METHOD FOR RAPID GENERATION OF AN INFECTIOUS RNA VIRUS

Abstract

The present invention relates to a method for rapid generation of an infectious RNA virus that completely eliminates the need of constructing a full-length c DNA, which covers the entire viral genome, cloning and propagating such full length c DNA.

Claims

1. A method for generating an infectious RNA virus comprising: a) introducing a promoter of DNA-dependent RNA polymerase in position 5 and optionally a terminator and a RNA polyadenylation sequence in position 3 of the entire genome of a RNA virus; b) amplifying the entire viral genome as prepared in step a) including said promoter and optionally said terminator and RNA polyadenylation sequence, in at least 2, 3, 4, 5 or 6 overlapping cDNA fragments; c) transfecting said cDNA fragments into a host cell, d) incubating the host cell of step c); and e) recovering the infectious RNA virus from said incubated host cell.

2. The method of claim 1, wherein said virus is a single stranded positive RNA virus.

3. (canceled)

4. The method of claim 1, wherein: said promoter of DNA-dependent RNA polymerase in position 5 is the human cytomegalovirus promoter (pCMV); and/or said optional terminator and RNA polyadenylation sequence is respectively the hepatitis delta ribozyme and the simian virus 40 polyadenylation signal (HDR/SV40 pA).

5. The method of claim 1, wherein step b) allows the production from 2 to 15 overlapping cDNA fragments.

6. The method of claim 1, wherein said host cell is selected from the group consisting of SW13 and BHK-21, HEK 293 and Vero cell lines.

7. The method of claim 1, wherein: step (c) is a step of direct transfection of the cDNA fragments obtained in step (b) as such, and said step (c) occurs directly after step (b).

8. The method of claim 1, wherein step c) is a step of transfecting plasmids or vectors comprising a cDNA fragment obtained in step (b), wherein each cDNA fragment is in individual and separate plasmid or vector.

9. The method of claim 1, wherein said method further comprises a step (b) after step (b) and prior to step (c) of purification of the overlapping cDNA fragments.

10. The method of claim 1, wherein step (d) of incubation lasts from 3 to 9 days.

11. The method of claim 1, wherein the transfected cDNA fragments of step (c) spontaneously recombine in the host cells during the incubation step (d).

12. The method of claim 1 wherein said method is used-for reverse genetic analysis.

13. The method of claim 1 wherein said method produces an infectious RNA virus-for the safe shipment of said infectious RNA virus.

14. A method for generating an infectious RNA virus in vivo comprising: a) introducing a promoter of DNA-dependent RNA polymerase in position 5 and optionally a terminator and a RNA polyadenylation sequence in position 3 of the entire genome of a RNA virus; b) amplifying the entire viral genome as prepared in step (a) including said promoter and optionally said terminator and RNA polyadenylation sequence, in at least 2, 3, 4, 5 or 6 overlapping cDNA fragments; c) inoculating said cDNA fragments into an animal model; and e) recovering the infectious RNA virus from a biological sample obtained from said animal.

15. The method of claim 2, wherein said virus is a virus selected from the group consisting of flavivirus, alphavirus and enterovirus.

16. The method according to claim 15, wherein said Flavivirus is selected from the group consisting of Japanese encephalitis viruses (JEV), West Nile virus (WNV); Dengue virus (DENV); Yellow fever virus (YFV); and Tick-borne encephalitis virus (TBEV).

17. The method according to claim 15, wherein said alphavirus is Chikungunya.

18. The method of claim 15, wherein said enterovirus is Coxsackie.

19. The method of claim 5, wherein step b) allows the production of 3, 4, 5 or 6 overlapping cDNA fragments.

20. The method of claim 9, wherein the purification step is through a chromatography column.

Description

FIGURES LEGENDS

[0104] FIG. 1: Universal strategy to rescue infectious single stranded positive RNA viruses

The entire viral genome, schematically represented in the figure (flaviviral genome), flanked respectively in 5 and 3 by the human cytomegalovirus promoter (pCMV) and the hepatitis delta ribozyme followed by the simian virus 40 polyadenylation signal (HDR/SV40 pA), was amplified by PCR in 3 overlapping cDNA fragments. Transfection of PCR products into permissive cells enabled the recovery of infectious viruses after 3 to 9 days. Horizontal blue arrows represent primers used to generate the 3 overlapping cDNA fragments.

