KR20230166042A - A full-length infectious clone of Zika virus or variants thereof and their uses - Google Patents

Abstract

One aspect relates to Zika virus infectious full-length clones or variants thereof and their uses. According to one aspect, a full-length Zika virus clone or derivative thereof containing the T7 bacterial phage promoter can be used to analyze the replication mechanism, life cycle, and pathogenicity of Zika virus. Therefore, the clone can be usefully used for screening of drugs for preventing or treating Zika virus infection and evaluating the effectiveness of diagnostic technology, and the genetic material, protein, or fragments thereof of the Zika virus recovered from the clone or a derivative thereof can be used to evaluate the effectiveness of Zika virus infection. It can be useful as a vaccine to prevent viruses.

Description

{A full-length infectious clone of Zika virus or variants thereof and their uses}

It relates to infectious full-length Zika virus clones or variants thereof and their uses.

Zika virus (ZIKV) is a virus with a 10.8-kb single-stranded positive RNA genome belonging to the Flavivirus genus of the Flaviviridae Family, with the 5' and 3' sides of the genome. It consists of an untranslated region (hereinafter abbreviated as “UTR”) located at the end and a single open reading frame (hereinafter abbreviated as “ORF”) located in between. This ORF creates a polyprotein consisting of approximately 3,400 amino acids, and this long single protein consists of three structural proteins (Capsid, prM, Envelope) and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, and NS4B). , NS5).

Zika virus, which was first discovered in Uganda, Africa in 1947, has received relatively little attention compared to other flavivirus viruses transmitted by mosquitoes until the early 2000s, but has recently spread rapidly in South America, Asia, and the Americas. It is spreading rapidly, and research is being conducted to develop control technology in each country. Zika virus infection is reported to cause symptoms such as skin rash, high fever, dizziness, anorexia nervosa, and joint pain, as well as neurological disorders such as microcephaly and encephalitis. It can also cause neonatal microcephaly and brain function impairment. The Zika virus can also spread between humans through sexual intercourse, so it can spread between humans during the asymptomatic incubation period.

The main vector of the Zika virus is the Aedes mosquito, and the population of this mosquito is increasing in Jeju, Korea, due to global warming, etc. It is reported that the population is increasing worldwide. This mosquito is especially abundant in residential areas, and unlike other species that feed mainly at dawn and dusk, it also feeds during the day, so it can become a social problem in large cities and places with a lot of foreigners.

Phylogenetically, Zika virus is largely divided into two strains: African and Asian. Although the two strains are genetically different, the Zika virus has only one serotype. In infection experiments using cell-based and mice, African strains are reported to be more infectious and pathogenic than Asian strains. Despite these differences in phenotype, recently reported cases of human infection with Zika virus belong to the Asian strain. . To date, it is not clear what genetic differences contribute to pathogenicity or why human infection cases are limited to Asian strains. The reverse genetics system-based Zika virus chimeric recombinant virus manufacturing technology can provide recombinant viruses that can be used to identify virulence factors that differ between Zika virus strains and analyze the characteristics of viral proteins. Furthermore, this recombinant virus can be used in the development of attenuated vaccine backbone vectors, where safety is important, and in antiviral drug screening.

RNA viruses do not have error-proof reading ability, such as RNA polymerase, which is a key element in replication. Because of this, the gene sequence changes rapidly during gene replication, so it is necessary to use viruses with homogeneous genes to study the virus life cycle at the molecular biology level. The reverse genetics system makes it possible to manufacture these viruses and utilizes them to study virus-host interactions, develop next-generation recombinant gene vaccines and antivirals, evaluate the effectiveness of vaccines and diagnostic technologies, and study the identification of pathogenic factors of Zika virus using animal models. makes possible. In addition, the viral cDNA clone obtained by the above technique can not only provide an antigen expression gene that can be used in the production of an mRNA vaccine, but can also be used for the expression of this antigen.

For the above purpose, a new recombinant Zika virus Con1 was created using the conserved gene sequence information of various Zika virus strains. In addition, by creating an infectious full-length clone using a cell-adapted virus of MR766, a representative Zika virus isolated in Uganda, Africa, it was used to identify how viral genes have changed and virulence factors have evolved over the past 70 years. Con1 showed more attenuated characteristics in cells than PRVABC59, an Asian virus strain. Chimeric clones were constructed using the infectious full-length cDNA clones for Con1 and cell-adapted MR766, and factors contributing to Zika virus pathogenicity were discovered. More specifically, chimeric clones (ZIKV-Con1/MR_NS5 and ZIKV-Con1/MR_NS1-5) obtained by substituting the gene encoding the non-structural protein (NS5 or NS1-NS5) of the highly pathogenic MR766 virus strain in the ZIKV Con1 full-length clone were created. Thus, a new Zika virus Con1 derivative clone with increased replication ability and pathogenicity than Con1 was produced. In addition, we analyzed the function of the 4 to 5 nucleotide (nt) gene sequence specifically present at the 3'-UTR end of African and Asian Zika viruses, and used this genetic information to produce and utilize recombinant viruses. was developed.

Korean Patent Publication No. 10-2015-0074714

One aspect is to provide a full-length clone of Zika virus containing the T7 bacterial phage promoter.

Another aspect is the nucleotide sequence encoding the capsid protein of the Zika virus, the nucleotide sequence encoding the envelope protein of the Zika virus, and the nucleotide sequence encoding the non-structural protein of the Zika virus in a full-length clone of the Zika virus containing the T7 bacterial phage promoter. Including,

The aim is to provide a subgenomic replicon of Zika virus containing a base sequence encoding the CMV promoter.

Another aspect is to provide a Zika virus minigenome containing a base sequence encoding the capsid protein of the Zika virus within a full-length Zika virus clone containing the T7 bacterial phage promoter.

Another aspect includes introducing into a cell a full-length clone of Zika virus or a derivative thereof comprising a T7 bacterial phage promoter;

treating the cells with a candidate agent;

Measuring the titer of Zika virus in cells treated with the drug; and

To provide a screening method for a drug for preventing or treating Zika virus infection, including the step of selecting a drug whose titer is reduced compared to a control group not treated with the drug.

Another aspect is to provide a Zika virus vaccine composition comprising genetic material of the Zika virus reconstructed from a full-length clone of the Zika virus or a derivative thereof containing a T7 bacterial phage promoter, a protein expressed by the genetic material, or a fragment thereof. .

Another aspect includes administering the genetic material of the Zika virus reconstructed from a full-length clone of the Zika virus or a derivative thereof containing a T7 bacterial phage promoter, a protein expressed by the genetic material, or a fragment thereof to an individual in need thereof. To provide a method for preventing or treating Zika virus infection, including:

Another aspect is the genetic material of the Zika virus restored from a full-length clone of the Zika virus or a derivative thereof containing a T7 bacterial phage promoter for the production of a drug for preventing or treating Zika virus infection, the protein expressed by the genetic material, or these It is to provide the purpose of the fragment.

One aspect provides a full-length clone of Zika virus containing a T7 bacterial phage promoter.

The "Zika virus" is a virus with a single-stranded positive RNA genome belonging to the Flavivirus genus of the Flaviviridae family, including 5'-UTR (untranslated region), 3'-UTR and ORF. It consists of an open reading frame. The ORF is a polyprotein with approximately 3,400 amino acids and consists of 3 structural proteins (Capsid, prM, Envelope) and 7 non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5). do.

The “promoter” refers to a nucleic acid sequence that controls the synthesis of a transcript by providing a recognition and binding site for RNA polymerase. The promoter may contain additional recognition sites or binding sites for additional factors involved in transcriptional regulation of the gene.

The “T7 bacterial phage promoter” is a promoter derived from the T7 bacterial phage, and refers to the DNA sequence to which the T7 bacterial phage-derived RNA polymerase binds to initiate transcription.

The “clone” refers to a copy of the same gene, and the “full-length clone” refers to a clone containing the full-length ZIKV gene. In one aspect, the full-length clone may include the full-length Zika virus gene.

According to one aspect, the clone can be used to analyze the replication mechanism, life cycle, and pathogenicity of Zika virus. Therefore, the clones and derivatives using them can be usefully used in the screening of drugs for preventing or treating Zika virus infection and in evaluating the effectiveness of diagnostic technologies, and the genetic material, proteins, or fragments thereof of the Zika virus recovered from the clones It can be useful as a vaccine to prevent Zika virus.

In one aspect, the clone may include a region cleaved by one or more enzymes selected from the group consisting of HDVRz (hepatitis delta virus ribozyme) and SacII. Since the clone includes the cleaved region, the template DNA can be linearized by the enzyme, and the linearized template DNA can terminate in vitro transcription by T7 RNA polymerase. Furthermore, the RNA produced through this can restore the Zika virus genome with the correct 3' terminal sequence through the action of HDVr.

Additionally, in one aspect, the clone may be prepared using the pBeloBAC11 vector as a template. The pBeloBAC11 vector is a bacterial artificial chromosome, which refers to an artificial DNA structure used to safely maintain and amplify a large genome clone of 100 kb or more in the form of a plasmid in E. coli. These bacterial artificial chromosomes can be used as a means to provide genes and specific promoters because they are easy to safely transport and maintain the entire genes and regulatory promoters of the organism being studied.

In one aspect, the Zika virus may be an Asian Zika virus, an African Zika virus, or a chimeric virus of the Asian Zika virus and the African Zika virus.

In one aspect, the Asian Zika virus may include a sequence that is commonly conserved in the Asian Zika virus sequence and the African Zika virus sequence (abbreviated as Con1). In addition, the Con1 has more than 95%, 96%, 97%, 98% or 99% homology to the nucleotide sequence (SEQ ID NO: 34) of PRVABC59 Zika virus (ATCC VR-1843) (GenBank number: KU501215.1). It may be. Specifically, the Asian Zika virus is one or more selected from the group consisting of a nucleotide sequence mutated from the nucleotide sequence encoding the capsid protein of the PRVABC59 Zika virus and a nucleotide sequence mutated from the nucleotide sequence encoding the replicase of the PRVABC59 Zika virus. It may include a base sequence.

More specifically, the nucleotide sequence mutated from the nucleotide sequence encoding the capsid protein of the PRVABC59 Zika virus may be a nucleotide sequence in which the 346th base of the PRVABC59 Zika virus nucleotide sequence (SEQ ID NO: 34) is substituted from C to T, The nucleotide sequence mutated from the nucleotide sequence encoding the replication enzyme of the PRVABC59 Zika virus may be a nucleotide sequence in which the 7939th base of the PRVABC59 Zika virus nucleotide sequence (SEQ ID NO: 34) is substituted from T to C, for example, The full-length clone of the Asian Zika virus, which includes a nucleotide sequence in which the 346th base of the PRVABC59 Zika virus nucleotide sequence (SEQ ID NO: 34) is substituted from C to T, and the 7939th base is substituted from T to C, is SEQ ID NO: It may be a polynucleotide consisting of 12 base sequences.

The “homology” is intended to indicate the degree of similarity with the wild-type base sequence. Comparison of such homology can be performed using a comparison program widely known in the art, and homology between two or more sequences can be determined. It can be calculated as a percentage (%).

According to one aspect, the Zika virus restored from the full-length clone of Con1 may be attenuated than the PRVABC59 Zika virus. The term “attenuation” refers to a phenomenon in which the proliferative power and pathogenicity of a virus are reduced.

In addition, in one aspect, the African Zika virus is a Zika virus adapted from MR766 Zika virus (ATCC VR-84) (GenBank number: KX830960) through subculture in cells, and MR766 Zika virus (ATCC VR-84) It may have more than 95%, 96%, 97%, 98%, or 99% homology with the nucleotide sequence (SEQ ID NO: 37) of (GenBank number: KX830960). Specifically, the African Zika virus may include a nucleotide sequence mutated from the nucleotide sequence encoding the non-structural protein NS3 of the MR766 Zika virus, for example, the nucleotide sequence encoding the non-structural protein NS3 of the MR766 Zika virus. The full-length clone of the African Zika virus containing a nucleotide sequence mutated from may be a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 36.

In one aspect, the Zika virus in the Con1 and MR766 clones may have a 3' terminal base sequence mutated. Specifically, the mutation is one in which the 3' terminal nucleotide sequence of the Asian Zika virus is replaced with the 3' terminal nucleotide sequence of the African Zika virus, or the 3' terminal nucleotide sequence of the African Zika virus is replaced with the 3' terminal nucleotide sequence of the Asian Zika virus. 'It may be replaced by the terminal base sequence. More specifically, the mutation is when the 3' terminal nucleotide sequence of the Zika virus is -GTCT-3', it is substituted with -TTTCT-3', or the 3' terminal nucleotide sequence of the Zika virus is -TTTCT-3. In the case of ', it may be replaced with -GTCT-3'. By substituting the 3' terminal base sequence of the Zika virus, different phenotypes can be displayed in mosquito-borne infections and animal experiments.

In one aspect, when the 3' terminal nucleotide sequence of the Con1 clone is substituted from -GTCT-3' to -TTTCT-3', the clone may be a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 13, and the MR766 If the 3' terminal nucleotide sequence of the clone is substituted from -TTTCT-3' to -GTCT-3', the clone may be a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 95.

