A d / yama2019 genetic evolution lineage influenza b virus reverse genetic manipulation system

Abstract

The application discloses a D / Yama2019 genetic evolution lineage influenza D virus reverse genetic manipulation system. Seven pairs of primers are designed to amplify DNA fragments corresponding to full-length genome PB2, PB1, P3, HEF, NP, M and NS segments of D / JY3002, and the DNA fragments are cloned into pHW2000 and pCC1-DualPro vectors to construct a plasmid, and the plasmid is co-transfected into a cell line to obtain artificially rescued D / Yama2019 genetic evolution lineage influenza D virus rD / JY3002. The genome sequence of rD / JY3002 is completely consistent with that of natural isolated strain D / JY3002, and the infection ability is equivalent, and the rD / JY3002 can be stably passaged for at least five generations. A unidirectional expression plasmid pPolI-D / JY3002-PB1-240-GFP-240 is constructed to obtain artificially rescued recombinant influenza D virus rD / JY3002-GFP carrying a green fluorescent reporter gene, and after the rD / JY3002-GFP infects host cells, the GFP expression amount is high and stable. The application establishes a high-efficiency and stable D / Yama2019 genetic evolution lineage influenza D virus reverse genetic manipulation system, and can be used for developing an influenza D virus vector to present an exogenous gene.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to a reverse genetic operating system for influenza virus type D of the D / Yama2019 genetic evolution lineage. Background Art

[0002] Influenza D virus, a new member of the Orthomyxoviridae family, was first isolated in the United States in 2011 from pigs with respiratory symptoms. Despite this, numerous epidemiological studies have shown that cattle are the primary natural reservoir for the virus. Influenza D virus circulates widely in cattle populations in nearly thirty countries, causing mild to moderate respiratory illness in cattle. Furthermore, specific antibodies have been detected in the sera of pigs, sheep, goats, horses, camels, deer, dogs, and humans, suggesting that influenza D virus may have multiple animal reservoirs and potentially infect humans.

[0003] The influenza D virus genome consists of seven negative-strand RNA segments: PB2, PB1, P3, HEF, NP, M, and NS. The three longest segments encode the viral polymerase complex subunits PB2, PB1, and P3, respectively, which catalyze the transcription and replication of viral genomic RNA (vRNA). The fourth segment encodes the major viral surface glycoprotein, the hemagglutinin-esterase-fusion protein (HEF), responsible for viral entry, fusion, and release. The fifth segment encodes the viral nucleoprotein (NP), which, together with the vRNA and polymerase complex, forms the core unit of viral replication—the genomic ribonucleoprotein complex (vRNP). The sixth segment encodes the viral matrix proteins DM1 and DM2. The shortest segment encodes the viral nonstructural proteins NS1 and NS2.

[0004] Based on the HEF genome segment sequences, influenza D viruses are divided into at least five genetic lineages: D / OK, D / 660, D / Yama2016, D / Yama2019, and D / CA2019. Influenza D viruses from the D / OK and D / 660 lineages are primarily found in cattle populations in Europe and the Americas. Influenza D viruses from the D / Yama2019 lineage have so far only been isolated from cattle populations in Asian countries such as China, Japan, and South Korea. Influenza D viruses from the D / Yama2016 and D / CA2019 lineages have only been reported in cattle populations in Japan and the United States, respectively.

[0005] The reverse genetics operating system is one of the most important tools for studying influenza virus biology. Negative sense strands and segmentation are two key features of the influenza virus genome. The establishment of an influenza virus reverse genetics operating system based entirely on exogenous plasmids focuses on constructing seven or eight bidirectional expression plasmids to simultaneously produce vRNA and mRNA for all viral genome segments, or constructing eleven or twelve unidirectional expression plasmids to produce vRNA for all viral genome segments and mRNA for four viral replication core unit proteins. The above strategy is also the most commonly used method for establishing reverse genetics operating systems for influenza viruses of different types, subtypes (or different genetic evolutionary lineages) and different strains.

[0006] The D / Yama2019 lineage of influenza D viruses is the predominant influenza D virus circulating in cattle populations in Asia. Importantly, studies have shown that influenza D viruses from this lineage are more potent in replication and pathogenicity than those from other lineages, but the specific mechanisms remain unclear. The lack of reverse genetics tools for influenza D viruses from the D / Yama2019 lineage has significantly hindered research on this lineage, including studies of viral replication and transmission, exploration of pathogenic mechanisms, and vaccine development. Summary of the Invention

[0007] At present, the technical difficulties in establishing a reverse genetic operating system for the D / Yama2019 genetic evolutionary lineage type D influenza virus are mainly reflected in the following aspects: First, obtaining the complete genome sequence of the D / Yama2019 genetic evolutionary lineage type D influenza virus strain, especially the accurate sequences at both ends of the viral genome; Second, constructing a bidirectional expression plasmid containing DNA fragments corresponding to the genome segments of the D / Yama2019 genetic evolutionary lineage type D influenza virus strain, especially the DNA fragments corresponding to the unstable HEF genome segments; Third, establishing suitable and effective reverse genetic operation methods and steps for influenza D virus.

