CN113637697A

CN113637697A - DENV-4 full-length infectious clone and construction method thereof

Info

Publication number: CN113637697A
Application number: CN202110790056.1A
Authority: CN
Inventors: 李义平; 杨炀
Original assignee: Sun Yat Sen University
Current assignee: Sun Yat Sen University
Priority date: 2021-07-13
Filing date: 2021-07-13
Publication date: 2021-11-12

Abstract

The invention constructs a dengue virus serotype 4 (DENV-4) full-length infectious clone pZG14D 4. The invention uses eukaryotic transcription vector pTight, and adds 7xTRE tetracycline response element in front of promoter to reduce the leakage expression of protein in bacteria, to construct DENV-4 infectious clone pZG14D 4. The system can successfully and efficiently save live viruses, virus positive cells can be observed the next day after transfection, and the titer of the viruses in the supernatant of the fifth day can reach 10⁶FFU/ml. The genomic cDNA clone which is continuously passaged five times in the bacteria has no base mutation and no impurity peak after being sequenced, and can still generate high-titer virus after being transfected into Huh7.5 cells (10)⁵FFU/ml), which indicates that the plasmid can stably replicate in bacteria and has genetic stability. The growth curve of the proliferation of the rescued progeny virus and the parental virus in vitro is equivalent.

Description

DENV-4 full-length infectious clone and construction method thereof

Technical Field

The invention relates to the field of genetic engineering, in particular to a DENV-4 full-length infectious clone and a construction method thereof.

Background

A series of diseases caused by infection with dengue virus (DENV) are currently viral diseases transmitted by important mosquitoes in tropical regions, and the transmission vectors are Aedes aegypti and Aedes albopictus. Before 1970, 9 countries had severe dengue epidemics, and dengue has now affected more than 100 countries in tropical and subtropical regions, with approximately 3.9 million cases of dengue infection occurring each year, with approximately 70% of cases appearing in asia. The world health organization estimates that the global incidence of disease observed has increased 30-fold over the past 50 years. Dengue virus today constitutes a significant threat to global public health, with about two-fifths of the world population at risk for dengue infection.

Globally, dengue viruses have four serotypes (DENV-1, DENV-2, DENV-3 and DENV-4) and can cause Dengue Fever (DF), Dengue Hemorrhagic Fever (DHF) and Dengue Shock Syndrome (DSS). Dengue fever is an acute febrile illness with headache, retroorbital pain, myalgia, arthralgia, rash, hemorrhagic symptoms and/or leukopenia. Hallmark features of DHF include thrombocytopenia, bleeding and plasma leakage signs, which may lead to hypotensive shock (DSS). Although the body can remain protective against this serotype for decades after infection, secondary infection with a different serotype increases the risk of severe dengue, which if left untreated, can be at a mortality rate of up to 20%. At present, there are no specific antiviral therapeutic drugs, and there are several vaccines used in local countries or regions or in clinical trials, but the phenomenon of Antibody-Dependent Enhancement (ADE) of dengue brings great challenges to the development and popularization of vaccines.

Dengue virus is a single positive-strand RNA-enveloped flavivirus whose 10.7kb genome contains a 5', 3' UTR and an open reading frame, which encodes a polyprotein that is cleaved post-translationally by host and viral proteases into three structural proteins (C, capsid; pr/M, membrane protein; E, envelope protein) and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS 5).

The reverse genetics system is an important means of studying viral life history from the molecular level of the viral genome, as well as pathogenesis, vaccine development and antiviral drug development. For RNA viruses, we routinely study them in vitro by establishing their full-length infectious clones and replicons. The development and marketing of HCV DAA drugs is a typical case of application of the HCV replicon system to antiviral drug screening.

Transfection of flavivirus genomic RNA into cell lines produces infectious viral particles, a phenomenon that has prompted the development of reverse genetics for flaviviruses. However, one of the problems that plague the construction of cDNA clones of flavivirus genomes and other viruses is that plasmids containing full-length or partial sequences of viruses are prone to fragment loss and recombination in transformed bacteria, and toxicity to bacteria is generated, so that stable clones cannot be obtained. Therefore, a series of approaches have been devised, including circumventing the bacterial amplification pathway: recombining in yeast to obtain clone; obtaining a full-length genome by using long-fragment PCR, and transcribing RNA in vitro to transfect to cells; obtaining a plasmid containing a full-length genome and a eukaryotic promoter by using a CPEC reaction (circular polymerase extension cloning technology), and transfecting the plasmid to a cell; obtaining a plasmid containing a full-length genome and a eukaryotic promoter by using a Gibson reaction, and transfecting the plasmid to a cell; several mutually overlapped PCR fragments containing full-length genome, eukaryotic promoter and 3' UTR terminal element are obtained by ISA reaction (infectious subgenomic amplicon), and then transfected to cells. Methods for amplification in bacteria include: modifying a sequence which predicts that a genome sequence can become a promoter in escherichia coli, and reducing the possibility of modification to stabilize the genome; inserting a splicing-removable intron in a ZIKV NS1 sequence in a eukaryote; adding a 7XTRE tetracycline response element in front of a eukaryotic promoter to reduce the leaky expression of the protein in bacteria, and the like.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a DENV-4 full-length infectious clone and a construction method thereof. The invention refers to a method for adding a 7XTRE tetracycline response element in front of a eukaryotic promoter, and constructs a stable and efficient DENV-4 full-length infectious clone.

