SARS-CoV-2 inactivated vaccine and preparation thereof

Abstract

Provided are SARS-Cov-2 inactivated vaccines and their preparation. Also provided are methods of treating or preventing or immunizing against a disease associated with SARS-Cov-2 viral infection, the method comprising administering to a subject a pharmaceutical composition or vaccine comprising a pharmaceutically effective amount of inactivated SARS-Cov-2 virus and a pharmaceutically acceptable carrier.

Description

Background Technology

[0001] The World Health Organization declared the coronavirus disease (COVID-19) outbreak a Public Health Emergency of International Concern on January 30, 2020, and a pandemic on March 11, 2020. It has been reported that approximately 80% of COVID-19 patients experience mild to moderate symptoms, with about 20% developing severe cases such as severe pneumonia, acute respiratory distress syndrome (ARDS), sepsis, and even death. The number of COVID-19 cases worldwide is growing at an alarming rate. As of April 10, 2020, the total number of confirmed cases worldwide had reached 1,623,173, and the death toll had risen to 97,236. Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is the pathogen of the current pandemic, belonging to the genus Betacoronavirus (β-CoV) of the family Coronaviridae. SARS-CoV-2, along with Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) and Middle East Respiratory Syndrome-associated Coronavirus (MERS-CoV), constitutes the three coronaviruses that pose the greatest threat to human life. SARS-CoV-2 contains a linear, single-stranded, positive-sense RNA genome encoding four structural proteins [including spike (S), envelope (E), membrane (M), and nucleocapsid (N)] (where S is the primary protective antigen that elicits highly effective neutralizing antibodies (NAb), 16 non-structural proteins (nsp1–nsp16), and several accessory proteins. Currently, there are no specific antiviral drugs or vaccines available for the emerging SARS-CoV-2. Therefore, the urgency of developing a vaccine is crucial for containing the pandemic and preventing future viral outbreaks.

[0002] Several types of SARS-CoV-2 vaccines, such as DNA- or RNA-based formulations, recombinant subunits containing viral epitopes, adenovirus-based vectors, and purified inactivated viruses, are under development. Purified inactivated viruses have traditionally been used in vaccine development, and such vaccines have been found to be safe and effective in preventing diseases caused by viruses such as influenza and poliovirus. However, because SARS-CoV-2 is a newly emerging virus, our understanding of its biological properties, transmission, and pathogenesis is limited, making vaccine development a significant challenge, let alone for emerging viruses. Summary of the Invention

[0003] This invention provides an inactivated vaccine against SARS-CoV-2 virus, its preparation, and its use. The inactivated vaccine of this invention induces SARS-CoV-2-specific neutralizing antibodies in animals, particularly mammals, including mice, rats, dogs, cats, non-human primates, and humans. These antibodies effectively neutralize at least ten representative SARS-CoV-2 strains worldwide, indicating a potentially broad neutralizing capacity against global SARS-CoV-2 strains. Therefore, the inactivated vaccine of this invention can be used to protect animals from SARS-CoV-2 infection, including but not limited to mice, rats, dogs, cats, non-human primates, and humans. In particular, the inactivated vaccine of this invention does not induce antibody-dependent enhancement. In other words, the inactivated vaccine of this invention induces an effective humoral response without immunopathology. Furthermore, the inactivated vaccine of this invention has been demonstrated to be safe through systematic reviews (including monitoring of clinical signs, blood biochemical indicators, and histopathological analysis).

[0004] In one aspect, the present invention provides an inactivated SARS-CoV-2 virus (SARS-CoV-2) or a derivative or related thereof, wherein the infectivity of SARS-CoV-2 is undetectable, and wherein the inactivated SARS-CoV-2 induces an immune response against the SARS-CoV-2 virus when administered to a patient.

[0005] In some embodiments, the SARS-CoV-2 virus or its derivatives or related substances are inactivated with a virus inactivating agent, preferably β-propiolactone and / or formalin. Preferably, the obtained inactivated SARS-CoV-2 virus or its derivatives or related substances are subsequently purified by ion exchange chromatography (IEC) and / or size exclusion chromatography (SEC). Preferably, the inactivated SARS-CoV-2 virus is treated with a non-restrictive endonuclease prior to size exclusion chromatography (SEC).

[0006] Preferably, the volume ratio of β-propiolactone to the virus harvesting fluid is 1:4000 to 1:6000, and the inactivation time at 4°C is 16 to 72 hours, more preferably 20 to 24 hours. More preferably, the inactivation time is 24 hours.

[0007] In some embodiments, the SARS-CoV-2 virus or its derivatives or related substances include a spike protein (S) having an amino acid sequence encoded by nucleotides 21508 to 25356 of SEQ ID NO:1 or a sequence having at least 95% identity with it.

[0008] In some embodiments, the SARS-CoV-2 virus or its derivatives or related substances include a nucleocapsid protein (N) having an amino acid sequence encoded by nucleotides 28246 to 29505 of SEQ ID NO:1 or a sequence having at least 95% identity with it.

[0009] In some embodiments, the SARS-CoV-2 virus or its derivatives or related substances include a membrane protein (M) having an amino acid sequence encoded by nucleotides 26495 to 27163 of SEQ ID NO:1 or having at least 95% identity with it.

[0010] In some implementations, the SARS-CoV-2 virus or its derivatives or related substances include ORF1a, which has the nucleotide sequence of nucleotides 238 to 13455 of SEQ ID NO:1 or a sequence having at least 95% identity with it.

[0011] In some embodiments, the SARS-CoV-2 virus or its derivatives or related substances include ORF1ab, which has the nucleotide sequence of nucleotides 13740 to 21527 of SEQ ID NO:1 or a sequence having at least 95% identity with it.

[0012] In some implementations, the SARS-CoV-2 virus or its derivatives or related substances include ORF3a, which has the nucleotide sequence of nucleotides 25365 to 26192 of SEQ ID NO:1 or a sequence having at least 95% identity with it.

[0013] Preferably, the SARS-CoV-2 virus or its derivatives or related substances include: ORF1a, having the nucleotide sequence of nucleotides 238 to 13455 of SEQ ID NO:1 or a sequence having at least 95% identity with it; and / or ORF1ab, having the nucleotide sequence of nucleotides 13740 to 21527 of SEQ ID NO:1 or a sequence having at least 95% identity with it; and / or spike protein (S), having the amino acid sequence encoded by nucleotides 21508 to 25356 of SEQ ID NO:1 or a sequence having at least 95% identity with it; and / or ORF3a, having the nucleotide sequence of nucleotides 25365 to 26192 of SEQ ID NO:1 or a sequence having at least 95% identity with it; and / or membrane protein (M), having the amino acid sequence encoded by nucleotides 238 to 1345 ... The amino acid sequence encoded by nucleotides 26495–27163 of SEQ ID NO:1 or a sequence having at least 95% identity with it; and / or nucleocapsid protein (N) having the amino acid sequence encoded by nucleotides 28246–29505 of SEQ ID NO:1 or a sequence having at least 95% identity with it.

[0014] Preferably, the SARS-CoV-2 virus or its derivatives or related substances include: ORF1a, having the nucleotide sequence of nucleotides 238 to 13455 of SEQ ID NO:1; ORF1ab, having the nucleotide sequence of nucleotides 13740 to 21527 of SEQ ID NO:1; spike protein (S), having the amino acid sequence encoded by nucleotides 21508 to 25356 of SEQ ID NO:1; ORF3a, having the nucleotide sequence of nucleotides 25365 to 26192 of SEQ ID NO:1; membrane protein (M), having the amino acid sequence encoded by nucleotides 26495 to 27163 of SEQ ID NO:1; and nucleocapsid protein (N), having the amino acid sequence encoded by nucleotides 28246 to 29505 of SEQ ID NO:1.

[0015] Preferably, the isolated SARS-CoV-2 virus or its derivatives or related substances have a genome comprising an RNA sequence having an inverse complementary sequence shown in any one of SEQ ID NO:1 to 3, or a sequence having at least 95% identity with it.

[0016] Preferably, the SARS-CoV-2 virus or its derivatives or related substances have a genome encoded by a sequence shown in any one of SEQ ID NO:1-3 or a sequence having at least 95% identity with it. Preferably, the SARS-CoV-2 virus or its derivatives or related substances have a genome encoded by a sequence shown in any one of SEQ ID NO:1-3, or have a genome comprising an RNA sequence having an inverse complementary sequence shown in any one of SEQ ID NO:1-3. The SARS-CoV-2 virus or its derivatives or related substances of the present invention exhibit high yields in Vero cells.

[0017] Preferably, before inactivation with β-propiolactone, the SARS-CoV-2 virus is reacted with cells at a density of 2 × 10⁻⁶ cells. 6 cells / mL to 10 × 10 6 Vero cells at a concentration of 1 cell / mL were cultured together in a bioreactor at 35–39°C for 3–7 days to obtain the virus harvest fluid.

[0018] In another aspect, the present invention provides a pharmaceutical composition or vaccine comprising a pharmaceutically effective amount of the inactivated SARS-CoV-2 virus or its derivatives or related substances, and a pharmaceutically acceptable carrier. The composition or vaccine is in a liquid or frozen state, such as a lyophilized state.

[0019] In some embodiments, the dosage of the pharmaceutical composition or vaccine is in the range of 0.5 to 8 μg based on protein weight, preferably in the range of 3 to 6 μg based on protein weight. In other words, each dose of vaccine provides complete protection against SARS-CoV-2 attack at a protein weight of 0.5 to 8 μg, preferably 3 to 6 μg based on protein weight.

[0020] In some embodiments, the SARS-CoV-2 virus is inactivated with β-propiolactone and / or formalin. Preferably, the inactivated SARS-CoV-2 virus is then purified by ion exchange chromatography (IEC) and / or size exclusion chromatography (SEC). Therefore, preferably, the pharmaceutical composition or vaccine comprises inactivated SARS-CoV-2 virus, which is then purified by ion exchange chromatography (IEC) and / or size exclusion chromatography (SEC). Preferably, prior to size exclusion chromatography (SEC), the inactivated SARS-CoV-2 virus is treated with a non-restrictive endonuclease.

[0021] Preferably, the volume ratio of β-propiolactone to the virus harvesting fluid is 1:4000 to 1:6000, and the inactivation time at 4°C is 16 to 72 hours, more preferably 20 to 24 hours. More preferably, the inactivation time is 24 hours.

[0022] Preferably, before inactivation with β-propiolactone, the SARS-CoV-2 virus is reacted with cells at a density of 2 × 10⁻⁶ cells. 6 cells / mL to 10 × 10 6 Vero cells at a concentration of 1 cell / mL were cultured together in a bioreactor at 35–39°C for 3–7 days to obtain the virus harvest fluid.

[0023] Pharmaceutically acceptable carriers may be Al(OH)3 adjuvants and / or CpG oligodeoxynucleotides. In some embodiments, inactivated SARS-CoV-2 virus is filtered through a membrane and then mixed with Al(OH)3 adjuvants and / or CpG oligodeoxynucleotides. Preferably, the membrane has a pore size of 0.2–0.25 μm, more preferably 0.22 μm.

[0024] In some implementations, the CpG oligodeoxynucleotide comprises at least two CpG units of at least 20 bp in length, and all nucleotides in the CpG oligodeoxynucleotide are sulfur-modified.

[0025] In some embodiments, the SARS-CoV-2 virus or its derivatives or related substances include a spike protein (S) having an amino acid sequence encoded by nucleotides 21508 to 25356 of SEQ ID NO:1 or a sequence having at least 95% identity with it.

[0026] In some embodiments, the SARS-CoV-2 virus or its derivatives or related substances include a nucleocapsid protein (N) having an amino acid sequence encoded by nucleotides 28246 to 29505 of SEQ ID NO:1 or a sequence having at least 95% identity with it.

[0027] In some embodiments, the SARS-CoV-2 virus or its derivatives or related substances include a membrane protein (M) having an amino acid sequence encoded by nucleotides 26495 to 27163 of SEQ ID NO:1 or a sequence having at least 95% identity with it.

[0028] In some implementations, the SARS-CoV-2 virus or its derivatives or related substances include ORF1a, which has the nucleotide sequence of nucleotides 238 to 13455 of SEQ ID NO:1 or a sequence having at least 95% identity with it.

[0029] In some embodiments, the SARS-CoV-2 virus or its derivatives or related substances include ORF1ab, which has the nucleotide sequence of nucleotides 13740 to 21527 of SEQ ID NO:1 or a sequence having at least 95% identity with it.

[0030] In some implementations, the SARS-CoV-2 virus or its derivatives or related substances include ORF3a, which has the nucleotide sequence of nucleotides 25365 to 26192 of SEQ ID NO:1 or a sequence having at least 95% identity with it.

[0031] Preferably, the SARS-CoV-2 virus or its derivatives or related substances include: ORF1a, having the nucleotide sequence of nucleotides 238 to 13455 of SEQ ID NO:1 or a sequence having at least 95% identity with it; and / or ORF1ab, having the nucleotide sequence of nucleotides 13740 to 21527 of SEQ ID NO:1 or a sequence having at least 95% identity with it; and / or spike protein (S), having the amino acid sequence encoded by nucleotides 21508 to 25356 of SEQ ID NO:1 or a sequence having at least 95% identity with it; and / or ORF3a, having the nucleotide sequence of nucleotides 25365 to 26192 of SEQ ID NO:1 or a sequence having at least 95% identity with it; and / or membrane protein (M), having the amino acid sequence encoded by nucleotides 238 to 1345 ... The amino acid sequence encoded by nucleotides 26495–27163 of SEQ ID NO:1 or a sequence having at least 95% identity with it; and / or nucleocapsid protein (N) having the amino acid sequence encoded by nucleotides 28246–29505 of SEQ ID NO:1 or a sequence having at least 95% identity with it.

[0032] Preferably, the SARS-CoV-2 virus or its derivatives or related substances include: ORF1a, having the nucleotide sequence of nucleotides 238 to 13455 of SEQ ID NO:1; ORF1ab, having the nucleotide sequence of nucleotides 13740 to 21527 of SEQ ID NO:1; spike protein (S), having the amino acid sequence encoded by nucleotides 21508 to 25356 of SEQ ID NO:1; ORF3a, having the nucleotide sequence of nucleotides 25365 to 26192 of SEQ ID NO:1; membrane protein (M), having the amino acid sequence encoded by nucleotides 26495 to 27163 of SEQ ID NO:1; and nucleocapsid protein (N), having the amino acid sequence encoded by nucleotides 28246 to 29505 of SEQ ID NO:1.

[0033] Preferably, the isolated SARS-CoV-2 virus or its derivatives or related substances have a genome comprising an RNA sequence having an inverse complementary sequence shown in any one of SEQ ID NO:1 to 3, or a sequence having at least 95% identity with it.

[0034] Preferably, the SARS-CoV-2 virus or its derivatives or related substances have a genome encoded by a sequence shown in any one of SEQ ID NO:1 to 3 or a sequence having at least 95% identity with it.

[0035] Preferably, the SARS-CoV-2 virus or its derivatives or related substances have a genome encoded by the sequence shown in any one of SEQ ID NO:1 to 3, or have a genome comprising an RNA sequence having an inverse complementary sequence shown in any one of SEQ ID NO:1 to 3.

[0036] The SARS-CoV-2 virus of this invention exhibits high yield in Vero cells.

[0037] Specifically, the pharmaceutical compositions or vaccines of the present invention provide complete protection against globally prevalent SARS-CoV-2 strains in non-human primates by evoking an effective humoral response without immunopathology.

[0038] The pharmaceutical compositions or vaccines of the present invention can elicit significantly higher S-specific antibody titers than those from serum of recovered COVID-19 patients. This observation, combined with the fact that antibodies targeting the N-type of SARS-CoV-2 do not provide protective immunity against the infection, suggests that the pharmaceutical compositions or vaccines of the present invention can elicit a more effective antibody response.

[0039] Previous experience developing SARS and MERS vaccine candidates has raised concerns about pulmonary immunopathology, which may be directly caused by type 2 helper T cell (Th2) responses or as a result of adverse drug reactions (ADE). While many vaccine-induced T-cell responses have proven crucial for acute viral clearance, protection against subsequent coronavirus infections is primarily mediated by humoral immunity. The “cytokine storm” caused by excessive T-cell responses has indeed highlighted the pathogenesis of COVID-19. Therefore, it is essential to carefully control T-cell responses induced by SARS-CoV-2 vaccines to avoid immunopathology. Against this backdrop, we systematically evaluated the safety of our vaccine in rhesus monkeys by documenting numerous clinical observations and biological indicators. No fever or weight loss was observed in any of the immunized rhesus monkeys, and all animals maintained normal appetite and mental status. Hematological and biochemical analyses, including blood biochemical tests, lymphocyte subset percentages (CD3+, CD4+, and CD8+), and key cytokines (TNF-α, IFN-γ, IL-2, IL-4, IL-5, and IL-6), showed no significant changes in the vaccinated group compared to the sham group and the placebo group. Furthermore, histopathological evaluation of various organs, including the lungs, heart, spleen, liver, kidneys, and brain, indicated that the vaccine of this invention did not induce any significant pathology in rhesus monkeys. No antibody-dependent enhancement of infection (ADE) was observed in any of the vaccinated rhesus monkeys.

[0040] This invention demonstrates the safety of the vaccine in rhesus monkeys; no enhanced infection or worsening of immunopathology was observed. This invention also demonstrates that administration of 6 μg of the vaccine per dose to rhesus monkeys provides complete protection against SARS-CoV-2 challenge.

[0041] On the other hand, the present invention provides a method for preparing an inactivated SARS-CoV-2 composition or vaccine, comprising using a virus inactivating agent, preferably β-propiolactone and / or formalin, to inactivate the SARS-CoV-2 virus.

[0042] In some embodiments, the SARS-CoV-2 virus is cultured in a large bioreactor, such as a Vero cell factory or fermenter, to obtain a virus harvest fluid, which is then inactivated with β-propiolactone for 24 hours. Preferably, the volume ratio of β-propiolactone to the virus harvest fluid is 1:4000 to 1:6000, and the inactivation time at 4°C is 16 to 72 hours, more preferably 20 to 24 hours. Preferably, the inactivation time is 24 hours.

[0043] In some implementation schemes, the volume ratio of formalin to virus harvesting fluid is 1:1000 to 1:4000, and the inactivation time at 4°C is 3 to 72 hours, preferably 3 to 13 hours.

[0044] Preferably, before inactivation with β-propiolactone, the SARS-CoV-2 virus is reacted with cells at a density of 2 × 10⁻⁶ cells. 6 cells / mL to 10 × 10 6 Vero cells at a concentration of 1 cell / mL were cultured together in a bioreactor at 35–39°C for 3–7 days to obtain the virus harvest fluid.

[0045] Preferably, the method further includes the step of purifying the inactivated SARS-CoV-2 virus using ion exchange chromatography (IEC) and / or size exclusion chromatography (SEC). Preferably, prior to size exclusion chromatography (SEC), the inactivated SARS-CoV-2 virus is treated with a non-restrictive endonuclease.

[0046] Preferably, the method further includes the step of mixing the inactivated SARS-CoV-2 virus with an Al(OH)3 adjuvant. In some embodiments, the inactivated SARS-CoV-2 virus is filtered through a membrane and then mixed with an Al(OH)3 adjuvant and / or CpG oligodeoxynucleotides. Preferably, the membrane has a pore size of 0.2–0.25 μm, more preferably 0.22 μm.

[0047] In some implementations, the CpG oligodeoxynucleotide comprises at least two CpG units of at least 20 bp in length, and all nucleotides in the CpG oligodeoxynucleotide are sulfur-modified.

[0048] In some implementations, for vaccine production, the SARS-CoV-2 virus is propagated in a bioreactor in 50–1000 liters of Vero cell culture, such as 50, 100, 200, 300, 400, 500, 600, or 700 liters, and inactivated using β-propiolactone and / or formalin. The virus is purified using centrifugation or deep filtration and two optimized chromatographic steps to produce a highly pure vaccine formulation. Furthermore, cryo-electron microscopy (cryo-EM) analysis reveals intact elliptical particles, 90–150 nm in diameter, dotted with corona spikes, representing the pre-fusion state of the virus.

[0049] In some embodiments, the SARS-CoV-2 virus or its derivatives or related substances include a spike protein (S) having an amino acid sequence encoded by nucleotides 21508 to 25356 of SEQ ID NO:1 or a sequence having at least 95% identity with it.

[0050] In some embodiments, the SARS-CoV-2 virus or its derivatives or related substances include a nucleocapsid protein (N) having an amino acid sequence encoded by nucleotides 28246-29505 of SEQ ID NO:1 or a sequence having at least 95% identity with it.

[0051] In some embodiments, the SARS-CoV-2 virus or its derivatives or related substances include a membrane protein (M) having an amino acid sequence encoded by nucleotides 26495 to 27163 of SEQ ID NO:1 or a sequence having at least 95% identity with it.

[0052] In some implementations, the SARS-CoV-2 virus or its derivatives or related substances include ORF1a, which has the nucleotide sequence of nucleotides 238 to 13455 of SEQ ID NO:1 or a sequence having at least 95% identity with it.

[0053] In some embodiments, the SARS-CoV-2 virus or its derivatives or related substances include ORF1ab, which has the nucleotide sequence of nucleotides 13740 to 21527 of SEQ ID NO:1 or a sequence having at least 95% identity with it.

[0054] In some implementations, the SARS-CoV-2 virus or its derivatives or related substances include ORF3a, which has the nucleotide sequence of nucleotides 25365 to 26192 of SEQ ID NO:1 or a sequence having at least 95% identity with it.

[0055] Preferably, the SARS-CoV-2 virus or its derivatives or related substances include: ORF1a, having the nucleotide sequence of nucleotides 238 to 13455 of SEQ ID NO:1 or a sequence having at least 95% identity with it; and / or ORF1ab, having the nucleotide sequence of nucleotides 13740 to 21527 of SEQ ID NO:1 or a sequence having at least 95% identity with it; and / or spike protein (S), having the amino acid sequence encoded by nucleotides 21508 to 25356 of SEQ ID NO:1 or a sequence having at least 95% identity with it; and / or ORF3a, having the nucleotide sequence of nucleotides 25365 to 26192 of SEQ ID NO:1 or a sequence having at least 95% identity with it; and / or membrane protein (M), having the amino acid sequence encoded by nucleotides 238 to 1345 ... The amino acid sequence encoded by nucleotides 26495–27163 of SEQ ID NO:1 or a sequence having at least 95% identity with it; and / or nucleocapsid protein (N) having the amino acid sequence encoded by nucleotides 28246–29505 of SEQ ID NO:1 or a sequence having at least 95% identity with it.

[0056] Preferably, the SARS-CoV-2 virus or its derivatives or related substances include: ORF1a, having the nucleotide sequence of nucleotides 238 to 13455 of SEQ ID NO:1; ORF1ab, having the nucleotide sequence of nucleotides 13740 to 21527 of SEQ ID NO:1; spike protein (S), having the amino acid sequence encoded by nucleotides 21508 to 25356 of SEQ ID NO:1; ORF3a, having the nucleotide sequence of nucleotides 25365 to 26192 of SEQ ID NO:1; membrane protein (M), having the amino acid sequence encoded by nucleotides 26495 to 27163 of SEQ ID NO:1; and nucleocapsid protein (N), having the amino acid sequence encoded by nucleotides 28246 to 29505 of SEQ ID NO:1.

[0057] Preferably, the isolated SARS-CoV-2 virus or its derivatives or related substances have a genome comprising an RNA sequence having the reverse complementary sequence shown in SEQ ID NO:1 or a sequence having at least 95% identity with it.

[0058] Preferably, the SARS-CoV-2 virus or its derivatives or related substances have a genome encoded by a sequence shown in any one of SEQ ID NO:1 to 3 or a sequence having at least 95% identity with it.

[0059] Preferably, the SARS-CoV-2 virus or its derivatives or related substances have a genome encoded by the sequence shown in any one of SEQ ID NO:1 to 3, or have a genome comprising an RNA sequence having the reverse complementary sequence shown in SEQ ID NO:1.

[0060] The SARS-CoV-2 virus of the present invention exhibits high yield in Vero cells. In another aspect, the present invention provides a method for treating or immunizing against diseases associated with SARS-CoV-2 virus infection, comprising administering to a subject a pharmaceutical composition or vaccine virus comprising a pharmaceutically effective amount of inactivated SARS-CoV-2 virus and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition or vaccine is administered via intradermal, subcutaneous, intramuscular, oral, or intranasal injection. Preferably, the pharmaceutical composition or vaccine is administered via intramuscular injection into the upper deltoid muscle.

[0061] Preferably, the disease includes, but is not limited to, pneumonia, severe acute respiratory infection, kidney failure, heart failure, adult respiratory distress syndrome (ARDS), liver injury, intestinal disease, or severe acute respiratory syndrome.

[0062] Specifically, the pharmaceutical compositions or vaccines of the present invention provide complete protection against globally prevalent SARS-CoV-2 strains in non-human primates by evoking an effective humoral response without immunopathology.

[0063] The pharmaceutical compositions or vaccines of the present invention can elicit significantly higher S-specific antibody titers than those from serum of recovered COVID-19 patients. This observation, combined with the fact that antibodies targeting the N-type of SARS-CoV-2 do not provide protective immunity against the infection, suggests that the pharmaceutical compositions or vaccines of the present invention can elicit a more effective antibody response.

[0064] Previous experience developing SARS and MERS vaccine candidates has raised concerns about pulmonary immunopathology, which could be directly caused by type 2 helper T cell (Th2) responses or as a result of adverse drug reactions (ADE). While many vaccine-induced T-cell responses have been demonstrated to be crucial for acute viral clearance, protection against subsequent coronavirus infections is primarily mediated by humoral immunity. The “cytokine storm” caused by excessive T-cell responses has indeed highlighted the pathogenesis of COVID-19. Therefore, it is essential to carefully control T-cell responses induced by SARS-CoV-2 vaccines to avoid immunopathology. Against this backdrop, we systematically evaluated the safety of our vaccine in rhesus monkeys by documenting numerous clinical observations and biological indicators. No fever or weight loss was observed in any of the rhesus monkeys after immunization, and all animals maintained normal appetite and mental status. Hematological and biochemical analyses, including blood biochemical tests, lymphocyte subset percentages (CD3+, CD4+, and CD8+), and key cytokines (TNF-α, IFN-γ, IL-2, IL-4, IL-5, and IL-6), showed no significant changes in the vaccinated group compared to the sham group and the placebo group. Furthermore, histopathological evaluation of various organs, including the lungs, heart, spleen, liver, kidneys, and brain, indicated that the vaccine of this invention did not induce any significant pathology in rhesus monkeys. No antibody-dependent enhancement of infection (ADE) was observed in any of the vaccinated rhesus monkeys.

[0065] This invention demonstrates the safety of the vaccine in rhesus monkeys; no enhancement of infection or worsening of immunopathology was observed. This invention also demonstrates that administration of the vaccine of this invention, with each dose containing 0.5–8 μg of protein, to rhesus monkeys improves complete protection against SARS-CoV-2 challenge. Preferably, the dosage of the pharmaceutical composition or vaccine is in the range of 2–6 μg by protein weight, and more preferably, in the range of 3–6 μg by protein weight.

[0066] In some embodiments, the SARS-CoV-2 virus is obtained from hospitalized patients. In some embodiments, the SARS-CoV-2 virus has a genomic sequence as shown in 2019-nCoV-BetaCoV / Wuhan / WIV04 / 2019 or EPI_ISL_412973, or a sequence having at least 95% identity with the sequences shown in 2019-nCoV-BetaCoV / Wuhan / WIV04 / 2019 and EPI_ISL_412973, preferably sequences having 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity with the sequences shown in 2019-nCoV-BetaCoV / Wuhan / WIV04 / 2019 or EPI_ISL_412973, respectively.

[0067] In another aspect, the present invention provides an isolated SARS-CoV-2 virus or a derivative or related thereof comprising a spike protein (S), wherein the spike protein (S) has an amino acid sequence encoded by nucleotides 21508 to 25356 of SEQ ID NO:1 or a sequence having at least 95% identity with it.

[0068] In some embodiments, the SARS-CoV-2 virus or its derivatives or related substances include a nucleocapsid protein (N) having an amino acid sequence encoded by nucleotides 28246 to 29505 of SEQ ID NO:1 or a sequence having at least 95% identity with it.

[0069] In some embodiments, the SARS-CoV-2 virus or its derivatives or related substances include a membrane protein (M) having an amino acid sequence encoded by nucleotides 26495 to 27163 of SEQ ID NO:1 or a sequence having at least 95% identity with it.

[0070] In some implementations, the SARS-CoV-2 virus or its derivatives or related substances include ORF1a, which has the nucleotide sequence of nucleotides 238 to 13455 of SEQ ID NO:1 or a sequence having at least 95% identity with it.

[0071] In some embodiments, the SARS-CoV-2 virus or its derivatives or related substances include ORF1ab, which has the nucleotide sequence of nucleotides 13740 to 21527 of SEQ ID NO:1 or a sequence having at least 95% identity with it.

[0072] In some implementations, the SARS-CoV-2 virus or its derivatives or related substances include ORF3a, which has the nucleotide sequence of nucleotides 25365 to 26192 of SEQ ID NO:1 or a sequence having at least 95% identity with it.

[0073] Preferably, the SARS-CoV-2 virus or its derivatives or related substances include: ORF1a, having the nucleotide sequence of nucleotides 238 to 13455 of SEQ ID NO:1 or a sequence having at least 95% identity with it; and / or ORF1ab, having the nucleotide sequence of nucleotides 13740 to 21527 of SEQ ID NO:1 or a sequence having at least 95% identity with it; and / or spike protein (S), having the amino acid sequence encoded by nucleotides 21508 to 25356 of SEQ ID NO:1 or a sequence having at least 95% identity with it; and / or ORF3a, having the nucleotide sequence of nucleotides 25365 to 26192 of SEQ ID NO:1 or a sequence having at least 95% identity with it; and / or membrane protein (M), having the amino acid sequence encoded by nucleotides 238 to 1345 ... The amino acid sequence encoded by nucleotides 26495–27163 of SEQ ID NO:1 or a sequence having at least 95% identity with it; and / or nucleocapsid protein (N) having the amino acid sequence encoded by nucleotides 28246–29505 of SEQ ID NO:1 or a sequence having at least 95% identity with it.

