TECHNICAL FIELD
The invention relates to a model for coronavirus infection. More particularly, it concerns animals infected with coronavirus that has been labeled with fluorescent protein.
BACKGROUND ART
Recently, a worldwide outbreak of Severe Acute Respiratory Syndrome (SARS) has caused a substantial number of deaths, disrupted travel plans, and placed thousands of people under quarantine. In fairly short order, using clinical specimens from patients in six countries, it was established that the infection is caused by a coronavirus. See, for example, Ksiazek, T. G., et al., New England J. Med. (2003) 348:1947-1958.
The members of the coronavirus family contain positive-sense RNA genomes of about 30 kb that cause respiratory or intestinal infections in a number of different species. See, for example, de Haan, C. A. M., et al., Virology (2002) 296:177-189. Based on antigenic and genetic criteria, they have been divided into three groups. The common feature of coronaviruses are essential genes encoding replication and structural functions. Interspersed among these genes are group-specific open reading frames (ORFs) that are homologous within each group but that differ among the groups.
The predominant essential gene (ORF) occupies about two-thirds of the genome and is located at the 5′ end of the genome. This gene is a replicase gene that encodes two large precursors, which are cleaved into products for RNA replication and transcription. The other common essential genes code for the four basic structural proteins N, M, E, and S. The nucleocapsid (N) protein packages the viral RNA, forming the core of the virion. This nucleocapsid core structure is surrounded by a lipid envelope in which the membrane (M) protein is most abundant. The small envelope (E) protein and the spike (S) protein are associated with the M protein. The S protein forms the viral peplomers that are involved in virus-cell and cell-cell fusion. These genes are located in the 3′ third of the viral genome. The identities and the locations of the group-specific genes vary, and all their functions have not yet been established. Group 2 viruses, to which mouse hepatitis virus (MHV) belongs, have two group-specific genes, gene 2a and a hemagglutinin-esterase (HE) ORF between ORF 1b and the S gene. Two additional group 2-specific genes, genes 4 and 5a, reside between the S and E genes.
MHV has a single-stranded, positive-sense RNA genome of approximately 31 kb. See, Kim, K. H., et al., J. Virol. (1995) 69:2313-2321. The 5′ end of the MHV genomic RNA contains a 72- to 77-nucleotide-long leader sequence. Downstream of the leader sequence are the MHV-specific genes, each of which is separated by a special short stretch of intergenic sequence. MHV infected cells produce seven major species of virus-specific subgenomic mRNAs. The coronavirus mRNAs are structurally polycistronic, yet produce monocistronic proteins. The coronavirus mRNAs share 3′ ends in a nested-set structure wherein each mRNA is progressively one gene longer than its 3′-neighboring gene, and only the 5′-most gene of each mRNA is translated. These subgenomic mRNAs are named according to their decreasing order of size from 1 to 7. The mRNA sequences are fused with leader sequence at their 5′ ends.
Serial undiluted passage of MHV strain JHM in DBT cells results in generation of defective interfering (DI) RNAs that can be classified into two types. One requires helper virus infection for replication. The other DI type includes DIssA, which is nearly genomic in size, replicates by itself in the absence of helper virus infection, and is packaged into MHV particles Almost all MHV mRNA synthesis is strongly inhibited in DIssA-replicating cells, whereas synthesis of mRNA 7 and its product, N protein, are not inhibited. RNase T1 oligonucleotide fingerprinting analysis of DIssA demonstrate that gene 1 and gene 7 of DIssA are essentially intact, whereas multiple deletions are present from genes 2 to 6. mRNA 7 is synthesized from DIssA template RNA but not from helper virus template RNA, and the gene 1 products and N protein are sufficient for the MHV RNA synthesis.
Thus, it will be sufficient to monitor replication if the DI type DIssA can be labeled. This is the approach illustrated in the present invention.
Fluorescent proteins have been used as fluorescent labels for a number of years. The originally isolated protein emitted green wavelengths and came to be called green fluorescent protein (GFP). Because of this, green fluorescent protein became a generic label for such fluorescent proteins in general, although proteins of various colors including red fluorescent protein (RFP), blue fluorescent protein (BFP) and yellow fluorescent protein (YFP) among others have been prepared. The nature of these proteins is discussed in, for example, U.S. Pat. Nos. 6,232,523; 6,235,967; 6,235,968; and 6,251,384 all incorporated herein by reference. These patents describe the use of fluorescent proteins of various colors to monitor tumor growth and metastasis in transgenic rodents which are convenient tumor models. In addition, these fluorescent proteins have been used to monitor expression mediated by promoters in U.S. application Ser. No. 09/812,710; to monitor infection by bacteria in U.S. Ser. No. 10/192,740 and to monitor cell sorting in U.S. provisional application 60/425,776. The use of fluorescent proteins of different colors to label the nucleus and cytoplasm of cells is disclosed in U.S. provisional applications 60/404,005 and 60/427,604 and mice which are labeled in all tissues, and thus have a consistent fluorescence of the same color are described in U.S. provisional application 60/445,583. All of these documents are incorporated herein by reference.
DISCLOSURE OF THE INVENTION
The invention provides an animal model wherein fluorescent labeled coronavirus are used to infect susceptible animal subjects, preferably rodents or rabbits, wherein the progress of infection—i.e., the replication of the coronavirus can be followed by monitoring the fluorescence. In a preferred embodiment, the animal is a transgenic animal which comprises tissues that fluoresce in a first color against which the fluorescence of the replicating coronavirus can be readily visualized. The model can be used to determine the effectiveness of vaccines and drugs by viewing, directly, the progress of infection with and without treatment or vaccination. The invention is illustrated below using the DIssA specific sequence from MHV as a model.
Thus, in one aspect, the invention is directed to a coronavirus labeled with a fluorescent protein such as GFP or RFP. In another aspect, the invention is directed to an animal infected with the labeled virus. In still another aspect, the invention is directed to methods to monitor the progress of infection, to evaluate the effectiveness of antiviral drugs, and to evaluate the effects of the vaccines using the animal models of the invention.
MODES OF CARRYING OUT THE INVENTION
The tools useful in the present invention are described in the U.S. patents and patent applications incorporated by reference above. Whole body imaging, the nature of fluorescent proteins useful in the invention, and methods to label entire animals have been described in these documents.
The disclosed method applicable to coronavirus of the various groups in the coronavirus family. Although in the illustrative example the virus is labeled with RFP and is viewed against a background of a nude mouse expressing GFP in all its tissues, neither the choice of these particular colors nor the use of a labeled animal as a subject is required.
Recombinant Coronavirus
The disclosed invention uses recombinant coronaviruses that are engineered to express a marker, such as a fluorescent protein. By infecting a model organism with the described recombinant coronavirus, one of ordinary skill in the art can use the recombinant virus to study the progression of viral replication in the host animal. Furthermore, the recombinant coronavirus model system has utility as an assay for identifying antiviral agents that slow or inhibit coronavirus replication.
Work by Cornelis, et al. has demonstrated that coronaviruses can by recombinantly engineered to express foreign genes without severe effects on viral replication. Cornelis, et al., J. Virol. (2003) 77(21):11312-11323, which is hereby incorporated by reference in its entirety. The results of this study suggest that position of the foreign gene within the viral genome may impact the viral replication of the recombinant virus vector. Specifically, Cornelis and coworkers observed that expression levels of the foreign gene increased when the foreign gene was inserted closer to the 3′ end of the viral genome. As such, in preferred embodiments of the invention, placement of the fluorescent protein coding sequence occurs toward the 3′ end of the viral genome.
Cornelis and coworkers utilized a murine coronavirus model for their study. The sequence of this virus is well known in the art. At least one variant of the human SARS virus has been sequenced. Marra, M. A., et al., “The Genome sequence of the SARS-associated coronavirus” Science 300 (5624), 1399-1404 (2003). This sequence is publicly available as Accession: NC 004718. The genomic sequence of this SARS variant is provided herein as SEQ ID NO: XX of Table I.