In order to achieve the purpose, the invention adopts the technical scheme that: providing a DENV-4 full-length infectious clone, wherein the sequence of the DENV-4 full-length infectious clone is SEQ ID NO:1 is shown.

As a preferred embodiment of the full-length infectious clone of the present invention, the full-length infectious clone is constructed as follows:

(1) the viral cDNA was divided into three fragments for PCR, PCR 1: 1-3384bp, PCR 2: 3385 and 69887 bp, PCR 3: 6989 10470bp, adding A tail to PCR product, connecting to T vector, and the corresponding cloning plasmids are pT-DV4-1, pT-DV4-2 and pT-DV 4-3;

(2) the 3' UTR of the classical strain DENV-4H241 was synthesized in the company: 10566 and 10664bp, after double digestion, the T4 ligase is connected to pT-DV4-3 vector after double digestion to obtain the cloned plasmid pT-DV4-3-3UTR, so that the 5' UTR of DENV-4H241 classical strain is respectively added before and after the genome is obtained: 1-36bp and 3' UTR: 10566 and 10664bp strain full-length sequence;

(3) construction of full-length infectious clones: the vector used is pTight vector, the vector contains pBR322 source high copy replication origin, hepta-tetracycline response element, cytomegalovirus eukaryotic promoter, hepatitis D virus ribozyme antigenome sequence, monkey virus 40 polyadenylation signal sequence, ampicillin resistance and other elements, and the competent cell used is C41; the cloning method comprises the following specific steps: the fragment of PCR1 and the fragment of PCR2 are fused into a fragment A through fusion PCR, the sequence of the genome on pT-DV4-3-3UTR is a fragment B, the fragment A and the fragment B are firstly recombined onto a linearized vector pTight through a homologous recombination mode, and finally the clone pZG14D4 with the full-length genome is obtained.

The invention also provides a recombinant virus prepared by the full-length infectious clone.

The invention also provides application of the full-length infectious clone in preparation of genetically stable dengue viruses.

The invention also provides application of the full-length infectious clone or the recombinant virus in preparation of a medicine for researching dengue fever, dengue hemorrhagic fever or dengue shock syndrome.

The invention also provides application of the full-length infectious clone or the recombinant virus in preparation of anti-dengue virus drugs.

The invention also provides the application of the full-length infectious clone or the recombinant virus in the preparation of dengue virus epitope.

The invention also provides the application of the full-length infectious clone or the recombinant virus in the aspect of preparing vaccines.

The invention has the beneficial effects that:

the invention constructs a dengue virus full-length infectious clone pZG14D 4. ZG14D4 is an epidemic strain of dengue virus in the Guangdong region, belonging to dengue virus serotype IV, which according to the Rico-Hesse dengue virus genotype nomenclature belongs to the Indonesian subtype. We constructed infectious clone pZG14D4 using the eukaryotic transcription vector pTight by adding a 7xTRE tetracycline response element in front of the promoter to reduce leaky expression of the protein in bacteria. The system can successfully and efficiently save live viruses, virus positive cells can be observed the next day after transfection, and the titer of the viruses in the supernatant of the fifth day can reach 10⁶FFU/ml. Clones which were serially passed five times in bacteria were sequenced without base mutation and without hetero-peak, transfected into Huh7.5 cells, and still able to produce high-titer virus (10)⁵FFU/ml), which indicates that the plasmid can stably replicate in bacteria and has genetic stability. The growth curve of the proliferation of the rescued progeny virus and the parental virus in vitro is equivalent.

Drawings

FIG. 1: the genome of DENV and the function of viral proteins. A single open reading frame encodes a polyprotein precursor that is co-cleaved post-translationally into three structural proteins (C, capsid; pr/M, membrane protein; E, envelope protein) and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS 5). The possible functions of these proteins during infection are depicted in the figure. The 5 'and 3' untranslated regions (UTRs) are simplified RNA secondary and tertiary structures. aa, an amino acid; c, capsid protein; CS, cyclization sequence; e, enveloping; m, a membrane; RdRp, RNA-dependent RNA polymerase; UAR, upstream AUG region; VR, variable region; SL, stem-loop.

FIG. 2: phylogenetic tree of the strains epidemic DENV-4 GZ14D 4. One dengue virus epidemic phylogenetic tree in 14 years, Guangdong province. Epidemic strain ZG14D4 belongs to dengue virus serotype IV, and the genotype belongs to Indonesia subtype according to the Rico-Hesse dengue virus genotype nomenclature.