[0074] Preferably, the SARS-CoV-2 virus or its derivatives or related substances include: ORF1a having the nucleotide sequence of nucleotides 238 to 13455 of SEQ ID NO:1; ORF1ab having the nucleotide sequence of nucleotides 13740 to 21527 of SEQ ID NO:1; spike protein (S) having the amino acid sequence encoded by nucleotides 21508 to 25356 of SEQ ID NO:1; ORF3a having the nucleotide sequence of nucleotides 25365 to 26192 of SEQ ID NO:1; membrane protein (M) having the amino acid sequence encoded by nucleotides 26495 to 27163 of SEQ ID NO:1; and nucleocapsid protein (N) having the amino acid sequence encoded by nucleotides 28246 to 29505 of SEQ ID NO:1.

[0075] Preferably, the isolated SARS-CoV-2 virus or its derivatives or related substances have a genome comprising an RNA sequence having an inverse complementary sequence shown in any one of SEQ ID NO:1 to 3, or a sequence having at least 95% identity with it.

[0076] Preferably, the SARS-CoV-2 virus or its derivatives or related substances have a genome encoded by a sequence shown in any one of SEQ ID NO:1 to 3 or a sequence having at least 95% identity with it.

[0077] Preferably, the SARS-CoV-2 virus or its derivatives or related substances have a genome encoded by the sequence shown in any one of SEQ ID NO:1 to 3, or have a genome comprising an RNA sequence having an inverse complementary sequence shown in any one of SEQ ID NO:1 to 3.

[0078] The isolated SARS-CoV-2 virus of this invention exhibited high yield in Vero cells.

[0079] The SARS-CoV-2 virus of the present invention exhibits high yield in Vero cells. Furthermore, the inactivated SARS-CoV-2 virus of the present invention demonstrates potent neutralizing activity against at least ten representative SARS-CoV-2 strains worldwide, indicating a potentially broader neutralizing capacity against SARS-CoV-2 strains globally, and inducing an effective humoral response in the absence of immunopathology.

[0080] On the other hand, the present invention provides isolated nucleic acids having the nucleotide sequence of nucleotides 21508 to 25356 of SEQ ID NO:1 or a sequence having at least 95% identity with it.

[0081] On the other hand, the present invention provides isolated nucleic acids having the nucleotide sequence of nucleotides 28246 to 29505 of SEQ ID NO:1 or a sequence having at least 95% identity with it.

[0082] On the other hand, the present invention provides isolated nucleic acids having the nucleotide sequence of nucleotides 26495 to 27163 of SEQ ID NO:1 or a sequence having at least 95% identity with it.

[0083] On the other hand, the present invention provides isolated nucleic acids having the nucleotide sequence of nucleotides 238 to 13455 of SEQ ID NO:1 or a sequence having at least 95% identity with it.

[0084] On the other hand, the present invention provides isolated nucleic acids having the nucleotide sequence of nucleotides 13740 to 21527 of SEQ ID NO:1 or a sequence having at least 95% identity with it.

[0085] On the other hand, the present invention provides isolated nucleic acids having the nucleotide sequence of nucleotides 25365 to 26192 of SEQ ID NO:1 or a sequence having at least 95% identity with it.

[0086] On the other hand, the present invention provides isolated nucleic acids comprising: the nucleotide sequence of nucleotides 238 to 13455 of SEQ ID NO:1 or a sequence having at least 95% identity with it; and / or the nucleotide sequence of nucleotides 13740 to 21527 of SEQ ID NO:1 or a sequence having at least 95% identity with it; and / or the nucleotide sequence of nucleotides 21508 to 25356 of SEQ ID NO:1 or a sequence having at least 95% identity with it; and / or the nucleotide sequence of nucleotides 25365 to 26192 of SEQ ID NO:1 or a sequence having at least 95% identity with it; and / or the nucleotide sequence of nucleotides 26495 to 27163 of SEQ ID NO:1 or a sequence having at least 95% identity with it; and / or the nucleotide sequence of nucleotides 28246 to 29505 of SEQ ID NO:1 or a sequence having at least 95% identity with it.

[0087] On the other hand, the present invention provides isolated nucleic acids comprising: the nucleotide sequence of nucleotides 238 to 13455 of SEQ ID NO:1; the nucleotide sequence of nucleotides 13740 to 21527 of SEQ ID NO:1; the nucleotide sequence of nucleotides 21508 to 25356 of SEQ ID NO:1; the nucleotide sequence of nucleotides 25365 to 26192 of SEQ ID NO:1; the nucleotide sequence of nucleotides 26495 to 27163 of SEQ ID NO:1; and the nucleotide sequence of nucleotides 28246 to 29505 of SEQ ID NO:1.

[0088] On the other hand, the present invention provides isolated nucleic acids having the nucleotide sequence of any one of SEQ ID NO:1 to 3, or a sequence having at least 95% identity with them.

[0089] On the other hand, the present invention provides isolated nucleic acids having the nucleotide sequence of any one of SEQ ID NO:1 to 3 or their reverse complementary sequence.

[0090] "At least 95%" means at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, at least 99.99%, and so on.

[0091] The sequence shown in SEQ ID NO:2 corresponds to the viral strain P1_CADDE-CD1716_hCoV-19 / Brazil / L70_CD1716 / 2020|Brazil_AM_Manaus|2020-12-16; the sequence shown in SEQ ID NO:3 corresponds to the viral strain ACC_20SF18530_GZ-GD_South America_2020-12-08. Attached Figure Description

[0092] Figure 1 Characterization of PiCoVacc is shown. (A) Maximum likelihood phylogenetic tree of SARS-CoV-2. The SARS-CoV-2 isolates used in this study are depicted and labeled with black lines. The continents from which the virus strains originated are colored differently. (B) Growth kinetics of PiCoVacc (CN2)P5 stock solution in Vero cells. (C) Assessment of the protein composition and purity of PiCoVacc by NuPAGE 4–12% Bis-Tris gel. (D) Representative electron micrographs of PiCoVacc.

[0093] Figure 2 This study demonstrates the neutralizing antibody responses induced by PiCoVacc immunization against 10 representative SARS-CoV-2 isolates. BALB / c mice and Wistar rats (n=10) were immunized with different doses of PiCoVacc or controls (adjuvant only, as sham models). Serum from COVID-19 recovered patients (RECOV) and uninfected individuals (NI) served as positive and negative controls, respectively. Antibody responses were analyzed in mice (A), humans (B), and rats (C). Top: SARS-CoV-2-specific IgG responses as measured by ELISA; Bottom: Neutralizing antibody titers as determined by micro-neutralization assay. The neutralizing activity profile induced by PiCoVacc was investigated in mice (D) and rats (E). Neutralization assays were performed against nine other SARS-CoV-2 isolates using mouse and rat serology collected 3 weeks post-vaccination. Dots represent individual animals and humans; dashed lines indicate detection limits; horizontal lines represent the geometric mean titer (GMT) at EC50 for each group.

[0094] Figure 3Immunogenicity and protective efficacy of PiCoVacc in non-human primates were demonstrated. Rhesus monkeys were immunized three times via intramuscular route with different doses of PiCoVacc, adjuvant alone (sham model), or placebo (n=4). SARS-CoV-2-specific IgG response (A) and neutralizing antibody titers (B) were measured. Dots represent individual rhesus monkeys; dashed lines indicate the limit of detection; horizontal lines represent the geometric mean titer (GMT) at EC50 for each group. The protective effect of PiCoVacc against SARS-CoV-2 challenge at week 3 post-immunization was evaluated in rhesus monkeys (C–F). Viral load was monitored in throat (C) and anal (D) swab samples collected from immunized rhesus monkeys on days 3, 5, and 7 post-inoculation. Viral load (E) was measured in different lobes of lung tissue from all immunized rhesus monkeys on day 7 post-infection. RNA was extracted and viral load was determined by qRT-PCR. All data are presented as mean ± SEM. Histopathological examination of lung tissue from all inoculated macaques was performed on day 7 post-infection (F). Lung tissue was collected and stained with hematoxylin and eosin.

[0095] Figure 4 Safety assessment of PiCoVacc in non-human primates is shown. Rhesus monkeys were immunized three times intramuscularly on days 0, 7, and 14 with low (1.5 μg per dose) or high (6 μg per dose) PiCoVacc, or adjuvant alone (sham), or placebo. (A and B) Hematological analysis of all four groups of rhesus monkeys (n=4). Percentages of lymphocyte subsets (including CD3+, CD4+, and CD8+) were monitored on day-1 (1 day before vaccination), day 18 (3 days after the second vaccination), and day 29 (7 days after the third vaccination) (A). Key cytokines, including TNF-α, IFN-γ, and IL-2, were examined on day-1, day 1 (the day of the first vaccination), day 4, day 18, and day 29 post-vaccination (B). Values ​​are mean ± standard deviation. (C) Histopathological assessment of lung tissue in the four groups of rhesus monkeys on day 29. Lung tissue was collected and stained with hematoxylin and eosin.

[0096] Figure 5 The amino acid sequence comparisons of SARS-CoV-2 are shown. The number and percentage of amino acid differences in S from the following SARS-CoV-2 isolates used in this study are shown (see Table 1 for details on these isolates).

[0097] Figure 6The purification of S, RBD, and N proteins is shown. (A) SDS-PAGE analysis of S, RBD, and N proteins. Lane 1: Molecular weight standard with relevant bands labeled; Lane 2: Recombinant S protein; Lane 3: Recombinant RBD protein; Lane 4: Recombinant N protein. (B) Size exclusion chromatograms of affinity-purified S, RBD, and N proteins. (Left) S protein data from a Superose 6 10 / 300 column are shown in red. (Middle) RBD protein data from a Superdex 200 10 / 300 column are shown in red. (Right) N protein data from a Superdex 200 10 / 300 column are shown in black.

[0098] Figure 7 The study demonstrated the monitoring of body weight and temperature to assess the safety of PiCoVacc in non-human primates. Rhesus monkeys were immunized three times intramuscularly on days 0, 7, and 14 with low-dose (1.5 μg per dose) or high-dose (6 μg per dose) PiCoVacc, or adjuvant alone (sham model), or placebo. Body weight and temperature were monitored at different time points.

[0099] Figure 8 The study demonstrated the monitoring of hematological parameters to assess the safety of PiCoVacc in non-human primates. Rhesus monkeys were immunized three times intramuscularly on days 0, 7, and 14 with low-dose (1.5 μg per dose) or high-dose (6 μg per dose) PiCoVacc, or adjuvant alone (sham model), or placebo. Several hematological parameters were measured at different time points: ALT (alanine aminotransferase), AST (aspartate aminotransferase), ALP (alkaline phosphatase), TBil (total bilirubin), GGT (gamma-glutamyl transferase), TP (total protein), Alb (albumin), Glb (globulin), A / G (albumin / globulin ratio), UREA (urea), Cre (creatinine), CK (creatine kinase), Glu (glucose), LDH (lactate dehydrogenase), CHO (total cholesterol), TG (triglycerides), Ca (calcium), P (phosphorus), and Na. + (sodium ion), K + (potassium ions), Cl - (Chloride ions).

[0100] Figure 9 This study demonstrates the monitoring of key cytokines to assess the safety of PiCoVacc in non-human primates. Rhesus monkeys were immunized three times intramuscularly at days 0, 7, and 14 with low-dose (1.5 μg per dose) or high-dose (6 μg per dose) PiCoVacc, or adjuvant alone (sham model), or placebo. IL-4, IL-5, and IL-6 were measured at different time points.

[0101] Figure 10 This study demonstrates the histopathological evaluation performed to assess the safety of PiCoVacc in non-human primates. Rhesus monkeys were immunized three times intramuscularly on days 0, 7, and 14 with low-dose (1.5 μg per dose) or high-dose (6 μg per dose) PiCoVacc, or adjuvant alone (sham model), or placebo. Histopathological evaluation of the brain, spleen, kidney, and heart was performed on day 29 in four groups of monkeys. Tissues were collected and stained with hematoxylin and eosin. Detailed Implementation

[0102] The descriptions of specific implementations and embodiments are for illustrative purposes only and not for limitation. Those skilled in the art will readily recognize that various non-critical parameters can be changed or modified to produce substantially similar results.

[0103] Example

[0104] Materials and methods

[0105] Facilities and Ethics Statement

[0106] All SARS-CoV-2 live virus experiments were conducted in the enhanced biosafety level 3 (P3+) facility of the Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences (CAMS), approved by the National Health Commission of the People's Republic of China. All mouse, rat, and macaque experiments were conducted in accordance with the guidelines for laboratory animal care and use issued by the Ministry of Science and Technology of the People's Republic of China.

[0107] Viral titration

[0108] SARS-CoV-2 viral titer was determined using a micro-dose cytopathic effect (CPE) assay. Serial 10-fold dilutions of virus-containing samples were mixed with Vero cells and then seeded into 96-well plates. After incubation at 36.5°C for 3–7 days in a 5% CO2 incubator, the presence of CPE was examined under a microscope. Viral titer was calculated using the Karber method.

[0109] vaccine preparation

[0110] SARS-CoV-2 coronavirus (CN2) was isolated from a COVID-19 infected patient and provided by the Zhejiang Provincial Center for Disease Control and Prevention. The virus was cultured in a bioreactor, inactivated with β-propiolactone for 24 hours, and then purified using ion-exchange chromatography (IEC) and size exclusion chromatography (SEC). Prior to IEC and SEC, the inactivated SARS-CoV-2 virus was treated with a non-restrictive endonuclease. The purified virus was mixed with Al(OH)3 adjuvant and used as a SARS-CoV-2 candidate vaccine. Prior to mixing with Al(OH)3 adjuvant, the inactivated SARS-CoV-2 virus was filtered through a membrane. Preferably, the membrane had a pore size of 0.22 μm. The resulting vaccine was named PiCoVacc, and its genome was encoded by the sequence shown in SEQ ID NO:1; or an RNA sequence having the reverse complementary sequence shown in SEQ ID NO:1.

[0111] RT-PCR

[0112] As previously described, total RNA was extracted from organs using the RNeasy mini kit (Qiagen) and the PrimerScript RT reagent kit (TaKaRa). The forward and reverse primers for RT-PCR targeting the SARS-CoV-2 envelope (E) gene were 5'-TCGTTTCGGAAGAGACAGGT-3' and 5'-GCGCAGTAAGGATGGCTAGT-3', respectively. The RT-PCR reaction conditions were 50°C for 30 min, followed by 40 cycles of 95°C for 15 min, 94°C for 15 s, and 60°C for 45 s.

[0113] Immunogenicity analysis of vaccines

[0114] Balb / c mice and Wistar rats were randomly divided into three groups and immunized intraperitoneally and intramuscularly with three doses (1.5 μg, 3 μg, and 6 μg / dose) of the test vaccine. All animals in each group were immunized twice (on day 0 and day 7). The control group was injected with saline. Blood was collected from the tail vein of the animals for antibody neutralization assays to analyze the immunogenicity of the vaccine.

[0115] Neutralization determination

[0116] Serum samples collected from immunized animals were inactivated at 56°C for 0.5 h and then serially diluted in two steps with cell culture medium. The diluted serum was then mixed with 100 TCID50. 50The viral suspension was mixed in a 1:1 ratio in 96-well plates and incubated at 36.5°C for 2 hours in a 5% CO2 incubator. Vero cells were then added to the serum-virus mixture, and the plates were incubated at 36.5°C for 5 days in a 5% CO2 incubator. Cytopathic effect (CPE) was recorded in each well under a microscope, and neutralizing titers were calculated at a 50% protective dilution.

[0117] Enzyme-linked immunosorbent assay (ELISA)

[0118] The SARS-CoV-2 antibody titer in serum samples collected from immunized animals was determined by indirect ELISA. 96-well microtiter plates were coated with 0.1 μg of purified S, M, and N proteins, respectively, and incubated overnight at 2–8°C. The plates were then blocked with 2% BSA at room temperature for 1 h. Diluted serum (1:100) was added to each well, and the plates were incubated at 37°C for 2 h. The plates were washed three times with PBS, and then incubated with HRP-bound goat anti-mouse antibody at 37°C for 1 h. The reaction was stopped by adding 2M H₂SO₄, and the plates were then developed using TMB. Final data were read at 450 / 630 nm using an ELISA reader.

[0119] Vaccine safety evaluation

[0120] The safety of the experimental SARS-CoV-2 vaccine was evaluated in rhesus monkeys. Four groups of rhesus monkeys (5 females and 5 males per group) were immunized three times on days 0, 7, and 14 with high-dose (6 μg / dose) vaccine, low-dose (1.5 μg / dose) vaccine, Al(OH)3 adjuvant, and saline, respectively. Data sets of several safety-related parameters, including clinical observation, body weight, and body temperature, were collected during and after immunization. Blood samples were also analyzed for lymphocyte subset percentages (CD3+, CD4+, and CD8+), key cytokines (TNF-α, IFN-γ, IL-2, IL-4, IL-5, and IL-6), and biochemical blood tests were performed. 60% of the monkeys were euthanized on day 18 post-immunization, and the remaining 40% were euthanized on day 29. Lung, heart, spleen, liver, kidney, and brain organs were collected for pathological analysis.

[0121] Attack determination on rhesus monkeys

[0122] Rhesus macaques (3-4 years old) were divided into four groups and administered intramuscular injections of high-dose (6 μg / dose) vaccine, medium-dose (3 μg / dose) vaccine, Al(OH)3 adjuvant, and physiological saline, respectively. All groups of animals received three immunizations (days 0, 7, and 14), followed by 10... 6 TCID 50 / ml SARS-CoV-2 virus is administered via the intratracheal route. Rhesus monkeys were euthanized and lung tissue was collected 7 days post-inoculation (dpi). Throat and anal swabs were collected on days 3, 5, and 7 post-inoculation (dpi). Blood samples were collected on days 0, 7, 14, and 21 post-immunization, and on days 3, 5, and 7 post-inoculation (dpi) for hematological analysis of SARS-CoV-2 and detection of neutralizing antibodies. Lung tissue was collected 7 days post-inoculation for RT-PCR assays and histopathological analysis.

[0123] Protein expression and purification

[0124] To express the prefused S extracellular domain, gene-encoding residues from positions 1 to 1208 of COVID-19S (GenBank: MN908947) were synthesized, featuring proline substitutions at residues 986 and 987, a "GSAS" substitution at the furin cleavage site (residues 682–685), and a 2×StrepTag, and cloned into the mammalian expression vector pCAGGS. To express COVID-19RBD, residues from positions 319 to 591 of COVID-19S were cloned upstream of the 2×StrepTag in the mammalian expression vector pCAGGS.

[0125] To express the N protein, residues 1–419 (GenBank: QHW06046.1) were cloned into the vector pET-28a containing a C-terminal 6×His. HEK Expi 293F cells (Thermo Fisher) were transiently transfected with the S extracellular domain and RBD using polyethyleneimine. The protein was purified from the filtered cell supernatant using StrepTactin resin (IBA) and then further purified by size exclusion chromatography using a Superose 6 10 / 300 column (GE Healthcare) or a Superdex 200 10 / 300 increase column (GE Healthcare) at 20 mM Tris pH 8.0 and 200 mM NaCl. The N protein was generated in BL21(DE3) cells by the introduction of IPTG. After sonication, the supernatant was loaded onto Ni-NTA according to the instructions (GE Healthcare) and eluted with elution buffer (20 mM Tris-HCl, 500 mM NaCl, 200 mM imidazole, pH 8.0). Size exclusion chromatography was then performed using a Superdex 200 10 / 300 Increase column (GE Healthcare).

[0126] Phylogenetic tree analysis

[0127] Fast sequences for COVID-19 were retrieved from the GISAID (https: / / www.gisaid.org / ), NCBI, and BIGD (https: / / bigd.big.ac.cn / ncov) databases. After quality control (removal of low-quality or short sequence lengths), 455 sequences were retained for phylogenetic analysis. Sequence alignment was performed using MAFFT by combining nine target sequences with sequences from public databases, and phylogenetic reconstruction was performed using IQtree with default parameters. The inferred maximum likelihood tree was plotted using ggtree.

[0128] Cryo-EM Sample Preparation

[0129] For cryo-EM sample preparation, 3 μL aliquots of purified virus particles were applied to a glow discharge C-flat R2 / 1Cu grid. As previously described, the grid was manually blotted at 100% relative humidity for 3 seconds for rapid freezing in liquid ethane (Vitrobot; FEI). All samples were examined using a Titan Krios microscope (FEI).

[0130] Example 1. Characterization and preparation of PiCoVacc.

[0131] To develop in vitro preclinical neutralization and challenge models for candidate SARS-CoV-2 vaccines, SARS-CoV-2 strains were isolated from bronchoalveolar lavage fluid (BALF) samples from 11 hospitalized patients (including 5 ICU patients), 4 from China, 4 from Italy, 1 from Switzerland, 1 from the UK, and 1 from Spain (Table 1). These patients were infected with SARS-CoV-2 during the most recent outbreak. The 11 samples containing SARS-CoV-2 strains were widely distributed on a phylogenetic tree constructed from all available sequences, which to some extent represents the circulating population (…). Figure 1 A and Figure 5The virus strain CN2 was selected for the development of a purified and inactivated SARS-CoV-2 virus vaccine (PiCoVacc), and 10 other strains (named CN1, CN3-CN5, and OS1-OS6) were selected as preclinical challenge strains. Some of these strains, including CN1 and OS1, are closely related to 2019-nCoV-BetaCoV / Wuhan / WIV04 / 2019 and EPI_ISL_412973, respectively, and can cause severe clinical symptoms, including respiratory failure requiring mechanical ventilation. The complete genome DNA sequence of the PiCoVacc strain is shown in SEQ ID NO:1, which corresponds to the RNA sequence of the PiCoVacc strain. Nucleotides 238–13455 of SEQ ID NO:1 encode ORF1a of the PiCoVacc strain; nucleotides 13740–21527 of SEQ ID NO:1 encode ORF1ab of the PiCoVacc strain; nucleotides 25365–26192 of SEQ ID NO:1 encode ORF3a of the PiCoVacc strain; nucleotides 26495–27163 of SEQ ID NO:1 encode the membrane protein (M) of the PiCoVacc strain; nucleotides 28246–29505 of SEQ ID NO:1 encode the nucleocapsid protein (P) of the PiCoVacc strain; nucleotides 21508–25356 of SEQ ID NO:1 encode the spike glycoprotein (S) of the PiCoVacc strain.

[0132] Table 1. Virus strain information

[0133]

[0134] To obtain a viral stock solution suitable for efficient growth in Vero cells to produce PiCoVacc, the CN3 strain plaque was first purified and passaged once in Vero cells to produce the P1 stock solution. Subsequently, four more passages were performed to produce P2–P5 stock solutions. Growth kinetics analysis of the P5 stock solution in Vero cells showed that at a temperature between 33°C and 37°C, and with a multiplicity of infection (MOI) of 0.0001–0.01, the stock solution replicated efficiently and reached 6–7 log5 values ​​at 3 or 4 days post-infection (dpi). 10 TCID 50 Peak titer per ml ( Figure 1B). To assess the genetic stability of PiCoVacc, five more passages were performed to obtain the P10 stock solution, whose whole genome was sequenced along with the whole genomes of the P1, P3, and P5 stock solutions. Compared to P1, only two amino acid substitutions occurred in the P5 and P10 stock solutions: Ala→Asp (E-A32D) at residue 32 (E) and Thr→Ile (nsp10-T49I) at residue 49 (nsp10) (Table 2). This indicates that the PiCoVacc CN2 strain has excellent genetic stability and no S mutations that could alter the NAb epitopes. To produce PiCoVacc, the virus was propagated in Vero cell cultures using a bioreactor and inactivated using β-propiolactone. The virus was purified using centrifugation and two optimized chromatographic steps to produce a high-purity PiCoVacc formulation. Figure 1 C). Therefore, for vaccine production, the virus was propagated in Vero cell cultures using a bioreactor and inactivated using β-propiolactone, followed by purification using centrifugation and two optimized chromatographic steps. Furthermore, cryo-electron microscopy (cryo-EM) analysis revealed intact elliptical particles with diameters of 90–150 nm, adorned with corona-like spikes, representing the pre-fusion state of the virus. Figure 1 D).

[0135] Table 2. Genetic stability analysis of PiCoV2. The initially determined sequence of PiCoV2 P1 stock solution was used as a reference sequence.

[0136]

[0137] Example 2. PiCoVacc immunization elicits neutralizing antibody responses against 10 representative SARS-CoV-2 isolates.

[0138] To assess the immunogenicity of PiCoVacc, BALB / c mice (n=10) were injected with various doses of PiCoVacc mixed with aluminum adjuvant (0 μg, 1.5 μg, 3 μg, or 6 μg per dose, with 0 μg in saline as a sham) on days 0 and 7. No inflammation or other adverse reactions were observed. Spike-, receptor-binding domain (RBD), and N-specific antibody responses were assessed by enzyme-linked immunosorbent assay (ELISA) 1–6 weeks post-initial immunization. Figure 6 SARS-CoV-2S and RBD-specific immunoglobulin G (IgG) rapidly appeared in the serum of vaccinated mice, reaching peak levels at week 6, with titers of 819,200 (>200 μg / ml) and 409,600 (>100 μg / ml), respectively. Figure 2A). RBD-specific IgG accounted for half of the S-induced antibody response, indicating that RBD is the major immunogen, which closely matches the serological characteristics of blood from recovered COVID-19 patients. Figure 2 A and Figure 2 B)(11). Surprisingly, in immunized mice, the amount of N-specific IgG induced was approximately 30-fold lower than that induced by antibodies targeting S or RBD. Interestingly, previous studies have shown that N-specific IgG is abundant in the serum of COVID-19 patients and could serve as a clinical diagnostic biomarker (11). Notably, PiCoVacc elicited a much higher titer of S-specific antibodies compared to the serum of COVID-19 convalescent patients. This observation, coupled with the fact that antibodies targeting the N of SARS-CoV-2 do not provide protective immunity against infection, suggests that PiCoVacc can elicit a more effective antibody response ( Figure 2 A and Figure 2 B). Next, we used the microneutralization assay (MN50) to measure SARS-CoV-2-specific neutralizing antibodies over a period of time. Similar to the S-specific IgG response, neutralizing antibody titers against the CN1 strain appeared in week 1 (12 for high-dose immunization), surged after booster immunization in week 2, and reached approximately 1,500 for low-dose and medium-dose immunizations and 3,000 for high-dose immunizations by week 7. Figure 2 A). In contrast, the spurious module did not produce a detectable SARS-CoV-2 specific antibody response ( Figure 2 A and Figure 2 B). Furthermore, in Wistar rats immunized using the same strategy, the immunogenicity assessment of PiCoVacc yielded similar results, with the maximum neutralizing titer reaching 2,048–4,096 at week 7. Figure 2 C). To investigate the neutralizing activity profile induced by PiCoVacc, we used mouse and rat serum collected 3 weeks post-vaccination to perform neutralization assays against nine other isolated SARS-CoV-2 strains. The neutralizing titers against these strains indicate that PiCoVacc can induce antibodies that may exhibit potent neutralizing activity against globally prevalent SARS-CoV-2 strains. Figure 2 (D and 2E).

[0139] Example 3. Immunogenicity and protective efficacy of PiCoVacc in non-human primates.

[0140] The immunogenicity and protective efficacy of PiCoVacc were evaluated in rhesus macaques (a non-human primate exhibiting COVID-19-like disease induced by SARS-CoV-2 infection). Macaques were immunized three times intramuscularly on days 0, 7, and 14 with either a moderate dose (3 μg per dose) or a high dose (6 μg per dose) of PiCoVacc (n=4). S-specific IgG and Nab (neutralizing antibodies) decreased in both vaccination groups at week 2 and increased to approximately 12,800 and approximately 50, respectively, at week 3 (before viral challenge), titers similar to those in the serum of recovered COVID-19 patients. Figure 3 A and Figure 3 B). Surprisingly, at week 3, the NAb titer (61) in the medium-dose immunization group was about 20% higher than that observed in the high-dose vaccination group (50), due to individual differences in the ability of one animal in the medium-dose group to elicit a titer about 10 times higher than the other three. Figure 3 B). Excluding this exception, the NAb titer in the medium-dose group decreased to 34, approximately 40% lower than that in the high-dose group. Subsequently, we conducted a challenge study in inoculated and control macaques, where 10 nucleotides of SARS-CoV-2CN1 were administered intratracheally on day 22 (one day after the third immunization). 6 TCID 50 The virus was directly injected into the lungs of animals to verify the protective effect. As expected, all control (sham and placebo) rhesus monkeys showed excessive viral genomic RNA copies (10^6) in the pharynx, perianal region, and lungs on days 3–7 post-inoculation (dpi). 4 ~10 6 / ml) and severe interstitial pneumonia ( Figure 3 C~ Figure 3 F). In contrast, all vaccinated macaques largely avoided SARS-CoV-2 infection, with very little pathological change in their lung tissue, likely due to the transmission of the virus via the tracheal route. 6 TCID 50 Caused by direct viral infection of the lungs, it requires a longer recovery time (more than a week). Figure 3 F). Viral load was significantly reduced in all vaccinated macaques, but slightly increased in the control animals from day 3 to 7 post-infection. Figure 3 C~ Figure 3 E). In all four macaques receiving the high dose, no viral load was detectable in the pharynx, perianal region, or lungs on day 7 post-infection. In the medium-dose immunization group, we did detect some viral spots in pharyngeal (3 / 4), perianal (2 / 4), and lung (1 / 4) specimens on day 7 post-infection, with a viral load reduction of approximately 95% compared to the sham group. Figure 3 C~ Figure 3 E). Interestingly, NAb titers in the vaccinated groups decreased by approximately 30% on day 3 post-infection to neutralize the virus, then rapidly increased from day 5 to day 7 post-infection to maintain their effective neutralizing efficacy. Higher NAb titers were observed on day 7 post-infection in the medium-dose vaccinated group (approximately 400 in 4 macaques; approximately 300 in 3 macaques if one outlier is discarded), likely due to lower levels of viral replication, indicating a longer time required for complete viral clearance. Despite the relatively low NAb titers observed in the medium-dose group prior to infection, providing partial protection, no antibody-dependent enhancement of infection (ADE) was observed in any of the vaccinated macaques.