In one aspect, the chimeric virus is a virus in which part of the nucleotide sequence of the Asian Zika virus is replaced with a part of the nucleotide sequence of the African Zika virus, or a part of the nucleotide sequence of the African Zika virus is replaced with the nucleotide sequence of the Asian Zika virus. It may be a virus in which part of the sequence has been replaced. Specifically, the chimeric virus is an Asian Zika virus in which the nucleotide sequence encoding the non-structural protein of the Asian Zika virus has been replaced with the nucleotide sequence encoding the non-structural protein of the African Zika virus, or the non-structural protein of the African Zika virus has been replaced. The coding nucleotide sequence may be an African Zika virus substituted with a nucleotide sequence encoding a non-structural protein of an Asian Zika virus, and the non-structural protein is selected from the group consisting of NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5. There can be more than one. More specifically, the chimeric virus is an Asian Zika virus in which the nucleotide sequence encoding the non-structural protein NS5 of the Asian Zika virus is replaced with the nucleotide sequence encoding the non-structural protein NS5 of the African Zika virus, or the nucleotide sequence encoding the non-structural protein NS5 of the African Zika virus. It is an Asian Zika virus in which the nucleotide sequence encoding the entire non-structural protein is substituted with the nucleotide sequence encoding the entire non-structural protein of the African Zika virus, or the nucleotide sequence encoding the entire non-structural protein of the African Zika virus is substituted with the nucleotide sequence encoding the entire non-structural protein of the African Zika virus. It may be an African Zika virus that has been replaced with a nucleotide sequence that encodes the entire non-structural protein.

According to one aspect, a chimeric Zika virus in which the nucleotide sequence encoding the non-structural protein NS5 of the Asian Zika virus is replaced with the nucleotide sequence encoding the non-structural protein NS5 of the African Zika virus, and the entire non-structural protein of the Asian Zika virus is encoded. Chimeric Zika virus, whose nucleotide sequence is substituted with a nucleotide sequence encoding the entire non-structural protein of the African Zika virus, may have increased replication ability or pathogenicity of the Zika virus compared to the Asian Zika virus, and the acetabular structure of the African Zika virus may be Chimeric Zika virus, in which the nucleotide sequence encoding the entire crude protein is replaced with the nucleotide sequence encoding the entire non-structural protein of the Asian Zika virus, may have a reduced replication ability or pathogenicity of the Zika virus compared to the African Zika virus.

In one aspect, when the nucleotide sequence encoding the non-structural protein NS5 of the Asian Zika virus in the clone is replaced with the nucleotide sequence encoding the non-structural protein NS5 of the African Zika virus, the clone has the nucleotide sequence of SEQ ID NO: 96. It may be a polynucleotide consisting of, and if the nucleotide sequence encoding the entire non-structural protein of the Asian Zika virus in the clone is replaced with the nucleotide sequence encoding the entire non-structural protein of the African Zika virus, the clone has the base of SEQ ID NO: 97. It may be a polynucleotide consisting of a sequence, and if the nucleotide sequence encoding the entire non-structural protein of the African Zika virus in the clone is replaced with the nucleotide sequence encoding the entire non-structural protein of the Asian Zika virus, the clone has SEQ ID NO: 98. It may be a polynucleotide consisting of a base sequence.

According to one example, to prepare a full-length Zika virus Con1 clone, three DNA fragments (Con1-1, Con1-2, and Con1-3) of the ZIKV-Con1 base sequence (SEQ ID NO: 1) were chemically synthesized, and pMW119 The pMW119-Con1-5'-3' intermediate vector (SEQ ID NO: 2) was constructed using the vector. Afterwards, the three DNA fragments of Con1 were sequentially inserted into the multi-cloning site (MCS) using restriction enzymes suitable for the pMW-T7-ZK-Con1 vector (SEQ ID NO: 3), and the pMW-T7-ZK-Con1 The vector (SEQ ID NO: 3) was subcloned into single copy pBeloBAC11 vector. An intermediate vector containing T7, HDVRz, and SacII restriction enzyme sites was constructed in the pBeloBAC11 vector, and the full-length pMW-T7-ZK-Con1 vector (SEQ ID NO: 3) was amplified using the linearized intermediate vector constructed as above as a template. The Zika virus gene (T7-ZK-Con1) was cloned using the In-Fusion method, resulting in pBAC-T7-ZK-Con1 (SEQ ID NO. 12) and pBAC-T7-ZK- whose terminal sequence was substituted with -TTTCT-3'. Con1(TTTCT) plasmid (SEQ ID NO: 13) was produced. To restore the produced full-length Zika virus clone, the pBAC-T7-ZK-Con1 plasmid (SEQ ID NO: 12) was linearized, the DNA was purified, and the transcribed RNA was purified and transduced into Vero E6 cells. The supernatant obtained after 3 to 5 days of culture was named P0, the supernatant obtained by infecting Vero E6 cells with P0 was named P1, and the supernatant obtained by infecting Vero E6 cells again with P1 was named P2. The viral titers of P0, P1, and P2 supernatants obtained by the above method were measured using plaque assay, and the amount of viral protein expression in cells was measured through Western blot analysis. As a result of measuring the viral titer, the titer of the P0 supernatant was significantly lower than that of P1 and P2, and as a result of measuring viral protein expression, it was confirmed that NS5, NS3, and capsid proteins were expressed in Con1 (see Example 1).

According to another example, as a result of comparing the nucleotide and amino acid sequences with Con1 produced in Example 1 and the Asian reference PRVABC59 virus strain (GenBank number: KU501215.1) (SEQ ID NO: 34), Con1 and PRVABC59 were There are differences in the capsid protein portion (I80T; the 80th amino acid isoleucine located in the Con1 capsid is substituted with threonine) and the replicase portion (A2611V; the 2611th amino acid alanine located in the Con1 replicase is substituted with valine) and the overall base sequence. It was confirmed that the homology was 98.5%. Afterwards, in order to compare and analyze virus titers, Vero E6 cells and A549 cells were infected with Con1 from restored P1 and PRVABC59 from transferred P2, respectively. Virus titer and plaque size were analyzed in both Vero E6 cells and A549 cells. The virus recovered from the ZIKV-Con1 full-length clone had a lower viral titer over time and a smaller plaque size than the PRVABC59 virus strain, confirming that the recombinant virus Con1 was attenuated compared to PRVABC59, the same Asian Zika virus strain (see Example 2) .

According to another example, to prepare a full-length Zika virus MR766 clone, three DNA fragments (MR766-1, MR766-2, and MR766-3) among the ZIKV-MR766 sequence (SEQ ID NO: 35) were amplified through PCR and produced as pBAC. -A linearized intermediate vector containing T7, HDVRz, and SacII sequences was constructed through inverse PCR using the T7-ZK-Con1 plasmid as a template, and the three amplified MR766 DNA fragments were cloned using the In-Fusion method, ultimately producing pBAC- T7-ZK-MR766 plasmid (SEQ ID NO: 36) was produced. When comparing the nucleotide and amino acid sequences of the produced recombinant MR766 and the reference MR766 virus strain (GenBank number: KX830960.1) (SEQ ID NO. 37), it was confirmed that a total of 5 nucleotides and 2 amino acid sequences were changed, and the restored rMR766 In order to compare the virus titers of MR766 distributed from ATCC, A549 cells were infected with the two viruses, and a plaque analysis was performed using the supernatant on the third day. As a result, there was no statistically significant difference in the virus titers of the two MR766s. It was confirmed that this was not the case (see Example 3).

According to another example, an experiment was conducted to confirm the effect of non-structural proteins on virus replication ability by constructing variants in which non-structural proteins were substituted for each other based on the ZIKV-Con1 clone and the ZIKV-MR766 clone and comparing the viral titers of the variants. was carried out. Specifically, pBAC-T7-ZK-Con1, which has the replication enzyme (NS5) among the non-structural proteins of MR766 or the entire non-structural protein (NS1-5) of MR766, based on the pBAC-T7-ZK-Con1 full-length clone (SEQ ID NO: 12). /MR_NS5 (SEQ ID NO: 96) and pBAC-T7-ZK-Con1/MR_NS1-5 (SEQ ID NO: 97), and the entire non-structural protein of Con1 (NS1) based on the pBAC-T7-ZK-MR766 full-length clone (SEQ ID NO: 36) pBAC-T7-ZK-MR766/Con1_NS1-5 (SEQ ID NO: 98) having -5) were each produced using the In-Fusion cloning method, the clones were restored, and comparative virus titer analysis was performed using the P1 supernatant. As a result, it was confirmed that the clone with the non-structural protein of MR766 had a higher virus titer than the clone with the non-structural protein of Con1, confirming that the non-structural protein of MR766, i.e. the replicase complex, had a superior replication ability than the replicase complex of Con1 ( See Example 5).

According to another example, mutants in which non-structural proteins were substituted based on the ZIKV-Con1 clone and ZIKV-MR766 clone were created and the mutants were infected with interferon α/β deficient mice to determine weight change, survival rate, and clinical effects of the mice. An experiment was conducted to determine the effect of non-structural proteins on viral pathogenicity by comparing symptoms, viral titers in serum, and viral titers in organs. As a result of observing the weight change, survival rate, and clinical symptoms of the mice, rMR766-infected mice restored from pBAC-T7-ZK-MR766 (SEQ ID NO: 36) and pBAC-T7-ZK-Con1/MR_NS1-5 (SEQ ID NO: 97) All of the Con1/MR_NS1-5-infected mice restored from pBAC-T7-ZK-Con1/MR_NS5 (SEQ ID NO: 96) died, and 4 out of 6 Con1/MR_NS5-infected mice restored from pBAC-T7-ZK-Con1/MR_NS5 died. All Con1-infected mice recovered from -ZK-Con1 (SEQ ID NO: 12) survived without any specific symptoms. As a result of observing the virus titer, there was no significant difference in titer in the case of Con1 (SEQ ID NO: 12) and Con1/MR_NS5 (SEQ ID NO: 96), but in the case of Con1/MR_NS1-5 (SEQ ID NO: 97), there was no significant difference in titer in Con1 (SEQ ID NO: 12). Compared to this, the virus titer was significantly higher in the kidney, liver, and day 3 serum, and in the case of rMR766 (SEQ ID NO: 36), the virus titer was significantly higher than that of other virus strains. Through the above experiment, it was confirmed that Con1 (SEQ ID NO: 12) in mice was attenuated compared to other virus strains, and the non-structural protein (NS5) of the African virus strain MR766 did not significantly contribute to replication ability, but showed a difference in pathogenicity. It was confirmed that it contributed greatly to this. Through this, it was confirmed that the pathogenicity of the Zika virus non-structural protein decreased as the African virus strain evolved into the Asian virus strain (see Example 6).

According to another example, based on the ZIKV-Con1 clone and the ZIKV-MR766 clone, mutants in which non-structural proteins were substituted were created and the mutants were infected with normal mice (C57BL/6) to determine the weight change, survival rate, and clinical symptoms of the mice. An experiment was performed to determine the effect of non-structural proteins on viral pathogenicity in a mouse model with intact immunity by comparing symptoms, viral titers in serum, and viral titers in organs. As a result of observing the weight change, survival rate, and clinical symptoms of the mice, both Con1 (SEQ ID NO: 12) infected mice and Con1/MR_NS5 (SEQ ID NO: 96) infected mice survived without any specific symptoms. In contrast, Con1/MR_NS1-5 (SEQ ID NO: 97) infected mice showed a significant change in body weight starting 7 days after infection, but all survived while recovering their body weight by day 15. All five mice infected with rMR766 (SEQ ID NO: 36) showed severe paralysis and died on days 7 and 8 of infection. As a result of observing the virus titer, a significant difference was found in the level and titer of viral RNA in serum for Con1 (SEQ ID NO: 12) and Con1/MR_NS5 (SEQ ID NO: 96), but no significance was found in all organs. In the case of Con1/MR_NS1-5 (SEQ ID NO: 97), the virus titer was significantly higher than that of Con1 (SEQ ID NO: 12) and Con1/MR_NS5 (SEQ ID NO: 96) in all organs and serum except testis, and rMR766 (SEQ ID NO: 36) ) had the highest virus titer compared to other virus strains. Seven days after infection, lesions were observed in the cerebral cortex of the mouse brain. In correlation with the level of viral RNA in the organ, no lesions were observed in Con1 (SEQ ID NO: 12) and Con1/MR_NS5 (SEQ ID NO: 96), but Con1/MR_NS1 In the case of mice infected with -5 (SEQ ID NO: 97) and rMR766 (SEQ ID NO: 36), lesions due to immune cell infiltration were clearly identified. Through the above experiment, it was confirmed that, similar to interferon α/β deficient mice, Con1 (SEQ ID NO: 12) was attenuated compared to other virus strains, and the non-structural protein (NS1-5) of MR766, an African virus strain, significantly contributed to replication ability. Thus, it was confirmed that there was a difference in pathogenicity. In addition, through the above experiment, it was confirmed that the interferon signal acts as an important factor in the difference in pathogenicity (see Example 7).

Another aspect is the nucleotide sequence encoding the capsid protein of the Zika virus, the nucleotide sequence encoding the envelope protein of the Zika virus, and the nucleotide sequence encoding the non-structural protein of the Zika virus in a full-length clone of the Zika virus containing the T7 bacterial phage promoter. Including,

A subgenomic replicon of Zika virus containing a base sequence encoding a CMV promoter is provided.

The terms “T7 bacterial phage promoter”, “Zika virus”, “full-length clone”, etc. may be within the above-mentioned range.