EXAMPLES

Example 1: ISA Method

[0105] Methods

[0106] Cells, Viruses, Infectious Clones and Antibodies

[0107] Baby hamster kidney (BHK-21) cells were grown at 37 C. with 5% CO2 in a minimal essential medium (Life Technologies) with 7% heat-inactivated foetal bovine serum (FBS; Life Technologies) and 1% Penicillin/Streptomycin (PS; 5000 U/mL and 5000 g/ml; Life Technologies). Human embryonic kidney 293 (HEK-293) cells and African green monkey kidney (VeroE6) cells were grown at 37 C. with 5% CO2 in the same medium than BHK-21 cells supplemented with 1% of non-essential amino acids (Life technologies). Human adrenal carcinoma (SW13) cells were grown at 37 C. with 5% CO2 in RPMI 1640 medium (Life Technologies) with 10% FBS and 1% PS. JEV genotype I strain JEV_CNS769_Laos_2009 (KC196115) was isolated in June 2009 from the cerebrospinal fluid of a patient in Laos16; YFV strain BOL 88/1999 (KF907504), isolated in 2009 from a human serum, was kindly provided by the National Center of Tropical Diseases (CENETROP), Santa-Cruz, Bolivia; DENV-4 strain Dak HD 34 460 (KF907503), isolated from a human serum, was kindly provided by Robert B Tesh from the Center for Biodefense and Emerging Infectious Diseases-Sealy Center for Vaccine Development (University of Texas Medical Branch, Galveston, Tex., USA); the infectious clone of JEV genotype III derived from the strain rp9 (DQ648597) was kindly provided by Yi-Ling Lin from the Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan; the infectious clone of WNV was derived from the strain

[0108] Ouganda 1937 (M12294); the infectious clone of TBEV was derived from the strain Oshima 5.10 (AB062063); the infectious clone of CV-B3 was derived from the strain 2679 (KJ489414). A JEV-specific immune serum (obtain after vaccination against JEV) and monoclonal DENV-specific antibodies17 were used to perform direct immunofluorescence assays.

[0109] Preparation of cDNA Fragments

[0110] The complete genome flanked respectively in 5 and 3 by the human cytomegalovirus promoter (pCMV) (SEQ ID No:1) and the hepatitis delta ribozyme followed by the simian virus 40 polyadenylation signal (HDR/SV40 pA) (SEQ ID No:2) was amplified by PCR in three overlapping DNA fragments of approximately 4.8 kb, 3.0 kb and 4.3 kb (4.8 kb, 2.9 kb and 5.2 kb for CHIKV, 4.8 kb, 4.1 kb and 3.4 kb for TBEV and 2.9 kb, 2.8 kb and 2.7 kb for CV-B3) (see Table 1 under).

[0111] For WNV, TBEV, JEV III and CHIKV, DNA fragments were obtained by PCR using infectious clones (for JEV III, a mutation was corrected using fusion PCR).

[0112] For JEV I (all DNA fragments), DENV-4 (first and third fragments) and YFV (first and third fragments), DNA fragments were synthesized de novo (Genscript) and amplified by PCR. Amplicons were produced using the Platinum PCR SuperMix High Fidelity kit (Life Technologies).