TABLE I
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(SEQ ID NO: XX)
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1atattaggtt tttacctacc caggaaaagc caaccaacct
cgatctcttg tagatctgtt
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61ctctaaacga actttaaaat ctgtgtagct gtcgctcggc
tgcatgccta gtgcacctac
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121gcagtataaa caataataaa ttttactgtc gttgacaaga
aacgagtaac tcgtccctct
|
181tctgcagact gcttacggtt tcgtccgtgt tgcagtcgat
catcagcata cctaggtttc
|
241gtccgggtgt gaccgaaagg taagatggag agccttgttc
ttggtgtcaa cgagaaaaca
|
301cacgtccaac tcagtttgcc tgtccttcag gttagagacg
tgctagtgcg tggcttcggg
|
361gactctgtgg aagaggccct atcggaggca cgtgaacacc
tcaaaaatgg cacttgtggt
|
421ctagtagagc tggaaaaagg cgtactgccc cagcttgaac
agccctatgt gttcattaaa
|
481cgttctgatg ccttaagcac caatcacggc cacaaggtcg
ttgagctggt tgcagaaatg
|
541gacggcattc agtacggtcg tagcggtata acactgggag
tactcgtgcc acatgtgggc
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601gaaaccccaa ttgcataccg caatgttctt cttcgtaaga
acggtaataa gggagccggt
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661ggtcatagct atggcatcga tctaaagtct tatgacttag
gtgacgagct tggcactgat
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721cccattgaag attatgaaca aaactggaac actaagcatg
gcagtggtgc actccgtgaa
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781ctcactcgtg agctcaatgg aggtgcagtc actcgctatg
tcgacaacaa tttctgtggc
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841ccagatgggt accctcttga ttgcatcaaa gattttctcg
cacgcgcggg caagtcaatg
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901tgcactcttt ccgaacaact tgattacatc gagtcgaaga
gaggtgtcta ctgctgccgt
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961gaccatgagc atgaaattgc ctggttcact gagcgctctg
ataagagcta cgagcaccag
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1021acacccttcg aaattaagag tgccaagaaa tttgacactt
tcaaagggga atgcccaaag
|
1081tttgtgtttc ctcttaactc aaaagtcaaa gtcattcaac
cacgtgttga aaagaaaaag
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1141actgagggtt tcatggggcg tatacgctct gtgtaccctg
ttgcatctcc acaggagtgt
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1201aacaatatgc acttgtctac cttgatgaaa tgtaatcatt
gcgatgaagt ttcatggcag
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1261acgtgcgact ttctgaaagc cacttgtgaa cattgtggca
ctgaaaattt agttattgaa
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1321ggacctacta catgtgggta cctacctact aatgctgtag
tgaaaatgcc atgtcctgcc
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1381tgtcaagacc cagagattgg acctgagcat agtgttgcag
attatcacaa ccactcaaac
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1441attgaaactc gactccgcaa gggaggtagg actagatgtt
ttggaggctg tgtgtttgcc
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1501tatgttggct gctataataa gcgtgcctac tgggttcctc
gtgctagtgc tgatattggc
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1561tcaggccata ctggcattac tggtgacaat gtggagacct
tgaatgagga tctccttgag
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1621atactgagtc gtgaacgtgt taacattaac attgttggcg
attttcattt gaatgaagag
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1681gttgccatca ttttggcatc tttctctgct tctacaagtg
cctttattga cactataaag
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1741agtcttgatt acaagtcttt caaaaccatt gttgagtcct
gcggtaacta taaagttacc
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1801aagggaaagc ccgtaaaagg tgcttggaac attggacaac
agagatcagt tttaacacca
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1861ctgtgtggtt ttccctcaca ggctgctggt gttatcagat
caatttttgc gcgcacactt
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1921gatgcagcaa accactcaat tcctgatttg caaagagcag
ctgtcaccat acttgatggt
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1981atttctgaac agtcattacg tcttgtcgac gccatggttt
atacttcaga cctgctcacc
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2041aacagtgtca ttattatggc atatgtaact ggtggtcttg
tacaacagac ttctcagtgg
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2101ttgtctaatc ttttgggcac tactgttgaa aaactcaggc
ctatctttga atggattgag
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2161gcgaaactta gtgcaggagt tgaatttctc aaggatgctt
gggagattct caaatttctc
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2221attacaggtg tttttgacat cgtcaagggt caaatacagg
ttgcttcaga taacatcaag
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2281gattgtgtaa aatgcttcat tgatgttgtt aacaaggcac
tcgaaatgtg cattgatcaa
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2341gtcactatcg ctggcgcaaa gttgcgatca ctcaacttag
gtgaagtctt catcgctcaa
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2401agcaagggac tttaccgtca gtgtatacgt ggcaaggagc
agctgcaact actcatgcct
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2461cttaaggcac caaaagaagt aacctttctt gaaggtgatt
cacatgacac agtacttacc
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2521tctgaggagg ttgttctcaa gaacggtgaa ctcgaagcac
tcgagacgcc cgttgatagc
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2581ttcacaaatg gagctatcgt tggcacacca gtctgtgtaa
atggcctcat gctcttagag
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2641attaaggaca aagaacaata ctgcgcattg tctcctggtt
tactggctac aaacaatgtc
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2701tttcgcttaa aagggggtgc accaattaaa ggtgtaacct
ttggagaaga tactgtttgg
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2761gaagttcaag gttacaagaa tgtgagaatc acatttgagc
ttgatgaacg tgttgacaaa
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2821gtgcttaatg aaaagtgctc tgtctacact gttgaatccg
gtaccgaagt tactgagttt
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2881gcatgtgttg tagcagaggc tgttgtgaag actttacaac
cagtttctga tctccttacc
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2941aacatgggta ttgatcttga tgagtggagt gtagctacat
tctacttatt tgatgatgct
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3001ggtgaagaaa acttttcatc acgtatgtat tgttcctttt
accctccaga tgaggaagaa
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3061gaggacgatg cagagtgtga ggaagaagaa attgatgaaa
cctgtgaaca tgagtacggt
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3121acagaggatg attatcaagg tctccctctg gaatttggtg
cctcagctga aacagttcga
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3181gttgaggaag aagaagagga agactggctg gatgatacta
ctgagcaatc agagattgag
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3241ccagaaccag aacctacacc tgaagaacca gttaatcagt
ttactggtta tttaaaactt
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3301actgacaatg ttgccattaa atgtgttgac atcgttaagg
aggcacaaag tgctaatcct
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3361atggtgattg taaatgctgc taacatacac ctgaaacatg
gtggtggtgt agcaggtgca
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3421ctcaacaagg caaccaatgg tgccatgcaa aaggagagtg
atgattacat taagctaaat
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3481ggccctctta cagtaggagg gtcttgtttg ctttctggac
ataatcttgc taagaagtgt
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3541ctgcatgttg ttggacctaa cctaaatgca ggtgaggaca
tccagcttct taaggcagca
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3601tatgaaaatt tcaattcaca ggacatctta cttgcaccat
tgttgtcagc aggcatattt
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3661ggtgctaaac cacttcagtc tttacaagtg tgcgtgcaga
cggttcgtac acaggtttat
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3721attgcagtca atgacaaagc tctttatgag caggttgtca
tggattatct tgataacctg
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3781aagcctagag tggaagcacc taaacaagag gagccaccaa
acacagaaga ttccaaaact
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3841gaggagaaat ctgtcgtaca gaagcctgtc gatgtgaagc
caaaaattaa ggcctgcatt
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3901gatgaggtta ccacaacact ggaagaaact aagtttctta
ccaataagtt actcttgttt
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3961gctgatatca atggtaagct ttaccatgat tctcagaaca
tgcttagagg tgaagatatg
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4021tctttccttg agaaggatgc accttacatg gtaggtgatg
ttatcactag tggtgatatc
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4081acttgtgttg taataccctc caaaaaggct ggtggcacta
ctgagatgct ctcaagagct
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4141ttgaagaaag tgccagttga tgagtatata accacgtacc
ctggacaagg atgtgctggt
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4201tatacacttg aggaagctaa gactgctctt aagaaatgca
aatctgcatt ttatgtacta
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4261ccttcagaag cacctaatgc taaggaagag attctaggaa
ctgtatcctg gaatttgaga
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4321gaaatgcttg ctcatgctga agagacaaga aaattaatgc