[0141] Example 4. Safety evaluation of PiCoVacc in non-human primates.

[0142] Previous experience developing SARS and MERS vaccine candidates has raised concerns about pulmonary immunopathology, which could be directly caused by type 2 helper T cell (Th2) responses or as a result of adverse drug reactions (ADE). While many vaccine-induced T-cell responses have been demonstrated to be crucial for acute viral clearance, protection against subsequent coronavirus infections is primarily mediated by humoral immunity. The “cytokine storm” caused by excessive T-cell responses has indeed highlighted the pathogenesis of COVID-19. Therefore, it is essential to carefully control T-cell responses induced by SARS-CoV-2 vaccines to avoid immunopathology. Against this backdrop, we systematically evaluated the safety of PiCoVacc in rhesus monkeys by documenting several clinical observations and biological indicators. Two groups of rhesus monkeys (n=10) were immunized intramuscularly with either a low dose (1.5 μg) or a high dose (6 μg), while two other groups (n=10) were immunized three times with adjuvant (sham) and saline (placebo), on days 0, 7, and 14. Following immunization with PiCoVacc, none of the macaques exhibited fever or weight loss, and all animals maintained normal appetite and mental status. Figure 7 Hematological and biochemical analyses, including blood biochemical tests, percentages of lymphocyte subsets (CD3+, CD4+, and CD8+), and key cytokines (TNF-α, IFN-γ, IL-2, IL-4, IL-5, and IL-6), showed no significant changes in the vaccinated group compared to the sham group and the placebo group. Figure 4 A~ Figure 4 B and Figures 8-9Furthermore, histopathological evaluation of various organs (including lungs, heart, spleen, liver, kidneys, and brain) in the four groups on day 29 indicated that PiCoVacc did not cause any significant pathological changes in rhesus monkeys. Figure 4 C and Figure 10 ).