[0113] The mixture (final volume: 50 L) consisted of 45 L of supermix, 2 L of DNA template at 1 ng/L (infectious clone or synthesized DNA fragment) and 200 nM of each primer. For DENV-4 and YFV, the second DNA fragment was obtained by RT-PCR from clarified cell supernatants. Nucleic acids were extracted using the EZ1 Virus Mini Kit v2 on the EZ1 Biorobot (both from Qiagen) according to the manufacturer's instructions and amplified with the Superscript III One-Step RT-PCR Platinum Taq Hifi kit (Life Technologies). The mixture (final volume: 50 L) contained 25 L of Reaction Mix, 2 L of nucleic acid extract, 100 nM of each primer, 1 L of Enzyme Mix and 20 L of Nuclease-Free Water. Assays were performed on a Biometra T-professional Standard Gradient thermocycler with the following conditions: 94 C. for 2 min followed by 40 cycles of 94 C. for 15 sec, 64 C. for 30 sec, 68 C. for 5 min and a preliminary step of 50 C. for 30 min for the RT-PCR. Size of the PCR products was verified by gel electrophoresis and purified using Amicon Ultra0.5 mL kit (Millipore) according to the manufacturer's instructions. When plasmid DNA was used as template, the complete removal of the template was ensured by a digestion step with the restriction enzyme Dpn1 (New England Biolabs) before transfection. To control the efficiency of this additional step, the inventors transfected (see below), as a control, only two cDNA fragments (the first and the second, 1 g final). These controls did not produce any infectious virus.

TABLE-US-00002 TABLE1 PrimersusedtoobtaincDANfragments cDNA SEQ SEQ Virus Fragment PrimerForward Position ID PrimerReverse Position ID JEVI I CACCCAACTGATCTTCAGCATCT 3 GAAGAATGATTCTGTAAGTGTCCAG 4054-4078 4 II CGTTGCCATGCCAATCTTAGCG 4002-4023 5 GGTGCTTGCGTCCTTCCACCAA 6983-7004 6 III CAAATGAGTATGGAATGCTGGAAAA 6932-6956 7 CTCAGGGTCAATGCCAGCGCTT 8 JEVII I GCCCACCGGAAGGAGCTGAC 9 CAGAGAGCAAATCCCTATGACGA 4078-4100 10 II CGTCACCATGCCAGTCTTAGCG 4001-4022 11 GCTTGGCAATCCAGTCAGTCCT 7004-7025 12 III CAAACGAGTACGGAATGCTAGAAA 6931-6954 13 CTCATGTTTGACAGCTTATCATCG 14 WNV I TCAATATTGGCCATTAGCCATATTAT 15 TGGATTGAACACTCCTGTAGACGC 4135-4158 16 II TGGTTGGAGTTGGAAGCCTCATC 4052-4074 17 GACCATGCCGTGGCCGGCC 7016-7034 18 III TGGACAAGACCAAGAATGACATTG 6920-6943 19 GTTACAAATAAAGCAATAGCATCACA 20 TBEV I CAGGGTTATTGTCTCATGAGCGGA 21 GCCACGCCCAGGAAGAGCATGA 4033-4054 22 II GGGCCCTCTGGAAATGGGGAGA 3892-3913 23 CAACCCAGGCTTGTCACCATCTTT 8003-8026 24 III GGGTGAGGTCGTGGACCTTGGA 7886-7907 25 CCTAGGAATTTCACAAATAAAGCATTTT 26 YFV I CACCCAACTGATCTTCAGCATCT 27 GCATGGAAGTGTCCTTTGAGTTCT 4071-4094 28 II GACTTGCAACGATGCTCTTTTGCA 4020-4043 29 GAGAGAGCATCGTCACAATGCC 7040-7061 30 III GATTCCATCCAGCACCGCACC 6964-6984 31 CTCAGGGTCAATGCCAGCGCTT 32 DENV-4 I GAATAAGGGCGACACGGAAATGT 33 TGAAGACAGCTTGTCCTGCACAA 34 II GATCATGGCTTGGAGGACCATTAT 3980-4003 35 GCTACTGCATAGAGCGTCCATG 6949-6970 36 III TTTACCAGGTAAAAACAGAAACCAC 6892-6916 37 CTCAGGGTCAATGCCAGCGCTT 38 JEVI I CACCCAACTGATCTTCAGCATCT 39 CATGGAACCATTCCCTATGGACT 1635-1657 40 6 II ACTGGATTGTGAACCAAGGAGTG 1560-1582 41 GAAGAATGATTCTGTAAGTGTCCAG 4054-4078 42 fragments III CGTTGCCATGCCAATCTTAGCG 4002-4023 43 AATATAACCCCGAGCGGCGATG 5511-5532 44 IV ATGTCACCAAACAGGGTGCCCAA 5440-5462 45 GGTGCTTGCGTCCTTCCACCAA 6983-7004 46 V CAAATGAGTATGGAATGCTGGAAAA 6932-6956 47 GCGCCGTGCTCCATTGATTCTG 8950-8971 48 VI GGCTGTGGGCACATTTGTCACG 8843-8864 49 CTCAGGGTCAATGCCAGCGCTT 50 CHIKV I CACCCAACTGATCTTCAGCATCT 51 CTGCTCGGGTGACCTGTCCTA 4050-4070 52 II TGAGATGTTTTTCCTATTCAGCAACT 3961-3986 53 AACAATGTGTTGACGAACAGAGTTA 6966-6990 54 III CTCCCTGCTGGACTTGATAGAG 6859-6880 55 CTCAGGGTCAATGCCAGCGCTT 56 CV-B3 I CACCCAACTGATCTTCAGCATCT 57 CCACACAACATGCGTACCAAGCA 2184-2206 58 II CAGGCGCTGGCGCTCCGACA 2148-2167 59 GTCTATGGTTATACTCTCTGAACA 4970-4994 60 III GACAGGAGGACACAAGTCAGAT 4921-4943 61 CTCAGGGTCAATGCCAGCGCTT 62