ctatatgcat ggatgttaga
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4381gccataatgg caaccatcca acgtaagtat aaaggaatta
aaattcaaga gggcatcgtt
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4441gactatggtg tccgattctt cttttatact agtaaagagc
ctgtagcttc tattattacg
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4501aagctgaact ctctaaatga gccgcttgtc acaatgccaa
ttggttatgt gacacatggt
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4561tttaatcttg aagaggctgc gcgctgtatg cgttctctta
aagctcctgc cgtagtgtca
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4621gtatcatcac cagatgctgt tactacatat aatggatacc
tcacttcgtc atcaaagaca
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4681tctgaggagc actttgtaga aacagtttct ttggctggct
cttacagaga ttggtcctat
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4741tcaggacagc gtacagagtt aggtgttgaa tttcttaagc
gtggtgacaa aattgtgtac
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4801cacactctgg agagccccgt cgagtttcat cttgacggtg
aggttctttc acttgacaaa
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4861ctaaagagtc tcttatccct gcgggaggtt aagactataa
aagtgttcac aactgtggac
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4921aacactaatc tccacacaca gcttgtggat atgtctatga
catatggaca gcagtttggt
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4981ccaacatact tggatggtgc tgatgttaca aaaattaaac
ctcatgtaaa tcatgagggt
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5041aagactttct ttgtactacc tagtgatgac acactacgta
gtgaagcttt cgagtactac
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5101catactcttg atgagagttt tcttggtagg tacatgtctg
ctttaaacca cacaaagaaa
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5161tggaaatttc ctcaagttgg tggtttaact tcaattaaat
gggctgataa caattgttat
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5221ttgtctagtg ttttattagc acttcaacag cttgaagtca
aattcaatgc accagcactt
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5281caagaggctt attatagagc ccgtgctggt gatgctgcta
acttttgtgc actcatactc
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5341gcttacagta ataaaactgt tggcgagctt ggtgatgtca
gagaaactat gacccatctt
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5401ctacagcatg ctaatttgga atctgcaaag cgagttctta
atgtggtgtg taaacattgt
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5461ggtcagaaaa ctactacctt aacgggtgta gaagctgtga
tgtatatggg tactctatct
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5521tatgataatc ttaagacagg tgtttccatt ccatgtgtgt
gtggtcgtga tgctacacaa
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5581tatctagtac aacaagagtc ttcttttgtt atgatgtctg
caccacctgc tgagtataaa
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5641ttacagcaag gtacattctt atgtgcgaat gagtacactg
gtaactatca gtgtggtcat
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5701tacactcata taactgctaa ggagaccctc tatcgtattg
acggagctca ccttacaaag
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5761atgtcagagt acaaaggacc agtgactgat gttttctaca
aggaaacatc ttacactaca
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5821accatcaagc ctgtgtcgta taaactcgat ggagttactt
acacagagat tgaaccaaaa
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5881ttggatgggt attataaaaa ggataatgct tactatacag
agcagcctat agaccttgta
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5941ccaactcaac cattaccaaa tgcgagtttt gataatttca
aactcacatg ttctaacaca
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6001aaatttgctg atgatttaaa tcaaatgaca ggcttcacaa
agccagcttc acgagagcta
|
6061tctgtcacat tcttcccaga cttgaatggc gatgtagtgg
ctattgacta tagacactat
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6121tcagcgagtt tcaagaaagg tgctaaatta ctgcataagc
caattgtttg gcacattaac
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6181caggctacaa ccaagacaac gttcaaacca aacacttggt
gtttacgttg tctttggagt
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6241acaaagccag tagatacttc aaattcattt gaagttctgg
cagtagaaga cacacaagga
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6301atggacaatc ttgcttgtga aagtcaacaa cccacctctg
aagaagtagt ggaaaatcct
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6361accatacaga aggaagtcat agagtgtgac gtgaaaacta
ccgaagttgt aggcaatgtc
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6421atacttaaac catcagatga aggtgttaaa gtaacacaag
agttaggtca tgaggatctt
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6481atggctgctt atgtggaaaa cacaagcatt accattaaga
aacctaatga gctttcacta
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6541gccttaggtt taaaaacaat tgccactcat ggtattgctg
caattaatag tgttccttgg
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6601agtaaaattt tggcttatgt caaaccattc ttaggacaag
cagcaattac aacatcaaat
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6661tgcgctaaga gattagcaca acgtgtgttt aacaattata
tgccttatgt gtttacatta
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6721ttgttccaat tgtgtacttt tactaaaagt accaattcta
gaattagagc ttcactacct
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6781acaactattg ctaaaaatag tgttaagagt gttgctaaat
tatgtttgga tgccggcatt
|
6841aattatgtga agtcacccaa attttctaaa ttgttcacaa
tcgctatgtg gctattgttg
|
6901ttaagtattt gcttaggttc tctaatctgt gtaactgctg
cttttggtgt actcttatct
|
6961aattttggtg ctccttctta ttgtaatggc gttagagaat
tgtatcttaa ttcgtctaac
|
7021gttactacta tggatttctg tgaaggttct tttccttgca
gcatttgttt aagtggatta
|
7081gactcccttg attcttatcc agctcttgaa accattcagg
tgacgatttc atcgtacaag
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7141ctagacttga caattttagg tctggccgct gagtgggttt
tggcatatat gttgttcaca
|
7201aaattctttt atttattagg tctttcagct ataatgcagg
tgttctttgg ctattttgct
|
7261agtcatttca tcagcaattc ttggctcatg tggtttatca
ttagtattgt acaaatggca
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7321cccgtttctg caatggttag gatgtacatc ttctttgctt
ctttctacta catatggaag
|
7381agctatgttc atatcatgga tggttgcacc tcttcgactt
gcatgatgtg ctataagcgc
|
7441aatcgtgcca cacgcgttga gtgtacaact attgttaatg
gcatgaagag atctttctat
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7501gtctatgcaa atggaggccg tggcttctgc aagactcaca
attggaattg tctcaattgt
|
7561gacacatttt gcactggtag tacattcatt agtgatgaag
ttgctcgtga tttgtcactc
|
7621cagtttaaaa gaccaatcaa ccctactgac cagtcatcgt
atattgttga tagtgttgct
|
7681gtgaaaaatg gcgcgcttca cctctacttt gacaaggctg
gtcaaaagac ctatgagaga
|
7741catccgctct cccattttgt caatttagac aatttgagag
ctaacaacac taaaggttca
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7801ctgcctatta atgtcatagt ttttgatggc aagtccaaat
gcgacgagtc tgcttctaag
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7861tctgcttctg tgtactacag tcagctgatg tgccaaccta
ttctgttgct tgaccaagct
|
7921cttgtatcag acgttggaga tagtactgaa gtttccgtta
agatgtttga tgcttatgtc
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7981gacacctttt cagcaacttt tagtgttcct atggaaaaac
ttaaggcact tgttgctaca
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8041gctcacagcg agttagcaaa gggtgtagct ttagatggtg
tcctttctac attcgtgtca
|
8101gctgcccgac aaggtgttgt tgataccgat gttgacacaa
aggatgttat tgaatgtctc
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8161aaactttcac atcactctga cttagaagtg acaggtgaca
gttgtaacaa tttcatgctc
|
8221acctataata aggttgaaaa catgacgccc agagatcttg
gcgcatgtat tgactgtaat
|
8281gcaaggcata tcaatgccca agtagcaaaa agtcacaatg
tttcactcat ctggaatgta
|
8341aaagactaca tgtctttatc tgaacagctg cgtaaacaaa
ttcgtagtgc tgccaagaag
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8401aacaacatac cttttagact aacttgtgct acaactagac
aggttgtcaa tgtcataact
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8461actaaaatct cactcaaggg tggtaagatt gttagtactt
gttttaaact tatgcttaag
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8521gccacattat tgtgcgttct tgctgcattg gtttgttata
tcgttatgcc agtacataca
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8581ttgtcaatcc atgatggtta cacaaatgaa atcattggtt
acaaagccat tcaggatggt
|
8641gtcactcgtg acatcatttc tactgatgat tgttttgcaa
ataaacatgc tggttttgac
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8701gcatggttta gccagcgtgg tggttcatac aaaaatgaca
aaagctgccc tgtagtagct
|
8761gctatcatta caagagagat tggtttcata gtgcctggct
taccgggtac tgtgctgaga
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8821gcaatcaatg gtgacttctt gcattttcta cctcgtgttt
ttagtgctgt tggcaacatt
|
8881tgctacacac cttccaaact cattgagtat agtgattttg
ctacctctgc ttgcgttctt
|
8941gctgctgagt gtacaatttt taaggatgct atgggcaaac
ctgtgccata ttgttatgac
|
9001actaatttgc tagagggttc tatttcttat agtgagcttc
gtccagacac tcgttatgtg
|
9061cttatggatg gttccatcat acagtttcct aacacttacc
tggagggttc tgttagagta
|
9121gtaacaactt ttgatgctga gtactgtaga catggtacat
gcgaaaggtc agaagtaggt
|
9181atttgcctat ctaccagtgg tagatgggtt cttaataatg