[0143] The current severe COVID-19 pandemic and the dramatic increase in deaths worldwide necessitate the urgent development of a SARS-CoV-2 vaccine, requiring a new epidemiological paradigm. The safety and efficacy of the vaccine are essential in both preclinical and clinical phases. Rhesus macaques, which mimic COVID-19-like symptoms after SARS-CoV-2 infection, appear very promising animal models for studying the disease. We provide extensive evidence for the safety of PiCoVacc in macaques; neither enhanced infection nor immunopathological deterioration was observed in our study. Our data also indicate that a dose of 6 μg of PiCoVacc provides complete protection against SARS-CoV-2 challenge in macaques. SEQUENCE LISTING <110> Beijing Sinovac Biotech Co., Ltd. <120> SARS-CoV-2 inactivated vaccine and its preparation <130> RYP2016252.8 <160> 3 <170> PatentIn version 3.5 <210> 1 <211> 29841 <212> DNA <213> coronavirus <400> 1 aaaccaacca actttcgatc tcttgtagat ctgttctcta aacgaacttt aaaatctgtg 60 tggctgtcac tcggctgcat gcttagtgca ctcacgcagt ataattaata actaattact 120 gtcgttgaca ggacacgagt aactcgtcta tcttctgcag gctgcttacg gtttcgtccg 180 tgttgcagcc gatcatcagc acatctaggt ttcgtccggg tgtgaccgaa aggtaagatg 240 gagagccttg tccctggttt caacgagaaa acacacgtcc aactcagttt gcctgtttta 300 caggttcgcg acgtgctcgt acgtggcttt ggagactccg tggaggaggt cttatcagag 360 gcacgtcaac atcttaaaga tggcacttgt ggcttagtag aagttgaaaa aggcgttttg 420 cctcaacttg aacagcccta tgtgttcatc aaacgttcgg atgctcgaac tgcacctcat 480 ggtcatgtta tggttgagct ggtagcagaa ctcgaaggca ttcagtacgg tcgtagtggt 540 gagacacttg gtgtccttgt ccctcatgtg ggcgaaatac cagtggctta ccgcaaggtt 600 cttcttcgta agaacggtaa taaaggagct ggtggccata gttacggcgc cgatctaaag 660 tcatttgact taggcgacga gcttggcact gatccttatg aagattttca agaaaactgg 720 aacactaaac atagcagtgg tgttacccgt gaactcatgc gtgagcttaa cggaggggca 780 tacactcgct atgtcgataa caacttctgt ggccctgatg gctaccctct tgagtgcatt 840 aaagaccttc tagcacgtgc tggtaaagct tcatgcactt tgtccgaaca actggacttt 900 attgacacta agaggggtgt atactgctgc cgtgaacatg agcatgaaat tgcttggtac 960 acggaacgtt ctgaaaagag ctatgaattg cagacacctt ttgaaattaa attggcaaag 1020 aaatttgaca ccttcaatgg ggaatgtcca aattttgtat ttcccttaaa ttccataatc 1080 aagactattc aaccaagggt tgaaaagaaa aagcttgatg gctttatggg tagaattcga 1140 tctgtctatc cagttgcgtc accaaatgaa tgcaaccaaa tgtgcctttc aactctcatg 1200 aagtgtgatc attgtggtga aacttcatgg cagacgggcg attttgttaa agccacttgc 1260 gaattttgtg gcactgagaa tttgactaaa gaaggtgcca ctacttgtgg ttacttaccc 1320 caaaatgctg ttgttaaaat ttattgtcca gcatgtcaca attcagaagt aggacctgag 1380 catagtcttg ccgaatacca taatgaatct ggcttgaaaa ccattcttcg taagggtggt 1440 cgcactattg cctttggagg ctgtgtgttc tcttatgttg gttgccataa caagtgtgcc 1500 tattgggttc cacgtgctag cgctaacata ggttgtaacc atacaggtgt tgttggagaa 1560 ggttccgaag gtcttaatga caaccttctt gaaatactcc aaaaagagaa agtcaacatc 1620 aatattgttg gtgactttaa acttaatgaa gagatcgcca ttattttggc atctttttct 1680 gcttccacaa gtgcttttgt ggaaactgtg aaaggtttgg attataaagc attcaaacaa 1740 attgttgaat cctgtggtaa ttttaaagtt acaaaaggaa aagctaaaaa aggtgcctgg 1800 aatattggtg aacagaaatc aatactgagt cctctttatg catttgcatc agaggctgct 1860 cgtgttgtac gatcaatttt ctcccgcact cttgaaactg ctcaaaattc tgtgcgtgtt 1920 ttacagaagg ccgctataac aatactagat ggaatttcac agtattcact gagactcatt 1980 gatgctatga tgttcacatc tgatttggct actaacaatc tagttgtaat ggcctacatt 2040 acaggtggtg ttgttcagtt gacttcgcag tggctaacta acatctttgg cactgtttat 2100 gaaaaactca aacccgtcct tgattggctt gaagagaagt ttaaggaagg tgtagagttt 2160 cttagagacg gttgggaaat tgttaaattt atctcaacct gtgcttgtga aattgtcggt 2220 ggacaaattg tcacctgtgc aaaggaaatt aaggagagtg ttcagacatt ctttaagctt 2280 gtaaataaat ttttggcttt gtgtgctgac tctatcatta ttggtggagc taaacttaaa 2340 gccttgaatt taggtgaaac atttgtcacg cactcaaagg gattgtacag aaagtgtgtt 2400 aaatccagag aagaaactgg cctactcatg cctctaaaag ccccaaaaga aattatcttc 2460 ttagagggag aaacacttcc cacagaagtg ttaacagagg aagttgtctt gaaaactggt 2520 gatttacaac cattagaaca acctactagt gaagctgttg aagctccatt ggttggtaca 2580 ccagtttgta ttaacgggct tatgttgctc gaaatcaaag acacagaaaa gtactgtgcc 2640 cttgcaccta atatgatggt aacaaacaat accttcacac tcaaaggcgg tgcaccaaca 2700 aaggttactt ttggtgatga cactgtgata gaagtgcaag gttacaagag tgtgaatatc 2760 acttttgaac ttgatgaaag gattgataaa gtacttaatg agaagtgctc tgcctataca 2820 gttgaactcg gtacagaagt aaatgagttc gcctgtgttg tggcagatgc tgtcataaaa 2880 actttgcaac foottctga attacttaca ccactgggca ttgatttaga tgagtggagt 2940 atggctacat actacttatt tgatgagtct ggtgagttta aattggcttc acatatgtat 3000 tgttctttct accctccaga tgaggatgaa gaagaaggtg attgtgaaga agaagagttt 3060 gagccatcaa ctcaatga gtatggtact gaagatgat accaaggtaa acctttggaa 3120 ttggtgcca cttctgctgc tcttcaacct gaagagagc aagagaga ttggttagat 3180 gatgagtc aaaactgt tggtcaaca gacggcagtg aggacaatca gacacact 3240 attcaaacaa ttgttgaggt tcaacctcaa ttagagatgg aacttacacc agttgttcag 3300 actattgag tgaatagtttt tagtggttat ttaaaactta ctgacaatgt atacattaaa 3360 aatgcagaca tgtggaaga agctaaaaag gtaaaaccaa cagtggttgt taatgcagcc 3420 aatgtttacc ttaacatgg aggaggtgtt gcaggagcct taaataggc tactacaat 3480 gccatgcaag ttgaatctga tgattacata gctactaatg gaccacttaa agtgggtggt 3540 agttgtgttt taagcggaca caatcttgct aacactgtc ttcatgttgt cggcccaaat 3600 gttaacaaag gtgagacat tcaacttt aagagtgctt atgaaaattt taatcagcac 3660 gaagttctac ttgcaccatt attatcagct ggtatttg gtgctgaccc atacattct 3720 ttaagagttt gtgtagatac tgttcgcaca atgtctact tagctctt tgataaaaat 3780 ctctatgaca aacttgtttc aagcttttg gaatgaaga gtgaaaagca agttgaacaa 3840 aagatcgctg agattcctaa agaggaagtt aagccatttta taactgaag taaaccttca 3900 gttgaacaga gaaacaga tgaaaaa atcaagctt gtgttgaga agttacaaca 3960 actctggaag aaactaagtt cctcacagaa aacttgttac tttatattga cattaatggc 4020 aatcttcatc cagattctgc cactcttgtt agtgacattg acatcactt cttaaagaaa 4080 gatgctccat atatagtggg tgatgttgtt caagggttg ttttaactgc tgtggttata 4140 cctactaaaa aggctggtgg cactactgaa atgctagcga aagctttgag aaagtgcca 4200 acagacaatt atattaccac ttaccggggt cagggtttaa atggttacac tgtagaggag 4260 gcaagacag tgcttaaaa gtgtaaagt gccttttaca ttctaccac tattatctct 4320 aatgagaagc aagaaatttct tggactgtt tcttggaatt tgcgagaat gcttgcacat 4380 gcagaagaaa cacgcaatt aatgcctgtc tgtgtggaaa ctaaagccat agtttcaact 4440 atacagcgta atataaggg tattaaaata cagagggtg tggttgatta tggtgctaga 4500 ttttactttt acaccagtaa aacaactgta gcgtcactta tcaacacact taacgatcta 4560 aatgaaactc ttgttacaat gccacttggc tatgtaacac atggcttaaa tttggaagaa 4620 gctgctcggt atatgagatc tctcaaagtg ccagctacag tttctgtttc ttcacctgat 4680 gctgttacag cgtataatgg ttatcttact tcttcttcta aaacacctga agaacatttt 4740 attgaaacca tctcacttgc tggttcctat aaagattggt cctattctgg acaatctaca 4800 caactaggta tagaatttct taagagaggt gataaaagtg tatattacac tagtaatcct 4860 accacattcc acctagatgg tgaagttatc acctttgaca atcttaagac acttctttct 4920 ttgagagaag tgaggactat taaggtgttt acaacagtag acaacattaa cctccacacg 4980 caagttgtgg acatgtcaat gacatatgga caacagtttg gtccaactta tttggatgga 5040 gctgatgtta ctaaaataaa acctcataat tcacatgaag gtaaaacatt ttatgtttta 5100 cctaatgatg acactctacg tgttgaggct ttgagtact accacacaac tgatcctagt 5160 tttctgggta ggtacatgtc agcattaaat cacactaaaa agtggaaata cccacaagtt 5220 aatggtttaa cttctattaa atgggcagat aacaactgtt atcttgccac tgcattgtta 5280 acactccaac aaatagagtt gaagtttaat ccacctgctc tacaagatgc ttattacaga 5340 gcaagggctg gtgaagctgc taacttttgt gcacttatct tagcctactg taataagaca 5400 gtaggtgagt taggtgatgt tagagaaaca atgagttact tgtttcaaca tgccaattta 5460 gattcttgca aaagagtctt gaacgtggtg tgtaaaactt gtggacaaca gcagacaacc 5520 cttaaggggtg tagaagctgt tatgtacatg ggcacacttt cttatgaaca atttaagaaa 5580 ggtgttcaga taccttgtac gtgtggtaaa caagctacaa aatatctagt acaacaggag 5640 tcaccttttg ttatgatgtc agcaccacct gctcagtatg aacttaagca tggtacattt 5700 acttgtgcta gtgagtacac tggtaattac cagtgtggtc actataaaca tataacttct 5760 aaagaaactt tgtattgcat agacggtgct ttacttacaa agtcctcaga atacaaaggt 5820 cctattacgg atgttttcta caaagaaaac agttacacaa caaccataaa accagttact 5880 tataaattgg atggtgttgt ttgtacagaa attgacccta agttggacaa ttattataag 5940 aaagacaatt cttatttcac agagcaacca attgatcttg taccaaacca accatatcca 6000 aacgcaagct tcgataattt taagtttgta tgtgataata tcaaatttgc tgatgattta 6060 aaccagttaa ctggttataa gaaacctgct tcaagagagc ttaaagttac attttccct 6120 gacttaaatg gtgatgtggt ggctattgat tataaacact acacaccctc ttttaagaaa 6180 ggagctaaat tgttacataa acctattgtt tggcatgtta acaatgcaac taataaagcc 6240 acgtataaac caaatacctg gtgtatacgt tgtctttgga gcacaaaacc agttgaaaca 6300 tcaaattcgt ttgatgtact gaagtcagag gacgcgcagg gaatggataa tcttgcctgc 6360 gaagatctaa aaccagtctc tgaagaagta gtggaaaatc ctaccataca gaaagacgtt 6420 cttgagtgta atgtgaaaac taccgaagtt gtaggagaca ttatacttaa accagcaaat 6480 aatagtttaa aaattacaga agaggttggc cacacagatc taatggctgc ttatgtagac 6540 aattctagtc ttactattaa gaaacctaat gaattatcta gagtattagg tttgaaaacc 6600 cttgctactc atggtttagc tgctgttaat agtgtccctt gggatactat agctaattat 6660 gctaagcctt ttcttaacaa agttgttagt acaactacta acatagttac acggtgttta 6720 aaccgtgttt gtactaatta tatgccttat ttctttactt tattgctaca attgtgtact 6780 tttactagaa gtacaaattc tagaattaaa gcatctatgc cgactactat agcaaagaat 6840 actgttaaga gtgtcggtaa atttgtcta gaggcttcat ttaattattt gaagtcacct 6900 aatttttcta aactgataaa tattataatt tggtttttac tattaagtgt ttgcctaggt 6960 tctttaatct actcaaccgc tgctttaggt gttttaatgt ctaatttagg catgccttct 7020 tactgtactg gttacagaga aggctatttg aactctacta atgtcactat tgcaacctac 7080 tgtactggtt ctataccttg tagtgtttgt cttagtggtt tagattcttt agacacctat 7140 ccttctttag aaactataca aattaccatt tcatctttta aatgggattt aactgctttt 7200 ggcttagttg cagagtggtt tttggcatat attcttttca ctaggttttt ctatgtactt 7260 ggattggctg caatcatgca attgtttttc agctattttg cagtacattt tattagtaat 7320 tcttggctta tgtggttaat attaatctt gtacaaatgg ccccgatttc agctatggtt 7380 agaatgtaca tcttctttgc atcattttat tatgtatgga aaagttatgt gcatgttgta 7440 gacggttgta attcatcaac ttgtatgatg tgttacaaac gtaatagagc aacaagagtc 7500 gaatgtacaa ctattgttaa tggtgttaga aggtcctttt atgtctatgc taatggaggt 7560 aaaggctttt gcaaactaca caattggaat tgtgttaatt gtgatacatt ctgtgctggt 7620 agtacattta ttagtgatga agttgcgaga gacttgtcac tacagtttaa aagaccaata 7680 aatcctactg accagtcttc ttacatcgtt gatagtgtta cagtgaagaa tggttccatc 7740 catctttact ttgataaagc tggtcaaaag acttatgaaa gacattctct ctctcatttt 7800 gttaacttag acaacctgag agctaataac actaaaggtt cattgcctat taatgttata 7860 gtttttgatg gtaaatcaaa atgtgaagaa tcatctgcaa aatcagcgtc tgtttactac 7920 agtcagctta tgtgtcaacc tatactgtta ctagatcagg cattagtgtc tgatgttggt 7980 gatagtgcgg aagttgcagt taaaatgttt gatgcttacg ttaatacgtt ttcatcaact 8040 tttaacgtac caatggaaaa actcaaaaca ctagttgcaa ctgcagaagc tgaacttgca 8100 aagaatgtgt ccttagacaa tgtcttatct acttttattt cagcagctcg gcaagggttt 8160 gttgattcag atgtagaaac taaagatgtt gttgaatgtc ttaaattgtc acatcaatct 8220 gacatagaag ttactggcga tagttgtaat aactatatgc tcacctataa caaagttgaa 8280 aacatgacac cccgtgacct tggtgcttgt attgactgta gtgcgcgtca tattaatgcg 8340 caggtagcaa aaagtcacaa cattgctttg atatggaacg ttaaagattt catgtcattg 8400 tctgaacaac tacgaaaaca aatacgtagt gctgctaaaa agaataactt accttttaag 8460 ttgacatgtg caactactag acaagttgtt aatgttgtaa caacaaagat agcacttaag 8520 ggtggtaaaa ttgttaataa ttggttgaag cagttaatta aagttacact tgtgttcctt 8580 tttgttgctg ctattttcta tttaataaca cctgttcatg tcatgtctaa acatactgac 8640 ttttcaagtg aaatcatagg atacaaggct attgatggtg gtgtcactcg tgacatagca 8700 tctacagata cttgttttgc taacaaacat gctgattttg acacatggtt tagtcagcgt 8760 ggtggtagtt atactaatga caaagcttgc ccattgattg ctgcagtcat aacaagagaa 8820 gtgggttttg tcgtgcctgg tttgcctggc acgatattac gcacaactaa tggtgacttt 8880 ttgcatttct tacctagagt ttttagtgca gttggtaaca tctgttacac accatcaaaa 8940 cttatagagt acactgactt tgcaacatca gcttgtgttt tggctgctga atgtacaatt 9000 tttaaagatg cttctggtaa gccagtacca tattgttatg ataccaatgt actagaaggt 9060 tctgttgctt atgaaagttt acgccctgac acacgttatg tgctcatgga tggctctatt 9120 attcaatttc ctaacaccta ccttgaaggt tctgttagag tggtaacaac ttttgattct 9180 gagtactgta ggcacggcac ttgtgaaaga tcagaagctg gtgtttgtgt atctactagt 9240 ggtagatggg tacttaacaa tgattattac agatctttac caggagtttt ctgtggtgta 9300 gatgctgtaa attacttac tatatgttt acaccactaa ttcaacctat tggtgctttg 9360 gacatatcag catctatagt agctggtggt attgtagcta tcgtagtaac atgccttgcc 9420 tactatttta tgaggtttag aagagctttt ggtgaataca gtcatgtagt tgcctttaat 9480 actttactat tccttatgtc attcactgta ctctgtttaa caccagttta ctcattctta 9540 cctggtgttt attctgttat ttacttgtac ttgacatttt atcttactaa tgatgtttct 9600 tttttagcac atattcagtg gatggttatg ttcacacctt tagtaccttt ctggataaca 9660 attgcttata tcatttgtat ttccacaaag catttctatt ggttctttag taattaccta 9720 aagagacgtg tagtctttaa tggtgtttcc tttagtactt ttgaagaagc tgcgctgtgc 9780 acctttttgt taataaaga aatgtatcta aagttgcgta gtgatgtgct attacctctt 9840 acgcaatata atagatactt agctctttat aataagtaca agtattttag tggagcaatg 9900 gatacaacta gctacagaga agctgcttgt tgtcatctcg caaaggctct caatgacttc 9960 agtaactcag gttctgatgt tctttaccaa ccaccacaaa cctctatcac ctcagctgtt 10020 ttgcagagtg gttttagaaa aatggcattc ccatctggta aagttgaggg ttgtatggta 10080 caagtaactt gtggtacaac tacacttaac ggtctttggc ttgatgacgt agtttactgt 10140 ccaagacatg tgatctgcac ctctgaagac atgcttaacc ctaattatga agatttactc 10200 attcgtaagt ctaatcataa tttcttggta caggctggta atgttcaact cagggttatt 10260 ggacattcta tgcaaaattg tgtacttaag cttaaggttg atacagccaa tcctaagaca 10320 cctaagtata agttgttcg cattcaacca ggacagactt tttcagtgtt agcttgttac 10380 aatggttcac catctggtgt ttaccaatgt gctatgaggc ccaatttcac tattaagggt 10440 tcattcctta atggttcatg tggtagtgtt ggttttaaca tagattatga ctgtgtctct 10500 ttttgttaca tgcaccatat ggaattacca actggagttc atgctggcac agacttagaa 10560 ggtaactttt atggaccttt tgttgacagg caaacagcac aagcagctgg tacggacaca 10620 actattacag ttaatgtttt agcttggttg tacgctgctg ttataaatgg agacaggtgg 10680 tttctcaatc gatttaccac aactcttaat gactttaacc ttgtggctat gaagtacaat 10740 tatgaacctc taacacaaga ccatgttgac atactaggac ctctttctgc tcaaactgga 10800 attgccgttt tagatatgtg tgcttcatta aaagaattac tgcaaaatgg tatgaatgga 10860 cgtaccatat tgggtagtgc tttattagaa gatgaattta caccttttga tgttgttaga 10920 caatgctcag gtgttacttt ccaaagtgca gtgaaaagaa caatcaaggg tacacaccac 10980 tggttgttac tcacaatttt gacttcactt ttagttttag tccagagtac tcaatggtct 11040 ttgttctttt ttttgtatga aaatgccttt ttaccttttg ctatgggtat tattgctatg 11100 tctgcttttg caatgatgtt tgtcaaacat aagcatgcat ttctctgttt gtttttgtta 11160 ccttctcttg ccactgtagc ttattttaat atggtctata tgcctgctag ttgggtgatg 11220 cgtattatga catggttgga tatggttgat actagtttgt ctggttttaa gctaaaagac 11280 tgtgttatgt atgcatcagc tgtagtgtta ctaatcctta tgacagcaag aactgtgtat 11340 gatgatggtg ctaggagagt gtggacactt atgaatgtct tgacactcgt ttataaagtt 11400 tattatggta atgctttaga tcaagccatt tccatgtggg ctcttataat ctctgttact 11460 tctaactact caggtgtagt tacaactgtc atgtttttgg ccagaggtat tgtttttatg 11520 tgtgttgagt attgccctat tttcttcata actggtaata cacttcagtg tataatgcta 11580 gtttattgtt tcttaggcta tttttgtact tgttactttg gcctcttttg tttactcaac 11640 cgctacttta gactgactct tggtgtttat gattacttag tttctacaca ggagtttaga 11700 tatatgaatt cacagggact actcccaccc aagaatagca tagatgcctt caaactcaac 11760 attaaattgt tgggtgttgg tggcaaacct tgtatcaaag tagccactgt acagtctaaa 11820 atgtcagatg taaagtgcac atcagtagtc ttactctcag ttttgcaaca actcagagta 11880 gaatcatcat ctaaattgtg ggctcaatgt gtccagttac acaatgacat tctcttagct 11940 aaagatacta ctgaagcctt tgaaaaaatg gtttcactac tttctgtttt gctttccatg 12000 cagggtgctg tagacataaa caagctttgt gaagaaatgc tggacaacag ggcaacctta 12060 caagctatag cctcagagtt tagttccctt ccatcatatg cagcttttgc tactgctcaa 12120 gaagcttatg agcaggctgt tgctaatggt gattctgaag ttgttcttaa aaagttgaag 12180 aagtctttga atgtggctaa atctgaattt gaccgtgatg cagccatgca acgtaagttg 12240 gaaaagatgg ctgatcaagc tatgacccaa atgtataaac aggctagatc tgaggacaag 12300 agggcaaaag ttactagtgc tatgcagaca atgcttttca ctatgcttag aaagttggat 12360 aatgatgcac tcaacaacat tatcaacaat gcaagagatg gttgtgttcc cttgaacata 12420 atacctta caacagcagc caaactaatg gttgtcatac cagactataa cacatataaa 12480 aatacgtgtg atggtacaac atttacttat gcatcagcat tgtgggaaat ccaacaggtt 12540 gtagatgcag atagtaaaat tgttcaactt agtgaaatta gtatggacaa ttcacctaat 12600 ttagcatggc ctcttattgt aacagcttta agggccaatt ctgctgtcaa attacagaat 12660 aatgagctta gtcctgttgc actacgacag atgtctttgtg ctgccggtac tacacaaact 12720 gcttgcactg atgacaatgc gttagcttac tacaacacaa caaagggagg tagttttgta 12780 cttgcactgt tatccgattt acaggatttg aaatgggcta gattccctaa gagtgatgga 12840 actggtacta tctatacaga actggaacca ccttgtaggt ttgttacaga cacacctaaa 12900 ggtcctaaag tgaagtattt atactttatt aaaggattaa acaacctaaa tagaggtatg 12960 gtacttggta gtttagctgc cacagtacgt ctacaagctg gtaatgcaac agaagtgcct 13020 gccaattcaa ctgtattatc tttctgtgct tttgctgtag atgctgctaa agcttacaaa 13080 gattatctag ctagtggggg acaaccaatc actaattgtg ttaagatgtt gtgtacacac 13140 attggtactg gtcaggcaat aacagttaca ccggaagcca atatggatca agaatccttt 13200 ggtggtgcat cgtgttgtct gtactgccgt tgccacatag atcatccaaa tcctaaagga 13260 ttttgtgact taaaaggtaa gtatgtacaa atacctacaa cttgtgctaa tgaccctgtg 13320 ggttttacac ttaaaaacac agtctgtacc gtctgcggta tgtggaaagg ttatggctgt 13380 agttgtgatc aactccgcga acccatgctt cagtcagctg atgcacaatc gtttttaaac 13440 gggtttgcgg tgtaagtgca gcccgtctta caccgtgcgg cacaggcact agtactgatg 13500 tcgtatacag ggcttttgac atctacaatg ataaagtagc tggttttgct aaattcctaa 13560 aaactaattg ttgtcgcttc caagaaaagg acgaagatga caatttaatt gattcttact 13620 ttgtagttaa gagacacact ttctctaact accaacatga agaaacaatt tataatttac 13680 ttaaggattg tccagctgtt gctaaacatg acttctttaa gtttagaata gacggtgaca 13740 tggtaccaca tatatcacgt caacgtctta ctaaatacac aatggcagac ctcgtctatg 13800 ctttaaggca ttttgatgaa ggtaattgtg acacattaaa agaaatactt gtcacataca 13860 attgttgtga tgatgattat ttcaataaaa aggactggta tgattttgta gaaaacccag 13920 atatattacg cgtatacgcc aacttaggtg aacgtgtacg ccaagctttg ttaaaaacag 13980 tacaattctg tgatgccatg cgaaatgctg gtattgttgg tgtactgaca ttagataatc 14040 aagatctcaa tggtaactgg tatgatttcg gtgatttcat acaaaccacg ccaggtagtg 14100 gagttcctgt tgtagattct tattattcat tgttaatgcc tatattaacc ttgaccaggg 14160 ctttaactgc agagtcacat gttgacactg acttaacaaa gccttacatt aagtgggatt 14220 tgttaaaata tgacttcacg gaagagaggt taaaactctt tgaccgttat tttaaatatt 14280 gggatcagac ataccaccca aattgtgtta actgtttgga tgacagatgc attctgcatt 14340 gtgcaaactt taatgtttta ttctctacag tgttcccacc tacaagtttt ggaccactag 14400 tgagaaaaat atttgttgat ggtgttccat ttgtagtttc aactggatac cacttcagag 14460 agctaggtgt tgtacataat caggatgtaa acttacatag ctctagactt agttttaagg 14520 aattacttgt gtatgctgct gaccctgcta tgcacgctgc ttctggtaat ctattactag 14580 ataaacgcac tacgtgcttt tcagtagctg cacttactaa caatgttgct tttcaaactg 14640 tcaaacccgg taattttaac aaagacttct atgactttgc tgtgtctaag ggtttcttta 14700 aggaaggaag ttctgttgaa ttaaaacact tcttctttgc tcaggatggt aatgctgcta 14760 tcagcgatta tgactactat cgttataatc taccaacaat gtgtgatatc agacaactac 14820 tatttgtagt tgaagttgtt gataagtact ttgattgtta cgatggtggc tgtattaatg 14880 ctaaccaagt catcgtcaac aacctagaca aatcagctgg ttttccattt aataaatggg 14940 gtaaggctag actttattat gattcaatga gttatgagga tcaagatgca cttttcgcat 15000 atacaaaacg taatgtcatc cctactataa ctcaaatgaa tcttaagtat gccattagtg 15060 caaagaatag agctcgcacc gtagctggtg tctctatctg tagtactatg accaatagac 15120 agttcatca aaaattattg aaatcaatag ccgccactag aggagctact gtagtaattg 15180 gaacaagcaa attctatggt ggttggcaca acatgttaaa aactgttat agtgatgtag 15240 aaaaccctca ccttatgggt tgggattatc ctaaatgtga tagagccatg cctaacatgc 15300 ttagaattat ggcctcactt gttcttgctc gcaaacatac aacgtgttgt agcttgtcac 15360 accgtttcta tagattagct aatgagtgtg ctcaagtatt gagtgaaatg gtcatgtgtg 15420 gcggttcact atatgttaaa ccaggtggaa cttcatcagg agatgccaca actgcttatg 15480 ctaatagtgt ttttaacatt tgtcaagctg tcacggccaa tgttaatgca cttttatcta 15540 ctgatggtaa caaaattgcc gataagtatg tccgcaattt acaacacaga ctttatgagt 15600 gtctctatag aatagagat gttgacacag actttgtgaa tgagttttac gcatatttgc 15660 gtaaacattt ctcaatgatg atactctctg acgatgctgt tgtgtgtttc aatagcactt 15720 atgcatctca aggtctagtg gctagcataa agaactttaa gtcagttctt tattatcaaa 15780 acaatgtttt tatgtctgaa gcaaaatgtt ggactgagac tgaccttact aaaggacctc 15840 atgaatttg ctctcaacat acaatgctag ttaaacaggg tgatgattat gtgtaccttc 15900 cttacccaga tccatcaaga atcctagggg ccggctgttt tgtagatgat atcgtaaaaa 15960 cagatggtac acttatgatt gaacggttcg tgtctttagc tatagatgct tacccactta 16020 ctaaacatcc taatcaggag tatgctgatg tctttcattt gtacttacaa tacataagaa 16080 agctacatga tgagttaaca ggacacatgt tagacatgta ttctgttatg cttactaatg 16140 ataacacttc aaggtattgg gaacctgagt tttatgaggc tatgtacaca ccgcatacag 16200 tcttacaggc tgttggggct tgtgttcttt gcaattcaca gacttcatta agatgtggtg 16260 cttgcatacg tagaccattc ttatgttgta aatgctgtta cgaccatgtc atatcaacat 16320 cacataaatt agtcttgtct gttaatccgt atgtttgcaa tgctccaggt tgtgatgtca 16380 cagatgtgac tcaactttac ttaggaggta tgagctatta ttgtaaatca cataaaccac 16440 ccattagttt tccattgtgt gctaatggac aagttttttgg tttatataaa aatacatgtg 16500 ttggtagcga taatgttact gactttaatg caattgcaac atgtgactgg acaaatgctg 16560 gtgattacat tttagctaac acctgtactg aaagactcaa gctttttgca gcagaaacgc 16620 tcaaagctac tgaggagaca tttaaactgt cttatggtat tgctactgta cgtgaagtgc 16680 tgtctgacag agaattacat ctttcatggg aagttggtaa acctagacca ccacttaacc 16740 gaaattatgt ctttactggt tatcgtgtaa ctaaaaacag taaagtacaa ataggagagt 16800 acaccttga aaaaggtgac tatggtgatg ctgttgttta ccgaggtaca acaacttaca 16860 aattaaatgt tggtgattat tttgtgctga catcacatac agtaatgcca ttaagtgcac 16920 ctacactagt gccacaagag cactatgtta gaattactgg cttataccca acactcaata 16980 tctcagatga gttttctagc aatgttgcaa attatcaaaa ggttggtatg caaaagtatt 17040 ctacactcca gggaccacct ggtactggta agagtcattt tgctattggc ctagctctct 17100 actacccttc tgctcgcata gtgtatacag cttgctctca tgccgctgtt gatgcactat 17160 gtgagaaggc attaaaatat ttgcctatag ataaatgtag tagaattata cctgcacgtg 17220 ctcgtgtaga gtgttttgat aaattcaaag tgaattcaac attagaacag tatgtcttttt 17280 gtactgtaaa tgcattgcct gagacgacag cagatatagt tgtctttgat gaaatttcaa 17340 tggccacaaa ttatgatttg agtgttgtca atgccagatt acgtgctaag cactatgtgt 17400 acattggcga ccctgctcaa ttacctgcac cacgcacatt gctaactaag ggcacactag 17460 aaccagaata tttcaattca gtgtgtagac ttatgaaaac tataggtcca gacatgttcc 17520 tcggaacttg tcggcgttgt cctgctgaaa ttgttgacac tgtgagtgct ttggtttatg 17580 ataataagct taaagcacat aaagacaaat cagctcaatg ctttaaaatg ttttataagg 17640 gtgttatcac gcatgatgtt tcatctgcaa ttaacaggcc acaaataggc gtggtaagag 17700 aattccttac acgtaaccct gcttggagaa aagctgtctt tatttcacct tataattcac 17760 agaatgctgt agcctcaaag atttgggac taccaactca aactgttgat tcatcacagg 17820 gctcagaata tgactatgtc atattcactc aaaccactga aacagctcac tcttgtaatg 17880 taaacagatt taatgttgct attaccagag caaaagtagg catactttgc ataatgtctg 17940 atagagacct tttgacaag ttgcaattta caagtcttga aattccacgt aggaatgtgg 18000 caactttaca agctgaaaat gtaacaggac tctttaaaga ttgtagtaag gtaatcactg 18060 ggttacatcc tacacaggca cctacacacc tcagtgttga cactaaattc aaaactgaag 18120 gtttatgtgt tgacatacct ggcataccta aggacatgac ctatagaaga ctcatctcta 18180 tgatgggttt taaaatgaat tatcaagtta atggttaccc taacatgttt atcacccgcg 18240 aagaagctat aagacatgta cgtgcatgga ttggcttcga tgtcgagggg tgtcatgcta 18300 ctagagaagc tgttggtacc aatttacctt tacagctagg tttttctaca ggtgttaacc 18360 tagttgctgt acctacaggt tatgttgata cacctaataa tacagatttt tccagagtta 18420 gtgctaaacc accgcctgga gatcaattta aacacctcat accacttatg tacaaaggac 18480 ttccttggaa tgtagtgcgt ataaagattg tacaaatgtt aagtgacaca cttaaaaatc 18540 tctctgacag agtcgtattt gtcttatggg cacatggctt tgagttgaca tctatgaagt 18600 atttgtgaa aataggacct gagcgcacct gttgtctatg tgatagacgt gccacatgct 18660 tttccactgc ttcagacact tatgcctgtt ggcatcattc tattggattt gattacgtct 18720 ataatccgtt tatgattgat gttcaacaat ggggttttac aggtaaccta caaagcaacc 18780 atgatctgta ttgtcaagtc catggtaatg cacatgtagc tagttgtgat gcaatcatga 18840 ctaggtgtct agctgtccac gagtgctttg ttaagcgtgt tgactggact attgaatatc 18900 ctataattgg tgatgaactg aagattaatg cggcttgtag aaaggttcaa cacatggttg 18960 ttaaagctgc attattagca gacaaattcc cagttcttca cgacattggt aaccctaaag 19020 ctattaagtg tgtacctcaa gctgatgtag aatggaagtt ctatgatgca cagccttgta 19080 gtgacaaagc ttataaaata gaagaattat tctattctta tgccacacat tctgacaaat 19140 tcacagatgg tgtatgccta ttttggaatt gcaatgcga tagatatcct gctaattcca 19200 ttgtttgtag atttgacact agagtgctat ctaaccttaa cttgcctggt tgtgatggtg 19260 gcagtttgta tgtaaataaa catgcattcc acacaccagc ttttgataaa agtgcttttg ttaatttaaa acaattacca tttttctatt actctgacag tccatgtgag tctcatggaa aacaagtagt gtcagatata gattatgtac cactaaagtc tgctacgtgt ataacacgtt gcaatttagg tggtgctgtc tgtagacatc atgctaatga gtacagattg tatctcgatg cttataacat gatgatctca gctggcttta gcttgtgggt ttacaaacaa tttgatactt ataacctctg gaacactttt acaagacttc agagttga aaatgtggct tttaatgttg fatheraggg acactttgat ggacaacagg gtgaagtacc agtttctatc attackaca ctgtttacac aaaagttgat ggtgttgatg tagattgtt tgaaataaa acaacattac ctgttaatgt agcatttgag ctttgggcta agcgcaacat taaaccagta ccagaggtga aaatactcaa taatttgggt gtggacattg ctgctaatac tgtgatctgg gactacaaaa gagatgctcc agcacatata tctactattg gtgtttgttc tatgactgac atagccaaga aaccaactga aacgatttgt gcaccactca ctgtcttttt tgatggtaga gttgatggtc 19980 aagtagactt atttagaat gcccgtaatg gtgttcttat tacagaaggt agtgttaaag 20040 gtttacaacc atctgtaggt cccaacaag ctagtcttaa tggagtcaca ttaattggag 20100 aagccgtaaa aacacagttc aattattata agaaagttga tggtgttgtc siacattac 20160 ctgaaactta ctttactcag agtagaatt taaacccagg agtcaatgg 20220 aaattgattt cttagaatta gctatggatg attcattga acggtataaa ttagaaggct 20280 atgccttcga acatacgtt tatggatt ttagtcatag tcagttaggt ggtttacatc 20340 tactgattgg actagctaaa cgttttaagg atcacctttt tgattagaa gattttattc 20400 ctatggacag tacagttaaa actatttca taacagatgc gcaacaggt tcatctaagt 20460 gtgtgtgttc tgttattgat ttattacttg atgattttgt tgaaataata aaatcccaag 20520 atttatctgt agtttctaag gttgtcaaag tgactattga ctatacagaa atttcattta 20580 tgctttggtg taagatggc catgtagaaa cattttaccc aaaattacaa tctagtcaag 20640 cgtggcaacc gggtgttgct atgcctaatc tttacaaaat gcaaagaatg ctattagaaa 20700 agtgtgacct tcaaaattat ggtgatagtg caacattacc taaaggcata atgatgaatg 20760 tcgcaaaata tactcaactg tgtcaatatt taaacacatt aacattagct gtaccctata 20820 atatgagagt tatacatttt ggtgctggtt ctgataaagg agttgcacca ggtacagctg 20880 ttttaagaca gtggttgcct acgggtacgc tgcttgtcga ttcagatctt aatgactttg 20940 tctctgatgc agattcaact ttgattggtg attgtgcaac tgtacataca gctaataaat 21000 gggatctcat tattagtgat atgtacgacc ctaagactaa aaatgttaca aaagaaaatg 21060 actctaaaga gggttttttc acttacattt gtgggtttat acaacaaaag ctagctcttg 21120 gaggttccgt ggctataaag ataacagaac attcttggaa tgctgatctt tataagctca 21180 tgggacactt cgcatggtgg acagcctttg ttactaatgt gaatgcgtca tcatctgaag 21240 catttttaat tggatgtaat tatcttggca aaccacgcga acaaatagat ggttatgtca 21300 tgcatgcaaa ttacatattt tggaggata caatccaat tcagttgtct tcctattctt tatttgacat gagtaaattt ccccttaat taaggggtac tgctgttatg tctttaaaag aaggtcaaat caatgatatg attttatctc ttcttagtaa aggtagactt attattagag 21540. aaaacaacag agttgttatt tctagtgatg ttcttgttaa caactaaacg aacaatgttt gtttttcttg ttttattgcc actagtctct agtcagtgtg ttaatcttac aaccagaact 21660. caattacccc ctgcatacac taattctttc acacgtggtg tttattaccc tgacaaagtt ttcagatcct cagttttaca ttcaactcag gacttgttct taccttctt ttccaatgtt acttggttcc atgctataca tgtctctggg accaatggta ctaagaggtt tgataaccct gtcctaccat ttaatgatgg tgtttatttt gcttccactg agagtctaa catataaga ggctggattt ttggtactac tttagattcg aagacccagt ccctacttat tgttaatac 21900. gctactaatg ttgttattaa agtctgtgaa tttcaatttt gtaatgatcc atttttgggt gtttattacc acaaaaacaa