[0114] Cell Transfection

[0115] 1 g final of either an equimolar mix of all cDNA fragments amplified by PCR or 1 g of infectious clone of CV-B3 was incubated with 12 l of Lipofectamine 2000 (Life Technologies) in 600 l of Opti-MEM medium (Life Technologies). According to the manufacturer's instructions, the mixture was added to a 12.5 cm2 culture flask of sub-confluent cells containing 1 mL of medium without antibiotics. After 4 hours of incubation, the cell supernatant was removed, cells were washed twice (HBSS; Life Technologies) and 3 mL of fresh medium was added. The cell supernatant was harvested when gross cytopathic effect (CPE) was observed (3-9 days depending on the cell type and the virus growth speed) or 9 days posttransfection for non cytopathic viruses, clarified by centrifugation, aliquoted and stored at 80 C. Each virus was then passaged four times using the same cell type except for the DENV-4 and YFV for which VeroE6 and HEK-293 were respectively used. Passages were performed by inoculating 333 L of clarified cell supernatantonto cells in a 12.5 cm2 culture flask containing 666 L of medium: after 2 hours of incubation, cells were washed twice (HBSS) and 3 mL of fresh medium was added. The cell supernatant was harvested after 2-6 days, clarified by centrifugation, aliquoted and stored at 80 C. Clarified cell supernatants (viruses stocks) were used to perform quantification of viral RNA, TCID50 assay, direct immunofluorescence assay and whole-genome sequencing.

[0116] Real Time PCR and RT-PCR Assays

[0117] To assess the production of infectious viruses and ensure that positive detection was not the result of cDNA contamination, viral RNA was quantified and compared with the quantity of detected cDNA using the Access RT-PCR Core Reagent kit (Promega) with or without the reverse transcriptase. RNA was extracted using the EZ1 mini virus 2.0 kit and the EZ1 Biorobot (both from Qiagen) according to the manufacturer's instructions. The mixture (final volume: 25 L) contained a standard quantity of AMV/Tfl 5 Reaction Buffer, 0.5 M of each primer, 0.5 L of dNTP Mix, 0.5 mM of MgSO4, 0.5 L of AMV reverse transcriptase (only for RT-PCR), 0.5 L of Tfl DNA polymerase, 15.5 L of Nuclease-Free Water and 2 L of extracted nucleic acids. Assays were performed using the CFX96 Touch Real-Time PCR Detection System (Biorad) with the following conditions: 50 C. for 15 min, 95 C. for 2 min, followed by 45 cycles of 95 C. for 15 sec, 60 C. for 40 sec. Data collection occurred during the 60 C. step. The difference between Cycle Threshold values (ct) obtained by Real time PCR and Real time RT-PCR assays has been used to assess viral RNA production. In addition, the amount of viral RNA expressed as dose detection limit (arbitrary unit; AU) was calculated from standard curves (nucleic acids from cell supernatants of cultured viruses were used as standard; five nucleic acid extracts were pooled and 10 l-aliquots were stored at 80 C.).