agcattacag agctctatca
|
9241ggagttttct gtggtgttga tgcgatgaat ctcatagcta
acatctttac tcctcttgtg
|
9301caacctgtgg gtgctttaga tgtgtctgct tcagtagtgg
ctggtggtat tattgccata
|
9361ttggtgactt gtgctgccta ctactttatg aaattcagac
gtgtttttgg tgagtacaac
|
9421catgttgttg ctgctaatgc acttttgttt ttgatgtctt
tcactatact ctgtctggta
|
9481ccagcttaca gctttctgcc gggagtctac tcagtctttt
acttgtactt gacattctat
|
9541ttcaccaatg atgtttcatt cttggctcac cttcaatggt
ttgccatgtt ttctcctatt
|
9601gtgccttttt ggataacagc aatctatgta ttctgtattt
ctctgaagca ctgccattgg
|
9661ttctttaaca actatcttag gaaaagagtc atgtttaatg
gagttacatt tagtaccttc
|
9721gaggaggctg ctttgtgtac ctttttgctc aacaaggaaa
tgtacctaaa attgcgtagc
|
9781gagacactgt tgccacttac acagtataac aggtatcttg
ctctatataa caagtacaag
|
9841tatttcagtg gagccttaga tactaccagc tatcgtgaag
cagcttgctg ccacttagca
|
9901aaggctctaa atgactttag caactcaggt gctgatgttc
tctaccaacc accacagaca
|
9961tcaatcactt ctgctgttct gcagagtggt tttaggaaaa
tggcattccc gtcaggcaaa
|
10021gttgaagggt gcatggtaca agtaacctgt ggaactacaa
ctcttaatgg attgtggttg
|
10081gatgacacag tatactgtcc aagacatgtc atttgcacag
cagaagacat gcttaatcct
|
10141aactatgaag atctgctcat tcgcaaatcc aaccatagct
ttcttgttca ggctggcaat
|
10201gttcaacttc gtgttattgg ccattctatg caaaattgtc
tgcttaggct taaagttgat
|
10261acttctaacc ctaagacacc caagtataaa tttgtccgta
tccaacctgg tcaaacattt
|
10321tcagttctag catgctacaa tggttcacca tctggtgttt
atcagtgtgc catgagacct
|
10381aatcatacca ttaaaggttc tttccttaat ggatcatgtg
gtagtgttgg ttttaacatt
|
10441gattatgatt gcgtgtcttt ctgctatatg catcatatgg
agcttccaac aggagtacac
|
10501gctggtactg acttagaagg taaattctat ggtccatttg
ttgacagaca aactgcacag
|
10561gctgcaggta cagacacaac cataacatta aatgttttgg
catggctgta tgctgctgtt
|
10621atcaatggtg ataggtggtt tcttaataga ttcaccacta
ctttgaatga ctttaacctt
|
10681gtggcaatga agtacaacta tgaacctttg acacaagatc
atgttgacat attgggacct
|
10741ctttctgctc aaacaggaat tgccgtctta gatatgtgtg
ctgctttgaa agagctgctg
|
10801cagaatggta tgaatggtcg tactatcctt ggtagcacta
ttttagaaga tgagtttaca
|
10861ccatttgatg ttgttagaca atgctctggt gttaccttcc
aaggtaagtt caagaaaatt
|
10921gttaagggca ctcatcattg gatgctttta actttcttga
catcactatt gattcttgtt
|
10981caaagtacac agtggtcact gtttttcttt gtttacgaga
atgctttctt gccatttact
|
11041cttggtatta tggcaattgc tgcatgtgct atgctgcttg
ttaagcataa gcacgcattc
|
11101ttgtgcttgt ttctgttacc ttctcttgca acagttgctt
actttaatat ggtctacatg
|
11161cctgctagct gggtgatgcg tatcatgaca tggcttgaat
tggctgacac tagcttgtct
|
11221ggttataggc ttaaggattg tgttatgtat gcttcagctt
tagttttgct tattctcatg
|
11281acagctcgca ctgtttatga tgatgctgct agacgtgttt
ggacactgat gaatgtcatt
|
11341acacttgttt acaaagtcta ctatggtaat gctttagatc
aagctatttc catgtgggcc
|
11401ttagttattt ctgtaacctc taactattct ggtgtcgtta
cgactatcat gtttttagct
|
11461agagctatag tgtttgtgtg tgttgagtat tacccattgt
tatttattac tggcaacacc
|
11521ttacagtgta tcatgcttgt ttattgtttc ttaggctatt
gttgctgctg ctactttggc
|
11581cttttctgtt tactcaaccg ttacttcagg cttactcttg
gtgtttatga ctacttggtc
|
11641tctacacaag aatttaggta tatgaactcc caggggcttt
tgcctcctaa gagtagtatt
|
11701gatgctttca agcttaacat taagttgttg ggtattggag
gtaaaccatg tatcaaggtt
|
11761gctactgtac agtctaaaat gtctgacgta aagtgcacat
ctgtggtact gctctcggtt
|
11821cttcaacaac ttagagtaga gtcatcttct aaattgtggg
cacaatgtgt acaactccac
|
11881aatgatattc ttcttgcaaa agacacaact gaagctttcg
agaagatggt ttctcttttg
|
11941tctgttttgc tatccatgca gggtgctgta gacattaata
ggttgtgcga ggaaatgctc
|
12001gataaccgtg ctactcttca ggctattgct tcagaattta
gttctttacc atcatatgcc
|
12061gcttatgcca ctgcccagga ggcctatgag caggctgtag
ctaatggtga ttctgaagtc
|
12121gttctcaaaa agttaaagaa atctttgaat gtggctaaat
ctgagtttga ccgtgatgct
|
12181gccatgcaac gcaagttgga aaagatggca gatcaggcta
tgacccaaat gtacaaacag
|
12241gcaagatctg aggacaagag ggcaaaagta actagtgcta
tgcaaacaat gctcttcact
|
12301atgcttagga agcttgataa tgatgcactt aacaacatta
tcaacaatgc gcgtgatggt
|
12361tgtgttccac tcaacatcat accattgact acagcagcca
aactcatggt tgttgtccct
|
12421gattatggta cctacaagaa cacttgtgat ggtaacacct
ttacatatgc atctgcactc
|
12481tgggaaatcc agcaagttgt tgatgcggat agcaagattg
ttcaacttag tgaaattaac
|
12541atggacaatt caccaaattt ggcttggcct cttattgtta
cagctctaag agccaactca
|
12601gctgttaaac tacagaataa tgaactgagt ccagtagcac
tacgacagat gtcctgtgcg
|
12661gctggtacca cacaaacagc ttgtactgat gacaatgcac
ttgcctacta taacaattcg
|
12721aagggaggta ggtttgtgct ggcattacta tcagaccacc
aagatctcaa atgggctaga
|
12781ttccctaaga gtgatggtac aggtacaatt tacacagaac
tggaaccacc ttgtaggttt
|
12841gttacagaca caccaaaagg gcctaaagtg aaatacttgt
acttcatcaa aggcttaaac
|
12901aacctaaata gaggtatggt gctgggcagt ttagctgcta
cagtacgtct tcaggctgga
|
12961aatgctacag aagtacctgc caattcaact gtgctttcct
tctgtgcttt tgcagtagac
|
13021cctgctaaag catataagga ttacctagca agtggaggac
aaccaatcac caactgtgtg
|
13081aagatgttgt gtacacacac tggtacagga caggcaatta
ctgtaacacc agaagctaac
|
13141atggaccaag agtcctttgg tggtgcttca tgttgtctgt
attgtagatg ccacattgac
|
13201catccaaatc ctaaaggatt ctgtgacttg aaaggtaagt
acgtccaaat acctaccact
|
13261tgtgctaatg acccagtggg ttttacactt agaaacacag
tctgtaccgt ctgcggaatg
|
13321tggaaaggtt atggctgtag ttgtgaccaa ctccgcgaac
ccttgatgca gtctgcggat
|
13381gcatcaacgt ttttaaacgg gtttgcggtg taagtgcagc
ccgtcttaca ccgtgcggca
|
13441caggcactag tactgatgtc gtctacaggg cttttgatat
ttacaacgaa aaagttgctg
|
13501gttttgcaaa gttcctaaaa actaattgct gtcgcttcca
ggagaaggat gaggaaggca
|
13561atttattaga ctcttacttt gtagttaaga ggcatactat
gtctaactac caacatgaag
|
13621agactattta taacttggtt aaagattgtc cagcggttgc
tgtccatgac tttttcaagt
|
13681ttagagtaga tggtgacatg gtaccacata tatcacgtca
gcgtctaact aaatacacaa
|
13741tggctgattt agtctatgct ctacgtcatt ttgatgaggg
taattgtgat acattaaaag
|
13801aaatactcgt cacatacaat tgctgtgatg atgattattt
caataagaag gattggtatg
|
13861acttcgtaga gaatcctgac atcttacgcg tatatgctaa
cttaggtgag cgtgtacgcc
|
13921aatcattatt aaagactgta caattctgcg atgctatgcg
tgatgcaggc attgtaggcg
|
13981tactgacatt agataatcag gatcttaatg ggaactggta
cgatttcggt gatttcgtac
|
14041aagtagcacc aggctgcgga gttcctattg tggattcata
ttactcattg ctgatgccca
|
14101tcctcacttt gactagggca ttggctgctg agtcccatat
ggatgctgat ctcgcaaaac
|
14161cacttattaa gtgggatttg ctgaaatatg attttacgga
agagagactt tgtctcttcg
|
14221accgttattt taaatattgg gaccagacat accatcccaa
ttgtattaac tgtttggatg
|
14281ataggtgtat ccttcattgt gcaaacttta atgtgttatt
ttctactgtg tttccaccta
|
14341caagttttgg accactagta agaaaaatat ttgtagatgg
tgttcctttt gttgtttcaa
|
14401ctggatacca ttttcgtgag ttaggagtcg tacataatca
ggatgtaaac ttacatagct
|
14461cgcgtctcag tttcaaggaa cttttagtgt atgctgctga
tccagctatg catgcagctt
|
14521ctggcaattt attgctagat aaacgcacta catgcttttc
agtagctgca ctaacaaaca
|
14581atgttgcttt tcaaactgtc aaacccggta attttaataa
agacttttat gactttgctg
|
14641tgtctaaagg tttctttaag gaaggaagtt ctgttgaact
aaaacacttc ttctttgctc
|
14701aggatggcaa cgctgctatc agtgattatg actattatcg
ttataatctg ccaacaatgt
|
14761gtgatatcag acaactccta ttcgtagttg aagttgttga
taaatacttt gattgttacg
|
14821atggtggctg tattaatgcc aaccaagtaa tcgttaacaa
tctggataaa tcagctggtt
|
14881tcccatttaa taaatggggt aaggctagac tttattatga
ctcaatgagt tatgaggatc
|
14941aagatgcact tttcgcgtat actaagcgta atgtcatccc
tactataact caaatgaatc
|
15001ttaagtatgc cattagtgca aagaatagag ctcgcaccgt
agctggtgtc tctatctgta
|
15061gtactatgac aaatagacag tttcatcaga aattattgaa
gtcaatagcc gccactagag
|
15121gagctactgt ggtaattgga acaagcaagt tttacggtgg
ctggcataat atgttaaaaa
|
15181ctgtttacag tgatgtagaa actccacacc ttatgggttg
ggattatcca aaatgtgaca
|
15241gagccatgcc taacatgctt aggataatgg cctctcttgt
tcttgctcgc aaacataaca
|
15301cttgctgtaa cttatcacac cgtttctaca ggttagctaa
cgagtgtgcg caagtattaa
|
15361gtgagatggt catgtgtggc ggctcactat atgttaaacc
aggtggaaca tcatccggtg
|
15421atgctacaac tgcttatgct aatagtgtct ttaacatttg
tcaagctgtt acagccaatg
|
15481taaatgcact tctttcaact gatggtaata agatagctga
caagtatgtc cgcaatctac
|
15541aacacaggct ctatgagtgt ctctatagaa atagggatgt
tgatcatgaa ttcgtggatg
|
15601agttttacgc ttacctgcgt aaacatttct ccatgatgat
tctttctgat gatgccgttg
|
15661tgtgctataa cagtaactat gcggctcaag gtttagtagc
tagcattaag aactttaagg
|
15721cagttcttta ttatcaaaat aatgtgttca tgtctgaggc
aaaatgttgg actgagactg
|
15781accttactaa aggacctcac gaattttgct cacagcatac
aatgctagtt aaacaaggag
|
15841atgattacgt gtacctgcct tacccagatc catcaagaat
attaggcgca ggctgttttg
|
15901tcgatgatat tgtcaaaaca gatggtacac ttatgattga
aaggttcgtg tcactggcta
|
15961ttgatgctta cccacttaca aaacatccta atcaggagta
tgctgatgtc tttcacttgt
|
16021atttacaata cattagaaag ttacatgatg agcttactgg
ccacatgttg gacatgtatt
|
16081ccgtaatgct aactaatgat aacacctcac ggtactggga
acctgagttt tatgaggcta
|
16141tgtacacacc acatacagtc ttgcaggctg taggtgcttg
tgtattgtgc aattcacaga
|
16201cttcacttcg ttgcggtgcc tgtattagga gaccattcct
atgttgcaag tgctgctatg
|
