CN115836124A - SARS-CoV-2 inactivated vaccine and its preparation - Google Patents

Abstract

Provides SARS-Cov-2 inactivated vaccine and its preparation. Also provided is a method of treating or preventing or immunizing a disease associated with SARS-Cov-2 virus 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

SARS-CoV-2 inactivated vaccine and its preparation

Background

The world health organization announced an outbreak of coronavirus disease (COVID-19) as a sudden public health event of international concern at 30/1/2020 and a pandemic at 11/3/2020. It has been reported that approximately 80% of patients with COVID-19 present mild to moderate symptoms, with approximately 20% presenting severe manifestations such as severe pneumonia, acute Respiratory Distress Syndrome (ARDS), sepsis and even death. The number of COVID-19 cases worldwide has increased at an alarming rate. By 10 days 4 months 2020, the total number of globally diagnosed cases has reached 1,623,173 cases, and the number of deaths has risen to 97,236. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a currently pandemic pathogen, and belongs to the genus beta-coronavirus (Betacononavirus, beta-CoV) of the family Coronaviridae. SARS-CoV-2, together with Severe acute respiratory syndrome coronavirus (SARS-CoV) and the middle east respiratory syndrome-associated coronavirus (MERS-CoV), constitute the three coronaviruses most threatening human life. SARS-CoV-2 contains a linear single-stranded sense RNA genome encoding 4 structural proteins [ including spike (S), envelope (E), membrane (M) and nucleocapsid (N) ] (where S is the main protective antigen that elicits highly neutralizing antibodies (NAb)), 16 non-structural proteins (nsp 1-nsp 16) and several accessory proteins. Currently, no specific antiviral drug or vaccine is available against the emerging SARS-CoV-2. Thus, the urgency for developing vaccines is critical to contain pandemics and prevent future viral outbreaks.

A variety of SARS-CoV-2 vaccine types, 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 been conventionally used for vaccine development, and it has been found that such vaccines can safely and effectively prevent diseases caused by viruses such as influenza virus and poliovirus. However, since SARS-CoV-2 is a new emerging virus, and our understanding of its biological nature, transmission and pathogenesis is limited, the development of vaccines against the virus has been a great challenge, let alone the new emerging virus.

Disclosure of Invention

The invention provides an inactivated vaccine aiming at SARS-CoV-2 virus, a preparation method and an application thereof. The inactivated vaccines of the present invention induce SARS-CoV-2 specific neutralizing antibodies in animals, particularly mammals, including mice, rats, dogs, cats, non-human primates, and humans. These antibodies effectively neutralized at least ten representative SARS-CoV-2 strains worldwide, suggesting that there may be a broad neutralizing capacity for SARS-CoV-2 strains worldwide. Thus, the inactivated vaccines of the present invention can be used to protect animals, including but not limited to mice, rats, dogs, cats, non-human primates, and humans, from SARS-CoV-2 challenge. In particular, the inactivated vaccines of the present invention do not induce antibody-dependent enhancement. In other words, the inactivated vaccine of the present invention induces an effective humoral response without immunopathology. In addition, the inactivated vaccine of the invention is safe after systematic evaluation (including monitoring clinical signs, blood biochemical indicators, histopathological analysis).

In one aspect, the 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.

In some embodiments, SARS-Cov-2 virus or a derivative or related thereof is inactivated with a virus inactivating agent, preferably beta-propiolactone and/or formalin. Preferably, the obtained inactivated SARS-Cov-2 virus or derivative or related thereof is subsequently purified using Ion Exchange Chromatography (IEC) and/or Size Exclusion Chromatography (SEC). Preferably, the inactivated SARS-Cov-2 virus is treated with a non-limiting endonuclease prior to Size Exclusion Chromatography (SEC).

Preferably, the volume ratio of beta-propiolactone to the virus harvest is 1. Further preferably, the inactivation time is 24 hours.

In some embodiments, the SARS-Cov-2 virus or derivative or related thereof comprises a spike protein (S) having an amino acid sequence encoded by nucleotides 21508-25356 of SEQ ID NO:1 or a sequence at least 95% identical thereto.

In some embodiments, SARS-Cov-2 virus or a derivative or related thereof comprises a nucleocapsid protein (N) having an amino acid sequence encoded by nucleotides 28246 to 29505 of SEQ ID NO:1 or a sequence at least 95% identical thereto.

In some embodiments, the SARS-Cov-2 virus or derivative or related thereof comprises a membrane protein (M) of sequence having or at least 95% identical to the amino acid sequence encoded by nucleotides 26495 to 27163 of SEQ ID NO: 1.

In some embodiments, SARS-Cov-2 virus or a derivative or related thereof comprises ORF1a having the nucleotide sequence of nucleotides 238 to 13455 of SEQ ID NO:1 or a sequence having at least 95% identity thereto.

In some embodiments, the SARS-Cov-2 virus or derivative or related thereof comprises ORF1ab having the nucleotide sequence of nucleotides 13740 to 21527 of SEQ ID No. 1 or a sequence that is at least 95% identical thereto.

In some embodiments, SARS-Cov-2 virus or a derivative or related thereof comprises ORF3a having the nucleotide sequence of nucleotides 25365 to 26192 of SEQ ID NO:1 or a sequence at least 95% identical thereto.

Preferably, the SARS-Cov-2 virus or derivative or related thereof comprises: ORF1a having the nucleotide sequence of nucleotides 238 to 13455 of SEQ ID NO. 1 or a sequence having at least 95% identity thereto; 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 thereto; and/or 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 thereto; 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 thereto; and/or a membrane protein (M) having the amino acid sequence encoded by nucleotides 26495 to 27163 of SEQ ID NO:1 or a sequence having at least 95% identity thereto; and/or 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 thereto.

Preferably, the SARS-Cov-2 virus or derivative or related thereof comprises: 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; a spike protein (S) having an 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; a membrane protein (M) having an amino acid sequence encoded by nucleotides 26495 to 27163 of SEQ ID NO: 1; and a nucleocapsid protein (N) having an amino acid sequence encoded by nucleotides 28246 to 29505 of SEQ ID NO. 1.

Preferably, the isolated SARS-CoV-2 virus or a derivative or related thereof has a genome comprising an RNA sequence having the reverse complement of any one of SEQ ID NOs 1 to 3, or a sequence at least 95% identical thereto.

Preferably, the SARS-CoV-2 virus or a derivative or related thereof has a genome encoded by a sequence represented by any one of SEQ ID NOs 1 to 3 or a sequence having at least 95% identity thereto. Preferably, the SARS-CoV-2 virus or a derivative or a related thereof has a genome encoded by a sequence shown in any one of SEQ ID NOS 1 to 3 or has a genome including an RNA sequence having a reverse complement sequence shown in any one of SEQ ID NOS 1 to 3. The SARS-Cov-2 virus or its derivative or related matter of the present invention shows high yield in Vero cell.

Preferably, SARS-Cov-2 virus is contacted with a cell density of 2X 10 prior to inactivation with beta-propiolactone ⁶ cells/mL to 10X 10 ⁶ cells/mL Vero cells, cultured together in a bioreactor at 35-39 ℃ for 3-7 days to obtain a virus harvest.

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 a derivative or related thereof and a pharmaceutically acceptable carrier. The composition or vaccine is in a liquid state or in a frozen state, e.g. in a lyophilized state.

In some embodiments, the dosage of the pharmaceutical composition or vaccine is in the range of 0.5 to 8 μ g by weight of protein, preferably in the range of 3 to 6 μ g by weight of protein. In other words, 0.5 μ g to 8 μ g per dose of vaccine by weight of protein gives complete protection against SARS-CoV-2 challenge, preferably 3 μ g to 6 μ g per dose of vaccine by weight of protein gives complete protection against SARS-CoV-2 challenge.

In some embodiments, SARS-Cov-2 virus is inactivated with beta-propiolactone and/or formalin. Preferably, the inactivated SARS-Cov-2 virus is subsequently purified by Ion Exchange Chromatography (IEC) and/or Size Exclusion Chromatography (SEC). Thus, preferably, the pharmaceutical composition or vaccine comprises an inactivated SARS-Cov-2 virus, which is purified by Ion Exchange Chromatography (IEC) and/or Size Exclusion Chromatography (SEC) after inactivation. Preferably, the inactivated SARS-Cov-2 virus is treated with a non-limiting endonuclease prior to Size Exclusion Chromatography (SEC).

Preferably, the volume ratio of beta-propiolactone to the virus harvest is 1. Preferably, the inactivation time is 24 hours.

Preferably, SARS-Cov-2 virus is contacted with a cell density of 2X 10 prior to inactivation with beta-propiolactone ⁶ cells/mL to 10X 10 ⁶ cells/mL Vero cells, cultured together in a bioreactor at 35-39 ℃ for 3-7 days to obtain a virus harvest.

The pharmaceutically acceptable carrier 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 contacted with Al (OH) ₃ Adjuvant and/or CpG oligodeoxynucleotide. Preferably, the pore size of the membrane is 0.2 to 0.25. Mu.m, preferably 0.22. Mu.m.

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

In some embodiments, SARS-Cov-2 virus or a derivative or related thereof comprises a spike protein (S) having an amino acid sequence encoded by nucleotides 21508 to 25356 of SEQ ID NO:1 or a sequence at least 95% identical thereto.

In some embodiments, SARS-Cov-2 virus or a derivative or related thereof comprises a nucleocapsid protein (N) having an amino acid sequence encoded by nucleotides 28246 to 29505 of SEQ ID NO:1 or a sequence at least 95% identical thereto.

In some embodiments, the SARS-Cov-2 virus or derivative or related thereof comprises a membrane protein (M) having an amino acid sequence encoded by nucleotides 26495 to 27163 of SEQ ID NO:1 or a sequence at least 95% identical thereto.

In some embodiments, SARS-Cov-2 virus or a derivative or related thereof comprises ORF1a having the nucleotide sequence of nucleotides 238 to 13455 of SEQ ID NO:1 or a sequence having at least 95% identity thereto.

In some embodiments, the SARS-Cov-2 virus or derivative or related thereof comprises ORF1ab having the nucleotide sequence of nucleotides 13740 to 21527 of SEQ ID NO:1 or a sequence having at least 95% identity thereto.

In some embodiments, SARS-Cov-2 virus or a derivative or related thereof comprises ORF3a having the nucleotide sequence of nucleotides 25365 to 26192 of SEQ ID NO:1 or a sequence having at least 95% identity thereto.

Preferably, the SARS-Cov-2 virus or derivative or related thereof comprises: ORF1a having the nucleotide sequence of nucleotides 238 to 13455 of SEQ ID NO. 1 or a sequence having at least 95% identity thereto; 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 thereto; and/or 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 thereto; 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 thereto; and/or a membrane protein (M) having the amino acid sequence encoded by nucleotides 26495 to 27163 of SEQ ID NO:1 or a sequence having at least 95% identity thereto; and/or 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 thereto.

Preferably, the SARS-Cov-2 virus or derivative or related thereof comprises: 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; a spike protein (S) having an 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; a membrane protein (M) having an amino acid sequence encoded by nucleotides 26495 to 27163 of SEQ ID NO: 1; and a nucleocapsid protein (N) having an amino acid sequence encoded by nucleotides 28246 to 29505 of SEQ ID NO. 1.

Preferably, the isolated SARS-CoV-2 virus or a derivative or related thereof has a genome comprising an RNA sequence having the reverse complement of any one of SEQ ID NOs 1 to 3, or a sequence at least 95% identical thereto.

Preferably, the SARS-CoV-2 virus or a derivative or related thereof has a genome encoded by a sequence represented by any one of SEQ ID NOs 1 to 3 or a sequence having at least 95% identity thereto.

Preferably, the SARS-CoV-2 virus or a derivative or a related thereof has a genome encoded by a sequence shown in any one of SEQ ID NOS 1 to 3 or has a genome including an RNA sequence having a reverse complement sequence shown in any one of SEQ ID NOS 1 to 3.

The SARS-Cov-2 virus of the invention shows high yield in Vero cells.

In particular, the pharmaceutical composition or vaccine of the invention provides complete protection against the globally prevalent SARS-CoV-2 strain in non-human primates by eliciting an effective humoral response without immunopathology.

The pharmaceutical composition or vaccine of the invention can elicit much higher S-specific antibody titers than sera from convalescent COVID-19 patients. This observation, combined with the fact that antibodies targeting the N of SARS-CoV-2 do not provide protective immunity against infection, indicates that the pharmaceutical composition or vaccine of the invention is capable of eliciting a more effective antibody response.

Previous experience in developing SARS and MERS vaccine candidates has led to concern over lung immunopathology that may be directly caused by a type 2 helper T cell (Th 2) response or as a result of (ADE). Although many vaccine-induced T cell responses have been shown to be critical for acute viral clearance, protection against subsequent coronavirus infection is primarily mediated by humoral immunity. The "cytokine storm" caused by excessive T cell responses has actually been shown to emphasize the pathogenesis of COVID 19. Therefore, the T cell response elicited by the SARS-CoV-2 vaccine must be well controlled to avoid immunopathology. In this context, we systematically evaluated the safety of the vaccines of the invention in macaques by recording a number of clinical observations and biological indicators. After immunization, no fever or weight loss occurs in the macaque, and the appetite and mental state of all animals are normal. Hematologic and biochemical analyses, including biochemical blood tests, lymphocyte subpopulation percentages (CD 3+, CD4+ and CD8 +) and key cytokines (TNF-. Alpha., IFN-. Gamma., IL-2, IL-4, IL-5 and IL-6) showed no significant change in the vaccinated groups compared to sham and placebo groups. Furthermore, histopathological evaluation of various organs including lung, heart, spleen, liver, kidney and brain indicates that the vaccine of the present invention does not cause any significant pathology in macaques. No antibody-dependent increase in infection (ADE) was observed for all vaccinated macaques.

The invention proves the safety of the vaccine in the macaque; no enhancement of infection and no deterioration of immunopathology were observed. The present invention also demonstrates that administration of 6 μ g of the vaccine of the present invention per dose to macaques provides complete protection against SARS-CoV-2 challenge.

In another aspect, the present invention provides a method of preparing an inactivated SARS-Cov-2 composition or vaccine comprising inactivating SARS-Cov-2 virus using a virus inactivating agent, preferably beta-propiolactone and/or formalin.

In some embodiments, the SARS-Cov-2 virus is cultured in a large bioreactor, e.g., a Viro cell factory or fermentor, to obtain a viral harvest and inactivated with beta-propiolactone for 24 hours. Preferably, the volume ratio of beta-propiolactone to the virus harvest is 1. Preferably, the inactivation time is 24 hours.

In some embodiments, the ratio of formalin to virus harvest by volume is 1 to 1000 to 4000, and the inactivation time is 3 to 72 hours, preferably 3 to 13 hours, at 4 ℃.

