos.
Raulisimo
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Ya se ha explicado que lo que se pretende es crear una "situación de pánico" para que la gente RECLAME la "restauración del Orden".
Lemavos
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Faltan aliens
Más esclavos de lo que somos ahora los que trabajamos en la empresa privada no se puede ser.
Muy peliculero todo.