16261accatgtcat ttcaacatca cacaaattag tgttgtctgt
taatccctat gtttgcaatg
|
16321ccccaggttg tgatgtcact gatgtgacac aactgtatct
aggaggtatg agctattatt
|
16381gcaagtcaca taagcctccc attagttttc cattatgtgc
taatggtcag gtttttggtt
|
16441tatacaaaaa cacatgtgta ggcagtgaca atgtcactga
cttcaatgcg atagcaacat
|
16501gtgattggac taatgctggc gattacatac ttgccaacac
ttgtactgag agactcaagc
|
16561ttttcgcagc agaaacgctc aaagccactg aggaaacatt
taagctgtca tatggtattg
|
16621ccactgtacg cgaagtactc tctgacagag aattgcatct
ttcatgggag gttggaaaac
|
16681ctagaccacc attgaacaga aactatgtct ttactggtta
ccgtgtaact aaaaatagta
|
16741aagtacagat tggagagtac acctttgaaa aaggtgacta
tggtgatgct gttgtgtaca
|
16801gaggtactac gacatacaag ttgaatgttg gtgattactt
tgtgttgaca tctcacactg
|
16861taatgccact tagtgcacct actctagtgc cacaagagca
ctatgtgaga attactggct
|
16921tgtacccaac actcaacatc tcagatgagt tttctagcaa
tgttgcaaat tatcaaaagg
|
16981tcggcatgca aaagtactct acactccaag gaccacctgg
tactggtaag agtcattttg
|
17041ccatcggact tgctctctat tacccatctg ctcgcatagt
gtatacggca tgctctcatg
|
17101cagctgttga tgccctatgt gaaaaggcat taaaatattt
gcccatagat aaatgtagta
|
17161gaatcatacc tgcgcgtgcg cgcgtagagt gttttgataa
attcaaagtg aattcaacac
|
17221tagaacagta tgttttctgc actgtaaatg cattgccaga
aacaactgct gacattgtag
|
17281tctttgatga aatctctatg gctactaatt atgacttgag
tgttgtcaat gctagacttc
|
17341gtgcaaaaca ctacgtctat attggcgatc ctgctcaatt
accagccccc cgcacattgc
|
17401tgactaaagg cacactagaa ccagaatatt ttaattcagt
gtgcagactt atgaaaacaa
|
17461taggtccaga catgttcctt ggaacttgtc gccgttgtcc
tgctgaaatt gttgacactg
|
17521tgagtgcttt agtttatgac aataagctaa aagcacacaa
ggataagtca gctcaatgct
|
17581tcaaaatgtt ctacaaaggt gttattacac atgatgtttc
atctgcaatc aacagacctc
|
17641aaataggcgt tgtaagagaa tttcttacac gcaatcctgc
ttggagaaaa gctgttttta
|
17701tctcacctta taattcacag aacgctgtag cttcaaaaat
cttaggattg cctacgcaga
|
17761ctgttgattc atcacagggt tctgaatatg actatgtcat
attcacacaa actactgaaa
|
17821cagcacactc ttgtaatgtc aaccgcttca atgtggctat
cacaagggca aaaattggca
|
17881ttttgtgcat aatgtctgat agagatcttt atgacaaact
gcaatttaca agtctagaaa
|
17941taccacgtcg caatgtggct acattacaag cagaaaatgt
aactggactt tttaaggact
|
18001gtagtaagat cattactggt cttcatccta cacaggcacc
tacacacctc agcgttgata
|
18061taaagttcaa gactgaagga ttatgtgttg acataccagg
cataccaaag gacatgacct
|
18121accgtagact catctctatg atgggtttca aaatgaatta
ccaagtcaat ggttacccta
|
18181atatgtttat cacccgcgaa gaagctattc gtcacgttcg
tgcgtggatt ggctttgatg
|
18241tagagggctg tcatgcaact agagatgctg tgggtactaa
cctacctctc cagctaggat
|
18301tttctacagg tgttaactta gtagctgtac cgactggtta
tgttgacact gaaaataaca
|
18361cagaattcac cagagttaat gcaaaacctc caccaggtga
ccagtttaaa catcttatac
|
18421cactcatgta taaaggcttg ccctggaatg tagtgcgtat
taagatagta caaatgctca
|
18481gtgatacact gaaaggattg tcagacagag tcgtgttcgt
cctttgggcg catggctttg
|
18541agcttacatc aatgaagtac tttgtcaaga ttggacctga
aagaacgtgt tgtctgtgtg
|
18601acaaacgtgc aacttgcttt tctacttcat cagatactta
tgcctgctgg aatcattctg
|
18661tgggttttga ctatgtctat aacccattta tgattgatgt
tcagcagtgg ggctttacgg
|
18721gtaaccttca gagtaaccat gaccaacatt gccaggtaca
tggaaatgca catgtggcta
|
18781gttgtgatgc tatcatgact agatgtttag cagtccatga
gtgctttgtt aagcgcgttg
|
18841attggtctgt tgaataccct attataggag atgaactgag
ggttaattct gcttgcagaa
|
18901aagtacaaca catggttgtg aagtctgcat tgcttgctga
taagtttcca gttcttcatg
|
18961acattggaaa tccaaaggct atcaagtgtg tgcctcaggc
tgaagtagaa tggaagttct
|
19021acgatgctca gccatgtagt gacaaagctt acaaaataga
ggaactcttc tattcttatg
|
19081ctacacatca cgataaattc actgatggtg tttgtttgtt
ttggaattgt aacgttgatc
|
19141gttacccagc caatgcaatt gtgtgtaggt ttgacacaag
agtcttgtca aacttgaact
|
19201taccaggctg tgatggtggt agtttgtatg tgaataagca
tgcattccac actccagctt
|
19261tcgataaaag tgcatttact aatttaaagc aattgccttt
cttttactat tctgatagtc
|
19321cttgtgagtc tcatggcaaa caagtagtgt cggatattga
ttatgttcca ctcaaatctg
|
19381ctacgtgtat tacacgatgc aatttaggtg gtgctgtttg
cagacaccat gcaaatgagt
|
19441accgacagta cttggatgca tataatatga tgatttctgc
tggatttagc ctatggattt
|
19501acaaacaatt tgatacttat aacctgtgga atacatttac
caggttacag agtttagaaa
|
19561atgtggctta taatgttgtt aataaaggac actttgatgg
acacgccggc gaagcacctg
|
19621tttccatcat taataatgct gtttacacaa aggtagatgg
tattgatgtg gagatctttg
|
19681aaaataagac aacacttcct gttaatgttg catttgagct
ttgggctaag cgtaacatta
|
19741aaccagtgcc agagattaag atactcaata atttgggtgt
tgatatcgct gctaatactg
|
19801taatctggga ctacaaaaga gaagccccag cacatgtatc
tacaataggt gtctgcacaa
|
19861tgactgacat tgccaagaaa cctactgaga gtgcttgttc
ttcacttact gtcttgtttg
|
19921atggtagagt ggaaggacag gtagaccttt ttagaaacgc
ccgtaatggt gttttaataa
|
19981cagaaggttc agtcaaaggt ctaacacctt caaagggacc
agcacaagct agcgtcaatg
|
20041gagtcacatt aattggagaa tcagtaaaaa cacagtttaa
ctactttaag aaagtagacg
|
20101gcattattca acagttgcct gaaacctact ttactcagag
cagagactta gaggatttta
|
20161agcccagatc acaaatggaa actgactttc tcgagctcgc
tatggatgaa ttcatacagc
|
20221gatataagct cgagggctat gccttcgaac acatcgttta
tggagatttc agtcatggac
|
20281aacttggcgg tcttcattta atgataggct tagccaagcg
ctcacaagat tcaccactta
|
20341aattagagga ttttatccct atggacagca cagtgaaaaa
ttacttcata acagatgcgc
|
20401aaacaggttc atcaaaatgt gtgtgttctg tgattgatct
tttacttgat gactttgtcg
|
20461agataataaa gtcacaagat ttgtcagtga tttcaaaagt
ggtcaaggtt acaattgact
|
20521atgctgaaat ttcattcatg ctttggtgta aggatggaca
tgttgaaacc ttctacccaa
|
20581aactacaagc aagtcaagcg tggcaaccag gtgttgcgat
gcctaacttg tacaagatgc
|
20641aaagaatgct tcttgaaaag tgtgaccttc agaattatgg
tgaaaatgct gttataccaa
|
20701aaggaataat gatgaatgtc gcaaagtata ctcaactgtg
tcaatactta aatacactta
|
20761ctttagctgt accctacaac atgagagtta ttcactttgg
tgctggctct gataaaggag
|
20821ttgcaccagg tacagctgtg ctcagacaat ggttgccaac
tggcacacta cttgtcgatt
|
20881cagatcttaa tgacttcgtc tccgacgcag attctacttt
aattggagac tgtgcaacag
|
20941tacatacggc taataaatgg gaccttatta ttagcgatat
gtatgaccct aggaccaaac
|
21001atgtgacaaa agagaatgac tctaaagaag ggtttttcac
ttatctgtgt ggatttataa
|
21061agcaaaaact agccctgggt ggttctatag ctgtaaagat
aacagagcat tcttggaatg
|
21121ctgaccttta caagcttatg ggccatttct catggtggac
agcttttgtt acaaatgtaa
|
21181atgcatcatc atcggaagca tttttaattg gggctaacta
tcttggcaag ccgaaggaac
|
21241aaattgatgg ctataccatg catgctaact acattttctg
gaggaacaca aatcctatcc
|
21301agttgtcttc ctattcactc tttgacatga gcaaatttcc
tcttaaatta agaggaactg
|
21361ctgtaatgtc tcttaaggag aatcaaatca atgatatgat
ttattctctt ctggaaaaag
|
21421gtaggcttat cattagagaa aacaacagag ttgtggtttc
aagtgatatt cttgttaaca
|
21481actaaacgaa catgtttatt ttcttattat ttcttactct
cactagtggt agtgaccttg
|
21541accggtgcac cacttttgat gatgttcaag ctcctaatta
cactcaacat acttcatcta
|
21601tgaggggggt ttactatcct gatgaaattt ttagatcaga
cactctttat ttaactcagg
|
21661atttatttct tccattttat tctaatgtta cagggtttca
tactattaat catacgtttg
|
21721gcaaccctgt catacctttt aaggatggta tttattttgc
tgccacagag aaatcaaatg
|
21781ttgtccgtgg ttgggttttt ggttctacca tgaacaacaa
gtcacagtcg gtgattatta
|
21841ttaacaattc tactaatgtt gttatacgag catgtaactt
tgaattgtgt gacaaccctt
|
21901tctttgctgt ttctaaaccc atgggtacac agacacatac
tatgatattc gataatgcat
|
21961ttaattgcac tttcgagtac atatctgatg ccttttcgct
tgatgtttca gaaaagtcag
|
22021gtaattttaa acacttacga gagtttgtgt ttaaaaataa
agatgggttt ctctatgttt
|
22081ataagggcta tcaacctata gatgtagttc gtgatctacc
ttctggtttt aacactttga
|
22141aacctatttt taagttgcct cttggtatta acattacaaa
ttttagagcc attcttacag
|
22201ccttttcacc tgctcaagac atttggggca cgtcagctgc
agcctatttt gttggctatt
|
22261taaagccaac tacatttatg ctcaagtatg atgaaaatgg
tacaatcaca gatgctgttg
|
22321attgttctca aaatccactt gctgaactca aatgctctgt
taagagcttt gagattgaca
|
22381aaggaattta ccagacctct aatttcaggg ttgttccctc
aggagatgtt gtgagattcc
|
22441ctaatattac aaacttgtgt ccttttggag aggtttttaa
tgctactaaa ttcccttctg
|
22501tctatgcatg ggagagaaaa aaaatttcta attgtgttgc
tgattactct gtgctctaca
|
22561actcaacatt tttttcaacc tttaagtgct atggcgtttc
tgccactaag ttgaatgatc
|
22621tttgcttctc caatgtctat gcagattctt ttgtagtcaa
gggagatgat gtaagacaaa
|
22681tagcgccagg acaaactggt gttattgctg attataatta
taaattgcca gatgatttca
|
22741tgggttgtgt ccttgcttgg aatactagga acattgatgc
tacttcaact ggtaattata
|
22801attataaata taggtatctt agacatggca agcttaggcc
ctttgagaga gacatatcta
|
22861atgtgccttt ctcccctgat ggcaaacctt gcaccccacc
tgctcttaat tgttattggc
|
22921cattaaatga ttatggtttt tacaccacta ctggcattgg
ctaccaacct tacagagttg
|
22981tagtactttc ttttgaactt ttaaatgcac cggccacggt
ttgtggacca aaattatcca
|
23041ctgaccttat taagaaccag tgtgtcaatt ttaattttaa
tggactcact ggtactggtg
|
23101tgttaactcc ttcttcaaag agatttcaac catttcaaca
atttggccgt gatgtttctg
|
23161atttcactga ttccgttcga gatcctaaaa catctgaaat
attagacatt tcaccttgcg
|
23221cttttggggg tgtaagtgta attacacctg gaacaaatgc
ttcatctgaa gttgctgttc
|
23281tatatcaaga tgttaactgc actgatgttt ctacagcaat
tcatgcagat caactcacac
