CN112011518A

CN112011518A - Construction and application of Zika virus ZG01 strain reverse genetic system

Info

Publication number: CN112011518A
Application number: CN201910460679.5A
Authority: CN
Inventors: 李义平; 陈奕奕
Original assignee: Sun Yat Sen University
Current assignee: Sun Yat Sen University
Priority date: 2019-05-30
Filing date: 2019-05-30
Publication date: 2020-12-01

Abstract

Zika virus (ZIKV) belongs to the Flaviviridae family of Flaviviridae, and in recent years Asian lineage viruses have spread from south America and others to various countries around the world. ZIKV infection is associated with guillain-barre syndrome and some birth defects, including microcephaly. The reverse genetics system is an important tool for studying the viral life cycle, pathogenesis, vaccine development and antiviral drugs, but the toxicity and instability of the cDNA clone of the flavivirus genome in transformed bacteria has been a major technical difficulty hindering the construction of infectious clones. The invention constructs a genetically stable eukaryotic initiated full-length infectious clone ZG01 of ZIKV. The ZG01 reverse genetic system provides a platform and research tool for researching ZIKV life cycle, ZIKV-host interaction, vaccine and antiviral drug development. Our construction method can also provide scientific reference for the construction and research of reverse genetics systems of other flaviviruses or RNA viruses.

Description

Construction and application of Zika virus ZG01 strain reverse genetic system

Technical Field

The invention belongs to the technical field of virus molecular biology and gene cloning, and particularly relates to construction and application of a Zika virus ZG01 strain.

Background

Zika virus (ZIKV) belongs to the Flaviviridae family of Flaviviridae, and in recent years Asian lineage viruses have spread from south America and others to various countries around the world. ZIKV infection is associated with guillain-barre syndrome and some birth defects, including microcephaly. The reverse genetics system is an important tool for studying the viral life cycle, pathogenesis, vaccine development and antiviral drugs, but the toxicity and instability of the cDNA clone of the flavivirus genome in transformed bacteria has been a major technical difficulty hindering the construction of infectious clones.

In terms of the reverse genetic system of ZIKV, in 2016, 6 months, shann et al, first reported the construction of a full-length infectious cDNA clone based on the FSS13025 strain, which is the only case of ZIKV whole genome assembled in a low-copy vector and directly amplified in escherichia coli. Tsetsarkin et al (2016) constructed an infectious clone of the strain Paraiba _01/2015 isolated in 2015 from a patient in Brazil, but this clone was attenuated in various cell lines relative to the original virus, limiting their utility in research to some extent. The differences between ZIKV asian lineage strains are not clear, so infectious clones of each virus are important to study spread of the virus. The lack of an infectious clonal system with phenotypic properties similar to that of the wild-type virus remains an obstacle to the study of the spread, pathogenesis and immune response mechanisms of the currently circulating strains of viruses.

Infectious clones published by Schwarz et al and Gadea et al are based on MR766 strain, the former constructs clones by inserting eukaryotic introns into NS1 protein, the latter realizes the rapid assembly of ZIKV infectious clones by ISA (infectious genomic clones) method, and at the same time, they try to insert GFP gene sequence into the middle of core protein to construct recombinant ZIKV capable of expressing GFP reporter gene. Weger-Lucarelli et al (2015) reported reverse genetics systems for the ZIKV PRVABC59 strain isolated from Podocosa and constructed a two-plasmid infectious cloning system that expressed ZIKV with replication efficiencies similar to wild-type viruses, and recombinant viruses showed comparable transmission rates in Aedes aegypti as wild-type and similar pathogenesis in AG129 mice. Widman et al (2017) reported a series of ZIKV reverse genetics systems, ranging from MR766 and H/PF/2013, to brazilian endemic strains such as SPH2015 and BeH819015, which were completed by directed ligation of palindromic structures formed by IIG-type cleavage using four genomic sequences. Liu et al (2017) first reported an infectious clone based on the ZIKV strain GZ01 in China, and during construction they inserted a eukaryotic intron-stabilizing plasmid near the junction of the E protein and NS1 protein, which intron had to be removed by an in vitro RNA self-splicing step after in vitro transcription.

Although several research groups have reported the construction of ZIKV full-length cDNA infectious clones in recent years, most are based on strains isolated before ZIKV explosive transmission in 2015, such as MR766, FSS13025 and H/PF/2013. Methods such as in vitro recombination of a multi-plasmid system and insertion of a eukaryotic intron into a genome are applied to the toxicity and instability of flavivirus genomes in bacterial hosts, but these methods are very complicated and time-consuming to perform in practice. Therefore, the development of a high-quality and high-efficiency ZIKV experimental model is a problem which needs to be improved and solved urgently.

Disclosure of Invention

Based on the above, the invention constructs a genetically stable eukaryotic initiated full-length infectious clone ZG01 of ZIKV. The ZG01 reverse genetic system provides a platform and research tool for researching ZIKV life cycle, ZIKV-host interaction, vaccine and antiviral drug development. Our construction method can also provide scientific reference for the construction and research of reverse genetics systems of other flaviviruses or RNA viruses.

In order to achieve the purpose, the technical scheme adopted by the invention comprises the following steps:

1. the ZIKV ZG01 strain was extracted from a patient urine specimen.