[0118] Tissue Culture Infectious Dose 50 (TCID50) Assay

[0119] For each determination, a 96-well plate culture containing 20,000 BHK-21 cells in 100 L of medium per well (added just before the inoculation) was inoculated with 50 L of serial 10-fold dilutions of clarified cell culture supernatants: each row included 6 wells of the same dilution and two negative controls. The plates were incubated for 7 days and read for absence or presence of CPE in each well. The determination of the TCID50/mL was performed using the method of Reed and Muench18.

[0120] Direct Immuno-Fluorescence Assay (dIFA)

[0121] Direct IFA were performed using 12.5 cm2 culture flasks of SW13 cells for JEV I and JEV III, and VeroE6 cells infected respectively 2 and 6 days before using clarified cell supernatant (see above: passage of viruses). The supernatant was removed and the cells washed twice (HBSS; Invitrogen), trypsinised, harvested and diluted (1/5) with fresh medium. After cytocentrifugation of 150 L of this cell suspension (3 min, 900 rpm; Cytospin, Thermo Scientific), the slides were dried, plunged 20 min in cold acetone for fixation, dried, incubated 30 min at 37 C. with appropriately diluted JEV-specific immune serum (see above) or monoclonal DENV-specific antibodies, washed twice with PBS, washed once with distilled water, dried, incubated 30 min at 37 C. with the appropriately diluted FITC-conjugated secondary antibody and Evans blue counterstain, washed twice with PBS, washed once with distilled water, dried, mounted and read using a fluorescence microscope.

[0122] Sequence Analysis of the Full-Length Genome

[0123] Complete genome sequencing was performed using the Ion PGM Sequencer19 (Life Technologies) and analyses conducted with the CLC Genomics Workbench 6 software. Virus supernatants were first clarified and treated with the Benzonase nuclease HC >99% (Novagen) at 37 C. overnight. Following RNA extraction (no RNA carrier was used; see above) using the EZ1 mini virus 2.0 kit and the EZ1 Biorobot (both from Qiagen), random amplification of nucleic acids was performed as previously described20. Amplified DNA was analysed using the Ion PGM Sequencer according to the manufacturer's instructions. The read obtained were trimmed: first using quality score, then by removing the primers used during the random amplification and finally at the 5 and 3 extremities by removing systematically 6 nucleotides. Only reads with a length greater than 29 nucleotides are used and mapped to the original genome sequence used as a reference. Mutation frequencies (proportion of viral genomes with the mutation) for each position were calculated simply as the number of reads with a mutation compared to the reference divided by the total number of reads at that site.

[0124] Results

[0125] The inventors developed a simple and versatile reverse genetics that facilitates the recovery of infectious RNA viruses from genomic DNA material without requiring cloning, propagation of cDNA into bacteria or in vitro RNA transcription. Their working hypothesis was that transfection of overlapping double-stranded DNA fragments, covering the entire genome of an RNA virus, into permissive cells would spontaneously enable recombination and synthesis of a DNA copy of the complete viral genome. By including at the 5 terminus of the first (5) DNA fragment, a promoter of DNA-dependent RNA polymerases, and at the 3 terminus of the last (3) DNA fragment a ribozyme sequence and a signal sequence for RNA poly-adenylation, the inventors anticipated that this genomic DNA copy would be transcribed as a full-length RNA genome with authentic 5 and 3 termini that would be efficiently exported out of the nucleus (in the case of a virus replicating in the cytoplasmic compartment).

[0126] The inventors first tested this hypothesis with 6 flaviviruses (i.e., arthropod-borne enveloped viruses with a single-stranded RNA genome of positive polarity that replicate in the cytoplasm of infected cells) that represent major flaviviral evolutionary lineages: two Japanese encephalitis viruses (JEV; genotype I (JEV I) and genotype III (JEV III)), one genotype 2 West Nile virus (WNV), one serotype 4 dengue virus (DENV-4), one wild-type strain of Yellow fever virus (YFV) and one Far-Eastern subtype Tick-borne encephalitis virus (TBEV) (Table 1).