[0008] In response to the above-mentioned deficiencies in the prior art, the present invention provides a reverse genetic operating system for influenza virus type D of the D / Yama2019 genetic evolution lineage (strain name: D / bovine / CHN / JY3002 / 2022 (D / JY3002)), including primers, plasmids, methods and applications for constructing the reverse genetic operating system.

[0009] The first object of the present invention is to provide a set of primers for amplifying full-length DNA fragments corresponding to the genome segments of influenza virus type D of the D / Yama2019 genetic evolution lineage, comprising primers having nucleotide sequences as shown in SEQ ID NOs. 1-14. The nucleotide sequences of the primers for amplifying the PB2 segment of the influenza virus type D strain D / JY3002 genome are shown in SEQ ID NOs. 1-2, the nucleotide sequences of the primers for amplifying the PB1 segment are shown in SEQ ID NOs. 3-4, the nucleotide sequences of the primers for amplifying the P3 segment are shown in SEQ ID NOs. 5-6, the nucleotide sequences of the primers for amplifying the HEF segment are shown in SEQ ID NOs. 7-8, the nucleotide sequences of the primers for amplifying the NP segment are shown in SEQ ID NOs. 9-10, the nucleotide sequences of the primers for amplifying the M segment are shown in SEQ ID NOs. 11-12, and the nucleotide sequences of the primers for amplifying the NS segment are shown in SEQ ID NOs. 13-14.

[0010] The second object of the present invention is to provide a primer set for amplifying a linearized vector for seamless cloning, which comprises a universal upstream primer with a nucleotide sequence as shown in SEQ ID NO.15 and segment-specific downstream primers with nucleotide sequences as shown in SEQ ID NO.16-22.

[0011] A third object of the present invention is to provide a pCC1-DualPro vector, the nucleotide sequence of which is shown in SEQ ID NO. 30. This vector can be used as a bidirectional expression plasmid for cloning DNA fragments corresponding to the HEF genomic segment of influenza virus D / JY3002. The bidirectional expression plasmid pHW2000 is a commonly used high-copy plasmid for constructing reverse genetic manipulation systems for influenza viruses. However, when attempting to clone and insert DNA fragments corresponding to the HEF genomic segment of influenza virus D into the pHW2000 plasmid, partial deletions of the inserted DNA fragments occur. To address this technical problem, the present invention constructs a bidirectional expression plasmid pCC1-DualPro with controllable copy number, enabling stable cloning and insertion of DNA fragments corresponding to the HEF genomic segment of influenza virus D into the pCC1-DualPro plasmid.

[0012] A fourth object of the present invention is to provide a unidirectional expression plasmid, pPolI-D / JY3002-PB1-240-GFP-240, carrying a green fluorescent reporter gene and a packaging signal for influenza virus D of the D / Yama2019 genetic evolutionary lineage. The nucleotide sequence of the plasmid is shown in SEQ ID NO. 31. This plasmid contains the GFP gene and DNA fragments corresponding to the sequences at both ends of the PB1 genomic segment of influenza virus D / JY3002. The sequences at both ends of the PB1 genomic segment can serve as packaging signal sequences to control the transcription, replication, and packaging of the corresponding genomic segment. The unidirectional expression plasmid, pPolI-D / JY3002-PB1-240-GFP-240, can transcribe into mutant recombinant vRNA containing the GFP gene. All constructed bidirectional expression plasmids containing DNA fragments corresponding to the influenza virus D / JY3002 genomic segment and this unidirectional expression plasmid are co-transfected into a cell line to rescue recombinant influenza virus D rD / JY3002-GFP carrying the green fluorescent reporter gene. The cell line was infected with rD / JY3002-GFP, and the infected cells could be observed to express green fluorescent protein under a fluorescence microscope.

[0013] A fifth object of the present invention is to provide a method for constructing a reverse genetic operating system for influenza virus type D of the D / Yama2019 genetic evolution lineage, comprising the following steps:

[0014] The DNA fragments corresponding to the PB2, PB1, P3, NP, M, and NS segments were seamlessly cloned into the pHW2000 vector, and the DNA fragment corresponding to the HEF segment was seamlessly cloned into the pCC1-DualPro vector to obtain seven bidirectional expression plasmids, which were then co-transfected into cell lines to obtain artificially rescued D / Yama2019 genetic evolutionary lineage D influenza virus;

[0015] The nucleotide sequences of the DNA fragments corresponding to the PB2, PB1, P3, NP, M and NS segments are shown in SEQ ID NOs. 23-25 ​​and 27-29, respectively; the nucleotide sequence of the DNA fragment corresponding to the HEF segment is shown in SEQ ID NO. 26; the nucleotide sequence of the pCC1-DualPro vector is shown in SEQ ID NO. 30.

[0016] Preferably, in the method described above, seven bidirectional expression plasmids and the unidirectional expression plasmid pPolI-D / JY3002-PB1-240-GFP-240 are co-transfected into the cell line during co-transfection; the nucleotide sequence of the unidirectional expression plasmid pPolI-D / JY3002-PB1-240-GFP-240 is shown in SEQ ID NO.31.

[0017] Preferably, the co-transfection into the cell line comprises the following steps:

[0018] S1. One day before transfection, plate 293T cells and MDCK cells at a ratio of 3:1 in a six-well cell culture plate. Culture in 5% CO2 at 37°C. When the cell density reaches 75%, begin transfection. Replace the old culture medium with 1.5 mL of DMEM supplemented with 10% fetal bovine serum but without antibiotics.

