Plasmids and method for obtaining viral particles

Abstract

The present invention describes a viral RNA expression plasmid and a method for obtaining viral particles based on the plasmids comprising transfecting animal cells with an expression vector or a set of expression vectors capable of expressing a nucleoprotein and RNA-dependent polymerase RNA; and transfecting the animal cell with an expression vector or a set of expression vectors with nucleotide sequences encoding recombinant RNA molecules.

Claims

1. A method of obtaining viral particles, the method comprising: a) transfecting each of a plurality of fish cells with a set of plasmids, which express: SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, and SEQ ID NO 10, wherein one of SEQ ID NOs 7, 8, 9, or 10 is cloned individually in each of the plasmids; b) once the cells are transfected in step a), co-transfecting the fish cells of step a) with a set of eight plasmids: pSS-URG/1, pSS-URG/2, pSS-URG/3, pSS-URG/4, pSS-URG/5, pSS-URG/6, pSS-URG/7, and pSS-URG/8, each plasmid having: a promoter sequence of Salmo salar internal transcribed sequences (ITS-1) region; a hammerhead ribozyme (HH rib) sequence; one coding sequence of infectious salmon anemia virus (ISAV) inserted in a cloning site sequence, wherein each pSS-URG/1 to 8 plasmid includes a different coding sequence of ISAV; a hepatitis virus ribozyme sequence; and a rabbit -globin transcription terminator sequence; and c) obtaining recombinant viral particles resulting from steps a) and b).

2. The method of claim 1, wherein the fish cells in steps a), b), and c) are cultured at 15-20 C.

3. The method of claim 1, wherein the recombinant viral particles express nucleic acids of single-stranded chimeric RNA of the ISAV.

4. The method of claim 1, wherein the recombinant viral particles express autogenous or exogenous proteins of the ISAV.

5. The method of claim 1, wherein the set of eight plasmids includes at least one recombinant plasmid comprising: a skeleton obtained from a pUC57 plasmid in which genomic sequences of viruses expressed as RNA can be introduced, the sequences being selected from a group consisting of: a promoter sequence of Salmo salar; a hammerhead ribozyme (HH rib) sequence; a cloning site sequence; a hepatitis virus ribozyme sequence; and a rabbit -globin transcription terminator sequence.

6. The method of claim 1, wherein the promoter sequence of Salmo salar is SEQ ID NO 1.

7. The method of claim 1, wherein the HH rib sequence is SEQ ID NO 2.

8. The method of claim 1, wherein the cloning site sequence is SEQ ID NO 3.

9. The method of claim 1, wherein the hepatitis virus ribozyme sequence is SEQ ID NO 4.

10. The method of claim 1, wherein the rabbit -globin transcription terminator sequence SEQ ID NO 5.

11. The method of claim 1, further comprising: cloning genetic sequences of an animal, plant, protist, fungal, bacterial, or viral origin into the plasmids using distant cut restriction enzyme SapI.

12. A set of expression plasmids including SEQ ID NOs 7, 8, and 9, wherein each of SEQ ID NOs 7, 8, and 9 is cloned individually in each expression plasmid.

13. An expression plasmid including SEQ ID NO 10.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1: Obtaining the plasmids to allow transcription of the 8 RNAv of ISAV901_09: (1) pSS-URG plasmids and the plasmid containing each of the ISAV genome segments are digested with the SapI restriction enzyme. The digestion products are visualized on a 1% agarose gel, and then purified. (2) The digested product corresponding to the ISAV genome segment and the linear pSS-URG plasmid are ligated with T4 ligase, and then this ligation is used to transform chemo-competent bacteria. (3) From the clones containing the expected recombinant plasmids, purification is carried out to confirm the correct insertion of the genome segment by sequencing.

(2) FIG. 2: Schematic design of pSS-URG, pSS-URG/S6-NotI and pSS-URG/S6-EGFP-HPR cassettes. The universal vector contains the sequence of: promoter of S. salar (SS prom); hammerhead ribozyme (HH rib); hepatitis virus ribozyme (HDV rib); rabbit -globin transcription terminator (Term). pSS-URG/S6-Not and pSS-URG/S6-EGFP-HPR plasmids contain the cDNA of antisense and inverted segment 6, which includes 5 and 3 UTRs and their modifications; NotI restriction site or the EGFP coding sequence.