|
23341cagcttggcg catatattct actggaaaca atgtattcca
gactcaagca ggctgtctta
|
23401taggagctga gcatgtcgac acttcttatg agtgcgacat
tcctattgga gctggcattt
|
23461gtgctagtta ccatacagtt tctttattac gtagtactag
ccaaaaatct attgtggctt
|
23521atactatgtc tttaggtgct gatagttcaa ttgcttactc
taataacacc attgctatac
|
23581ctactaactt ttcaattagc attactacag aagtaatgcc
tgtttctatg gctaaaacct
|
23641ccgtagattg taatatgtac atctgcggag attctactga
atgtgctaat ttgcttctcc
|
23701aatatggtag cttttgcaca caactaaatc gtgcactctc
aggtattgct gctgaacagg
|
23761atcgcaacac acgtgaagtg ttcgctcaag tcaaacaaat
gtacaaaacc ccaactttga
|
23821aatattttgg tggttttaat ttttcacaaa tattacctga
ccctctaaag ccaactaaga
|
23881ggtcttttat tgaggacttg ctctttaata aggtgacact
cgctgatgct ggcttcatga
|
23941agcaatatgg cgaatgccta ggtgatatta atgctagaga
tctcatttgt gcgcagaagt
|
24001tcaatggact tacagtgttg ccacctctgc tcactgatga
tatgattgct gcctacactg
|
24061ctgctctagt tagtggtact gccactgctg gatggacatt
tggtgctggc gctgctcttc
|
24121aaataccttt tgctatgcaa atggcatata ggttcaatgg
cattggagtt acccaaaatg
|
24181ttctctatga gaaccaaaaa caaatcgcca accaatttaa
caaggcgatt agtcaaattc
|
24241aagaatcact tacaacaaca tcaactgcat tgggcaagct
gcaagacgtt gttaaccaga
|
24301atgctcaagc attaaacaca cttgttaaac aacttagctc
taattttggt gcaatttcaa
|
24361gtgtgctaaa tgatatcctt tcgcgacttg ataaagtcga
ggcggaggta caaattgaca
|
24421ggttaattac aggcagactt caaagccttc aaacctatgt
aacacaacaa ctaatcaggg
|
24481ctgctgaaat cagggcttct gctaatcttg ctgctactaa
aatgtctgag tgtgttcttg
|
24541gacaatcaaa aagagttgac ttttgtggaa agggctacca
ccttatgtcc ttcccacaag
|
24601cagccccgca tggtgttgtc ttcctacatg tcacgtatgt
gccatcccag gagaggaact
|
24661tcaccacagc gccagcaatt tgtcatgaag gcaaagcata
cttccctcgt gaaggtgttt
|
24721ttgtgtttaa tggcacttct tggtttatta cacagaggaa
cttcttttct ccacaaataa
|
24781ttactacaga caatacattt gtctcaggaa attgtgatgt
cgttattggc atcattaaca
|
24841acacagttta tgatcctctg caacctgagc ttgactcatt
caaagaagag ctggacaagt
|
24901acttcaaaaa tcatacatca ccagatgttg atcttggcga
catttcaggc attaacgctt
|
24961ctgtcgtcaa cattcaaaaa gaaattgacc gcctcaatga
ggtcgctaaa aatttaaatg
|
25021aatcactcat tgaccttcaa gaattgggaa aatatgagca
atatattaaa tggccttggt
|
25081atgtttggct cggcttcatt gctggactaa ttgccatcgt
catggttaca atcttgcttt
|
25141gttgcatgac tagttgttgc agttgcctca agggtgcatg
ctcttgtggt tcttgctgca
|
25201agtttgatga ggatgactct gagccagttc tcaagggtgt
caaattacat tacacataaa
|
25261cgaacttatg gatttgttta tgagattttt tactcttaga
tcaattactg cacagccagt
|
25321aaaaattgac aatgcttctc ctgcaagtac tgttcatgct
acagcaacga taccgctaca
|
25381agcctcactc cctttcggat ggcttgttat tggcgttgca
tttcttgctg tttttcagag
|
25441cgctaccaaa ataattgcgc tcaataaaag atggcagcta
gccctttata agggcttcca
|
25501gttcatttgc aatttactgc tgctatttgt tacdatctat
tcacatcttt tgcttgtcgc
|
25561tgcaggtatg gaggcgcaat ttttgtacct ctatgccttg
atatattttc tacaatgcat
|
25621caacgcatgt agaattatta tgagatgttg gctttgttgg
aagtgcaaat ccaagaaccc
|
25681attactttat gatgccaact actttgtttg ctggcacaca
cataactatg actactgtat
|
25741accatataac agtgtcacag atacaattgt cgttactgaa
ggtgacggca tttcaacacc
|
25801aaaactcaaa gaagactacc aaattggtgg ttattctgag
gataggcact caggtgttaa
|
25861agactatgtc gttgtacatg gctatttcac cgaagtttac
taccagcttg agtctacaca
|
25921aattactaca gacactggta ttgaaaatgc tacattcttc
atctttaaca agcttgttaa
|
25981agacccaccg aatgtgcaaa tacacacaat cgacggctct
tcaggagttg ctaatccagc
|
26041aatggatcca atttatgatg agccgacgac gactactagc
gtgcctttgt aagcacaaga
|
26101aagtgagtac gaacttatgt actcattcgt ttcggaagaa
acaggtacgt taatagttaa
|
26161tagcgtactt ctttttcttg ctttcgtggt attcttgcta
gtcacactag ccatccttac
|
26221tgcgcttcga ttgtgtgcgt actgctgcaa tattgttaac
gtgagtttag taaaaccaac
|
26281ggtttacgtc tactcgcgtg ttaaaaatct gaactcttct
gaaggagttc ctgatcttct
|
26341ggtctaaacg aactaactat tattattatt ctgtttggaa
ctttaacatt gcttatcatg
|
26401gcagacaacg gtactattac cgttgaggag cttaaacaac
tcctggaaca atggaaccta
|
26461gtaataggtt tcctattcct agcctggatt atgttactac
aatttgccta ttctaatcgg
|
26521aacaggtttt tgtacataat aaagcttgtt ttcctctggc
tcttgtggcc agtaacactt
|
26581gcttgttttg tgcttgctgc tgtctacaga attaattggg
tgactggcgg gattgcgatt
|
26641gcaatggctt gtattgtagg cttgatgtgg cttagctact
tcgttgcttc cttcaggctg
|
26701tttgctcgta cccgctcaat gtggtcattc aacccagaaa
caaacattct tctcaatgtg
|
26761cctctccggg ggacaattgt gaccagaccg ctcatggaaa
gtgaacttgt cattggtgct
|
26821gtgatcattc gtggtcactt gcgaatggcc ggacactccc
tagggcgctg tgacattaag
|
26881gacctgccaa aagagatcac tgtggctaca tcacgaacgc
tttcttatta caaattagga
|
26941gcgtcgcagc gtgtaggcac tgattcaggt tttgctgcat
acaaccgcta ccgtattgga
|
27001aactataaat taaatacaga ccacgccggt agcaacgaca
atattgcttt gctagtacag
|
27061taagtgacaa cagatgtttc atcttgttga cttccaggtt
acaatagcag agatattgat
|
27121tatcattatg aggactttca ggattgctat ttggaatctt
gacgttataa taagttcaat
|
27181agtgagacaa ttatttaagc ctctaactaa gaagaattat
tcggagttag atgatgaaga
|
27241acctatggag ttagattatc cataaaacga acatgaaaat
tattctcttc ctgacattga
|
27301ttgtatttac atcttgcgag ctatatcact atcaggagtg
tgttagaggt acgactgtac
|
27361tactaaaaga accttgccca tcaggaacat acgagggcaa
ttcaccattt caccctcttg
|
27421ctgacaataa atttgcacta acttgcacta gcacacactt
tgcttttgct tgtgctgacg
|
27481gtactcgaca tacctatcag ctgcgtgcaa gatcagtttc
accaaaactt ttcatcagac
|
27541aagaggaggt tcaacaagag ctctactcgc cactttttct
cattgttgct gctctagtat
|
27601ttttaatact ttgcttcacc attaagagaa agacagaatg
aatgagctca ctttaattga
|
27661cttctatttg tgctttttag cctttctgct attccttgtt
ttaataatgc ttattatatt
|
27721ttggttttca ctcgaaatcc aggatctaga agaaccttgt
accaaagtct aaacgaacat
|
27781gaaacttctc attgttttga cttgtatttc tctatgcagt
tgcatatgca ctgtagtaca
|
27841gcgctgtgca tctaataaac ctcatgtgct tgaagatcct
tgtaaggtac aacactaggg
|
27901gtaatactta tagcactgct tggctttgtg ctctaggaaa
ggttttacct tttcatagat
|
27961ggcacactat ggttcaaaca tgcacaccta atgttactat
caactgtcaa gatccagctg
|
28021gtggtgcgct tatagctagg tgttggtacc ttcatgaagg
tcaccaaact gctgcattta
|
28081gagacgtact tgttgtttta aataaacgaa caaattaaaa
tgtctgataa tggaccccaa
|
28141tcaaaccaac gtagtgcccc ccgcattaca tttggtggac
ccacagattc aactgacaat
|
28201aaccagaatg gaggacgcaa tggggcaagg ccaaaacagc
gccgacccca aggtttaccc
|
28261aataatactg cgtcttggtt cacagctctc actcagcatg
gcaaggagga acttagattc
|
28321cctcgaggcc agggcgttcc aatcaacacc aatagtggtc
cagatgacca aattggctac
|
28381taccgaagag ctacccgacg agttcgtggt ggtgacggca
aaatgaaaga gctcagcccc
|
28441agatggtact tctattacct aggaactggc ccagaagctt
cacttcccta cggcgctaac
|
28501aaagaaggca tcgtatgggt tgcaactgag ggagccttga
atacacccaa agaccacatt
|
28561ggcacccgca atcctaataa caatgctgcc accgtgctac
aacttcctca aggaacaaca
|
28621ttgccaaaag gcttctacgc agagggaagc agaggcggca
gtcaagcctc ttctcgctcc
|
28681tcatcacgta gtcgcggtaa ttcaagaaat tcaactcctg
gcagcagtag gggaaattct
|
28741cctgctcgaa tggctagcgg aggtggtgaa actgccctcg
cgctattgct gctagacaga
|
28801ttgaaccagc ttgagagcaa agtttctggt aaaggccaac
aacaacaagg ccaaactgtc
|
28861actaagaaat ctgctgctga ggcatctaaa aagcctcgcc
aaaaacgtac tgccacaaaa
|
28921cagtacaacg tcactcaagc atttgggaga cgtggtccag
aacaaaccca aggaaatttc
|
28981ggggaccaag acctaatcag acaaggaact gattacaaac
attggccgca aattgcacaa
|
29041tttgctccaa gtgcctctgc attctttgga atgtcacgca
ttggcatgga agtcacacct
|
29101tcgggaacat ggctgactta tcatggagcc attaaattgg
atgacaaaga tccacaattc
|
29161aaagacaacg tcatactgct gaacaagcac attgacgcat
acaaaacatt cccaccaaca
|
29221gagcctaaaa aggacaaaaa gaaaaagact gatgaagctc
agcctttgcc gcagagacaa
|
29281aagaagcagc ccactgtgac tcttcttcct gcggctgaca
tggatgattt ctccagacaa
|
29341cttcaaaatt ccatgagtgg agcttctgct gattcaactc
aggcataaac actcatgatg
|
29401accacacaag gcagatgggc tatgtaaacg ttttcgcaat
tccgtttacg atacatagtc
|
29461tactcttgtg cagaatgaat tctcgtaact aaacagcaca
agtaggttta gttaacttta
|
29521atctcacata gcaatcttta atcaatgtgt aacattaggg
aggacttgaa agagccacca
|
29581cattttcatc gaggccacgc ggagtacgat cgagggtaca
gtgaataatg ctagggagag
|
29641ctgcctatat ggaagagccc taatgtgtaa aattaatttt
agtagtgcta tccccatgtg
|
29701attttaatag cttcttagga gaatgacaaa aaaaaaaaaa
aaaaaaaaaa a
|
While placement of the fluorescent protein within the coronavirus genome is preferred, additional preferred embodiments of the invention provide for the construction of virus-fluorescent fusion proteins that permit one of ordinary skill in the art to follow viral reproduction in an animal model. Either viral structural proteins or non-structural proteins can be used as fusion protein partners. Preferred structural proteins for use as fusion protein partners include but are not limited to a nucleocapsid phosphoprotein, a spike glycoprotein, a membrane glycoprotein, a small envelope protein, or a hemagglutinin-esterase glycoprotein. Sequences for each of these proteins have been disclosed in the art for a variety of coronaviruses, including the murine and SARS strains.