Preferably, SARS-Cov-2 virus is contacted with a cell density of 2X 10 prior to inactivation with beta-propiolactone ⁶ cells/mL to 10X 10 ⁶ cells/mL Vero cells, cultured together in a bioreactor at 35-39 ℃ for 3-7 days to obtain a virus harvest.

Preferably, the method further comprises the step of purifying the inactivated SARS-Cov-2 virus by Ion Exchange Chromatography (IEC) and/or Size Exclusion Chromatography (SEC). Preferably, the inactivated SARS-Cov-2 virus is treated with a non-limiting endonuclease prior to Size Exclusion Chromatography (SEC).

Preferably, the method further comprises contacting the inactivated SARS-Cov-2 virus with Al (OH) ₃ And (3) mixing the adjuvant. In some embodiments, the inactivated SARS-Cov-2 virus is filtered through a membrane and then contacted with Al (OH) ₃ Adjuvant and/or CpG oligodeoxynucleotide. Preferably, the pore size of the membrane is 0.2 to 0.25. Mu.m, preferably 0.22. Mu.m.

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

In some embodiments, to produce a vaccine, SARS-Cov-2 virus is propagated in 50 to 1000 liters of Vero cell culture, e.g., 50, 100, 200, 300, 400, 500, 600, 700 liters of Vero cell culture, using a bioreactor and inactivated using beta-propiolactone and/or formalin. The virus is purified using centrifugation or depth filtration and two optimized chromatographic steps, resulting in a highly pure vaccine preparation. In addition, cryo-electron microscope (cryo-EM) analysis showed intact ellipsoidal particles, 90-150 nm in diameter, interspersed with coronary spikes, representing the pre-fusion state of the virus.

In some embodiments, SARS-Cov-2 virus or a derivative or related thereof comprises a spike protein (S) having an amino acid sequence encoded by nucleotides 21508 to 25356 of SEQ ID NO:1 or a sequence at least 95% identical thereto.

In some embodiments, SARS-Cov-2 virus or a derivative or related thereof comprises a nucleocapsid protein (N) having an amino acid sequence encoded by nucleotides 28246-29505 of SEQ ID NO:1 or a sequence at least 95% identical thereto.

In some embodiments, the SARS-Cov-2 virus or derivative or related thereof comprises a membrane protein (M) having an amino acid sequence encoded by nucleotides 26495 to 27163 of SEQ ID NO:1 or a sequence at least 95% identical thereto.

In some embodiments, SARS-Cov-2 virus or a derivative or related thereof comprises ORF1a having the nucleotide sequence of nucleotides 238 to 13455 of SEQ ID NO:1 or a sequence having at least 95% identity thereto.

In some embodiments, the SARS-Cov-2 virus or derivative or related thereof comprises ORF1ab having the nucleotide sequence of nucleotides 13740 to 21527 of SEQ ID NO:1 or a sequence having at least 95% identity thereto.

In some embodiments, SARS-Cov-2 virus or a derivative or related thereof comprises ORF3a having the nucleotide sequence of nucleotides 25365 to 26192 of SEQ ID NO:1 or a sequence at least 95% identical thereto.

Preferably, the SARS-Cov-2 virus or derivative or related thereof comprises: ORF1a having the nucleotide sequence of nucleotides 238 to 13455 of SEQ ID NO. 1 or a sequence having at least 95% identity thereto; 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 thereto; and/or 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 thereto; 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 thereto; and/or a membrane protein (M) having the amino acid sequence encoded by nucleotides 26495 to 27163 of SEQ ID NO:1 or a sequence having at least 95% identity thereto; and/or 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 thereto.

Preferably, the SARS-Cov-2 virus or derivative or related thereof comprises: 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; a spike protein (S) having an 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; a membrane protein (M) having an amino acid sequence encoded by nucleotides 26495 to 27163 of SEQ ID NO: 1; and a nucleocapsid protein (N) having an amino acid sequence encoded by nucleotides 28246 to 29505 of SEQ ID NO: 1.

Preferably, the isolated SARS-CoV-2 virus or a derivative or related thereof has a genome comprising an RNA sequence having the reverse complement of SEQ ID NO:1 or a sequence having at least 95% identity thereto.

Preferably, the SARS-CoV-2 virus or a derivative or related thereof has a genome encoded by a sequence represented by any one of SEQ ID NOs 1 to 3 or a sequence having at least 95% identity thereto.

Preferably, the SARS-CoV-2 virus or a derivative or a related thereof has a genome encoded by a sequence shown in any one of SEQ ID NOS: 1 to 3 or has a genome including an RNA sequence having a reverse complement sequence shown in SEQ ID NO: 1.

The SARS-Cov-2 virus of the invention shows high yield in Vero cells. In another aspect, the present invention provides a method of treating or immunizing against a disease 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 route of administration of the pharmaceutical composition or vaccine is intradermal, subcutaneous, intramuscular, oral, or intranasal vaccination. Preferably, the pharmaceutical composition or vaccine is administered by intramuscular injection into the upper part of the deltoid muscle.

Preferably, the disease includes, but is not limited to, pneumonia, severe acute respiratory infection, renal failure, heart failure, adult Respiratory Distress Syndrome (ARDS), liver injury, bowel disease, or severe acute respiratory syndrome.

In particular, the pharmaceutical composition or vaccine of the invention provides complete protection against the globally prevalent SARS-CoV-2 strain in a non-human primate by eliciting an effective humoral response without immunopathology.

The pharmaceutical composition or vaccine of the invention can elicit much higher S-specific antibody titers than sera from convalescent COVID-19 patients. This observation, combined with the fact that antibodies targeting the N of SARS-CoV-2 do not provide protective immunity against infection, indicates that the pharmaceutical composition or vaccine of the invention is capable of eliciting a more effective antibody response.

Previous experience in developing SARS and MERS vaccine candidates has led to concern over lung immunopathology, either directly as a result of a type 2 helper T cell (Th 2) response or as a result of (ADE). Although many vaccine-induced T cell responses have been shown to be critical for acute viral clearance, protection against subsequent coronavirus infection is primarily mediated by humoral immunity. The "cytokine storm" caused by excessive T cell responses has actually been shown to emphasize the pathogenesis of COVID 19. Therefore, the T cell response elicited by the SARS-CoV-2 vaccine must be well controlled to avoid immunopathology. In this context, we systematically evaluated the safety of the vaccines of the invention in macaques by recording a number of clinical observations and biological indicators. After immunization, no fever or weight loss occurs in the macaque, and the appetite and mental state of all animals are normal. Hematologic and biochemical analyses, including biochemical blood tests, lymphocyte subpopulation percentages (CD 3+, CD4+ and CD8 +) and key cytokines (TNF-. Alpha., IFN-. Gamma., IL-2, IL-4, IL-5 and IL-6) showed no significant change in the vaccinated groups compared to sham and placebo groups. Furthermore, histopathological evaluation of various organs including lung, heart, spleen, liver, kidney and brain indicates that the vaccine of the present invention does not cause any significant pathology in macaques. No antibody-dependent increase in infection (ADE) was observed for all vaccinated macaques.

The invention proves the safety of the vaccine in the macaque; no enhancement of infection and no deterioration of immunopathology were observed. The present invention also demonstrates that administration of a dose of the vaccine of the present invention of 0.5-8 μ g protein per dose to macaques, preferably in a dose range of 2-6 μ g protein by weight, preferably in a dose range of 3-6 μ g protein by weight, can improve the complete protection against the SARS-CoV-2 challenge.

In some embodiments, the SARS-Cov-2 virus is obtained from a hospitalized patient. 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, respectively, or a sequence with at least 95% identity to the sequence shown in 2019-nCoV-BetaCoV/Wuhan/WIV04/2019 and EPI _ ISL _412973, respectively, preferably a sequence with 96%, 97%, 98%, 99%, 99.5%, 99.9% identity to the sequence shown in 2019-nCoV-BetaCoV/Wuhan/WIV04/2019 or EPI _ ISL _412973, respectively.

In another aspect, the 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 at least 95% identical thereto.

In some embodiments, SARS-Cov-2 virus or a derivative or related thereof comprises a nucleocapsid protein (N) having an amino acid sequence encoded by nucleotides 28246 to 29505 of SEQ ID NO:1 or a sequence at least 95% identical thereto.

In some embodiments, the SARS-Cov-2 virus or derivative or related thereof comprises a membrane protein (M) having an amino acid sequence encoded by nucleotides 26495 to 27163 of SEQ ID NO:1 or a sequence at least 95% identical thereto.

In some embodiments, SARS-Cov-2 virus or a derivative or related thereof comprises ORF1a having the nucleotide sequence of nucleotides 238 to 13455 of SEQ ID NO:1 or a sequence having at least 95% identity thereto.

In some embodiments, the SARS-Cov-2 virus or derivative or related thereof comprises ORF1ab having the nucleotide sequence of nucleotides 13740 to 21527 of SEQ ID NO:1 or a sequence having at least 95% identity thereto.

In some embodiments, SARS-Cov-2 virus or a derivative or related thereof comprises ORF3a having the nucleotide sequence of nucleotides 25365 to 26192 of SEQ ID NO:1 or a sequence at least 95% identical thereto.

Preferably, the SARS-Cov-2 virus or derivative or related thereof comprises: ORF1a having the nucleotide sequence of nucleotides 238 to 13455 of SEQ ID NO. 1 or a sequence having at least 95% identity thereto; 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 thereto; and/or 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 thereto; 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 thereto; and/or a membrane protein (M) having the amino acid sequence encoded by nucleotides 26495 to 27163 of SEQ ID NO:1 or a sequence having at least 95% identity thereto; and/or 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 thereto.

Preferably, the SARS-Cov-2 virus or derivative or related thereof comprises: 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; a spike protein (S) having an 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; a membrane protein (M) having an amino acid sequence encoded by nucleotides 26495 to 27163 of SEQ ID NO: 1; and a nucleocapsid protein (N) having an amino acid sequence encoded by nucleotides 28246 to 29505 of SEQ ID NO. 1.

Preferably, the isolated SARS-CoV-2 virus or a derivative or related thereof has a genome comprising an RNA sequence having the reverse complement of any one of SEQ ID NOs 1 to 3, or a sequence at least 95% identical thereto.

Preferably, the SARS-CoV-2 virus or a derivative or related thereof has a genome encoded by a sequence represented by any one of SEQ ID NOs 1 to 3 or a sequence having at least 95% identity thereto.

Preferably, the SARS-CoV-2 virus or a derivative or a related thereof has a genome encoded by a sequence shown in any one of SEQ ID NOS 1 to 3 or has a genome including an RNA sequence having a reverse complement sequence shown in any one of SEQ ID NOS 1 to 3.

The isolated SARS-Cov-2 virus of the invention exhibits high yield in Vero cells.

The SARS-Cov-2 virus of the invention shows high yield in Vero cells. And after the SARS-Cov-2 virus is inactivated, the SARS-Cov-2 virus shows strong neutralization activity to at least ten representative SARS-Cov-2 strains in the world, which indicates that the SARS-Cov-2 virus can have wider neutralization capability to the SARS-Cov-2 strains in the world and can induce effective humoral response without immune pathology.

In another aspect, the invention provides an isolated nucleic acid having a nucleotide sequence of nucleotides 21508 to 25356 of SEQ ID NO. 1 or a sequence having at least 95% identity thereto.

In another aspect, the invention provides an isolated nucleic acid having the nucleotide sequence of nucleotides 28246 to 29505 of SEQ ID NO. 1 or a sequence having at least 95% identity thereto.

In another aspect, the invention provides an isolated nucleic acid having the nucleotide sequence of nucleotides 26495 to 27163 of SEQ ID NO. 1 or a sequence having at least 95% identity thereto.

In another aspect, the invention provides an isolated nucleic acid having a nucleotide sequence of nucleotides 238 to 13455 of SEQ ID NO. 1 or a sequence having at least 95% identity thereto.

In another aspect, the invention provides an isolated nucleic acid having a nucleotide sequence of nucleotides 13740 to 21527 of SEQ ID NO. 1 or a sequence having at least 95% identity thereto.

In another aspect, the invention provides an isolated nucleic acid having a nucleotide sequence of nucleotides 25365 to 26192 of SEQ ID NO. 1 or a sequence having at least 95% identity thereto.

In another aspect, the invention provides an isolated nucleic acid comprising: 1 or a sequence having at least 95% identity thereto; and/or the nucleotide sequence of nucleotides 13740 to 21527 of SEQ ID NO. 1 or a sequence having at least 95% identity thereto; and/or the nucleotide sequence of nucleotides 21508 to 25356 of SEQ ID NO. 1 or a sequence having at least 95% identity thereto; and/or the nucleotide sequence of nucleotides 25365 to 26192 of SEQ ID NO. 1 or a sequence with at least 95% identity thereto; and/or the nucleotide sequence of nucleotides 26495 to 27163 of SEQ ID NO. 1 or a sequence having at least 95% identity thereto; and/or the nucleotide sequence of nucleotides 28246 to 29505 of SEQ ID NO. 1 or a sequence having at least 95% identity thereto.

In another aspect, the invention provides an isolated nucleic acid comprising: the 238 th to 13455 th nucleotide of SEQ ID NO. 1; 1, the 13740 th to 21527 th nucleotides of SEQ ID NO; 1, nucleotide sequence of 21508-25356 nucleotides of SEQ ID NO; a nucleotide sequence of 25365 to 26192 nucleotides of SEQ ID NO. 1; and the nucleotide sequence of nucleotides 26495 to 27163 of SEQ ID NO. 1; the 28246 th to 29505 th nucleotide sequence of SEQ ID NO. 1.

In another aspect, the invention provides an isolated nucleic acid having the nucleotide sequence of any one of SEQ ID NOs 1 to 3, or a sequence having at least 95% identity thereto.

In another aspect, the present invention provides an isolated nucleic acid having the nucleotide sequence of any one of SEQ ID NOs 1 to 3 or the reverse complement thereof.

By "at least 95%" is meant 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.

2 corresponds to the viral strain P1_ CADDE-CD1716_ hCoV-19/Brazil/L70_ CD1716/2020 pre-existing 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.

Drawings

Figure 1 shows the characterization of PiCoVacc. (A) SARS-CoV-2 maximum likelihood phylogenetic tree. The SARS-CoV-2 isolates used in this study are depicted and labeled by black lines. The strains of the virus are from different continents. (B) Growth kinetics of PicoVacc (CN 2) P5 stock in Vero cells. (C) Assessment of the protein composition and purity of PicoVacc by NuPAGE 4-12% Bis-Tris gels. (D) representative Electron micrographs of PiCoVacc.