2. Three fragments covering the entire viral genome were amplified by PCR and then assembled into pBR322 derived low copy vector pTight. The genome has a CMV promoter at the 5 'end and a HDVr at the 3' end, which are used to initiate DNA transcription in eukaryotic cells and to remove non-viral sequences by self-cleaving activity, respectively. The pTight vector contains a heptad tetracycline response element to reduce toxicity of the flavivirus genome to competent cells. Since the fragment A can not be directly assembled into a vector, the fragment A is divided into two sections of A1 and A2, ECP (E. coli promoter Escherichia coli) silent mutation is introduced into an E protein of the A2 fragment, and a full-length clone of ZIKV ZG01 is constructed (figure 1)

3. Obtaining high-efficiency stable infectious clone by utilizing adaptive mutation, inoculating ZIKV virus extracted from urine of a patient to newborn rat brain, obtaining tissue suspension containing the virus from the newborn rat brain after 7-12 days, inoculating the tissue suspension to Vero cells, and obtaining 10 after passage^7.5High titer ZIKV of PFU/ml, reverse transcription using the same as a template, PCR synthesis of a fragment covering the ORF and most of the UTR of ZIKV, sequencing, and PCR synthesisFragments covering the ORF of ZIKV and most of the UTR were formed and sequenced. After alignment of the sequencing results with the original ZG01 sequence, we identified four nucleotide mutations (C2178T/G2913A/T4991C/T10561C) (Table 1), all four mutations were added simultaneously to the full-length cDNA clone ZG01, named ZG01_4 m. Vero cells were transfected with ZG01_4m, in which ZG01_4m showed efficient viral replication and a higher infection rate. The resulting recombinant virus ZG01_4m was transfected. And (3) passaging twice in Vero cells to obtain a second-generation virus, extracting virus supernatant RNA and sequencing, wherein the added mutation exists stably, and no new mutation is found, so that the ZG01_4m recombinant virus can keep genetic stability after passaging in the cells.

4. Comparing the replication curve of the recombinant ZG01_4m virus in vero cells with the replication curve of the parental virus on cells and the replication curve of the neonatal mouse brain, the replication efficiency of the virus after the recombinant virus infects a cell line in vitro is equivalent to that of the parental virus. (FIG. 2 FIG. 3)

5. The plasmid is transformed by Turbo competent cells, 4-5 monoclonals are picked, mixed culture is carried out in a liquid culture medium, then the plasmid is extracted, the step is repeated five times, a fifth generation pZG01_4m plasmid is obtained and named as pZG01_4m-G5, DNA sequencing is carried out, the sequence of the fifth generation pZG01_4m plasmid is completely consistent with pZG01_4m which is not amplified in the bacteria for multiple times, and the ZG01_4m clone has high genetic stability in Turbo bacteria (figure 4).

Drawings

FIG. 1 is a schematic representation of the construction of ZIKV full-length infectious clones.

The ZIKV virus strain ZG01 genome, the nucleotide position of restriction endonuclease site and the cloning step. Three PCR fragments, A, B and C, covering the entire ZIKV viral genome were first synthesized and then assembled into pBR 322-derived low copy vector pTight. The genome has a CMV promoter at the 5 'end and a HDVr at the 3' end, which are used to initiate DNA transcription in eukaryotic cells and to remove non-viral sequences by self-cleaving activity, respectively. The pTight vector contains a heptad tetracycline response element to reduce toxicity of the flavivirus genome to competent cells. Fragment a, which cannot be directly assembled into the vector, was divided into two segments a1 and a2, and ECP (e.coli promoter e.coli) silent mutations were introduced into the E protein of fragment a 2. Nucleotide positions in the figure are referenced to ZIKV ZG01 strain, GenBank ID: KY 379148.

FIG. 2 is an adaptive mutation in the high titer ZG01 gene over multiple passages.

(A) The ZG01 strain genome, contained the ECP1 mutation and four adaptive mutations found from in vivo and in vitro passages. (B-E) Single adaptive mutation or combination of mutations increased pZG01 post-transfection virus yield. Infectious clonal plasmids were transfected into Vero cells, cell culture supernatants were collected at the time points shown, and infectious titers in the supernatants were determined using the plaque assay. Three independent replicates were performed and the data are shown as Mean ± SD. Data statistics are shown as significant (p) using the T test<0.05;, ((p) means very marked<0.01). L.o.d. (limitation of detection), indicating the detection minimum (10)^1.05PFU/ml) (F) post-transfection ZIKV positive cells were observed by immunofluorescence experiments. Mu.g of infectious cloning plasmid was co-transfected with 0.5. mu.g of pTet-off into Vero cells and immunofluorescence experiments were performed on the third day. Green and blue represent ZIKV protein and nucleus, respectively (Hoechst 33342 staining). (G) Plaques form the experimental image. The plot is that Vero cells were infected after dilution of cell supernatant at day three post transfection, pZG 01-4 m dilution 1/10000, pMR766 dilution 1/100000, overlaid on semi-solid medium and stained with 1% crystal violet.

FIG. 3 is a graph showing that adaptive mutations found in vivo and in vitro passages enhance replication and virus production of infectious clones.

(a-B) Vero cells and a549 cells were infected with parental virus and ZG01_4m virus (MOI ═ 0.01). Cell culture supernatants were collected 1-5 days post infection and infectious titers in the supernatants were determined using the plaque assay. Three independent replicates were performed and the data are shown as Mean ± SD. Data statistics used T-test, which indicates significant (p <0.05), which indicates very significant ((p <0.01), l.o.d. (limitation of detection), which indicates minimal detection (101.05PFU/ml)

FIG. 4 shows that ZG01_4M was stably passable in host bacteria.