FIG. 3: schematic representation of the construction of the full-length infectious clone pZG14D4 of DENV-4. Schematic representation of infectious clone of the DENV-4 strain ZG14D 4. Comprises a heptatetracycline response element, a cytomegalovirus (CMVmin) eukaryotic promoter at the 5' end of a genome, a full-length sequence of a strain, an antigenome sequence of hepatitis delta virus ribozyme (HDVr) and a monkey virus 40(SV40) polyadenylation signal sequence.

FIG. 4: construction procedure of a full-Length infectious clone of DENV-4. DENV-4 strain ZG14D4 genome and cloning steps. Firstly, two PCR fragments A and B covering the whole DENV-4 virus genome are obtained by PCR, and then the fragment A and the fragment B are firstly recombined onto a linearized vector pTight in a homologous recombination mode to obtain a full-length clone.

FIG. 5: the DENV-4 full-length infectious clone rescued live virus. (A) pZG14D4 transfected cells were observed by immunofluorescence experiments. Mu.g of pZG14D4 was co-transfected with 0.5. mu.g of pTet-off into Huh7.5 cells, and six hours after transfection, the cells on

days

2, 3, 4 and 5 were subjected to immunofluorescence experiments, respectively, with the amount of the cells being 0 hour after the cell change. Green and blue represent DENV protein and nucleus, respectively. (B) The DENV-4 infectious clonal plasmid efficiently rescues live virus. Mu.g of pZG14D4 was co-transfected with 0.5. mu.g of pTet-off into Vero cells and Huh7.5 cells, and supernatants were harvested at 3, 5 and 7 days (daysposttransfection) after transfection, respectively, and were diluted ten-fold to infect Vero cells and immunofluorescence assay was performed 48h after infection, and virus supernatant titers were determined by FFU.

FIG. 6: the DENV-4 full-length infectious clone was stable after serial passages in bacteria. (A) The infectious clone plasmid (P0) is transformed into C41 competent cells, and plasmid is extracted by shaking to obtain P1 generation plasmid, and the plasmid is continuously passed for five times and five times according to the method to obtain P5 generation plasmid. (B) Mu.g of P5 generation infectious clone plasmid and 0.5. mu.g of pTet-off were co-transfected into Huh7.5 cells, supernatants were harvested at 2, 3, 4, and 5 days (days-transfection) after transfection, respectively, and were diluted ten-fold to infect Vero cells, immunofluorescence experiments were performed 48h after infection, and virus supernatant titers were determined by FFU.

FIG. 7: the growth curve of the saved progeny virus and parental virus in the cells of the DENV-4 infectious clone. (A) Growth curves of progeny virus and parental virus in huh7.5 cells. (B) Growth of progeny virus and parental virus in Vero cells. The progeny virus rescued by the DENV-4 full-length infectious clone and the parental virus infect Huh7.5 and Vero cells at the MOI of 0.05, supernatant is collected at 1, 2, 3, 4 and 5 days (days-infection) after infection respectively, the Vero cells are infected by ten-fold stepwise dilution of the supernatant, an immunofluorescence experiment is carried out 48h after infection, and the titer of the virus supernatant is determined by FFU. Three independent replicates were performed and the data are shown as Mean ± SD. Data statistics used T-test, indicates significant (p <0.05), indicates very significant ((p <0.01), l.o.d., limitation of detection), and indicates minimal detection (101 FFU/ml).

Detailed Description

To more clearly illustrate the technical solutions of the present invention, the following embodiments are further described, but the present invention is not limited thereto, and these embodiments are only some examples of the present invention.

The materials and methods adopted by the invention are as follows:

materials (I) and (II)

Experimental cell lines: the cell line used in the present invention, Huh7.5, was provided by professor Charlie Rice, university of Zhongshan, 293T, Vero, BHK-21, and Zhang, university of A549, university of Zhongshan.

Plasmid: the cloning vector pTight was supplied by professor Yueh, institut of health, Taiwan.

Second, method

(1) Virus in vitro amplification (DENV)

The popular strains of DENV Guangzhou are all from the subject group of yellow professor, Zhongshan medical college, Zhongshan university. Vero cells were first plated at 5X 10 per well⁵Spreading the cells on 6-well plate, culturing at 37 deg.C for about 16 hr, and standingAnd (4) adhering to the wall. At the time of infection, the virus solution was diluted with serum-free medium DMEM so that the virus MOI of each well at the time of infection became 0.01 and the volume of the solution reached 800 μ l, just covering the cell surface. 37 ℃ and 5% CO₂Incubating for 2 hr, adsorbing virus, removing supernatant, washing with PBS for 1-2 times, adding 2.5ml DMEM containing 2% FBS, 37 deg.C, and 5% CO₂After culturing for 72 hours under the conditions, the cell supernatant (i.e., the amplified virus solution) was collected, filtered through a 0.45 μm filter, and stored in a refrigerator at-80 ℃.

(2) RNA extraction

1) Adding 1ml or 750 μ l Trizol (the addition of the Trizol is insufficient and can cause DNA pollution of extracted RNA) into a sample (a proper amount of cells or 250 μ l of virus), repeatedly sucking or covering, violently mixing (fully breaking cells), and performing lysis at room temperature for 5min (standing for separation of nucleic acid-protein complex);

(after this step, pause at-80 ℃ C.)