FIG. 2 shows that PicoVacc immunization elicits a neutralizing antibody response 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 a sham model). Sera from COVID19 convalescent patients (RECOV) and uninfected persons (NI) served as positive and negative controls, respectively. Antibody responses were analyzed in mice (A), humans (B) and rats (C). The upper graph is as follows: SARS-CoV-2 specific IgG response measured by ELISA; the following figures: neutralizing antibody titers were determined by microneutralization assay. The PicoVacc-induced spectrum of neutralizing activity was studied in mice (D) and rats (E). Neutralization assays were performed on the other 9 isolated SARS-CoV-2 strains using mouse and rat sera collected 3 weeks after vaccination. Dots represent individual animals and humans; the dashed line represents the detection limit; horizontal lines indicate the Geometric Mean Titer (GMT) of EC50 per group.

Figure 3 shows the immunogenicity and protective efficacy of PiCoVacc in non-human primates. Macaques were immunized 3 times by intramuscular route with different doses of either PiCoVacc or adjuvant only (sham) or placebo (n = 4). SARS-CoV-2 specific IgG response (A) and neutralizing antibody titers (B) were measured. Points represent single macaques; the dotted line represents the limit of detection; horizontal lines indicate the Geometric Mean Titer (GMT) of EC50 per group. The protective effect of PicoVacc on SARS-CoV-2 challenge at week 3 after immunization was evaluated in macaques (C-F). Throat (C) and anus (D) swab samples collected from vaccinated macaques on days 3, 5 and 7 post-vaccination were monitored for viral load. The viral load (E) of the different lung lobes of the lung tissue of all vaccinated macaques was measured on day 7 post-infection. RNA was extracted and viral load was determined by qRT-PCR. All data are expressed as mean ± SEM. Lung histology examination of all vaccinated macaques on day 7 post-infection (F). Lung tissue was collected and stained with hematoxylin and eosin.

Figure 4 shows the safety assessment of PiCoVacc in non-human primates. Macaques were immunized 3 times intramuscularly on days 0, 7 and 14 with either a low dose (1.5 μ g per dose) or a high dose (6 μ g per dose) of either PiCoVacc or adjuvant only (sham) or placebo. (a and B) hematological analysis of all four groups of rhesus macaques (n = 4). The percentage (a) of lymphocyte subpopulations (including CD3+, CD4+ and CD8 +) was 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). Critical cytokines, including TNF- α, IFN- γ and IL-2 (B), were examined on day-1, day 1 (day of first vaccination), day 4, day 18 and day 29 post-vaccination. Values are mean ± standard deviation. (C) day 29, lung pathology assessments of four groups of macaques. Lung tissue was collected and stained with hematoxylin and eosin.

FIG. 5 shows a comparison of the amino acid sequences of SARS-CoV-2. The number and percentage of amino acid differences in S of the following SARS-CoV-2 isolates used in this study are shown (for detailed information on these strains, see Table 1).

FIG. 6 shows purification of S, RBD and N proteins. (A) SDS-PAGE analysis of S, RBD and N proteins. Lane 1: molecular weight standards, labeled bands of interest; lane 2: recombinant S protein; lane 3: recombinant RBD protein; lane 4: a recombinant N protein. (B) Size exclusion chromatograms of affinity purified S, RBD and N proteins. (left) S protein data from Superose 6/300 column is shown in red. RBD protein data from Superdex 200/300 column is shown in red. (right) N protein data from Superdex 200 10/300 column is shown in black.

Figure 7 shows monitoring of body weight and body temperature to assess the safety of PiCoVacc in non-human primates. Macaques were immunized intramuscularly 3 times on days 0, 7 and 14 with either a low dose (1.5 μ g per dose) or a high dose (6 μ g per dose) of either PicoVacc or adjuvant only (sham) or placebo. Body weight and body temperature were monitored at different time points.

Figure 8 shows monitoring of hematological indicators to assess the safety of PiCoVacc in non-human primates. Macaques were immunized 3 times intramuscularly with either a low dose (1.5 μ g per dose) or a high dose (6 μ g per dose) of either PiCoVacc or adjuvant only (sham) or placebo on days 0, 7 and 14. Several hematological indicators were measured at different time points. ALT (alanine aminotransferase), AST (aspartate aminotransferase), ALP (alkaline phosphatase), TBil (total bilirubin), GGT (gamma-glutamyl transpeptidase), TP (total protein), alb (albumin), glb (globulin), A/G (albumin/globulin ratio), UREA (haematurin), cre (creatinine), CK (creatine kinase), glu (glucose), LDH (lactate dehydrogenase), CHO (total cholesterol), TG (triglyceride), ca (calcium), P (phosphorus), na (Na), and the like ⁺ (sodium ion), K ⁺ (Potassium ion) and Cl ^- (chloride ion).

Figure 9 shows monitoring of key cytokines to assess the safety of PiCoVacc in non-human primates. Macaques were immunized 3 times intramuscularly on days 0, 7 and 14 with either a low (1.5 μ g per dose) or high (6 μ g per dose) dose of either PicoVacc or adjuvant only (sham) or placebo. IL4, IL5 and IL6 were measured at different time points.

Figure 10 shows histopathological evaluation performed to assess the safety of PiCoVacc in non-human primates. Macaques were immunized intramuscularly 3 times on days 0, 7 and 14 with either a low dose (1.5 μ g per dose) or a high dose (6 μ g per dose) of either PicoVacc or adjuvant only (sham) or placebo. The brains, spleens, kidneys and hearts of four groups of macaques were histopathologically evaluated on day 29. Tissues were collected and stained with hematoxylin and eosin.

Detailed Description

The description of specific embodiments and examples are provided for purposes of illustration and not for limitation. Those skilled in the art will readily recognize a variety of non-critical parameters that may be changed or modified to produce substantially similar results.

Examples

Materials and methods

Statement of facility and ethics

All SARS-CoV-2 live virus experiments were performed in the enhanced biosafety level 3 (P3 +) facility of the national institute of medicine and sciences (CAMS) laboratory animal science institute approved by the national health and welfare Committee of the people's republic of China. All the experiments of mice, rats and macaques were performed according to the regulations of the "guidelines for nursing and using laboratory animals" of the department of science and technology of the people's republic of China.

Viral titration

SARS-CoV-2 virus titer was determined by microdose cytopathic effect (CPE) assay. The virus-containing samples diluted 10-fold in series were mixed with Vero cells and then seeded in 96-well culture plates. 5% CO at 36.5 deg.C ₂ After 3 to 7 days in the incubator, the cells were examined under a microscope for the presence of CPE. Viral titers were calculated using the Karber method.

Vaccine preparation

SARS-CoV-2 coronavirus (CN 2) was isolated from a patient infected with COVID-19 and was supplied by the Zhejiang province disease prevention and control center. The virus was cultured in a bioreactor, inactivated with beta-propiolactone for 24 hours, and then purified by Ion Exchange Chromatography (IEC) and Size Exclusion Chromatography (SEC). Inactivated SARS-CoV-2 virus is treated with non-limiting nucleic acid prior to Ion Exchange Chromatography (IEC) and Size Exclusion Chromatography (SEC)And (4) performing endonuclease treatment. Purified virus and Al (OH) ₃ Adjuvant mixing, and using as candidate vaccine of SARS-CoV-2. In combination with Al (OH) ₃ Inactivated SARS-CoV-2 virus was filtered through a membrane prior to adjuvant mixing. Preferably, the membrane has a pore size of 0.22 μm. The resulting vaccine was designated as PicoVacc, whose genome was encoded by the sequence shown in SEQ ID NO. 1; or an RNA sequence having the reverse complement sequence shown in SEQ ID NO. 1.

RT-PCR

Total RNA was extracted from organs using RNeasy mini kit (Qiagen) and PrimerScript RT reagent kit (TaKaRa) as described previously. The forward and reverse primers for SARS-CoV-2 envelope (E) gene used in RT-PCR were 5 '-TCGTTTCGGAAGAGACAGGGT-3' and 5 '-GCGCGCAGTAAGGATGGCTAGT-3', respectively. The RT-PCR reaction conditions were 50 ℃ for 30min, then 95 ℃ for 15min, 94 ℃ for 15s, 60 ℃ for 45s, and 40 cycles.

Vaccine immunogenicity assay

Balb/c mice, wistar rats were randomized into three groups and immunized intraperitoneally and intramuscularly with three doses (1.5. Mu.g, 3. Mu.g, 6. Mu.g/dose) of the test vaccine, respectively. All animals grouped were immunized twice (on days 0 and 7). The control group was injected with physiological saline. Animals were bled from the tail vein and then subjected to an antibody neutralization assay to analyze the immunogenicity of the vaccine.

Neutralization assay

Serum samples collected from immunized animals were inactivated at 56 ℃ for 0.5h and serially diluted in cell culture medium in two steps. The diluted serum was mixed with 100TCID ₅₀ The virus suspension of (a) was mixed in a 96-well plate at a ratio of 1 ₂ Incubate in the incubator at 36.5 ℃ for 2 hours. Vero cells were then added to the serum-virus mixture and plates were incubated at 5% CO ₂ Incubate at 36.5 ℃ for 5 days in an incubator. Cytopathic effect (CPE) was recorded microscopically for each well and neutralization titers were calculated at dilutions of 50% protective conditions.

Enzyme-linked immunosorbent assay (ELISA)

The SARS-CoV-2 antibody titer of a serum sample collected from the immunized animal is determined by an indirect ELISA assay. The purified S eggs were used in an amount of 0.1. Mu.g eachThe 96-well microtiter plate was coated with albumin, M protein, N protein, overnight at 2-8 ℃ and blocked with 2% BSA at room temperature for 1h. Diluted serum (1, 100) was added to each well, washed 3 times with pbs at 37 ℃ 2h, and then incubated with HRP-conjugated goat anti-mouse antibody for 1h at 37 ℃. Addition of 2M H ₂ SO ₄ To stop the reaction, the plate was then developed using TMB and the final data read by ELISA reader at 450/630 nm.

Vaccine safety evaluation

The safety of the SARS-CoV-2 test vaccine was evaluated in macaques. On days 0, 7 and 14, high dose (6. Mu.g/dose) vaccine, low dose (1.5. Mu.g/dose) vaccine, al (OH) were administered ₃ Four groups of macaques (5 female monkeys and 5 male monkeys/group) were immunized 3 times with adjuvant and saline. Data sets of some safety-related parameters were collected during and after immunization, including clinical observations, body weight, body temperature. The collected blood samples were also analyzed for lymphocyte subpopulation percentages (CD 3+, CD4+, and CD8 +), key cytokines (TNF- α, IFN- γ, IL-2, IL-4, IL-5, IL-6), and biochemical blood tests. 60% of the monkeys were euthanized at day 18 post-immunization, and the remaining 40% were euthanized at day 29. Lung, heart, spleen, liver, kidney and brain organs were collected for pathological analysis.

Challenge assay for rhesus monkeys

The rhesus macaques (3-4 years old) are divided into four groups, and high dose (6 mug/dose) vaccine, medium dose (3 mug/dose) vaccine and Al (OH) are injected into the muscle respectively ₃ Adjuvants and physiological saline. All animals in the cohort were immunized 3 times ( day 0, 7 and 14) and then 10 days ⁶ TCID ₅₀ The/ml SARS-CoV-2 virus was challenged by the intratracheal route. Macaques were euthanized 7 days after inoculation (day post inoculation, dpi) and lung tissue was collected. Throat and anal swabs were collected on days 3, 5, and 7 post inoculation (dpi). Blood samples were collected at 0, 7, 14 and 21 days post immunization (dpi) and 3, 5 and 7 days post inoculation (dpi) for hematological analysis of SARS-CoV-2 and detection of neutralizing antibodies. Lung tissue was collected 7 days after inoculation for RT-PCR assay and histopathological assay.

Protein expression and purification

To express the prefusion S ectodomain, gene-encoded residues 1-1208 of COVID-19S (GenBank: MN 908947) were synthesized with proline substitutions at residues 986 and 987, "GSAS" substitutions at the furin cleavage sites (residues 682-685) and 2 × StrepTag and cloned into the mammalian expression vector pCAGGS. To express COVID-19RBD, residues 319-591 of COVID-19S were cloned upstream of the 2 × StrepTag of the mammalian expression vector pCAGGS.

For the expression of the N protein, residues 1 to 419 (GenBank: QHW 06046.1) were cloned into the C-terminal 6 XHis-containing vector pET-28 a. HEK Expi 293F cells (Thermo Fisher) were transiently transfected with the S ectodomain 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 either Superose 6/300 column (GE Healthcare) or Superdex 200/300 addition (Increate) column (GE Healthcare) under conditions of 20mM Tris pH8.0, 200mM NaCl. The N protein was produced in BL21 (DE 3) by introducing 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, 500mM NaCl, 200mM imidazole, pH 8.0) followed by size exclusion chromatography using Superdex 200/300 Increatase column (GE Healthcare).

Phylogenetic tree analysis

The Fasta sequence of COVID-19 was retrieved from the databases of GISAID (https:// www.gisaid.org /), NCBI and BIGD (https:// bigd.big.ac.cn/ncov). After quality control (removal of low quality or short sequence length sequences), 455 sequences were retained for phylogenetic analysis. By combining 9 target sequences with sequences from public databases, we performed sequence alignments using MAFFT and phylogenetic reconstruction using IQtree with default parameters. The inferred maximum likelihood tree is plotted using a ggtree.

Cryo-EM sample preparation

For cryo-EM sample preparation, 3 μ L aliquots of purified virus particles were applied to a glow discharge C-flat R2/1Cu grid. The grids were blotted manually at 100% relative humidity for 3 seconds for flash freezing in liquid ethane (Vitrobot; FEI) as previously described. All samples were examined on a TitanKrios microscope (FEI).

Example 1 characterization and preparation of PicoVacc.

To develop an in vitro preclinical neutralization and challenge model for the candidate SARS-CoV-2 vaccine, SARS-CoV-2 strains were isolated from bronchoalveolar lavage (BALF) samples from 11 hospitalized patients (including 5 ICU patients), of which 4 in China, 4 in Italy, 1 in Switzerland, 1 in the UK, 1 in Spain (Table 1). These patients were infected with SARS-CoV-2 during the last outbreak. 11 samples containing SARS-CoV-2 strain were spread widely over phylogenetic trees constructed from all available sequences, which to some extent represented circulating populations (FIGS. 1A and 5). Strain CN2 was selected for purification of inactivated SARS-CoV-2 virus vaccine development (PiCoVacc), and the other 10 strains (designated CN1, CN3-CN5 and OS1-OS 6) were selected as preclinical challenge strains. Some of these strains, including CN1, OS1, are closely related to 2019-nCoV-BetaCoV/Wuhan/WIV04/2019 and EPI _ ISL _412973, respectively, can cause severe clinical symptoms, including respiratory failure, requiring mechanical ventilation. The entire genomic DNA sequence of the PiCoVacc strain is shown in SEQ ID NO. 1, which corresponds to the RNA sequence of the PiCoVacc strain. The 238 th to 13455 th nucleotides of SEQ ID NO. 1 encode ORF1a of the PicoVacc strain; the 13740 th to 21527 th nucleotides of SEQ ID NO. 1 encode ORF1ab of the PicoVacc strain; the 25365-26192 nucleotides of SEQ ID NO. 1 encode ORF3a of the PicoVacc strain; the 26495 th to 27163 th nucleotides of SEQ ID NO. 1 encode the membrane protein (M) of the PicoVacc strain; nucleotides 28246 to 29505 of SEQ ID NO. 1 encode the nucleocapsid protein (P) of the PicoVacc strain; nucleotides 21508 to 25356 of SEQ ID NO:1 encode the spike glycoprotein (S) of the PicoVacc strain.