(A) Survival curves of wild type C57 newborn mice after infection with virus. Newborn mice born for 1-2 days were injected intracranially with 60000PFU of recombinant ZG01 — 4M (n-8), parental ZG01 (n-6) or recombinant MR766 (n-5). An equal volume of PBS (n-5) was used for negative control.

(B) Fluorescent quantitative PCR was used to determine the relative amount of viral RNA in C57 brains. Viral RNA levels were normalized to brain GAPDH levels and three independent replicates were performed with data shown as Mean ± SD.

(C) Plaque formation experiments virus titers were determined in C57 brains and three independent replicates were performed, with data shown as Mean ± SD.

(D) And (3) carrying out immunohistochemical detection on the wild C57 newborn mouse brain coronal section to obtain ZIKV. Black arrows indicate positive cells. Scale bar: 100 μm

(E) Wild type C57 neonatal mouse brain coronal section HE staining. The black line indicates the cortical extent. Scale bar: 500 μm

Detailed Description

The present invention is further explained with reference to the following specific examples, but it should be noted that the following examples are only illustrative of the present invention and should not be construed as limiting the present invention, and all technical solutions similar or equivalent to the present invention are within the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are commercially available products.

1) The viral sequences were analyzed.

2) Viral RNA was extracted from the virus solution using TRIzol method. Adding 200ul of virus liquid into an RNase-free EP tube, adding 750ul of TRIzol reagent, gently mixing, incubating at room temperature for 10 minutes, adding 200ul of chloroform, shaking, mixing, and standing at room temperature for 3 minutes. At 4 ℃ 12000g were centrifuged for 15 minutes. Taking the centrifuged supernatant to a new RNase-free EP tube, adding isopropanol and 1ul glycogen which are equal in volume, reversing, uniformly mixing, and standing at room temperature for 10 minutes. At 4 ℃ 12000g were centrifuged for 10 min. The supernatant was aspirated, 1ml of 75% ethanol was added, the mixture was shaken and mixed, and then, 7500g of the mixture was centrifuged at 4 ℃ for 5 minutes. The supernatant was aspirated off, the tube was allowed to stand until the RNA was dried, and 20ul RNase-free water was added. After the concentration was measured, the sample was stored in an ultra-low temperature refrigerator.

3) cDNA fragments covering the complete viral genome are synthesized from the viral RNA by reverse primers, 9ul of the RNA in the previous step is taken, 1ul of 10mM dNTP mix and 2.5ul of reverse transcription primers are added, the mixture is mixed evenly and reacted for 5 minutes at 65 ℃, and the mixture is placed on ice for 1 minute. 4ul of 5 XBuffer, 1ul of 100mM DTT, 1ul of RNase inhibitor and 1.5ul of SuperScript III reverse transcriptase were added, mixed well, reacted at 50 ℃ for 60 minutes and at 70 ℃ for 15 minutes to obtain cDNA. And (3) performing segmented PCR on the basis of the virus cDNA, sequencing the PCR product, and assembling a complete virus genome sequence according to a sequencing result.

4) And (3) predicting the prokaryotic promoter of the escherichia coli.

5) Transforming plasmid and amplifying. The ligation product was taken 5ul and dissolved in 100ul competent C41 cells and incubated in ice for 30 min. After heat shock at 42 ℃ for 45 seconds, the cells were placed on ice for recovery for 2min, 500. mu.l of LB medium without ampicillin was added, shaking culture was carried out at 37 ℃ and 220rpm for 1 hour, centrifugation was carried out at 4000rpm for 3 minutes, about 100. mu.l of the supernatant, suspended cells were left, applied to LB solid medium containing 100ug/ml of ampicillin, and cultured at 37 ℃ for 16 hours. A single colony was picked, added to LB liquid medium containing 100ug/ml ampicillin, and subjected to shaking culture at 37 ℃ and 220rpm for 16 hours for colony identification. For plasmids pZG01, 100ng of the plasmid was dissolved in 100ul of competent Turbo cells and ice-cooled for 30 minutes. After heat shock at 42 ℃ for 45 seconds, the cells were placed on ice for 2 minutes, 500. mu.l of ampicillin-free SOC medium was added, shaking culture was carried out at 28 ℃ and 220rpm for 1 hour, centrifugation was carried out at 4000rpm for 3 minutes, and about 100. mu.l of the supernatant, which was resuspended, was applied to 2 XYT solid medium containing 15ug/ml of ampicillin, and cultured at 28 ℃ for 20 hours. Single colonies were picked, added to 2 XYT liquid medium containing 15ug/ml ampicillin, and subjected to shaking culture at 28 ℃ and 220rpm for 20 hours for plasmid extraction.