caaaagttgg atggaaagtg agttcagagt ttattctagt gcgaataatt gcacttttga atatgtctct cagccttttc ttatggacct tgaaggaaaa 22080 cagggtaatt tcaaaaatct tagggaattt gtgtttaaga atattgatgg ttattttaaa 22140 atatattcta agcacacgcc tattaattta gtgcgtgatc tccctcaggg tttttcggct 22200 ttagaaccat tggtagattt gccaataggt attaacatca ctaggtttca aactttactt 22260 gctttacata gaagttattt gactcctggt gattcttctt caggttggac agctggtgct 22320 gcagcttatt atgtgggtta tcttcaacct aggacttttc tattaaaata taatgaaaat 22380 ggaaccatta cagatgctgt agactgtgca cttgaccctc tctcagaaac aaagtgtacg 22440 ttgaaatcct tcactgtaga aaaaggaatc tatcaaactt ctaactttag agtccaacca 22500 acagaatcta ttgttagatt tcctaatatt acaaacttgt gcccttttgg tgaagttttt 22560 aacgccacca gatttgcatc tgtttatgct tggaacagga agagaatcag caactgtgtt 22620 gctgattatt ctgtcctata taattccgca tcattttcca cttttaagtg ttatggagtg 22680 tctcctacta aattaaatga tctctgcttt actaatgtct atgcagattc atttgtaatt 22740 agaggtgatg aagtcagaca aatcgctcca gggcaaactg gaaagattgc tgattataat 22800 tataaattac cagatgattt tacaggctgc gttatagctt ggaattctaa caatcttgat 22860 tctaaggttg gtggtaatta taattacctg tatagattgt ttaggaagtc taatctcaaa 22920 ccttttgaga gagatatttc aactgaaatc tatcaggccg gtagcacacc ttgtaatggt 22980 gttgaaggtt ttaattgtta ctttccttta caatcatatg gtttccaacc cactaatggt 23040 gttggttacc aaccatacag agtagtagta cttctttttg aacttctaca tgcaccagca 23100 actgtttgtg gacctaaaaa gtctactaat ttggttaaaa acaaatgtgt caatttcaac 23160 ttcaatggtt taacaggcac aggtgttctt actgagtcta acaaaaagtt tctgcctttc 23220 caacaatttg gcagagacat tgctgacact actgatgctg tccgtgatcc acagacactt 23280 gagattcttg acattacacc atgttctttt ggtggtgtca gtgtataac accaggaaca 23340 aatacttcta accaggttgc tgttctttat caggatgtta actgcacaga agtccctgtt 23400 gctattcatg cagatcaact tactcctact tggcgtgttt attctacagg ttctaatgtt 23460 tttcaaacac gtgcaggctg tttaataggg gctgaacatg tcaacaactc atatgagtgt 23520 gacataccca ttggtgcagg tatatgcgct agttatcaga ctcagactaa ttctcctcgg 23580 cgggcacgta gtgtagctag tcaatccatc attgcctaca ctatgtcact tggtgcagaa 23640 aattcagttg cttactctaa taactctatt gccataccca caaattttac tattagtgtt 23700 accacagaaa ttctaccagt gtctatgacc aagacatcag tagattgtac aatgtacatt 23760 tgtggtgatt caactgaatg cagcaatctt ttgttgcaat atggcagttt ttgtacacaa 23820 ttaaaccgtg ctttaactgg aatagctgtt gaacaagaca aaaacaccca agaagtttttt 23880 gcacaagtca aacaaattta caaaacacca ccaattaaag atttggtgg ttttaattttt 23940 tcacaaatat taccagatcc atcaaaacca agcaagaggt catttattga agatctactt 24000 ttcaacaaag tgacacttgc agatgctggc ttcatcaaac aatatggtga ttgccttggt 24060 gatattgctg ctagagacct catttgtgca caaaagttta acggccttac tgttttgcca 24120 cctttgctca cagatgaaat gattgctcaa tacacttctg cactgttagc gggtacaatc 24180 acttctggtt ggacctttgg tgcaggtgct gcattacaaa taccatttgc tatgcaaatg 24240 gcttataggt ttaatggtat tggagttaca cagaatgttc tctatgagaa ccaaaaattg 24300 attgccaacc aatttaatag tgctattggc aaaattcaag actcactttc ttccacagca 24360 agtgcacttg gaaaacttca agatgtggtc aaccaaaatg cacaagcttt aaacacgctt 24420 gttaaacaac ttagctccaa ttttggtgca atttcaagtg ttttaaatga tatcctttca 24480 cgtcttgaca aagttgaggc tgaagtgcaa attgataggt tgatcacagg cagacttcaa 24540 agtttgcaga catatgtgac tcaacaatta attagagctg cagaaatcag agcttctgct 24600 aatcttgctg ctactaaaaat gtcagagtgt gtacttggac aatcaaaaag agttgatttt 24660 tgtggaaagg gctatcatct tatgtccttc cctcagtcag cacctcatgg tgtagtcttc 24720 ttgcatgtga cttatgtccc tgcacaagaa aagaacttca caactgctcc tgccatttgt 24780 catgatggaa aagcacactt tcctcgtgaa gtgtcttg ttcaatgg cacacactgg 24840 tttgtacac aaggaattt tttgaacca caatcatta ctacagacaa cacattgtg 24900 tctggtaact gtgatgttgt ataggatt gtcacaca cagtttatga tccttttgcaa 24960 cctgaattag actcattca ggaggagtta gataaatatt ttagaatca tacatcacca 25020 gatgttgatt taggtgacat ctctggcatt aatgctcag ttgtaaacat tcaaaagaa 25080 attgaccgcc tcaatgaggt tgccagaat ttaatgaat ctctcatcga tctcagaa 25140 cttggaaagt atgagcagta tataaaatgg ccatggtaca tttggctagg ttttagct 25200 ggcttgattg ccatagtaat ggtgacaatt atgctttgct gtatgaccag ttgctgtagt 25260 tgtctcagg gctgttgttc tgtggatcc tgctgcaaat ttgatgaaga cgactctgag 25320 ccagtgctca aaggagtca attackattac actaaacga acttatggat ttgtttatga 25380 gatctcac attggact gtactttga agcaggtga atcaggt gctactcctt 25440 cagattttgt tcgcgctact gcaacgatac cgatacaagc ctcactccct ttcggatggc 25500 ttattgttgg cgttgcactt cttgctgttt ttcagagcgc ttccaaaatc ataaccctca 25560 aaaagagatg gcaactagca ctctccaagg gtgttcactt tgtttgcaac ttgctgttgt 25620 tgtttgtaac agtttactca caccttttgc tcgttgctgc tggccttgaa gccccttttc 25680 tctatcttta tgctttagtc tacttcttgc agagtataaa ctttgtaaga ataataatga 25740 ggctttggct ttgctggaaa tgccgttcca aaaacccatt actttatgat gccaactatt 25800 ttctttgctg gcatactaat tgttacgact attgtatacc ttacaatagt gtaacttctt 25860 caattgtcat tacttcaggt gatggcacaa caagtcctat ttctgaacat gactaccaga 25920 ttggtggtta tactgaaaaa tgggaatctg gagtaaaaga ctgtgttgta ttacacagtt 25980 acttcacttc agactattac cagctgtact caactcaatt gagtacagac actggtgttg 26040 aacatgttac cttcttcatc tacaataaaa ttgttgatga gcctgaagaa catgtccaaa 26100 ttcacacaat cgacggttca tccggagttg ttaatccagt aatggaacca atttatgatg 26160 aaccgacgac gactactagc gtgcctttgt aagcacaagc tgatgagtac gaacttatgt 26220 actcattcgt ttcggaagag acaggtacgt taatagttaa tagcgtactt ctttttcttg 26280 ctttcgtggt attcttgcta gttacactag ccatccttac tgcgcttcga ttgtgtgcgt 26340 actgctgcaa tattgttaac gtgagtcttg taaaaccttc tttttacgtt tactctcgtg 26400 ttaaaaatct gaattcttct agagttcctg atcttctggt ctaaacgaac taaatattat 26460 attagttttt ctgtttggaa ctttaatttt agccatggca gattccaacg gtactattac 26520 cgttgaagag cttaaaaagc tccttgaaca atggaaccta gtaataggtt tcctattcct 26580 tacatggatt tgtcttctac aatttgccta tgccaacagg aataggtttt tgtatataat 26640 taagttaatt ttcctctggc tgttatggcc agtaacttta gcttgttttg tgcttgctgc 26700 tgtttacaga ataaattgga tcaccggtgg aattgctatc gcaatggctt gtcttgtagg 26760 cttgatgtgg ctcagctact tcattgcttc tttcagactg tttgcgcgta cgcgttccat 26820 gtggtcattc aatccagaaa ctaacattct tctcaacgtg ccactccatg gcactattct 26880 gaccagaccg cttctagaaa gtgaactcgt aatcggagct gtgatccttc gtggacatct 26940 tcgtattgct ggacaccatc taggacgctg tgacatcaag gacctgccta aagaaatcac 27000 tgttgctaca tcacgaacgc tttcttatta caaattggga gcttcgcagc gtgtagcagg 27060 tgactcaggt ttgctgcat acagcgcta caggattggc aactataaat taaacacaga 27120 ccattccagt agcagtgaca atattgcttt gcttgtacag taagtgacaa cagatgtttc 27180 atctcgttga ctttcaggtt actatagcag agatattact aattattatg aggactttta 27240 aagtttccat ttggaatctt gattacatca taaacctcat attaaaaat ttatctaagt 27300 cactaactga gaataaatat tctcaattag atgaagagca accaatggag attgattaaa 27360 cgaacatgaa aattattctt ttcttggcac tgataacact cgctacttgt gagctttatc 27420 actaccaaga gtgtgttaga ggtacaacag tacttttaaa agaaccttgc tcttctggaa 27480 catacgaggg caattcacca tttcatcctc tagctgataa caaatttgca ctgacttgct 27540 ttagcactca atttgctttt gcttgtcctg acggcgtaaa acacgtctat cagttacgtg 27600 ccagatcagt ttcacctaaa ctgttcatca gacaagagga agttcaagaa ctttactctc 27660 caatttttct tattgttgcg gcaatagtgt ttataacact ttgcttcaca ctcaaaagaa 27720 agacagaatg attgaacttt cattaattga cttctatttg tgctttttag cctttctgct 27780 attccttgtt ttaattatgc ttattatctt ttggttctca cttgaactgc aagatcataa 27840 tgaaacttgt cacgcctaaa cgaacatgaa atttcttgtt ttcttaggaa tcatcacaac 27900 tgtagctgca tttcaccaag aatgtagttt acagtcatgt actcaacatc aaccatatgt 27960 agttgatgac ccgtgtccta ttcacttcta ttctaaatgg tatattagag taggagctag 28020 aaaatcagca cctttaattg aattgtgcgt ggatgaggct ggttctaaat cacccattca 28080 gtacatcgat atcggtaatt atacagtttc ctgttcacct tttacaatta attgccagga 28140 acctaaattg ggtagtcttg tagtgcgttg ttcgttctat gaagaactttt tagagtatca 28200 tgacgttcgt gttgttttag atttcatcta aacgaacaaa ctaaaatgtc tgataatgga ccccaaaatc agcgaaatgc accccgcatt acgtttggtg gaccctcaga ttcaactggc agtaaccaga atggagaacg cagtggggcg cgatcaaaac aacgtcggcc ccaaggttta cccaatata ctgcgtcttg gttcaccgct ctcactcaac atggcaagga agaccttaa ttccctcgag gacaaggcgt tccaattaac accaatagca gtccagatga ccaaattggc 28560. tactaccgaa gagctaccag acgaattcgt ggtggtgacg gtaaatgaa agatctcagt ccagatggt atttctacta cctaggaact gggccagaag ctggacttcc ctatggtgct 28680. aacaaagacg gcatcatatg ggttgcaact gagggagcct tgaatacacc aaaagatcac attggcaccc gcaatcctgc tacaatgct gcaatcgtgc tacaacttcc tcaaggaaca acattgccaa aaggcttcta cgcagaaggg agcagaggcg gcagtcaagc ctcttctcgt tcctcatcac gtagtcgcaa cagttcaaga aattcaactc caggcagcag tagggaact tctcctgcta gaatggctgg caatggcggt gatgctgctc ttgctttgct gctgcttgac 28920 agattgaacc agcttgagag caaaatgtct ggtaaaggcc aacaacaaca aggccaaact 28980 gtcactaaga aatctgctgc tgaggcttct aagaagcctc ggcaaaaacg tactgccact 29040 aaagcataca atgtaacaca agctttcggc agacgtggtc cagaacaaac ccaaggaaat 29100 tttggggacc aggaactaat cagacaagga actgattaca aacattggcc gcaaattgca 29160 caatttgccc ccagcgcttc agcgttcttc ggaatgtcgc gcattggcat ggaagtcaca 29220 ccttcgggaa cgtggttgac ctacacaggt gccatcaaat tggatgacaa agatccaaat 29280 ttcaaagatc aagtcatttt gctgaataag catattgacg catacaaaac attcccacca 29340 acagagccta aaaaggacaa aaagaagaag gctgatgaaa ctcaagcctt accgcagaga 29400 cagaagaaac agcaaactgt gactcttctt cctgctgcag atttggatga tttctccaaa 29460 caattgcaac aatccatgag cagtgctgac tcaactcagg cctaaactca tgcagaccac 29520 acaaggcaga tgggctatat aaacgttttc gctttccgt ttacgatata tagtctactc 29580 ttgtgcagaa tgaattctcg taactacata gcacaagtag atgtagttaa ctttaatctc 29640 acatagcaat ctttaatcag tgtgtaacat tagggaggac ttgaaagagc caccacattt 29700 tcaccgaggc cacgcggagt acgatcgagt gtacagtgaa caatgctagg gagagctgcc 29760 tatatggaag agccctaatg tgtaaaatta attttagtag tgctatcccc atgtgatttt 29820 aatagcttct taggagaatg a 29841 <210> 2 <211> 29606 <212> DNA <213> Coronavirus <220> <221> misc_feature <222> (3454)..(3741) <223> n is a, c, g, or t <220> <221> misc_feature <222> (16192)..(16193) <223> n is a, c, g, or t <220> <221> misc_feature <222> (20480)..(20750) <223> n is a, c, g, or t <220> <221> misc_feature <222> (21084)..(21323) <223> n is a, c, g, or t <220> <221> misc_feature <222> (22262)..(22479) <223> n is a, c, g, or t <220> <221> misc_feature <222> (22815)..(23081) <223> n is a, c, g, or t <220> <221> misc_feature <222> (24918)..(24919) <223> n is a, c, g, or t <400> 2 agatctgttc tctaaacgaa ctttaaaatc tgtgtggctg tcactcggct gcatgcttag 60 tgcactcacg cagtataatt aataactaat tactgtcgtt gacaggacac gagtaactcg 120 tctatcttct gcaggctgct tacggtttcg tccgtgttgc agccgatcat cagcacatct 180 aggttttgtc cgggtgtgac cgaaaggtaa gatggagagc cttgtccctg gtttcaacga 240 gaaaacacac gtccaactca gtttgcctgt tttacaggtt cgcgacgtgc tcgtacgtgg 300 ctttggagac tccgtggagg aggtcttatc agaggcacgt caacatctta aagatggcac 360 ttgtggctta gtagaagttg aaaaaggcgt tttgcctcaa cttgaacagc cctatgtgtt 420 catcaaacgt tcggatgctc gaactgcacc tcatggtcat gttatggttg agctggtagc 480 agaactcgaa ggcattcagt acggtcgtag tggtgagaca cttggtgtcc ttgtccctca 540 tgtgggcgaa ataccagtgg cttaccgcaa ggttcttctt cgtaagaacg gtaataaagg 600 agctggtggc catagttacg gcgccgatct aaagtcattt gacttaggcg acgagcttgg 660 cactgatcct tatgaagact ttcaagaaaa ctggaacact aaacatagca gtggtgttac 720 ccgtgaactc atgcgtgagc ttaacggagg ggcatacact cgctatgtcg ataacaactt 780 ctgtggccct gatggctacc ctcttgagtg cattaaagac cttctagcac gtgctggtaa 840 agcttcatgc actttgtccg aacaactgga ctttattgac actaagaggg gtgtatactg 900 ctgccgtgaa catgagcatg aaattgcttg gtacacggaa cgttctgaaa agagctatga 960 attgcagaca ccttttgaaa ttaaattggc aaagaaattt gacaccttca atggggaatg 1020 tccaaatttt gtatttccct taaattccat aatcaagact attcaaccaa gggttgaaaa 1080 gaaaaagctt gatggcttta tgggtagaat tcgatctgtc tatccagttg cgtcaccaaa 1140 tgaatgcaac caaatgtgcc tttcaactct catgaagtgt gatcattgtg gtgaaacttc 1200 atggcagacg ggcgattttg ttaaagccac ttgcgaattt tgtggcactg agaatttgac 1260 taaagaaggt gccactactt gtggttactt accccaaaat gctgttgtta aaatttattg 1320 tccagcatgt cacaattcag aagtaggacc tgagcatagt cttgccgaat accataatga 1380 atctggcttg aaaaccattc ttcgtaaggg tggtcgcact attgcctttg gaggctgtgt 1440 gttctcttat gttggttgcc ataacaagtg tgcctattgg gttccacgtg ctagcgctaa 1500 cataggttgt aaccatacag gtgttgttgg agaaggttcc gaaggtctta atgacaacct 1560 tcttgaaata ctccaaaaag agaaagtcaa catcaatatt gttggtgact ttaaacttaa 1620 tgaagagatc gccattattt tggcatcttt ttctgcttcc acaagtgctt ttgtggaaac 1680 tgtgaaaggt ttggattata aagcattcaa acaaattgtt gaatcctgtg gtaattttaa 1740 agttacaaaa ggaaaagcta aaaaaggtgc ctggaatatt ggtgaacaga aatcaatact 1800 gagtcctctt tatgcatttg catcagaggc tgctcgtgtt gtacgatcaa ttttctcccg 1860 cactcttgaa actgctcaaa attctgtgcg tgttttacag aaggccgcta taacaatact 1920 agatggaatt tcacagtatt cactgagact cattgatgct atgatgttca catctgattt 1980 ggctactaac aatctagttg taatggccta cattacaggt ggtgttgttc agttgacttc 2040 gcagtggcta actaacatct ttggcactgt ttgaaaaa ctcaaacccg tccttgattg 2100 gcttgaagag aagtttaagg aaggtgtaga gtttcttaga gacggttggg aaattgttaa 2160 attatctca acctgtgctt gtgaaattgt cggtggacaa attgtcacct gtgcaaagga 2220 aattaaggag agtgttcaga cattctttaa gcttgtaaat aaatttttgg ctttgtgtgc 2280 tgactctatc attattggtg gagctaaact taaagccttg aatttaggtg aaacatttgt 2340 cacgcactca aagggattgt acagaaagtg tgttaaatcc agagaagaaa ctggcctact 2400 catgcctcta aaagccccaa aagaaattat cttcttagag ggagaaacac ttcccacaga 2460 agtgttaaca gaggaagttg tcttgaaaac tggtgattta caaccattag aaaacctac 2520 tagtgaagct gttgaagctc cattggttgg tacaccagtt tgtattaacg ggcttatgtt 2580 gctcgaaatc aaagacacag aaaagtactg tgcccttgca cctaatatga tggtaacaaa 2640 caataccttc acactcaaag gcggtgcacc aacaaaggtt acttttggtg atgatactgt 2700 gatagaagtg caaggttaca agagtgtgaa tatcacttttt gaacttgatg aaaggattga 2760 taaagtactt aatgagaagt gctctgccta tacagttgaa ctcggtacag aagtaaatga 2820 gttcgcctgt gttgtggcag atgctgtcat aaaaactttg caaccagtat ctgaattact 2880 tacaccactg ggcattgatt tagatgagtg gagtatggct acatactact tatttgatga 2940 gtctggtgag tttaaattgg cttcacatat gtattgttct ttttaccctc cagatgagga 3000 tgaagaagaa ggtgattgtg aagaagaaga gtttgagcca tcaactcaat atgagtatgg 3060 tactgaagat gattaccaag gtaaaccttt ggaatttggt gccacttctg ctgctcttca 3120 acctgaagaa gagcaagaag aagattggtt agatgatgat agtcaacaaa ctgttggtca 3180 acaagacggc agtgaggaca atcagacaac tactattcaa acaattgttg aggttcaacc 3240 tcaattagag atggaactta caccagttgt tcagactatt gaagtgaata gttttagtgg 3300 ttatttaaaa cttactgaca atgtatacat taaaaatgca gacattgtgg aagaagctaa 3360 aaaggtaaaa ccaacagtgg ttgttaatgc agccaatgtt taccttaaac atggaggagg 3420 tgttgcagga gccttaaata aggctactaa caannnnnnn nnnnnnnnnn nnnnnnnnnn 3480 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 3540 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 3600 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 3660 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 3720 nnnnnnnnnn nnnnnnnnnn nctttgataa aaatctctat gacaaacttg ttttaagctt 3780 tttggaaatg aagagtgaaa agcaagttga acaaaagatc gctgagattc ctaaagagga 3840 agttaagcca tttataactg aaagtaaacc ttcagttgaa cagagaaaac aagatgataa 3900 gaaaatcaaa gcttgtgttg aagaagttac aacaactctg gaagaaacta agttcctcac 3960 agaaaacttg ttactttata ttgacattaa tggcaatctt catccagatt ctgccactct 4020 tgttagtgac attgacatca ctttcttaaa gaaagatgct ccatatatag tgggtgatgt 4080 tgttcaagag ggtgttttaa ctgctgtggt tatacctact aaaaaggctg gtggcactac 4140 tgaaatgcta gcgaaagctt tgagaaaagt gccaacagac aattatataa ccacttaccc 4200 gggtcagggt ttaaatggtt acactgtaga ggaggcaaag acagtgctta aaaagtgtaa 4260 aagtgccttt tacattctac catctattat ctctaatgag aagcaagaaa ttcttggaac 4320 tgtttcttgg aatttgcgag aaatgcttgc acatgcagaa gaaacacgca aattaatgcc 4380 tgtctgtgtg gaaactaaag ccatagtttc aactatacag cgtaaatata agggtattaa 4440 aatacaagag ggtgtggttg attatggtgc tagattttac ttttacacca gtaaaacaac 4500 tgtagcgtca cttatcaaca cacttaacga tctaaatgaa actcttgtta caatgccact 4560 tggctatgta acacatggct taaatttgga agaagctgct cggtatatga gatctctcaa 4620 agtgccagct acagtttctg tttcttcacc tgatgctgtt acagcgtata atggttatct 4680 tacttcttct tctaaaacac ctgaagaaca ttttattgaa accatctcac ttgctggttc 4740 ctataaagat tggtcctatt ctggacaatc tacacaacta ggtatagaat ttcttaagag 4800 aggtgataaa agtgtatatt acactagtaa tcctaccaca ttccacctag atggtgaagt tatcaccttt gacaatctta agacacttct ttctttgaga gaagtgagga ctattaaggt gtttacaaca gtagacaaca ttaacctcca cacgcaagtt gtggacatgt caatgacata tggacaacag tttggtccaa cttatttgga tggagctgat gttactaaaa taaaacctca 5100. 5100. 5100. 5100. 5100. 5100. 5100. 5100. 5100. 5100 ggcttttgag tactaccaca caactgatcc tagttttctg ggtaggtaca tgtcagcatt aaatcacact aaaaagtgga aatacccaca agttaatggt ttaacttcta ttaaatgggc agtaacaac tgttatcttg ccactgcatt gttaacactc cacaaatag agttgaagtt taatccacct gctctacaag atgcttatta cagagcaagg gctggtgaag ctgctaactt ttgtgcactt atcttagcct actgtaataa gacagtaggt gagttaggtg atgttaga 5400 5460. aacaatgagt tacttgtttc aacatgccaa tttagattct tgcaaaagag tcttgaacgt ggtgtgtaaa acttgtggac aacagcagac aacccttaag ggtgtagaag ctgttatgta 5520 catgggcaca ctttcttatg aacaatttaa gaaaggtgtt cagatacctt gtacgtgtgg 5580 taaacaagct acacaatatc tagtacaaca ggagtcacct tttgttatga tgtcagcacc 5640 acctgctcag tatgaactta agcatggtac atttacttgt gctagtgagt acactggtaa 5700 ttaccagtgt ggtcactata aacatataac ttctaaagaa actttgtatt gcatagacgg 5760 tgctttactt acaaaagtcct cagaatacaa aggtcctatt acggatgttt tctacaaaga 5820 aaacagttac acaacaacca taaaaccagt tacttataaa ttggatggtg ttgtttgtac 5880 agaaattgac cctaagttgg acaattatta taagaaagac aattcttatt tcacagagca 5940 accaattgat cttgtaccaa accaaccata tccaaacgca agcttcgata atttaagtt 6000 tgtatgtgat aatatcaaat ttgctgatga tttaaaccag ttaactggtt ataagaaacc 6060 tgcttcaaga gagcttaaag ttacattttt ccctgactta aatggtgatg tggtggctat 6120 tgattataaa cactacacac cctctttaa gaaaggagct aaattgttac ataaacctat 6180 tgtttggcat gttaacaatg caactaataa agccacgtat aaaccaaata cctggtgtat 6240 acgttgtctt tggagcaca aaccggttga aacatcaaat tcgtttgatg tactgaagtc 6300 aggatgcg cagggaatgg ataatcttgc ctgcgagat ctaaaaccag tctctgaaga 6360 agtagtggaa atcctacca tacagaaaga cgttcttgag tgtaatgtga aaactaccga 6420 agttgtagga gatatac ttaaaccagc aaataatagt ttaaaaatta cagaagaggt 6480 tggccacaca gatctaatgg ctgcttatgt agacaatctct agtcttacta ttaagaaacc 6540 taatgaatta tctgagtgt taggttga aacccttgct actcatggtt tagctgctgt 6600 taatagtgtc ccttgggata ctatagctaa ttatgctaag ccttttctta acaaagttgt 6660 tagtacaact actacatag ttacacggtg tttaaaccgt gtttgtacta attatatgcc 6720 ttatttcttt actttattgc tacattgtg tactttact agaagtaca attctagaat 6780 taaagcatct atgccgacta ctatagcaaa gatactgtt aagagtgtcg gtaaattttg 6840 tcagaggct tcatttaatt attgaagtc acctatttt tctaactga taatattat 6900 aatttggttt ttactattaa gtgtttgcct aggttcttta atctactcaa ccgctgcttt 6960 aggtgtttta atgtctaatt taggcatgcc ttcttactgt actggttaca gagaaggcta 7020 tttgaactct actaatgtca ctattgcaac ctactgtact ggttctatac cttgtagtgt 7080 ttgtcttagt ggtttagatt ctttagacac ctatccttct ttagaaacta tacaaattac 7140 catttcatct tttaaatggg atttaactgc ttttggctta gttgcagagt ggtttttggc 7200 atatattctt ttcactaggt ttttctatgt acttggattg gctgcaatca tgcaattgtt 7260 tttcagctat tttgcagtac attttattag taattcttgg cttatgtggt taataattaa 7320 tcttgtacaa atggccccga tttcagctat ggttagaatg tacatcttct ttgcatcatt 7380 ttattatgta tggaaaagtt atgtgcatgt tgtagacggt tgtaattcat caacttgtat 7440 gatgtgttac aaacgtaata gagcaacaag agtcgaatgt acaactattg ttaatggtgt 7500 tagaaggtcc ttttatgtct atgctaatgg aggtaaaggc ttttgcaaac tacacaattg 7560 gaattgtgtt aattgtgata cattctgtgc tggtagtaca tttattagtg atgaagttgc 7620 gagagacttg tcactacagt ttaaaagacc aataaatcct actgaccagt cttcttacat 7680 cgttgatagt gttacagtga agaatggttc catccatctt tactttgata aagctggtca 7740 aaagacttat gaaagacatt ctctctctca ttttgttaac ttagacaacc tgagagctaa 7800 taacactaaa ggttcattgc ctattaatgt tatagttttt gatggtaaat caaaatgtga 7860 agaatcatct gcaaaatcag cgtctgttta ctacagtcag cttatgtgtc aacctatact 7920 gttactagat caggcattag tgtctgatgt tggtgatagt gcggaagttg cagttaaaat 7980 gtttgatgct tacgttaata cgttttcatc aacttttaac gtaccaatgg aaaaactcaa 8040 aacactagtt gcaactgcag aagctgaact tgcaaagaat gtgtccttag acaatgtctt 8100 atctactttt atttcagcag ctcggcaagg gtttgttgat tcagatgtag aaactaaaga 8160 tgttgttgaa tgtcttaaat tgtcacatca atctgacata gaagttactg gcgatagttg 8220 taataactat atgctcacct ataacaaagt tgaaaacatg acaccccgtg accttggtgc 8280 ttgtattgac tgtagtgcgc gtcatattaa tgcgcaggta gcaaaaagtc acaacattgc 8340 tttgatatgg aacgttaaag atttcatgtc attgtctgaa caactacgaa aacaaatacg 8400 tagtgctgct aaaaagaata acttaccttt tagttgaca tgtgcaacta ctagacaagt 8460 tgttaatgtt gtaacaacaa agatagcact taagggtggt aaaattgtta ataattggtt 8520 gaagcagtta attaaagtta cacttgtgtt cctttttgtt gctgctattt tctatttaat 8580 aacacctgtt catgtcatgt ctaaacatac tgacttttca agtgaaatca taggatacaa 8640 ggctattgat ggtggtgtca ctcgtgacat agcatctaca gatacttgtt ttgctaacaa 8700 acatgctgat tttgacacat ggtttagcca gcgtggtggt agttatacta atgacaaagc 8760 ttgcccattg attgctgcag tcataacaag agaagtgggt tttgtcgtgc ctggtttgcc 8820 tggcacgata ttacgcacaa ctaatggtga cttttgcat ttcttaccta gagtttttag 8880 tgcagttggt aacatctgtt acacaccatc aaaacttata gagtacactg actttgcaac 8940 atcagcttgt gttttggctg ctgaatgtac aatttttaaa gatgcttctg gtaagccagt 9000 accatattgt tatgatacca atgtactaga aggttctgtt gcttatgaaa gtttacgcc 9060 tgacacacgt tatgtgctca tggatggctc tattattcaa tttcctaaca cctaccttga 9120 aggttctgtt aggtggtaa caacttttga ttctgagtac tgtaggcacg gcacttgtga aagatcagaa gctggtgttt gtgtatctac tagtggtaga tgggtactta acaatgatta ttacagatct ttaccaggag ttttctgtgg tgtagtgct gtaaatttac ttactataat gtttacacca ctaattcaac ctattggtgc tttggacata tcagcatcta tagtagctgg tggtattgta gctatcgtag taacatgcct tgcctactat tttatgaggt ttagaagagc ttttggtgaa tacagtcatg tagttgcctt taatacttta ctattcctta tgtcattcac tgtactctgt ttaacaccag tttactcatt cttacctggt gtttattctg ttatttactt gtacttgaca ttttatctta ctaatgatgt ttctttttta gcacatattc agtggatggt father cctttagtac ctttctggat aacaattgct fathercattt gtatttccac aaagcatttc tattggttct ttagtaatta cctaaagaga cgtgtagtct ttaatggtgt ttcctttagt acttttgaag aagctgcgct gtgcaccttt ttgttaaata aagaaatgta tctaaagttg cgtagtgatg tgctattacc tcttacgcaa father acttagctct ttataataag tacaagtatt ttagtggagc aatggataca actagctaca gagaagctgc 9900 ttgttgtcat ctcgcaaagg ctctcaatga cttcagtaac tcaggttctg atgttcttta 9960 ccaaccacca caaacctcta tcacctcagc tgttttgcag agtggtttta gaaaaatggc 10020 attcccatct ggtaaagttg agggttgtat ggtacaagta acttgtggta caactacact 10080 taacggtctt tggcttgatg acgtagttta ctgtccaaga catgtgatct gcacctctga 10140 agacatgctt aaccctaatt atgaagattt actcattcgt aagtctaatc ataatttctt 10200 ggtacaggct ggtaatgttc aactcagggt tattggacat tctatgcaaa attgtgtact 10260 taagcttaag gttgatacag ccaatcctaa gacacctaag tataagtttg ttcgcattca 10320 accaggacag actttttcag tgttagcttg ttacaatggt tcaccatctg gtgtttacca 10380 atgtgctatg aggcccaatt tcactattaa gggttcattc cttaatggtt catgtggtag 10440 tgttggtttt aacatagatt atgactgtgt ctctttttgt tacatgcacc atatggaatt 10500 accaactgga gttcatgctg gcacagactt agaaggtaac ttttatggac cttttgttga 10560 caggcaaaca gcacaagcag ctggtacgga cacaactatt acagttaatg ttttagcttg 10620 gttgtacgct gctgttataa atggagacag gtggttctc aatcgattta ccacaactct 10680 taatgacttt aaccttgtgg ctatgaagta caattatgaa cctctaacac aagaccatgt 10740 tgacatacta ggacctcttt ctgctcaaac tggaattgcc gttttagata tgtgtgcttc 10800 attaaaagaa ttactgcaaa atggtatgaa tggacgtacc atattgggta gtgctttatt 10860 agaagatgaa tttacacctt ttgatgttgt tagacaatgc tcaggtgtta ctttccaaag 10920 tgcagtgaaa agaacaatca agggtacaca ccactggttg ttactcacaa ttttgacttc 10980 acttttagtt ttagtccaga gtactcaatg gtctttgttc ttttttttgt atgaaaatgc 11040 cttttacct tttgctatgg gtattattgc tatgtctgct tttgcaatga tgtttgtcaa 11100 acataagcat gcatttctct gtttgttttt gttaccttct cttgccactg tagcttattt 11160 tatatggtc tatatgcctg ctagttgggt gatgcgtatt atgacatggt tggatatggt 11220 tgatactagt ttgaagctaa aagactgtgt tatgtatgca tcagctgtag tgttactaat 11280 ccttatgaca gcaagaactg tgtatgatga tggtgctagg agagtgtgga cacttatgaa 11340 tgtcttgaca ctcgtttata aagtttatta tggtaatgct ttagatcaag ccatttccat 11400 gtgggctctt ataatctctg ttacttctaa ctactcaggt gtagttacaa ctgtcatgtt 11460 tttggccaga ggtattgttt ttatgtgtgt tgagtattgc cctattttct tcataactgg 11520 taatacactt cagtgtataa tgctagttta ttgtttctta ggctattttt gtacttgtta 11580 ctttggcctc ttttgtttac tcaaccgcta ctttagactg actcttggtg tttatgatta 11640 cttagtttct acacaggagt ttagatatat gaattcacag ggactactcc cacccaagaa 11700 tagcatagat gccttcaaac tcaacattaa attgttgggt gttggtggca aaccttgtat 11760 caaagtagcc actgtacagt ctaaaatgtc agatgtaaag tgcacatcag tagtcttact 11820 ctcagttttg caacaactca gagtagaatc atcatctaaa ttgtgggctc aatgtgtcca 11880 gttacacaat gacattctct tagctaaaga tactactgaa gcctttgaaa aaatggtttc 11940 actactttct gttttgcttt ccatgcaggg tgctgtagac ataaacaagc tttgtgaaga 12000 aatgctggac aacagggcaa ccttacaagc tatagcctca gagtttagtt cccttccatc 12060 atatgcagct tttgctactg ctcaagaagc ttatgagcag gctgttgcta atggtgattc 12120 tgaagttgtt cttaaaaagt tgaagaagtc tttgaatgtg gctaaatctg aatttgaccg 12180 tgatgcagcc atgcaacgta agttggaaaa gatggctgat caagctatga cccaaatgta 12240 taaacaggct agatctgagg acaagagggc aaaagttact agtgctatgc agacaatgct 12300 tttcactatg cttagaaagt tggataatga tgcactcaac aacattatca acaatgcaag 12360 agatggttgt gttcccttga acataatacc tcttacaaca gcagccaaac taatggttgt 12420 cataccagac tataacacat ataaaaatac gtgtgatggt acaacattta cttatgcatc 12480 agcattgtgg gaaatccaac aggttgtaga tgcagatagt aaaattgttc aacttagtga 12540 aattagtatg gacaattcac ctaatttagc atggcctctt attgtaacag ctttaagggc 12600 caattctgct gtcaaattac agaataatga gcttagtcct gttgcactac gacagatgtc 12660 ttgtgctgcc ggtactacac aaactgcttg cactgatgac aatgcgttag cttattacaa 12720 cacaacaaag ggaggtaggt ttgtacttgc actgttatcc gatttacagg atttgaaatg 12780 ggctagattc cctaagagtg atggaactgg tactatctat acagaactgg aaccaccttg 12840 taggtttgtt acagacacac ctaaaggtcc taaagtgaag tatttatact ttattaaagg 12900 attaaacaac ctaaatagag gtatggtact tggtagttta gctgccacag tacgtctaca 12960 agctggtaat gcaacagaag tgcctgccaa ttcaactgta ttatctttct gtgcttttgc 13020 tgtagatgct gctaaagctt acaaagatta tctagctagt gggggacaac caatcactaa 13080 ttgtgttaag atgttgtgta cacacactgg tactggtcag gcaataacag ttacaccgga 13140 agccaatatg gatcaagaat cctttggtgg tgcatcgtgt tgtctgtact gccgttgcca 13200 catagatcat ccaaatccta aaggattttg tgacttaaaa ggtaagtatg tacaaatacc 13260 tacaacttgt gctaatgacc ctgtgggttt tacacttaaa aacacagtct gtaccgtctg 13320 cggtatgtgg aaaggttatg gctgtagttg tgatcaactc cgcgaaccca tgcttcagtc 13380 agctgatgca caatcgtttt taaacgggtt tgcggtgtaa gtgcagcccg tcttacaccg 13440 tgcggcacag gcactagtac tgatgcgta tacagggctt ttgacatcta caatgataaa 13500 gtagctggtt ttgctaaatt cctaaaaact aattgttgtc gcttccaaga aaaggacgaa 13560 gatgacaatt taattgattc ttactttgta gttaagagac acactttctc taactaccaa 13620 catgaagaaa caatttataa tttacttaag gattgtccag ctgttgctaa acatgacttc 13680 tttaagttta gaatagacgg tgacatggta ccacatatat cacgtcaacg tcttactaaa 13740 tacacaatgg cagacctcgt ctatgcttta aggcattttg atgaaggtaa ttgtgataca 13800 ttaaaagaaa tacttgtcac atacaattgt tgtgatgatg attatttcaa taaaaaggac 13860 tggtatgatt ttgtagaaaa cccagatata ttacgcgtat acgccaactt aggtgaacgt 13920 gtacgccaag ctttgttaaa aacagtacaa ttctgtgatg ccatgcgaaa tgctggtatt 13980 gttggtgtac tgacattaga taatcaagat ctcaatggta actggtatga tttcggtgat 14040 ttcatacaaa ccacgccagg tagtggagtt cctgttgtag attcttatta ttcattgtta 14100 atgcctatat taaccttgac cagggcttta actgcagagt cacatgttga cactgactta 14160 acaaagcctt acattaagtg ggatttgtta aaatatgact tcacggaagagaggttaaaa 14220 ctctttgacc gttatttaa atattgggat cagacatacc acccaaattg tgttaactgt 14280 ttggatgaca gatgcattct gcattgtgca aactttaatg ttttattctc tacagtgttc 14340 ccacttacaa gttttggacc actagtgaga aaaatatttg ttgatggtgt tccatttgta 14400 14460 catagctcta gacttagttt taggaatta cttgtgtatg ctgctgaccc tgctatgcac 14520 gctgcttctg gtaatctatt actagataaa