(3) FIG. 3: RT-PCR of the 6-NotI-HPR segment from ASK salmon cells transfected with pSS-URG/S6-Not-HPR plasmids. Agarose gel electrophoresis of RT-PCR products of segment 6 at selected post transfection times. ASK cells were transfected using Fugene 6 (Promega), the plasmid used was pSS-URG/segment 6.

(4) FIG. 4: Reverse genetics to obtain recombinant ISA virus: ASK cells are transfected with 8 pSS-URG/S1-8 plasmids, which allow transcription of the 8 vRNA (ISAV genome), together with 4 plasmids that allow expression of the proteins that form the cRNP (PB2, PB1, PA and NP). Co-transfection of these 12 plasmids in the same cell to allow the formation of the 8 cRNP in the nucleus, which are the minimum unit required to form an ISAV viral particle. These cRNPs allow transcription and replication of the vRNA, allowing synthesis of all proteins that form the virus and the generation of new cRNPs, thus forming recombinant viral particles.

(5) FIG. 5: RT-PCR of 6-NotI-HPR segment from ST swine cells (a) and HEK-293 human cells (b), both transfected with pSS-URG/S6-NotI-HPR plasmid using Fugene 6 (Promega). Agarose gel electrophoresis of RT-PCR products of segment 6 at selected post transfection times.

(6) FIG. 6: Electron Microscopy Analysis of recombinant ISAV from sections of infected ASK cells. Cytoplasmic membrane with ISAV particles budding from infections with: (A) WT ISAV 901_09, (B) rISAVS6-EGFP-HPR and (C) rISAV 901_09. (D) Endosomal section showing rISAV 901_09 particles inside endosomes, which correspond to the initial steps of the fusion of viral and endosomal membranes. Bar: 200 nm.

EXAMPLES

(7) ISAV 901_09 Strain Genome Adapted to Cell Culture

(8) The complete genome of a virus isolate adapted to cell culture, such as ISAV 901_09 (HPR 1c), was sequenced.

(9) Alignments between the noncoding regions (UTR) of the 5 and 3 ends of complete sequences of the six ISAV isolates, two Scots (390/98 and 982/08), one Norwegian (Glesvr/2/90), two Canadian (NBISA01 and RPC NB 98-049), and one Chilean (ADL-PM 3205 ISAV) have high conservation at the ends of each viral genome segment, allowing to design universal primers described in Table I. The primers were used to amplify the genome of the Chilean ISAV 901_09 strain. The result of sequencing the eight viral genome segments is shown in Table IV. The sizes of the eight viral segments range from 2267 bp to 906 bp for segments 1 and 8, respectively.

(10) The sequence of the 3UTR regions ranged from 7 nucleotides in segment 6 to 48 nucleotides in segment 3, and there were no differences in the size of each 3UTR end previously described for the 6 genomes analyzed, except for the addition of a nucleotide at the 3UTR end of segment 7. The sequences of the 5UTR ends of ISAV 901_09 range from 67 nucleotides in segment 4 to 147 nucleotides in segment 3. The alignment of the UTR regions also indicates that ISAV 901_09 has a high similarity with the ISAV Glesr/2/90 strain (between 97% and 98% identity).

(11) Universal Vector Design for ISAV Reverse Genetics

(12) In order to achieve a vector which expresses segments of full-length viral RNA without additional nucleotides, taking advantage of advances in synthetic biology, an innovative design was made integrating elements previously used in reverse genetics of RNA viruses, such as Hammerhead ribozymes and delta () hepatitis virus, together with genome elements of the Salmo salar species. The designed vector was called pSS-URG (plasmid for Salmo salar Universal Reverse Genetic). The correctly connected components contained by the vector and ordered from left to right are: As a promoter ITS-1 region of Salmo salar, a sequence of Hammerhead ribozyme, the ribozyme of delta () hepatitis virus, and the sequences of the two ribozymes incorporate two cutting sites for Sap I enzyme (New England Biolabs), and finally incorporated as transcription terminator is rabbit beta globin terminator (FIG. 2). This vector would allow cloning, without incorporating additional sequences, any viral segment, through the use of a distant cut enzyme, such as Sap I. Thus, this study presents a vector to be the base of the reverse genetics system for the ISA virus.