[0019] S2. Take 1 μg of each of the seven bidirectional expression plasmids or the seven bidirectional expression plasmids and the unidirectional expression plasmid pPolI-D / JY3002-PB1-240-GFP-240, add 200 μL of Opti-MEM and mix thoroughly. Then add 20 μL of PEI-25K transfection reagent and let the mixture stand for 25 minutes. Then add the mixture to the six-well cell culture plate prepared in step S1 and culture at 37°C for 6-9 hours.

[0020] S3. Replace the old culture medium with 1.7 mL of DMEM containing 100 U / mL penicillin and 100 μg / mL streptomycin, without fetal bovine serum. Incubate at 37°C for 36-42 hours, then supplement with 500 μL of DMEM containing 100 U / mL penicillin, 100 μg / mL streptomycin, and 1 μg / mL TPCK-trypsin. Continue incubating for 3-4 days, supplementing with 200-400 μL of DMEM containing 100 U / mL penicillin, 100 μg / mL streptomycin, and 0.1 μg / mL TPCK-trypsin as appropriate.

[0021] S4. Wait until the HA titer of the cell supernatant reaches 2 3 -2 6 HA units / 25μL, collect the cell supernatant, and obtain the artificially rescued D / Yama2019 genetic evolution lineage type D influenza virus.

[0022] The co-transfection reverse genetics operation steps of the present invention are different from those of other types of influenza viruses and are improved and optimized according to the replication characteristics of influenza D virus. Because the rescue and detoxification time of influenza D virus is significantly later than that of influenza A and influenza B viruses, usually requiring 72 hours, while the latter are generally between 24-48 hours, it is necessary to ensure that the cells after the transfection plasmid have a good state to support the rescue and detoxification of influenza D virus. The purpose of selecting the transfection plasmid to replace the culture medium with DMEM culture medium containing antibiotics but not containing fetal bovine serum after 6-9 hours, and selecting to supplement TPCK-trypsin after 36-42 hours of cultivation is to ensure that the cells have a good growth state. However, when rescuing influenza A and influenza B viruses, the culture medium during the transfection plasmid is the culture medium without fetal bovine serum, and the liquid change after 6-9 hours is replaced with the culture medium containing TPCK-trypsin.

[0023] The sixth object of the present invention is to provide an artificially rescued D / Yama2019 genetic evolution lineage type D influenza virus constructed using the construction method of the D / Yama2019 genetic evolution lineage type D influenza virus reverse genetic operating system.

[0024] The seventh object of the present invention is to provide a D / Yama2019 genetic evolution lineage type D influenza virus reverse genetic operating system reagent set, which comprises the primer set for amplifying the full-length DNA fragment of the D / Yama2019 genetic evolution lineage type D influenza virus genome segment, the primer set for amplifying the linearized vector for seamless cloning, the pCC1-DualPro vector and the unidirectional expression plasmid pPolI-D / JY3002-PB1-240-GFP-240.

[0025] The present invention uses the cDNA reverse transcribed from viral RNA as a template, and uses seven pairs of primers to amplify the DNA fragments corresponding to the PB2, PB1, P3, HEF, NP, M and NS segments of the full-length genome of D / JY3002. The DNA fragments are seamlessly cloned into the pHW2000 vector and the pCC1-DualPro vector to construct the plasmids pHW2000-D / JY3002-PB2, pHW2000-D / JY3002-PB1, pHW2000-D / JY3002-P3, and pCC1-DualPro. C1-DualPro-D / JY3002-HEF, pHW2000-D / JY3002-NP, pHW2000-D / JY3002-M, and pHW2000-D / JY3002-NS were co-transfected into cell lines to generate the rescued D / Yama2019 lineage D influenza virus, rD / JY3002. Whole-genome sequencing revealed that the genome sequence of rD / JY3002 was identical to that of the naturally isolated strain D / JY3002. Infection and passage experiments demonstrated that the rescued influenza D virus possessed comparable infectivity to the naturally isolated strain, and that the rescued virus could be stably passaged for at least five generations. Using the constructed unidirectional expression plasmid pPolI-D / JY3002-PB1-240-GFP-240, a recombinant influenza D virus carrying a green fluorescent reporter gene, rD / JY3002-GFP, was artificially rescued. After infection of host cells, GFP expression was high and stable. This invention establishes an efficient and stable reverse genetics system for influenza D viruses of the D / Yama2019 genetic evolutionary lineage, which can be used to develop influenza D virus vectors for delivering exogenous genes. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1are the two end sequences of each genome segment of influenza D virus strain D / JY3002 obtained by high-depth high-throughput sequencing; wherein, Figures A-G are 100 bp sequences of 5' end and 3' end of PB2, PB1, P3, HEF, NP, M and NS genome segments of influenza D virus of different genetic evolution lineages (D / OK, D / 660 and D / Yama2019), respectively; the third line in the sequence alignment chart shows the two end sequences of the genome segment of the published but incomplete influenza D virus strain D / JY3002, and the fourth line is the complete two end sequences of the genome segment of the influenza D virus strain D / JY3002 obtained in Example 1, and the red box part of the chart shows the non-coding region sequence of the two ends of the genome segment of the influenza D virus strain D / JY3002.

