Reverse genetics system for establishing completely plasmid-based tilapia lake virus (TiLV)

Abstract

The present disclosure relates to the establishment of a reverse genetic system for a fully plasmid-based tilapia lake virus (TiLV), and the use of the reverse genetic system for rescuing recombinant TiLV and reporting TiLV. The disclosure also relates to plasmids for rescuing TiLVs as well as recombinant TiLVs and reporter TiLVs rescued using the methods disclosed herein.

Description

[0001] This application claims priority to U.S. Non-Provisional Patent Application No. 18 / 442,171, the entire contents of which are incorporated herein by reference.

[0002] Reference to a sequence listing

[0003] This application contains a sequence listing, identified as Sequence_Listing_P25681US00.xml; size: 93 bytes; creation date: February 1, 2024, which is hereby incorporated by reference in its entirety. Technical Field

[0004] The present disclosure relates to methods for establishing a tilapia lake virus reverse genetics system. Background Art

[0005] Tilapia lake virus (TiLV), the causative agent of tilapia lake virus disease (TiLVD), is a novel virus first identified in Israel in 2014. It is a ten-segment, single-stranded, negative-sense RNA virus classified by the International Committee on Taxonomy of Viruses as belonging to the family Amnoonviridae, genus Tilapinevirus, and species Tilapinevirus tilapiae. Since the initial description of the virus and disease, cases have been reported in Asia, Africa, and the Americas. Recently, China, the largest tilapia producer, reported its first natural TiLV infection. Tilapia is one of the most important sources of protein, particularly in low- and middle-income countries. TiLV can cause mortality rates of up to 90% in tilapia, and there is no treatment or commercially available vaccine for TiLV infection. Therefore, TiLV poses a serious threat to the global tilapia industry and food safety.

[0006] Currently, scientific knowledge and research related to TiLV are very limited, with many important gaps in knowledge that need to be filled. The replication and molecular pathogenesis of the virus remain largely unknown. Reverse genetics (RG), a method for generating viruses entirely from cloned cDNA, is a powerful tool for studying viruses and developing vaccines. However, there is currently no reverse genetics system (RGS) for TiLV, hindering our efforts to understand its pathogenesis and develop new vaccines or antiviral drugs.

[0007] There are several challenges in establishing an RGS for TiLV: (i) TiLV is a fish RNA virus, but rescuing the virus using TiLV-susceptible fish-derived cells is difficult, primarily because previous systems for rescuing related viruses utilize bidirectional transcription systems, which require knowledge of the promoter sequence of the relevant fish RNA polymerase I. However, information on the promoter of relevant fish RNA polymerase I is currently lacking; (ii) the majority of TiLV viral genes and the functions of their corresponding viral proteins are unknown, posing a significant obstacle to the establishment of an RGS; and (iii) the TiLV genome consists of ten segments, and the large number of segments also increases the difficulty of rescuing the virus.

[0008] Therefore, it is very necessary to establish the RGS of this virus. Summary of the Invention

[0009] This disclosure relates to the development of a TiLV recombinant gene sequence sequence (RGS) that can rescue recombinant TiLV even when most viral proteins are unknown. This is the first RGS for TiLV. Furthermore, this disclosure relates to TiLV labeled with a reporter gene, which can be used to monitor viral infection in vivo and in vitro.

[0010] In a first aspect, the present invention provides a method for rescuing recombinant TiLV, comprising:

[0011] i) preparing a plurality of expression plasmids for expressing the ten genomic segments of TiLV, wherein each expression plasmid of the plurality of expression plasmids comprises at least one cDNA sequence of the ten genomic segments of TiLV;

[0012] ii) transfecting co-cultured Vero cells and TiLV-sensitive fish-derived cells with multiple expression plasmids;

[0013] iii) culturing the transfected cells obtained in step ii) at a temperature of about 28° C. to rescue the recombinant TiLV; and selectively rescuing

[0014] iv) Reassortant TiLV.

[0015] In certain embodiments, the plurality of expression plasmids are bidirectional expression plasmids that express all viral gRNAs and viral mRNAs of TiLV.

[0016] In certain embodiments, the cDNA sequences of the ten genomic segments express all viral gRNAs and viral mRNAs of TiLV.

[0017] In certain embodiments, the cDNA sequences of the ten genomic segments of TiLV are at least 90% identical to SEQ ID NOs: 1-10, respectively.

[0018] In certain embodiments, the cDNA sequences of the ten genomic segments of TiLV are at least 90% identical to SEQ ID NOs: 11-20, respectively.

[0019] In certain embodiments, the cDNA sequences of the ten genomic segments of TiLV comprise or consist of SEQ ID NOs: 1-10, respectively.

[0020] In certain embodiments, the cDNA sequences of the ten genomic segments of TiLV comprise or consist of SEQ ID NOs: 11-20, respectively.