6) And (4) culturing and transfecting the cells. When the cell growth reaches about 80% of the density, the cell is passaged. After washing off the medium, the medium was washed 1 time with 1ml of PBS solution, PBS was discarded, 1ml of 0.25% trypsin was added, and the mixture was allowed to stand at 37 ℃ for 5 minutes. Digestion was stopped by adding 2ml of complete medium, passaged at a ratio of 1:5, and cultured in a 5% CO2 incubator at 37 ℃. The cell complete medium was changed to 1ml of Opti-MEM medium for each well of 12-well plates at 37 ℃ and 5% CO before transfection₂Culturing under the condition. 0.1ug of plasmid was added to 100ul of Opti-MEM medium, 0.25ul Lipofectamine 2000 was added to 100ul Opti-MEM medium, and the mixture was incubated at room temperature for 10 minutes. The plasmid-containing Opti-MEM medium was added to the Lipofectamine 2000-containing medium and incubated at room temperature for 20 minutes. The transfection solution was added to 12-well plates and incubated at 37 ℃ for 4 hours. The Opti-MEM medium mixed with the transfection solution was discarded, and 1ml of DMEM medium containing 10% FBS was added.

7) Immunofluorescence. An indirect immunofluorescence assay was performed to detect viral protein expression in Vero cells transfected with ZIKV full-length infectious clonal plasmid. Vero cells transfected with ZIKV full length infectious clonal plasmid in 24-well plates. At the indicated time points, cell supernatants were aspirated and cells were fixed in 3.7% formaldehyde for 15 min at room temperature. After 1 hour incubation in PBS blocking buffer containing 1% BSA, blocking buffer containing 2% anti-flavivirus mouse monoclonal antibody was added and incubated for 2 hours at room temperature. After the blocking buffer containing the antibody was aspirated, the cells were washed with PBS for 5 minutes for 3 times. Blocking buffer containing 2% Alexa Fluor 488 anti-mouse IgG was added and incubated for 1 hour at room temperature. After the blocking buffer containing the antibody was aspirated, the cells were washed with PBS for 5 minutes for 3 times. Observed under a fluorescent microscope.

8) Plaque formation experiments. The infectious titer of ZIKV was determined using a plaque assay. Vero cells were plated at 2X 105 per well in 12-well plates 16-20 hours prior to infection experiments. The virus-containing culture supernatant was serially diluted 10-fold, from 101 to 107-fold, in DMEM. For each dilution, 300ul of the diluted virus solution was added to wells of a 12-well plate of Vero cells cultured to about 90% confluence. The cells with the virus solution were incubated at 37 ℃ and 5% CO₂Incubate for 2 hours under conditions and shake the well plate every 15 minutes to ensure that the virus dilution completely covers the cell monolayer to obtain a uniform infection. After incubation, after washing the cell monolayer with serum-free DMEM medium to remove unadsorbed virus, 1ml of semi-solid containing medium was added per well and the plates were incubated at 37 ℃ and 5% CO₂Cultured under the conditions for 5 days. The semi-solid medium was removed and the plates were washed twice with PBS, fixed with 5% formaldehyde and allowed to stand at room temperature for 60 minutes. After removing the fixing solution, the wells are washedPlates were stained with 1% crystal violet for 1 min and washed twice with PBS. The visible plaques were counted and the virus titer was calculated accordingly.

9) And (4) carrying out a suckling mouse experiment. C57BL/6 wild-type suckling mice born for 1-2 days were injected intracranially with ZIKV diluent (60000 PFU/mouse) and the negative control group was injected with an equal volume of PBS solution containing 0.5% BSA. Daily observations were made as to whether neurological symptoms or death occurred. C57BL/6 wild-type suckling mice were sacrificed by cervical dislocation after viral injection and severe neurological symptoms. The brains were dissected out, half for plaque formation experiments and half for real-time fluorescent quantitative PCR reactions. All samples were stored at-80 ℃. After the tissue sample was weighed, a PBS solution containing 0.5% BSA was added at a ratio of 1g/10ml, and ground with a homogenizer at 4 ℃ for 2 minutes. Half of the homogenized extract was used for viral RNA extraction, and the other half was centrifuged at 14000g for 15 minutes at 4 ℃ and the supernatant was used for plaque formation experiments.

10) And (5) identifying a virus sequence. Collecting cell supernatant or mouse brain grinding fluid in a virus infection experiment, extracting RNA, and performing RT-PCR by using the RNA as a template. PCR was performed using cDNA as template, with five fragments covering almost the entire ZG01 genome (nucleotide positions 21-10703), and the PCR products were sequenced and compared to the plasmid sequence. The primers are shown in appendix II.

11) And (3) carrying out real-time fluorescence quantitative PCR reaction. Tissue RNA was extracted by TRIzol method and the concentration was measured. 1ug of RNA was used as template, 4ul of 5 xqRTSuperMix (Vazyme) was added, and RNase-free ddH was added₂O to 20ul, reacting at 50 ℃ for 15 minutes, and inactivating at 85 ℃ for 2 minutes to obtain cDNA. Taking 1ul cDNA product, adding 2ul of 6.5ul ddH2O, 10ddH2O 2 XchamQSYBR qPCR Master Mix (Vazyme), ZIKV genome primer or GPADH gene primer, mixing evenly, and carrying out circular reaction according to the instruction. ZIKV genomic data were normalized to GAPDH gene and RNA relative copy number was calculated as Ct model.

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 has been 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, and all the technical solutions of the present invention belong to the protection scope of the present invention.