The “CMV promoter” is a promoter derived from cytomegalovirus and refers to a DNA sequence for expressing a target gene in a plasmid vector, and the promoter can initiate transcription in animal cells.

The “subgenomic replicon” refers to a plasmid that contains only a portion of the structural protein base sequence of a virus and thus has no viral infectivity but is capable of autonomous viral replication.

In one aspect, the replicon may further include a reporter gene. Specifically, the reporter gene may be a luciferase gene, and more specifically, the reporter gene may be a Renilla luciferase gene. Additionally, the replicon may further include a gene expressing a target protein instead of the reporter gene.

According to one aspect, the subgenomic replicon contains a luciferase gene, and the degree of viral replication can be measured by measuring the activity of the luciferase. Therefore, the subgenomic replicon can be usefully used to measure viral replication capacity.

In one aspect, the replicon may include part of the base sequence of the Asian Zika virus, and specifically may include part of the polynucleotide consisting of the base sequence of SEQ ID NO: 12. More specifically, when the Zika virus in the replicon is the Asian Zika virus (Con1), the replicon may be a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 55.

In one aspect, the Zika virus in the replicon may have a 3' terminal base sequence mutated. Specifically, the mutation may be a replacement of the 3' terminal nucleotide sequence of the Asian Zika virus with the 3' terminal nucleotide sequence of the African Zika virus. More specifically, the mutation may be the 3' terminal nucleotide sequence of the Zika virus. In the case of -GTCT-3', it may be substituted with -TTTCT-3'. By substituting the 3' terminal nucleotide sequence of the Zika virus in the replicon, changes in the replication ability of the virus from the replicon can be confirmed.

In one aspect, when the Zika virus in the replicon is an Asian Zika virus and the 3' terminal nucleotide sequence of the Zika virus is substituted from -GTCT-3' to -TTTCT-3', the replicon may be a polynucleotide consisting of the base sequence of SEQ ID NO: 56.

According to one embodiment, when the 3'-terminal sequence of the Asian Zika virus is replaced with the 3'-terminal sequence of the African Zika virus, it is confirmed whether changes occur in the replication, translation, RNA stability and proliferation of the Zika virus. To this end, a subgenomic replicon (SEQ ID NO: 55 or 56) and minigenome (SEQ ID NO: 77 or 78) were constructed based on the pBAC-T7-ZK-Con1 full-length clone (SEQ ID NO: 12), and RNA was analyzed by decay analysis. Stability was compared, and virus proliferation ability was compared using plaque analysis. As a result of comparing the viral replication ability according to the substitution of the 3' terminal sequence with the created subgenomic replicon, in the case of the NS5_GAA subgenomic replicon, luciferase is expressed through replication in a form in which the activity of the replicase has been lost. Activity was significantly reduced. In addition, as a result of comparing the degree of viral protein translation according to substitution of the 3' terminal sequence with the produced minigenome, it was confirmed that there was no significant change in luciferase activity. In the case of viral RNA stability and virus proliferation, it was confirmed that no significant changes occurred due to 3' terminal sequence substitution (see Example 4).

Another aspect provides a minigenome of Zika virus comprising a nucleotide sequence encoding the capsid protein of Zika virus in a full-length clone of Zika virus containing a T7 bacterial phage promoter.

The terms “T7 bacterial phage promoter,” “Zika virus,” “full-length clone,” etc. may be within the scope described above.

The “minigenome” refers to a genome containing only a portion of the base sequence of a virus, which is non-viral infective but capable of expressing self-encoded foreign proteins. Specifically, the minigenome may include a nucleotide sequence encoding the viral 5'UTR and the N-terminus 38 of the capsid protein, luciferase, and 3'UTR.

In one aspect, the minigenome may further include a reporter gene. Specifically, the reporter gene may be a luciferase gene, and more specifically, the reporter gene may be a Renilla luciferase gene.

According to one aspect, the minigenome contains a luciferase gene, and the amount of viral protein expression can be measured by measuring the activity of the luciferase. Therefore, the minigenome can be usefully used to measure the degree of viral protein translation.

In one aspect, the minigenome may include part of the base sequence of the Asian Zika virus, and specifically may include part of the polynucleotide consisting of the base sequence of SEQ ID NO: 12. More specifically, when the Zika virus in the minigenome is an Asian Zika virus, the minigenome may be a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 77.

In one aspect, the Zika virus in the minigenome may have a 3' terminal base sequence mutated. Specifically, the mutation may be a replacement of the 3' terminal nucleotide sequence of the Asian Zika virus with the 3' terminal nucleotide sequence of the African Zika virus. More specifically, the mutation may be the 3' terminal nucleotide sequence of the Zika virus. In the case of -GTCT-3', it may be substituted with -TTTCT-3'. By substituting the 3' terminal nucleotide sequence in the minigenome, the degree of translation of the Zika virus restored from the minigenome can be changed.

In one aspect, when the Zika virus in the minigenome is an Asian Zika virus and the 3' terminal nucleotide sequence of the Zika virus is substituted from -GTCT-3' to -TTTCT-3', the minigenome has the sequence It may be a polynucleotide consisting of the base sequence number 78.

Another aspect includes introducing into a cell a full-length clone of Zika virus or a derivative thereof comprising a T7 bacterial phage promoter;

treating the cells with a candidate agent;

Measuring the titer of Zika virus in cells treated with the drug; and

It provides a screening method for a drug for preventing or treating Zika virus infection, comprising the step of selecting a drug whose titer is reduced compared to a control group not treated with the drug.

The terms “T7 bacterial phage promoter,” “Zika virus,” “full-length clone,” etc. may be within the scope described above.

According to one aspect, the method involves treating cells into which the full-length clone of the Zika virus or a derivative thereof is introduced with a candidate drug, and if the virus titer decreases compared to a control group not treated with the candidate drug, the candidate drug is applied to the Zika virus. By selecting drugs for preventing or treating infectious diseases, they can be effectively used to screen candidate drugs. In addition, since the full-length clone of Zika virus or a derivative thereof may include the base sequence of an Asian Zika virus, an African Zika virus, or a chimeric Zika virus, the screening method can be used to prevent or treat infections caused by various Zika viruses. It can be effectively used for screening of pharmaceuticals.

The term “derivative thereof” refers to any recombinant clone that can be derived from a full-length clone of Zika virus containing the T7 bacterial phage promoter. Specifically, the derivative may be a subgenomic replicon or minigenome.

The “cell” refers to any cell that can be infected with Zika virus. Specifically, the cells may be animal cells, and more specifically, the cells may be human cells.

The “introduction” refers to the process of infecting the cell by transfecting the full-length clone of the Zika virus into the cell. The clone may be introduced into the cell by physical, chemical or biological means. The physical means may include gene guns, calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, etc. The chemical means may include colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles and liposomes. Additionally, the biological means may include the use of DNA or RNA vectors. Specifically, the introduction can be performed with lipofectamine.

The term “titer” refers to the level at which a virus proliferates or the amount of virus. By measuring the titer, the replicative ability or pathogenicity of the virus can be measured, and the titer can be measured using a plaque assay.

The "Zika virus infection" refers to an acute infectious disease caused by infection with the Zika virus, and is one selected from the group consisting of fever, rash, vomiting, joint pain, arthritis, headache, muscle pain, eye pain, non-purulent conjunctivitis, and conjunctival injection. It may be accompanied by the above symptoms. Additionally, it is known that in pregnant women, Zika virus infection can cause neonatal microcephaly.

The term “prevention” refers to any action that suppresses or delays the onset of Zika virus infection, and the term “treatment” refers to any action that improves or beneficially changes Zika virus infection by administering medication.

The term “administration” refers to the physical introduction of a medicament into a subject using any of a variety of methods and delivery systems known to those skilled in the art.

The term “individual” refers to a subject in need of prevention or treatment of Zika virus infection. Specifically, the subject is a human or mammal such as a primate, mouse, rat, dog, cat, horse, pig, rabbit, or cow. It can be.

Another aspect provides a Zika virus vaccine composition comprising genetic material of the Zika virus reconstructed from a full-length clone of the Zika virus or a derivative thereof containing a T7 bacterial phage promoter, a protein expressed by the genetic material, or a fragment thereof.

The terms “T7 bacterial phage promoter,” “Zika virus,” “full-length clone,” “derivative thereof,” etc. may be within the scope described above.

According to one aspect, the genetic material of the Zika virus, the protein expressed by the genetic material, or fragments thereof can induce an immune response in an individual to generate immunity to the Zika virus. Therefore, the vaccine composition can be usefully used as a vaccine for preventing Zika virus infection.

The “genetic material” refers to a material that causes the expression of individual genetic traits in an organism and may include DNA and RNA. Specifically, the genetic material may be the genetic material of the Zika virus, and more specifically, the genetic material may be the RNA of the Zika virus.

The “vaccine” refers to a pharmaceutical composition containing an antigen that induces an immunological response in animals and can induce immunity to a disease caused by the antigen. Specifically, the vaccine may contain Zika virus antigen.

The term “antigen” refers to a molecule with one or more epitopes that stimulates the host's immune system to produce a secretory, humoral, or cellular antigen-specific response, and may include a complete protein, part of a protein, peptide, fusion protein, or sugar. It may be proteins, lipids, carbohydrates, nucleic acids, polysaccharides, allergenic substances, tissues, cells, and combinations thereof.The antigen may be a natural product purified from a virus, or may be artificially produced through methods such as genetic recombination. The antigen may be a virion, which is a complete virus particle, an incomplete virus particle, a viral structural protein, a viral non-structural protein, a whole pathogen, a protein or glycoprotein derived from a pathogen, an infection protection antigen, a neutralizing epitope, etc., and may be an infectious agent. It may include those that have and those that have lost their infectivity (inactivated antigens).The inactivated antigens can be physically (X-ray irradiation, UV irradiation, heat, ultrasound), chemically (formalin, betapropiolactone, binary ethylene). It may be inactivated by manipulation (imine, mercury, alcohol, chlorine), etc. Specifically, the antigen may be an antigen of the Zika virus, and more specifically, the antigen may be the genetic material of the Zika virus, the genetic material. It may be one or more selected from the group consisting of expressed proteins and fragments thereof.

The “vaccine composition” may further include one or more selected from the group consisting of a pharmaceutically acceptable carrier, diluent, and immunity enhancer.

The term “pharmaceutically acceptable” means medically or veterinarily suitable for use in humans or animals without excessive toxicity, irritation, allergic reactions, or complications.

The carrier may be, for example, colloidal suspension, powder, saline solution, lipid, liposome, microsphere or nano-spherical particle. They may form complexes or associate with delivery vehicles and may be used in the art as lipids, liposomes, microparticles, gold, nanoparticles, polymers, condensation agents, polysaccharides, polyamino acids, dendrimers, saponins, adsorption enhancers or fatty acids. It can be transported in vivo using known delivery systems.

The immune enhancing agent refers to a substance that can enhance an individual's immune response. The immune response may be phagocytosis by macrophages, antigen presentation by dendritic cells, humoral immune response by B cells, or cellular immune response by T cells.

When the vaccine composition is formulated, it can be prepared using commonly used diluents or excipients such as lubricants, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, fillers, extenders, binders, wetting agents, disintegrants, and surfactants. there is. Solid preparations for oral administration may include tablets, pills, powders, granules, capsules, etc., and such solid preparations may contain at least one excipient in the vaccine composition, such as starch, calcium carbonate, sucrose ( It can be prepared by mixing sucrose, lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc can also be used. Liquid preparations for oral use include suspensions, oral solutions, emulsions, and syrups. In addition to the commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. . Preparations for parenteral administration may include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. As a base for suppositories, witepsol, macrogol, tween 61, cacao, laurin, glycerogeratin, etc. can be used, and when manufacturing in the form of eye drops, known diluents or excipients can be used. there is. When the vaccine composition is formulated as an injection, the injection may be an aqueous solvent such as physiological saline solution or Ringer's solution, vegetable oil, higher fatty acid ester (e.g., ethyl oleate, etc.), alcohol (e.g., ethanol, It can be manufactured using non-aqueous solvents such as benzyl alcohol, propylene glycol, glycerin, etc., and stabilizers to prevent deterioration (e.g. ascorbic acid, sodium bisulfite, sodium pyrosulfite, BHA, Pharmaceutical carriers such as tocopherol, EDTA, etc.), emulsifiers, buffers for pH adjustment, and preservatives to inhibit microbial growth (e.g., phenylmercuric nitrate, thimerosal, benzalkonium chloride, phenol, cresol, benzyl alcohol, etc.) may include.

The vaccine composition is for external use on the skin or intraperitoneal injection, intrarectal injection, subcutaneous injection, intravenous injection, intramuscular injection, intraarterial injection, intramedullary injection, intracardiac injection, intrathecal injection, transdermal injection, intranasal injection, and enteral injection. Parenteral and oral administration may be possible, such as by injection, topical injection, sublingual injection, or intrathoracic injection.

The vaccine composition can be administered in a pharmaceutically effective amount. At this time, the term “pharmaceutically effective amount” refers to an amount sufficient to demonstrate the effect of the vaccine and an amount that does not cause side effects or a serious or excessive immune response. The exact administration concentration may vary depending on the antigen to be administered; It can be easily determined by a person skilled in the art depending on factors well known in the medical field, such as the patient's age, weight, health, gender, patient's sensitivity to the drug, administration route, and administration method, and may be administered once to several times. For example, the vaccine composition may be administered daily or every other day at a dose of 0.001 mg/kg to 1,000 mg/kg, and may be administered divided into 1 to 3 times per day.