TABLE 1 Virus Strain information

To obtain efficient growth for P production in Vero cellsViral stocks of iCoVacc, CN3 strain was first plaque purified and passaged once in Vero cells to produce Pl stocks. After this, four additional passages were performed to produce P2-P5 stocks. Analysis of the growth kinetics of P5 stocks in Vero cells showed that at temperatures between 33 ℃ and 37 ℃ and at multiplicity of infection (MOI) between 0.0001 and 0.01, the stocks replicated efficiently and reached 6 to 7log at 3 or 4 days post infection (dpi) ₁₀ TCID ₅₀ Peak titer in ml (FIG. 1B). To assess the genetic stability of the PicoVacc, additional 5 passages were performed to obtain P10 stock, and the whole genome was sequenced along with the whole genome of P1, P3 and P5 stocks. Compared to P1, only two amino acid substitutions occurred in P5 and P10 stock solutions, ala → Asp (E-a 32D) at residue 32E and Thr → Ile (nsp 10-T49I) at residue 49 nsp10 (table 2), indicating that the PiCoVacc CN2 strain has excellent genetic stability without any S mutations that could alter the NAb epitope. To produce PicoVacc, the virus was propagated in Vero cell culture using a bioreactor and inactivated with β -propiolactone. The virus was purified using centrifugation and two optimized chromatography steps, resulting in a highly pure PiCoVacc preparation (fig. 1C). Thus, to produce a vaccine, the virus was propagated in Vero cell culture using a bioreactor and inactivated using β -propiolactone, followed by purification of the virus using centrifugation and two optimized chromatographic steps. In addition, cryo-electron microscope (cryo-EM) analysis showed intact ellipsoidal particles of 90-150 nm diameter interspersed with coronary spikes, representing the pre-fusion state of the virus (FIG. 1D).

Table 2 genetic stability analysis of PiCoV2. Initially determined PicoV ₂ The sequence of the P1 stock was used as a reference sequence.

Example 2 PicoVacc immunization elicited a neutralizing antibody response against 10 representative SARS-CoV-2 isolates.

To assess the immunogenicity of the PiCoVacc, groups of BALB/c mice (n = 10) were injected with multiple doses of PiCoVacc (0 μ g, 1.5 μ g or 3 μ g or 6 μ g per dose, 0 μ g in physiological saline as a sham model) mixed with aluminum adjuvant on days 0 and 7. No inflammation or other adverse effects were observed. Spike-, receptor-binding domain (RBD) -and N-specific antibody responses were assessed by enzyme-linked immunosorbent assay (ELISA) 1-6 weeks after the initial immunization (fig. 6). SARS-CoV-2S and RBD-specific immunoglobulin G (IgG) appeared rapidly in the sera of vaccinated mice and peaked at week 6 with titers of 819,200 (> 200. Mu.g/ml) and 409,600 (> 100. Mu.g/ml), respectively (FIG. 2A). RBD-specific IgG accounts for half of the S-induced antibody response, indicating that RBD is the major immunogen, matching closely the serological characteristics of the blood of convalescent COVID-19 patients (fig. 2A and 2B) (11). Surprisingly, the amount of induced N-specific IgG is about 30-fold lower in immunized mice than antibodies targeting S or RBD. Interestingly, previous studies have shown that N-specific IgG is present in high levels in the serum of patients with COVID-19 and can be used as one of the clinical diagnostic markers (11). Notably, the PiCoVacc elicited much higher S-specific antibody titers than the sera 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 is able to elicit a more efficient antibody response (fig. 2A and 2B). Next, we measured SARS-CoV-2 specific neutralizing antibodies over time using a micro-neutralization assay (MN 50). Similar to the S-specific IgG response, neutralizing antibody titers against the CN1 strain appeared at week 1 (12 for high dose immunization), proliferated after booster at week 2, and reached approximately 1,500 for low and medium dose and 3,000 for high dose at week 7, respectively (fig. 2A). In contrast, the mock-up did not produce a detectable SARS-CoV-2 specific antibody response (FIGS. 2A and 2B). Furthermore, immunogenicity assessment of PicoVacc in Wistar rats using the same immunization strategy yielded similar results, with maximal neutralization titers reaching 2,048-4,096 at week 7 (FIG. 2C). To study the PicoVacc-induced spectrum of neutralizing activity, we performed neutralization assays on 9 other isolated SARS-CoV-2 strains using mouse and rat sera collected 3 weeks after vaccination. Neutralization titers against these strains indicate that PicoVacc is able to elicit antibodies that may exhibit potent neutralizing activity against the globally circulating SARS-CoV-2 strain (FIGS. 2D and 2E).

Example 3 immunogenicity and protective efficacy of PicoVacc in non-human primates.

The immunogenicity and protective efficacy of PicoVacc was evaluated in rhesus monkeys (Macacamulatta), a non-human primate exhibiting a COVID-19-like disease caused by SARS-CoV-2 infection. Macaques were immunized 3 times intramuscularly (n = 4) on days 0, 7 and 14, using either a medium dose (3 μ g per dose) or a high dose (6 μ g per dose) of PiCoVacc. The S-specific IgG and Nab (neutralizing antibodies) of both vaccinated groups decreased at week 2 and increased to about 12,800 and about 50, respectively, at week 3 (before virus challenge), with titers similar to those of convalescent COVID-19 patient sera (fig. 3A and 3B). Unexpectedly, at week 3 NAb titers in the medium dose immunization group (61) were about 20% higher than those 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 titers that were about 10-fold higher than the other three (fig. 3B). Excluding this exception, NAb titers in the medium dose group were reduced to 34, which was about 40% lower than the high dose group. Subsequently, we performed a challenge study in which 10 of SARS-CoV-2CN1 was administered by the intratracheal route on day 22 (one day after the third immunization) in vaccinated and control macaques ⁶ TCID ₅₀ The vaccine is directly inoculated into the lung of an animal to verify the protective effect. As expected, all control (sham and placebo) macaques developed an excessive number of copies of viral genomic RNA in the pharynx, perianal and lungs on days 3-7 post-inoculation (dpi) (10) ⁴ ～10 ⁶ /ml) and severe interstitial pneumonia (fig. 3C-3F). In contrast, all vaccinated macaques largely avoided SARS-CoV-2 infection, with very little change in lung histopathology, probably 10 by intratracheal route ⁶ TCID ₅₀ Direct inoculation of the virus into the lungs caused it took longer (more than one week) to recover completely (fig. 3F). Viral load was significantly reduced in all vaccinated macaques, but slightly increased in control animals at days 3-7 post infection (fig. 3C-3E). Receiving high dosesAll four macaques did not detect viral load in pharynx, perianal and lungs on day 7 post-infection. In the medium dose immunization group, we did partially detect viral plaques from pharyngeal (3/4), anal (2/4) and pulmonary (1/4) specimens on day 7 post-infection, while viral load was reduced by about 95% compared to the sham group (fig. 3C-3E). Interestingly, NAb titers in vaccinated groups dropped by about 30% 3 days post-infection to neutralize the virus, and then increased rapidly from day 5 to 7 post-infection to maintain its potent neutralizing efficacy. Higher NAb titers were observed in the medium dose vaccinated group at day 7 post-infection (4 macaques about 400; if one outlier was discarded, 3 macaques about 300) compared to the high dose vaccinated group (titers about 145), which may be due to lower levels of viral replication, indicating that a longer time was required to completely clear the virus. Although relatively low NAb titers were observed in the pre-infection moderate dose group, providing partial protection, no antibody-dependent infection enhancement (ADE) was observed for any vaccinated macaques.

Example 4 safety assessment of PicoVacc in non-human primates.

Previous experience in developing SARS and MERS vaccine candidates has led to concern over lung immunopathology that may be directly caused by a type 2 helper T cell (Th 2) response or as a result of (ADE). Although many vaccine-induced T cell responses have been shown to be critical for acute viral clearance, protection against subsequent coronavirus infection is primarily mediated by humoral immunity. The "cytokine storm" caused by excessive T cell responses has actually been shown to emphasize the pathogenesis of COVID 19. Therefore, the T cell response elicited by the SARS-CoV-2 vaccine must be well controlled to avoid immunopathology. In this context, we systematically evaluated the safety of PiCoVacc in macaques by recording several clinical observations and biological indicators. Two groups of macaques (n = 10) were immunized intramuscularly with either a low dose (1.5 μ g) or a high dose (6 μ g), and two other groups of macaques (n = 10) were immunized 3 times on days 0, 7, and 14 with adjuvant (sham) and saline (placebo). After immunization of PicoVacc, all macaques showed no fever and weight loss, and all animals were normal for appetite and mental status (FIG. 7). Hematologic and biochemical analyses, including biochemical blood tests, lymphocyte subpopulation percentages (CD 3+, CD4+ and CD8 +) and key cytokines (TNF-. Alpha., IFN-. Gamma., IL-2, IL-4, IL-5 and IL-6) showed no significant change in the vaccinated groups compared to sham (sham) and placebo (FIGS. 4A-4B and 8-9). Furthermore, histopathological evaluation of various organs (including lung, heart, spleen, liver, kidney and brain) of the 4 groups at day 29 showed that PiCoVacc did not cause any significant pathology in macaques (fig. 4C and fig. 10).

The current severe prevalence of COVID19 and the dramatic increase in worldwide deaths requires the urgent development of SARS-CoV-2 vaccines, which require a new epidemic paradigm. The safety and efficacy of vaccines are essential in both preclinical studies and clinical trials. Rhesus monkeys that mimic COVID-19-like symptoms following SARS-CoV-2 infection appear to be very promising as animal models for studying the disease. We provide extensive evidence for the safety of PiCoVacc in macaques; in our study, neither an enhancement of infection nor a worsening of immunopathology was observed. Our data also show that macaques at 6. Mu.g/dose of PiCoVacc have a complete protective effect against SARS-CoV-2 challenge.

SEQUENCE LISTING

<110> Beijing Koxing vitamin technology Co., ltd

<120> SARS-CoV-2 inactivated vaccine and preparation thereof

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<170> PatentIn version 3.5

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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 cagtatctga attacttaca ccactgggca ttgatttaga tgagtggagt 2940

atggctacat actacttatt tgatgagtct ggtgagttta aattggcttc acatatgtat 3000

tgttctttct accctccaga tgaggatgaa gaagaaggtg attgtgaaga agaagagttt 3060

gagccatcaa ctcaatatga gtatggtact gaagatgatt accaaggtaa acctttggaa 3120

tttggtgcca cttctgctgc tcttcaacct gaagaagagc aagaagaaga ttggttagat 3180

gatgatagtc aacaaactgt tggtcaacaa gacggcagtg aggacaatca gacaactact 3240

attcaaacaa ttgttgaggt tcaacctcaa ttagagatgg aacttacacc agttgttcag 3300

actattgaag tgaatagttt tagtggttat ttaaaactta ctgacaatgt atacattaaa 3360

aatgcagaca ttgtggaaga agctaaaaag gtaaaaccaa cagtggttgt taatgcagcc 3420

aatgtttacc ttaaacatgg aggaggtgtt gcaggagcct taaataaggc tactaacaat 3480

gccatgcaag ttgaatctga tgattacata gctactaatg gaccacttaa agtgggtggt 3540

agttgtgttt taagcggaca caatcttgct aaacactgtc ttcatgttgt cggcccaaat 3600

gttaacaaag gtgaagacat tcaacttctt aagagtgctt atgaaaattt taatcagcac 3660

gaagttctac ttgcaccatt attatcagct ggtatttttg gtgctgaccc tatacattct 3720

ttaagagttt gtgtagatac tgttcgcaca aatgtctact tagctgtctt tgataaaaat 3780

ctctatgaca aacttgtttc aagctttttg gaaatgaaga gtgaaaagca agttgaacaa 3840

aagatcgctg agattcctaa agaggaagtt aagccattta taactgaaag taaaccttca 3900

gttgaacaga gaaaacaaga tgataagaaa atcaaagctt gtgttgaaga agttacaaca 3960

actctggaag aaactaagtt cctcacagaa aacttgttac tttatattga cattaatggc 4020

aatcttcatc cagattctgc cactcttgtt agtgacattg acatcacttt cttaaagaaa 4080

gatgctccat atatagtggg tgatgttgtt caagagggtg ttttaactgc tgtggttata 4140

cctactaaaa aggctggtgg cactactgaa atgctagcga aagctttgag aaaagtgcca 4200

acagacaatt atataaccac ttacccgggt cagggtttaa atggttacac tgtagaggag 4260

gcaaagacag tgcttaaaaa gtgtaaaagt gccttttaca ttctaccatc tattatctct 4320

aatgagaagc aagaaattct tggaactgtt tcttggaatt tgcgagaaat gcttgcacat 4380

gcagaagaaa cacgcaaatt aatgcctgtc tgtgtggaaa ctaaagccat agtttcaact 4440

atacagcgta aatataaggg tattaaaata caagagggtg 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 tttgagtact 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

cttaagggtg 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 atttttccct 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 attttgtcta 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 aattaatctt 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 atttacttac taatatgttt 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 taaataaaga 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 agtttgttcg 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

atacctctta 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 atgtcttgtg ctgccggtac tacacaaact 12720

gcttgcactg atgacaatgc gttagcttac tacaacacaa caaagggagg taggtttgta 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

agtttcatca aaaattattg aaatcaatag ccgccactag aggagctact gtagtaattg 15180

gaacaagcaa attctatggt ggttggcaca acatgttaaa aactgtttat 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 aaatagagat 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

atgaattttg 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 aagtttttgg 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

acacctttga 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 tatgtctttt 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 attttgggac taccaactca aactgttgat tcatcacagg 17820

gctcagaata tgactatgtc atattcactc aaaccactga aacagctcac tcttgtaatg 17880

taaacagatt taatgttgct attaccagag caaaagtagg catactttgc ataatgtctg 17940

atagagacct ttatgacaag 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

attttgtgaa 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 gcaatgtcga tagatatcct gctaattcca 19200