[0021] In certain embodiments, the fish-derived cell is an E11 cell derived from Channa striatus.

[0022] In certain embodiments, the co-cultured cells are Vero E6 cells and E11 cells.

[0023] In a second aspect, the present invention provides a method for rescuing a reporter gene-tagged TiLV, comprising:

[0024] i) preparing an expression plasmid comprising a cDNA sequence of one genomic segment of TiLV and a reporter gene tag sequence, wherein the reporter gene tag sequence is integrated at the 3' end of the open reading frame (ORF) of the genomic segment, and a certain number of nucleotides at the 3' end of the ORF are inserted after the sequence tag; and preparing a plurality of expression plasmids, each of the plurality of expression plasmids comprising a cDNA sequence of at least one genomic segment of the remaining nine genomic segments of TiLV;

[0025] ii) transfecting all the expression plasmids prepared in step i) into co-cultured Vero cells and fish-derived cells sensitive to TiLV; and

[0026] iii) culturing the transfected cells obtained in step ii) at a temperature of about 28° C., thereby rescuing the reporter gene-tagged TiLV.

[0027] In certain embodiments, the expression plasmid is a bidirectional expression plasmid that expresses all viral gRNAs and viral mRNAs of TiLV.

[0028] In certain embodiments, the cDNA sequences of the ten genomic segments of TiLV are at least 90% identical to SEQ ID NOs: 1-10, respectively.

[0029] In certain embodiments, the cDNA sequences of the ten genomic segments of TiLV are at least 90% identical to SEQ ID NOs: 11-20, respectively.

[0030] In certain embodiments, the cDNA sequences of the ten genomic segments of TiLV comprise or consist of SEQ ID NOs: 1-10, respectively.

[0031] In certain embodiments, the cDNA sequences of the ten genomic segments of TiLV comprise or consist of SEQ ID NOs: 11-20, respectively.

[0032] In certain embodiments, the fish-derived cell is an E11 cell derived from Channa fasciatus.

[0033] In certain embodiments, the co-cultured cells are Vero E6 cells and E11 cells.

[0034] In certain embodiments, the reporter gene tag sequence is HiBiT, GFP, NanoLuc, or mCherry.

[0035] In a third aspect, provided herein is a recombinant, rearranged, and reporter TiLV rescued according to any one of the methods of the embodiments described herein.

[0036] In a fourth aspect, provided herein are vaccines comprising the attenuated or inactivated recombinant and reporter TiLV described herein, as well as one or more expression plasmids described herein, and viral proteins expressed by one or more expression plasmids described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] The above and other objects and features of the present disclosure will become apparent from the following description of the present disclosure when taken in conjunction with the accompanying drawings.

[0038] Figure 1. Schematic diagram of the reverse genetics system for TiLV rescue. (A) The designed plasmid uses the pcDNA3.1 vector as a backbone. The transcription unit contains a CMV promoter, a mouse RNA polymerase I terminator, a cDNA sequence of a TiLV genomic fragment (in reverse direction), a human RNA polymerase I promoter, and a BGH polyadenylation signal. (B) Ten plasmids capable of producing all viral gRNAs and viral mRNAs are transfected into co-cultured Vero E6 and E11 cells. Recombinant or reporter viruses are then rescued.

[0039] Figure 2Schematic diagram of the TiLV genome (based on the TiLV-Israel-HK strain). Genomic features include a 5-nucleotide sequence at the 3' end (uni-5), the main open reading frame (ORF), and a 6-nucleotide sequence at the 5' end (uni-6). The polyadenylation signal sequence within the TiLV genome segment is indicated.

[0040] Figure 3 .Rescue of HiBiT-tagged reporter TiLV. (A) Schematic diagram of the HP5-HiBiT fusion containing HP5, HiBiT, and self-cleaving polypeptide 2A. The HiBiT sequence is integrated after the 50th or 100th amino acid of HP5, denoted as aa50 or aa100, respectively. The 80 nucleotide sequence at the 3' end of the HP5 ORF is integrated downstream of HiBiT, denoted as 80. (B) RT-PCR amplification of the full-length genomic segment 5 of the reporter virus. PCR products were separated on a 1% agarose gel. M = Marker (NEB1kb Plus DNA Ladder), T1 = recombinant TiLV-1, T(S5) = rearranged TiLV-1 (S5), P3 = 3rd generation reporter virus, P4 = 4th generation reporter virus, P5 = 5th generation reporter virus.

[0041] Figure 4 Immunofluorescence assay detecting HiBiT-tagged viral proteins in E11 cells. E11 cells were mock-infected and infected with recombinant TiLV-1 (Til-4-2011), recombinant TiLV-2 (TiLV-Israel-HK), or reporter TiLV. Two days later, cells were fixed and HiBiT-tagged proteins were visualized using a mouse anti-HiBiT antibody as the primary antibody and an Alexa Fluor 488 goat anti-mouse IgG antibody as the secondary antibody. Arrows point to representative fluorescent staining in E11 cells. DETAILED DESCRIPTION

[0042] definition

[0043] Unless expressly stated otherwise, the use of the singular herein includes the plural (and vice versa).