(13) Once the pSS-URG plasmid is synthesized, subcloning of the eight genome segments of ISAV was achieved from synthetic genomes using distant cut enzyme Sap I (Data not shown). In addition to cloning the eight viral segments, as a genetic marker and in order to prove that generated viruses are recombinant agents and do not correspond to a contamination of the procedure, two genetic elements were inserted in the HPR area of the universal vector containing segment 6. The first genetic variant corresponds to the insertion in the HPR area of a sequence of nine nucleotides with the cutting site for the NotI enzyme, calling this new vector pSS-URG/S6-NotI-HPR. A second genetic variant corresponds to the product of cloning the sequence which codes for EGFP using previously created NotI site, thus the new vector called pSS-URG/S6-EGFP-HPR is obtained.

(14) Analysis of Functionality for pSS-URG Vector by Ex Vivo Transcription Trial

(15) To determine whether all the elements included in the vector allow the expression of viral RNA in salmon cells, and due to the uncertainty of functionality of the promoter suggested in ITS-1 region of Salmo salar, ASK cells were transfected with pSS-URG/S6-NotI-HPR plasmid in an ex vivo transcription trial. To determine the existence of a transcription process, the functionality analysis was made by detecting the RNAV at times 0, 3, 6, 9, 12 and 15 after transfection (hpt) through RT-PCR. The reverse transcription reaction was made using a single first complementary to the Not I restriction site. Surprisingly, the analysis result can display a PCR product from three hpt, which increases in intensity until 15 hpt (FIG. 3). This result would indicate that from transfected pSS-URG/S6-NotI-HPR plasmid, the cell is generating an RNA having the NotI restriction site, and therefore ITS-1 region of Salmo salar corresponds to a promoter element. To prove the generation of a viral RNA without additional nucleotides, a RT-PCR was carried out with specific primers for each ribozyme, the results showed that it was not possible to obtain an amplification product with primers that recognize sequences of ribozymes in any point of kinetics, indicating that generated RNA has no additional regions, such as, for example, ribozymes (data not shown).

(16) These results suggest the use of the ITS-1 region and the inclusion in pSS-URG vector to express any type of RNA inside the cells. For example, RNA can be expressed as interfering RNA, silencing RNA or also micro RNAs.

(17) Obtaining Recombinant ISA Virus (ISAVr)

(18) As it has been reported for Influenza virus, the functional minimum unit of the virus corresponds to the ribonucleoprotein (RNP) complex, which consists of viral RNA that is bound by multiple copies of NP and by the viral polymerase including PB1, PB2 and PA subunits. In order to form the RNP complexes in salmon cells, ORFs of segments 1 to 4 of ISAV901_09 were cloned into expression vectors commanded by the Cytomegalovirus promoter. Thus, using the pTriEx-3 vector (Novagen), ORFs of segments 1, 2 and 4 were cloned, obtaining PTRIEX3-PB2, PTRIEX PB1, PTRIEX3-PA vectors. Besides, using the pCI-neo vector (Promega), segment 3 was cloned generating pCI-neo-NP vector (Data not shown). Transfection of these vectors into salmon cells allows expression of recombinant proteins PB2, PB1, PA and NP, respectively.

(19) Generation of ISAVr.sup.S6-NotI-HPR

(20) For the generation of ISAVr S6-NotI-HPR, ASK cells were cotransfected with twelve plasmids, four of which correspond to expression vectors PTRIEX3-PB2, PTRIEX PB1, PTRIEX3 PA and pCI-neo-NP, and the remaining eight correspond to plasmids for pSS-URG reverse genetics with each of the eight genome segments of ISAV901_09 as DNA, replacing native segment 6 by Seg6-NotI-HPR. In order to amplify and determine the presence of recombinant virus, after transfection of cells, two blind passages were made in ASK cells infecting with the supernatants obtained from the previous transfections. On the one hand, the presence of RNAV of segment 6 (NotI/HPR) was detected, through RT-PCR, obtaining a product of an expected 306 bp size, both in the RNA extracted from the transfection supernatant and from the two subsequent passages, which suggests the presence of infectious viruses. The second passage supernatant was used to infect a greater amount of ASK cells. From the infected cells, which showed an obvious cytopathic effect (data not shown), the recombinant virus was visualized by transmission electron microscopy. FIG. 6 shows spherical particles similar to virus with diameters near 100 nm, which suggests that these correspond to the recombinant viruses. Therefore, it was possible to detect a recombinant ISAVr.sup.S6-NotI-HPR virus in infected cells, with replicative activity and reproducible cytopathic effect in passages subsequent to their generation.