cgcactacgt gcttttcagt agctgcactt 14580 actaacaatg ttgcttttca aactgtcaaa cccggtaatt ttaacaaaga cttctatgac 14640 tttgctgtgt ctaagggttt ctttaaggaa ggaagttctg ttgaattaaa acacttcttc 14700 tttgctcagg atggtaatgc tgctatcagc gattatgact actatcgtta taatctacca 14760 acaatgtgtg atatcagaca actactattt gtagttgaag ttgttgataa gtactttgat 14820 tgttacgatg gtggctgtat taatgctaac caagtcatcg tcaacaacct agacaaatca 14880 gctggttttc catttaataa atggggtaag gctagacttt attatgattc aatgagttat 14940 gaggatcaag atgcactttt cgcatataca aaacgtaatg tcatccctac tataactcaa 15000 atgaatctta agtatgccat tagtgcaaag aatagagctc gcaccgtagc tggtgtctct 15060 atctgtagta ctatgaccaa tagacagttt catcaaaaat tattgaaatc aatagccgcc 15120 actagaggag ctactgtagt aattggaaca agcaaattct atggtggttg gcacaacatg 15180 ttaaaaactg tttatagtga tgtagaaaac cctcacctta tgggttggga ttatcctaaa 15240 tgtgatagag ccatgcctaa catgcttaga attatggcct cacttgttct tgctcgcaaa 15300 catacaacgt gttgtagctt gtcacaccgt ttctatagat tagctaatga gtgtgctcaa 15360 gtattgagtg aaatggtcat gtgtggcggt tcactatatg ttaaaccagg tggaacctca 15420 tcaggagatg ccacaactgc ttatgctaat agtgttttta acatttgtca agctgtcacg 15480 gccaatgtta atgcactttt atctactgat ggtaacaaaa ttgccgataa gtatgtccgc 15540 aatttacaac acagacttta tgagtgtctc tatagaaata gagatgttga cacagacttt 15600 gtgaatgagt tttacgcata tttgcgtaaa catttctcaa tgatgatact ctctgacgat 15660 gctgttgtgt gtttcaatag cacttatgca tctcaaggtc tagtggctag cataaagaac 15720 tttaagtcag ttctttatta tcaaaacaat gtttttatgt ctgaagcaaa atgttggact 15780 gagactgacc ttactaaag acctcatgaa ttttgctctc aacatacaat gctagttaaa 15840 cagggtgatg attatgtgta ccttccttac ccagatccat caagaatcct aggggccggc 15900 tgttttgtag atgatatcgt aaaaaacagat ggtacactta tgattgaacg gttcgtgtct 15960 ttagctatag atgcttaccc acttactaaaa catcctaatc aggagtatgc tgatgtcttt 16020 catttgtact tacaatacat aagaaagcta catgatgagt taacaggaca catgttagac 16080 atgtattctg ttatgcttac taatgataac acttcaaggt attgggaacc tgagttttat 16140 gaggctatgt acacaccgca tacagtctta caggctgttg gggcttgtgt tnnttgcaat 16200 tcacagactt cattaagatg tggtgcttgc atacgtagac cattcttatg ttgtaaatgc 16260 tgttacgacc atgtcatatc aacatcacat aaattagttt tgtctgttaa tccgtatgtt 16320 tgcaatgctc caggttgtga tgtcacagat gtgactcaac tttacttagg aggtatgagc 16380 tattattgta aatcacataa accacccatt agttttccat tgtgtgctaa tggacaagtt 16440 tttggtttat ataaaaatac atgtgttggt agcgataatg ttactgactt taatgcaatt 16500 gcaacatgtg actggacaaa tgctggtgat tacattttag ctaacacctg tactgaaaga 16560 ctcaagcttt ttgcagcaga aacgctcaaa gctactgagg agacatttaa actgtcttat 16620 ggtattgcta ctgtacgtga agtgctgtct gacagagaat tacatctttc atgggaagtt 16680 ggtaaaccta gaccaccact taaccgaaat tatgtcttta ctggttatcg tgtaactaaa 16740 aacagtaaag tacaaatagg agagtacacc tttgaaaaag gtgactatgg tgatgctgtt 16800 gttaccgag gtacaacaac ttacaaatta aatgttggtg attattttgt gctgacatca 16860 catacagtaa tgccattaag tgcacctaca ctagtgccac aagagcacta tgttagaatt 16920 actggcttat acccaacact caatatctca gatgagtttt ctagcaatgt tgcaaattat 16980 caaaaggttg gtatgcaaaa gtattctaca ctccagggac cacctggtac tggtaagagt 17040 cattttgcta ttggcctagc tctctactac cttctgctc gcatagtgta tacagcttgc 17100 tctcatgccg ctgttgatgc actatgtgag aaggcattaa aatatttgcc tatagataaa 17160 tgtagtagaa ttatacctgc acgtgctcgt gtagattgtt ttgataaatt caaagtgaat 17220 tcaacattag aacagtatgt cttttgtact gtaaatgcat tgcctgagac gacagcagat 17280 atagttgtct ttgatgaaat ttcaatggcc acaaattatg atttgagtgt tgtcaatgcc 17340 agattacgtg ctaagcacta tgtgtacatt ggcgaccctg ctcaattacc tgcaccacgc 17400 acattgctaa ctaagggcac actagaacca gaatatttca attcagtgtg tagacttatg 17460 aaaactatag gtccagacat gttcctcgga acttgtcggc gttgtcctgc tgaaattgtt 17520 gacactgtga gtgctttggt ttatgataat aagcttaaag cacataaaga caaatcagct 17580 caatgcttta aaatgtttta taagggtgtt atcacgcatg atgtttcatc tgcaattaac 17640 aggccacaaa tagcgtggt aagagaattc cttacacgta accctgcttg gagaaaagct 17700 gtctttattt caccttataa ttcacagaat gctgtagcct caaagatttt gggactacca 17760 actcaaactg ttgattcatc acagggctca gaatatgact atgtcatatt cactcaaacc 17820 actgaaacag ctcactcttg taatgtaaac agatttaatg ttgctattac cagagcaaaa 17880 gtaggcatac tttgcataat gtctgataga gacctttatg acaagttgca atttacaagt 17940 cttgaaattc cacgtaggaa tgtggcaact ttacaagctg aaaatgtaac aggactcttt 18000 aaagattgta gtaaggtaat cactgggtta catcctacac aggcacctac acacctcagt 18060 gttgacacta aattcaaaac tgaaggttta tgtgttgaca tacctggcat acctaaggac 18120 atgacctata gaagactcat ctctatgatg ggttttaaaa tgaattatca agttaatggt 18180 taccctaaca tgttatcac ccgcgaagaa gctataagac atgtacgtgc atggattggc 18240 ttcgatgtcg aggggtgtca tgctactaga gaagctgttg gtaccaattt acctttacag 18300 ctaggttttt ctacaggtgt taacctagtt gctgtaccta caggttatgt tgatacacct 18360 aataatacag atttttccag agttagtgct aaaccaccgc ctggagatca attaaacac 18420 ctcataccac ttatgtacaa aggacttcct tggaatgtag tgcgtataaa gattgtacaa 18480 atgttaagtg acacacttaa aaatctctct gacagagtcg tatttgtctt atgggcacat 18540 ggctttgagt tgacatctat gaagtatttt gtgaaaatag gacctgagcg cacctgttgt 18600 ctatgtgata gacgtgccac atgcttttcc actgcttcag acacttatgc ctgttggcat 18660 cattctattg gatttgatta cgtctataat ccgtttatga ttgatgttca acaatggggt 18720 tttacaggta acctacaaag caaccatgat ctgtattgtc aagtccatgg taatgcacat 18780 gtagctagtt gtgatgcaat catgactagg tgtctagctg tccacgagtg ctttgttaag 18840 cgtgttgact ggactattga atatcctata attggtgatg aactgaagat taatgcggct 18900 tgtagaaagg ttcaacacat ggttgttaaa gctgcattat tagcagacaa attcccagtt 18960 cttcacgaca ttggtaaccc taaagctatt aagtgtgtac ctcaagctga tgtagaatgg 19020 aagttctatg atgcacagcc ttgtagtgac aaagcttata aaatagaaga attattctat 19080 tcttatgcca cacattctga caaattcaca gatggtgtat gcctattttg gaattgcaat 19140 gtcgatagat atcctgctaa ttccattgtt tgtagatttg acactagagt gctatctaac 19200 cttaacttgc ctggttgtga tggtggcagt ttgtatgtaa ataaacatgc attccacaca 19260 ccagcttttg ataaaagtgc ttttgttaat ttaaaacaat taccattttt ctattactct 19320 gacagtccat gtgagtctca tggaaaacaa gtagtgtcag atatagatta tgtaccacta 19380 aagtctgcta cgtgtataac acgttgcaat ttaggtggtg ctgtctgtag acatcatgct 19440 aatgagtaca gattgtatct cgatgcttat aacatgatga tctcagctgg ctttagcttg 19500 tgggtttaca aacaatttga tacttataac ctctggaaca cttttacaag acttcagagt 19560 ttagaaaatg tggcttttaa tgttgtaaat aagggacact ttgatggaca acagggtgaa 19620 gtaccagttt ctatcattaa taacactgtt tacacaaaag ttgatggtgt tgatgtagaa 19680 ttgtttgaaa ataaaacaac attacctgtt aatgtagcat ttgagctttg ggctaagcgc 19740 aacattaaac cagtaccaga ggtgaaaata ctcaataatt tgggtgtgga cattgctgct 19800 aatactgtga tctgggacta caaaagagat gctccagcac atatatctac tattggtgtt 19860 tgttctatga ctgacatagc caagaaacca actgaaacga tttgtgcacc actcactgtc 19920 ttttttgatg gtagagttga tggtcaagta gacttattta gaaatgcccg taatggtgtt 19980 cttattacag aaggtagtgt taaaggttta caaccatctg taggtcccaa acaagctagt 20040 cttaatggag tcacattaat tggagaagcc gtaaaaacac agttcaatta ttataagaaa 20100 gttgatggtg ttgtccaaca attacctgaa acttacttta ctcagagtag aaatttacaa 20160 gaatttaaac ccaggagtca aatggaaatt gatttcttag aattagctat ggatgaattc 20220 attgaacggt ataaattaga aggctatgcc ttcgaacata tcgtttatgg agattttagt 20280 catagtcagt taggtggttt acatctactg attggactag ctaaacgttt taaggaatca 20340 ccttttgaat tagaagattt tattcctatg gacagtacag ttaaaaacta tttcataaca 20400 gatgcgcaaa caggttcatc taagtgtgtg tgttctgtta ttgatttatt acttgatgat 20460 tttgttgaaa taataaaatn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 20520 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 20580 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 20640 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 20700 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn atatttaaac 20760 acattaacat tagctgtacc ctataatatg agagttatac attttggtgc tggttctgat 20820 aaaggagttg caccaggtac agctgtttta agacagtggt tgcctacggg tacgctgctt 20880 gtcgattcag atcttaatga ctttgtctct gatgcagatt caactttgat tggtgattgt 20940 gcaactgtac atacagctaa taaatgggat ctcattatta gtgatatgta cgaccctaag 21000 actaaaaatg ttacaaaaga aaatgactct aaagagggtt ttttcactta catttgtggg 21060 tttatacaac aaagctagc tctnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 21120 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 21180 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 21240 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 21300 nnnnnnnnnn nnnnnnnnnn nnntttattt gacatgagta aatttcccct taaattaagg 21360 ggtactgctg ttatgtcttt aaaagaaggt caaatcaatg atatgatttt atctcttctt 21420 agtaaaggta gacttataat tagagaaaac aacagagttg ttatttctag tgatgttctt 21480 gttaacaact aaacgaacaa tgtttgtttt tcttgtttta ttgccactag tctctagtca 21540 gtgtgttaat tttacaaaca gaactcaatt accctctgca tacactaatt ctttcacacg 21600 tggtgttat taccctgaca aagttttcag atcctcagtt ttacattcaa ctcaggactt 21660 gttcttacct ttcttttcca atgttacttg gttccatgct atacatgtct ctgggaccaa 21720 tggtactaag aggtttgata accctgtcct accatttaat gatggtgttt atttgcttc 21780 cactgagaag tctaacataa taagaggctg gatttttggt actactttag attcgaagac 21840 ccagtcccta cttattgtta ataacgctac taatgttgtt attaaagtct gtgaatttca 21900 attttgtaat tatccatttt tgggtgttta ttaccacaaa aacaacaaaa gttggatgga 21960 aagtgagttc agagtttatt ctagtgcgaa taattgcact tttgaatatg tctctcagcc 22020 ttttcttatg gaccttgaag gaaaacaggg taatttcaaa aatcttagtg aatttgtgtt 22080 taagaatatt gatggttatt ttaaaatata ttctaagcac acgcctatta atttagtgcg 22140 tgatctccct cagggttttt cggctttaga accattggta gatttgccaa taggtattaa 22200 catcactagg tttcaaactt tacttgcttt acatagaagt tatttgactc ctggtgattc 22260 tnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 22320 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 22380 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 22440 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnt agatttccta atattacaaa 22500 cttgtgccct tttggtgaag tttttaacgc caccagattt gcatctgttt atgcttggaa 22560 caggaagaga atcagcaact gtgttgctga ttattctgtc ctatataatt ccgcatcatt 22620 ttccactttt aagtgttatg gagtgtctcc tactaaatta aatgatctct gctttactaa 22680 tgtctatgca gattcatttg taattagagg tgatgaagtc agacaaatcg ctccagggca 22740 aactggaacg attgctgatt ataattataa attaccagat gattttacag gctgcgttat 22800 agcttggaat tctannnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 22860 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 22920 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 22980 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 23040 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn naaaagtcta ctaatttggt 23100 taaaaacaaa tgtgtcaatt tcaacttcaa tggtttaaca ggcacaggtg ttcttactga 23160 gtctaacaaa aagtttctgc ctttccaaca atttggcaga gacattgctg acactactga 23220 tgctgtccgt gatccacaga cacttgagat tcttgacatt acaccatgtt cttttggtgg 23280 tgtcagtgtt ataacaccag gaacaaatac ttctaaccag gttgctgttc tttatcaggg 23340 tgttaactgc acagaagtcc ctgttgctat tcatgcagat caacttactc ctacttggcg 23400 tgtttattct acaggttcta atgtttttca aacacgtgca ggctgtttaa taggggctga 23460 atatgtcaac aactcatatg agtgtgacat acccattggt gcaggtatat gcgctagtta 23520 tcagactcag actaattctc ctcggcgggc acgtagtgta gctagtcaat ccatcattgc 23580 ctacactatg tcacttggtg cagaaaattc agttgcttac tctaataact ctattgccat 23640 acccacaaat tttactatta gtgttaccac agaaattcta ccagtgtcta tgaccaagac 23700 atcagtagat tgtacaatgt acatttgtgg tgattcaact gaatgcagca atcttttgtt 23760 gcaatatggc agtttttgta cacaattaaa ccgtgcttta actggaatag ctgttgaaca 23820 agacaaaaac acccaagaag tttttgcaca agtcaaacaa atttacaaaa caccaccaat 23880 taaagatttt ggtggtttta atttttcaca aatattacca gatccatcaa aaccaagcaa 23940 gaggtcattt attgaagatc tacttttcaa caaagtgaca cttgcagatg ctggcttcat 24000 caaacaatat ggtgattgcc ttggtgatat tgctgctaga gacctcattt gtgcacaaaa 24060 gtttaacggc cttactgttt tgccaccttt gctcacagat gaaatgattg ctcaatacac 24120 ttctgcactg ttagcgggta caatcacttc tggttggacc tttggtgcag gtgctgcatt 24180 acaaatacca tttgctatgc aaatggctta taggtttaat ggtattggag ttacacagaa 24240 tgttctctat gagaaccaaa aattgattgc caaccaattt aatagtgcta ttggcaaaat 24300 tcaagactca ctttcttcca cagcaagtgc acttggaaaa cttcaagatg tggtcaacca 24360 aaatgcacaa gctttaaaca cgcttgttaa acaacttagc tccaattttg gtgcaatttc 24420 aagtgtttta aatgatatcc tttcacgtct tgacaaagtt gaggctgaag tgcaaattga 24480 taggttgatc acaggcagac ttcaaagttt gcagacatat gtgactcaac aattaattag 24540 agctgcagaa atcagagctt ctgctaatct tgctgctatt aaaatgtcag agtgtgtact 24600 tggacaatca aaaagagttg atttttgtgg aaagggctat catcttatgt ccttccctca 24660 gtcagcacct catggtgtag tcttcttgca tgtgacttat gtccctgcac aagaaaagaa 24720 cttcacaact gctcctgcca tttgtcatga tggaaaagca cactttcctc gtgaaggtgt 24780 cttgtttca aatggcacac actggttgt aacacaaagg aatttttatg aaccacaaat 24840 cattactaca gacaacacat ttgtgtctgg taactgtgat gttgtaatag gaattgtcaa 24900 caacacagtt tatgatcnnt tgcaacctga attagactca ttcaaggagg agttagataa 24960 atattttaag aatcatacat caccagatgt tgatttaggt gacatctctg gcattaatgc 25020 ttcatttgta aacattcaaa aagaaattga ccgcctcaat gaggttgcca agaatttaaa tgaatctctc atcgatctcc aagaacttgg aaagtatgag cagtatataa aatggccatg gtacatttgg ctaggtttta tagctggctt gattgccata gtaatggtga caattatgct ttgctgtatg accagttgct gtagttgtct caagggctgt tgttcttgtg gatcctgctg 25260 caaatttgat gaagacgact ctgagccagt gctcaaagga gtcaaattac attack aacgaactta tggatttgtt tatgagaatc ttcacaattg gaactgtac tttgaagcaa ggtgaatca aggatgctac tccttcagat tttgttcgcg ctactgcaac gataccgata caagcctcac tccctttcgg atggcttatt gttggcgttg cacttcttgc tgtttttcag agcgcttcca aaatcataac cctcaaaaag agatggcaac tagcactctc caagggtgtt cactttgttt gcaacttgct gttgttgttt gtaacagttt actcacacct tttgctcgtt gctgctggcc ttgaagcccc ttttctctat ctttatgctt tagtctactt cttgcagagt 25680 ataactttg tagged aatgaggctt tggctttgct ggaaatgccg ttccaaaaac ccattacttt atgatgccaa ctattttctt tgctggcata ctaattgtta cgactattgt 25800 ataccttaca atagtgtaac ttcttcaatt gtcattactt caggtgatgg cacaacaagt 25860 cctatttctg aacatgacta ccagattggt ggttatactg aaaaatggga atctggagta 25920 aaagactgtg ttgtattaca cagttacttc acttcagact attaccagct gtactcaact 25980 caattgagta cagacactgg tgttgaacat gttaccttct tcatctacaa taaattgtt 26040 gatgagcctg aagaacatgt ccaaattcac acaatcgacg gttcacccgg agttgttaat 26100 ccagtaatgg aaccaattta tgatgaaccg acgacgacta ctagcgtgcc tttgtaagca 26160 caagctgatg agtacgaact tatgtactca ttcgtttcgg aagagacagg tacgttaata 26220 gttaatagcg tacttcttt tcttgctttc gtggtattct tgctagttac actagccatc 26280 cttactgcgc ttcgattgtg tgcgtactgc tgcaatattg ttaacgtgag tcttgtaaaa 26340 ccttcttttt acgtttactc tcgtgttaaa aatctgaatt cttctagagt tcctgatctt 26400 ctggtctaaa cgaactaaat attachment tttttctgtt tggaacttta atttagcca tggcagattc caacggtact attaccgttg aagagcttaa aaagctcctt caacggtact acctagtaat aggtttccta ttccttacat ggatttgtct tctacaattt gcctatgcca acaggaatag gtttttgtat father taattttcct ctggctgtta tggccagtaa ctttagcttg ttttgtgctt gctgctgttt acagaataaa ttggatcacc ggtggaattg ctatcgcaat ggcttgtctt gtaggcttga tgtggctcag ctacttcatt gcttctttca gactgtttgc gcgtacgcgt tccatgtggt cattcaatcc agaaactaac attcttctca acgtgccact ccatggcact attctgacca gaccgcttct agaaagtgaa ctcgtaatcg gagctgtgat ccttcgtgga catcttcgta ttgctggaca ccatctagga cgctgtgaca tcaaggacct gcctaaaga atcactgttg ctacatcacg aacgctttct tattacaaat tgggagcttc gcagcgtgta gcaggtgact caggttttgc tgcatacagt cgctacagga 27060 ttggcaacta taattaaac acagaccatt ccagtagcag tgacaatatt gctttgcttg tacagtaagt ghaacagat gttcatctc gttgactttc agttactat agcagagata 27180 ttactaatta ttatgaggac tttaaagtt tccatttgga atcttgatta catcataac 27240 ctcaatatta aaatttatc tagtcacta actgagaata atattctca attagatgaa 27300 gagcaaccaa tggagattga ttaaacgaac atgaaaatta ttctttctt ggcactgata 27360 acacctcgcta cttgtgagct ttcactac caagagtgtg ttagaggtac aacagtactt 27420 ttaaaagaac cttgctcttc tggaacatac gaggcaatt caccattca tcctctagct 27480 gataacaaat ttgcactgac tgctttagc actcaatttg cttttgctg tcctgacggc 27540 gtaaaacacg tctatcagtt acgtgccaga tcagttcac ctaaactgtt catcagacaca 27600 gaggaagttc aagaacttta ctctccaattt ttcttattg ttgcggcaat agtgtttata 27660 acacttgct tcacactca agaaagaca gatgattga acttcatta attgacttct 27720 atttgtgctt tttagccttt ctgctattcc ttgttttaat tatgcttatt atctttttggt 27780 tctcacttga actgcaagat cataatgaaa cttgtcacgc ctaaacgaac atgaaatttc 27840 ttgttttctt aggaatcatc acaactgtag ctgcatttca ccaagaatgt agtttacagt 27900 catgtactca acatcaacca tatgtagttg atgacccgtg tcctattcac ttctattcta 27960 aatggtatat tagagtagga gctagaaaat cagcaccttt aattgaattg tgcgtggatg 28020 aggctggttc taaatcaccc attcagtaca tcgatatcgg taattataca gtttcctgtt 28080 taccttttac aattaattgc cagaaaccta aattgggtag tcttgtagtg cgttgttcgt 28140 tctatgaaga cttttagag tatcatgacg ttcgtgttgt tttagatttc atctaaacga 28200 acaaacaaac taaaatgtct gataatggac cccaaaatca gcgaaatgca ccccgcatta 28260 tgtttggtgg accctcagat tcaactggca gtaaccagaa tggagaacgc agtggggcgc 28320 gatcaaaaca acgtcggccc caaggtttac ccaataatac tgcgtcttgg ttcaccgctc 28380 tcactcaaca tggcaaggaa gaccttaaat tccctcgagg acaaggcgtt ccaattaaca 28440 ccaatagcag tcgagatgac caaattggct actaccgaag agctaccaga cgaattcgtg 28500 28560 ggccagaagc tggacttcccc tatggtgcta aaaagacgg catcatatgg gttgcaactg 28620 agggagcctt gaatacacca aaagatcaca ttggcacccg caatcctgct aacaatgctg 28680 caatcgtgct acaacttcct caaggaacaa cattgccaaa aggcttctac gcagaaggga 28740 gcagaggcgg cagtcaagcc tctttcgtt cctcatcacg tagtcgcaac agttcaagaa 28800 attcaactcc aggcagctct aaacgaactt ctcctgctag aatggctggc aatggcggtg 28860 atgctgctct tgctttgctg ctgcttgaca gattgaacca gcttgagagc aaaatgtctg 28920 gtaaaggcca acaacaacaa ggccaaactg tcactaagaa atctgctgct gaggcttcta 28980 agaagcctcg gcaaaaacgt actgccaacta aagcatacaa tgtaacacaa gctttcggca 29040 gacgtggtcc agaacaaacc caaggaaatt ttggggacca ggactaatc agacaaggaa 29100 ctgattacaa acattggccg caaattgcac aatttgcccc cagcgcttca gcgttcttcg 29160 gaatgtcgcg cattggcatg gaagtcacac cttcgggaac gtggttgacc tacacaggtg 29220 gaatgtcgcg cattggcatg gaagtcacac cttcgggaac gtggttgacc tacacaggtg 29220 ccatcaaatt ggatgacaaa gatccaaatt tcaaagatca agtcattttg ctgaataagc 29280 ccatcaaatt ggatgacaaa gatccaaatt tcaaagatca agtcattttg ctgaataagc 29280 atattgacgc atacaaaaca ttcccaccaa cagagcctaa aaaggacaaa aagaagaagg 29340 atattgacgc atacaaaaca ttcccaccaa cagagcctaa aaaggacaaa aagaagaagg 29340 ctgatgaaac tcaagcctta ccgcagagac agaagaaaca gcaaactgtg actcttcttc 29400 ctgatgaaac tcaagcctta ccgcagagac agaagaaaca gcaaactgtg actcttcttc 29400 ctgctgcaga tttggatgat ttctccaaac aattgcaaca atccatgagc agtgctgact 29460 ctgctgcaga tttggatgat ttctccaaac aattgcaaca atccatgagc agtgctgact 29460 caactcaggc ctaaactcat gcagaccaca caaggcagat gggctatata aacgttttcg 29520 caactcaggc ctaaactcat gcagaccaca caaggcagat gggctatata aacgttttcg 29520 cttttccgtt tacgatatat agtctactct tgtgcagaat gaattctcgt aactacatag 29580 cttttccgtt tacgatatat agtctactct tgtgcagaat gaattctcgt aactacatag 29580 [[ID=1,13]] cacaagtaga tgtagttaac tttaat 29606 cacaagtaga tgtagttaac tttaat 29606 <210> 3<210> 3 <211> 29885<211> 29885 <212> DNA<212> DNA <213> 冠状病毒<213> Coronavirus <220><220> <221> misc_feature <221> misc_feature <222> (1)..(26) <222> (1)..(26) <223> n is a, c, g, or t <223> n is a, c, g, or t [[ID=,32]]<220> <220> <221> misc_feature <221> misc_feature <222> (6882)..(7027) <222> (6882)..(7027) <223> n is a, c, g, or t <223> n is a, c, g, or t <220> <220> <221> misc_feature <221> misc_feature <222> (22333)..(22489) <223> n is a, c, g, or t <220> <221> misc_feature <222> (27210)..(27428) <223> n is a, c, g, or t <220> <221> misc_feature <222> (29849)..(29885) <223> n is a, c, g, or t <400> 3 nnnnnnnnn nnnnnnnnn nnnnnnacaa accaaccaac tttcgatctc ttgtagatct 60 gttctctaaa cgaactttaa aatctgtgtg gctgtcactc ggctgcatgc tgtgcact 120 cacgcagtat attaataac taattactgt cgttgacagg acacgagtaa ctcttctatc 180 ttctgcaggc tgcttacggt ttcgtccgtg ttgcagccga tcatcagcac atctaggttt 240 tgtccgggtg tgaccgaaag gtaagatgga gagccttgtc cctggtttca acgagaaaac 300 acacgtccaa ctcagtttgc ctgttttaca ggttcgcgac gtgctcgtac gtggctttgg 360 agactccgtg gaggaggtct tatcagaggc acgtcaacat cttaaagatg gcacttgtgg 420 cttagtagaa gttgaaaaag gcgttttgcc tcaacttgaa cagccctatg tgttcatcaa 480 acgttcggat gctcgaactg cacctcatgg tcatgttatg gttgagctgg tagcagaact 540 cgaaggcatt footcggtc gtagtggtga gacacttggt gtccttgtcc ctcatgtggg 600 cgaaatacca gtggcttacc gcaaggttct tcttcgtaag aacggtaata aaggagctgg 660 tggccatagt tacggcgccg atctaaagtc atttgactta ggcgacgagc ttggcactga 720 780 actcatgcgt gagcttaacg gagggggcata cactcgctat gtcgataaca acttctgtgg 840 ccctgatggc taccctcttg agtgcattaa agaccttcta gcacgtgctg gtaaagcttc 900 atgcactttg tccgaacaac tggactttat tgacactaag aggggtgtat actgctgccg 960 tgaacatgag catgaaattg cttggtacac ggaacgttct gaaaagagct atgaattgca 1020 gacacctttt gaaattaaat tggcaaagaa atttgacatc ttcaatgggg aatgtccaaa 1080 ttttgtattt cccttaaatt ccataatcaa gactattcaa ccaagggttg aaaagaaaaa 1140 gcttgatggc tttatgggta gaattcgatc tgtctatcca gttgcgtcac caaatgaatg 1200 caaccaaatg tgcctttcaa ctctcatgaa gtgtgatcat tgtggtgaaa cttcatggca 1260 gacgggcgat tttgttaaag ccacttgcga attttgtggc actgagaatt tgactaaaga 1320 aggtgccact acttgtggtt acttacccca aaatgctgtt gttaaaattt attgtccagc 1380 atgtcacaat tcagaagtag gacctgagca tagtcttgcc gaataccata atgaatctgg 1440 cttgaaaacc attcttcgta agggtggtcg cactattgcc tttggaggct gtgtgttctc 1500 ttatgttggt tgccataaca agtgtgccta ttgggttcca cgtgctagcg ctaacatagg 1560 ttgtaaccat acaggtgttg ttggagaagg ttccgaaggt cttaatgaca accttcttga 1620 aatactccaa aaagagaaag tcaacatcaa tattgttggt gactttaaac ttaatgaaga 1680 gatcgccatt attttggcat ctttttctgc ttccacaagt gcttttgtgg aaactgtgaa 1740 aggtttggat tataaagcat tcaaacaaat tgttgaatcc tgtggtaatt ttaaagttac 1800 aaaaggaaaa gctaaaaaag gtgcctggaa tattggtgaa cagaaatcaa tactgagtcc 1860 tctttatgca tttgcatcag aggctgctcg tgttgtacga tcaattttct cccgcactct 1920 tgaaactgct caaaattctg tgcgtgtttt agaaggcc gctataacaa tactagatgg 1980 aatttcacag tattcactga gactcattga tgctatgatg ttcacatctg atttggctac 2040 taacaatcta gttgtaatgg cctacattac aggtggtgtt gttcagttga cttcgcagtg 2100 gctaactaac atctttggca ctgtttatga aaaactcaaa cccgtccttg attggcttga 2160 agaaagttt aaagaaggtg tagagtttct tagagacggt tgggaaattg ttaaatttat 2220 ctcaacctgt gcttgtgaaa ttgtcggtgg acaaattgtc acctgtgcaa aggaaattaa 2280 ggagagtgtt cagacattct ttaagcttgt aaaataattt ttggctttgt gtgctgactc 2340 tatcattatt ggtggagcta aacttaaagc cttgaattta ggtgaaacat ttgtcacgca 2400 ctcaaaggga ttgtacagaa agtgtgttaa atccagagaa gaaactggcc tactcatgcc 2460 tctaaaagcc ccaaaagaaa ttatcttctt agaggagaa acacttccca cagaagtgtt 2520 2580 agctgttgaa gctccattgg ttggtacacc agtttgtatt aacgggctta tgttgctcga 2640 aatcaagac acagaaagt actgtgccct tgcacctaat atgatgtaa ctacaatac 2700 cttcacactc aaaggcggtg caccacaaa ggttacttt ggtgatgaca ctgtgataga 2760 agtgcaaggt tacagagtg tgaatcac tttgaactt gatgaaagga ttgataagt 2820 acttaatgag aagtgctctg cctatacagt tgaacctcggt acagaagtaa atgagttcgc 2880 ctgtgttgtg gcagatgctg tcataaaaac tttgcaacca gtatctgaat tacttacacc 2940 actgggcatt gatttagatg agtggatt ggctacatac tacttatttg atgagtctgg 3000 tgagtttaaa ttggctcac atatgtattg ttcttttac cctccagatg aggatgaaga 3060 agaaggtgat tgtgagaag agagttttga gccatcact caatgagt atggtactga 3120 agatgattac caagtaac ctttggaatt tgtgccact tctgctgctc tcaacctga 3180 frequency frequency ggagatt gggttagatga tgagtcaaactgttg gtxaag 3240 cggcagtgag gatatcaga acaccatt tcaacatt gttgaggttc aaccctcatt 3300 agagatggaa cttacaccag ttgttcagac tattgaagtg atagtttta gtggttattt 3360 aaaacttact gacaatgtat acattaaaaa tgcagacatt gtggaagaag ctaaaaaggt 3420 aaaaccaaca gtggttgtta atgcagccaa tgtttacctt aaacatggag gaggtgttgc 3480 aggagcctta aataaggcta ctaacaatgc catgcaagtt gaatctgatg attacatagc 3540 tactaatgga ccacttaaag tgggtggtag ttgtgtttta agcggacaca atcttgctaa 3600 acactgtctt catgttgtcg gcccaaatgt taacaaaggt gaagacattc aacttcttaa 3660 gagtgcttat gaaaatttta atcagcacga agttctactt gcaccattat tatcagctgg 3720 tatttttggt gctgacccta tacattcttt aagagtttgt gtatagactg ttcgcacaaa 3780 tgtctactta gctgtctttg ataaaaatct ctatgataaa cttgtttcaa gctttttgga 3840 aatgaagagt gaaaagcaag ttgaacaaaa gatcgctgag attcctaaag aggaagttaa 3900 gccatttata actgaaagta aactttcagt tgaacagaga aaacaagatg ataagaaaat 3960 caaagcttgt gttgaagaag ttacaacaac tctggagaaa actaagttcc tcacagaaaa 4020 cttgttactt tatattgaca ttaatggcaa tcttcatcca gattctgcca ctcttgttag 4080 tgacattgac atcactttct taaagaaaga tgctccatat atagtgggtg atgttgttca 4140 agagggtgtt ttaactgctg tggttatacc tactaaaaag gctggtggca ctactgaaat 4200 gctagcgaaa gctttgagaa aagtgccaac agacaattat ataaccactt acccgggtca 4260 gggtttaaat ggttacactg tagaggaggc aaagacagtg cttaaaaagt gtaaaagtgc 4320 cttttacatt ctaccatcta ttatctctaa tgagaagcaa gaaattcttg gaactgtttc 4380 ttggaatttg cgagaaatgc ttgcacatgc agaagaaaca cgcaaattaa tgcctgtctg 4440 tgtggaaact aaagccatag tttcaactat acagcgtaaa tataagggta ttaaaataca 4500 agagggtgtg gttgattatg gtgctagatt ttacttttac accagtaaaa caactgtagc 4560 gtcacttatc aacacactta acgatctaaa tgaaactctt gttacaatgc cacttggcta 4620 tgtaacacat ggcttaaatt tggaagaagc tgctcggtat atgagatctc tcaaagtgcc 4680 agctacagtt tctgtttctt cacctgatgc tgttacagcg tataatggtt atcttacttc 4740 ttcttctaaa acacctgaag aacattttat tgaaaccatc tcacttgctg gttcctataa 4800 agattggtcc tattctggac aatctacaca actaggtata gaatttctta agagaggtga 4860 taaaagtgta tattacacta gtaatcctac cacattccac ctagatggtg aagttatcac 4920 ctttgacaat cttaagacac ttctttcttt gagagaagtg aggactatta aggtgtttac 4980 aacagtagac aacattaacc tccacacgca agttgtggac atgtcaatga catatggaca 5040 acagtttggt ccaacttatt tggatggagc tgatgttact aaaataaaac ctcataattc 5100 acatgaaggt aaaacatttt atgttttacc taatgatgac actctacgtg ttgaggcttt 5160 tgagtactac cacacaactg atcctagttt tctgggtagg tacatgtcag cattaaatca 5220 cactaaaaat tggaaatacc cacaagttaa tggtttaact tctattaaat gggcagataa 5280 caactgttat cttgccactg cattgttaac actccaacaa atagagttga agtttaatcc 5340 acctgctcta caagatgctt attacagagc aagggctggt gaagctgcta acttttgtgc 5400 acttatctta gcctactgta ataagacagt aggtgagtta ggtgatgtta gagaaacaat 5460 gagttacttg tttcaacatg ccaatttaga ttcttgcaaa agagtcttga acgtggtgtg 5520 taaaacttgt ggacaacagc agacaaccct taagggtgta gaagctgtta tgtacatggg cacactttct tatgaacaat ttaagaaagg tgttcagata ccttgtacgt gtggtaaaca agctacaaaa tatctagtac aacaggagtc accttttgtt atgatgtcag caccacctgc tcagtatgaa cttaagcatg gtacatttac ttgtgctagt gtacactg gtaattacca gtgtggtcac fatherly father agaaactttg father acggtgcttt acttacaaag tcctcagaat acaaaggtcc tattacggat gttttctaca aagaaaacag ttacacaaca accataaaac cagttactta taattggat ggtgttgttt gtacagaaat tgaccctaag ttggacaatt attack agcaattct tatttcacag agcaaccaat tgatcttgta ccaaaccaac catatccaaa cgcaagcttc gataatttta agtttgtatg tgataatatc aaatttgctg atgatttaaa ccagttaact ggttataaga aacctgcttc aagagagctt aaagttacat ttttccctga cttaaatggt gatgtggtgg ctattgatta 6240. acaccctctt ttaagaagg agctaaattg ttacataaac ctattgtttg gcatgttaac aatgcaacta ataaagccac gtataaacca aatacctggt gtatacgttg tctttggagc acaaaaccag ttgaaacatc aaattcgttt gatgtactga agtcagagga cgcgcaggga atggataatc ttgcctgcga agatctaaaa ccagtctctg aagaagtagt ggaaaatcct accatacaga aagacgttct tgagtgtaat gtgaaaacta ccgaagttgt aggagacatt attack attack attack tagtttaaaa attack aggttggcca cacagatcta atggctgctt atgtagacaa ttctagtctt actattaga aacctaatga attack gtattaggtt tgaaaaccct tgctactcat ggtttagctg ctgttaatag tgtcccttgg gatactatag ctaattatgc tagcctttt cttaacaaag ttgttagtac aactactaac atagttacac ggtgtttaaa ccgtgtttgt actattata tgccttattt ctttacttta ttgctacaat tgtgtacttt tactagaagt acaaattcta gattaaagc atctatgccg actacttag caaagaatac tgttaagagt gnnnnnnnnn nnnnnnnnnn 6960. 6960. 6960. 6960. 6960. 6960. 6960. 6960. 6960. 