2) Add 200. mu.l chloroform, mix vigorously to make the color uniform pink, and let stand at room temperature for 2min (visible separation). And (3) Trizol: the chloroform ratio is generally 5: 1;

3) centrifuge at 12000rpm for 15min at 4 deg.C (confirm that the EP tube is covered with the lid and the refrigerated centrifuge is opened to pre-cool). There are three layers in the EP tube at this time: the upper aqueous phase is RNA, the middle layer is DNA precipitate, and the lower organic phase is protein;

4) taking the supernatant to another new EP tube (wiping off water vapor on the tube wall, and ensuring that the tube wall is unstable in layering and cannot shake, and selecting a small-range pipettor to slowly suck, and enabling the gun head to face the new EP tube to see clearly); if the requirement on the purity of RNA is high, centrifuging at 12000rpm for 15min, and re-taking the supernatant (selection);

5) adding isopropanol with the same volume, turning upside down, mixing, and standing at-20 deg.C for more than 20 min; [ 0.25ml of isopropyl alcohol and 0.25ml of high salt solution (0.8M sodium citrate and 1.2M NaCl) were added to the aqueous phase per 1ml of TRIzol and mixed and centrifuged to leave proteoglycan and polysaccharide in the solution and precipitate pure RNA with high efficiency ]

6) Centrifuging at 12000rpm at 4 deg.C for 15 min;

7) the supernatant was discarded (it was poured directly and then sucked dry upside down), and 1ml of 75% ethanol (DEPC-treated RNase free water: anhydrous ethanol ═ 1: 3v/v), the pellet was allowed to rise, washed gently upside down, and centrifuged at 7500rpm at 4 ℃ for 5 min. Repeating the steps once;

8) pouring most of ethanol, centrifuging slightly, sucking dry by a liquid transfer machine, drying RNA precipitate at room temperature for 3-5 min (excessive drying can cause great reduction of RNA solubility), adding a proper amount (20-50 mu l) of DEPC (diethyl phthalate) for dissolving, measuring RNA quality and concentration, and carrying out 2 mu l agarose gel electrophoresis.

(3) Reverse transcription of viral fragment RNA

RT (which is carried out on ice during preparation) is prepared according to the following components, and in order to ensure the accuracy of the preparation of the reaction liquid and reduce errors caused during split charging, the reaction liquid is prepared according to a slightly larger volume of the actual dosage.

Composition (I)	Volume of
		gene specific primer(10μM)	2.5μl
RNA	9μl
		dNTP(10mM)	1μl

Heat shock at 65 deg.C for 2min, placing on ice for 5min, and adding the following components:

composition (I)	Volume of
		5×First-StrandBuffer	4μl
DTT(0.1M)	1μl
		RNAsin(20-40U/μl)	0.5μl
SuperScriptⅢ(200U/μl)	2μl

Treating at 60 deg.C for 50min (reverse transcription process) and 70 deg.C for 15min (reverse transcriptase inactivation process) on PCR instrument, and storing at 4 deg.C.

Then 1. mu.l of RNAse H (1-4U/. mu.l) and 1. mu.l of RNAseT (1000U/. mu.l) were added and treated at 37 ℃ for 20min in order to remove excess RNA.

Example 1 phylogenetic Tree of the circulating strain, strain ZG14D4, DENV-4

Inoculating a clinical virus sample to Vero cells for culture amplification to obtain a virus with higher titer, extracting virus RNA, inverting to obtain cDNA, amplifying to a sequence (10529bp) covering most of genome by three-section PCR, and sequencing to identify the strain. According to the nomenclature of the Rico-Hesse dengue virus genotypes, the E protein gene sequences of the DENV-4 genotypes and representative strains of the DENV1-4 are downloaded on GenBank, and are compared with the E protein sequence of the strains by MEGA7 software, and a phylogenetic tree is constructed by a neighbor-joining (NJ) method. From the evolutionary tree, it can be seen that the circulating strain ZG14D4 belongs to dengue virus serotype IV, and the genotype belongs to indonesian subtype (fig. 2).

EXAMPLE 2 construction of full-Length infectious clone pZG14D4

When constructing the full length, the inventor adds 5 'UTR (1-36bp) and 3' UTR (10566-10664bp) of the DENV-4H241 classical strain before and after the full length respectively, and the cloned elements comprise a heptatetracycline response element, a cytomegalovirus (CMVmin) eukaryotic promoter, a full-length sequence of the strain, an antigenome sequence of hepatitis delta virus ribozyme (HDVr) and a polyadenylation signal sequence of monkey virus 40(SV40) (figure 3).

The construction of full-length infectious clone pZG14D4 included the following steps:

(1) the cDNA is divided into three segments for PCR, PCR 1(1-3384bp), PCR 2(3385 and 6987bp), PCR 3 (69888 and 10470bp), PCR products are added with A tail and then are connected to a T vector, and the corresponding cloning plasmids are pT-DV4-1, pT-DV4-2 and pT-DV4-3 respectively.