[0044] As used herein, the term "about" or "approximately" refers to a variation of ±10%, ±7%, ±5%, ±3%, ±1%, or ±0% of a nominal value, unless otherwise stated or inferred.

[0045] As used herein, the term "rescuing viruses" encompasses processes well known to those skilled in the art, ie, the process of generating viral clones from viral genomes.

[0046] In the context of two or more nucleic acid or polypeptide sequences, "percent identity" refers to the percentage of identical nucleotides or amino acids contained in the two or more sequences. A specific identity of nucleotides or amino acids can refer to, for example, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity.

[0047] As used herein, "mutation" refers to a change in a nucleic acid relative to a reference sequence (generally the normal or "wild-type" sequence occurring in nature) and includes translocations, deletions, insertions, and substitutions.

[0048] The method disclosed herein rescues TiLV through a reverse genetics technique in which the virus is rescued in cells using multiple expression plasmids containing fragments of the TiLV genome.

[0049] In a first aspect, provided herein is a method for rescuing TiLV, comprising:

[0050] i) preparing a plurality of expression plasmids for expressing the ten genomic segments of TiLV, wherein each expression plasmid of the plurality of expression plasmids comprises at least one complementary DNA (cDNA) sequence of the ten genomic segments of TiLV;

[0051] ii) transfecting the multiple expression plasmids into co-cultured Vero cells and fish-derived cells sensitive to TiLV;

[0052] iii) culturing the transfected cells obtained in step ii) at a temperature of about 28° C. to rescue the recombinant TiLV; and selectively rescuing

[0053] iv) Reassortant TiLV.

[0054] The method is described in detail below.

[0055] Expression plasmid

[0056] Multiple expression plasmids are used to generate all viral gRNAs and viral mRNAs of TiLV.

[0057] The expression plasmid is a bidirectional expression plasmid.

[0058] It should be noted that bidirectional expression plasmids contain at least two promoters that drive expression in different directions (i.e., 5' to 3', 3' to 5') in the same plasmid. Typically, one of the promoters is an RNA polymerase I promoter, while the other is an RNA polymerase II promoter. This is useful because the RNA polymerase I promoter can be used to express uncapped viral RNA, while the RNA polymerase II promoter can be used to transcribe mRNA, which can then be translated into viral proteins, thereby allowing the simultaneous expression of viral RNA and viral proteins in the same plasmid.

[0059] The RNA polymerase I and RNA polymerase II promoters used in the expression plasmid may be derived from organisms of the same taxonomic class as the host cell, or may be derived from organisms of a different taxonomic class than the host cell.

[0060] To construct the expression plasmid, the cDNA of each of the ten genomic segments of TiLV can be synthesized first, and then the cDNA can be inserted into a vector using conventional techniques known in the art.

[0061] The expression plasmid should contain all the genomic segments of TiLV. An expression plasmid can contain one, two, three, four, five or more genomic segments.

[0062] In certain embodiments, the cDNA sequences of the ten genomic segments of TiLV are at least 90%, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 1-10 or SEQ ID NOs: 11-20, respectively. The multiple expression plasmids should express all viral gRNAs and viral mRNAs of TiLV to rescue recombinant TiLV.

[0063] In certain embodiments, the cDNA sequences of the ten genomic segments of TiLV comprise or consist of SEQ ID NOs: 1-10 or SEQ ID NOs: 11-20, respectively.

[0064] Cell transfection

[0065] The expression plasmid can be introduced into the host cell using any technique known to those skilled in the art. For example, the expression plasmid can be introduced into the host cell using methods such as electroporation, DEAE-dextran, calcium phosphate coprecipitation, cationic liposomes, or viral vectors. In certain embodiments, liposome-based transfection reagents, such as lipofectamine 3000, are preferred. Once transfected, the host cell will begin to express the viral RNA fragments and viral proteins.

[0066] In certain embodiments, the cells used to express the ten genomic segments of TiLV are Vero cells, which are co-cultured with fish-derived cells that are sensitive to TiLV. Vero cells include VeroJCRB0111, Vero CCL-81, Vero 76, or VeroE6 cells.

[0067] In certain embodiments, fish-derived cells susceptible to TiLV include E11, SSN-1, TiB (derived from tilapia brain), FHM (derived from fathead minnow), CIK (derived from grass carp kidney), CCB (derived from carp brain), and ZF4 (derived from zebrafish).

[0068] When cells are used as hosts, cell culture conditions (eg, temperature, cell density, pH, etc.) have great flexibility for different cell lines and can be modified according to the specific application.

