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. 4 is a pictorial representation of neighbor joining trees illustrating putative phylogenetic relationships between SARS-CoV and other human and animal куда нельзя лететь отдыхать из за коронавируса coronaviruses. Amino acid sequences of the indicated SARS-CoV proteins were compared with those куда нельзя лететь отдыхать из за коронавируса 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); куда нельзя лететь отдыхать из за коронавируса 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 куда нельзя лететь отдыхать из за коронавируса 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 куда нельзя лететь отдыхать из за коронавируса 2.0.1. 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. 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. 5B is a higher magnification photomicrograph of lung tissue from the same SARS patient (?250).
Syncytial cells show no conspicuous viral inclusions. 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 куда нельзя лететь отдыхать из за коронавируса using feline anti-FIPV-1 ascitic fluid. Immunoalkaline phosphatase with naphthol-fast red substrate and hematoxylin куда нельзя лететь отдыхать из за коронавируса counter stain was used. 6A–B are electronmicrographs illustrating ultrastructural characteristics of a coronavirus-infected cell in bronchoalveolar lavage (BAL) from a SARS patient. 6A is an electronmicrograph of a coronavirus-infected cell. Numerous intracellular and extracellular particles are present; virions куда нельзя лететь отдыхать из за коронавируса are indicated by the arrowheads. 6B is a higher magnification electronmicrograph of the area seen at the arrow in FIG. 7A–C illustrate the organization of the SARS-CoV genome.
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, куда нельзя лететь отдыхать из за коронавируса 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). 7B is an куда нельзя лететь отдыхать из за коронавируса 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.
7C is a digitized image куда нельзя лететь отдыхать из за коронавируса 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. 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. 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 куда нельзя лететь отдыхать из за коронавируса 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,куда нельзя лететь отдыхать из за коронавируса 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 куда нельзя лететь отдыхать из за коронавируса 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 (куда нельзя лететь отдыхать из за коронавируса encoded by nucleic acid 27,074 to 27,262 of SEQ ID NO: куда нельзя лететь отдыхать из за коронавируса 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,куда нельзя лететь отдыхать из за коронавируса 120 to 29,385 of SEQ ID NO: 1). SEQ ID NOs: 13–15 куда нельзя лететь отдыхать из за коронавируса 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 куда нельзя лететь отдыхать из за коронавируса assays.
Abbreviations 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 куда нельзя лететь отдыхать из за коронавируса 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.