6960 nnnnnnnnn nnnnnnnnn nnnnnnnnn nnnnnnnnn nnnnnnnnn nnnnnnnnn 7020 nnnnnnntct aatttaggca tgccttctta ctgtactggt tacagagaag gctatttgaa 7080 ctctactaat gtcactattg caacctactg tactggttct ataccttgta gtgtttgtct 7140 tagtggttta gattctttag acacctatcc ttctttagaa actatacaaa ttaccatttc 7200 atcttttaaa tgggatttaa ctgctttgg cttagttgca gagtggtttt tggcatatat 7260 tcttttcact aggtttttct atgtacttgg attggctgca atcatgcaat tgtttttcag 7320 ctattttgca gtacatttta ttagtaattc ttggcttatg tggttaataa ttaatcttgt 7380 acaaatggcc ccgatttcag ctatggttag aatgtacatc ttctttgcat cattttatta 7440 tgtatggaaa agttatgtgc atgttgtaga cggttgtaat tcatcaactt gtatgatgtg 7500 ttacaaacgt atagagcaa caagagtcga atgtacaact attgttaatg gtgttagaag 7560 gtcctttat gtctatgcta atggaggtaa aggcttttgc aaactacaca attggaattg 7620 tgttaattgt gatacattct gtgctggtag tacatttatt agtgatgaag ttgcgagaga 7680 cttgtcacta cagtttaaaa gaccaataaa tcctactgac cagtcttctt acatcgttga 7740 tagtgttaca gtgaagaatg gttccatcca tctttacttt gataaagctg gtcaaaagac 7800 ttatgaaaga cattctctct ctcattttgt taacttagac aacctgagag ctaataacac 7860 taaaggttca ttgcctatta atgttatagt ttttgatggt aaatcaaaat gtgaagaatc 7920 atctgcaaaa tcagcgtctg tttactacag tcagcttatg tgtcaaccta tactgttact 7980 agatcaggca ttagtgtctg atgttggtga tagtgcggaa gttgcagtta aaatgtttga 8040 tgcttacgtt aatacgtttt catcaacttt taacgtacca atggaaaaac tcaaaacact 8100 agttgcaact gcagaagctg aacttgcaaa gaatgtgtcc ttagacaatg tcttatctac 8160 ttttattca gcagctcggc aagggtttgt tgattcagat gtagaaacta aagatgttgt 8220 tgaatgtctt aaattgtcac atcaatctga catagaagtt actggcgata gttgtaataa 8280 ctatatgctc acctataaca aagttgaaaa catgacaccc cgtgaccttg gtgcttgtat 8340 tgactgtagt gcgcgtcata ttaatgcgca ggtagcaaaa agtcacaaca ttgctttgat 8400 atggaacgtt aaagatttca tgtcattgtc tgaacaacta cgaaaacaaa tacgtagtgc 8460 tgctaaaaag aataacttac cttttaagtt gacatgtgca actactagac aagttgttaa 8520 tgttgtaaca acaaagatag cacttaaggg tggtaaaatt gttaataatt ggttgaagca 8580 gttaattaaa gttacacttg tgttcctttt tgttgctgct attttctatt taataacacc 8640 tgttcatgtc atgtctaaac atactgactt ttcaagtgaa atcataggat acaaggctat 8700 tgatggtggt gtcactcgtg acatagcatc tacagatact tgttttgcta acaaacatgc 8760 tgattttgac acatggttta gccagcgtgg tggtagttat actaatgaca aagcttgccc 8820 attgattgct gcagtcataa caagagaagt gggttttgtc gtgcctggtt tgcctggcac 8880 gatattacgc acaactaatg gtgacttttt gcatttctta cctagagttt ttagtgcagt 8940 tggtaacatc tgttacacac catcaaaact tagagagtac actgactttg caacatcagc 9000 ttgtgttttg gctgctgaat gtacaatttt taaagatgct tctggtaagc fòtccata 9060 ttgttatgat accaatgtac tagaaggttc tgttgcttat gaaagtttac gccctgacac 9120 acgttatgtg ctcatggatg gctctattat tcaatttcct aacacctacc ttgaaggttc 9180 tgttagagtg gtaacaactt ttgattctga gtactgtagg cacggcactt gtgaaagatc 9240 agaagctggt gtttgtgtat ctactagtgg tagatgggta cttaacaatg attattacag 9300 atctttacca ggagttttct gtggtgtaga tgctgtaaat ttacttacta atatgtttac 9360 accactaatt caacctattg gtgctttgga catatcagca tctatagtag ctggtggtat 9420 tgtagctatc gtagtaacat gccttgccta ctattttatg aggtttagaa gagctttttgg 9480 tgaatacagt catgtagttg cctttaatac tttactattc cttatgtcat tcactgtact 9540 ctgttaaca ccagtttact cattcttacc tggtgttat tctgttattt acttgtactt 9600 gacattttat cttactaatg atgtttcttt tttagcacat attcagtgga tggttatgtt 9660 cacaccttta gtacctttct ggataacaat tgcttatatc atttgtattt ccacaaagca 9720 tttctattgg ttctttagta attacctaaa gagacgtgta gtctttaatg gtgtttcctt 9780 tagtacttt gaagaagctg cgctgtgcac cttttgtta aataaagaaa tgtatctaaa 9840 gttgcgtagt gatgtgctat tacctcttac gcaatataat agatacttag ctctttataa 9900 tagtacaag tattttagtg gagcaatgga tacaactagc tacagaag ctgcttgttg 9960 tcatctcgca aaggctctca atgacttcag taactcaggt tctgatgttc tttaccaacc 10020 accacaaacc tctatcacct cagctgtttt gcagagtggt tttagaaaaa tggcattccc 10080 atctggtaaa gttgagggtt gtatggtaca agtaacttgt ggtacaacta cacttaacgg 10140 tctttggctt gatgacgtag tttactgtcc aagacatgtg atctgcacct ctgaagacat 10200 gcttaaccct aattatgaag atttactcat tcgtaagtct aatcataatt tcttggtaca 10260 ggctggtaat gttcaactca gggttattgg acattctatg caaaattgtg tacttaagct 10320 tagggttgat acagccaatc ctaagacacc tagtataag tttgttcgca ttcaaccagg 10380 agaactttt tcagtgttag cttgttacaa tggttcacca tctggtgttt accaatgtgc 10440 tatgaggccc aatttcacta ttaagggttc attccttaat ggttcatgtg gtagtgttgg 10500 ttttaacata gattatgact gtgtctcttt ttgttacatg caccatatgg aattaccaac 10560 tggagttcat gctggcacag acttagaagg taacttttat ggaccttttg ttgacaggca 10620 aacagcacaa gcagctggta cggacacaac tattacagtt aatgttttag cttggttgta 10680 cgctgctgtt ataaatggag acaggtggtt tctcaatcga tttaccacaa ctcttaatga 10740 ctttaacctt gtggctatga agtacaatta tgaacctcta acacaagacc atgttgacat 10800 actaggacct ctttctgctc aaactggaat tgccgtttta gatatgtgtg cttcattaaa 10860 agaattactg caaaatggta tgaatggacg taccatattg ggtagtgctt tattagaaga 10920 tgaatttaca ccttttgatg ttgttagaca atgctcaggt gttactttcc aaagtgcagt 10980 gaaaagaaca atcaagggta cacaccactg gttgttactc acaattttga cttcactttt 11040 agttttagtc cagagtactc aatggtcttt gttctttttt ttgtatgaaa atgccttttt 11100 accttttgct atgggtatta ttgctatgtc tgcttttgca atgatgtttg tcaaacataa 11160 gcatgcattt ctctgtttgt ttttgttacc ttctcttgcc actgtagctt attttaatat 11220 ggtctatatg cctgctagtt gggtgatgcg tattatgaca tggttggata tggttgatac 11280 tagtttgaag ctaaaagact gtgttatgta tgcatcagct gtagtgttac taatccttat 11340 gacagcaaga actgtgtatg atgatggtgc taggagagtg tggacactta tgaatgtctt 11400 gacactcgtt tataaagttt attatggtaa tgctttagat caagccattt ccatgtgggc 11460 tcttataatc tctgttactt ctaactactc aggtgtagtt acaactgtca tgtttttggc 11520 cagaggtatt gtttttatgt gtgttgagta ttgccctatt ttcttcataa ctggtaatac 11580 acttcagtgt ataatgctag tttattgttt cttaggctat ttttgtactt gttactttgg 11640 cctcttttgt ttactcaacc gctactttag actgactctt ggtgtttatg attacttagt 11700 ttctacacag gagtttagat atatgaattc acagggacta ctcccaccca agaatagcat 11760 agatgccttc aaactcaaca ttaaattgtt gggtgttggt ggcaaacctt gtatcaaagt 11820 agccactgta cagtctaaaa tgtcagatgt aaagtgcaca tcagtagtct tactctcagt 11880 tttgcaacaa ctcagagtag aatcatcatc taaattgtgg gctcaatgtg tccagttaca 11940 caatgacatt ctcttagcta agatactac tgaagccttt gaaaaaatgg ttcactact 12000 ttctgttttg ctttccatgc agggtgctgt agacataaac aagctttgtg aagaaatgct 12060 ggacaacagg gcaaccttac aagctatagc ctcagagtttt agttcctc catcatatgc 12120 agcttttgct actgctcaag aagcttatga gcaggctgtt gctaatggtg attctgaagt 12180 tgttcttaaa aagttgaaga agtctttgaa tgtgctaaa tctgaatttg accgtgatgc 12240 agccatgcaa cgtaagttgg aaagatggc tgatcaagct atgacccaa tgtataaaca 12300 ggctagatct gaggacaaga gggcaaagt tactagtgct atgcagacaa tgctttcac 12360 tatgcttaga aagttgata atgatgcact storm atcacaatg tatgcttag 12420 ttgtgttccc ttgaactaa tacctactac aacagcagcc aaactaatgg ttgtcatacc 12480 agactataac acatataaaa atacgtgtga tgtacaaca tttacttatg catcagcatt 12540 gtgggaaatc cacaggttg tagagcaga tgtaaatt gttcaactta gtgaaattag 12600 tatggacaat tcacctaatt tagcatggcc tcttattgta acagctttaa gggccaattc 12660 tgctgtcaaa ttacagaata atgagcttag tcctgttgca ctacgacaga tgtcttgtgc 12720 tgccggtact acaacaactg cttgcactga tgacaatgcg ttagcttact acaacacaac 12780 aaagggaggt aggtttgtac ttgcactgtt atccgattta caggatttga aatgggctag 12840 attccctaag agtgatggaa ctggtactat ctatacagaa ctggaccac cttgtaggtt 12900 tgttacagac acacctaaag gtcctaaagt gaagtattta tactttatta aagattaaa 12960 caacctaaat agaggtatgg tacttggtag tttagctgcc acagtacgtc tacaagctgg 13020 taatgcaaca gaagtgcctg ccaattcaac tgtattatct ttctgtgctt ttgctgtaga 13080 tgctgctaaa gcttacaaag attatctagc tagtggggga caaccaatca ctaattgtgt 13140 taagatgttg tgtacacaca ctggtactgg tcaggcaata acagttacac cggaagccaa 13200 tatggatcaa gaatcctttg gtggtgcatc gtgttgtctg tactgccgtt gccacataga 13260 tcatccaaat cctaaaggat tttgtgactt aaaaggtaag tatgtacaaa tacctacaac 13320 ttgtgctaat gaccctgtgg gttttacact taaaacaca gtctgtaccg tctgcggtat 13380 gtggaaaggt tatggctgta gttgtgatca actccgcgaa cccatgctc attcagctga 13440 tgcacaatcg ttttaacg gtttgcggt gtaagtgcag cccgtcttac accgtgcggc 13500 acaggcacta gtactgatgt cgtatacagg gcttttgaca tctacaatga taaagtagct 13560 ggttttgcta aattcctaaa actaattgt tgtcgcttcc aaaaagga cgaagatgac 13620 aatttaattg attcttactt tgtagttaag agacacactt tctctaacta ccacatgaa 13680 gaaacaattt ataatttact taggattgt ccagctgttg ctaaacatga cttctttaag 13740 tttagaatag acggtgacat ggtaccacat atatcacgtc aacgtcttac taaatacaca 13800 atggcagacc tcgtctatgc tttaggcat tttgatgaag gtaattgtga cacattaaaa 13860 gaatacttg tcacatacaa ttgttgtgat gatgattatt tcataaaaa ggactggtat 13920 gattttgtag aaaacccaga tatattacgc gtatacgcca acttaggtga acgtgtacgc 13980 caagctttgt taaaaacgt acaattctgt gatgccatgc gaaatgctgg tattgttggt 14040 gtactgacat tagataatca agatctcaat ggtaactggt atgatttcgg tgatttcata 14100 caaaccacgc caggtagtgg agttcctgtt gtagattctt attattcatt gttaatgcct 14160 atattaacct tgaccagggc tttaactgca gagtcacatg ttgacactga cttaacaaag 14220 ccttacatta agtgggattt gttaaaatat gacttcacgg aagagaggtt aaaactcttt 14280 gaccgttatt ttaaatattg ggatcagaca taccacccaa attgtgttaa ctgtttggat 14340 gacagatgca ttctgcattg tgcaaacttt aatgttttat tctctacagt gttcccactt 14400 acaagttttg gaccactagt gagaaaaata tttgttgatg gtgttccatt tgtagtttca 14460 actggatacc acttcagaga gctaggtgtt gtacataatc aggatgtaaa cttacatagc 14520 tctagactta gttttaagga attacttgtg tatgctgctg acctgctat gcacgctgct 14580 tctggtaatc tattactaga taaacgcact acgtgctttt cagtagctgc acttactaac 14640 aatgttgctt ttcaaactgt caaacccggt aattttaaca aagacttcta tgactttgct 14700 gtgtctaagg gtttctttaa ggaaggaagt tctgttgaat taaaacactt cttctttgct 14760 caggatggta atgctgctat cagcgattat gactactatc gttataatct accaacaatg 14820 tgtgatatca gacaactact atttgtagtt gaagttgttg ataagtactt tgattgttac 14880 gatggtggct gtattaatgc taaccaagtc atcgtcaaca acctagacaa atcagctggt 14940 tttccattta ataaatgggg taaggctaga ctttattatg attcaatgag ttatgaggat 15000 caagatgcac tttcgcata tacaaaacgt aatgtcatcc ctactataac tcaaatgaat 15060 cttaagtatg cattagtgc aaagaataga gctcgcaccg tagctggtgt ctctatctgt 15120 agtactatga ccaatagaca gtttcatcaa aaattattga aatcaatagc cgccactaga 15180 ggagctactg tagtaattgg aacaagcaaa ttctatggtg gttggcacaa catgttaaaa 15240 actgtttata gtgatgtaga aaaccctcac cttatgggtt gggattatcc taaatgtgat 15300 agagccatgc ctaacatgct tagaattatg gcctcacttg ttcttgctcg caaacataca 15360 acgtgttgta gcttgtcaca ccgtttctat agattagcta atgagtgtgc tcaagtattg 15420 agtgaaatgg tcatgtgtgg cggttcacta tatgttaaac caggtggaac ctcatcagga 15480 gatgccacaa ctgcttatgc taatagtgtt tttaacattt gtcaagctgt cacggccaat 15540 gttaatgcac ttttatctac tgatggtaac aaaattgccg ataagtatgt ccgcaattta 15600 caacacagac tttatgagtg tctctataga aatagagatg ttgacacaga ctttgtgaat 15660 gagttttacg catatttgcg taaacatttc tcaatgatga tactctctga cgatgctgtt 15720 gtgtgtttca atagcactta tgcatctcaa ggtctagtgg ctagcataaa gaactttaag 15780 tcagttcttt attatcaaaa caatgttttt atgtctgaag caaaatgttg gactgagact 15840 gaccttacta aaggacctca tgaattttgc tctcaacata caatgctagt taaacagggt 15900 gatgattatg tgtaccttcc ttacccagat ccatcaagaa tcctaggggc cggctgtttt 15960 gtagatgata tcgtaaaaac agatggtaca cttatgattg aacggttcgt gtctttagct 16020 atagatgctt acccacttac taaacatcct aatcaggagt atgctgatgt ctttcatttg 16080 tacttacaat acataagaaa gctacatgat gagttaacag gacacatgtt agacatgtat 16140 tctgttatgc ttactaatga taacacttca aggtattggg aacctgagtt ttatgaggct 16200 atgtacacac cgcatacagt cttacaggct gttggggctt gtgttctttg caattcacag 16260 acttcattaa gatgtggtgc ttgcatacgt agaccattct tatgttgtaa atgctgttac 16320 gaccatgtca tatcaacatc acataaatta gtcttgtctg ttaatccgta tgtttgcaat 16380 gctccaggtt gtgatgtcac agatgtgact caactttact taggaggtat gagctattat 16440 tgtaaatcac ataaaccacc cattagtttt ccattgtgtg ctaatggaca agtttttggt 16500 ttatataaaa atacatgtgt tggtagcgat aatgttactg actttaatgc aattgcaaca 16560 tgtgactgga caaatgctgg tgattacatt ttagctaaca cctgtactga aagactcaag 16620 ctttttgcag cagaaacgct caaagctact gaggagacat ttaaactgtc ttatggtatt 16680 gctactgtac gtgaagtgct gtctgacaga gaattacatc tttcatggga agttggtaaa 16740 cctagaccac cacttaaccg aaattatgtc tttactggtt atcgtgtaac taaaaacagt 16800 aaagtacaaa taggagagta cacctttgaa aaaggtgact atggtgatgc tgttgtttac 16860 cgaggtacaa caacttacaa attaaatgtt ggtgattatt ttgtgctgac atcacataca 16920 gtaatgccat taagtgcacc tacactagtg ccacaagagc actatgttag aattactggc 16980 ttatacccaa cactcaatat ctcagatgag ttttctagca atgttgcaaa ttatcaaaag 17040 gttggtatgc aaaagtattc tacactccag ggaccacctg gtactggtaa gagtcatttt 17100 gctattggcc tagctctcta ctacccttct gctcgcatag tgtatacagc ttgctctcat 17160 gccgctgttg atgcactatg tgagaaggca ttaaaatatt tgcctataga taaatgtagt 17220 agaattatac ctgcacgtgc tcgtgtagag tgttttgata aattcaaagt gaattcaaca 17280 ttagaacagt atgtcttttg tactgtaaat gcattgcctg agacgacagc agatatagtt 17340 gtctttgatg aaatttcaat ggccacaaat tatgatttga gtgttgtcaa tgccagatta 17400 cgtgctaagc actatgtgta cattggcgac cctgctcaat tacctgcacc acgcacattg 17460 ctaactaagg gcacactga accagaat ttcaatcag tgtgtagact tatgaaaact 17520 ataggtccag acatgttcct cggaacttgt cggcgttgtc ctgctgaaat tgttgacact 17580 gtgagtgctt tggtttatga taataagctt aaagcacata aagacaaatc agctcaatgc 17640 tttaaaatgt tttataaggg tgttatcacg catgatgttt catctgcaat taacaggcca 17700 caaataggcg tggtaagaga attccttaca cgtaaccctg cttggagaaa agctgtcttt 17760 atttcacctt atattcaca gatgctgta gcctcaaga ttttgggact accaactcaa 17820 actgttgatt catcacagggg ctcagaatat gactatgtca tattcactca aaccactgaa 17880 acagctcact cttgtaatgt aacagattt aatgttgcta ttaccagagc aaagtaggc 17940 atactttgca taatgtctga tagagacctt tatgacaagt tgcaatttac aagtcttgaa 18000 attccacgta ggaatgtggc aactttacaa gctgaaatg taacaggact ctttaagat 18060 tgtagtagg taatcactgg gttacactcct acacaggcac ctacacacct cagtgttgac 18120 actaaattca aaactgaagg tttatgtgtt gacatacctg gcatacctaa ggacatgacc 18180 tatagaagac tcatctctat gatgggtttt aaaatgaatt atcaagttaa tggttaccct 18240 aacatgttta tcacccgga agaagctata agacatgtac gtgcatggat tggcttcgat 18300 gtcgaggggt gtcatgctac tagagaagct gttggtacca atttaccttt acagctaggt 18360 ttttctacag gtgttaacct agttgctgta cctacaggtt atgttgatac acctaataat 18420 acagattttt ccagagttag tgctaaacca ccgcctggag atcaatttaa acacctcata 18480 ccacttatgt acaaaggact tccttggaat gtagtgcgta taaagattgt acaaatgtta 18540 agtgacacac ttaaaaatct ctctgacaga gtcgtatttg tcttatgggc acatggcttt 18600 gagttgacat ctatgaagta ttttgtgaaa ataggacctg agcgcacctg ttgtctatgt 18660 gatagacgtg ccacatgctt ttccactgct tcagacactt atgcctgttg gcatcattct 18720 attggatttg attacgtcta taatccgttt atgattgatg ttcaacaatg gggttttaca 18780 ggtaacctac aaagcaacca tgatctgtat tgtcaagtcc atggtaatgc acatgtagct 18840 agttgtgatg caatcatgac taggtgtcta gctgtccacg agtgctttgt taagcgtgtt 18900 gactggacta ttgaatatcc tataattggt gatgaactga agattaatgc ggcttgtaga 18960 aaggttcaac acatggttgt taaagctgca ttattagcag acaaattccc agttcttcac 19020 gacattggta accctaaagc tattaagtgt gtacctcaag ctgatgtaga atggaagttc 19080 tatgatgcac agccttgtag tgacaaagct tataaaatag aagaattatt ctattcttat 19140 gccacacatt ctgacaaatt cacagatggt gtatgcctat tttggaattg caatgtcgat 19200 agatatcctg ctaattccat tgtttgtaga tttgacacta gagtgctatc taaccttaac 19260 ttgcctggtt gtgatggtgg cagtttgtat gtaataaac atgcattcca cacaccagct 19320 tttgataaaa gtgcttttgt taatttaaaa caattaccat ttttctatta ctctgacagt 19380 ccatgtgagt ctcatggaaa acaagtagtg tcagatatag attatgtacc actaaagtct 19440 gctacgtgta taacacgttg caatttaggt ggtgctgtct gtagacatca tgctaatgag 19500 tacagattgt atctcgatgc ttacatg atgatctcag ctggctttag cttgtggtt 19560 tacaacaat ttgactta taacctgg aacacttta cagacttca gagtttagaa 19620 aatgtggctt ttaatgttgt aaataggga cactttgatg vakaacaggg tgaagtacca 19680 gtttctatca ttaatacac tgtttacaca aaagttgatg gtgttgatgt agaattgttt 19740 gaaaaayaaa cacattacc tgttaatgta gcatttgagc tttgggctaa gcgcaacatt 19800 aaaccagtac cagaggtgaa atactcaat aatttgggtg tggacattgc tgctaatact 19860 gtgatctggg actacaaag agatgctcca gcacatatat ctactattgg tgtttgttct 19920 atgactgaca tagccaagaa accactgaa acgatttgtg caccactcac tgtctttttt 19980 gatggtagag ttgatggtca agtagactta tttagaatg cccgtaatgg tgttcttatt 20040 acagaaggta gtgttaaagg tttacaacca tctgtaggtc acacaaggc tagtcttaat 20100 ggagtcacat taattggaga agccgtaaaa acacagttca attattata gaagttgat 20160 ggtgttgtcc aacaattacc tgaaacttac tttactcaga gtagaattt acagaattt 20220 aaacccagga gtcaaatgga aattgatttc ttagaattag ctatggatga attcattgaa 20280 cggtataaat tagaaggcta tgccttcgaa catatcgttt atggagattt tagtcatagt 20340 cagttaggtg gtttacatct actgattgga ctagctaaac gttttaagga atcacctttt 20400 gaattagaag attttattcc tatggacagt acagttaaaa actatttcat aacagatgcg 20460 caaacaggtt catctaagtg tgtgtgttct gttattgatt tattacttga tgattttgtt 20520 gaaataataa aatcccaaga tttatctgta gtttctaagg ttgtcaaagt gactattgac 20580 tatacagaaa tttcatttat gctttggtgt aaagatggcc atgtagaaac atttaccca 20640 aaattacaat ctagtcaagc gtggcaaccg ggtgttgcta tgcctaatct ttacaaaatg 20700 caaagaatgc tattagaaaa gtgtgacctt caaaattatg gtgatagtgc aacattacct 20760 aaaggcataa tgatgaatgt cgcaaaatat actcaactgt gtcaatattt aaacacatta 20820 acattagctg taccctataa tatgagagtt atacattttg gtgctggttc tgataaagga 20880 gttgcaccag gtacagctgt tttaagacag tggttgccta cgggtacgct gcttgtcgat 20940 tcagatctta atgactttgt ctctgatgca gattcaactt tgattggtga ttgtgcaact 21000 gtacatacag ctaataaatg ggatctcatt attagtgata tgtacgaccc taagactaaa 21060 aatgttacaa aagaaaatga ctctaaagag ggttttttca cttacatttg tgggtttata 21120 caacaaaagc tagctcttgg aggttccgtg gctataaaga taacagaaca ttcttggaat 21180 gctgatcttt ataagctcat gggacacttc gcatggtgga cagcctttgt tactaatgtg 21240 aatgcgtcat catctgaagc atttttaatt ggatgtaatt atcttggcaa accacgcgaa 21300 caaatagatg gttatgtcat gcatgcaaat tacatatttt ggaggaatac aaatccaatt 21360 cagttgtctt cctattcttt atttgacatg agtaaatttc cccttaaatt aaggggtact 21420 gctgttatgt ctttaaaaga aggtcaaatc aatgatatga tttatctct tcttagtaaa 21480 ggtagactta taattagaga aaacaacaga gttgttattt ctagtgatgt tcttgttaac 21540 aactaaacga acaatgtttg ttttctttgt tttattgcca ctagtctcta gtcagtgtgt 21600 taattttaca accagaactc aattaccccc tgcatacact aattctttca cacgtggtgt 21660 ttattaccct gacaaagttt tcagatcctc agttttacat tcaactcagg acttgttctt 21720 acctttcttt tccaatgtta cttggttcca tgctatacat gtctctggga ccaatggtac 21780 taagaggtt gctaaccctg tcctaccatt taatgatggt gttattttg cttccactga 21840 gaagtctaac ataataagag gctggatttt tggtactact ttagattcga agacccagtc 21900 cctacttatt gttaataacg ctactaatgt tgttattaaa gtctgtgaat ttcaattttg 21960 taatgatcca ttttgggtg tttattacca caaaaacaac aaaagttgga tggaaagtga 22020 gttcagagtt tattctagtg cgaataattg cacttttgaa tatgtctctc agccttttct 22080 tatggacctt gaaggaaaac agggtaattt caaaaatctt agggaatttg tgtttaagaa 22140 tattgatggt tattttaaaa tatattctaa gcacacgcct attaatttag tgcgtggtct 22200 ccctcagggt tttcggctt tagaaccatt ggtagatttg ccaataggta ttaacatcac 22260 taggtttcaa actttacata gaagttattt gactcctggt gattcttctt caggttggac 22320 agctggtgct gcnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 22380 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 22440 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnt ctaactttag 22500 agtccaacca acagaatcta ttgttagatt tcctaatatt acaaacttgt gcccttttgg 22560 tgaagttttt aacgccacca gatttgcatc tgtttatgct tggaacagga agagaatcag 22620 caactgtgtt gctgattatt ctgtcctata taattccgca tcattttcca cttttaagtg 22680 ttatggagtg tctcctacta aattaaatga tctctgcttt actaatgtct atgcagattc 22740 atttgtaatt agaggtgatg aagtcagaca aatcgctcca gggcaaactg gaaatattgc 22800 tgattataat tataaattac cagatgattt tacaggctgc gttatagctt ggaattctaa 22860 caatcttgat tctaaggttg gtggtaatta taattacctg tatagattgt ttaggaagtc 22920 taatctcaaa ccttttgaga gagatatttc aactgaaatc tatcaggccg gtagcacacc 22980 ttgtaatggt gttaaaggtt ttaattgtta ctttcctttta caatcatatg gtttccaacc cacttatggt gttggttacc aaccatacag tagtagta ctttcttttg aacttctaca 23100 23160. tgcaccagca actgtttgtg gacctaaaa gtctactaat ttggttaaaa acaaatgtgt 23220. ttcaatggtt taacaggcac aggtgttctt actgagtcta acaaaaagtt tctgcctttc caacaatttg gcagagacat tgctgacact actgatgctg tccgtgatcc 23340. acagacactt gagattcttg acattacacc atgttctttt ggtggtgtca gtgttataac accaggaca aatacttcta accaggttgc tgttctttat cagggtgtta actgcacaga agtccctgtt gctattcatg cagatcaact tactcctact tggcgtgttt attctacagg 23460 ttctaatgtt tttcaaacac gtgcaggctg tttaataggg gctgaacatg tcaacaactc atatgagtgt gacataccca ttggtgcagg fathergcgct agttatcaga ctcagactaa ttctcctcgg cgggcacgta gtgtagctag tcaatccatc attgcctaca ctatgtcact tggtgtagaa aattcagttg cttactctaa taactctatt gccataccca caaattttac 23700 tattagtgtt accacagaaa ttctaccagt gtctatgacc aagacatcag tagattgtac 23760 aatgtacatt tgtggtgatt caactgaatg cagcaatctt ttgttgcaat atggcagttt 23820 ttgtacacaa ttaaaccgtg ctttaactgg aatagctgtt gaacaagaca aaaacaccca 23880 agaagtttt gcacaagtca aacaaattta caaaacacca ccaattaaag atttggtgg 23940 ttttaattt tcacaaatat taccagatcc atcaaaacca agcaagaggt catttattga 24000 agatctactt ttcaacaaag tgacacttgc agatgctggc ttcatcaaac aatatggtga 24060 ttgccttggt gatattgctg ctagagacct cattgtgca caaaagttta acggccttac 24120 tgttttgcca cctttgctca cagatgaaat gattgctcaa tacacttctg cactgttagc 24180 gggtacaatc acttctggtt ggacctttgg tgcaggtgct gcattacaaa taccattgc 24240 tatgcaaatg gcttataggt ttaatggtat tggagttaca cagaatgttc tctatgagaa 24300 ccaaaaattg attgccaacc aatttaatag tgctattggc aaaattcaag actcactttc 24360 ttccacagca agtgcacttg gaaaacttca agatgtggtc aaccaaatg cacaagcttt 24420 aaacacgctt gttaaacaac ttagctccaa ttttgtgca atttcaagtg ttttaatga 24480 tatcctttca cgtcttgaca aagttgaggc tgagtgcaa attgataggt tgatcacagg 24540 cagacttca agtttgcaga catatgtgac tcaatta attagagctg cagaatcag 24600 agcttctgct aatcttgctg ctactaaaat gtcagagtgt gtacttggac atcaaaag 24660 agttgattt tgtgaagg gctatcatct tatgtccttc cctcagtcag cacctcatgg 24720 tgtagtcttc ttgcatgtga cttatgtccc tgcacaagaa aagaacttca caactgctcc 24780 tgccattgt catgatggaa agcacactt tcctcgtgaa gtgtcttg ttcaatgg 24840 cacacactgg ttgtacac aaaggaattt tttgacca caaatcatta ctacagacaa 24900 cacatttgtg tctggtaact gtgatgttgt ataggaatt gtcaacaca cagtttatga 24960 tcctttgcaa cctgaattag actcattca ggaggagtta gataatatt ttagaatca 25020 tacatcacca gatgttgatt taggtgacat ctctggcatt aatgcttcag ttgtaaaacat 25080 tcaaaaagaa attgaccgcc tcaatgaggt tgccaagaat ttaaatgaat ctctcatcga 25140 25200 ttttatagct ggcttgattg ccatagtaat ggtgacaatt atgctttgct gtatgaccag 25260 ttgctgtagt tgtctcaagg gctgttgttc ttgtggatcc tgctgcaaat ttgatgaaga 25320 cgactctgag ccagtgctca aaggagtcaa attacattac acataaacga acttatggat 25380 25440 gctactcctt cagattttgt tcgcgctact gcaacgatac cgatacaagc ctcactccct 25500 ttcggatggc ttatgttgg cgttgcactt cttgctgttt ttcatagcgc ttccaaaatc 25560 ataaccctca aaaagagatg gcaactagca ctctccaagg gtgttcactt tgtttgcaac 25620 ttgctgttgt tgtttgtaac agtttactca caccttttgc tcgttgctgc tggccttgaa 25680 25740 ataataatga ggctttggct ttgctggaaa tgccgttcca aaaacccatt actttatgat 25800 gccaactatt ttctttgctg gcatactaat tgttacgact attgtatacc ttacaatagt 25860 gtaacttctt caattgtcat tactttaggt gatggcacaa caagtcctat ttctgaacat 25920 gactaccaga ttggtggtta tactgaaaaa tgggaatctg gagtaaaaga ctgtgttgta 25980 ttacacagtt acttcacttc agactattac cagctgtact caactcaatt gagtacagac 26040 actggtgttg aacatgttac cttcttcatc tacaataaaa ttgttgatga gcctgaagaa 26100 catgtccaaa ttcacacaat cgacggttca tccggagttg ttaatccagt aatggaacca 26160 atttatgatg aaccgacgac gactactagc gtgcctttgt aagcacaagc tgatgagtac 26220 gaacttatgt actcattcgt ttcggaagag acaggtacgt taatagttaa tagcgtactt 26280 ctttttcttg ctttcgtggt attcttgcta gttacactag ccatccttac tgcgcttcga 26340 ttgtgtgcgt actgctgcaa tattgttaac gtgagtcttg taaaaccttc tttttacgtt 26400 tactctcgtg ttaaaaatct gaattcttct agagttcttg atcttctggt ctaaacgaac 26460 taaatattat attagttttt ctgtttggaa ctttaatttt agccatggca gattccaacg 26520 gtactattac cgttgaagag cttaaaaagc tccttgaaca atggaaccta gtaataggtt 26580 tcctattcct tacatggatt tgtcttctac aatttgccta tgccaacagg aataggtttt 26640 tgtatataat taagttaatt ttcctctggc tgttatggcc agtaacttta gcttgttttg 26700 tgcttgctgc tgtttacaga ataaattgga tcaccggtgg aattgctatc gcaatggctt 26760 gtcttgtagg cttgatgtgg ctcagctact tcattgcttc tttcagactg tttgcgcgta 26820 cgcgttccat gtggtcattc aatccagaaa ctaacattct tctcaacgtg ccactccatg 26880 gcactattct gaccagaccg cttctagaaa gtgaactcgt aatcggagct gtgatccttc 26940 gtggacatct tcgtattgct ggacaccatc taggacgctg tgacatcaag gacctgccta 27000 aagaaatcac tgttgctaca tcacgaacgc tttcttatta caaattggga gcttcgcagc 27060 gtgtagcagg tgactcaggt tttgctgcat acagtcgcta caggattggc aactataaat 27120 taaacacaga ccattccagt agcagtgaca atattgcttt gcttgtacag taagtgacaa 27180 cagatgtttc atctcgttga ctttcaggtn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 27240 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 27300 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 27360 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 27420 nnnnnnnntc actaccaaga gtgtgttaga ggtacaacag tacttttaaa agaaccttgc 27480 tcttctggaa catacgaggg caattcacca tttcatcctc tagctgataa caaatttgca 27540 ctgacttgct ttagcactca atttgctttt gcttgtcctg acggcgtaaa acacgtctat 27600 cagttacgtg ccagatcagt ttcacctaaa ctgttcatca gacaagagga agttcaagaa 27660 ctttactctc caatttttct tattgttgcg gcaatagtgt ttataacact ttgcttcaca 27720 ctcaaaagaa agacagaatg attgaacttt cattaattga cttctatttg tgctttttag 27780 cctttctgct attccttgtt ttaattatgc ttattatctt ttggttctca cttgaactgc 27840 aagatcataa tgaaacttgt cacgcctaaa cgaacatgaa atttcttgtt ttcttaggaa 27900 tcatcacaac tgtagctgca tttcaccaag aatgtagttt acagtcatgt actcaacatc 27960 aaccatatgt agttgatgac ccgtgtccta ttcacttcta ttctaaatgg tatattagag 28020 taggagctag aaaatcagca cctttaattg aattgtgcgt ggatgaggct ggttctaaat 28080 cacccattca gtacatcgat atcggtaatt atacagtttc ctgtttacct tttacaatta 28140 attgccagga acctaaattg ggtagtcttg tagtgcgttg ttcgttctat gaagaactttt 28200 tagagtatca tgacgttcgt gttgttttag attttatcta aacgaacaaa ctaaaatgtc 28260 tgataatgga ccccaaaatc agcgaaatgc accccgcatt acgtttggtg gaccctcaga 28320 ttcaactggc agtaaccaga atggagaacg cagtggggcg cgatcaaaac aacgtcggcc 28380 ccaaggttta cccaataata ctgcgtcttg gttcaccgct ctcactcaac atggcaagga 28440 agaccttaaa ttccctcgag gacaaggcgt tccaattaac accaatagca gtccagatga 28500 ccaaattggc tactaccgaa gagctaccag acgaattcgt ggtggtgacg gtaaaatgaa 28560 agatctcagt ccagatggt atttctacta cctaggact gggccagaag ctggacttcc 28620 ctatggtgct aaaaagacg gcatcatatg ggttgcaact gagggagcct tgaatacacc 28680 aaaagatcac attgcaccc gcaatcctgc taacaatgct gcaatcgtgc tacaacttcc 28740 tcaggaaca acatgccaa aaggctctta cgcagaaggg agcagaggcg gcagtcaagc 28800 ctcttctcgt tcctcatcac gtagtcgcaa cagttcaaga attcaactc caggcagcag 28860 tagggaatt tctcctgcta gatggctgg caatggcggt gatgctgctc tgctttgct 28920 gctgcttgac agattgacc agcttgagag caaatgtct ggtaaaggcc aaaacaaca 28980 agccaaact gtcactaaga aatctgctgc tgaggctct aagaagccctc ggcaaaaacg 29040 tactgccact aaagcataca atgtacaca agcttcggc agacgtggtc cagacaac 29100 ccaaggaat ttggggacc aggaactaat cagaxagga actgattaca aacattggcc 29160 gcaaattgca caatttgccc ccagcgcttc agcgttcttc ggaatgtcgc gcattggcat 29220 ggaagtcaca ccttcggggaa cgtggttgac ctacacaggt gccatcaaat tggatgacaa 29280 agatccaaat ttcaaagatc aagtcatttt gctgaataag catattgacg catacaaaac 29340 attcccacca acagagccta aaaaggacaa aaagaagaag gctgatgaaa ctcaagcctt 29400 accgcagaga cagaagaaac agcaaactgt gactcttctt cctgctgcag atttggatga 29460 tttctccaaa caattgcaac aatccatgag cagtgctgac tcaactcagg cctaaactca 29520 tgcagaccac acaaggcaga tgggctatat aaacgttttc gctttccgt ttacgatata 29580 tagtctactc ttgtgcagaa tgaattctcg taactacata gcacaagtag atgtagttaa 29640 ctttaatctc acatagcaat ctttaatcag tgtgtaacat tagggaggac ttgaaagagc 29700 caccacattt tcaccgaggc cacgcggagt acgatcgagt gtacagtgaa caatgctagg 29760 gagagctgcc tatatggaag agccctaatg tgtaaaatta attttagtag tgctatcccc 29820 atgtgatttt aatagcttct taggaann nnnnnnnnn nnnnnnnnn nnnnnnnnn 29880 nnnnn 29885