ttgtttgtag atttgacact agagtgctat ctaaccttaa cttgcctggt tgtgatggtg 19260

gcagtttgta tgtaaataaa catgcattcc acacaccagc ttttgataaa agtgcttttg 19320

ttaatttaaa acaattacca tttttctatt actctgacag tccatgtgag tctcatggaa 19380

aacaagtagt gtcagatata gattatgtac cactaaagtc tgctacgtgt ataacacgtt 19440

gcaatttagg tggtgctgtc tgtagacatc atgctaatga gtacagattg tatctcgatg 19500

cttataacat gatgatctca gctggcttta gcttgtgggt ttacaaacaa tttgatactt 19560

ataacctctg gaacactttt acaagacttc agagtttaga aaatgtggct tttaatgttg 19620

taaataaggg acactttgat ggacaacagg gtgaagtacc agtttctatc attaataaca 19680

ctgtttacac aaaagttgat ggtgttgatg tagaattgtt tgaaaataaa acaacattac 19740

ctgttaatgt agcatttgag ctttgggcta agcgcaacat taaaccagta ccagaggtga 19800

aaatactcaa taatttgggt gtggacattg ctgctaatac tgtgatctgg gactacaaaa 19860

gagatgctcc agcacatata tctactattg gtgtttgttc tatgactgac atagccaaga 19920

aaccaactga aacgatttgt gcaccactca ctgtcttttt tgatggtaga gttgatggtc 19980

aagtagactt atttagaaat gcccgtaatg gtgttcttat tacagaaggt agtgttaaag 20040

gtttacaacc atctgtaggt cccaaacaag ctagtcttaa tggagtcaca ttaattggag 20100

aagccgtaaa aacacagttc aattattata agaaagttga tggtgttgtc caacaattac 20160

ctgaaactta ctttactcag agtagaaatt tacaagaatt taaacccagg agtcaaatgg 20220

aaattgattt cttagaatta gctatggatg aattcattga acggtataaa ttagaaggct 20280

atgccttcga acatatcgtt tatggagatt ttagtcatag tcagttaggt ggtttacatc 20340

tactgattgg actagctaaa cgttttaagg aatcaccttt tgaattagaa gattttattc 20400

ctatggacag tacagttaaa aactatttca taacagatgc gcaaacaggt tcatctaagt 20460

gtgtgtgttc tgttattgat ttattacttg atgattttgt tgaaataata aaatcccaag 20520

atttatctgt agtttctaag gttgtcaaag tgactattga ctatacagaa atttcattta 20580

tgctttggtg taaagatggc 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 tggaggaata caaatccaat tcagttgtct tcctattctt 21360

tatttgacat gagtaaattt ccccttaaat taaggggtac tgctgttatg tctttaaaag 21420

aaggtcaaat caatgatatg attttatctc ttcttagtaa aggtagactt ataattagag 21480

aaaacaacag agttgttatt tctagtgatg ttcttgttaa caactaaacg aacaatgttt 21540

gtttttcttg ttttattgcc actagtctct agtcagtgtg ttaatcttac aaccagaact 21600

caattacccc ctgcatacac taattctttc acacgtggtg tttattaccc tgacaaagtt 21660

ttcagatcct cagttttaca ttcaactcag gacttgttct tacctttctt ttccaatgtt 21720

acttggttcc atgctataca tgtctctggg accaatggta ctaagaggtt tgataaccct 21780

gtcctaccat ttaatgatgg tgtttatttt gcttccactg agaagtctaa cataataaga 21840

ggctggattt ttggtactac tttagattcg aagacccagt ccctacttat tgttaataac 21900

gctactaatg ttgttattaa agtctgtgaa tttcaatttt gtaatgatcc atttttgggt 21960

gtttattacc acaaaaacaa caaaagttgg atggaaagtg agttcagagt ttattctagt 22020

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 ctttcttttg 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 gtgttataac 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 agaagttttt 23880

gcacaagtca aacaaattta caaaacacca ccaattaaag attttggtgg ttttaatttt 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 ctactaaaat gtcagagtgt gtacttggac aatcaaaaag agttgatttt 24660

tgtggaaagg gctatcatct tatgtccttc cctcagtcag cacctcatgg tgtagtcttc 24720

ttgcatgtga cttatgtccc tgcacaagaa aagaacttca caactgctcc tgccatttgt 24780

catgatggaa aagcacactt tcctcgtgaa ggtgtctttg tttcaaatgg cacacactgg 24840

tttgtaacac aaaggaattt ttatgaacca caaatcatta ctacagacaa cacatttgtg 24900

tctggtaact gtgatgttgt aataggaatt gtcaacaaca cagtttatga tcctttgcaa 24960

cctgaattag actcattcaa ggaggagtta gataaatatt ttaagaatca tacatcacca 25020

gatgttgatt taggtgacat ctctggcatt aatgcttcag ttgtaaacat tcaaaaagaa 25080

attgaccgcc tcaatgaggt tgccaagaat ttaaatgaat ctctcatcga tctccaagaa 25140

cttggaaagt atgagcagta tataaaatgg ccatggtaca tttggctagg ttttatagct 25200

ggcttgattg ccatagtaat ggtgacaatt atgctttgct gtatgaccag ttgctgtagt 25260

tgtctcaagg gctgttgttc ttgtggatcc tgctgcaaat ttgatgaaga cgactctgag 25320

ccagtgctca aaggagtcaa attacattac acataaacga acttatggat ttgtttatga 25380

gaatcttcac aattggaact gtaactttga agcaaggtga aatcaaggat 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 tttgctgcat acagtcgcta caggattggc aactataaat taaacacaga 27120

ccattccagt agcagtgaca atattgcttt gcttgtacag taagtgacaa cagatgtttc 27180

atctcgttga ctttcaggtt actatagcag agatattact aattattatg aggactttta 27240

aagtttccat ttggaatctt gattacatca taaacctcat aattaaaaat 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 gaagactttt tagagtatca 28200

tgacgttcgt gttgttttag atttcatcta aacgaacaaa ctaaaatgtc tgataatgga 28260

ccccaaaatc agcgaaatgc accccgcatt acgtttggtg gaccctcaga ttcaactggc 28320

agtaaccaga atggagaacg cagtggggcg cgatcaaaac aacgtcggcc ccaaggttta 28380

cccaataata ctgcgtcttg gttcaccgct ctcactcaac atggcaagga agaccttaaa 28440

ttccctcgag gacaaggcgt tccaattaac accaatagca gtccagatga ccaaattggc 28500

tactaccgaa gagctaccag acgaattcgt ggtggtgacg gtaaaatgaa agatctcagt 28560

ccaagatggt atttctacta cctaggaact gggccagaag ctggacttcc ctatggtgct 28620

aacaaagacg gcatcatatg ggttgcaact gagggagcct tgaatacacc aaaagatcac 28680

attggcaccc gcaatcctgc taacaatgct gcaatcgtgc tacaacttcc tcaaggaaca 28740

acattgccaa aaggcttcta cgcagaaggg agcagaggcg gcagtcaagc ctcttctcgt 28800

tcctcatcac gtagtcgcaa cagttcaaga aattcaactc caggcagcag taggggaact 28860

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 gcttttccgt 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 ttatgaaaaa ctcaaacccg tccttgattg 2100

gcttgaagag aagtttaagg aaggtgtaga gtttcttaga gacggttggg aaattgttaa 2160

atttatctca 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 aacaacctac 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 tatcactttt 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 4860

tatcaccttt gacaatctta agacacttct ttctttgaga gaagtgagga ctattaaggt 4920

gtttacaaca gtagacaaca ttaacctcca cacgcaagtt gtggacatgt caatgacata 4980

tggacaacag tttggtccaa cttatttgga tggagctgat gttactaaaa taaaacctca 5040

taattcacat gaaggtaaaa cattttatgt tttacctaat gatgacactc tacgtgttga 5100

ggcttttgag tactaccaca caactgatcc tagttttctg ggtaggtaca tgtcagcatt 5160

aaatcacact aaaaagtgga aatacccaca agttaatggt ttaacttcta ttaaatgggc 5220

agataacaac tgttatcttg ccactgcatt gttaacactc caacaaatag agttgaagtt 5280

taatccacct gctctacaag atgcttatta cagagcaagg gctggtgaag ctgctaactt 5340

ttgtgcactt atcttagcct actgtaataa gacagtaggt gagttaggtg atgttagaga 5400

aacaatgagt tacttgtttc aacatgccaa tttagattct tgcaaaagag tcttgaacgt 5460

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 acaaagtcct cagaatacaa aggtcctatt acggatgttt tctacaaaga 5820

aaacagttac acaacaacca taaaaccagt tacttataaa ttggatggtg ttgtttgtac 5880

agaaattgac cctaagttgg acaattatta taagaaagac aattcttatt tcacagagca 5940

accaattgat cttgtaccaa accaaccata tccaaacgca agcttcgata attttaagtt 6000

tgtatgtgat aatatcaaat ttgctgatga tttaaaccag ttaactggtt ataagaaacc 6060

tgcttcaaga gagcttaaag ttacattttt ccctgactta aatggtgatg tggtggctat 6120

tgattataaa cactacacac cctcttttaa gaaaggagct aaattgttac ataaacctat 6180

tgtttggcat gttaacaatg caactaataa agccacgtat aaaccaaata cctggtgtat 6240

acgttgtctt tggagcacaa aaccggttga aacatcaaat tcgtttgatg tactgaagtc 6300

agaggatgcg cagggaatgg ataatcttgc ctgcgaagat ctaaaaccag tctctgaaga 6360

agtagtggaa aatcctacca tacagaaaga cgttcttgag tgtaatgtga aaactaccga 6420

agttgtagga gacattatac ttaaaccagc aaataatagt ttaaaaatta cagaagaggt 6480

tggccacaca gatctaatgg ctgcttatgt agacaattct agtcttacta ttaagaaacc 6540

taatgaatta tctagagtgt taggtttgaa aacccttgct actcatggtt tagctgctgt 6600

taatagtgtc ccttgggata ctatagctaa ttatgctaag ccttttctta acaaagttgt 6660

tagtacaact actaacatag ttacacggtg tttaaaccgt gtttgtacta attatatgcc 6720

ttatttcttt actttattgc tacaattgtg tacttttact agaagtacaa attctagaat 6780

taaagcatct atgccgacta ctatagcaaa gaatactgtt aagagtgtcg gtaaattttg 6840

tctagaggct tcatttaatt atttgaagtc acctaatttt tctaaactga taaatattat 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 taagttgaca 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 ctttttgcat ttcttaccta gagtttttag 8880

tgcagttggt aacatctgtt acacaccatc aaaacttata gagtacactg actttgcaac 8940

atcagcttgt gttttggctg ctgaatgtac aatttttaaa gatgcttctg gtaagccagt 9000

accatattgt tatgatacca atgtactaga aggttctgtt gcttatgaaa gtttacgccc 9060

tgacacacgt tatgtgctca tggatggctc tattattcaa tttcctaaca cctaccttga 9120

aggttctgtt agagtggtaa caacttttga ttctgagtac tgtaggcacg gcacttgtga 9180

aagatcagaa gctggtgttt gtgtatctac tagtggtaga tgggtactta acaatgatta 9240

ttacagatct ttaccaggag ttttctgtgg tgtagatgct gtaaatttac ttactaatat 9300

gtttacacca ctaattcaac ctattggtgc tttggacata tcagcatcta tagtagctgg 9360

tggtattgta gctatcgtag taacatgcct tgcctactat tttatgaggt ttagaagagc 9420

ttttggtgaa tacagtcatg tagttgcctt taatacttta ctattcctta tgtcattcac 9480

tgtactctgt ttaacaccag tttactcatt cttacctggt gtttattctg ttatttactt 9540

gtacttgaca ttttatctta ctaatgatgt ttctttttta gcacatattc agtggatggt 9600

tatgttcaca cctttagtac ctttctggat aacaattgct tatatcattt gtatttccac 9660

aaagcatttc tattggttct ttagtaatta cctaaagaga cgtgtagtct ttaatggtgt 9720

ttcctttagt acttttgaag aagctgcgct gtgcaccttt ttgttaaata aagaaatgta 9780

tctaaagttg cgtagtgatg tgctattacc tcttacgcaa tataatagat acttagctct 9840

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 gtggtttctc 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

ctttttacct tttgctatgg gtattattgc tatgtctgct tttgcaatga tgtttgtcaa 11100

acataagcat gcatttctct gtttgttttt gttaccttct cttgccactg tagcttattt 11160

taatatggtc 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 tgatgtcgta 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 tcacggaaga gaggttaaaa 14220

ctctttgacc gttattttaa atattgggat cagacatacc acccaaattg tgttaactgt 14280

ttggatgaca gatgcattct gcattgtgca aactttaatg ttttattctc tacagtgttc 14340

ccacttacaa gttttggacc actagtgaga aaaatatttg ttgatggtgt tccatttgta 14400

gtttcaactg gataccactt cagagagcta ggtgttgtac ataatcagga tgtaaactta 14460

catagctcta gacttagttt taaggaatta 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 ttactaaagg acctcatgaa ttttgctctc aacatacaat gctagttaaa 15840

cagggtgatg attatgtgta ccttccttac ccagatccat caagaatcct aggggccggc 15900

tgttttgtag atgatatcgt aaaaacagat ggtacactta tgattgaacg gttcgtgtct 15960

ttagctatag atgcttaccc acttactaaa 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

gtttaccgag 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 ccttctgctc 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 taggcgtggt 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 tgtttatcac ccgcgaagaa gctataagac atgtacgtgc atggattggc 18240

ttcgatgtcg aggggtgtca tgctactaga gaagctgttg gtaccaattt acctttacag 18300

ctaggttttt ctacaggtgt taacctagtt gctgtaccta caggttatgt tgatacacct 18360

aataatacag atttttccag agttagtgct aaaccaccgc ctggagatca atttaaacac 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 aaaagctagc 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

tggtgtttat taccctgaca aagttttcag atcctcagtt ttacattcaa ctcaggactt 21660

gttcttacct ttcttttcca atgttacttg gttccatgct atacatgtct ctgggaccaa 21720

tggtactaag aggtttgata accctgtcct accatttaat gatggtgttt attttgcttc 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

ctttgtttca aatggcacac actggtttgt 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 25080