(2) The DENV-4H241 classical strain 3' UTR (10566-10664bp) is synthesized by the company, and after double digestion (MluI-NsiI), T4 ligase is connected to pT-DV4-3 vector which is subjected to double digestion (MluI-NsiI), so as to obtain the cloned plasmid pT-DV4-3-3 UTR. Thus, we obtained the full-length sequences of the strains with the 5 'UTR (1-36bp) and the 3' UTR (10566-10664bp) of the DENV-4H241 classical strain added before and after the genome respectively.

(3) Full-length infectious clones were constructed. The vector used was a pTight vector comprising a pBR 322-derived high copy replication origin, a heptatetracycline response element, a cytomegalovirus (CMVmin) eukaryotic promoter, an antigenome sequence of hepatitis delta virus ribozyme (HDVr), a monkey virus 40(SV40) polyadenylation signal sequence, an ampicillin resistance element and the like, and the competent cell used was C41.

Initially, we planned to clone the three fragments cloned on the T vector into pTight vector by homologous recombination plus enzymatic ligation. We first homologously recombined the fragment from PCR1 into pTight vector to obtain clone pTight DV 4-1. Then the fragment of PCR2 and pTight DV4-1 are subjected to double digestion (AflII-RsrII), the recovered fragment is connected by T4 ligase, and transformed, and when colonies are identified, correct clones cannot be grown, and the method cannot obtain transformants of the fragment (1-6937 bases) on vectors.

Thus, we adjusted the cloning method to the specific steps: the fragment of PCR1 and the fragment of PCR2 are fused into fragment A through fusion PCR, the sequence of the genome on pT-DV4-3-3UTR is fragment B, the fragment A and the fragment B are firstly recombined onto a linearized vector pTight through a homologous recombination mode, and finally the clone pZG14D4 with the full-length genome is obtained (figure 4), and the sequence of the clone is shown as SEQ ID NO: 1.

Example 3 pZG14D4 rescues live viruses with high efficacy

pZG14D4 is a eukaryotic promoter, and the plasmid can be directly transfected into cells for expression with pTet-off. The co-transfer plasmid is required because a heptatetracycline response element is added before the pZG14D4 CMV-min promoter, which serves to reduce the toxicity of the clone to bacteria due to transcription in bacteria, to promote the transcriptional expression of the promoter when the rTA protein expressed from the pTet-off plasmid binds to the operator region in cells, and to shut down the system when DOX is added. Full-length infectious clone plasmid pZG14D4 was co-transfected with pTet-off into Vero and Huh7.5 cells, and we found that virus-positive cells were observed the next day after transfection, and that the virus titer in the supernatant reached 10 on the fifth day⁶At the FFU/ml level (fig. 5), the system was able to successfully and efficiently rescue live virus.

Example 4 stability of 4 pZG14D4 after serial passages in bacteria

Because of the instability of the flavivirus genome in bacteria, we serially passaged the pZG14D4 plasmid in bacteria to explore the genetic stability of the plasmid. The infectious clone plasmid (P0) was transformed into C41 competent cells, the plasmid was extracted by shaking to obtain plasmid generation P1, and the plasmid generation P5 was obtained by five serial passages (FIG. 6A), and the full length was sequenced without base mutation. The P5 generation infectious clone plasmid was co-transfected into Huh7.5 cells with pTet-off and still able to produce high titers of virus (10)⁵FFU/ml) (FIG. 6B), indicating that the plasmid is stably replicated in bacteria and is genetically stable.

Example 5 growth curves of rescued progeny and parental viruses in vitro

To evaluate the proliferation efficiency of the progeny virus rescued from infectious clone versus the parental virus in vitro, we infected Huh7.5 and Vero cells with both viruses at the same MOI, collected cell supernatants each day 1-5 days post infection, and measured the virus titer in the supernatants to plot the virus growth curves (fig. 7). In Huh7.5 cells, the virus can reach the peak of the titer on the third day, and in Vero cells, the virus can reach the peak of the titer on the fourth day, and the titer is equivalent. Therefore, we can consider that the saved progeny virus and the parental virus have the same proliferation efficiency in vitro.

Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

SEQUENCE LISTING

<110> Zhongshan university

<120> DENV-4 full-length infectious clone and construction method thereof

<130> 2021.06.04

<160> 1

<170> PatentIn version 3.3

<210> 1

<211> 10653

<212> DNA

<213> Artificial Synthesis

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tttttaatag acggaccaga cacctccgaa tgccccaatg agcgaagagc atggaacttt 2880

tttgaggtag aagactatgg atttggcatg tttacgacca acatatggat gaaattccga 2940

gaaggaagtt cagaagtgtg tgaccataga ctaatgtcgg cggcaatcaa agatcagaaa 3000

gctgtgcatg ctgacatggg ttattggata gagagctcaa aaaaccagac ctggcagata 3060

gagaaagcat cccttattga agtgaaaaca tgtctgtggc ccaaaaccca cacgctgtgg 3120

agcaatggag tgctggaaag tcagatgctc attccaagat catatgcagg ccccttttca 3180

cagcacaatt accgtcaggg ctatgccacg caaaccgtgg gcccatggca cctaggcaaa 3240

ttggagatag actttggaga atgccccggg acaacagtcg caattcggga ggattgtgac 3300

cacagaggcc catctttgag aaccaccact gcatctggaa aactggtcac gcaatggtgc 3360

tgccgctcct gcacgatgcc tcccttaagg ttcttgggag aagatggatg ttggtatggg 3420

atggagatca ggcctctgag tgaaaaagaa gagaacatgg tcaaatcaca ggtaacggcc 3480

ggacagggca catcagaaac tttttctatg gggctgttat gcctgacctt gtttgtggaa 3540

gaatgcttga ggagaagagt tactaggaag cacatgatat tggttgtggt aatcaccttt 3600

tgcgctatca tcctaggagg tctcacatgg atggacttac tacgagccct tatcatgtta 3660

ggggatacta tgtctggtag aataggagga cagatccacc tagccatcat ggcagtgttt 3720

aagatgtcac caggatacgt gctgggtgtg tttttaagga aacttacttc aagagagaca 3780

gcgctgatgg taataggaat ggccatgaca acggtgtttt caatcccaca tgaccttatg 3840

gaactcattg atggaatatc attggggttg atattactaa aaatagtaac acattttgac 3900

aacacccaag tgggaaccct agccctttcc ttgacttttt taagatcaac aataccatta 3960

gtcatggctt ggaggaccat tatggctgtg ttctttgtgg tcacactcat tcctttgtgc 4020

aggacaagct gtcttcaaaa acagtcccac tgggtagaaa taacagcact catcctagga 4080

gcccaggctt tgccagtgta cctaatgact ctcatgaagg gagcctcaag gagatcttgg 4140

cctctcaatg agggcataat ggctgtaggt ttggtgagcc tcttaggaag cgccctcttg 4200

aagaatgatg ttcctttggc tggcccaatg gtggcaggag gcttgcttct agcggcttac 4260

gtaatgagtg gtagctcagc agacctgtca ctagagaaag ctgccaacgt gcagtgggat 4320

gaaatggcgg acataactgg ctcaagccca atcatagaag tgaagcagga tgaagatggc 4380

tctttttcca tacgggacgt cgaggaaacc aatatgatga ctctcttggt gaaactggca 4440

ctgataacag tatcaggtct ttaccccttg gcaattccag tcacaatggc attgtggtat 4500

atttggcaag tgaagacaca aagatcagga gctctgtggg acgtcccctc acccgctgcc 4560

acccagaaag ccacattgtc tgaaggagtg tataggatta tgcaaagagg gctgtttggg 4620

aaaactcaag ttggagtagg aatacacatg gaaggcgtat tccacacaat gtggcatgta 4680

acaaggggat cagtgatctg tcatgagaca gggagattag agccatcttg ggctgacgtc 4740

agaaacgaca tgatatcata cggtggggga tggaggctcg gagacaaatg ggacaaagaa 4800

gaagatgttc aggtcttagc catagaacca gggaaaaatc ccaaacatgt ccaaacgaaa 4860

cctggccttt tcaagaccct aactggagaa attggagcag taactctaga tttcaaaccc 4920

ggaacgtctg gctctcccat cattaacaag aaagggaaag ttattggact ctacggaaat 4980

ggagtggtta ccaaatcagg tgattatgtc agtgccataa cgcaagccga gagaattggt 5040

gagccagact atgaagtaga tgaggacatt tttcgaaaga aaagattaac cataatggat 5100

ttacaccccg gagctggaaa gacaaaaaga atcctcccat caatagtcag agaggcttta 5160

aaaaggaggc tgcgaaccct gattttggct cccacgagag tggtggcggc cgagatggaa 5220

gaggccctac gtggactgcc aatccgttat cagactccag ctgtgaaatc agagcacaca 5280

ggaagagaga ttgtagacct catgtgtcat gcaaccttta caacaagact tttgtcatca 5340

accagggttc ctaattataa cctcatagtg atggatgaag cacatttcac tgacccttgc 5400

agtgtcgcgg ctagaggata catttcaacc agggtggaaa tgggagaggc agcagctatc 5460

ttcatgactg caacccctcc tggagcgaca gatcccttcc cccagagcaa cagcccaata 5520

gaagacattg agagggaaat tccagaaagg tcatggaaca cagggttcga ctggataaca 5580