[0027] Figure 2 is the electrophoresis result of the products of amplifying the DNA fragments (PB2, PB1, P3, HEF, NP, M and NS) corresponding to the genome segments of influenza D virus D / JY3002 using the seven groups of primer sequences (SEQ ID NO. 1-14) designed and synthesized; "M" on the rightmost lane represents Marker, used to indicate the size of the DNA fragments.

[0028] Figure 3 is a colony PCR identification electrophoretogram; wherein, A is the electrophoretogram of PCR identification of the colonies obtained by seamlessly cloning the DNA fragments corresponding to the HEF genome segment of D / JY3002 into pHW2000 vector; lanes 1-7 represent 7 different colony samples; B is the electrophoretogram of PCR identification of the colonies obtained by seamlessly cloning the DNA fragments corresponding to the HEF genome segment of D / JY3002 into pCC1-DualPro vector; lanes 1-2 represent 2 different colony samples; "M" represents Marker, used to indicate the size of the DNA fragments.

[0029] Figure 4This is a map of the bidirectional expression plasmid pCC1-DualPro; the bidirectional expression plasmid pCC1-DualPro is a low-copy plasmid with controllable copy number, containing the CMV promoter (Human cytomegalovirus immediate early promoter) and the BGH poly A terminator (Bovine growth hormone polyadenylation signal), which are used to transcribe and produce the corresponding viral mRNA; the Human Pol I promoter (Human RNA polymerase I promoter) and Murine Pol I terminator (Murine RNA polymerase I termination signal) are embedded in the opposite direction, which are used to transcribe and produce the corresponding viral vRNA.

[0030] Figure 5 Maps of bidirectional expression plasmids containing DNA fragments corresponding to influenza virus D / JY3002 genome segments; A is the map of pHW2000-D / JY3002-PB2, B is the map of pHW2000-D / JY3002-PB1, C is the map of pHW2000-D / JY3002-P3, D is the map of pCC1-DualPro-D / JY3002-HEF, E is the map of pHW2000-D / JY3002-NP, F is the map of pHW2000-D / JY3002-M, and G is the map of pHW2000-D / JY3002-NS.

[0031] Figure 6 The infection titers of artificially rescued influenza virus rD / JY3002 and different passages are shown; A is the infection titer of artificially rescued influenza virus rD / JY3002 and naturally isolated influenza virus D / JY3002 (TCID 50 ); B is the infectious titer of artificially rescued influenza D viruses of different passages (rD / JY3002-P1 to P5); "ns" indicates no significant difference.

[0032] Figure 7This is a map of the unidirectional expression plasmid pPolI-D / JY3002-PB1-240-GFP-240; the unidirectional expression plasmid pPolI-D / JY3002-PB1-240-GFP-240 contains DNA fragments corresponding to 240bp sequences at the 5' and 3' ends of the influenza virus D / JY3002 PB1 genomic segment, embedded between the Human Pol I promoter (Human RNA polymerase I promoter) and the Murine Pol I terminator (Murine RNA polymerase I termination signal, abbreviated as PolI Ter. in the figure). This plasmid can transcribe a mutant recombinant vRNA containing the GFP gene.

[0033] Figure 8 Figure 3: Artificially rescued expression and infection of recombinant influenza D virus rD / JY3002-GFP; A: After recombinant influenza D virus rD / JY3002-GFP-P0 and rD / JY3002-GFP-P1 infected MDCK cells, the infected cells expressed green fluorescent protein as observed under a fluorescence microscope; B: The infection titer of rD / JY3002-GFP-P0 and rD / JY3002-GFP-P1. DETAILED DESCRIPTION

[0034] The following examples are provided to further illustrate the present invention, but are not intended to limit the present invention.

[0035] Example 1

[0036] 1. Amplification of the full-length genome sequence of influenza virus D / JY3002 and the corresponding DNA fragments of each genome segment

[0037] We previously published the incomplete genome sequence of influenza virus D / JY3002 on GenBank (https: / / www.ncbi.nlm.nih.gov / nuccore / ?term=influenza%20D%20virus%20D / bovine / CHN / JY3002) (accession numbers: OR685126-OR685132). To establish a reverse genetics system for D / JY3002, this example used high-throughput sequencing to obtain the full-length genome sequence of influenza virus strain D / bovine / CHN / JY3002 / 2022 (D / JY3002), belonging to the D / Yama 2019 lineage. Specifically, we obtained the precise sequences at both ends of the genome segments. Then, seven pairs of primers (SEQ ID NO.1-NO.14) were designed based on the full-length genome sequence of D / JY3002, and DNA fragments corresponding to the full-length genome segments PB2, PB1, P3, HEF, NP, M and NS of D / JY3002 were amplified.