Model
The disclosed invention uses recombinant coronaviruses that are engineered to express a marker, such as a fluorescent protein. By infecting a model organism with the described recombinant coronavirus, one of ordinary skill in the art can use the recombinant virus to study the progression of viral replication in the host animal. Furthermore, the recombinant coronavirus model system has utility as an assay for identifying antiviral agents that slow or inhibit coronavirus replication.
The label used in the various aspects of the invention is a fluorescent protein. The native gene encoding the seminal protein in this class, green fluorescent protein (GFP) has been cloned from the bioluminescent jellyfish Aequorea victoria (Morin, J., et al., J. Cell Physiol (1972) 77:313-318). The availability of the gene has made it possible to use GFP as a marker for gene expression. The original GFP itself is a 283 amino acid protein with a molecular weight of 27 kD. It requires no additional proteins from its native source nor does it require substrates or cofactors available only in its native source in order to fluoresce. (Prasher, D. C., et al., Gene (1992) 111:229-233; Yang, F., et al., Nature Biotechnol (1996) 14:1252-1256; Cody, C. W., et al., Biochemistry (1993) 32:1212-1218.) Mutants of the original GFP gene have been found useful to enhance expression and to modify excitation and fluorescence, so that “GFP” in various colors, including reds and blues has been obtained. GFP-S65T (wherein serine at 65 is replaced with threonine) is particularly useful in the present invention method and has a single excitation peak at 490 nm. (Heim, R., et al., Nature (1995) 373:663-664); U.S. Pat. No. 5,625,048. Other mutants have also been disclosed by Delagrade, S., et al., Biotechnology (1995) 13:151-154; Cormack, B., et al., Gene (1996) 173:33-38 and Cramer, A., et al., Nature Biotechnol (1996) 14:315-319. Additional mutants are also disclosed in U.S. Pat. No. 5,625,048. By suitable modification, the spectrum of light emitted by the GFP can be altered. Thus, although the term “GFP” is often used in the present application, the proteins included within this definition are not necessarily green in appearance. Various forms of GFP exhibit colors other than green and these, too, are included within the definition of “GFP” and are useful in the methods and materials of the invention. In addition, it is noted that green fluorescent proteins falling within the definition of “GFP” herein have been isolated from other organisms, such as the sea pansy, Renilla reniformis. Any suitable and convenient form of GFP can be used to modify the infectious agents useful in the invention, both native and mutated forms.
In order to avoid confusion, the simple term “fluorescent protein” will be used; in general, this is understood to refer to the fluorescent proteins which are produced by various organisms, such as Renilla and Aequorea as well as modified forms of these native fluorescent proteins which may fluoresce in various visible colors, such as red, yellow, and cobalt, which are exhibited by red fluorescent protein (RFP), yellow fluorescent protein (YFP) or cobalt fluorescent protein (CFP), respectively. In general, the terms “fluorescent protein” and “GFP” or “RFP” are used interchangeably.
Because fluorescent proteins are available in a variety of colors, imaging with respect to more than a single color can be done simultaneously. For example, two different infective agents or three different infective agents each expressing a characteristic fluorescence can be administered to the organism and differential effects of proposed treatments evaluated. In addition, a single infectious organism could be labeled constitutively with a single color and a different color used to produce a fusion with a gene product either intracellular or that is secreted. Thus, the nucleotide sequence encoding a fluorescent protein having a color different from that used to label the organism per se can be inserted at a locus to be studied or as a fusion protein in a vector with a protein to be studied. Two-color imaging will be used to visualize targeting of the virus to particular sites in the model, such as the lungs. Further, one or more infective agents can each be labeled with a single color, a gene of interest with another color, and the host model tissue with a third color. For example, fluorescence-expressing coronavirus models will enable visualization of viral reproduction by whole body imaging.
The method of the disclosed invention can be used, to monitor the replication of the recombinant coronaviruses discussed above and the affect various antiviral agents such as chemotherapeutic agents and antiviral vaccines have on coronavirus reproduction.
The methods of the invention utilize infectious agents which have been modified to express the nucleotide sequence encoding a fluorescent protein, preferably of sufficient fluorescence intensity that the fluorescence can be seen in the subject without the necessity of any invasive technique. While whole body imaging is preferred because of the possibility of real-time observation, endoscopic techniques, for example, can also be employed or, if desired, tissues or organs excised for direct or histochemical observation.
The nucleotide sequence encoding the fluorescent protein may be introduced into the infectious agent by direct modification, such as modification of a viral genome to locate the fluorescent protein encoding sequence in a suitable position under the control sequences endogenous to the virus, or may be introduced into microbial systems using appropriate expression vectors.
The appropriately modified infectious agent is then administered to the subject in a manner which mimics, if desired, the route of infection believed used by the agent or by an arbitrary route. Administration may be by injection, gavage, oral, by aerosol into the respiratory system, by suppository, by contact with a mucosal surface in general, or by any suitable means known in the art to introduce infectious agents.
Although endoscopy can be used as well as excision of individual tissues, it is particularly convenient to visualize the migration of infective agent and infected cells in the intact animal through fluorescent imaging. This permits real-time observation and monitoring of progression of infection on a continuous basis, in particular, in model systems, in evaluation of potential anti-infective drugs and protocols. Thus, the inhibition of infection observed directly in test animals administered a candidate drug or protocol in comparison to controls which have not been administered the drug or protocol indicates the efficacy of the candidate and its potential as a treatment. In subjects being treated for infection, the availability of fluorescent imaging permits those devising treatment protocols to be informed on a continuous basis of the advisability of modifying or not modifying the protocol. In one embodiment, to screen for effective antiviral agents, recombinant coronaviruses that express fluorescently-labeled viral proteins are injected into a murine model to follow viral reproduction. Sites of viral infection are highly fluorescent and readily visualized by blue light excitation in a light box with a CCD camera and a GFP filter.
Suitable vertebrate subjects for use as models are preferably mammalian subjects, most preferably convenient laboratory animals such as rabbits, rats, mice, and the like. For closer analogy to human subjects, primates could also be used. Any appropriate vertebrate subject can be used, the choice being dictated mainly by convenience and similarity to the system of ultimate interest. Ultimately, the vertebrate subjects can be humans.
The following examples are offered to illustrate but not to limit the invention.
EXAMPLE 1
A. Background
A dual-color fluorescence imaging model of tumor-host interaction based on an RFP-expressing tumor growing in GFP transgenic mice, enabling dual-color visualization of the tumor-stroma interaction including tumor angiogenesis and infiltration of lymphocytes in the tumor has been described. Transgenic mice expressing the GFP under the control of a chicken beta-actin promoter and cytomegalovirus enhancer were used as the host (Okabe, M., et al., FEBS Lett (1997) 407:315-319). All of the tissues from this transgenic line fluoresce green under blue excitation light. RFP-expressing B16F0 (B16F0-RFP) mouse melanoma cells were transduced with the pLNCX2-DsRed-2-RFP plasmid. The B16F0-RFP tumor and GFP-expressing host cells could be clearly imaged simultaneously. High-resolution dual-color images enabled resolution of the tumor cells and the host tissues down to the single cell level. Host cells including fibroblasts, tumor infiltrating lymphocytes, dendritic cells, blood vessels and capillaries that express GFP, could be readily distinguished from the RFP-expressing tumor cells. This dual-color fluorescence imaging system should facilitate studies for understanding tumor-host interaction during tumor growth and tumor angiogenesis. The dual-colored chimeric system also provides a powerful tool to analyze and isolate tumor infiltrating lymphocytes and other host stromal cells interacting with the tumor for therapeutic and diagnostic/analytic purposes. The principles of this model are used in the dual-color imageable RFP-MHV-GFP-host infectious model of the invention.
B. Methods
Viruses and cells: The methods of de Haan, et al., Virol. (2002) 296:177-189 are followed. The MHV-A59 temperature-sensitive (ts) mutant LA16, the plaque-cloned MHV-JHM, and virus sample obtained after 19 undiluted passages of original plaque-cloned MHV-JHM (JHM19th) are employed. Mouse DBT cells are used for RNA transfection and propagation of viruses.