gactaccaag ggaaaactgt gtggtttgtt cccagcataa aagctggaaa tgacattgca 5640

aattgtttga gaaagtcggg aaagagagtg atccagttga gcaggaaaac ctttgacacg 5700

gagtatccaa agacgaaact cacggactgg gatttcgtgg tcaccacaga tatatctgaa 5760

atgggggcca atttcagagc tgggagagtg atagacccca ggagatgcct caagcctgtt 5820

atcctaacag atgggccaga gagagttatt ctagcaggtc caatcccagt aactccagca 5880

agtgccgctc agagaagagg gcgaataggt aggaatccag cacaggaaga tgatcaatat 5940

gttttctccg gagacccact aaaaaatgat gaagatcatg ctcactggac agaagcaaag 6000

atgctgcttg acaatatcta cacccctgaa gggataattc caacactgtt tggtccggaa 6060

agggaaaaaa cccaagccat tgatggagag tttcgcctca gaggggaaca aaggaagact 6120

tttgtggaat taatgaagag aggagacctt ccggtgtggc tgagctacaa ggtagcttct 6180

gccggcatct cctacaaaga tcgagaatgg tgcttcacag gagaaaggaa taaccaaatt 6240

ttagaagaaa acatggaggt tgaaatttgg actaaagagg gagaaaagaa aaagctaagg 6300

ccaaaatggt tggatgcacg tgtgtacgct gatcccatgg ctttgaagga tttcaaggag 6360

tttgccagtg gaagaaagag cataactctc gacatcctaa cagagattgc cagtttgcca 6420

acttaccttt cctctaaggc caagctggct cttgacaaca tagtcatgct ccacacaaca 6480

gaaaaaggag ggagggccta ccaacacgcc ctgaacgaac tcccggagtc actggaaacg 6540

ctcatgcttg tagctctact aggtgctatg acagcaggca tcttcctgtt tttcatgcag 6600

gggaaaggaa taggaaaatt gtcaatgggt ctaatagcca tagccgtggc tagtggcttg 6660

ctctgggtag cagaaattca gccccagtgg atagcagctt caatcatact ggagttcttt 6720

cttatggtgc tgttgatacc agaaccagaa aaacaaagga ccccacaaga caatcaattg 6780

atctacgtca tattgaccat tctcaccatc atcggtctta tagcagccaa tgagatgggg 6840

ctgattgaaa aaacaaaaac tgactttggg ttttaccagg taaaaacaga aacaactatt 6900

cttgatgtgg atttgagacc agcctcagca tggacactct atgcggtagc caccaccatt 6960

ttgactccca tgctgagaca caccatagaa aacacgtctg ccaacctatc tctagcagcc 7020

attgccaacc aagcagctgt tttaatggga cttggaaaag gatggccgct ccacagaatg 7080

gaccccggtg tgccgctgtt agcaatagga tgttattctc aagtgaaccc aacaaccctg 7140

acagcatcct tagtcatgct ttttgtccat tatgccataa taggtccagg attgcaggca 7200

aaagccacac gagaggccca gaaaaggaca gctgctggga tcatgaagaa ccccacggtg 7260

gacgggataa cagtaataga tctagaacca atatcgtatg acccaaaatt tgaaaagcaa 7320

ttagggcaag tcatgttact agtcctgtgt gctggacaat tgcttttgat gagaacaaca 7380

tgggctctct gcgaagtctt gactttggcc acaggaccag ccatgacctt gtgggagggc 7440

aacccgggaa ggttttggaa cacgaccata gctgtatcca cagccaacat tttcagagga 7500

agctatttgg caggagctgg actagctttt tcgctcataa agaatgtaca aacccctagg 7560

aggggaactg ggactacagg agagacactg ggagagaagt ggaagagaca gttaaactca 7620

ttagacagaa aagagtttga agagtacaaa agaagtggaa tactagaagt ggataggact 7680

gaagccaagt ctgccctaaa agatggatcc aaaatcaagc atgcggtgtc cagagggtct 7740

agtaagatca gatggattgt tgaaagaggg atggtaaagc cgaaagggaa ggtcgtggat 7800

cttggttgcg ggaggggagg atggtcctat tacatggcga cactcaagaa cgtgactgaa 7860

gtgaaaggat atacaaaagg aggtccagga catgaggaac caatccccat ggctacttat 7920

ggctggaact tggtcaaact ccattcaggg gttgatgtgt tttacaaacc cactgagcag 7980

gtggacacct tgctctgtga cattggggag tcgtcctcta atccgacaat agaggaagga 8040

aggacattga gagtcttgaa gatggtagag ccatggctct cttcaaaacc tgagttctgc 8100

atcaaagtcc tcaaccccta catgccaaca gttatagaag agctggagaa actccagaga 8160

agatatggcg gaagcctcgt cagatgccct ttatccagga attccaccca tgagatgtat 8220

tgggtgtcag gtgcgtcggg aaacatcgtg agttctgtga acacaatatc aaagatgctg 8280

ttgaacaggt tcacaacaag gcataggaaa cccacttatg agaaggacgt ggatcttggg 8340

gcaggaacga gaagtgtctc cactgaaaca gaaaaaccag acatgacagt tattgggaga 8400

aggcttcaga gattgcaaga agagcacaag gaaacctggc actatgacca ggaaaaccca 8460

tacagaacct gggcgtatca tggaagctat gaagctccct cgacaggctc agcatcctcc 8520

atggtgaacg gggtagtaaa actgctgaca aaaccctggg atgtgattcc aatggtgacc 8580