Another aspect is administering the genetic material of the Zika virus, which is reconstructed from a full-length clone of the Zika virus or a derivative thereof containing the T7 bacterial phage promoter, the protein expressed by the genetic material, or a fragment thereof, to an individual in need thereof. Provides a method for preventing or treating Zika virus infection comprising the following steps:

The terms “T7 bacterial phage promoter”, “Zika virus”, “full-length clone”, “derivative thereof”, “genetic material”, “individual”, “administration”, “Zika virus infection”, “prevention”, “treatment” etc. may be within the range described above.

According to one aspect, the genetic material of the Zika virus, the protein expressed by the genetic material, or fragments thereof can induce an immune response in an individual to generate immunity to the Zika virus. Therefore, the method can be useful for preventing or treating Zika virus infection.

Another aspect is the genetic material of the Zika virus restored from a full-length clone of the Zika virus or a derivative thereof containing a T7 bacterial phage promoter for the production of a drug for preventing or treating Zika virus infection, the protein expressed by the genetic material, or these Provides the use of fragments of .

The terms “Zika virus infection”, “Zika virus”, “prevention”, “treatment”, “T7 bacterial phage promoter”, “full-length clone”, “derivative thereof”, “genetic material”, etc. may be within the scope of the foregoing. .

According to one aspect, full-length Zika virus clones and derivatives thereof containing the T7 bacterial phage promoter can be used to analyze the replication mechanism, life cycle, and pathogenicity of Zika virus. Therefore, the clone can be usefully used in the screening of drugs for preventing or treating Zika virus infection and in assessing the effectiveness of diagnostic technology, and the genetic material, protein, or fragments thereof of the Zika virus recovered from the clone or derivative thereof can be used to It can be useful as a vaccine to prevent viruses.

Figure 1 shows a genealogy created using reference virus strains and a total of 38 Zika virus strain genes for constructing ZIKV-Con1 clones using conserved sequences between Asian and African strains.
Figure 2 shows a schematic diagram of the genome structure of ZIKV-Con1, the restriction enzyme positions used for cloning, the gene fragment, the promoter used, and the ribozyme used to cut the terminal sequence.
Figure 3a shows the supernatant (passage number 0, P0) produced after transducing RNA obtained through an in vitro transcription process into Vero E6 cells to restore the Con1 full-length clone, and the supernatant produced after infecting new Vero E6 cells with P0 (P1). ) and the Zika virus titer measured by plaque analysis for the supernatant (P2) obtained through the same process (ND, meaning not detected below the detection limit).
Figure 3b shows the results of Western blot analysis of viral proteins (NS5, NS3, and capsid) in cells infected with the restored Zika virus.
Figure 4 shows the nucleotide and amino acid differences between ZIKV-Con1 and PRVABC59, a reference Zika virus strain of the Asian lineage.
Figure 5a shows the results of comparative analysis of virus titers by infecting Vero E6 cells and A549 cells with PRVABC59 virus strain (P2) and restored ZIKV-Con1 (P1), respectively (* indicates P <0.05; ** indicates P <0.01; *** indicates P < 0.001).
Figure 5b shows the results of comparing the virus titer by substituting two amino acids toward PRVABC59 based on the Con1 clone to confirm the effect of the amino acid difference between the PRVABC59 virus strain and ZIKV-Con1 on the virus titer (*** indicates P <0.001; ns not statistically significant).
Figure 5c shows the results of comparative analysis of plaque sizes of PRVABC59 virus strain and ZIKV-Con1 (*** indicates P <0.001).
Figure 6a shows the full-length pBAC-T7-ZK-MR766 produced based on three pieces of DNA fragments obtained by amplifying the African Zika virus strain MR766 to P4 through passage in Vero E6 cells and using viral RNA present in the P4 supernatant. represents a clone.
Figure 6b shows the results of comparing the nucleotide and amino acid sequences of MR766 (GenBank KX830960.1) and the produced rMR766.
Figure 7 shows the results of comparing the difference in titers between restored MR766 and purchased MR766 at 72 hours after infection in A549 cells (ns is not statistically significant).
Figure 8 shows a schematic diagram of a subgenomic replicon each having two different 3'-terminal sequences constructed based on the CMV promoter.
Figure 9 shows 2'-CMA (2', known as a universal replicase inhibitor) to verify the subgenomic replicon. Renilla luciferase activity decreases over time after treatment with C-methyl adenosine), confirming that replication is inhibited by the drug (*** indicates P <0.001).
Figure 10 shows the results of confirming the effect of the 3'-end sequence on replication using a verified subgenomic replicon (* is P <0.05; *** is P <0.001; ns is not statistically significant) .
Figure 11 shows a schematic diagram of each T7 promoter-based minigenome with different 3'-terminal sequences.
Figure 12 shows the results of confirming the effect of changes in the 3'-terminal sequence on viral protein translation through luciferase activity (ns is not statistically significant).
Figure 13a is an analysis of the decomposition rate of isotopically labeled 3'-UTR (428 nt and 429 nt) in Vero E6 cell lysate by comparing the isotope signal intensity, showing the Zika virus according to the 3'-terminal sequence change. By comparing the differences in production rates and RNA decomposition patterns of derived subgenomic flavivirus RNA (sfRNA) 1, 2, and 3 (sfRNA1, sfRNA2, and sfRNA3), it was confirmed that the two terminal sequences did not significantly affect 3'-UTR stability. It is a result.
Figure 13b is an analysis of the degradation rate of the last stem-loop portion (82 nt and 83 nt) of the isotopically labeled 3'-UTR in Vero E6 cell lysate by comparing the isotope signal intensity, 3'-end This result confirms that differences in 4 to 5 nt sequences do not significantly affect the rate of stem-loop RNA degradation.
Figure 13c is an analysis of the degradation rate of isotopically labeled 3'-UTR (428 nt and 429 nt) in mosquito-derived C6/36 cell lysate by comparing the isotope signal intensity, which is related to the 3'-end sequence change. By comparing the differences in production rates and RNA decomposition patterns of Zika virus-derived sfRNA1 and sfRNA2, it was confirmed that the two terminal sequences did not significantly affect 3'-UTR stability.
Figure 13d is an analysis of the degradation rate of the last stem-loop portion (82 nt and 83 nt) of the isotopically labeled 3'-UTR in mosquito-derived C6/36 cell lysate by comparing the isotope signal intensity, 3 This result confirms that differences in the 4 to 5 nt sequence of the '-terminal sequence do not significantly affect the rate of stem-loop RNA decomposition.
Figure 14a shows the results of comparing and analyzing virus titers over time using a plaque assay after infecting Vero E6 cells and A549 cells with P1 supernatant obtained through restoration of Con1 full-length clones with different 3'-UTR terminal sequences (ns is no statistical significance).
Figure 14b shows the results of comparing and analyzing virus titers over time using a plaque assay after infecting Vero E6 cells and A549 cells with P1 supernatant obtained through restoration of rMR766 full-length clones with different 3'-UTR terminal sequences (ns is no statistical significance).
Figure 15 shows a clone in which the replicase (NS5) or the entire non-structural protein (NS1-5) among the non-structural proteins of the ZIKV-Con1 clone was replaced with the replicase or non-structural protein of MR766 (Con1/MR_NS5 and Con1/MR_NS1-5, respectively) and A schematic diagram of a clone (rMR766/Con1_NS1-5) in which the entire non-structural protein of the MR766 clone was replaced with the non-structural protein of Con1 is shown.
Figure 16a shows the results of measuring the viral titers of Con1, Con1/MR_NS5, Con1/MR_NS1-5, and rMR766 in the Vero E6 cell line on day 3 after infection by plaque assay (** indicates P <0.01; ** * indicates P <0.001; ns indicates no statistical significance).
Figure 16b shows the results of measuring the viral titers of rMR766 and rMR766/Con1_NS1-5 in Vero E6 cell line by plaque assay on the 3rd day after infection (*** indicates P <0.001).
Figure 17 shows a schematic diagram of the experimental schedule for mice (interferon α/β deficient mice, A129 mice) for comparative pathogenicity analysis of Con1, Con1/MR_NS5, Con1/MR_NS1-5, and rMR766.
Figure 18a shows the results of comparative analysis of mouse body weight changes after infecting A129 mice with Con1, Con1/MR_NS5, Con1/MR_NS1-5, and rMR766, respectively (* is P <0.05; ** is P <0.01; * ** indicates P < 0.001).
Figure 18b is a schematic diagram of the mouse (A129) infection experiment schedule for comparative analysis of pathogenicity of Con1, Con1/MR_NS5, Con1/MR_NS1-5, and rMR766. After infecting A129 mice with each virus, paralysis and survival rate of mice were measured. The comparison results are shown (* is P <0.05; ** is P <0.01).
Figure 19 shows blood samples collected on the 3rd day-post-infection (dpi) and 5th day (5 dpi) after infection of A129 mice with Con1, Con1/MR_NS5, Con1/MR_NS1-5, and rMR766, respectively. The results of comparative analysis of virus titers in blood using plaque assay are shown (** is P <0.01; *** is P <0.001; ns is not statistically significant; LOD, limit of detection).
Figures 20a and 20b show the organs (spleen, testis, kidney, brain, and liver) extracted after infecting mice with Con1, Con1/MR_NS5, Con1/MR_NS1-5, and rMR766, respectively, on the 5th day after infection. The results of comparative analysis of the viral RNA copy number (Figure 20a) and the relative RNA copy number level (fold indicated above the bar) compared to Con1 are shown (Figure 20b) (* indicates P <0.05; ** indicates P <0.01; *** indicates P <0.001; ns indicates no statistical significance).
Figure 21 is a schematic diagram of the mouse (C57BL/6) infection experiment schedule for comparative analysis of the pathogenicity of Con1, Con1/MR_NS5, Con1/MR_NS1-5, and rMR766, where F and M represent male and female mice, respectively, and Mab-5A3 is the type. I refers to a monoclonal antibody that binds to the interferon receptor and blocks interferon signaling.
Figure 22a shows the results of measuring the change in body weight of the mouse after infecting C57BL/6 mice with Con1, Con1/MR_NS5, Con1/MR_NS1-5, and rMR766, respectively (* is P <0.05; ** is P <0.01 ;*** indicates P<0.001).
Figure 22b shows the results of comparing the survival rate (determination of death when 20% of the initial body weight is reduced) of the mice after infecting C57BL/6 mice with Con1, Con1/MR_NS5, Con1/MR_NS1-5, and rMR766, respectively (** is P < 0.01).
Figure 23 shows the results of comparative analysis of the degree of clinical symptoms of mice after infecting C57BL/6 mice with Con1, Con1/MR_NS5, Con1/MR_NS1-5, and rMR766, respectively (Con1/MR_NS5 and Con1/MR_NS1-5 shows similar symptoms to Con1 (not shown).
Figure 24 shows the virus in the blood after infecting C57BL/6 mice with Con1, Con1/MR_NS5, Con1/MR_NS1-5, and rMR766, respectively, and collecting blood on the 4th day (4 dpi) and 7th day (7 dpi) after infection. The results of comparative analysis of RNA copy numbers are shown (* is P <0.05; ** is P <0.01; *** is P <0.001; ns is not statistically significant; LOD, limit of detection.).
Figures 25a and 25b show the organs (spleen, testis, kidney, brain, and liver) extracted after infecting mice with Con1, Con1/MR_NS5, Con1/MR_NS1-5, and rMR766, respectively, on the 7th day after infection. The results of comparative analysis of the viral RNA copy number (Figure 25a) and the relative RNA copy number level (fold indicated above the bar) compared to Con1 are shown (Figure 25b) (* indicates P <0.05; ** indicates P <0.01; *** indicates P <0.001; ns indicates no statistical significance).
Figure 26 shows that after infecting C57BL/6 mice with Con1, Con1/MR_NS5, Con1/MR_NS1-5, and rMR766, the brain was removed on the 7th day of infection, and the degree of inflammatory cell infiltration in the cerebral cortex was measured by H&E. Analyzed by staining, it shows differences in lesions in brain tissue caused by infection with each virus.
Figure 27a is a schematic diagram of the experimental schedule for intracerebral injection (ic) of each virus (10 ⁴ PFU) into suckling mice (ICR mice) to compare and evaluate the safety of Con1 with MR766, showing the number of animals per group, analysis items, and Indicates the measurement period.
Figure 27b shows the results of measuring the change in body weight of suckling mice for 14 days after ic injection of MR766 (heat inactivated MR766), MR766, and Con1 into ICR mice as shown in Figure 27a, and the symbols for data points The markings are the same as in Figure 27a (*** indicates P <0.001).
Figure 27c shows the results of analyzing the survival rate for 14 days after ic injection of MR766, MR766, and Con1 inactivated by heat treatment into ICR mice as shown in Figure 27a. Death was determined based on a loss of more than 25% of the initial body weight. , and the symbol for the data point is shown in the same way as in Figure 27a (*** indicates P <0.001).
Figure 27d shows the results of analyzing the viral RNA copy number and titer in brain tissue 6 and 7 days after MR766 and Con1 were injected into ICR mice by intracerebral injection as in Figure 27a.