[0069] In certain embodiments, prior to transfection of the expression plasmid, the co-cultured cells are cultured at approximately 32° C.-37° C., e.g., 33° C.-37° C., 34° C.-37° C., 35° C.-37° C., 36° C.-37° C., 32° C.-36° C., 32° C.-35° C., 32° C.-34° C., or 32° C.-33° C. Typically, the cells are cultured at about 37° C.

[0070] In certain embodiments, cells are transfected with a total amount of 5 μg to 15 μg of plasmids, wherein each plasmid comprises 0.5-1.5 μg. In certain embodiments, the total amount of all plasmids used to transfect cells is 6 μg to 15 μg, 7 μg to 15 μg, 8 μg to 15 μg, 9 μg to 15 μg, 10 μg to 15 μg, 11 μg to 15 μg, 12 μg to 15 μg, 13 μg to 15 μg, 14 μg to 15 μg, etc.

[0071] In certain embodiments, following transfection of the plasmid, the cells are cultured at a temperature of about 28°C, eg, 30°C, 29°C, 27°C, 26°C, 25°C, or lower.

[0072] Virus harvest

[0073] In certain embodiments, the methods of the present invention further comprise harvesting and isolating the recombinant virus by methods known in the art.

[0074] In certain embodiments, the rescued recombinant TiLV can be passaged in fish-derived cells that are susceptible to TiLV, such as E11, SSN-1, and TiB cells.

[0075] Reporter viruses are powerful tools for monitoring viral infections in cultured cells and their animal hosts. They have proven particularly useful for screening antiviral drugs in vitro or evaluating the efficacy of vaccines or therapeutics in vivo. Therefore, in a second aspect, provided herein is a method for rescuing reporter gene-tagged TiLV, comprising:

[0076] i) preparing an expression plasmid, the plasmid comprising a cDNA sequence of one genomic segment of TiLV and a reporter gene tag sequence, the reporter gene tag sequence being integrated at the 3' end of the open reading frame (ORF) of the genomic segment, and a certain number of nucleotides at the 3' end of the ORF being inserted after the sequence tag; and preparing a plurality of expression plasmids, each of the plurality of expression plasmids comprising a cDNA sequence of at least one genomic segment of the remaining nine genomic segments of TiLV;

[0077] ii) transfecting all the expression plasmids prepared in step i) into co-cultured Vero cells and fish-derived cells sensitive to TiLV; and

[0078] iii) culturing the transfected cells obtained in step ii) at a temperature of about 28° C., thereby rescuing the reporter gene-tagged TiLV.

[0079] In certain embodiments, the self-cleaving polypeptide 2A is inserted between the open reading frame (ORF) and the tag.

[0080] Examples of integrating tags into genomic fragments can be found in Figure 3 In plasmid HP5-2A-HiBiT-80 in Figure A, 80 nucleotides near the 3' end of the ORF of genomic segment 5 are added to the 3' end of the HiBiT tag. It is important to note that the nucleotide sequence added to the 3' end of the HiBiT tag is associated with the packaging signal sequence of the genomic segment and should partially (or completely) contain the packaging signal sequence of the genomic segment.

[0081] In certain embodiments, the expression plasmid is a bidirectional expression plasmid that expresses all viral gRNAs and viral mRNAs of TiLV.

[0082] In certain embodiments, the cDNA sequences of the ten genomic segments of TiLV are at least 90% identical to SEQ ID NOs: 1-10, respectively.

[0083] In certain embodiments, the cDNA sequences of the ten genomic segments of TiLV are at least 90% identical to SEQ ID NOs: 11-20, respectively.

[0084] In certain embodiments, the cDNA sequences of the ten genomic segments of TiLV comprise or consist of SEQ ID NOs: 1-10, respectively.

[0085] In certain embodiments, the cDNA sequences of the ten genomic segments of TiLV comprise or consist of SEQ ID NOs: 11-20, respectively.

[0086] In certain embodiments, the fish-derived cell is an E11 cell derived from Channa fasciatus.

[0087] In certain embodiments, the co-cultured cells are Vero E6 cells and E11 cells.

[0088] In certain embodiments, the reporter gene tag is HiBiT, GFP, NanoLuc, or mCherry. The most preferred reporter gene tag is the HiBiT tag. The reporter gene tag can be detected by methods known in the art.

[0089] In a third aspect, provided herein are recombinant, rearranged, and reporter TiLVs rescued by any of the methods disclosed herein.

[0090] Recombinant and reporter viruses are valuable tools for studying TiLV replication, pathogenesis, and host-virus interactions. They can be used to investigate the function of specific viral genes or to study the effects of viral infection on cells and organisms.

[0091] In a fourth aspect, provided herein are vaccines comprising the attenuated or inactivated recombinant and reporter TiLV described herein, as well as one or more expression plasmids described herein, and viral proteins expressed by one or more expression plasmids described herein.