[0038] 1.1 Obtaining the full-length genome sequence of influenza virus D / JY3002 by high-throughput sequencing

[0039] Influenza virus D / JY3002 was propagated using MDCK (Madin Darby Canine Kidney, ATCC: CCL-34) cells, and 9.6 mL of virus supernatant sample (HA titer was 2 8 HA units / 25μL), centrifuged at 12000rpm for 10 minutes to remove cell debris, filtered the sample with a 0.45μm syringe filter, and then concentrated to 0.3mL using a 100kDa centrifugal concentration filter. Viral RNA was extracted using the TRIzol method, and a high-throughput sequencing library was prepared according to the instructions of the BGI MGIEasy Respiratory Microbial Genome Amplification Kit. Finally, high-throughput sequencing was completed using the BGI G99 sequencer to obtain the full-length genome sequence of the D / JY3002 virus strain, especially the accurate sequences at both ends of the genome segment ( Figure 1 The nucleotide sequences of the full-length genomic segments PB2, PB1, P3, HEF, NP, M, and NS of D / JY3002 are shown in SEQ ID NOs. 23-29, respectively.

[0040] 1.2 Amplification of DNA fragments corresponding to D / JY3002 genomic segments

[0041] The extracted viral RNA was reverse transcribed into cDNA using a Vazyme HiScript III 1st Strand cDNA Synthesis kit and used as a template for amplifying the DNA fragment corresponding to the D / JY3002 genomic segment. Then, the full-length fragment of the corresponding genomic segment was amplified using a high-fidelity DNA polymerase using seven pairs of primers (SEQ ID NO. 1-14). Figure 2 ). Among them, the nucleotide sequences of primers used to amplify the PB2 segment of the influenza D virus strain D / JY3002 genome are shown in SEQ ID NOs. 1-2, the nucleotide sequences of primers for amplifying the PB1 segment are shown in SEQ ID NOs. 3-4, the nucleotide sequences of primers for amplifying the P3 segment are shown in SEQ ID NOs. 5-6, the nucleotide sequences of primers for amplifying the HEF segment are shown in SEQ ID NOs. 7-8, the nucleotide sequences of primers for amplifying the NP segment are shown in SEQ ID NOs. 9-10, the nucleotide sequences of primers for amplifying the M segment are shown in SEQ ID NOs. 11-12, and the nucleotide sequences of primers for amplifying the NS segment are shown in SEQ ID NOs. 13-14.

[0042] The primer sequences are as follows:

[0043] SEQ ID NO.1: 5'-AGCATAAGCAGAGGATGTCACTACTATTAACGC-3',

[0044] SEQ ID NO.2: 5'-CCGCCGGGTTATTAGCAGTAGCAAGAGGATTTTTTCAATGTG-3';

[0045] SEQ ID NO.3: 5'-GGAGCATAAGCAGAGGATTTTATAACAATGGA-3',

[0046] SEQ ID NO.4: 5'-CCGCCGGGTTATTAGCAGTAGCAAGAGGATTTTTC-3';

[0047] SEQ ID NO.5: 5'-AGCATAAGCAGGAGATTTAGAAATGTCTAGTAT-3',

[0048] SEQ ID NO.6: 5'-CCGCCGGGTTATTAGCAGTAGCAAGGAGATTTTTAA-3';

[0049] SEQ ID NO.7: 5'-AGCATAAGCAGGAGATTTTCAAAGATGTTTTTG-3',

[0050] SEQ ID NO.8: 5'-CCGCCGGGTTATTAGCAGTAGCAAGGAGATTTTTTCTAAGAT-3';

[0051] SEQ ID NO.9: 5'-AGCATAAGCAGGAGATTATTAAGCAATATGGAC-3',

[0052] SEQ ID NO.10: 5'-CCGCCGGGTTATTAGCAGTAGCAAGGAGATTTTTTGTTAAAT-3';

[0053] SEQ ID NO.11: 5'-GGAGCATAAGCAGAGGATAATTTTTGACGCAATG-3',

[0054] SEQ ID NO.12: 5'-CCGCCGGGTTATTAGCAGTAGCAAGAGGATTTTTTCGCGA-3';

[0055] SEQ ID NO.13: 5'-GGAGCATAAGCAGGGGTGTACAATTTCAATATG-3',

[0056] SEQ ID NO. 14: 5'-CCGCCGGGTTATTAGCAGTAGCAAGGGGTTTTTTCATACT-3'.

[0057] The PCR amplified products were separated by electrophoresis in 1% agarose gel ( Figure 2 ), and the DNA fragment size was consistent with the expected size. An agarose gel containing the target DNA fragment was cut and purified using a gel DNA recovery kit (Omega). The DNA concentration was measured and stored in a refrigerator at 4°C. This served as the target insert gene for subsequent seamless cloning into the corresponding plasmid vector.

[0058] 2. Construction of a bidirectional expression plasmid containing DNA fragments corresponding to the D / JY3002 genome segment

[0059] The bidirectional expression plasmids pHW2000 and pCC1-DualPro were linearized by PCR amplification, and the PCR products were purified and recovered using a gel DNA recovery kit (Omega).

[0060] A universal upstream primer (SEQ ID NO. 15) and specific downstream primers corresponding to the genomic segments (SEQ ID NO. 16-22) were designed and used. PCR reactions were performed using the bidirectional expression plasmid pHW2000 or pCC1-DualPro as templates and high-fidelity DNA polymerase to obtain linearized pHW2000 and pCC1-DualPro, respectively.