Hereinafter, the present invention will be described in more detail through examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

Reference example

Reference Example 1. Zika virus and cell culture

Zika virus MR766 virus strain (VR-84) and PRVABC59 virus strain (VR-1843) were distributed by ATCC. Each virus stock was cultured in Vero E6 cells (green monkey kidney cells; 2 x 10 ⁶ cells/100-mm plate) cultured in DMEM (Dulbecco's modified eagle's medium) supplemented with 2% fetal bovine serum (FBS). It was propagated in

Vero E6, A549, and Vero cells were subcultured at 37°C in the presence of 5% CO ₂ in DMEM supplemented with 10% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin, and C6/36 cells were subcultured with 10% FBS. , subcultured at 28°C in DMEM supplemented with 100 U/ml penicillin and 100 μg/ml streptomycin in the presence of 5% CO ₂ .

Reference Example 2. Generation of Zika virus family tree

As of May 2016, the majority of the 264 Zika virus genome sequences stored in the GenBank database were not reported as full-length sequences, so the conserved sequences were analyzed by dividing the UTR portion and ORF portion. Among these, 12 sequences corresponded to the complete genome and were used to construct both UTRs, and 38 sequences were used to construct the conserved ORF sequence. In determining the conserved sequences of UTR and ORF, if specific nucleotides were not given, nucleotides were determined based on the original Ugandan virus strain (NC_012532.1). And for the 5'-UTR and 3'-UTR parts where in/del occurred, the sequence with the largest number of cases was selected based on the longest part. A family tree was created by aligning 38 sequences based on the amino acid sequence of the ORF among the Zika virus genomes used (see Figure 1).

Reference Example 3. Virus infection method

Vero E6 cells and A549 cells were seeded in a 60-mm plate at a cell number of ⁸ Infected cells were washed with PBS and cultured in medium supplemented with 2% FBS for a certain period of time, and the infected cell culture was stored to analyze the virus titer level.

To inject the virus into the cerebral cortex of suckling mice, 3-day-old ICR mice were used, and for the intracranial injection method, a 10-ul dose of virus was injected into the left cerebral cortex of the mouse head at a depth of approximately 2 mm using an insulin syringe. (10 ⁴ PFU) was injected. Infected mice were observed for 14 days to observe changes in body weight, and the survival rate (death was determined when the mouse lost 25% of its initial weight) was analyzed. In addition, to analyze the significance of differences between groups, weight change was statistically analyzed using Student's t -analysis, and survival rate was statistically analyzed using Log-rank (Mantel-Cox) analysis.

Reference Example 4. Virus titer analysis method

The culture medium of the infected cells was serially diluted 10-fold in serum-free DMEM and infected with Vero cells cultured in a 6-well plate (3 x 10 ⁵ /well). Afterwards, the cells were incubated with the virus at 37°C for 2 hours, and the infected cells were washed with PBS and cultured at 37°C in DMEM supplemented with 1% agarose, 2% FBS, 1% penicillin, and streptomycin in the presence of 5% CO ₂ . After 3 days of infection, cells were fixed with 10% formaldehyde and stained using 1% crystal violet solution.

Reference Example 5. Virus pathogenicity analysis method

A129 mice lacking interferon α/β receptors were infected with each restored virus (P1, 100 PFU/100 μl) via the footpad route and observed for a total of 15 days. With 12 animals per group, survival rate and clinical symptoms were observed for 6 animals in each group. Blood was collected from 6 animals in each group on the 3rd and 5th days after infection, and organs were removed on the 5th day. To observe changes in body weight, the weight of the mice was measured every day, and when the body weight decreased to less than 80% of the comparison group, they were ethically killed.

In addition, normal mouse C57BL/6 was infected with each restored virus (P1, 10 ⁴ PFU/100ul) by intraperitoneal route and observed for a total of 15 days. There were 10 animals per group, blood was collected on the 4th and 7th days after infection, and organs were removed on the 7th day. The survival rate and clinical symptoms of 5 mice in each group were observed, the weight of the mice was measured daily to check weight changes, and ethically killed when the body weight decreased below 80%.

Nursing mice were infected with the restored Con1 virus (P1, 10 ⁴ PFU/10ul) by intracranial administration, and 3 out of 10 mice per group were analyzed for viral RNA levels and titers in the brain on the 6th and 7th days after infection, and the remaining mice were infected. Weight changes in 7 animals were observed for a total of 14 days.

Example

Example 1. Production and restoration of Zika virus Con1 full-length clone

In order to prepare a full-length Zika virus Con1 clone, three DNA fragments (Con1-1, Con1- 2 and Con1-3) were chemically synthesized. Because the flavivirus gene is unstable in E. coli , a low copy number vector, pMW119, was used as a vector to clone the fragment. The pMW119 contains the T7 promoter, 51 nt of Zika virus 5'-UTR terminal sequence, multi-cloning site (MCS) including restriction enzymes NheI, ApaLI, KasI and SfiI, and 2,392 nt of Zika virus 3'-UTR terminal sequence (8,416 to 10,806 nt) and an EciI restriction enzyme site for linearization were cloned using the In-Fusion method to construct the pMW119-Con1-5'-3' intermediate vector (SEQ ID NO: 2).

For the In-Fusion cloning, an intermediate vector DNA fragment was first prepared by inverse PCR, the DNA fragments were mixed with 5x Infusion premix, reacted at 50°C for 15 minutes to transform into E. coli , and then the DNA was extracted. did.

Next, the pMW-T7-ZK-Con1 vector (SEQ ID NO: 3) was constructed by sequentially inserting the three DNA fragments of Con1 using restriction enzymes suitable for MCS. The primer sequences used to construct pMW-T7-ZK-Con1 are shown in Table 1 below.

Gene/vector primer Sequence (5'-3') ^a note Con1-B Con1-B_F tcg agc tcg gta ccc ggg taa tac gac tca cta tag AGT TGT TGA TCT GTG TGA ATC AG (SEQ ID NO: 4) Primer set for amplifying Con1-B cDNA and cloning into pMW119 for construction of pMW119-Con1-5'-3' cassette vector. Con1-B_R tga tta cgc c aa gct t gg cgg aga att ccg cgg AGA AAC CAT GGA TTT CCC CAC (SEQ ID NO: 5) Con1-1 Con1-1_F GAG TTT GAA GCG AAA GCT AGC (SEQ ID NO: 6) Primer set for Con1-1 cDNA amplification Con1-1_R GGG CAT T GT GCA C TC CCT G (SEQ ID NO: 7) Con1-2 Con1-2_F GCA GGG A GT GCA C AA TGC C (SEQ ID NO: 8) Primer set for Con1-2 cDNA amplification Con1-2_R CTT TAA AGT T GG CGC C CA TCT C (SEQ ID NO: 9) Con1-3 Con1-3_F GAT G GG CGC C AA CTT TAA AGC (SEQ ID NO: 10) Primer set for Con1-3 cDNA amplification Con1-3_R TCC TCA TAT TTC ACT GGC CTC C (SEQ ID NO: 11)

^a Zika virus sequences are indicated in uppercase letters, and other sequences are indicated in lowercase letters. In-Fusion cloning positions are indicated in bold, and restriction element positions are underlined.

Additionally, the pMW-T7-ZK-Con1 vector (SEQ ID NO: 3) was subcloned into the single copy pBeloBAC11 vector. First, an intermediate vector containing T7, HDVRz, and SacII restriction enzyme sites was constructed in the pBeloBAC11 vector. The full-length Zika virus gene (T7-ZK-Con1) amplified using the pMW-T7-ZK-Con1 vector (SEQ ID NO. 3) as a template was cloned into the linearized intermediate vector prepared as above using the In-Fusion method, and finally pBAC. -T7-ZK-Con1 (SEQ ID NO: 12) and pBAC-T7-ZK-Con1 (TTTCT) plasmid (SEQ ID NO: 13) whose terminal sequence was substituted with -TTTCT-3' were constructed (see Figure 2). The primer sequences used to prepare pBAC-T7-ZK-Con1 and its variants are shown in Table 2 below.

Gene/vector primer Sequence (5'-3') ^a note HDVr HDVr_F ggg tcg gca tgg cat ctc cac ctc ctc gcg gtc cga cct ggg cta (SEQ ID NO: 14) Primer sets to prepare full-length HDVr sequences by primer hybridization and extension HDVr_R ctt ctc cct tag cct acc gaa gta gcc cag gtc gga ccg cga gga (SEQ ID NO: 15) T7-HDVr T7-HDVr_F taa tac gac tca cta tag ggg tcg gca tgg cat ctc (SEQ ID NO: 16) Primer set for manufacturing T7-HDVr using HDVr as a template T7-HDVr_R ctt ctc cct tag cct acc g (SEQ ID NO: 17) pBAC-Vec cassette pBAC-Vec_F agg cta agg gag aag ccg cgg aag ctt gag tat tct ata gtg tca c (SEQ ID NO: 18) Primer set for producing linearized pBAC-Vec cassette through inverse PCR using the pSARS-REF-Feo plasmid as a template. pBAC-Vec_R tag tga gtc gta tta ggc gcc tga tgc ggt att ttc (SEQ ID NO: 19) T7-ZK-Con1 T7-ZK-Con1_F taa tac gac tca cta tag AGT TGT TG (SEQ ID NO: 20) Primer set to prepare T7 promoter tag full-length Con1 cDNA with -GTCT or -TTTCT 3' terminal sequence using pMW-T7-ZK-Con1 as a template. ZK-Con1(GTCT)_R atg cca tgc cga ccc AGA CCC ATG GAT TTC CCC AC (SEQ ID NO: 21) ZK-Con1(TTTCT)_R atg cca tgc cga ccc AGA AACC ATG GAT TTC CCC AC (SEQ ID NO: 22) pBAC-T7-HDVr cassette pBAC-T7-HDVr_F ggg tcg gca tgg cat ctc (SEQ ID NO: 23) Primer set for producing linearized pBAC-T7-HDVr cassette through inverse PCR using the pBAC-T7-HDVr vector as a template. pBAC-T7-HDVr_R tag tga gtc gta tta ggc gcc (SEQ ID NO: 24) GAA substitution in NS5 mut_F GAA TGG CAG TCA GTG GAG CTG CTT GCG TTG TGA AGC CAA T (SEQ ID NO: 25) Primer set to introduce GAA substitutions in the NS5 active site mut_R ATT GGC TTC ACA ACG CAA GCA GCT CCA CTG ACT GCC ATT C (SEQ ID NO: 26) ZK-MR766_NS5 MR-NS5_F CTT GGT CAA GAG ACG T GG AGG TGG GAC (SEQ ID NO: 27) Primer set to prepare ZK-MR766_NS5 using pBAC-T7-ZK-MR766 as a template MR-NS5_R CAT TAA GAT TGG TGC TTA CAA CAC TCC GG (SEQ ID NO: 28) pBAC-T7-ZK-Con1△NS5 ZK-Con1-Vec_F GCA CCA ATC TTA ATG TTG TCA GG (SEQ ID NO: 29) Primer set to prepare linearized pBAC-T7-ZK-Con1 lacking the NS5 coding gene through inverse PCR using pBAC-T7-ZK-Con1 as a template (finally, pBAC- To produce T7-ZK-Con1/MR_NS5.) ZK-Con1-Vec_R ACG TCT CTT GAC CAA G CC AGC (SEQ ID NO: 30) ZK-MR766_NS1-5 MR-NS1-5_F ACA GCC GTC TCT GCT GAC GTG GGG TGC (SEQ ID NO: 31) Primer set for preparing ZK-MR766_NS1-5 using pBAC-T7-ZK-MR766 as a template pBAC-T7-ZK-Con1△NS1-5 ZK-Con1-Vec_R_2 AGC AGA GAC GGC TGT GGA TAA G (SEQ ID NO: 32) Primer set to prepare linearized pBAC-T7-ZK-Con1 lacking the NS1-5 coding gene through inverse PCR using pBAC-T7-ZK-Con1 as a template (finally, ZK-MR766_NS1-5 and In-Fusion cloning) To produce pBAC-T7-ZK-Con1/MR_NS1-5.)

^a Zika virus sequences are indicated in uppercase letters, and other sequences are indicated in lowercase letters. In-Fusion cloning positions are indicated in bold.

To restore the produced full-length Zika virus clone, pBAC-T7-ZK-Con1 plasmid (SEQ ID NO: 12) was linearized by reacting with SacII restriction enzyme at 37°C for 4 hours, and DNA was purified by phenol/chloroform extraction and DNA precipitation using isopropanol. was purified. Afterwards, in vitro transcription and capping were performed using the mMESSAGE mMACHINE T7 Transcription kit. After purifying pure RNA using Trizol, Vero E6 cells (2 x 10 ⁶ cells/100-mm-plate) were transduced using lipofectamine. 4 hours after transfection, the medium was replaced with fresh medium, and after 2 days, a certain amount of cells and supernatant were placed on a new plate and cultured. The supernatant obtained after 3 days of culture was named P0. The P0 was infected with Vero ^E6 cells (2 ⁶ cells/100-mm-plate) and the resulting supernatant was named P2.