[0127] Entire genomes were amplified by PCR in 3 DNA fragments of approximately 4 kb, each with 70-100 bp overlapping regions. The first and last fragments were flanked respectively in 5 and 3 by the human cytomegalovirus promoter (pCMV) and the hepatitis delta ribozyme followed by the simian virus 40 polyadenylation signal (HDR/SV40 pA) (FIG. 1). PCR products were column-purified, and 1 g of an equimolar mix of all fragments was transfected into SW13 and/or BHK-21 cell lines, which, ensure efficient recovery of flaviviral infectious genomes. Cell supernatant media from these infectious cultures were serially passaged four times using the same cell types, enabling the isolation of JEV I, JEV III, TBEV and WNV. For more demanding viruses, isolation could be achieved by passaging in additional permissive cells (e.g., DENV-4: VeroE6 cells; YFV: HEK-293 cells). Virus replication after four serial passages was demonstrated for each virus using a combination of the following criteria: [0128] (i) production of viral genomes in cell supernatant medium using real time RT-PCR methods, [0129] (ii) production of infectious particles in cell supernatant medium using TCID50 assays, [0130] (iii) detection of cytopathic effect (CPE), [0131] (iv) detection of viral antigens by direct immunofluorescence assays, and [0132] (v) complete viral genome sequencing using next generation sequencing (NGS) method.

[0133] The robustness, flexibility and versatility of the methods were further challenged as follows. Firstly, the inventors decreased the size and increased the number of overlapping fragments combined for transfection. This was exemplified in the case of JEV I, for which the ISA method generated infectious viruses, when using up to 6 overlapping amplicons of approximately 2 kb. Secondly, they applied the ISA method to viruses with a single-stranded RNA genome of positive polarity that belong to different families: Chikungunya virus (CHIKV, an enveloped virus, family Togaviridae) and Coxsackievirus B3 (CV-B3, a nonenveloped virus, family Picornaviridae). Again, infectious viruses could be isolated following transfection and four passages in HEK-293 cells (CHIKV) or BGM cells (CV-B3) (Table 2 under). Furthermore, the inventors used as a control the CV-B3 obtained following transfection of a plasmid-bearing infectious genome and they obtained similar results in terms of infectivity and sequence data (Table 2).

TABLE-US-00003 TABLE 2 Characterization of the recovered viruses Origin of the material used to Real produce subgenomic Cell line used time RT- amplicons for Cell line used PCR Log10 Virus Srain I II III transfection during passages (U.A) TCID50/ml CPE JEV JEV I DNS DNS DNS BHK-21 BHK-21 1.32E+08 5.8 Yes SW13 SW13 1.52E+07 5.2 Yes SW13* SW13* 9.33E+06 2.8* Yes JEV III I.C. I.C. I.C. BHK-21 BHK-21 3.77E+07 6.1 Yes SW13 SW13 4.04E+06 4.8 Yes Chimeric JEV DNS I.C. I.C. BHK-21 BHK-21 9.33E+07 6.7 Yes I/JEV III SW13 SW13 1.00E+07 6.8 Yes Chimeric JEV I.C. DNS DNS BHK-21 BHK-21 6.58E+07 6.6 Yes III/JEV I SW13 SW13 3.06E+07 6.4 Yes WNV Ouganda I.C. I.C. I.C. BHK-21 BHK-21 5.73E+07 5.3 Yes TBEV Oshima 5.10 I.C. I.C. I.C. BHK-21 BHK-21 3.28E+08 9.1 Yes DENV-4 Dak HD 34 DNS Viral DNS SW13 VeroE6 6.59E+04 N/A No 460 RNA YFV BOL 88/1999 DNS Viral DNS SW13 HEK 1.42E+05 5.2 Yes RNA CHIKV OPYI I.C. I.C. I.C. HEK-293 HEK-293 2.01E+07 7 Yes CV-B3 2679 I.C. I.C. I.C. SW13 BGM 4.64E+07 7.4 Yes CV-B3.sup. 2679.sup. Not obtained by SW13.sup. BGM.sup. 9.33E+07 7.4.sup. Yes PCR.sup. Substitutions Substitutions per per site after 4 site after 4 dN/dS dN/dS passages passages Virus Srain dIFA (all mutations) (fixed mutations) (all mutations) (fixed mutations) JEV JEV I N/A 3.273 N/A 1.27E+03 7.29E04 Positive 0.409 N/A 7.29E+04 9.11E05 N/A N/A N/A N/A N/A JEV III N/A 1.286 1.143 1.54E03 1.45E03 Positive 0.536 N/A 6.37E04 Chimeric JEV N/A 0.404 1.571 1.36E03 3.64E04 I/JEV III N/A 1.19 1.589 9.10E04 7.28E04 Chimeric JEV N/A 0.268 0.268 2.73E04 2.73E04 III/JEV I N/A 5.357 3.178 1.00E03 6.38E04 WNV Ouganda N/A 0.268 N/A 4.55E04 2.73E04 TBEV Oshima 5.10 N/A 3.214 N/A 7.20E04 9.00E05 DENV-4 Dak HD 34 Positive 0.436 0.535 8.45E04 5.63E04 460 YFV BOL 88/1999 N/A 0.818 0.818 4.63E04 4.63E04 CHIKV OPYI N/A 2.24 N/A 4.21E04 CV-B3 2679 N/A N/A N/A 2.70E04 CV-B3.sup. 2679.sup. N/A N/A N/A