TABLE 1 adaptive mutations in the high titer ZG01 Gene over multiple passages

Sequence listing

<110> Zhongshan university

Construction and application of <120> Zika virus strain ZG01 reverse genetic system

<160> 1

<170> SIPOSequenceListing 1.0

<210> 2

<211> 10691

<212> DNA

<213> isolation of Zika virus ZG01 (Zika virus)

<400> 2

cagactgcga cagttcgagt ttgaagcgaa agctagcaac agtatcaaca ggttttattt 60

tggatttgga aacgagagtt tctggtcatg aaaaacccaa aaaagaaatc cggaggattc 120

cggattgtca atatgctaaa acgcggagta gcccgtgtga gcccctttgg gggcttgaag 180

aggctgccag ccggacttct gctgggtcat gggcccatca ggatggtctt ggcgattcta 240

gcctttttga gattcacggc aatcaagcca tcactgggtc tcatcaatag atggggttca 300

gtggggaaaa aagaggctat ggaaataata aagaagttca agaaagatct ggctgccatg 360

ctgagaataa tcaatgctag gaaggagaag aagagacgag gcgcagatac tagtgtcgga 420

attgttggcc tcctgctgac cacagctatg gcagcggagg tcactagacg tgggagtgca 480

tactatatgt acttggacag aaacgatgct ggggaggcca tatcttttcc aaccacattg 540

gggatgaata agtgttatat acagatcatg gatcttggac acatgtgtga tgccaccatg 600

agctatgaat gccctatgct ggatgagggg gtggaaccag atgacgtcga ttgttggtgc 660

aacacgacgt caacttgggt tgtgtacgga acctgccatc acaaaaaagg tgaagcacgg 720

agatctagaa gagctgtgac gctcccttcc cattccacta ggaagctgca aacgcggtcg 780

caaacctggt tggaatcaag agaatacaca aagcacttga ttagagtcga aaattggata 840

ttcaggaacc ctggcttcgc gttagcagca gctgccatcg cttggctttt gggaagctca 900

acgagccaaa aagtcatata cttggtcatg atactgctga ttgccccggc atacagcatc 960

aggtgcatag gagtcagcaa tagggacttt gtggaaggta tgtcaggtgg gacttgggtc 1020

gatgttgtct tggaacatgg aggttgtgtc accgtaatgg cacaggacaa accgactgtc 1080

gacatagagc tggttacaac aacagtcagc aacatggcgg aggtaagatc ctactgctat 1140

gaggcatcaa tatcagacat ggcttcggac agccgctgcc caacacaagg tgaagcctac 1200

cttgacaagc aatcagacac tcaatatgtc tgcaaaagaa cgttagtgga cagaggctgg 1260

ggaaatggat gtggactttt tggcaaaggg agcctggtga catgcgctaa gtttgcatgc 1320

tccaagaaaa tgaccgggaa gagcatccag ccagagaatc tggagtaccg gataatgctg 1380

tcagttcatg gctcccagca cagtgggatg atcgttaatg acacaggaca tgaaactgat 1440

gagaatagag cgaaagttga gataacgccc aattcaccaa gagccgaagc caccctgggg 1500

ggttttggaa gcctaggact tgattgtgaa ccgaggacag gccttgactt ttcagatttg 1560

tattacttga ctatgaataa caagcactgg ttggttcaca aggagtggtt ccacgacatt 1620

ccattacctt ggcacgcagg ggcagacacc ggaactccac actggaacaa caaagaagca 1680

ctggtagagt tcaaggacgc acatgccaaa aggcaaactg tcgtggttct agggagtcaa 1740

gaaggagcag ttcacacggc ccttgctgga gctctggagg ctgagatgga tggagcaaag 1800

ggaaggctgt cctctggcca cttgaaatgt cgcctgaaaa tggataaact tagattgaag 1860

ggcgtgtcat actccttgtg tactgcagcg ttcacattca ccaagatccc ggctgaaaca 1920

ctgcacggga cagtcacagt ggaggtacag tacgcaggga cagatggacc ttgcaaggtt 1980

ccagctcaga tggcggtgga catgcaaact ctgaccccag ttgggaggtt gataaccgct 2040

aaccccgtaa tcactgaaag cactgagaac tctaagatga tgctggaact tgatccacca 2100

tttggggact cttacattgt cataggagtc ggggagaaga agatcaccca ccactggcac 2160

aggagtggca gcaccattgg aaaagcattt gaagccactg tgagaggtgc caagagaatg 2220

gcagtcttgg gagacacagc ctgggacttt ggatcagttg gaggcgctct caactcattg 2280

ggcaagggca tccatcaaat ttttggagca gctttcaaat cattgtttgg aggaatgtcc 2340

tggttctcac aaattctcat tggaacgttg ctgatgtggt tgggtctgaa cacaaagaat 2400

ggatctattt cccttatgtg cttggcctta gggggagtgt tgatcttctt atccacagcc 2460

gtctctgctg atgtggggtg ctcggtggac ttctcaaaga aggagacgag atgcggtaca 2520

ggggtgtttg tctataacga cgttgaagcc tggagggaca ggtacaagta ccatcctgac 2580

tccccccgta gattggcagc agcagtcaag caagcctggg aagatggtat ctgcgggatc 2640

tcctctgttt caagaatgga aaacatcatg tggagatcag tagaagggga gctcaacgca 2700

atcctggaag agaatggagt tcaactgacg gtcgttgtgg gatctgtaaa aaaccccatg 2760

tggagaggtc cacagagatt gcccgtgcct gtgaacgagc tgccccacgg ctggaaggct 2820