[0092] Example

[0093] Materials and methods

[0094] Cells and viruses

[0095] Vero E6 cells (ATCC, CRL-1586) and E11 cells (provided by Dr. Sven M. Bergmann) were cultured in MEMα medium (Gibco) supplemented with 10% fetal bovine serum (Gibco) at the indicated temperatures (5% CO ). A TiLV isolate (provided by Professor Eran Bacharach) was passaged and propagated in E11 cells, and its complete genome was determined using the following method (this sequenced TiLV strain was designated TiLV-Israel-HK).

[0096] The ten genomic segments of the tilapia lake virus isolate (Til-4-2011) are shown in SEQ ID NOs: 1-10, and the ten genomic segments of the tilapia lake virus isolate (TiLV-Israel-HK) are shown in SEQ ID NOs: 11-20.

[0097] The primers used to amplify mRNA, complementary RNA (cRNA), genomic RNA ends, and the full-length TiLV genome fragment are listed in Table 1.

[0098] Table 1. Primer list

[0099]

[0100]

[0101] plasmids

[0102] The cDNAs for each TiLV segment (SEQ ID NOs: 1-10) were synthesized and inserted into the pcDNA3.1 vector, flanked by a 222-bp human RNA polymerase I (Pol I) promoter sequence and a 34-bp mouse terminator sequence. These plasmids were synthesized by BGI and designated pPolI-TiLV-S1, pPolI-TiLV-S2, pPolI-TiLV-S3, pPolI-TiLV-S4, pPolI-TiLV-S5, pPolI-TiLV-S6, pPolI-TiLV-S7, pPolI-TiLV-S8, pPolI-TiLV-S9, and pPolI-TiLV-S10, respectively. The synthetic sequence of the TiLV segment was essentially identical to the sequence of the TiLV reference genome (NCBI accession number: GCF_001630085.1), wherein the R (G or A) at nucleotide position 481 of the reference genome segment 3 was selected as G during synthesis, the R at nucleotide position 661 was selected as A during synthesis, and the R at nucleotide position 703 was selected as G during synthesis.

[0103] In addition, three plasmids were constructed using overlap extension PCR cloning based on our TiLV genome sequencing results. This method replaced TiLV segments 2, 5, and 6 in plasmids pPolI-TiLV-S2, pPolI-TiLV-S5, and pPolI-TiLV-S6 with the corresponding segments from the TiLV-Israel-HK strain (SEQ ID NO:12, SEQ ID NO:15, and SEQ ID NO:16). The resulting plasmids were named pPolI-TiLV-S2-2, pPolI-TiLV-S5-2, and pPolI-TiLV-S6-2, respectively.

[0104] The plasmid used to rescue the reporter virus was synthesized by BGI and named pPolI-HP5-2A-HiBiT80 (SEQ ID NO: 67). All plasmids used for transfection were extracted using the QIAGEN Plasmid Extraction Kit (Plasmid Maxi or Midi Kit).

[0105] Determination of the complete viral genome

[0106] The 5' and 3' terminal sequences of TiLV genomic fragments were determined using the 3' rapid amplification of cDNA ends (RACE) technique with a 5' / 3' RACE kit (Generation 2, Roche). First, total RNA was extracted from TiLV-infected E11 cells using TRIzol reagent (Invitrogen) and polyadenylated using Poly(A) polymerase (NEB). First-strand cDNA was synthesized using the polyadenylated RNA as a template using oligo(dT)-anchored primers (see Table 1). This cDNA was then used as a template for PCR amplification of the terminal sequences using PCR anchor primers and fragment-specific primers (see Table 1). All target PCR products were purified using a QIAquick Gel Extraction Kit (QIAGEN) and subjected to Sanger sequencing.

[0107] Based on the 3' RACE sequencing results, primers were designed (see Table 1) to amplify the full-length TiLV fragment. Similarly, the target PCR product was purified and subjected to Sanger sequencing.

[0108] Determination of the polyadenylation signal sequence of TiLV using 3' RACE technique

[0109] Total RNA was isolated from E11 cells infected with wild-type TiLV (TiLV-Israel-HK), and the 3' end sequence of the viral mRNA was determined by 3' RACE. All other steps were identical to those described above, except that the total RNA was not polyadenylated.

[0110] Rescue of recombinant, rearranged, and reporter TiLV from cloned cDNA

[0111] Vero E6 and E11 cells (ratio approximately 4:1) were seeded in a T25 cell culture flask and cultured overnight at 37°C. The co-cultured cells were transfected with 10 plasmids (1.25 μg each) using approximately 2 μL of Lipofectamine 3000 (Invitrogen) per μg of plasmid DNA. The transfected cells were then cultured at 28°C.

[0112] Sanger sequencing of recombinant, rearranged, and reporter TiLV genome segments 5 and 6

[0113] The rescued virus was passaged on E11 cells. On day 3 post-infection, total RNA was isolated from the infected E11 cells (at least the third passage of virus) using TRIzol reagent, and cDNA was synthesized using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). Touchdown PCR was performed using this cDNA as a template to amplify the full length of fragments 5 and 6. Finally, the desired PCR products were purified and subjected to Sanger sequencing.