Claims

1. An inactivated SARS-CoV-2 virus, wherein the infectivity of the inactivated SARS-CoV-2 is undetectable, and wherein the inactivated SARS-CoV-2 induces an immune response against the SARS-CoV-2 virus when administered to a subject. The SARS-CoV-2 virus mentioned above includes: ORF1a, whose nucleotide sequence is shown as nucleotides 238 to 13455 of SEQ ID NO: 1; and / or ORF1ab, whose nucleotide sequence is shown as nucleotides 13740-21527 of SEQ ID NO: 1; and / or Spike protein S, whose amino acid sequence is encoded by nucleotides 21508–25356 of SEQ ID NO: 1; and / or ORF3a, whose nucleotide sequence is shown as nucleotides 25365-26192 of SEQ ID NO: 1; and / or Membrane protein M, whose amino acid sequence is encoded by nucleotides 26495-27163 of SEQ ID NO: 1; and / or Nucleocapsid protein N, whose amino acid sequence is encoded by nucleotides 28246-29505 of SEQ ID NO: 1; The genomic DNA sequence of the SARS-CoV-2 virus is shown in SEQ ID NO:

1.

2. The inactivated SARS-CoV-2 virus according to claim 1, wherein the inactivated SARS-CoV-2 virus is obtained by inactivating the SARS-CoV-2 virus with a virus inactivating agent.

3. The inactivated SARS-CoV-2 virus according to claim 2, wherein the virus inactivating agent is β-propiolactone and / or formalin.

4. The inactivated SARS-CoV-2 virus according to any one of claims 1 to 3, wherein the inactivated SARS-CoV-2 virus is further purified by ion exchange chromatography and / or size exclusion chromatography.

5. The inactivated SARS-CoV-2 virus according to any one of claims 1 to 3, wherein the inactivated SARS-CoV-2 virus is treated with a non-restrictive endonuclease prior to size exclusion chromatography.

6. The inactivated SARS-CoV-2 virus according to any one of claims 1 to 3, wherein, Before inactivation with β-propiolactone, the SARS-CoV-2 virus was mixed with cells at a density of 2 × 10⁻⁶ cells. 6 cells / mL to 10 × 10 6 Vero cells per mL were cultured together in a bioreactor at 35–39°C for 3–7 days to obtain the virus harvest fluid.

7. The inactivated SARS-CoV-2 virus according to any one of claims 1 to 3, wherein, The volume ratio of β-propiolactone to virus harvest fluid is 1:4000~1:6000, and the inactivation time at 4℃ is 16~72 hours.

8. The inactivated SARS-CoV-2 virus according to claim 7, wherein the inactivation time is 20 to 24 hours.

9. The inactivated SARS-CoV-2 virus according to claim 8, wherein the inactivation time is 24 hours.

10. The inactivated SARS-CoV-2 virus according to any one of claims 1 to 3, wherein the SARS-CoV-2 virus comprises: ORF1a, whose nucleotide sequence is shown as nucleotides 238 to 13455 of SEQ ID NO: 1; ORF1ab, whose nucleotide sequence is shown as nucleotides 13740-21527 of SEQ ID NO: 1; The spike protein S has an amino acid sequence encoded by nucleotides 21508 to 25356 of SEQ ID NO: 1; ORF3a, whose nucleotide sequence is shown as nucleotides 25365-26192 of SEQ ID NO: 1; Membrane protein M, whose amino acid sequence is encoded by nucleotides 26495-27163 of SEQ ID NO: 1; as well as Nucleocapsid protein N, whose amino acid sequence is encoded by nucleotides 28246-29505 of SEQ ID NO:

1.

11. The inactivated SARS-CoV-2 virus according to any one of claims 1 to 3, wherein the SARS-CoV-2 virus has a genome, the genome comprising an RNA sequence of the reverse complementary sequence shown in SEQ ID NO:

1.

12. The inactivated SARS-CoV-2 virus according to any one of claims 1 to 3, wherein the SARS-CoV-2 virus has a genome encoded by the sequence shown in SEQ ID NO:

1.

13. A pharmaceutical composition or vaccine comprising a pharmaceutically effective amount of inactivated SARS-CoV-2 virus and a pharmaceutically acceptable vector; The SARS-CoV-2 virus mentioned above includes: ORF1a, whose nucleotide sequence is shown as nucleotides 238 to 13455 of SEQ ID NO: 1; and / or ORF1ab, whose nucleotide sequence is shown as nucleotides 13740-21527 of SEQ ID NO: 1; and / or Spike protein S, whose amino acid sequence is encoded by nucleotides 21508–25356 of SEQ ID NO: 1; and / or ORF3a, whose nucleotide sequence is shown as nucleotides 25365-26192 of SEQ ID NO: 1; and / or Membrane protein M, whose amino acid sequence is encoded by nucleotides 26495-27163 of SEQ ID NO: 1; and / or Nucleocapsid protein N, whose amino acid sequence is encoded by nucleotides 28246-29505 of SEQ ID NO: 1; The genomic DNA sequence of the SARS-CoV-2 virus is shown in SEQ ID NO:

1.

14. The pharmaceutical composition or vaccine according to claim 13, wherein the inactivated SARS-CoV-2 virus is obtained by inactivating the SARS-CoV-2 virus with a virus inactivating agent.

15. The pharmaceutical composition or vaccine according to claim 14, wherein the virus inactivating agent is β-propiolactone and / or formalin.

16. The pharmaceutical composition or vaccine according to claim 13, wherein the pharmaceutical composition or the vaccine is in a liquid or frozen state.

17. The pharmaceutical composition or vaccine according to claim 13, wherein the pharmaceutical composition or the vaccine is in a liquid or lyophilized state.

18. The pharmaceutical composition or vaccine according to claim 13, wherein the dosage of the pharmaceutical composition or the vaccine is in the range of 0.5 to 8 μg based on protein weight.

19. The pharmaceutical composition or vaccine according to claim 18, wherein the dosage of the pharmaceutical composition or the vaccine is in the range of 3 to 6 μg based on protein weight.

20. The pharmaceutical composition or vaccine according to claim 13, wherein the pharmaceutically acceptable carrier is an Al(OH)3 adjuvant and / or CpG oligodeoxynucleotide.

21. The pharmaceutical composition or vaccine according to claim 13, wherein the inactivated SARS-CoV-2 virus is purified by ion exchange chromatography and / or size exclusion chromatography after inactivation.

22. The pharmaceutical composition or vaccine of claim 21, wherein the inactivated SARS-CoV-2 virus is treated with a non-restrictive endonuclease prior to size exclusion chromatography.

23. The pharmaceutical composition or vaccine according to claim 21, wherein, Before inactivation with β-propiolactone, the SARS-CoV-2 virus was mixed with cells at a density of 2 × 10⁻⁶ cells. 6 cells / mL to 10 × 10 6 Vero cells per mL were cultured together in a bioreactor at 35–39°C for 3–7 days to obtain the virus harvest fluid.

24. The pharmaceutical composition or vaccine according to claim 21, wherein, The volume ratio of β-propiolactone to virus harvest fluid is 1:4000~1:6000, and the inactivation time at 4℃ is 16~72 hours.

25. The pharmaceutical composition or vaccine according to claim 24, wherein the inactivation time is 20 to 24 hours.

26. The pharmaceutical composition or vaccine according to claim 25, wherein the inactivation time is 24 hours.

27. The pharmaceutical composition or vaccine according to claim 21, wherein the inactivated SARS-CoV-2 virus is filtered through a membrane and then mixed with an Al(OH)3 adjuvant and / or CpG oligodeoxynucleotides, wherein the membrane has a pore size of 0.2 to 0.25 μm.

28. The pharmaceutical composition or vaccine according to claim 27, wherein the membrane has a pore size of 0.22 μm.

29. The pharmaceutical composition or vaccine according to claim 13, wherein the SARS-CoV-2 virus comprises: ORF1a, whose nucleotide sequence is shown as nucleotides 238 to 13455 of SEQ ID NO: 1; ORF1ab, whose nucleotide sequence is shown as nucleotides 13740-21527 of SEQ ID NO: 1; The spike protein S has an amino acid sequence encoded by nucleotides 21508 to 25356 of SEQ ID NO: 1; ORF3a, whose nucleotide sequence is shown as nucleotides 25365-26192 of SEQ ID NO: 1; Membrane protein M, whose amino acid sequence is encoded by nucleotides 26495-27163 of SEQ ID NO: 1; as well as Nucleocapsid protein N, whose amino acid sequence is encoded by nucleotides 28246-29505 of SEQ ID NO:

1.

30. The pharmaceutical composition or vaccine of claim 13, wherein the SARS-CoV-2 virus has a genome comprising an RNA sequence of the reverse complementary sequence shown in SEQ ID NO:

1.

31. The pharmaceutical composition or vaccine according to claim 13, wherein the SARS-CoV-2 virus has a genome encoded by the sequence shown in SEQ ID NO:

1.

32. A method for preparing an inactivated SARS-CoV-2 composition or vaccine, comprising inactivating SARS-CoV-2 virus with a virus inactivating agent; The SARS-CoV-2 virus mentioned above includes: ORF1a, whose nucleotide sequence is shown as nucleotides 238 to 13455 of SEQ ID NO: 1; and / or ORF1ab, whose nucleotide sequence is shown as nucleotides 13740-21527 of SEQ ID NO: 1; and / or Spike protein S, whose amino acid sequence is encoded by nucleotides 21508–25356 of SEQ ID NO: 1; and / or ORF3a, whose nucleotide sequence is shown as nucleotides 25365-26192 of SEQ ID NO: 1; and / or Membrane protein M, whose amino acid sequence is encoded by nucleotides 26495-27163 of SEQ ID NO: 1; and / or Nucleocapsid protein N, whose amino acid sequence is encoded by nucleotides 28246-29505 of SEQ ID NO: 1; The genomic DNA sequence of the SARS-CoV-2 virus is shown in SEQ ID NO:

1.

33. The method for preparing an inactivated SARS-CoV-2 composition or vaccine according to claim 32, wherein the virus inactivating agent is β-propiolactone and / or formalin.

34. The method for preparing an inactivated SARS-CoV-2 composition or vaccine according to claim 32, wherein the inactivated SARS-CoV-2 virus is further purified by ion exchange chromatography and / or size exclusion chromatography after inactivation.

35. The method for preparing an inactivated SARS-CoV-2 composition or vaccine according to claim 32, wherein the inactivated SARS-CoV-2 virus is treated with a non-restrictive endonuclease prior to size exclusion chromatography.

36. The method for preparing an inactivated SARS-CoV-2 composition or vaccine according to claim 32, wherein, Before inactivation with β-propiolactone, the SARS-CoV-2 virus was mixed with cells at a density of 2 × 10⁻⁶ cells. 6 cells / mL to 10 × 10 6 Vero cells per mL were cultured together in a bioreactor at 35–39°C for 3–7 days to obtain the virus harvest fluid.

37. The method for preparing an inactivated SARS-CoV-2 composition or vaccine according to claim 36, wherein, The volume ratio of β-propiolactone to virus harvest fluid is 1:4000~1:6000, and the inactivation time at 4℃ is 16~72 hours.

38. The method for preparing an inactivated SARS-CoV-2 composition or vaccine according to claim 37, wherein the inactivation time is 20 to 24 hours.

39. The method for preparing an inactivated SARS-CoV-2 composition or vaccine according to claim 38, wherein the inactivation time is 24 hours.

40. The method for preparing an inactivated SARS-CoV-2 composition or vaccine according to claim 36, wherein, The volume ratio of formalin to virus harvesting fluid is 1:1000 to 1:4000, and the inactivation time at 4°C is 3 to 72 hours.

41. The method for preparing an inactivated SARS-CoV-2 composition or vaccine according to claim 40, wherein the inactivation time is 3 to 13 hours.

42. The method for preparing an inactivated SARS-CoV-2 composition or vaccine according to claim 32, wherein, The inactivated SARS-CoV-2 virus is filtered through a membrane and then mixed with Al(OH)3 adjuvant and / or CpG oligodeoxynucleotides, wherein the membrane has a pore size of 0.2~0.25 μm.

43. The method for preparing an inactivated SARS-CoV-2 composition or vaccine according to claim 42, wherein the membrane has a pore size of 0.22 μm.

44. The method for preparing an inactivated SARS-CoV-2 composition or vaccine according to claim 32 further comprises the step of mixing the inactivated SARS-CoV-2 virus with Al(OH)3 adjuvant and / or CpG oligodeoxynucleotides.

45. The method for preparing an inactivated SARS-CoV-2 composition or vaccine according to claim 32, wherein the inactivated SARS-CoV-2 virus is filtered through a membrane and then mixed with an Al(OH)3 adjuvant and / or CpG oligodeoxynucleotides, wherein the membrane has a pore size of 0.20~0.25 μm.

46. ​​The method for preparing an inactivated SARS-CoV-2 composition or vaccine according to claim 45, wherein the membrane has a pore size of 0.22 μm.

47. The method for preparing an inactivated SARS-CoV-2 composition or vaccine according to claim 32, wherein a bioreactor is used to propagate the SARS-CoV-2 virus in 50 to 1000 liters of Vero cell culture and inactivation is performed using β-propiolactone.

48. The method for preparing an inactivated SARS-CoV-2 composition or vaccine according to claim 32, wherein deep filtration or centrifugation and two optimized chromatographic steps are used to purify the virus.

49. The method for preparing an inactivated SARS-CoV-2 composition or vaccine according to claim 32, wherein the SARS-CoV-2 virus comprises: ORF1a, whose nucleotide sequence is shown as nucleotides 238 to 13455 of SEQ ID NO: 1; ORF1ab, whose nucleotide sequence is shown as nucleotides 13740-21527 of SEQ ID NO: 1; The spike protein S has an amino acid sequence encoded by nucleotides 21508 to 25356 of SEQ ID NO: 1; ORF3a, whose nucleotide sequence is shown as nucleotides 25365-26192 of SEQ ID NO: 1; Membrane protein M, whose amino acid sequence is encoded by nucleotides 26495-27163 of SEQ ID NO: 1; as well as Nucleocapsid protein N, whose amino acid sequence is encoded by nucleotides 28246-29505 of SEQ ID NO:

1.

50. The method for preparing an inactivated SARS-CoV-2 composition or vaccine according to claim 32, wherein the SARS-CoV-2 virus has a genome comprising an RNA sequence of the reverse complementary sequence shown in SEQ ID NO:

1.

51. The method for preparing an inactivated SARS-CoV-2 composition or vaccine according to claim 32, wherein the SARS-CoV-2 virus has a genome encoded by the sequence shown in SEQ ID NO:

1.

52. Use of the pharmaceutical composition or vaccine of any one of claims 13 to 31 in the preparation of a medicament for treating or immunizing a subject against a disease associated with SARS-CoV-2 infection.

53. The use according to claim 52, wherein the pharmaceutical composition or the vaccine is administered via intradermal, subcutaneous, intramuscular, oral, or intranasal administration.

54. The use according to claim 53, wherein the pharmaceutical composition or the vaccine is administered by intramuscular injection into the upper deltoid muscle of the subject.

55. The use according to claim 52, wherein the disease includes, but is not limited to, pneumonia or pneumonia syndrome, severe acute respiratory infection, renal failure, heart failure, adult respiratory distress syndrome (ARDS), liver injury, intestinal disease, or severe acute respiratory syndrome.

56. The use according to claim 52, wherein the subject is an animal.

57. The use according to claim 56, wherein the subject is a mammal, including mice, rats, dogs, cats, non-human primates, and humans.

58. The use according to claim 52, wherein the pharmaceutical composition or the vaccine provides complete protection against SARS-CoV-2 infection in non-human primates by inducing an effective humoral response without immunopathology.

59. The use according to claim 52, wherein the pharmaceutical composition or the vaccine does not produce antibody-dependent enhancement of infection.

60. The use according to claim 52, wherein the pharmaceutical composition or the vaccine, at a dose of 0.5 to 8 μg, produces complete protection against SARS-CoV-2 challenge in a subject.

61. The use according to claim 60, wherein the vaccine produces complete protection against SARS-CoV-2 challenge in subjects at a dose of 2-6 μg.

62. An isolated SARS-CoV-2 virus comprising a spike protein S, the amino acid sequence of which is encoded by nucleotides 21508 to 25356 of SEQ ID NO: 1; wherein the genomic DNA sequence of the SARS-CoV-2 virus is shown in SEQ ID NO:

1.

63. The isolated SARS-CoV-2 virus according to claim 62 further comprises nucleocapsid protein N, the amino acid sequence of which is encoded by nucleotides 28246 to 29505 of SEQ ID NO:

1.

64. The isolated SARS-CoV-2 virus according to claim 62, further comprising membrane protein M, the amino acid sequence of which is encoded by nucleotides 26495 to 27163 of SEQ ID NO:

1.

65. The isolated SARS-CoV-2 virus according to claim 62, further comprising ORF1a, the nucleotide sequence of which is shown as nucleotides 238 to 13455 of SEQ ID NO:

1.

66. The isolated SARS-CoV-2 virus according to claim 62, further comprising ORF1ab, the nucleotide sequence of which is shown as nucleotides 13740 to 21527 of SEQ ID NO:

1.

67. The isolated SARS-CoV-2 virus according to claim 62, further comprising ORF3a, the nucleotide sequence of which is shown as nucleotides 25365 to 26192 of SEQ ID NO:

1.

68. The isolated SARS-CoV-2 virus according to claim 62, further comprising: ORF1a, whose nucleotide sequence is shown as nucleotides 238 to 13455 of SEQ ID NO: 1; and / or ORF1ab, whose nucleotide sequence is shown as nucleotides 13740-21527 of SEQ ID NO: 1; and / or ORF3a, whose nucleotide sequence is shown as nucleotides 25365-26192 of SEQ ID NO: 1; and / or Membrane protein M, whose amino acid sequence is encoded by nucleotides 26495-27163 of SEQ ID NO: 1; and / or Nucleocapsid protein N, whose amino acid sequence is encoded by nucleotides 28246-29505 of SEQ ID NO:

1.

69. The isolated SARS-CoV-2 virus according to claim 62, further comprising: ORF1a, whose nucleotide sequence is shown as nucleotides 238 to 13455 of SEQ ID NO: 1; ORF1ab, whose nucleotide sequence is shown as nucleotides 13740-21527 of SEQ ID NO: 1; ORF3a, whose nucleotide sequence is shown as nucleotides 25365-26192 of SEQ ID NO: 1; Membrane protein M, whose amino acid sequence is encoded by nucleotides 26495-27163 of SEQ ID NO: 1; as well as Nucleocapsid protein N, whose amino acid sequence is encoded by nucleotides 28246-29505 of SEQ ID NO:

1.

70. The isolated SARS-CoV-2 virus according to claim 62, wherein the isolated SARS-CoV-2 virus has a genome comprising an RNA sequence of the reverse complementary sequence shown in SEQ ID NO: 1; or has a genome encoded by the sequence shown in SEQ ID NO: 1.

Images