tgggggaaat cgtacttcgt cagagcagca aagacaaata acagctttgt cgtggatggt 2880

gacacactga aggaatgccc actcaaacat agagcatgga acagctttct tgtggaggat 2940

catgggttcg gggtatttca cactagtgtc tggctcaagg ttagagaaga ttattcatta 3000

gagtgtgatc cagccgttat tggaacagct gttaagggaa aggaggctgt acacagtgat 3060

ctaggctact ggattgagag tgagaagaat gacacatgga ggctgaagag ggcccatctg 3120

atcgagatga aaacatgtga atggccaaag tcccacacat tgtggacaga tggaatagaa 3180

gagagtgatc tgatcatacc caagtcttta gctgggccac tcagccatca caataccaga 3240

gagggctaca ggacccaaat gaaagggcca tggcacagtg aagagcttga aattcggttt 3300

gaggaatgcc caggcactaa ggtccacgtg gaggaaacat gtggaacaag aggaccatct 3360

ctgagatcaa ccactgcaag cggaagggtg atcgaggaat ggtgctgcag ggagtgcaca 3420

atgcccccac tgtcgttccg ggctaaagat ggctgttggt atggaatgga gataaggccc 3480

aggaaagaac cagaaagcaa cttagtaagg tcaatggtga ctgcaggatc aactgatcac 3540

atggaccact tctcccttgg agtgcttgtg atcctgctca tggtgcagga agggctgaag 3600

aagagaatga ccacaaagat catcataagc acatcaatgg cagtgctggt agctatgatc 3660

ctgggaggat tttcaatgag tgacctggct aagcttgcaa ttttgatggg tgccaccttc 3720

gcggaaatga acactggagg agatgtagct catctggcgc tgatagcggc attcaaagtc 3780

agaccagcgt tgctggtatc tttcatcttc agagctaatt ggacaccccg tgaaagcatg 3840

ctgctggcct tggcctcgtg tcttttgcaa actgcgatct ccgccttgga aggcgacctg 3900

atggttctca tcaatggttt tgctttggcc tggttggcag tacgagcgat ggttgttcca 3960

cgcactgata acatcaccct agcaatcctg gctgctctga caccactggc ccggggcaca 4020

ctgcttgtgg cgtggagagc aggccttgct acttgcgggg ggtttatgct cctctctctg 4080

aagggaaaag gcagtgtgaa gaagaactta ccatttgtca tggccctggg actaaccgct 4140

gtgaggctgg tcgaccccat caacgtggtg ggactgctgt tgctcacaag gagtgggaag 4200

cggagctggc cccctagcga agtactcaca gctgttggcc tgatatgcgc attggctgga 4260

gggttcgcca aggcagatat agagatggct gggcccatgg ccgcggtcgg tctgctaatt 4320

gtcagttacg tggtctcagg aaagagtgtg gacatgtaca ttgaaagagc aggtgacatc 4380

acatgggaaa aagatgcgga agtcactgga aacagtcccc ggctcgatgt ggcgctagat 4440

gagagtggtg atttctccct ggtggaggat gacggtcccc ccatgagaga gatcatactc 4500

aaggtggtcc tgatgaccat ctgtggcatg aacccaatag ccataccctt tgcagctgga 4560

gcgtggtacg tatacgtgaa gactggaaaa aggagtggtg ctctatggga tgtgcctgct 4620

cccaaggaag taaaaaaggg ggagaccaca gatggagtgt acagagtaat gactcgtaga 4680

ctgctaggtt caacacaagt tggagtggga gttatgcaag agggggtctt tcacactatg 4740

tggcacgtca caaaaggatc cgcgctgaga agcggtgaag ggagacttga tccatactgg 4800

ggagatgtca agcaggatct ggtgtcatac tgtggtccat ggaagctaga tgccgcctgg 4860

gacgggcaca gcgaggtgca gctcttggcc gtgccccccg gagagagagc gaggaacatc 4920

cagactctgc ccggaatatt taagacaaag gatggggaca ttggagcggt tgcgctggat 4980

tacccagcag gaacttcagg atctccaatc ctagacaagt gtgggagagt gataggactt 5040

tatggcaatg gggtcgtgat caaaaatggg agttatgtta gtgccatcac ccaagggagg 5100

agggaggaag agactcctgt tgagtgcttc gagccttcga tgctgaagaa gaagcagcta 5160

actgtcttag acttgcatcc tggagctggg aaaaccagga gagttcttcc tgaaatagtc 5220

cgtgaagcca taaaaacaag actccgtact gtgatcttag ctccaaccag ggttgtcgct 5280

gctgaaatgg aggaggccct tagagggctt ccagtgcgtt atatgacaac agcagtcaat 5340

gtcacccact ctggaacaga aatcgtcgac ttaatgtgcc atgccacctt cacttcacgt 5400

ctactacagc caattagagt ccccaactat aatctgtata ttatggatga ggcccacttt 5460

acagatccct caagtatagc agcaagagga tacatttcaa caagggttga gatgggcgag 5520

gcggctgcca tcttcatgac cgccacgcca ccaggaaccc gtgacgcatt tccggactcc 5580

aactcaccaa ttatggacac cgaagtggaa gtcccagaga gagcctggag ctcaggcttt 5640

gattgggtga cggatcattc tggaaaaaca gtttggtttg ttccaagcgt gaggaacggc 5700

aatgagatcg cagcttgtct gacaaaggct ggaaaacggg tcatacagct cagcagaaag 5760

acttttgaga cagagttcca gaaaacaaaa catcaagagt gggactttgt cgtgacaact 5820

gacatttcag agatgggcgc caactttaaa gctgaccgtg tcatagattc caggagatgc 5880

ctaaagccgg tcatacttga tggcgagaga gtcattctgg ctggacccat gcctgtcaca 5940