[0114] Next-generation sequencing of recombinant TiLV

[0115] Total RNA was isolated from E11 cells infected with recombinant TiLV-1 (Til-4-2011). Library construction, Illumina sequencing (151 bp paired-end sequencing), and basic de novo assembly were performed at the PanorOmic Sciences Center of the University of Hong Kong.

[0116] Immunofluorescence assay to detect and report TiLV

[0117] Mouse anti-HiBiT monoclonal antibody (Promega) was used to detect cells reporting TiLV infection. Cells were fixed with pre-chilled methanol (Sigma) at -20°C for 15 minutes and then blocked with 1% bovine serum albumin (Sigma) containing 0.3% Triton X-100 (Sigma) for 1 hour at room temperature. Subsequently, cells were incubated with anti-HiBiT antibody (1:1000 dilution) for 2 hours at room temperature. After three consecutive washes with PBS, cells were incubated with Alexa Fluor 488 goat anti-mouse IgG antibody (Invitrogen) at a dilution of 1:1000 for 1 hour at room temperature. Finally, cells were washed three times with PBS, and images were acquired using a Nikon ECLIPSE Ti2-E inverted microscope.

[0118] result

[0119] Establishment of TiLV reverse genetics system

[0120] In the process of establishing a TiLV reverse genetics system, we tested the susceptibility of numerous mammalian cell lines (including but not limited to HEK293T, Calu-3, Vero E6, and BHK21) to TiLV. Ultimately, we found that TiLV could replicate in Vero E6 cells. Therefore, we hypothesized that TiLV could be rescued by transfecting Vero E6 cells. Because available information on the function of TiLV viral proteins is very limited, we employed a bidirectional transcription system used to rescue influenza viruses. This strategy allows for the simultaneous expression of viral genomic RNA (gRNA) and viral mRNA in TiLV-susceptible cells.

[0121] First, we designed a plasmid in which the cDNA of each TiLV fragment was inserted between the RNA polymerase I promoter and the RNA polymerase II promoter ( Figure 1A Insertion of 10 TiLV segment cDNAs will result in the transcription of 10 viral gRNAs and all viral mRNAs. Ten plasmids containing fragments of the TiLV reference genome (GCF_001630085.1, TiLV isolate Til-4-2011) were synthesized and transfected into Vero E6 cells co-cultured with fish-derived E11 cells ( Figure 1B, Table 2). The transfected cells were cultured at 28°C, and the supernatant was collected at different time points to detect the presence of virus in the transfected cells. Starting from 7 days after transfection, the transfected cells were detected to produce recombinant viruses in quantities sufficient to be detected in E11 cells. The recombinant virus was then passaged on E11 cells, and two methods were used to verify that the recombinant virus was indeed derived from the cloned cDNA and was the designed virus (Til-4-2011): (i) The full-length cDNA sequence of recombinant virus segments 5 and 6 was amplified by RT-PCR and sequenced. Sequence analysis confirmed that the sequences of recombinant virus segments 5 and 6 were identical to the cDNA sequence of the plasmid used to rescue the virus. (ii) Total RNA was isolated from E11 cells infected with the recombinant virus and subjected to second-generation sequencing (raw sequencing data are available at NCBI and can be obtained through BioProject No. PRJNA1010714). De novo assembly was performed using the obtained data, and ten of the 15 most highly covered discontinuous long sequences / scaffolds were highly similar (approximately 100%) to TiLV genomic fragments (Table 3). Taken together, these results indicate that we have indeed rescued TiLV using the cloned cDNA.

[0122] Table 2. Plasmids used to generate recombinant, rearranged, and reporter TiLVs

[0123]

[0124] Table 3. Summary of the first 15 discontinuous long sequences / scaffolds assembled from scratch

[0125]

[0126]

[0127] *Number of reads that can be mapped to the de novo assembled TiLV genome × 151 / scaffold length

[0128] To further validate the reliability and practicality of our established system, we performed whole-genome sequencing on a wild-type TiLV strain originating in Israel (designated TiLV-Israel-HK). Since we had successfully rescued TiLV using our developed system, we hypothesized that TiLV could produce complementary RNA (cRNA) during replication. By adding a poly(A) tail to the TiLV gRNA and cRNA, the 3'-terminal sequences of the gRNA and cRNA could be determined using 3' RACE technology. This allowed us to determine the 5'- and 3'-terminal sequences of TiLV genomic fragments. Combining amplification of the full-length cDNA sequences of each TiLV fragment and sequencing, we were able to assemble the complete genome of the TiLV-Israel-HK strain (SEQ ID NOs: 11-20; GenBank accession numbers: OQ437054-OQ437063). Our sequence analysis revealed that segments 2, 5, and 6 exhibited numerous differences compared to the TiLV reference genome, while the sequences of the remaining seven segments were nearly identical to the TiLV reference genome. Therefore, we constructed three new plasmids based on the newly sequenced TiLV-Israel-HK segments 2, 5, and 6. We then transfected co-cultured Vero E6 and E11 cells with the previously synthesized plasmids (segments 1, 3, 4, and 7 to 10) and the three new plasmids (Table 2). The newly rescued virus was passaged in E11 cells, and the full-length cDNA sequences of segments 5 and 6 were amplified and sequenced. Sequence analysis of the amplified fragments showed that the new recombinant TiLV (TiLV-Israel-HK) was also successfully rescued.