(21) Generation of ISAVr.sup.S6-EGFP-HPR

(22) In order to generate recombinant ISA virus containing a reporter gene, such as EGFP, in order to facilitate ex vivo monitoring and discard that results are artifactual results or contamination, ASK cells were co-transfected with twelve plasmids simultaneously: four of them correspond to expression vectors PTRIEX3-PB2, PTRIEX-PB1, PTRIEX3 PA and pCI-neo-NP; the remaining eight plasmids correspond to vectors pSS-URG/1, pSS-URG/2, pSS-URG/3, pSS-URG/4, pSS-URG/5, pSS-URG/7 and pSS-URG/8; also incorporating segment 6 with vector pSS-URG/S6-EGFP-HPR; the virus which contains EGFP in the HPR area of the protein is called ISAVr.sup.S6-EGFP-HPR.

(23) To determine whether recombinant viral particles were generated after transfection, the culture supernatant (passage 0, P0) was analyzed 7 days after transfection (dpt). For this purpose, it was initially detected by RT-PCR RNAv of Segment 6, as well as the EGFP coding sequence in a second PCR product, and finally an area containing both part of Segment 6 and the EGFP. The results showed that RT-PCR products for Segment 6 have a different migration distance of the PCR products for the native virus (300 bp) and the recombinant virus having EGFP in the HE protein (1,000 bp). The RT-PCR of the EGFP coding sequence has a 500 bp product, which is not observed in the native virus analyzed. For RT-PCR of S6-EGFP, an amplification product of 800 bp was obtained for the recombinant virus as expected.

(24) Infectivity of ISAVr.sup.S6-EGFP-HPR in ASK Cells

(25) To determine whether the supernatant of ASK cells transfected with twelve plasmids indeed contains the viral variant ISAVr.sup.S6-EGFP-HPR with the characteristics of an infectious agent, EGFP fluorescence was used as a reporter. The ASK cells infected with the supernatant that would contain the first progeny ISAVr.sup.S6-EGFP-HPR were analyzed under confocal microscope 7 days after infection. The results show that it is possible to visualize cells emitting green fluorescence attributable to EGFP, corresponding to the first passage of the ISAVr.sup.S6-EGFP-HPR virus. Distribution of the EGFP mark is found mainly in the cytoplasm and towards the plasma membrane, fluorescence being not observed in the cell nucleus. To confirm that the supernatant of transfected cells (passage 0) contains the ISAVr.sup.S6-EGFP-HPR virus with lytic capacity, a lysis plaque trial was carried out on ASK cells. The result of the lysis plaque trial showed that the recombinant virus has the ability to generate lysis plaques like the wild virus, obtaining a virus titre in the order of 110.sup.4 PFU/m L.

(26) Stability of ISAVr.sup.S6-EGFP-HPR

(27) Subsequently, the ability of this recombinant virus to maintain infectiousness and fluorescence was assessed in cell culture. Four blind passages of infection in ASK cells were carried out with 7-day gaps. Then, in each supernatant of the recombinant virus passages, a RT-PCR was carried out to detect RNAv both of Segment 6 and of the coding sequence for EGFP, and a region of the EGFP-S6 hybrid sequence. The result made possible to visualize a PCR product of 500 bp EGFP and EGFP-S6 hybrid sequences of 800 bp, indicating the presence of segment 6 containing the EGFP gene in all supernatants analyzed. The PCR product of segment 6 shows a 300 bp product in the supernatant of infected ASK cells with ISAV901_09 wild virus, as expected, in contrast to the 1,000 bp of the PCR product obtained in each of the four passages of ISAVr.sup.S6-EGFP-HPR virus, whose larger size is the result of having incorporated the EGFP gene in segment 6.

(28) To determine that each of the four passages not only had a virus with infectivity, but also was capable of fluorescing, indicating the correct folding of HE with EGFP in the HPR area, an analysis was carried out by confocal microscopy in infected ASK cells. Confocal microscopy showed cells that emit green fluorescence in all passages analyzed, increasing in each passage the abundance of fluorescent cells. These results suggest that the region of the HE protein elected to incorporate EGFP is not affected by the incorporation of this ORF, thus allowing the generation of a chimeric recombinant ISA virus capable of replicating, infecting and spreading in multiple passages without losing the ability to fluoresce. Titration of the fourth blind passage made to ISAVr.sup.S6-EGFP-HPR virus by qRT-PCR in real time resulted in a titre of 3.6310.sup.6 copies Seg 8/mL and a value of 6.510.sup.5 PFU/mL obtained by lysis plaque trial, showing a lysis plaque size similar to that observed after conducting plaque trial on ISAV901_09 wild virus.