[0061] The primer sequences are as follows:

[0062] SEQ ID NO.15: 5'-TACTGCTAATAACCCGGCGGCCCAAAATGCCG-3';

[0063] SEQ ID NO.16 (PB2): 5'-TGACATCCTCTGCTTATGCTCCCCCCCAACTTCGGAGGTCGA-3',

[0064] SEQ ID NO.17(PB1): 5'-AAATCCTCTGCTTATGCTCCCCCCCAACTTCGGAGGTCGA-3',

[0065] SEQ ID NO.18(P3): 5'-CTAAATCTCCTGCTTATGCTCCCCCCCAACTTCGGAGGTCGA-3',

[0066] SEQ ID NO.19 (HEF): 5'-GAAAATCTCCTGCTTATGCTCCCCCCCAACTTCGGAGGTCGA-3',

[0067] SEQ ID NO. 20 (NP): 5'-AATAATCTCCTGCTTATGCTCCCCCCCAACTTCGGAGGTCGA-3', SEQ ID NO. 21 (M): 5'-ATATCCTCTGCTTATGCTCCCCCCCAAACTTCGGAGGTCGA-3', SEQ ID NO. 22 (NS): 5'-TACACCCCTGCTTATGCTCCCCCCCAACTTCGGAGGTCGA-3'.

[0068] In order to improve the connection efficiency, overcome the limitation of enzyme cutting sites, and avoid the insertion of exogenous sequences, this embodiment uses homologous recombinase (Sanggong Seamless Cloning Kit, Seamless Cloning Kit) to connect the DNA fragment corresponding to the D / JY3002 genomic segment and the linearized bidirectional expression plasmid. The connection product was transformed into DH5α competent cells, and the bacterial solution was spread on an LB culture plate containing ampicillin (or chloramphenicol) and inverted and cultured overnight at 37°C. Monoclonal colonies were picked and inoculated into 5mL of LB liquid culture medium containing antibiotics, and cultured on a shaker at 37°C overnight. Colony PCR verification was performed using specific primers (SEQ ID NO.1-14).

[0069] When attempting to seamlessly clone the DNA fragment corresponding to the D / JY3002 HEF genomic segment into the pHW2000 vector, and then performing PCR verification and Sanger sequencing on the obtained colonies, it was found that the DNA fragment corresponding to the HEF genomic segment ligated into pHW2000 was deleted, e.g. Figure 3 As shown in A, lanes 1-7 are 7 randomly selected colonies, among which no specific DNA bands were detected in lanes 1-2, indicating that colonies 1-2 are negative colonies; while DNA bands were detected in lanes 3-7, but the size was significantly smaller than the size of the complete HEF gene band (as shown in Figure 1). Figure 3 The results of the experiment were as follows (lanes 1-2 in B), indicating that the HEF gene cloned into colonies 3-7 was deleted. Sanger sequencing further confirmed this result. In order to solve this problem, taking into account the instability of the DNA fragment corresponding to the HEF genomic segment in the high-copy plasmid, the low yield of single-copy or low-copy plasmids, and the need for bidirectional expression characteristics, this embodiment newly modified and optimized a bidirectional expression vector pCC1-DualPro with controllable copy number (after adding an inducer, it will convert from a low-copy plasmid to a high-copy plasmid), and seamlessly cloned the DNA fragment corresponding to the D / JY3002 HEF genomic segment into this vector ( Figure 4 ), the nucleotide sequence of which is shown in SEQ ID NO.30. The obtained colonies were then verified by PCR and Sanger sequencing, and the results showed that the DNA fragment corresponding to the HEF genomic segment connected to pCC1-DualPro was complete and correct ( Figure 3(B, lanes 1-2) The modified bidirectional expression plasmid pCC1-DualPro has a controllable copy number, and DNA fragments corresponding to the HEF genomic segment can be stably cloned and inserted into the pCC1-DualPro plasmid. DNA fragments corresponding to other genomic segments of D / JY3002 were seamlessly cloned into the pHW2000 vector. The resulting colonies were verified by PCR and Sanger sequencing. Ultimately, bidirectional expression plasmids containing DNA fragments corresponding to each genomic segment of D / JY3002 were obtained. The constructed plasmids were named pHW2000-D / JY3002-PB2, pHW2000-D / JY3002-PB1, pHW2000-D / JY3002-P3, pCC1-DualPro-D / JY3002-HEF, pHW2000-D / JY3002-NP, pHW2000-D / JY3002-M, and pHW2000-D / JY3002-NS ( Figure 5 ).

[0070] 3. Artificial rescue of influenza D virus and virus infection and passage

[0071] 3.1 Artificial rescue of influenza D virus

[0072] (1) One day before transfection, 293T cells and MDCK cells were plated in a 3:1 ratio in a six-well cell culture plate and co-cultured in a 5% CO2, 37°C incubator. When the cell density reached 75%, transfection was started.

[0073] (2) Before transfection, place the required reagents at room temperature to keep them consistent with room temperature, and replace the old cell culture medium with 1.5 mL of DMEM culture medium containing 10% fetal bovine serum but without antibiotics (this step is one of the key steps and is crucial for maintaining a good cell state after transfection).

[0074] (3) Take 1 μg of each of the seven plasmids constructed above, then add 200 μL Opti-MEM and mix thoroughly.

[0075] (4) Slowly drip 20 μL of PEI-25K transfection reagent into the tube, gently pipette 6 times, and let it stand at room temperature for 25 minutes.