[0129] Determination of TiLV polyadenylation signal

[0130] The polyadenylation signal of influenza virus is a string of uridine sequences located near the 5' end of the genomic RNA. However, sequence analysis of the TiLV genome did not reveal a similar structure. This discrepancy led us to further investigate the polyadenylation signal in the TiLV genome. Given that the viral mRNA of influenza virus has a Poly(A) tail, we hypothesized that the viral mRNA of TiLV might also exhibit this feature. Finally, we determined the 3' end sequence of 10 TiLV mRNAs by 3' RACE technology. Sequence analysis showed that the polyadenylation signal sequence of the 10 TiLV fragments was 3'-CCC UUU-5' or 3'-CUC UUU-5'. The position of the polyadenylation signal sequence of fragment 5 is very unique, with 14 nucleotides after its polyadenylation signal. In contrast, the remaining 9 fragments showed a high degree of consistency, with each fragment having 7 nucleotides after the polyadenylation signal sequence ( Figure 2 ).

[0131] Rearrangement of TiLV to the rescue

[0132] Phylogenomics studies have provided evidence of TiLV rearrangement, particularly for segments 5 and 6. To provide direct experimental evidence of TiLV genome segment rearrangement, we transfected co-cultured Vero E6 and E11 cells with a single plasmid containing TiLV-Israel-HK segment 5 or segment 6 and nine plasmids containing the TiLV-4-2011 segment (Table 2). Successful rescue of the rearranged viruses was confirmed by Sanger sequencing. These results demonstrate that our established system can rescue rearranged viruses and further confirm its practicality and versatility.

[0133] HiBiT-tagged reporter TiLV rescue

[0134] Reporter viruses are powerful tools for monitoring viral infection in cells and their hosts. They have proven particularly useful for screening antiviral drugs in vitro and for evaluating the efficacy of vaccines or therapeutics in vivo. However, inserting reporter genes into viral genomes, particularly (segmented) RNA viruses, can negatively impact key biological functions, making the development of reliable reporter viruses challenging. Therefore, we chose to rescue the reporter TiLV by inserting a HiBiT tag, a small 11-amino acid peptide that allows for convenient detection and quantification of HiBiT-tagged proteins.

[0135] Initially, we attempted to add the HiBiT sequence directly to the C-terminus of the predicted ORF of TiLV genomic segment 5 ( Figure 3 A). Despite repeated attempts, we were unable to rescue the reporter virus. We then selected two insertion sites ( Figure 3 A), we predicted that they would have little effect on the function of viral proteins, but this approach also proved unsuccessful. Given that the genome of influenza virus contains a segment-specific packaging signal sequence that is crucial for the packaging of viral RNA into virions, we hypothesized that a similar packaging signal might exist in TiLV. To test this hypothesis, we designed a plasmid (pPolI-HP5-2A-HiBiT80) in which the terminal 80 nucleotides of the ORF of segment 5 were added after the HiBiT sequence to restore the packaging signal ( Figure 3 A). Using this plasmid and nine other unmodified plasmids, we successfully rescued a reporter virus. This reporter virus was able to be passaged in E11 cells and caused significant cytopathic effects, similar to those of the wild-type virus and other rescued viruses. We amplified the full-length sequence of segment 5 of the reporter virus by RT-PCR. Gel electrophoresis imaging showed that, as expected, the size of the full-length segment 5 of the reporter TiLV was larger than that of the full-length segments 5 of the other unmodified rescued viruses ( Figure 3B). This verifies that our modification was successfully integrated into the TiLV genome. In addition, fragment 5 of the 3rd, 4th, and 5th generation reporter viruses was sequenced, and the sequencing results showed that all sequences were identical to the cDNA in the plasmid pPolI-HP5-2A-HiBiT80, and the 80-nucleotide repeat sequence also remained intact. We concluded from these results that the rescued reporter virus was stable in serial passages (at least 5 generations). In addition, we also performed immunofluorescence tests on the reporter virus. Figure 4 As shown, HiBiT-tagged proteins were readily detected in E11 cells infected with the reporter virus using anti-HiBiT antibodies.