[0134] Summary of the different viruses produced in this study: the specific name of the strain, the origin of the initial material (DNS, De Novo Synthesis; I.C., Infectious Clone; or Viral RNA) used as the template for production of the first (I), second (II) and third (III) fragment, the cell line used for the transfection and the passages, the relative quantification of the amount of viral RNA and infectious titres in cell supernatants at the fourth passage by real time RT-PCR and TCID50 assay, the presence or absence of cytopathic effect (CPE) as well as the research of viral antigens by direct immunofluorescence assay (dIFA). Complete viral genome sequences were obtained using NGS technology. dN and dS correspond respectively to the number of non-synonymous substitutions per non-synonymous site and the number of synonymous substitutions per synonymous site. * Results obtained by transfection of six overlapping fragments. Results obtained by transfecting directly the CV-B3 plasmid-bearing infectious clone. N/A and AU mean not available and arbitrary unit respectively.

[0135] Thirdly, the inventors demonstrated the capability of ISA method to generate genetically modified viruses in days. This was exemplified by the PCR-based correction of a frame-shift mutation (1915del) in fragment one of a defective JEV III infectious clone and the subsequent recovery of the corresponding virus (Supplementary Methods). They were also able to produce chimeric viruses by exchanging the first DNA fragment (encoding structural proteins) of genotype I and III JEVs. Despite 11 mismatches in the overlapping region of the first two fragments, transfection resulted in the production of intergenotypic JEV I/JEV III and JEV III/JEV I chimeras. Analysis of complete genomic sequences established at the fourth passage, using NGS, showed that the genetic drift (rate of sequence change) was modest (ranging from 1.45E-03 to 9.00E-05 substitutions per site when considering fixed mutations). A majority of non-synonymous mutations, the presence of shared mutations amongst the different JEV strains (7/85), and the non-random distribution of mutations (at frequency above 10%) along the genome (with both hot spots and highly conserved regions) denoted adaptation to the cell culture conditions.

[0136] The mutation rate varied according to the cells used for isolation and, as expected, was higher in viruses derived from low-passage strains than in those derived from culture-adapted strains. In conclusion, the ISA method is a very simple procedure with which to expedite production of infectious genetically modified RNA viruses within days, with perfect control of the viral sequences and starting from a variety of initial sources including pre-existing infectious clones, viral RNA or de novo synthesized DNA genomic sequences. This technique has the future potential to generate the design of large reverse genetics experiments for RNA viruses, on a scale that could not previously have been considered. It also has the capacity, specifically to modulate the characteristics of the viruses recovered from experimental procedures. Additionally, because DNA subgenomic fragments can conveniently be obtained by PCR, this method has the potential to conserve the genetic diversity of viral populations13 when starting from viral RNA. Error-prone PCR may be also be used to create artificial viral heterogeneity, e.g. for facilitating the selection of adapted viruses15 under various experimental selection conditions and, conversely, high-fidelity polymerases and clonal amplification templates may be used to control the degree of clonality of the viruses produced.