tgaatctctc atcgatctcc aagaacttgg aaagtatgag cagtatataa aatggccatg 25140

gtacatttgg ctaggtttta tagctggctt gattgccata gtaatggtga caattatgct 25200

ttgctgtatg accagttgct gtagttgtct caagggctgt tgttcttgtg gatcctgctg 25260

caaatttgat gaagacgact ctgagccagt gctcaaagga gtcaaattac attacacata 25320

aacgaactta tggatttgtt tatgagaatc ttcacaattg gaactgtaac tttgaagcaa 25380

ggtgaaatca aggatgctac tccttcagat tttgttcgcg ctactgcaac gataccgata 25440

caagcctcac tccctttcgg atggcttatt gttggcgttg cacttcttgc tgtttttcag 25500

agcgcttcca aaatcataac cctcaaaaag agatggcaac tagcactctc caagggtgtt 25560

cactttgttt gcaacttgct gttgttgttt gtaacagttt actcacacct tttgctcgtt 25620

gctgctggcc ttgaagcccc ttttctctat ctttatgctt tagtctactt cttgcagagt 25680

ataaactttg taagaataat aatgaggctt tggctttgct ggaaatgccg ttccaaaaac 25740

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 taaaattgtt 26040

gatgagcctg aagaacatgt ccaaattcac acaatcgacg gttcacccgg agttgttaat 26100

ccagtaatgg aaccaattta tgatgaaccg acgacgacta ctagcgtgcc tttgtaagca 26160

caagctgatg agtacgaact tatgtactca ttcgtttcgg aagagacagg tacgttaata 26220

gttaatagcg tacttctttt tcttgctttc gtggtattct tgctagttac actagccatc 26280

cttactgcgc ttcgattgtg tgcgtactgc tgcaatattg ttaacgtgag tcttgtaaaa 26340

ccttcttttt acgtttactc tcgtgttaaa aatctgaatt cttctagagt tcctgatctt 26400

ctggtctaaa cgaactaaat attatattag tttttctgtt tggaacttta attttagcca 26460

tggcagattc caacggtact attaccgttg aagagcttaa aaagctcctt gaacaatgga 26520

acctagtaat aggtttccta ttccttacat ggatttgtct tctacaattt gcctatgcca 26580

acaggaatag gtttttgtat ataattaagt taattttcct ctggctgtta tggccagtaa 26640

ctttagcttg ttttgtgctt gctgctgttt acagaataaa ttggatcacc ggtggaattg 26700

ctatcgcaat ggcttgtctt gtaggcttga tgtggctcag ctacttcatt gcttctttca 26760

gactgtttgc gcgtacgcgt tccatgtggt cattcaatcc agaaactaac attcttctca 26820

acgtgccact ccatggcact attctgacca gaccgcttct agaaagtgaa ctcgtaatcg 26880

gagctgtgat ccttcgtgga catcttcgta ttgctggaca ccatctagga cgctgtgaca 26940

tcaaggacct gcctaaagaa atcactgttg ctacatcacg aacgctttct tattacaaat 27000

tgggagcttc gcagcgtgta gcaggtgact caggttttgc tgcatacagt cgctacagga 27060

ttggcaacta taaattaaac acagaccatt ccagtagcag tgacaatatt gctttgcttg 27120

tacagtaagt gacaacagat gtttcatctc gttgactttc aggttactat agcagagata 27180

ttactaatta ttatgaggac ttttaaagtt tccatttgga atcttgatta catcataaac 27240

ctcataatta aaaatttatc taagtcacta actgagaata aatattctca attagatgaa 27300

gagcaaccaa tggagattga ttaaacgaac atgaaaatta ttcttttctt ggcactgata 27360

acactcgcta cttgtgagct ttatcactac caagagtgtg ttagaggtac aacagtactt 27420

ttaaaagaac cttgctcttc tggaacatac gagggcaatt caccatttca tcctctagct 27480

gataacaaat ttgcactgac ttgctttagc actcaatttg cttttgcttg tcctgacggc 27540

gtaaaacacg tctatcagtt acgtgccaga tcagtttcac ctaaactgtt catcagacaa 27600

gaggaagttc aagaacttta ctctccaatt tttcttattg ttgcggcaat agtgtttata 27660

acactttgct tcacactcaa aagaaagaca gaatgattga actttcatta attgacttct 27720

atttgtgctt tttagccttt ctgctattcc ttgttttaat tatgcttatt atcttttggt 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 ctttttagag 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

gtggtgacgg taaaatgaaa gatctcagtc caagatggta tttctactac ctaggaactg 28560

ggccagaagc tggacttccc tatggtgcta acaaagacgg catcatatgg gttgcaactg 28620

agggagcctt gaatacacca aaagatcaca ttggcacccg caatcctgct aacaatgctg 28680

caatcgtgct acaacttcct caaggaacaa cattgccaaa aggcttctac gcagaaggga 28740

gcagaggcgg cagtcaagcc tcttctcgtt 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 actgccacta aagcatacaa tgtaacacaa gctttcggca 29040

gacgtggtcc agaacaaacc caaggaaatt ttggggacca ggaactaatc agacaaggaa 29100

ctgattacaa acattggccg caaattgcac aatttgcccc cagcgcttca gcgttcttcg 29160

gaatgtcgcg cattggcatg gaagtcacac cttcgggaac gtggttgacc tacacaggtg 29220

ccatcaaatt ggatgacaaa gatccaaatt tcaaagatca agtcattttg ctgaataagc 29280

atattgacgc atacaaaaca ttcccaccaa cagagcctaa aaaggacaaa aagaagaagg 29340

ctgatgaaac tcaagcctta ccgcagagac agaagaaaca gcaaactgtg actcttcttc 29400

ctgctgcaga tttggatgat ttctccaaac aattgcaaca atccatgagc agtgctgact 29460

caactcaggc ctaaactcat gcagaccaca caaggcagat gggctatata aacgttttcg 29520

cttttccgtt tacgatatat agtctactct tgtgcagaat gaattctcgt aactacatag 29580

cacaagtaga tgtagttaac tttaat 29606

<210> 3

<211> 29885

<212> DNA

<213> coronavirus

<220>

<221> misc_feature

<222> (1)..(26)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (6882)..(7027)

<223> n is a, c, g, or t

<220>

<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

nnnnnnnnnn nnnnnnnnnn nnnnnnacaa accaaccaac tttcgatctc ttgtagatct 60

gttctctaaa cgaactttaa aatctgtgtg gctgtcactc ggctgcatgc ttagtgcact 120

cacgcagtat aattaataac 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 cagtacggtc gtagtggtga gacacttggt gtccttgtcc ctcatgtggg 600

cgaaatacca gtggcttacc gcaaggttct tcttcgtaag aacggtaata aaggagctgg 660

tggccatagt tacggcgccg atctaaagtc atttgactta ggcgacgagc ttggcactga 720

tccttatgaa gattttcaag aaaactggaa cactaaacat agcagtggtg ttacccgtga 780

actcatgcgt gagcttaacg gaggggcata 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 acagaaggcc gctataacaa tactagatgg 1980

aatttcacag tattcactga gactcattga tgctatgatg ttcacatctg atttggctac 2040

taacaatcta gttgtaatgg cctacattac aggtggtgtt gttcagttga cttcgcagtg 2100

gctaactaac atctttggca ctgtttatga aaaactcaaa cccgtccttg attggcttga 2160

agagaagttt aaggaaggtg tagagtttct tagagacggt tgggaaattg ttaaatttat 2220

ctcaacctgt gcttgtgaaa ttgtcggtgg acaaattgtc acctgtgcaa aggaaattaa 2280

ggagagtgtt cagacattct ttaagcttgt aaataaattt ttggctttgt gtgctgactc 2340

tatcattatt ggtggagcta aacttaaagc cttgaattta ggtgaaacat ttgtcacgca 2400

ctcaaaggga ttgtacagaa agtgtgttaa atccagagaa gaaactggcc tactcatgcc 2460

tctaaaagcc ccaaaagaaa ttatcttctt agagggagaa acacttccca cagaagtgtt 2520

aacagaggaa gttgtcttga aaactggtga tttacaacca ttagaacaac ctactagtga 2580

agctgttgaa gctccattgg ttggtacacc agtttgtatt aacgggctta tgttgctcga 2640

aatcaaagac acagaaaagt actgtgccct tgcacctaat atgatggtaa ctaacaatac 2700

cttcacactc aaaggcggtg caccaacaaa ggttactttt ggtgatgaca ctgtgataga 2760

agtgcaaggt tacaagagtg tgaatatcac ttttgaactt gatgaaagga ttgataaagt 2820

acttaatgag aagtgctctg cctatacagt tgaactcggt acagaagtaa atgagttcgc 2880

ctgtgttgtg gcagatgctg tcataaaaac tttgcaacca gtatctgaat tacttacacc 2940

actgggcatt gatttagatg agtggagtat ggctacatac tacttatttg atgagtctgg 3000

tgagtttaaa ttggcttcac atatgtattg ttctttttac cctccagatg aggatgaaga 3060

agaaggtgat tgtgaagaag aagagtttga gccatcaact caatatgagt atggtactga 3120

agatgattac caaggtaaac ctttggaatt tggtgccact tctgctgctc ttcaacctga 3180

agaagagcaa gaagaagatt ggttagatga tgatagtcaa caaactgttg gtcaacaaga 3240

cggcagtgag gacaatcaga caactactat tcaaacaatt gttgaggttc aacctcaatt 3300

agagatggaa cttacaccag ttgttcagac tattgaagtg aatagtttta 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 gtagatactg 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 tctggaagaa 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 5580

cacactttct tatgaacaat ttaagaaagg tgttcagata ccttgtacgt gtggtaaaca 5640

agctacaaaa tatctagtac aacaggagtc accttttgtt atgatgtcag caccacctgc 5700

tcagtatgaa cttaagcatg gtacatttac ttgtgctagt gagtacactg gtaattacca 5760

gtgtggtcac tataaacata taacttctaa agaaactttg tattgcatag acggtgcttt 5820

acttacaaag tcctcagaat acaaaggtcc tattacggat gttttctaca aagaaaacag 5880

ttacacaaca accataaaac cagttactta taaattggat ggtgttgttt gtacagaaat 5940

tgaccctaag ttggacaatt attataagaa agacaattct tatttcacag agcaaccaat 6000

tgatcttgta ccaaaccaac catatccaaa cgcaagcttc gataatttta agtttgtatg 6060

tgataatatc aaatttgctg atgatttaaa ccagttaact ggttataaga aacctgcttc 6120

aagagagctt aaagttacat ttttccctga cttaaatggt gatgtggtgg ctattgatta 6180

taaacactac acaccctctt ttaagaaagg agctaaattg ttacataaac ctattgtttg 6240

gcatgttaac aatgcaacta ataaagccac gtataaacca aatacctggt gtatacgttg 6300

tctttggagc acaaaaccag ttgaaacatc aaattcgttt gatgtactga agtcagagga 6360

cgcgcaggga atggataatc ttgcctgcga agatctaaaa ccagtctctg aagaagtagt 6420

ggaaaatcct accatacaga aagacgttct tgagtgtaat gtgaaaacta ccgaagttgt 6480

aggagacatt atacttaaac cagcaaataa tagtttaaaa attacagaag aggttggcca 6540

cacagatcta atggctgctt atgtagacaa ttctagtctt actattaaga aacctaatga 6600

attatctaga gtattaggtt tgaaaaccct tgctactcat ggtttagctg ctgttaatag 6660

tgtcccttgg gatactatag ctaattatgc taagcctttt cttaacaaag ttgttagtac 6720

aactactaac atagttacac ggtgtttaaa ccgtgtttgt actaattata tgccttattt 6780

ctttacttta ttgctacaat tgtgtacttt tactagaagt acaaattcta gaattaaagc 6840

atctatgccg actactatag caaagaatac tgttaagagt gnnnnnnnnn nnnnnnnnnn 6900

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 6960

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 7020

nnnnnnntct aatttaggca tgccttctta ctgtactggt tacagagaag gctatttgaa 7080

ctctactaat gtcactattg caacctactg tactggttct ataccttgta gtgtttgtct 7140

tagtggttta gattctttag acacctatcc ttctttagaa actatacaaa ttaccatttc 7200

atcttttaaa tgggatttaa ctgcttttgg 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 aatagagcaa caagagtcga atgtacaact attgttaatg gtgttagaag 7560

gtccttttat 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

ttttatttca 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 tatagagtac actgactttg caacatcagc 9000

ttgtgttttg gctgctgaat gtacaatttt taaagatgct tctggtaagc cagtaccata 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 gagcttttgg 9480

tgaatacagt catgtagttg cctttaatac tttactattc cttatgtcat tcactgtact 9540

ctgtttaaca ccagtttact cattcttacc tggtgtttat tctgttattt acttgtactt 9600

gacattttat cttactaatg atgtttcttt tttagcacat attcagtgga tggttatgtt 9660

cacaccttta gtacctttct ggataacaat tgcttatatc atttgtattt ccacaaagca 9720

tttctattgg ttctttagta attacctaaa gagacgtgta gtctttaatg gtgtttcctt 9780

tagtactttt gaagaagctg cgctgtgcac ctttttgtta aataaagaaa tgtatctaaa 9840

gttgcgtagt gatgtgctat tacctcttac gcaatataat agatacttag ctctttataa 9900

taagtacaag tattttagtg gagcaatgga tacaactagc tacagagaag 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 taagtataag tttgttcgca ttcaaccagg 10380

acagactttt 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 aagatactac tgaagccttt gaaaaaatgg tttcactact 12000

ttctgttttg ctttccatgc agggtgctgt agacataaac aagctttgtg aagaaatgct 12060

ggacaacagg gcaaccttac aagctatagc ctcagagttt agttcccttc catcatatgc 12120

agcttttgct actgctcaag aagcttatga gcaggctgtt gctaatggtg attctgaagt 12180

tgttcttaaa aagttgaaga agtctttgaa tgtggctaaa tctgaatttg accgtgatgc 12240

agccatgcaa cgtaagttgg aaaagatggc tgatcaagct atgacccaaa tgtataaaca 12300

ggctagatct gaggacaaga gggcaaaagt tactagtgct atgcagacaa tgcttttcac 12360

tatgcttaga aagttggata atgatgcact caacaacatt atcaacaatg caagagatgg 12420

ttgtgttccc ttgaacataa tacctcttac aacagcagcc aaactaatgg ttgtcatacc 12480

agactataac acatataaaa atacgtgtga tggtacaaca tttacttatg catcagcatt 12540

gtgggaaatc caacaggttg tagatgcaga tagtaaaatt gttcaactta gtgaaattag 12600

tatggacaat tcacctaatt tagcatggcc tcttattgta acagctttaa gggccaattc 12660

tgctgtcaaa ttacagaata atgagcttag tcctgttgca ctacgacaga tgtcttgtgc 12720

tgccggtact acacaaactg cttgcactga tgacaatgcg ttagcttact acaacacaac 12780

aaagggaggt aggtttgtac ttgcactgtt atccgattta caggatttga aatgggctag 12840

attccctaag agtgatggaa ctggtactat ctatacagaa ctggaaccac cttgtaggtt 12900

tgttacagac acacctaaag gtcctaaagt gaagtattta tactttatta aaggattaaa 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 taaaaacaca gtctgtaccg tctgcggtat 13380