[0136] In our experiments, we first tested the susceptibility of multiple mammalian cell lines to TiLV and demonstrated for the first time that TiLV can replicate in Vero E6 cells. Second, to address the unknown functions of most TiLV viral proteins, we designed plasmids that express all viral gRNAs and viral mRNAs. Third, to address the large number of TiLV genomic fragments, we co-cultured Vero E6 cells with E11 cells, which are the most sensitive cell type to TiLV. We also increased the number of cells and the amount of plasmid used for transfection to improve rescue efficiency.

[0137] The successful development of a reverse genetics system for TiLV suggests that the replication mechanism of TiLV is similar to that of influenza virus. Our genome sequencing results indicate that viral cRNA is also present during TiLV replication. However, the exact replication mechanism requires further investigation. Furthermore, TiLV exhibits several unique properties. For example, by sequencing the terminal sequences of the viral mRNA, we identified a polyadenylation signal sequence in the TiLV genome. However, this signal sequence differs from that found in other RNA viruses.

[0138] The significance of this invention lies in the fact that we have successfully established, for the first time, a completely plasmid-based reverse genetics system for TiLV. Overall, this reverse genetics system will facilitate further research into the replication and molecular pathogenesis of TiLV. Most importantly, it will pave the way for the development of new vaccines and antiviral drugs.

Claims

1. A method for rescuing recombinant tilapia lake virus (TiLV), comprising: i) preparing a plurality of expression plasmids for expressing the ten genomic segments of TiLV, wherein each expression plasmid of the plurality of expression plasmids comprises at least one complementary DNA (cDNA) sequence of the ten genomic segments of TiLV; ii) transfecting the multiple expression plasmids into co-cultured Vero cells and fish-derived cells sensitive to TiLV; iii) culturing the transfected cells obtained in step ii) at a temperature of about 28° C. to rescue the recombinant TiLV; and selectively rescuing iv) Reassortant TiLV. 2 . The method according to claim 1 , wherein the plurality of expression plasmids are bidirectional expression plasmids that express all viral genomic RNA (gRNA) and viral messenger RNA (mRNA) of TiLV. 3 . The method according to claim 1 , wherein the cDNA sequences of the ten genomic segments of TiLV are at least 90% identical to SEQ ID NOs: 1-10, respectively. 4 . The method according to claim 1 , wherein the cDNA sequences of the ten genomic segments of TiLV are at least 90% identical to SEQ ID NOs: 11-20, respectively. 5 . The method according to claim 1 , wherein the cDNA sequences of the ten genomic segments of TiLV comprise or consist of SEQ ID NOs: 1-10, respectively. 6 . The method according to claim 1 , wherein the cDNA sequences of the ten genomic segments of TiLV comprise or consist of SEQ ID NOs: 11-20, respectively. The method according to claim 1 , wherein the fish-derived cells are E11 cells derived from Channa striatus . The method according to claim 1 , wherein the co-cultured cells are Vero E6 cells and E11 cells.

9. A method for rescuing a reporter gene-labeled TiLV, the method comprising: i) preparing an expression plasmid comprising a cDNA sequence of one genomic segment of TiLV and a reporter tag sequence, wherein the reporter tag sequence is integrated at the 3' end of the open reading frame (ORF) of the genomic segment, and a certain number of nucleotides at the 3' end of the ORF are inserted after the sequence tag; and preparing a plurality of expression plasmids, each of the plurality of expression plasmids comprising a cDNA sequence of at least one genomic segment of the remaining nine genomic segments of TiLV; ii) transfecting all the expression plasmids prepared in step i) into co-cultured Vero cells and fish-derived cells sensitive to TiLV; as well as iii) culturing the transfected cells obtained in step ii) at a temperature of about 28° C., thereby rescuing the reporter gene-tagged TiLV.

10. The method according to claim 9, wherein the expression plasmid is a bidirectional expression plasmid that expresses all viral gRNAs and viral mRNAs of TiLV.

11. The method of claim 9, wherein the cDNA sequences of the ten genomic segments of TiLV are at least 90% identical to SEQ ID NOs: 1-10, respectively.

12. The method of claim 9, wherein the cDNA sequences of the ten genomic segments of TiLV are at least 90% identical to SEQ ID NOs: 11-20, respectively.

13. The method according to claim 9, wherein the cDNA sequences of the ten genomic segments of TiLV comprise or consist of SEQ ID NOs: 1-10, respectively.

14. The method according to claim 9, wherein the cDNA sequences of the ten genomic segments of TiLV comprise or consist of SEQ ID NOs: 11-20, respectively. The method according to claim 9 , wherein the fish-derived cells are E11 cells derived from Channa fasciatus. The method according to claim 9 , wherein the co-cultured cells are Vero E6 cells and E11 cells.

17. The method according to claim 9, wherein the reporter gene tag sequence is HiBiT, GFP, NanoLuc or mCherry.

18. Recombinant, rearranged and reporter TiLV rescued according to the method of claim 1 or 9.

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