(29) Infectivity of ISAVr.sup.S6-EGFP-HPR in Salmonid Cell Lines

(30) In order to determine whether by incorporating a sequence in the HPR area of the ISAVr.sup.S6-EGFP-HPR virus, this acquires the ability to infect other salmonid species or lose infectivity in permissive cells (ASK cells), an infection kinetics was carried out in RTG-2, CSE-119 and ASK cells. The ex vivo trial was conducted for 7 days using the fourth passage of the fluorescent recombinant virus and compared to the ISAV901_09 wild virus, and the fourth passage of a wild recombinant virus generated for this trial rISAV.sup.901_9 (MOI of 0.01). The analysis at 0, 2, 4 and 7 dpi by qRT-PCR quantifying the number of copies of segment 8 in each supernatant showed that none of the three viruses analyzed had the ability to replicate in RTG-2 cells or in CSE-119 cells.

(31) In contrast, the infection kinetics carried out on ASK cells showed that initially the ISAV 901_09 virus has a larger number of copies than recombinant viruses, rISAV.sup.901 and rISAV.sup.S6-EGFP-HPR. On the second day after infection, however, an increase occurs in the number of copies of the recombinant viruses, reaching titres near 1,000 segment 8/mL, with values similar to the wild virus. These results suggest that the incorporation of EGFP in the HPR area of HE protein does not alter the replicative behavior of the fluorescent recombinant virus in ASK cells, and does not extend the host range at least in the ex vivo trials in RTG-2 cells or in CSE-119 cells.

(32) These results can lead to the conclusion that it is possible to incorporate into the pSS-URG plasmid a sequence encoding both for a viral protein and for an exogenous or chimeric protein, thus achieving the generation of a modified or chimeric recombinant ISA virus; these modifications would not alter or affect its infectious or propagation characteristics.

(33) Functionality of pSS-URG Plasmid in Salmon, Swine and Human Cell Lines

(34) To determine the ability of the pSS-URG/S6-NotI-HPR vector for transcribing segment 6 as RNAv, it was transfected into ASK cells using Fugene 6 (Promega) at a 1:6 ratio and according to manufacturers' specifications. 0 hpt is considered as the time when adding the transfection mix to the cells. The initial incubation takes place for 3 hours at 16 C. F. Once the incubation time had elapsed, the transfection mix was removed and the cells were washed twice with PBS, this being a 3-hpt time. At each point of the transfection kinetics, which occurs at 0, 3, 6, 9, 12 and 15 hpt, the cells are removed for extraction of total RNA, possible contaminating DNA was removed with DNase I. With the RNA obtained, RNAv of segment 6 NotI was detected by RT-PCR using specific primers for segment 6 NotI. The analysis allows to observe a PCR product of expected 306 bps size from 3 hpt, which increases in intensity until 15 hpt (FIG. 3). Therefore, it is proved that the pSS-URG plasmid is functional.

(35) Surprisingly, the ability to transcribe the viral RNA is not restricted to salmon cells cultured at 16 C. Using the same procedure (with incubations at 37 C.), but conducted only at 12 hpt, functionality was observed in human cell line HEK 293 and swine cell line ST, incubated at 37 C. (FIG. 5). This is reflected in obtaining a RT-PCR product of the expected size which allows to conclude that the vector is functional in salmon cell lines incubated at 16 C. and mammal cell lines incubated at 37 C., being this a tool that would allow the expression of any RNA in cells or tissues of vertebrate animals, whether cold-blooded or warm-blooded.

(36) ISAVr.sup.S6-EGFP-HPR: Correlation Between the Number of Viral Copies and Measured Fluorescence

(37) Since the ISAVr.sup.S6-EGFP-HPR virus has similar characteristics to the wild virus when infecting ASK cells, and also has the advantage of monitoring the infection by incorporating EGFP as reporter, the goal is to determine whether there is correlation between the viral load and fluorescence in the supernatant of infection caused by this recombinant virus. The analysis carried out on serial dilutions of fluorescent recombinant virus through qRT-PCR and fluorescence quantitation established that there is a direct relationship showing an increased fluorescence detected when the viral titer of the solution increases, showing a fluorescence intensity of 500 units/mL for a titer of 210.sup.6 copies/mL.