[0137] Finally, the method of the invention has the potential to revolutionise the safety and security of future exchanges of RNA viruses between scientific institutions, by the separate shipment at room temperature of simple, on-infectious, DNA subgenomic fragments that, could then be combined and transfected by the recipient institute, enabling rapid, simple and safe recovery of the infectious viral strain.

Example 2: Method ISA with cDNA Fragments in Individual and Separate Plasmids

[0138] The inventors further illustrated the ISA method in the specific embodiment where step c) is a step of transfection of plasmids or vectors comprising a cDNA fragment obtained in step b), wherein each cDNA fragment is in individual and separate plasmid or vector.

[0139] This experiment was performed using three plasmids containing the same fragments of the Japanese Encephalitis virus genome (Genotype I, Laos strain) as those previously used for recovering infectious virus by the ISA method after PCR amplification.

[0140] The three plasmids were linearised by digestion with the restriction enzyme Fse I and directly transfected in equimolar quantity (1 g final) into SW13 cells without prior PCR amplification. After 9 days and 1 passage, the virus was successfully recovered from culture.

Example 3: Application of the Method ISA In Vivo

[0141] Overlapping fragments covering the entire genome of RNA viruses and flanked respectively at 5 and 3 by promoter of DNA-dependent RNA polymerase and terminator/RNA polyadenylation signal were prepared using the method of the invention.

[0142] These DNA fragments were directly inoculated to live animals and allowed to recover infectious virus from several animal samples. In addition, clinical surveillance of animals (appearance of symptom and significant weight loss) allowed to observed typical signs of infection.

a) Experiment 1: Tick-Borne Encephalitis Virus (TBEV; Flavivirus)

[0143] The inventors used a wild-type strain of tick-borne encephalitis virus (strain Oshima 5.10 (GenBank accession number AB062063)). They applied the method of the invention to DNA overlapping fragments.

[0144] Five-weeks-old C57Bl/6J female mice were inoculated with three DNA overlapping fragments.

[0145] The clinical course of the viral infection was monitored by following [0146] (i) the clinical manifestations of the disease (shivering, humpback, dirty eyes, hemi- or tetra-paresia, hemiplegia or tetraplegia); and [0147] (ii) the weight of the mice exactly as described by Fabritus L et al., 2015, Attenuation of Tick-Borne Encephalitis Virus Using Large-Scale Random Codon Re-encoding. PLoS Pathog 11(3).

[0148] Brains and spleens were collected from sacrificed mice 14 days post-inoculation. Brains and spleens were grounded and centrifuged. The resulting supernatant was used to assess the presence of infectious virus.

[0149] The presence of infectious virus was assessed using molecular (real time RT-PCR) and classical cell culture methods (isolation of infectious viruses).

[0150] Using an initial amount of DNA ranging between 2 to 5 g, and two different inoculation routes (intraperitoneal and intradermal injections), infectious viruses were detected from both brains and spleens. Clinical manifestations (significant weight losses and symptoms) of the diseases were also observed.

b) Experiment 2: Intracerebral Inoculation of Suckling Mice

[0151] The inventors used wild-type strains of tick-borne encephalitis virus (strain Oshima 5.10 (GenBank accession number AB062063)) and Japanese encephalitis (JEV_CNS769_Laos_2009 (GenBank accession number KC196115)). They used the method of the invention to generate the DNA overlapping fragments.

[0152] DNA overlapping fragments were used diluted in PBS or were mixed with a transfection reagent.

[0153] Suckling OF1 mice were inoculated by intracerebral injection of DNA overlapping fragments. The clinical course of the viral infection was monitored by following the clinical manifestation of the disease (shivering, lethargy). Brains were collected from sacrificed mice 6-12 days post-inoculation. Brains were grounded and centrifuged. The resulting supernatant was used to assess the presence of infectious virus.

[0154] The presence of infectious virus was assessed using molecular (real time RT-PCR) and classical cell culture methods (isolation of infectious viruses).

[0155] Using 2 g of DNA, infectious viruses were detected in brains for both viruses (TBEV and JEV) and with or without addition of transfection reagent. Clinical manifestations of the diseases were also observed.