catgccagcg ctgcccagag gagggggcgc ataggcagga atcccaacaa acctggagat 6000

gagtatctgt atggaggtgg gtgcgcagag actgacgaag accatgcaca ttggcttgaa 6060

gcaagaatgc tccttgacaa tatttacctc caagatggcc tcatagcctc gctctatcga 6120

cctgaggccg acaaagtagc agccattgag ggagagttca agcttaggac ggagcaaagg 6180

aagacctttg tggaactcat gaaaagagga gatcttcctg tttggctggc ctatcaggtt 6240

gcatctgccg gaataaccta cacagataga agatggtgct ttgatggcac gaccaacaac 6300

accataatgg aagacagtgt gccggcagag gtgtggacca gacacggaga gaaaagagtg 6360

ctcaaaccga ggtggatgga cgccagagtt tgttcagatc atgcggccct gaagtcattc 6420

aaggagtttg ccgctgggaa aagaggagcg gcttttggag tgatggaagc cctgggaaca 6480

ctgccaggac acatgacaga gagattccag gaagccattg acaacctcgc tgtgctcatg 6540

cgggcagaga ctggaagcag gccttacaaa gccgcggcag cccaattgcc ggagacccta 6600

gagaccatta tgcttttggg gttgctggga acagtctcgc tgggaatctt cttcgtcttg 6660

atgaggaaca agggcatagg gaagatgggc tttggaatgg tgactcttgg ggccagcgca 6720

tggctcatgt ggctctcgga aattgagcca gccagaattg catgtgtcct cattgttgtg 6780

ttcctattgc tggtggtgct catacctgag ccagaaaagc aaagatctcc ccaggacaac 6840

caaatggcaa tcatcattat ggtagcagta ggtcttctgg gcttgattac cgccaatgaa 6900

ctcggatggt tggagagaac aaagagtgac ctaagccatc taatgggaag gagagaggag 6960

ggggcaacca taggattctc aatggacatt gacctgcggc cagcctcagc ttgggccatc 7020

tatgctgcct tgacaacttt cattacccca gccgtccaac atgcagtgac cacctcatac 7080

aacaactact ccttaatggc gatggccacg caagctggag tgttgtttgg tatgggcaaa 7140

gggatgccat tctacgcatg ggactttgga gtcccgctgc taatgatagg ttgctactca 7200

caattaacac ccctgaccct aatagtggcc atcattttgc tcgtggcgca ctacatgtac 7260

ttgatcccag ggctgcaggc agcagctgcg cgtgctgccc agaagagaac ggcagctggc 7320

atcatgaaga accctgttgt ggatggaata gtggtgactg acattgacac aatgacaatt 7380

gacccccaag tggagaaaaa gatgggacag gtgctactca tagcagtagc cgtctccagc 7440

gccatactgt cgcggaccgc ctgggggtgg ggggaggctg gggccctgat cacagccgca 7500

acttccactt tgtgggaagg ctctccgaac aagtactgga actcctctac agccacttca 7560

ctgtgtaaca tttttagggg aagttacttg gctggagctt ctctaatcta cacagtaaca 7620

agaaacgctg gcttggtcaa gagacgtggg ggtggaacag gagagaccct gggagagaaa 7680

tggaaggccc gcttgaacca gatgtcggcc ctggagttct actcctacaa aaagtcaggc 7740

atcaccgagg tgtgcagaga agaggcccgc cgcgccctca aggacggtgt ggcaacggga 7800

ggccatgctg tgtcccgagg aagtgcaaag ctgagatggt tggtggatcg gggatacctg 7860

cagccctatg gaaaggtcat tgatcttgga tgtggcagag ggggctggag ttactacgcc 7920

gccaccatcc gcaaagttca agaagtgaaa ggatacacaa aaggaggccc tggtcatgaa 7980

gaacccgtgt tggtgcaaag ctatgggtgg aacatagtcc gtcttaagag tggggtggac 8040

gtctttcata tggcggctga gccgtgtgac acgttgctgt gtgacatagg tgagtcatca 8100

tctagtcctg aagtggaaga agcacggacg ctcagagtcc tctccatggt gggggattgg 8160

cttgaaaaaa gaccaggagc cttttgcata aaagtgttgt gcccatacac cagcactatg 8220

atggaaaccc tggagcgact gcagcgtagg tatgggggag gactggtcag agtgccactc 8280

tcccgcaact ctacacatga gatgtactgg gtctctggag cgaaaagcaa caccataaaa 8340

agtgtgtcca ccacgagcca gctcctcttg gggcgcatgg acgggcctag gaggccagtg 8400

aaatatgagg aggatgtgaa tctcggctct ggcacgcggg ctgtggcaag ctgcgctgaa 8460

gctcccaaca tgaagatcat tggtaaccgc attgaaagga tccgcagtga gcacgcggaa 8520

acgtggttct ttgacgagaa ccacccatat aggacatggg cttaccatgg aagctatgag 8580

gcccccacac aagggtcagc gtcctctcta ataaacgggg ttgtcaggct cctgtcaaaa 8640

ccctgggatg tggtgactgg agtcacagga atagccatga ccgacaccac accgtatggt 8700

cagcaaagag ttttcaagga aaaagtggac actagggtgc cagaccccca agaaggcact 8760

cgtcaggtta tgagcatggt ctcttcctgg ttgtggaaag agctaggcaa acacaaacgg 8820

ccacgagtct gtaccaaaga agagttcatc aacaaggttc gtagcaatgc agcattaggg 8880

gcaatatttg aagaggaaaa agagtggaag actgcagtgg aagctgtgaa cgatccaagg 8940

ttctgggctc tagtggacaa ggaaagagag caccacctga gaggagagtg ccagagttgt 9000

gtgtacaaca tgatgggaaa aagagaaaag aaacaagggg aatttggaaa ggccaagggc 9060