[0076] (5) Gently add the above mixture to a six-well cell culture plate and incubate in a 37°C incubator for 6-9 hours. (6) Replace the old culture medium with 1.7 mL of DMEM culture medium containing antibiotics (100 U / mL penicillin and 100 μg / mL streptomycin, the same below) and without fetal bovine serum. After incubation at 37°C for 36-42 hours, add 500 μL of DMEM culture medium containing antibiotics and 1 μg / mL TPCK-trypsin (this step is different from the reverse genetic operation steps of other types of influenza viruses. It is an improved and optimized operation step based on the replication characteristics of influenza D virus).

[0077] (7) After culturing at 37°C for 3-4 days, the virus in the cell supernatant was detected by hemagglutination test. If the culture medium turned yellow during the process, 200-400 μL of DMEM culture medium containing antibiotics and 0.1 μg / mL TPCK-trypsin could be added. Normally, the HA titer of the cell supernatant can reach 2 3 -2 6 HA units / 25μL. In this example, the HA titer of the cell supernatant 5 days after transfection was 2 6 HA units / 25μL.

[0078] (8) The cell supernatant was collected and labeled as the P0 virus, named rD / JY3002, thereby obtaining the artificially rescued influenza D virus rD / JY3002.

[0079] 3.2 rD / JY3002 infection and passage

[0080] The results of whole genome sequencing showed that the genome sequence of rD / JY3002 was completely consistent with the genome sequence of the naturally isolated strain D / JY3002.

[0081] In order to confirm the successful rescue of influenza virus type D rD / JY3002, the infectivity and passage ability of rD / JY3002 were further verified. 600 μL / well P0 virus was inoculated into MDCK cells (six-well plate) and incubated in a 37°C incubator for 1.5-2 hours. The inoculum was then discarded and replaced with virus culture medium (DMEM culture medium containing antibiotics and 0.5 μg / mL TPCK-trypsin). Five days after inoculation, the virus titer in the cell supernatant was measured. The infectious amount of rD / JY3002 in the cell supernatant was determined using the Reed-Muench method and was 5.01 x 10 6 TCID 50 / mL(TCID 50 : 50% Tissue Culture Infectious Dose, half cell culture infectious dose) Figure 6A in the figure). Then, the cell supernatant was collected and labeled as the P1 generation virus, named rD / JY3002-P1. 0.01 MOI of rD / JY3002-P1 was inoculated into MDCK cells (six-well plates). Five days after inoculation, the cell supernatant was collected and labeled as the P2 generation virus, named rD / JY3002-P2, and its infectious titer was determined. Similarly, different generations of artificially rescued influenza D viruses were harvested, namely: rD / JY3002-P1, rD / JY3002-P2 (6.76x10 6 TCID 50 / mL)、rD / JY3002-P3(1.08x 10 6 TCID 50 / mL)、rD / JY3002-P4(5.01x 10 6 TCID 50 / mL) and rD / JY3002-P5 (5.25 x 10 6 TCID 50 / mL)( Figure 6 By comparison, it was found that under the same 0.01 MOI (Multiplicity of infection) virus dose and the same culture conditions, the infectious titer of rD / JY3002-P2 was similar to that of the natural isolated strain D / JY3002 (1.00 x 10 7 TCID 50 / mL) had no significant difference ( Figure 6 A in the figure indicates that the infection ability of artificially rescued influenza D viruses is comparable to that of naturally isolated influenza D viruses; there is no significant difference in the infection titer of artificially rescued influenza D viruses of different generations ( Figure 6 B), suggesting that artificially rescued influenza D viruses can be stably propagated for at least five generations. These results confirm that this example successfully established a reverse genetic manipulation system for influenza D virus D / JY3002, a genetically evolved lineage of D / Yama2019.

[0082] 4. Artificial rescue of recombinant influenza virus rD / JY3002-GFP carrying a green fluorescent reporter gene

[0083] 4.1 Design and synthesis of the unidirectional expression plasmid pPolI-D / JY3002-PB1-240-GFP-240

[0084] The sequences at both ends of the influenza virus genome segment, including the non-coding region sequences at both ends and the partial coding region sequences, can serve as viral packaging signal sequences to control the transcription, replication, and packaging of the corresponding genome segment. Accordingly, when the influenza virus packaging signal sequences are added to the two ends of the foreign gene to form an artificially modified genome segment, this genome segment can also be packaged into virions, thereby producing recombinant influenza virus. Recombinant influenza virus can infect host cells and express foreign genes in host cells. In this embodiment, 60bp, 120bp, 180bp, 240bp, or 300bp at the 5' and 3' ends of the influenza virus D / JY3002 PB1 genome segment are selected as the assumed packaging signal sequences, and the green fluorescent reporter gene GFP is inserted between the assumed packaging signal sequences at both ends. Then, the DNA fragment corresponding to this artificially modified genome segment is embedded between the HumanPol I promoter and the Murine Pol I terminator in opposite directions to design and form a series of unidirectional expression plasmids. The results showed that when the 240bp sequences at the 5' and 3' ends of the influenza virus D / JY3002 PB1 genome segment were selected as the putative packaging signal sequences to construct a unidirectional expression plasmid, the GFP expression level was high and most stable after the recombinant influenza virus was used to infect host cells. This unidirectional expression plasmid was named pPolI-D / JY3002-PB1-240-GFP-240 ( Figure 7 ), the nucleotide sequence of which is shown in SEQ ID NO.31.