cagttggcca tgacagacac aacccctttt gggcaacaaa gagtgttcaa ggagaaggtg 8640

gataccagaa caccacaacc aaaacccggc acacgaatga ttatgaccac gacagccaat 8700

tggctgtggg ctctcctcgg gaagaagaaa agccccagat tgtgtacaag ggaagagttc 8760

atctcaaaag ttaggtcaaa tgcagccata ggcgcagtct ttcaggaaga acaaggatgg 8820

acatcagcca gtgaagctgt gaatgacagc cgattctggg aactggttga caaagaaagg 8880

gctctgcacc aggaaggaaa atgtgaatcg tgtgtctata acatgatggg aaagcgtgag 8940

aaaaaattag gagagtttgg tagggccaag ggaagccgag caatctggta catgtggcta 9000

ggggcgcggt tcctggaatt tgaagccctg ggtttcttga atgaagatca ctggtttagc 9060

agagaaaact catggagtgg agtggaaggg gaaggtctgc acagattggg atatatcttg 9120

gaggatatag acaagcagga aggagatcta atatatgctg acgacacagc aggctgggac 9180

acgagaatca ctgaggatga ccttttaaat gaagaactga tcacagaaca gatggccccc 9240

catcacaaga ccctagccaa agccattttc aaactaacct atcaaaacaa agtggtgaaa 9300

gtcctcagac ccacaccgaa aggagcggtg atggacatta tatccaggaa agaccaaaga 9360

ggtagtggac aagttggaac atatggttta aacacattca ctaacatgga agttcaactt 9420

atccgccaaa tggaagctga aggagtcatc acacaagacg acatgcagaa cccaaaaggg 9480

ttgaaagaaa gagttgagaa atggctgaca gagtgtggcg tcgacaggtt gaagaggatg 9540

gcaatcagtg gagatgattg cgtggtgaag cccctagatg agaggttcag cacctccctc 9600

ctcttcttga acgatatggg aaaggtgagg aaagacatcc cgcagtggga accatccaag 9660

ggatggaaaa actggcaaga ggttcctttt tgctcccacc attttcataa gatctttatg 9720

aaagatggcc gctcactagt tgttccatgc agaaaccagg atgaactgat aggaagagcc 9780

agaatctcgc aaggggctgg atggagttta agagaaacag cctgtttggg caaagcttac 9840

gcccaaatgt ggtcgcttat gtatttccat agaagggacc tacgtttagc ctccatggcc 9900

atatgttcag cagttccaac ggaatggttt ccaacaagca ggacaacatg gtcaatccac 9960

gctcatcacc agtggatgac cactgaagac atgctcaaag tgtggaacag ggtgtggata 10020

gaagataacc ccaatatgac tgacaagact ccagtccatt cgtgggaaga cataccttac 10080

ctagggaaaa gagaggattt gtggtgtgga tccctgattg gactctcttc cagagccacc 10140

tgggcgaaga acattcacac ggccataacc caggtcagga atctgatcgg aaaagaggac 10200

tacgtggatt acatgccagt catgaaaaga tacagcgctc cttccgaaag tgaaggagtt 10260

ctgtaattac taataacaaa caccaaagag accattgaag tcaggccact tgtgccacgg 10320

cttgagcaaa ccgtgctgcc tgtagctccg ccaataatgg gaggcgtaaa attcccaggg 10380

aggccatgcg ccacggaagc tgtacgcgtg gcatattgga ctagcggtta gaggagaccc 10440

ctcccatcac tgacaaaacg cagcaacaaa gggggcccga agccaggagg aagctgtact 10500

tctggtggaa ggactagagg ttagaggaga cccccccaac acaaaaacag catattgacg 10560

ctgggaaaga ccagagatcc tgctgtctct gcaacatcaa tccaggcaca gagcgccgcg 10620

agatggattg gtgttgttga tccaacaggt tct 10653

Claims

1. A full-length infectious clone of DENV-4, wherein the sequence of the full-length infectious clone of DENV-4 is SEQ ID NO:1 is shown.

2. The full-length infectious clone of claim 1, wherein said full-length infectious clone is constructed by the following method:

3. A recombinant virus produced from the full length infectious clone of claim 1.

4. Use of the full-length infectious clone of claim 1 for the preparation of genetically stable dengue virus.

5. Use of the full length infectious clone of claim 1 or the recombinant virus of claim 3 for the manufacture of a medicament for the study of dengue fever, dengue hemorrhagic fever, or dengue shock syndrome.

6. Use of the full-length infectious clone of claim 1 or the recombinant virus of claim 3 for the preparation of a medicament against dengue virus.

7. Use of the full length infectious clone of claim 1 or the recombinant virus of claim 3 for the preparation of a vaccine for studying dengue virus epitopes.

8. Use of the full length infectious clone of claim 1 or the recombinant virus of claim 3 for the preparation of a vaccine.