gtggaaaggt tatggctgta gttgtgatca actccgcgaa cccatgcttc agtcagctga 13440

tgcacaatcg tttttaaacg ggtttgcggt gtaagtgcag cccgtcttac accgtgcggc 13500

acaggcacta gtactgatgt cgtatacagg gcttttgaca tctacaatga taaagtagct 13560

ggttttgcta aattcctaaa aactaattgt tgtcgcttcc aagaaaagga cgaagatgac 13620

aatttaattg attcttactt tgtagttaag agacacactt tctctaacta ccaacatgaa 13680

gaaacaattt ataatttact taaggattgt ccagctgttg ctaaacatga cttctttaag 13740

tttagaatag acggtgacat ggtaccacat atatcacgtc aacgtcttac taaatacaca 13800

atggcagacc tcgtctatgc tttaaggcat tttgatgaag gtaattgtga cacattaaaa 13860

gaaatacttg tcacatacaa ttgttgtgat gatgattatt tcaataaaaa ggactggtat 13920

gattttgtag aaaacccaga tatattacgc gtatacgcca acttaggtga acgtgtacgc 13980

caagctttgt taaaaacagt 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 accctgctat 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 ttttcgcata tacaaaacgt aatgtcatcc ctactataac tcaaatgaat 15060

cttaagtatg ccattagtgc 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 gcacactaga accagaatat ttcaattcag 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 ataattcaca gaatgctgta gcctcaaaga ttttgggact accaactcaa 17820

actgttgatt catcacaggg ctcagaatat gactatgtca tattcactca aaccactgaa 17880

acagctcact cttgtaatgt aaacagattt aatgttgcta ttaccagagc aaaagtaggc 17940

atactttgca taatgtctga tagagacctt tatgacaagt tgcaatttac aagtcttgaa 18000

attccacgta ggaatgtggc aactttacaa gctgaaaatg taacaggact ctttaaagat 18060

tgtagtaagg taatcactgg gttacatcct acacaggcac ctacacacct cagtgttgac 18120

actaaattca aaactgaagg tttatgtgtt gacatacctg gcatacctaa ggacatgacc 18180

tatagaagac tcatctctat gatgggtttt aaaatgaatt atcaagttaa tggttaccct 18240

aacatgttta tcacccgcga 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 gtaaataaac 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 ttataacatg atgatctcag ctggctttag cttgtgggtt 19560

tacaaacaat ttgatactta taacctctgg aacactttta caagacttca gagtttagaa 19620

aatgtggctt ttaatgttgt aaataaggga cactttgatg gacaacaggg tgaagtacca 19680

gtttctatca ttaataacac tgtttacaca aaagttgatg gtgttgatgt agaattgttt 19740

gaaaataaaa caacattacc tgttaatgta gcatttgagc tttgggctaa gcgcaacatt 19800

aaaccagtac cagaggtgaa aatactcaat aatttgggtg tggacattgc tgctaatact 19860

gtgatctggg actacaaaag agatgctcca gcacatatat ctactattgg tgtttgttct 19920

atgactgaca tagccaagaa accaactgaa acgatttgtg caccactcac tgtctttttt 19980

gatggtagag ttgatggtca agtagactta tttagaaatg cccgtaatgg tgttcttatt 20040

acagaaggta gtgttaaagg tttacaacca tctgtaggtc ccaaacaagc tagtcttaat 20100

ggagtcacat taattggaga agccgtaaaa acacagttca attattataa gaaagttgat 20160

ggtgttgtcc aacaattacc tgaaacttac tttactcaga gtagaaattt acaagaattt 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 attttaccca 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 ttttatctct tcttagtaaa 21480

ggtagactta taattagaga aaacaacaga gttgttattt ctagtgatgt tcttgttaac 21540

aactaaacga acaatgtttg tttttcttgt 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

taagaggttt gctaaccctg tcctaccatt taatgatggt gtttattttg cttccactga 21840

gaagtctaac ataataagag gctggatttt tggtactact ttagattcga agacccagtc 21900

cctacttatt gttaataacg ctactaatgt tgttattaaa gtctgtgaat ttcaattttg 21960

taatgatcca tttttgggtg 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 ttttcggctt 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 ctttccttta caatcatatg gtttccaacc 23040

cacttatggt gttggttacc aaccatacag agtagtagta ctttcttttg aacttctaca 23100

tgcaccagca actgtttgtg gacctaaaaa gtctactaat ttggttaaaa acaaatgtgt 23160

caatttcaac ttcaatggtt taacaggcac aggtgttctt actgagtcta acaaaaagtt 23220

tctgcctttc caacaatttg gcagagacat tgctgacact actgatgctg tccgtgatcc 23280

acagacactt gagattcttg acattacacc atgttctttt ggtggtgtca gtgttataac 23340

accaggaaca aatacttcta accaggttgc tgttctttat cagggtgtta actgcacaga 23400

agtccctgtt gctattcatg cagatcaact tactcctact tggcgtgttt attctacagg 23460

ttctaatgtt tttcaaacac gtgcaggctg tttaataggg gctgaacatg tcaacaactc 23520

atatgagtgt gacataccca ttggtgcagg tatatgcgct agttatcaga ctcagactaa 23580

ttctcctcgg cgggcacgta gtgtagctag tcaatccatc attgcctaca ctatgtcact 23640

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

agaagttttt gcacaagtca aacaaattta caaaacacca ccaattaaag attttggtgg 23940

ttttaatttt tcacaaatat taccagatcc atcaaaacca agcaagaggt catttattga 24000

agatctactt ttcaacaaag tgacacttgc agatgctggc ttcatcaaac aatatggtga 24060

ttgccttggt gatattgctg ctagagacct catttgtgca caaaagttta acggccttac 24120

tgttttgcca cctttgctca cagatgaaat gattgctcaa tacacttctg cactgttagc 24180

gggtacaatc acttctggtt ggacctttgg tgcaggtgct gcattacaaa taccatttgc 24240

tatgcaaatg gcttataggt ttaatggtat tggagttaca cagaatgttc tctatgagaa 24300

ccaaaaattg attgccaacc aatttaatag tgctattggc aaaattcaag actcactttc 24360

ttccacagca agtgcacttg gaaaacttca agatgtggtc aaccaaaatg cacaagcttt 24420

aaacacgctt gttaaacaac ttagctccaa ttttggtgca atttcaagtg ttttaaatga 24480

tatcctttca cgtcttgaca aagttgaggc tgaagtgcaa attgataggt tgatcacagg 24540

cagacttcaa agtttgcaga catatgtgac tcaacaatta attagagctg cagaaatcag 24600

agcttctgct aatcttgctg ctactaaaat gtcagagtgt gtacttggac aatcaaaaag 24660

agttgatttt tgtggaaagg gctatcatct tatgtccttc cctcagtcag cacctcatgg 24720

tgtagtcttc ttgcatgtga cttatgtccc tgcacaagaa aagaacttca caactgctcc 24780

tgccatttgt catgatggaa aagcacactt tcctcgtgaa ggtgtctttg tttcaaatgg 24840

cacacactgg tttgtaacac aaaggaattt ttatgaacca caaatcatta ctacagacaa 24900

cacatttgtg tctggtaact gtgatgttgt aataggaatt gtcaacaaca cagtttatga 24960

tcctttgcaa cctgaattag actcattcaa ggaggagtta gataaatatt ttaagaatca 25020

tacatcacca gatgttgatt taggtgacat ctctggcatt aatgcttcag ttgtaaacat 25080

tcaaaaagaa attgaccgcc tcaatgaggt tgccaagaat ttaaatgaat ctctcatcga 25140

tctccaagaa cttggaaagt atgagcagta tataaaatgg ccatggtaca tttggctagg 25200

ttttatagct ggcttgattg ccatagtaat ggtgacaatt atgctttgct gtatgaccag 25260

ttgctgtagt tgtctcaagg gctgttgttc ttgtggatcc tgctgcaaat ttgatgaaga 25320

cgactctgag ccagtgctca aaggagtcaa attacattac acataaacga acttatggat 25380

ttgtttatga gaatcttcac aattggaact gtaactttga agcaaggtga aatcaaggat 25440

gctactcctt cagattttgt tcgcgctact gcaacgatac cgatacaagc ctcactccct 25500

ttcggatggc ttattgttgg cgttgcactt cttgctgttt ttcatagcgc ttccaaaatc 25560

ataaccctca aaaagagatg gcaactagca ctctccaagg gtgttcactt tgtttgcaac 25620

ttgctgttgt tgtttgtaac agtttactca caccttttgc tcgttgctgc tggccttgaa 25680

gccccttttc tctatcttta tgctttagtc tacttcttgc agagtataaa ctttgtaaga 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 gaagactttt 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 ccaagatggt atttctacta cctaggaact gggccagaag ctggacttcc 28620

ctatggtgct aacaaagacg gcatcatatg ggttgcaact gagggagcct tgaatacacc 28680

aaaagatcac attggcaccc gcaatcctgc taacaatgct gcaatcgtgc tacaacttcc 28740

tcaaggaaca acattgccaa aaggcttcta cgcagaaggg agcagaggcg gcagtcaagc 28800

ctcttctcgt tcctcatcac gtagtcgcaa cagttcaaga aattcaactc caggcagcag 28860

taggggaatt tctcctgcta gaatggctgg caatggcggt gatgctgctc ttgctttgct 28920

gctgcttgac agattgaacc agcttgagag caaaatgtct ggtaaaggcc aacaacaaca 28980

aggccaaact gtcactaaga aatctgctgc tgaggcttct aagaagcctc ggcaaaaacg 29040

tactgccact aaagcataca atgtaacaca agctttcggc agacgtggtc cagaacaaac 29100

ccaaggaaat tttggggacc aggaactaat cagacaagga actgattaca aacattggcc 29160

gcaaattgca caatttgccc ccagcgcttc agcgttcttc ggaatgtcgc gcattggcat 29220

ggaagtcaca ccttcgggaa 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 gcttttccgt 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 taggagaann nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 29880

nnnnn 29885

Claims (66)

1. An inactivated SARS-Cov-2 virus or a derivative or related thereof, 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.

2. An inactivated SARS-Cov-2 virus or a derivative or related thereof according to claim 1, wherein the inactivated SARS-Cov-2 virus is obtained by inactivation of SARS-Cov-2 virus with a virus inactivating agent, preferably beta-propiolactone and/or formalin.

3. An inactivated SARS-Cov-2 virus or a derivative or related thereof according to claim 1 or 2, wherein the inactivated SARS-Cov-2 virus is further purified by ion exchange chromatography and/or size exclusion chromatography.

4. An inactivated SARS-Cov-2 virus or a derivative or related thereof according to claim 1 or 2, wherein the inactivated SARS-Cov-2 virus is treated with a non-limiting endonuclease prior to size exclusion chromatography.

5. An inactivated SARS-Cov-2 virus or a derivative or related thereof according to claim 1 or 2, wherein the SARS-Cov-2 virus is associated with a cell density of 2 x 10 prior to inactivation with beta-propiolactone ⁶ cells/mL to 10X 10 ⁶ cells/mL Vero cells, cultured together in a bioreactor at 35-39 ℃ for 3-7 days to obtain a virus harvest.

6. An inactivated SARS-Cov-2 virus or a derivative or related thereof according to claim 1 or 2, wherein the volume ratio of β -propiolactone to the viral harvest is 1 to 4000 to 6000, the inactivation time is 16 to 72 hours, preferably 20 to 24 hours, more preferably 24 hours at 4 ℃.

7. An inactivated SARS-Cov-2 virus or a derivative or related thereof according to claim 1 or 2, wherein the SARS-Cov-2 virus or derivative or related thereof comprises:

ORF1a having the nucleotide sequence of nucleotides 238 to 13455 of SEQ ID NO. 1 or a sequence having at least 95% identity thereto; 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 thereto; and/or

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 thereto; 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 thereto; and/or

Membrane protein M having the amino acid sequence encoded by nucleotides 26495 to 27163 of SEQ ID NO. 1 or a sequence having at least 95% identity thereto; and/or

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 thereto.

8. An inactivated SARS-Cov-2 virus or a derivative or related thereof according to claim 1 or 2, wherein the SARS-Cov-2 virus or derivative or related thereof comprises:

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 an 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 an amino acid sequence encoded by nucleotides 26495 to 27163 of SEQ ID NO. 1; and

nucleocapsid protein N having an amino acid sequence encoded by nucleotides 28246 to 29505 of SEQ ID NO. 1.

9. An inactivated SARS-Cov-2 virus or a derivative or related thereof according to claim 1 or 2, wherein the SARS-Cov-2 virus or derivative or related thereof has a genome comprising an RNA sequence having the reverse complement of any one of SEQ ID NOs 1 to 3, or a sequence having at least 95% identity thereto.

10. An inactivated SARS-CoV-2 virus or a derivative or related thereof according to claim 1 or 2, wherein the SARS-CoV-2 virus or derivative or related thereof has a genome encoded by the sequence shown by any of SEQ ID NOs 1 to 3 or a sequence having at least 95% identity thereto.

11. The inactivated SARS-CoV-2 virus or a derivative or related thereof according to claim 1 or 2, wherein the SARS-CoV-2 virus or derivative or related thereof has a genome encoded by a sequence shown in any one of SEQ ID NOs 1 to 3 or has a genome comprising an RNA sequence having a reverse complement sequence shown in any one of SEQ ID NOs 1 to 3.

12. A pharmaceutical composition or vaccine comprising a pharmaceutically effective amount of inactivated SARS-Cov-2 virus or a derivative or related thereof, and a pharmaceutically acceptable carrier.

13. Pharmaceutical composition or vaccine according to claim 12, wherein the inactivated SARS-Cov-2 virus or a derivative or related thereof is obtained by inactivation of SARS-Cov-2 virus with a virus inactivating agent, preferably beta-propiolactone and/or formalin.

14. The pharmaceutical composition or vaccine according to claim 12, wherein the pharmaceutical composition or the vaccine is in a liquid or frozen state, preferably in a liquid or lyophilized state, more preferably the dosage of the pharmaceutical composition or the vaccine is in the range of 0.5-8 μ g by weight of protein, preferably in the range of 3-6 μ g by weight of protein.

15. The pharmaceutical composition or vaccine of claim 12, wherein the pharmaceutically acceptable carrier is Al (OH) ₃ Adjuvants and/or CpG oligodeoxynucleotides.

16. The pharmaceutical composition or vaccine of claim 12, wherein the inactivated SARS-Cov-2 virus or derivative or related thereof is purified by ion exchange chromatography and/or size exclusion chromatography after inactivation.