agccgcgcca tctggtatat gtggctgggg gctagatttc tagagttcga agcccttgga 9120

ttcttgaacg aggatcactg gatggggaga gagaactcag gaggtggtgt tgaagggctg 9180

ggattacaaa gactcggata tgtcctagaa gagatgagtc gtataccagg aggaaggatg 9240

tatgcagatg acactgctgg ctgggacacc cgcattagca ggtttgatct ggagaatgaa 9300

gctctaatca ccaaccaaat ggagaaaggg cacagggcct tggcattggc cataatcaag 9360

tacacatacc aaaacaaagt ggtaaaggtc cttagaccag ctgaaaaagg gaaaacagtt 9420

atggacatta tttcgagaca agaccaaagg gggagcggac aagttgtcac ttacgctctt 9480

aacacattta ccaacctagt ggtgcaactc attcggaata tggaggctga ggaagttcta 9540

gagatgcaag acttgtggct gctgcggagg tcagagaaag tgaccaactg gttgcagagc 9600

aacggatggg ataggctcaa acgaatggca gtcagtggag atgattgcgt tgtgaagcca 9660

attgatgata ggtttgcaca tgccctcagg ttcttgaatg atatgggaaa agttaggaag 9720

gacacacaag agtggaaacc ctcaactgga tgggacaact gggaagaagt tccgttttgc 9780

tcccaccatt tcaacaagct ccatctcaag gacgggaggt ccattgtggt tccctgccgc 9840

caccaagatg aactgattgg ccgggcccgc gtctctccag gggcgggatg gagcatccgg 9900

gagactgctt gcctagcaaa atcatatgcg caaatgtggc agctccttta tttccacaga 9960

agggacctcc gactgatggc caatgccatt tgttcatctg tgccagttga ctgggttcca 10020

actgggagaa ctacctggtc aatccatgga aagggagaat ggatgaccac tgaggacatg 10080

cttgtggtgt ggaacagagt gtggattgag gagaacgacc acatggaaga caagacccca 10140

gttacgaaat ggacagacat tccctatttg ggaaaaaggg aagacttgtg gtgtggatct 10200

ctcatagggc acagaccgcg caccacctgg gctgagaaca ttaaaaacac agtcaacatg 10260

gtgcgcagga tcataggtga tgaagaaaag tacatggatt acctatccac ccaagttcgc 10320

tacttgggtg aagaagggtc tacacctgga gtgctgtaag caccaatctt aatgttgtca 10380

ggcctgctag tcagccacag cttggggaaa gctgtgcagc ctgtgacccc cccaggagaa 10440

gctgggaaac caagcctata gtcaggccga gaacgccatg gcacggaaga agccatgctg 10500

cctgtgagcc cctcagagga cactgagtca aaaaacccca cgcgcttgga ggcgcaggat 10560

gggaaaagaa ggtggcgacc ttccccaccc ttcaatctgg ggcctgaact ggagatcagc 10620

tgtggatctc cagaagaggg actagtggtt agaggagacc ccccggaaaa cgcaaaacag 10680

catattgacg t 10691

Claims

1. A Zika virus strain ZIKV ZG01, wherein the full-length nucleotide sequence is GenBank ID: KY 379148.

2. The strain ZIKV ZG01 of claim 1, wherein four mutations (C2178T/G2913A/T4991C/T10561C) are added to construct a highly efficient full-length infectious clone ZG01_4m with four mutations and capable of stable inheritance.

3. The strain ZIKV ZG01 of claim 1, wherein mutation of ECP1 alone results in a transformant containing a full-length clone of ZG01 and a full-length clone of ZG01 pZG01 containing a mutation of ECP1 (see fig. 1).

4. The highly efficient full-length infectious clone ZG01_4m of claim 2, wherein the transfected Vero cells can be replicated to obtain highly efficient infectious viruses.

5. Use of the Zika virus strain and sequence of claim 1, the infectious clone of claim 2 or 3, or a clone carrying any one or more mutations therein, in culturing other strains to establish a ZIKV cell infection model.

6. Use of the Zika virus strain and sequence of claim 1, the infectious clone of claim 2 or 3, or a clone carrying any one or more mutations therein, in constructing various recombinant viruses, and culturing other strains using the mutations to establish a ZIKV cell infection model.

7. A Zika virus strain and sequence according to claim 1, an infectious clone according to claim 2 or 3, or a clone carrying any one or more mutations therein, constructed recombinant viruses, and uses thereof for vaccine and drug development and production.

8. A Zika virus strain and sequence according to claim 1, an infectious clone according to claim 2 or 3, or a clone carrying any one or more mutations therein, for drug target discovery and treatment in Zika therapy.