The P0, P1, and P2 supernatants obtained by the above method were sequentially diluted 10-fold with the virus in each supernatant in serum-free DMEM and infected with Vero cells (3 x 10 ⁵ cells/6 well-plate). Two hours after infection, the wells were washed three times with PBS and overlaid with a mixture of complete DMEM and 1% low-melting agarose containing 2% FBS and 1% penicillin/streptomycin. After 4 days, the number of plaques was counted and the virus titer was analyzed. To use it as a negative control when comparing restored virus titers and virus replication, the GDD sequence present in the active site of Zika virus replicase NS5 was replaced with GAA using the QuikChange II XL Site-Directed Mutagenesis kit (Agilent). Con1 (Con1(NS5_GAA), SEQ ID NO: 33), which lost replication enzyme activity, was prepared. In addition, the amount of viral protein expression in cells was measured through Western blot analysis.

As a result of measuring the virus titer, it was confirmed that the titer of the P0 supernatant was significantly lower than that of P1 and P2, and that the virus titer was not measured in the case of the negative control Con1 (NS5_GAA) (SEQ ID NO. 33) (see Figure 3a). In addition, the P2 virus obtained in the above experiment was infected with Huh7 cells, another cell line, and the level of viral RNA in the supernatant, the level of intracellular viral RNA, and the expression of the viral proteins NS5, NS3, and capsid protein were confirmed through Western blot (Figure see 3b)

Through the above experiment, a new recombinant virus Con1 containing the conserved sequence of Zika virus was prepared, the Con1 was restored using Vero E6 cells, and the viral titer of the restored Con1 was confirmed.

Example 2. Comparative analysis of virus titer between Con1 and Asian Zika virus strain PRVABC59

Since Con1 produced through Example 1 belongs to the Asian lineage in the phylogenetic tree analysis, the reference PRVABC59 virus strain (GenBank number: KU501215.1) (SEQ ID NO: 34), an Asian lineage distributed from ATCC, and the nucleotide and amino acid sequences were used. compared.

As a result of the comparison, it was confirmed that Con1 and PRVABC59 differed from Con1 in the capsid protein portion (I80T) and replicase portion (A2611V) and had an overall nucleotide sequence homology of 98.5% (see Figure 4).

Afterwards, to comparatively analyze the virus titer, Vero E6 and ^A549 cells (8 Two hours after infection, the cells were washed with PBS, and the supernatant was collected in new medium at each hour (24, 48, and 72 hours), and virus titer and plaque size were analyzed by plaque assay.

As a result of the analysis, it was confirmed that the virus recovered from the ZIKV-Con1 full-length clone had a reduced virus titer at each measurement time compared to the PRVABC59 virus strain in both Vero E6 cells and A549 cells (see Figure 5a). In addition, as a result of comparing the virus proliferation rate after substituting the two amino acids that differed from PRVABC59 with the PRVABC59 amino acid in the Con1 clone, it was confirmed that the Con1 virus titer did not change due to these amino acid substitutions (see Figure 5b). Additionally, it was confirmed that the plaque size of Con1 was smaller than that of PRVABC59 (see Figure 5c).

Through the above experiment, it was verified that the new recombinant virus Con1 containing the conserved sequence of Zika virus showed attenuated characteristics compared to PRVABC59, an Asian Zika virus strain, due to several sequence differences in the Con1 gene.

Example 3. Construction and restoration of cell-adapted Zika virus rMR766 full-length clone

To produce the full-length Zika virus MR766 clone, the MR766 virus strain distributed from ATCC was used. After extracting RNA from the supernatant (P4) after four passages in Vero E6, cDNA was synthesized through RT-PCR, and 1 to 8,032 nt, 8,012 to 9,288 nt, and 9,272 to 9,272 nt of the ZIKV-MR766 sequence (SEQ ID NO: 35). Three DNA fragments (MR766-1, MR766-2, and MR766-3) corresponding to the 11,807 nt position were amplified through PCR. Afterwards, a linearized intermediate vector containing T7, HDVRz, and SacII sequences was constructed through inverse PCR using the pBAC-T7-ZK-Con1 plasmid (SEQ ID NO. 12) as a template, and the three amplified MR766 DNA fragments were incubated with In-Fusion. By cloning using this method, pBAC-T7-ZK-MR766 plasmid (SEQ ID NO: 36) was finally produced (see Figure 6a). When comparing the nucleotide and amino acid sequences of the produced recombinant MR766 and the reference MR766 virus strain (GenBank number: KX830960.1) (SEQ ID NO: 37), it was confirmed that a total of 5 nucleotides and 2 amino acid sequences were changed (see Figure 6b). ). The primer sequences used to prepare pBAC-T7-ZK-MR766 and its variants are shown in Table 3 below.

Gene/vector primer Sequence (5'-3') ^a note MR766-1 MR766-1_F taa tac gac tca cta tag AGT TGT TGA TCT GTG TGA GTC AGA CTG C (SEQ ID NO: 38) Primer set for amplifying MR766-1 cDNA fragment MR766-1_R GTT CCA CCC ATA GCT TTG CAC CA (SEQ ID NO: 39) MR766-2 MR766-2_F GTG CAA AGC TAT GGG TGG AAC (SEQ ID NO: 40) Primer set for amplifying MR766-2 cDNA fragment MR766-2_R GTG TCC CAG CCA GCA GT G TCA T (SEQ ID NO: 41) MR766-3 MR766-3_F ACT GCT GGC TGG GAC AC C (SEQ ID NO: 42) Primer set for amplifying MR766-3 cDNA fragment MR766-3_R gat gcc atg ccg acc c AG AAA CCA TGG ATT TCC CCA C (SEQ ID NO: 43) pBAC-T7-HDVr cassette pBAC-T7-HDVr_F_2 ggg tcg gca tgg cat ctc cac ct (SEQ ID NO: 44) Primer set to prepare a linearized pBAC-T7-HDVr cassette through inverse PCR using the pBAC-T7-HDVr vector as a template (finally, pBAC-T7- through MR766-1, 2, 3 DNA fragments and In-Fusion cloning) To produce ZK-rMR766.) pBAC-T7-HDVr_R_2
tat agt gag tcg tat ta g gcg cct gat gc (SEQ ID NO: 45) ZK-Con1_NS1-5 Con1-NS1-5_F TCT GCT GAT GTG GGG TGC TC (SEQ ID NO: 46) Primer set to prepare ZK-Con1_NS1-5 using pBAC-T7-ZK-Con1 as a template Con1-NS1-5_R ATT GGT GCT TAC AGC AC T CCA G (SEQ ID NO: 47) pBAC-T7-ZK-MR766 △NS1-5 ZK-MR766-Vec_F GTG CTG TAA GCA CCA AT T TTA GTG (SEQ ID NO: 48) A primer set to prepare linearized Pbac-T7-ZK-MR766 with a deletion of the NS1-5 coding gene through inverse PCR using pBAC-T7-ZK-rMR766 as a template (finally, the ZK-Con1_NS1-5 DNA fragment and In- To produce pBAC-T7-ZK-rMR766/Con1_NS1-5 through Fusion cloning.) ZK-MR766-Vec_R CCC CAC ATC AGC AGA AAC AG (SEQ ID NO: 49)

^a Zika virus sequences are indicated in uppercase letters, and other sequences are indicated in lowercase letters. In-Fusion cloning positions are indicated in bold.

Additionally, a comparison of virus titers between restored rMR766 and MR766 distributed from ATCC was conducted. After infecting ^A549 cells (8 As a result of the analysis, there was no statistically significant difference in the virus titers of the two MR766s (see Figure 7).

Through the above experiment, a cell-adapted full-length Zika virus clone rMR766 containing the MR766 gene was prepared, and the viral titer of the restored rMR766 was confirmed.

Example 4. Analysis of the effect of changes in the 3'-terminal sequence on the virus through Zika virus full-length clones and derivatives

Zika viruses from Africa and Asia all have a common 3'-terminal sequence (-GTCT-3'), but the MR766 virus strain is known to have a specific 3'-terminal sequence (-TTTCT-3'). Accordingly, the following experiment was performed to determine whether changes occurred in replication, translation, RNA stability, and proliferation of Zika virus when the 3'-terminal sequence was replaced.

(1) Subgenomic replicon construction and analysis of the impact of 3'-end sequence changes using it

To determine the effect that changes in the 3'-terminal sequence may have on virus replication, a subgenomic replicon (sgRep) was created based on the pBAC-T7-ZK-Con1 full-length clone (SEQ ID NO: 12). Produced.

To prepare the subgenomic replicon, a CMV promoter-based full-length clone was first produced. Specifically, each ZK-Con1-HDVr DNA having a different terminal sequence was obtained through PCR using pBAC-T7-ZK-Con1 (SEQ ID NO: 12) and pBAC-T7-ZK-Con (TTTCT) (SEQ ID NO: 13) as templates. Fragments (SEQ ID NOs. 50 and 51) were prepared, and pBAC-CMV-5'-3' was linearized through inverse PCR using pSARS-REP-Feo (vector with SARS-CoV subgenomic replicon cDNA) as a template. pBAC-CMV-ZK-Con1 (SEQ ID NO: 53) and pBAC-CMV-ZK-Con1 (TTTCT) (SEQ ID NO: 54) were produced by cloning with an intermediate vector (SEQ ID NO: 52) and In-Fusion method. Afterwards, the FMDV 2A lyase gene sequence was added to enable independent processing between the Renilla luciferase gene sequence and the Puromycin gene sequence through sequential bridge PCR. The final prepared Puro-2A-Rluc-2A DNA fragment and the linearized sgRep intermediate vector excluding structural proteins (including capsid protein N-terminal C and envelope protein C-terminal E30 coding sequences) were converted to In-Fusion through inverse PCR. pBAC-CMV-ZK-Con1_sgRep (SEQ ID NO: 55) and pBAC-CMV-ZK-Con1_sgRep (TTTCT) (SEQ ID NO: 56) were produced by cloning using this method (see FIG. 8). The primer sequences used to construct pBAC-CMV-ZK-Con1 and subgenomic replicons are shown in Table 4 below.

Gene/vector primer Sequence (5'-3') ^a note CMV-ZK-Con1-HDVr CMV-ZK-Con1-HDVr_F cgt tta gtg aac cgt AGT TGT TGA TCT GTG TGA ATC AGA C (SEQ ID NO: 57) Primer set for preparing CMV promoter full-length Con1 cDNA containing the HDVr gene using pBAC-T7-ZK-Con1 and pBAC-T7-ZK-Con1(TTTCT) as templates. CMV-ZK-Con1-HDVr_R caa cta gaa ggc aca g ct tct ccc tta gcc tac cga ag (SEQ ID NO: 58) pBAC-CMV-5′-3′ cassette pBAC-CMV_F ctg tgc ctt cta gtt g cc ag (SEQ ID NO: 59) A primer set to prepare a linearized pBAC-CMV-5'-3'cassette through inverse PCR using the pSARS-REF-Feo plasmid as a template (finally using the CMV-ZK-Con1-HDVr DNA fragment and In-Fusion cloning) To produce pBAC-CMV-ZK-Con1 and pBAC-CMV-ZK-Con1(TTTCT).) pBAC-CMV_R acg gtt cac taa acg agc tct g (SEQ ID NO: 60) FMDV 2A FMDV 2A_F gtg aaa cag act ttg aat ttt gac ctt ctt aag ctg gcg gga gac (SEQ ID NO: 61) Primer set for preparing FMDV 2A gene (2A) by primer hybridization and extension FMDV 2A_R ggg ccc ggg gtt gga ctc gac gtc tcc cgc cag ctt aag aag (SEQ ID NO: 62) Puro Puromycin_F atg acc gag tac aag ccc ac (SEQ ID NO: 63) Primer set for preparing cDNA encoding the puromycin resistance gene (Puro) derived from pLenti CMV/TO Puro empty (w175-1) plasmid (Addgene plasmid #17482) Puromycin_R ggc acc ggg ctt gcg g (SEQ ID NO: 64) P-2A Puro-2A_F ccg caa gcc cgg tgc c gt gaa aca gac ttt gaa ttt tga cc (SEQ ID NO: 65) Primer set for producing P-2A cDNA by fusing Puro and FMDV 2A (2A) genes 2A-Rluc_R cac ctt gga agc cat ggg c cc ggg gtt gga c (SEQ ID NO: 66) Puro-2A Puromycin_F atg acc gag tac aag ccc ac (SEQ ID NO: 63) Primer set for preparing cDNA of Puro fused to FMDV 2A Puro2A-Rluc_R cac ctt gga agc cat ggg (SEQ ID NO: 67) Rluc 2A-Rluc_F gcc cat ggc ttc caa ggt g ta cga c (SEQ ID NO: 68) Primer set for preparing cDNA encoding Renilla luciferase gene (Rluc) from pRL-TK plasmid (Takara Bio) Rluc-2A_R cgt acg ctg ctc gtt ctt cag (SEQ ID NO: 69) R-2A Rluc-2A_F ctg aag aac gag cag cgt acg gtg aaa cag act ttg aat ttt gac c (SEQ ID NO: 70) Primer set for producing R-2A cDNA by fusing Rluc and FMDV 2A (2A) genes 2A-E30_R CAG ACC CAA CCA CAT ggg ccc ggg gtt gga c (SEQ ID NO: 71) Rluc-2A 2A-Rluc_F gcc cat ggc ttc caa ggt g ta cga c (SEQ ID NO: 68) Primer set for preparing cDNA of Rluc fused to FMDV 2A 2A-Rluc2A-E30_R CAG ACC CAA CCA cat ggg c (SEQ ID NO: 72) Puro-2A-Rluc-2A Puromycin_F atg acc gag tac aag ccc ac (SEQ ID NO: 63) Primer set for producing Puro-2A-Rluc-2A gene 2A-Rluc2A-E30_R CAG ACC CAA CCA cat ggg c (SEQ ID NO: 72) sgRep cassette E30_F atg TGG TTG GGT CTG AAC ACA AAG (SEQ ID NO: 73) Primer set to prepare a linearized subgenomic replicon cassette through inverse PCR using pBAC-CMV-ZK-Con1 and pBAC-CMV-ZK-Con1(TTTCT) as templates, (ultimately Puro-2A-Rluc- To produce pBAC-CMV-ZK-Con1-sgRep and pBAC-CMV-ZK-Con1-sgRep(TTTCT) through 2A DNA fragment and In-Fusion cloning.) C38-Puro_R CTT GTA CTC GGT CAT cag aag tcc ggc tgg cag (SEQ ID NO: 74)

^a Zchyrus sequences are indicated in uppercase letters, and other sequences are indicated in lowercase letters. In-Fusion cloning positions are indicated in bold.