17. The pharmaceutical composition or vaccine of claim 16, wherein the inactivated SARS-Cov-2 virus or derivative or related thereof is treated with a non-limiting endonuclease prior to size exclusion chromatography.

18. The pharmaceutical composition or vaccine of claim 16, wherein the SARS-Cov-2 virus or derivative or related thereof is associated with a cell density of 2 x 10 prior to inactivation with beta-propiolactone ⁶ cells/mL to 10X 10 ⁶ cells/mL Vero cells, cultured together in a bioreactor at 35-39 ℃ for 3-7 days to obtain a virus harvest.

19. The pharmaceutical composition or vaccine according to claim 16, wherein the volume ratio of β -propiolactone to the viral harvest is 1.

20. The pharmaceutical composition or vaccine of claim 16,wherein the inactivated SARS-Cov-2 virus or a derivative or related thereof is filtered through a membrane and then contacted with Al (OH) ₃ Adjuvant and/or CpG oligodeoxynucleotide, the pore size of the membrane is 0.2-0.25 μm, preferably 0.22 μm.

21. The pharmaceutical composition or vaccine of claim 12, wherein the SARS-Cov-2 virus or derivative or related thereof comprises:

ORF1a having the nucleotide sequence of nucleotides 238 to 13455 of SEQ ID NO. 1 or a sequence having at least 95% identity thereto; 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 thereto; and/or

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 thereto; 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 thereto; and/or

Membrane protein M having the amino acid sequence encoded by nucleotides 26495 to 27163 of SEQ ID NO. 1 or a sequence having at least 95% identity thereto; and/or

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 thereto.

22. The pharmaceutical composition or vaccine of claim 12, wherein the SARS-Cov-2 virus or derivative or related thereof comprises:

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 an 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 an amino acid sequence encoded by nucleotides 26495 to 27163 of SEQ ID NO. 1; and

nucleocapsid protein N having an amino acid sequence encoded by nucleotides 28246 to 29505 of SEQ ID NO. 1.

23. The pharmaceutical composition or vaccine of claim 12, wherein the SARS-CoV-2 virus or derivative or related thereof has a genome comprising an RNA sequence having the reverse complement of any one of SEQ ID NOs 1-3, or a sequence at least 95% identical thereto.

24. The pharmaceutical composition or vaccine of claim 12, wherein the SARS-CoV-2 virus or derivative or related thereof has a genome encoded by a sequence set forth in any one of SEQ ID NOs 1 or a sequence at least 95% identical thereto.

25. A method of preparing an inactivated SARS-Cov-2 composition or vaccine comprising inactivating a SARS-Cov-2 virus with a virus inactivating agent, preferably beta-propiolactone and/or formalin.

26. A method of making an inactivated SARS-Cov-2 composition or vaccine according to claim 25, wherein the inactivated SARS-Cov-2 virus is further purified by ion exchange chromatography and/or size exclusion chromatography after inactivation.

27. A method of making an inactivated SARS-Cov-2 composition or vaccine according to claim 25, wherein the inactivated SARS-Cov-2 virus is treated with a non-limiting endonuclease prior to size exclusion chromatography.

28. A method of making an inactivated SARS-Cov-2 composition or vaccine according to claim 25, wherein beta propiolactone is being used to kill the virusBefore the survival, the SARS-Cov-2 virus and the cell density are 2X 10 ⁶ cells/mL to 10X 10 ⁶ cells/mL Vero cells, cultured together in a bioreactor at 35-39 ℃ for 3-7 days to obtain a virus harvest.

29. A method of preparing an inactivated SARS-Cov-2 composition or vaccine according to claim 28, wherein the volume ratio of β -propiolactone to the viral harvest is 1.

30. A method of preparing an inactivated SARS-Cov-2 composition or vaccine according to claim 28, wherein the volume ratio of formalin to virus harvest is 1.

31. The method for preparing inactivated SARS-Cov-2 composition or vaccine according to claim 25, wherein the inactivated SARS-Cov-2 virus is filtered through a membrane and then contacted with Al (OH) ₃ Adjuvant and/or CpG oligodeoxynucleotide, the pore size of the membrane is 0.2-0.25 μm, preferably 0.22 μm.

32. The method for preparing an inactivated SARS-Cov-2 composition or vaccine according to claim 25, further comprising contacting the inactivated SARS-Cov-2 virus with Al (OH) ₃ A step of mixing an adjuvant and/or a CpG oligodeoxynucleotide.

33. The method for preparing inactivated SARS-Cov-2 composition or vaccine according to claim 25, wherein the inactivated SARS-Cov-2 virus is filtered through a membrane and then contacted with Al (OH) ₃ Adjuvant and/or CpG oligodeoxynucleotide, wherein the pore size of the membrane is 0.20-0.25 μm, preferably the pore size of the membrane is 0.22 μm.

34. A method of preparing an inactivated SARS-Cov-2 composition or vaccine according to claim 25, wherein the SARS-Cov-2 virus is propagated in 50 to 1000 litres of Vero cell culture using a bioreactor and inactivated using β -propiolactone.

35. The method of making an inactivated SARS-Cov-2 composition or vaccine according to claim 25, wherein the virus is purified using depth filtration or centrifugation, and two optimized chromatography steps.

36. The method of making an inactivated SARS-Cov-2 composition or vaccine according to claim 25, wherein the SARS-Cov-2 virus comprises:

ORF1a having the nucleotide sequence of nucleotides 238 to 13455 of SEQ ID NO. 1 or a sequence having at least 95% identity thereto; 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 thereto; and/or

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 thereto; 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 thereto; and/or

Membrane protein M having the amino acid sequence encoded by nucleotides 26495 to 27163 of SEQ ID NO. 1 or a sequence having at least 95% identity thereto; and/or

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 thereto.

37. The method of making an inactivated SARS-Cov-2 composition or vaccine according to claim 25, wherein the SARS-Cov-2 virus comprises:

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 an 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 an amino acid sequence encoded by nucleotides 26495 to 27163 of SEQ ID NO. 1; and

nucleocapsid protein N having an amino acid sequence encoded by nucleotides 28246 to 29505 of SEQ ID NO. 1.

38. A method of making an inactivated SARS-Cov-2 composition or vaccine according to claim 25, wherein the SARS-Cov-2 virus has a genome comprising an RNA sequence having the reverse complement of any one of SEQ ID NOs 1 to 3, or a sequence at least 95% identical thereto.

39. The method for preparing an inactivated SARS-Cov-2 composition or vaccine according to claim 25, wherein the SARS-Cov-2 virus has a genome encoded by the sequence shown by any of SEQ ID NOs 1 to 3 or a sequence having at least 95% identity thereto.

40. A method of making an inactivated SARS-Cov-2 composition or vaccine according to claim 25, wherein the SARS-Cov-2 virus has a genome encoded by a sequence set forth in any one of SEQ ID NOs 1 to 3 or has a genome comprising an RNA sequence having a reverse complement sequence set forth in any one of SEQ ID NOs 1 to 3.

41. A method of treating or immunizing a subject against a disease associated with SARS-Cov-2 infection, comprising administering to the subject a pharmaceutically effective amount of the pharmaceutical composition or vaccine of any one of claims 12 to 24.

42. A method of treating or immunizing a disease associated with SARS-Cov-2 infection in a subject according to claim 41, wherein the pharmaceutical composition or the vaccine is administered by intradermal, subcutaneous, intramuscular, oral or intranasal vaccination, preferably by intramuscular injection into the upper triangle of the subject.

43. A method of treating or immunizing a disease associated with SARS-Cov-2 infection in a subject according to claim 41, 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.

44. A method of treating or immunizing a disease associated with SARS-Cov-2 infection in a subject according to claim 41, wherein the subject is an animal, preferably a mammal, including mouse, rat, dog, cat, non-human primate and human.

45. A method of treating or immunizing a disease associated with SARS-CoV-2 infection in a subject according to claim 41, wherein the pharmaceutical composition or the vaccine provides complete protection of the non-human primate against SARS-CoV-2 infection by eliciting an effective humoral response without the immunopathology.

46. A method of treating or immunizing a disease associated with SARS-Cov-2 infection in a subject according to claim 41, wherein the pharmaceutical composition or the vaccine does not produce antibody-dependent enhancement of infection.

47. A method of treating or immunizing a disease associated with SARS-Cov-2 infection in a subject according to claim 41, wherein the pharmaceutical composition or the vaccine produces complete protection against SARS-Cov-2 challenge in the subject at a dose of 0.5 to 8 μ g, preferably the vaccine produces complete protection against SARS-Cov-2 challenge in the subject at a dose of 2 to 6 μ g.

48. The inactivated SARS-Cov-2 virus of any one of claims 1 to 11, the pharmaceutical composition or vaccine of any one of claims 12 to 25, the method of preparing an inactivated SARS-Cov-2 composition or vaccine of any one of claims 26 to 40, the method of treating or immunizing against a disease associated with SARS-Cov-2 infection of any one of claims 41 to 47, wherein the SARS-Cov-2 virus has a genomic sequence as shown in 2019-nCoV-BetaCoV/Wuhan/WIV04/2019 or EPI _ ISL _412973, respectively, or a sequence with at least 95% identity to a sequence as shown in 2019-nCoV-BetaCoV/Wuhan/WIV04/2019 or EPI _ ISL _412973, preferably a sequence with at least 95%, 99%, 99.99%, 99%, 99.97%, 99%, 99.7%, 99%, 99.9%, 99%, or 99% identity to a sequence as shown in 2019-nCoV-BetaCoV/Wuhan/WIV 04/WIV/2019 or EPI _ ISL _412973, respectively.

49. An isolated SARS-CoV-2 virus or a derivative or related thereof comprising a spike protein S having an amino acid sequence encoded by nucleotides 21508 to 25356 of SEQ ID No. 1 or a sequence at least 95% identical thereto.

50. An isolated SARS-CoV-2 virus or a derivative or related thereof according to claim 49 further comprising a nucleocapsid protein N having the amino acid sequence encoded by nucleotides 28246 to 29505 of SEQ ID NO. 1 or a sequence at least 95% identical thereto.

51. An isolated SARS-CoV-2 virus or a derivative or related thereof as claimed in claim 49 further comprising a membrane protein M having the amino acid sequence encoded by nucleotides 26495 to 27163 of SEQ ID NO:1 or a sequence at least 95% identical thereto.

52. An isolated SARS-CoV-2 virus or a derivative or related thereof according to claim 49 further comprising ORF1a having the nucleotide sequence of nucleotides 238 to 13455 of SEQ ID NO 1 or a sequence having at least 95% identity thereto.

53. An isolated SARS-CoV-2 virus or a derivative or related thereof according to claim 49 further comprising an ORF1ab having the nucleotide sequence of nucleotides 13740 to 21527 of SEQ ID NO 1 or a sequence having at least 95% identity thereto.

54. An isolated SARS-CoV-2 virus or a derivative or related thereof according to claim 49 further comprising ORF3a having the nucleotide sequence of nucleotides 25365 to 26192 of SEQ ID NO. 1 or a sequence having at least 95% identity thereto.

55. The isolated SARS-CoV-2 virus or derivative or related thereof according to claim 49, wherein the isolated SARS-CoV-2 virus or derivative or related thereof comprises:

ORF1a having the nucleotide sequence of nucleotides 238 to 13455 of SEQ ID NO. 1 or a sequence having at least 95% identity thereto; 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 thereto; and/or

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 thereto; 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 thereto; and/or

Membrane protein M having the amino acid sequence encoded by nucleotides 26495 to 27163 of SEQ ID NO. 1 or a sequence having at least 95% identity thereto; and/or

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 thereto.

56. The isolated SARS-CoV-2 virus or derivative or related thereof according to claim 49, wherein the isolated SARS-CoV-2 virus or derivative or related thereof comprises:

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 an 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 an amino acid sequence encoded by nucleotides 26495 to 27163 of SEQ ID NO. 1; and

nucleocapsid protein N having an amino acid sequence encoded by nucleotides 28246 to 29505 of SEQ ID NO. 1.

57. An isolated SARS-CoV-2 virus or a derivative or related thereof according to claim 49 wherein the isolated SARS-CoV-2 virus or a derivative or related thereof has a genome comprising an RNA sequence having the reverse complement sequence set forth in any one of SEQ ID NOS 1 to 3, or a sequence having at least 95% identity thereto; or a genome having a sequence represented by any one of SEQ ID NOs 1 to 3 or a sequence having at least 95% identity thereto.

58. An isolated nucleic acid comprising the nucleotide sequence of nucleotides 21508-25356 of SEQ ID No. 1 or a sequence having at least 95% identity thereto.

59. An isolated nucleic acid comprising the nucleotide sequence of nucleotides 28246 to 29505 of SEQ ID No. 1 or a sequence having at least 95% identity thereto.

60. An isolated nucleic acid comprising the nucleotide sequence of nucleotides 26495 to 27163 of SEQ ID No. 1 or a sequence having at least 95% identity thereto.

61. An isolated nucleic acid comprising the nucleotide sequence of nucleotides 238 to 13455 of SEQ ID No. 1 or a sequence having at least 95% identity thereto.

62. An isolated nucleic acid comprising the nucleotide sequence of nucleotides 13740 to 21527 of SEQ ID No. 1 or a sequence having at least 95% identity thereto.

63. An isolated nucleic acid comprising the nucleotide sequence of nucleotides 25365 to 26192 of SEQ ID No. 1 or a sequence having at least 95% identity thereto.

64. An isolated nucleic acid, comprising:

1 or a sequence having at least 95% identity thereto; and/or

1, or a sequence having at least 95% identity thereto; and/or

1, nucleotide sequence of 21508-25356 or a sequence with at least 95% identity thereto; and/or

1, nucleotide sequence of 25365-26192 nucleotides of SEQ ID NO. 1 or a sequence with at least 95% identity thereto; and/or

The nucleotide sequence of nucleotides 26495 to 27163 of SEQ ID NO. 1 or a sequence having at least 95% identity thereto; and/or

The nucleotide sequence of 28246 to 29505 nucleotides of SEQ ID NO. 1 or a sequence having at least 95% identity thereto.

65. An isolated nucleic acid, comprising:

the 238 th to 13455 th nucleotide of SEQ ID NO. 1;

1, the 13740 th to 21527 th nucleotides of SEQ ID NO;

1, nucleotide sequence of 21508-25356 nucleotides of SEQ ID NO;

a nucleotide sequence of 25365 to 26192 nucleotides of SEQ ID NO. 1;

the nucleotide sequence of 26495 to 27163 of SEQ ID NO. 1; and

the 28246 th to 29505 th nucleotide sequence of SEQ ID NO. 1.

66. An isolated nucleic acid comprising the nucleotide sequence of any one of SEQ ID NOs 1-3, or a sequence having at least 95% identity thereto, or the reverse complement thereof.

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