The methods of Kim, K. H., J. Virol. (1995) 69:2313-2321 are followed, in the following sections:
Preparation of virus-specific intracellular RNA and Northern (RNA) blotting: Virus-specific RNAs are extracted from virus-infected cells. 1.5 mg of intracellular RNA is denatured and electrophoresed through a 1% agarose gel containing formaldehyde. The separated RNA was blotted onto nylon filters. The RNA on the filters is hybridized with 32P-labeled probes specific for the various regions of MHV RNA.
RNA transcription and transfection: Plasmids are linearized by XbaI digestion and transcribed in vitro with T7 RNA polymerase. Lipofection is used for RNA transfection.
Isolation of clones containing the DIssA-specific sequence: For the amplification of a DIssA-related subgenomic RNA, cDNA is first synthesized from intracellular RNA, using as a primer oligonucleotide 1116 (5′-CTGAAACTCTTTTCCCT-3′)(SEQ ID NO: XX), which binds to positive-strand MHV mRNA 7 at nucleotides 250 to 267 from the 5′ end of mRNA 7. MHV-specific cDNA is then incubated with oligonucleotide 78 (5′-AGCTTTACGTACCCTCTCTACTATAAAACTCTTGTAGTTT-3′)(SEQ ID NO: XX), which binds to antileader sequence of MHV RNA, in PCR buffer (0.05 M KCl, 0.01 M Tris hydrochloride [pH 8.3], 0.0025 M MgCl2, 0.01% gelatin, 0.17 mM of each deoxynucleoside triphosphate, 5 U of Taq polymerase [Promega]) at 93.8° C. for 30 s, 37.8° C. for 45 s, and 72.8° C. for 100 s for 25 cycles DIssA subgenomic RNA were separated by agarose gelelectrophoresis and hybridized with a probe which corresponds to 1.5 to 1.7 kb from the 3′ end of MHV genomic RNA. This probe hybridizes with all MHV mRNAs. The DIssA subgenomic RNA-specific RT-PCR product is eluted from the gel and cloned into the TA cloning vector (Invitrogen). Clones containing DIssA-specific sequence are isolated by colony hybridization using the probe that was used for Southern blot analysis. For amplification of DIssA RNA, cDNA is first synthesized from gel-purified DIssA RNA by using oligonucleotide 1116 as a primer. DIssA-specific cDNA is then incubated with oligonucleotide 10121 (5′-GAAGGGTTGTATGTGTTG-3′)(SEQ ID NO: XX), which binds to negative strand MHV RNA at nucleotides 798 to 815 from the 5′ end of gene 2, in PCR buffer under the PCR conditions described above. The DIssA-specific RT-PCR product is eluted from the preparative gel and cloned into the TA cloning vector. Clones containing DIssA-specific sequences are isolated by colony hybridization using the probe which hybridizes at MHV gene 2-1.
Construction of Mouse Hepatitis Full-Length cDNA linked to RFP: DIssA is a naturally occurring self-replicating DI RNA with nearly intact genes 1 and 7 of the MHV as noted above. We will flank gene 1 and gene 7 of the cDNA of MHV, plus the RFP gene in bacterial artificial chromosome (BAC) pBeloBACII, at its 5′ end by the CMV immediate-early promoter and at its 3′ end followed by poly(A) tail in turn followed by the hepatitis delta virus ribozyme and the bovine GH termination and polyadenylation sequences pBAC-MHV-RFP (see, Almazan, F., et al, PNAS (2000) 97:5516-5521).
Transfection and Recovery of an Infectious Virus from a cDNA Clone: The methods of Almazan, F., et al., PNAS (2000) 97:5516-5521 are used in this procedure. The mouse DBT cells are used for transfected by pBAC-MHV-RFP. After an incubation period of 2 days, the cell supernatant was harvested and passaged six times on fresh DBT cells. Virus present in the cell supernatant was analyzed by plaque tritation and RT-PCR.
RFP Expression Vectors (See, Yang, M., Proc. Natl. Acad. Sci. USA (2002) 99:3824-3829). The pLNCX2 vectors is purchased from CLONTECH Laboratories, Inc. (Palo Alto, Cailf.). The pLNCX2 vector contains the neomycin resistance gene for antibiotic selection in eukaryotic cells. The red fluorescent protein (RFP), (DsRed2, CLONTECH Laboratories, Inc., Palo Alto, Cailf.), is inserted in the pLNCX2 vector at the Egl II and Not I sites.
RFP vector production (See, Yang, M., Proc. Natl. Acad. Sci. USA (2002) 99:3824-3829). For retroviral transduction, PT67, an NIH3T3-derived packaging cell line, expressing the 10 Al viral envelope, is purchased from CLONTECH Laboratories, Inc. PT67 cells are cultured in DME (Irvine Scientific, Santa Ana, Calif.) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Gemini Bio-products, Calabasas, Calif.). For vector production, packaging cells (PT67), at 70% confluence, are incubated with a precipitated mixture of DOTAP™ reagent (Boehringer Mannheim), and saturating amounts of pLEIN-GFP or pLNCX2-DsRed-2-RFP plasmid for 18 hours. Fresh medium is replenished at this time. The cells are examined by fluorescence microscopy 48 hours post-transfection. For selection, the cells are cultured in the presence of 500 μg/ml- 2000 μg/ml of G418 increased in a step-wise manner (Life Technologies, Grand Island, N.Y.) for seven days.
Dual-color imaging of virus-host interaction: After infection of recombinant coronavirus to the GFP transgenic mice, the fresh tissues are cut into ˜1 mm3 pieces. The tissues are digested with trypsin/EDTA at 37 C.° for 10 minutes before examination. After trypsinization, tissues are put on precleaned microscope slides (Fisher Scientific, Pittsburgh, Pa.) and covered with a cover slip (Fisher Scientific). The tissues are pressed to become thin enough by pushing the cover slip to display the intact vasculature on the slides. The GFP-fluorescing host cells that are infected with the coronavirus can be readily observed under fluorescence microscopy. Laser-based systems will be used for whole-body dual-color imaging of the chimeric system (please see below). All fluorescence results will be confirmed by standard immunohistochemical techniques to identify host all types infected by the RFP-MHV.
Fluorescence imaging (See, Yang, M., Proc. Natl. Acad. Sci. USA (2002) 99:3824-3829). A Leica fluorescence stereo microscope model LZ12 equipped with a mercury 50W lamp power supply is used for initial lower resolution imaging. For visualization of both GFP and RFP fluorescence simultaneously, excitation is produced through a D425/60 band pass filter and 470 DCXR dichroic mirror. Emitted fluorescence is collected through a long pass filter GG475 (Chroma Technology, Brattleboro, Vt.). Macroimaging is carried out in a light box (Lightools Research, Encinitas, Calif.). Fluorescence excitation of both GFP and RFP tumors is produced in the lightbox through an interference filter (440+/−20 nm) using slit fiber optics. Fluorescence is observed through a 520 nm long pass filter. Images from the microscope and light box are captured on a Hamamatsu C5810 3-chip cool color CCR camera (Hamamatsu Photonics Systems, Bridgewater, N.J.). Laser-based imaging is carried out with the Spectra Physics model 3941-M1BB dual photon laser, Photon Technology Intl. model GL-3300 nitrogen laser and the Photon Technology Intl. model GL-302 dye laser. Images are processed for contrast and brightness and analyzed with the use of Image Pro Plus 4.0 software (Media Cybernetics, Silver Springs, Md.). High resolution images of 1024×724 pixels are captured directly on an IBM PC or continuously through video output on a high resolution Sony VCR model SLV-R1000 (Sony Corp., Tokyo Japan).
Multiphoton confocal microscopy (Wang, W., et al., Cancer Research (2002) 6278-6288). The dual photon laser (Spectra-Physics model 3941-M1BB) is also used with the Radiance 2000 multiphoton system (Bio-Rad, Hercules, Calif.) at 960 nm, the optimal wavelength for GFP fluorescence. The images are collected using Bio-Rad's Lasersharp 2000 software. Excitation is confmed only to the optical section being observed. No excitation of the fluorophore will occur at 960 run wavelength not in the plane of focus. The Millenia, Tsunami Ti:Sapphire laser, an accessory for the Spectra Physics model 3941-M1BB dual photon laser, has long wavelength optics (beyond 1,000 nm) for RFP multiphoton imaging. Images are processed with Image Pro Plus 4.0 software.
Spectral resolution. Spectral imaging, is the generation of images containing a high-resolution optical spectrum at every pixel, to “unmix” the viral RFP signal from that of the GFP-labeled host. The standard GFP-mouse imaging system (long-pass emission filter) is modified by replacing the usual color camera with the cooled monochrome camera (Roper Scientific CCD thermo-cooled digital camera) and a liquid crystal tunable filter (CRI, Inc., Woburn, Mass.) positioned in front of a conventional macro-lens. Typically, a series of images is taken every 10 nm from 500 to 650 nm and assembled automatically in memory into a spectral “stack.” Using pre-defined GFP or RFP and autofluorescence spectra, the image can be resolved into different images using a linear combination chemometrics-based algorithm that generates images containing only the autofluorescence signals or only the GFP or RFP signals, now visible against essentially a black background. Using spectral autofluorescence subtraction, sensitivity is enhanced due to improvements in signal to noise ratio. The advantages provided by the GFP- or RFP-labeled tumor models, which allow noninvasive, and highly selective imaging, are further enhanced by using wavelength-selective imaging techniques and analysis to image tumors on deep organs such as the lung (personal communication, Richard Levenson, CRI, Inc., Woburn, Mass.).
Depth of imaging: External visualization of single cells or microscopic colonies of viral infected cells on internal organs is one goal of this application. Imaging of this power requires reducing scatter of excitation and emission light. Multiphoton and single photon lasers will be used for deeper penetration in the living animal. Confocal microscopy will also be used in conjunction with the multiphoton laser. The relatively high wave length of the excitation light, about 470 nm (960 nm for GFP dual photon and about 1,220 nm for RFP dual photon), will not damage tissue. The multiphoton confocal system will highly limit the irradiation area further protecting the host tissues. Skin-flaps also greatly reduce scatter which we have already shown to enable external single-cell imaging. Use of the long wave length Ds-Red-2-RFP also reduces scatter.
C. Results
The infected mice are treated with various drug regimens and evaluated for replication of the virus with and without the presence of the drug. Drugs that succeed in reducing viral replication are identified as successful candidates as therapeutic agents.
Similarly, mice subjected to immunization procedures to be tested are challenged after immunization with infectious levels of MHV coronavirus. The ability of the subject to resist infection after exposure is then evaluated.