Additionally, an experiment was performed to analyze the effect of changes in the 3'-terminal sequence on virus replication using the subgenomic replicon system constructed as described above. Prior to this, 500 ng of subgenomic replicon (pBAC-CMV-ZK-Con1_sgRep) (SEQ ID NO: 55) was added to Huh7 cells (6-well plate, 3 x 10 ⁵ cells/well) using lipofectamine. Transduction was performed, and the replicon system was verified using 2'-CMA (2'-C-Methyladenosine), a universal replicase inhibitor. Afterwards, the effect of changes in the 3'-terminal sequence on virus replication was confirmed through luciferase activity.

As a result of the experiment, it was confirmed that when Huh7 cells transduced with the subgenomic replicon were treated with 2'-CMA, virus replication was inhibited as time passed from 24 to 48 hours, and the subgenomic replica was produced. The cloning ability of cones was verified (see Figure 9).

In addition, as a result of confirming the effect on virus replication according to changes in the 3'-end sequence in the subgenomic replicon verified as above, in the case of the subgenomic replicon with the 3'-end sequence of MR766, In the 48-hour period, there was a slight difference in luciferase activity between the subgenomic replicon with the 3'-end sequence of Con1, and in the case of the NS5_GAA subgenomic replicon (SEQ ID NO: 75), luciferase activity Activity was significantly reduced (see Figure 10).

(2) Minigenome production and analysis of the impact of 3'-end sequence changes using it

To determine the effect that changes in the 3'-terminal sequence may have on viral protein translation, a minigenome was constructed.

To prepare the minigenome, first, pBAC-T7-ZK-Con1 (SEQ ID NO: 12) and pBAC-T7-ZK-Con1 (TTTCT) (SEQ ID NO: 13) were used as templates to produce the N-terminal C38 coding gene of the capsid protein. An intermediate vector (SEQ ID NO: 76) was created by linearizing the 5'-UTR and 3'-UTR containing the sequence by inverse PCR using the F primer (SEQ ID NO: 82) and R primer (SEQ ID NO: 83), and an intermediate vector (SEQ ID NO: 76) was created at the coding position. Renilla luciferase gene sequence was cloned using the In-Fusion method. Afterwards, pcDNA3.1_Con1-minigenome-Rluc (SEQ ID NO: 77) and pcDNA3.1_Con1-minigenome (TTTCT)-Rluc (SEQ ID NO: 78) were created by subcloning into the pcDNA3.1 vector (see FIG. 11). Primer sequences used for minigenome production are shown in Table 5 below.

Gene/vector primer Sequence (5'-3') ^a note Renilla luciferase Rluc_F CCA GCC GGA CTT CTG atg gct tcc aag gtg tac gac (SEQ ID NO: 79) Primer set for preparing cDNA encoding Renilla luciferase gene (Rluc) from pRL-TK plasmid (Takara Bio) Rluc_R CAT TAA GAT TGG TGC tta cgt acg ctg ctc gtt ctt cag (SEQ ID NO: 80) pBAC-T7-5'-3' cassette 3'-UTR_F GCA CCA ATC TTA ATG TTG TCA GGC C (SEQ ID NO: 81) Primer set to prepare linearized pBAC-T7-5'-3' cassette through inverse PCR using pBAC-T7-ZK-Con1 and pBAC-T7-ZK-Con1(TTTCT) as templates. C38_R CAG AAG TCC GGC TGG CAG (SEQ ID NO: 82) T7-Rluc-HDVr T7-Rluc-HDVr_F taa tac gac tca cta tag AGT TGT TGA TCT GTG TG (SEQ ID NO: 83) Primer set for preparing T7 promoter Rluc cDNA containing the HDVr gene using pBAC-T7-ZK-Con1-minigenome-Rluc and minigenome(TTTCT)-Rluc as templates. T7-Rluc-HDVr_R ccg cgg ctt ctc cct tag (SEQ ID NO: 84) pcDNA3.1
cassette 3.1_F agg gag aag ccg cgg ctt ctg agg cgg aaa gaa cca gct g (SEQ ID NO: 85) Primer set to create pcDNA3.1 cassette
(Finally, use In-Fusion cloning with the T7-Rluc-HDVr gene to produce pcDNA3.1_Con1-minigenome-Rluc and pcDNA3.1_Con1-minigenome-Rluc(TTTCT).) 3.1_R tag tga gtc gta tta gcg tat atc tgg ccc gta cat c (SEQ ID NO: 86)

^a Zika virus sequences are indicated in uppercase letters, and other sequences are indicated in lowercase letters. In-Fusion cloning positions are indicated in bold.

Additionally, an experiment was performed to analyze the effect of changes in the 3'-terminal sequence on viral protein translation using the minigenome produced as described above. For this purpose, 1 μg of minigenome (pBAC-ZK-Con1-minigenome) RNA was transduced into Huh7 cells (6-well plate, 3 x 10 ⁵ cells/well), and luciferase activity was compared after 24 hours. .

As a result of the experiment, no significant difference was measured in the luciferase activity of the minigenome having the 3'-end sequence of Con1 and the minigenome having the 3'-end sequence of MR766 (see FIG. 12). Through this, it was confirmed that changes in the 3'-terminal sequence did not significantly affect viral protein translation.

(3) Analysis of the effect of changes in 3’-terminal sequence on viral RNA stability and virus proliferation

The following experiment was performed to determine whether the 3'-terminal sequence could affect viral RNA stability and viral replication.

First, decay analysis was performed to determine the effect of 3'-terminal sequence on viral RNA stability. For this purpose, a Forward primer (SEQ ID NO: 87) containing the T7 promoter sequence and a Reverse primer (SEQ ID NO: 88) containing the 3'-end sequence of Con1 (-GTCT-3') and the 3'-end sequence of MR766 ( 3'-UTR DNA (-GTCT-3') (SEQ ID NO: 90) and 3'-UTR DNA (-TTTCT-3') using a reverse primer (SEQ ID NO: 89) containing -TTTCT-3') SEQ ID NO: 91) was amplified. Afterwards, in vitro transcription was performed in the presence of isotope α ³² P-GTP using the in vitro T7 Megascript kit to produce isotopically labeled 3'-UTR RNA with 428 nt or 429 nt. In addition, using the forward primer (SEQ ID NO: 92) containing the T7 promoter sequence and the reverse primer of SEQ ID NO: 88 or 89, an isotopically labeled 3'-UTR stem-loop (3') having 82 nt or 83 nt -SL) was produced (SEQ ID NOs: 93 and 94). 10 pmol of 3'-UTR RNA synthesized as above was mixed with 40 μg of Vero E6 cell lysate and reacted at 37°C for 0, 1, 2, and 4 hours. In addition, because Zika virus is a mosquito-borne virus, decay analysis was additionally performed using C6/36 cells, a mosquito cell line. For this purpose, 10 pmol of 3'-UTR RNA synthesized as above was mixed with 10 μg of C6/36 cell lysate and reacted at 37°C for 0, 5, 10, 20, and 60 minutes. After each reaction time, RNA was extracted and loaded on an 8% polyacrylamide gel containing 8M Urea, and signal intensity was analyzed through phosphoimage analysis.

As a result of the analysis, no significant difference was observed in RNA stability between the 3'-end sequence of Con1 and the 3'-end sequence of MR766 (see Figures 13a to 13d).

Second, an experiment was performed to determine the effect of the 3'-terminal sequence on virus proliferation. pBAC-T7-ZK-Con1 (SEQ ID NO: 12), pBAC-T7-ZK-Con1 (TTTCT) (SEQ ID NO: 13), pBAC-T7-ZK-MR766 (SEQ ID NO: 36) and pBAC-T7-ZK-MR766 ( GTCT) (SEQ ID NO: 95) full-length clone P1 virus was restored, and Vero E6 and A549 cells ( ⁸ analyzed.

As a result of the experiment, no significant difference was observed in RNA stability between the 3'-end sequence of Con1 and the 3'-end sequence of MR766 (see FIGS. 13a to 13d), and pBAC-T7-ZK-Con1 (SEQ ID NO: 12) There was no significant difference in the virus titer of pBAC-T7-ZK-Con1(TTTCT) (SEQ ID NO: 13) (see Figure 14a), and pBAC-T7-ZK-MR766 (SEQ ID NO: 36) and pBAC-T7- The virus titer of ZK-MR766 (GTCT) (SEQ ID NO: 95) also showed no significant difference (see Figure 14b). Through this, it was confirmed that changes in the 3'-terminal sequence did not significantly affect viral RNA stability and virus proliferation.

(4) Sintering

Through the above experiments, it was confirmed that the 3'-terminal sequence replacement did not affect virus replication ability, protein translation, viral RNA stability, and virus proliferation.

Example 5. Construction of ZIKV-Con1 clone-based variants and ZIKV-MR766 clone-based variants and comparative analysis of their virus titers

Based on the ZIKV-Con1 clone and the ZIKV-MR766 clone, variants in which non-structural proteins were replaced were created, and an experiment was performed to determine the effect of the non-structural proteins on virus replication ability by comparing the virus titers of the variants.

Specifically, pBAC-T7-ZK-Con1, which has the replication enzyme (NS5) among the non-structural proteins of MR766 or the entire non-structural protein (NS1-5) of MR766, based on the pBAC-T7-ZK-Con1 full-length clone (SEQ ID NO: 12). /MR_NS5 (SEQ ID NO: 96) and pBAC-T7-ZK-Con1/MR_NS1-5 (SEQ ID NO: 97), and the entire non-structural protein of Con1 (NS1) based on the pBAC-T7-ZK-MR766 full-length clone (SEQ ID NO: 36) -5) pBAC-T7-ZK-rMR766/Con1_NS1-5 (SEQ ID NO: 98) were each produced using the In-Fusion cloning method (see FIG. 15).

Afterwards, the clones were restored, and a comparative analysis of virus titers was performed using the P1 supernatant. To compare titers, Vero ^E6 cells (8 did.

As a result, the titer of Con1/MR_NS5 (SEQ ID NO: 96) in Vero E6 cells did not show a statistically significant difference compared to the titer of Con1 (SEQ ID NO: 12), but Con1/MR_NS1-5 (SEQ ID NO: 97) and In the case of rMR766 (SEQ ID NO: 36), the titer was significantly higher than that of Con1 (SEQ ID NO: 12) in the 72-hour period after infection, and the titer of Con1/MR_NS1-5 (SEQ ID NO: 97) was higher than that of rMR766 (SEQ ID NO: 36). It was confirmed that it was significantly lower than (see Figure 16a). On the contrary, in the case of rMR766/Con1_NS1-5 (SEQ ID NO: 98), in which rMR766 (SEQ ID NO: 36) was replaced with the entire non-structural protein of Con1 (SEQ ID NO: 12), the titer was significantly lower than that of rMR766 (SEQ ID NO: 36). Confirmed (see FIG. 16b).

Through the above experiment, it was confirmed that the clone with the non-structural protein of MR766 had a higher viral titer than the clone with the non-structural protein of Con1, thereby verifying that the non-structural protein of MR766, i.e., the replicase complex, had a superior replication ability than the replicase complex of Con1. .

Example 6. Pathogenicity analysis by virus strain using interferon-deficient mice

To compare the pathogenicity of Con1 (SEQ ID NO: 12), Con1/MR_NS5 (SEQ ID NO: 96), Con1/MR_NS1-5 (SEQ ID NO: 97), and rMR766 (SEQ ID NO: 36) viruses, each virus was infected in mice. , experiments were conducted to measure changes in body weight, survival rate, clinical symptoms, viral titer in serum, and viral titer in organs of mice.

Specifically, the footpad of interferon α/β deficient mice was infected with 100 PFU/100 μl of each virus, and the weight change, survival rate, and clinical symptoms of the mice were observed daily for a total of 15 days, and body weight was not reduced to less than 80%. In this case, death was recorded. The experiment was conducted with 12 animals in each group. Survival rate and clinical symptoms were observed for 6 animals in each group. Blood was collected from 6 animals in each group on the 3rd day after infection, and blood was collected on the 5th day and organs (spleen, testis, Kidney, brain, and liver) were removed (see Figure 17). Using collected blood, the viral titer present in the serum was measured by plaque assay, the viral titer in the extracted organs was analyzed by qPCR, and the viral RNA titer was expressed as a relative change based on Con1.

As a result of observing the weight change, survival rate, and clinical symptoms of mice, rMR766 (SEQ ID NO: 36)-infected mice began to die from the 6th day, and all died by reducing to less than 80% of the initial body weight on the 7th day. All mice infected with Con1/MR_NS1-5 (SEQ ID NO: 97) in which the crude protein was substituted showed severe paralysis of the hind limbs and one mouse died on the 7th day, 2 mice died on the 8th day, and 3 mice died on the 9th day. . In the case of Con1/MR_NS5 (SEQ ID NO: 96), 1 animal died on the 8th day with mild symptoms of hind limb paralysis, 2 animals on the 9th day, and 1 animal on the 11th day decreased to less than 80% of the initial weight. Accordingly, a total of 4 mice were killed ethically, and the remaining 2 mice all survived until the 21st day. Finally, it was confirmed that all Con1 (SEQ ID NO: 12) infected mice survived until day 15 without any specific symptoms or significant body weight changes (see FIGS. 18A and 18B).

As a result of measuring the virus titer in the serum of blood collected on the 3rd and 5th days, the cell experiment results in Example 5 showed that the virus titer of Con1/MR_NS1-5 (SEQ ID NO: 97) was Con1 (SEQ ID NO: 12). ) and Con1/MR_NS5 (SEQ ID NO: 96), which were significantly higher than the titers, there was no significant difference in the virus titer in serum 3 days after infection. In the case of rMR766 (SEQ ID NO: 36), on day 3, similar to the cell experiment results in Example 5, the virus titer was confirmed to be significantly higher than that of other virus strains (see FIG. 19).

As a result of measuring the virus titer in the organs (spleen, testis, kidney, brain, and liver) removed on the 5th day, the cell experiment results in Example 5 showed that the virus titer of Con1/MR_NS1-5 (SEQ ID NO: 97) was Unlike the titers of Con1 (SEQ ID NO: 12) and Con1/MR_NS5 (SEQ ID NO: 96), which were significantly higher, the virus titers of Con1 (SEQ ID NO: 12) and Con1/MR_NS1-5 (SEQ ID NO: 97) were found only in the kidney and liver. A significant difference appeared. In the case of rMR766 (SEQ ID NO: 36), the virus titer was confirmed to be significantly higher than that of other virus strains in all other organs, except that the titer was similar to Con1/MR_NS1-5 (SEQ ID NO: 97) in the spleen. (see Figures 20a and 20b).

Through the above experiment, it was confirmed that the non-structural protein NS5 of MR766, an African virus strain, does not significantly affect replication ability in mice, but significantly contributes to differences in pathogenicity. As the African virus strain evolved into an Asian virus strain, Zika virus It was confirmed that the pathogenicity of non-structural proteins was reduced.

Example 7. Pathogenicity analysis by virus strain using normal mice

To compare the pathogenicity of Con1 (SEQ ID NO: 12), Con1/MR_NS5 (SEQ ID NO: 96), Con1/MR_NS1-5 (SEQ ID NO: 97), and rMR766 (SEQ ID NO: 36) virus strains in a general mouse model (C57BL/6). , After infecting mice with each virus, experiments were performed to measure changes in body weight, survival rate, clinical symptoms, virus titer in serum, and virus titer in organs of the mouse. Since Zika virus infection does not occur easily in a general mouse model, 2 mg of IFNAR antibody was injected intraperitoneally 1 day before infection, 0.5 mg 1 day after infection, and 0.5 mg 4 days after infection.

Infection with the virus was specifically conducted through the intraperitoneal route with each virus at 10 ⁴ PFU/100 μl, and the weight change, survival rate, and clinical symptoms of the mice were observed daily for a total of 15 days, and the body weight was reduced to less than 80%. In this case, death was recorded. The experiment was conducted with 10 animals in each group, 5 animals in each group were observed for survival rate and clinical symptoms, 5 animals in each group had blood collected on the 4th day after infection, and blood was collected on the 7th day and organs (spleen, testis, Kidney, brain, and liver) were removed (see Figure 24). Using collected blood, the viral RNA copy number and titer present in the serum were measured by plaque assay, the viral RNA copy number in the extracted organs was analyzed by qPCR, and the viral RNA level was expressed as a relative change based on Con1.

As a result of observing the weight change, survival rate, and clinical symptoms of mice, unlike the results of interferon α/β deficient mice, only rMR766 (SEQ ID NO: 36) infected mice began to die from the 7th day with symptoms of paralysis of the hind limbs. All died after reducing to less than 80% of their initial weight on the 8th day. In the case of Con1/MR_NS1-5 (SEQ ID NO: 97), there was a significant difference compared to Con1-infected mice from the 7th day of infection, but there was a tendency to gradually recover body weight. On the other hand, it was confirmed that all Con1 (SEQ ID NO: 12) and Con1/MR_NS5 (SEQ ID NO: 96) infected mice survived until day 15 without any specific symptoms or significant body weight changes (see FIGS. 22A and 22B).

As a result of measuring the virus titer in the serum of blood collected on the 4th and 7th days, unlike the interferon α/β deficient mouse of Example 6, the viral RNA level and virus titer in the serum were 4 and 7 days after infection. It was confirmed that Con1/MR_NS5 (SEQ ID NO: 96), Con1/MR_NS1-5 (SEQ ID NO: 97), and rMR766 (SEQ ID NO: 36) all showed significant differences compared to Con1 (SEQ ID NO: 12) infected mice. In the case of rMR766 (SEQ ID NO: 36), similar to Example 6, it was confirmed that the virus titer was significantly higher than that of other virus strains (see FIG. 24).

In addition, as a result of measuring the viral titer in the organs (spleen, testis, kidney, brain, and liver) removed on the 7th day, unlike the viral titer in the serum, the significance of Con1 and Con1/MR_NS5 could not be confirmed in all organs, but testis It was confirmed that the viral RNA level of Con1/MR_NS1-5 (SEQ ID NO: 97) was significantly higher than that of Con1 (SEQ ID NO: 12) and Con1/MR_NS5 (SEQ ID NO: 96) in all organs except. Similar to the above experiment, rMR766 (SEQ ID NO: 36) was confirmed to have a significantly higher virus titer than other virus strains (see FIGS. 25A and 25B).

Additionally, lesions due to infection of the cerebral cortex were confirmed through H&E staining in the brain extracted on day 7. Consistent with the mouse survival rate and body weight change, the infiltration of many immune cells around blood vessels was confirmed in the cerebral cortex of rMR766 (SEQ ID NO: 36) infected mice, and Con1/MR_NS1-5 (SEQ ID NO: 97) Additionally, lesions were identified. In addition, lesions, although slight, were confirmed in Con1/MR_NS5 (SEQ ID NO: 96) infected mice, but it was confirmed that Con1 showed no difference in lesions compared to mock infection (see FIG. 26).

Through experiments with the normal mice and interferon α/β deficient mice, it was confirmed that interferon receptors and their downstream signals play an important role in virulence, and similarly to Example 6, It was confirmed that the pathogenicity of Zika virus non-structural proteins decreased as the virus strain evolved into an Asian virus strain.

Example 8. Safety evaluation experiment of Con1 using suckling mice

To confirm the safety of Con1 (SEQ ID NO: 12) in suckling mice, an experiment was conducted using the MR766 (ATCC, VR-84) virus as a control. 10 ⁴ PFU of the MR766 virus strain was heat inactivated at 56°C for 30 minutes and used as a negative control, and 10 ⁴ PFU of the Con1 and MR766 virus strains were each injected into the cerebral cortex of suckling mice at a volume of 10-μl. By injection, changes in body weight and survival rate were measured for 2 weeks. A total of 10 mice were administered per group. For 3 mice, the RNA level and titer of the virus present in the mouse brain on the 7th day after infection were analyzed using qPCR and plaque analysis, and for the remaining 7 mice, changes in body weight and survival rate were measured (Figure 27a).

As a result of measuring the change in body weight for each virus injection group, body weight increased to a similar level compared to the control group until the 4th day of infection, but decreased from the 5th day, and very serious clinical symptoms accompanied by weight loss occurred on the 6th day of infection ( withdrawal, decreased motility, etc.) and all mice died. However, in the case of Con1, the body weight gradually increased and was maintained for up to 2 weeks, and although there was a large difference in body weight compared to the control group, it was confirmed that no death occurred except for one animal on the 14th day of infection (see Figure 27b).

Based on the above weight change, a weight loss of more than 25% was set as the criterion for death and the change in survival rate was observed. As a result of observing the change in survival rate, 1 mouse infected with the MR766 virus strain died after 5 days and all 6 mice after 6 days after infection, regardless of weight loss. . However, 3 mice infected with the Con1 virus strain died due to weight loss of more than 25% after 8 days of infection, 2 mice died on the 9th day, and 2 mice died on the 10th day due to weight loss (see Figure 27c).

Then, on the 7th day of infection, the level and titer of viral RNA in the brain were analyzed. In the case of the MR766 infection group, all mice died on the 6th day, and the viral RNA and titer in the brain of the mice that died on the 6th day were analyzed. In the Con1 infection group, the viral RNA and titer in the brain were analyzed on the 7th day. As a result, it was confirmed that the viral RNA level and titer in the brain of the MR766 virus strain infection group was higher than that of the Con1 virus strain (see Figure 27d).

Through the above experiment, it was confirmed that the Con1 virus strain also has virulence, but is relatively attenuated compared to the MR766 virus strain.

Claims (27)

Full-length clone of Zika virus containing the T7 bacterial phage promoter.
The full-length clone of Zika virus according to claim 1, wherein the clone includes a region cleaved by one or more enzymes selected from the group consisting of HDVRz and SacII.
The clone according to claim 1, wherein the clone is prepared using the pBeloBAC11 vector as a template.
The clone of claim 1, wherein the Zika virus is an Asian Zika virus, an African Zika virus, or a chimeric virus of the Asian Zika virus and the African Zika virus.
The clone according to claim 4, wherein the Asian Zika virus includes sequences commonly conserved in Asian and African Zika virus sequences.
The clone according to claim 5, wherein the clone is a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 12.
The clone according to claim 5, wherein the Zika virus recovered from the clone is attenuated than the PRVABC59 Zika virus.
The clone according to claim 4, wherein the African Zika virus comprises a nucleotide sequence mutated from the nucleotide sequence encoding the non-structural protein NS3 of the MR766 Zika virus.
The clone according to claim 8, wherein the clone is a polynucleotide consisting of the base sequence of SEQ ID NO: 36.
The clone according to claim 1, wherein the Zika virus in the clone has a 3' terminal base sequence mutated.
The method of claim 10, wherein the mutation is a substitution of -TTTCT-3' when the 3' terminal nucleotide sequence of the Zika virus is -GTCT-3', or the 3' terminal nucleotide sequence of the Zika virus is -TTTCT- In the case of 3', the clone is substituted with -GTCT-3'.
The clone according to claim 11, wherein the clone is a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 13 or 95.
The method of claim 4, wherein the chimeric virus is an Asian Zika virus in which a nucleotide sequence encoding a non-structural protein of an Asian Zika virus is replaced with a nucleotide sequence encoding a non-structural protein of an African Zika virus, or an acetabulum sequence of an African Zika virus. A clone, which is an African Zika virus in which the nucleotide sequence encoding the crude protein is replaced with the nucleotide sequence encoding the non-structural protein of the Asian Zika virus.
The clone according to claim 13, wherein the non-structural protein is at least one selected from the group consisting of NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5.
The clone according to claim 13, wherein the clone is a polynucleotide consisting of one of the base sequences of SEQ ID NOs: 96 to 98.
The method of claim 13, wherein the Asian Zika virus in which the nucleotide sequence encoding the non-structural protein of the Asian Zika virus is substituted with the nucleotide sequence encoding the non-structural protein of the African Zika virus has the replication capacity of the Zika virus or the Asian Zika virus. Clones with increased pathogenicity.
The method of claim 13, wherein the African Zika virus in which the nucleotide sequence encoding the non-structural protein of the African Zika virus is replaced with the nucleotide sequence encoding the non-structural protein of the Asian Zika virus has a replication capacity of Zika virus or higher than that of the African Zika virus. Clones with reduced pathogenicity.
The clone of claim 1 includes a base sequence encoding a capsid protein of the Zika virus, a base sequence encoding an envelope protein of the Zika virus, and a base sequence encoding a non-structural protein of the Zika virus,
A subgenomic replicon of Zika virus containing a base sequence encoding the CMV promoter.
The replicon according to claim 18, wherein the replicon is a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 55.
The replicon according to claim 18, wherein the 3' terminal base sequence of the Zika virus in the replicon is mutated.
The replicon according to claim 20, wherein the replicon is a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 56.
A minigenome of Zika virus containing a base sequence encoding the capsid protein of Zika virus in the clone of claim 1.
The minigenome of claim 22, wherein the minigenome is a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 77.
The minigenome of claim 22, wherein the 3' terminal nucleotide sequence of the Zika virus in the minigenome is mutated.
The minigenome of claim 24, wherein the minigenome is a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 78.
Introducing the clone or derivative thereof of claim 1 into a cell;
treating the cells with a candidate agent;
Measuring the titer of Zika virus in cells treated with the drug; and
A screening method for a drug for preventing or treating Zika virus infection, comprising the step of selecting a drug whose titer is reduced compared to a control group not treated with the drug.
A Zika virus vaccine composition comprising genetic material of the Zika virus reconstructed from the clone of claim 1 or a derivative thereof, a protein expressed by the genetic material, or a fragment thereof.