[0085] 4.2 Artificial rescue of recombinant influenza D virus rD / JY3002-GFP

[0086] Following the steps in 3.1, seven bidirectional expression plasmids containing DNA fragments corresponding to the D / JY3002 genomic segments and the above-mentioned unidirectional expression plasmid pPolI-D / JY3002-PB1-240-GFP-240 were co-transfected into cells. Five days after transfection, the HA titer of the cell supernatant reached 2 6 HA units / 25μL. The cell supernatant was collected and labeled as P0 virus, named rD / JY3002-GFP, and its infectious titer was determined to be 7.94x 10 4 TCID 50 / mL( Figure 8 B). After MDCK cells (six-well plate) were infected with 0.05 MOI of rD / JY3002-GFP for 48 hours, the infected cells could be observed to express green fluorescent protein ( Figure 8 5 days after infection, the cell supernatant was collected and labeled as P1 virus, named rD / JY3002-GFP-P1, and its infectious titer was determined to be 3.16 x 10 6 TCID 50 / mL( Figure 8 B). After 0.05 MOI of rD / JY3002-GFP-P1 was inoculated into new MDCK cells (six-well plate) for 48 hours, the infected cells could still express green fluorescent protein ( Figure 8 The above results further confirm that this example successfully established a reverse genetics system for influenza D virus of the D / Yama2019 genetic evolution lineage, which can be used to develop influenza D virus vectors to present exogenous genes.

Claims

1. A pCC1-DualPro vector, characterized in that The nucleotide sequence is shown in SEQ ID NO.

30.

2. A unidirectional expression plasmid pPolI-D / JY3002-PB1-240-GFP-240 carrying a green fluorescent reporter gene and a D / Yama2019 lineage influenza virus packaging signal, characterized in that: The nucleotide sequence is shown as SEQ ID NO.

31.

3. A method for constructing a reverse genetic operating system for influenza virus type D of the D / Yama2019 genetic evolution lineage, characterized in that: The following steps are involved: The DNA fragments corresponding to the PB2, PB1, P3, NP, M, and NS segments were seamlessly cloned into the pHW2000 vector, and the DNA fragment corresponding to the HEF segment was seamlessly cloned into the pCC1-DualPro vector to obtain seven bidirectional expression plasmids, which were then co-transfected into cell lines to obtain artificially rescued D / Yama2019 genetic evolutionary lineage D influenza virus; The nucleotide sequences of the DNA fragments corresponding to the PB2, PB1, P3, NP, M and NS segments are shown in SEQ ID NOs.23-25 ​​and SEQ ID NOs.27-29, respectively; the nucleotide sequence of the DNA fragment corresponding to the HEF segment is shown in SEQ ID NO.26; the nucleotide sequence of the pCC1-DualPro vector is shown in SEQ ID NO.

30.

4. The method according to claim 3, wherein During co-transfection, seven bidirectional expression plasmids and the unidirectional expression plasmid pPolI-D / JY3002-PB1-240-GFP-240 were co-transfected into the cell line; the nucleotide sequence of the unidirectional expression plasmid pPolI-D / JY3002-PB1-240-GFP-240 is shown in SEQ ID NO.

31.

5. The method according to claim 3 or 4, characterized in that The co-transfection into the cell line comprises the following steps: S1. One day before transfection, plate 293T cells and MDCK cells at a ratio of 3:1 in a six-well cell culture plate. Culture in 5% CO2 at 37°C. When the cell density reaches 75%, begin transfection. Replace the old culture medium with 1.5 mL of DMEM supplemented with 10% fetal bovine serum but without antibiotics. S2. Take 1 μg of each of the seven bidirectional expression plasmids described in claim 3 or the seven bidirectional expression plasmids described in claim 4 and the unidirectional expression plasmid pPolI-D / JY3002-PB1-240-GFP-240, add 200 μL of Opti-MEM and mix thoroughly. Add 20 μL of PEI-25K transfection reagent dropwise and let the mixture stand for 25 minutes. Add the mixture to the six-well cell culture plate in step S1 and culture at 37°C for 6-9 hours. S3. Replace the old culture medium with 1.7 mL of FBS-free DMEM supplemented with 100 U / mL penicillin and 100 µg / mL streptomycin. Incubate at 37°C for 36-42 hours, then supplement with 500 µL of DMEM supplemented with 100 U / mL penicillin, 100 µg / mL streptomycin, and 1 µg / mL TPCK-trypsin. Continue incubating for 3-4 days, supplementing with 200-400 µL of DMEM supplemented with 100 U / mL penicillin, 100 µg / mL streptomycin, and 0.1 µg / mL TPCK-trypsin as appropriate. S4. Wait until the HA titer of the cell supernatant reaches 2 3 -2 6 HA units / 25 μL, and the cell supernatant was collected to obtain the artificially rescued D / Yama2019 genetic evolutionary lineage D influenza virus.

6. A D / Yama2019 genetic evolution lineage influenza virus reverse genetics system reagent set, characterized in that: The invention comprises a primer set consisting of primers with nucleotide sequences as shown in SEQ ID NO.1-14, a primer set consisting of a universal upstream primer with a nucleotide sequence as shown in SEQ ID NO.15 and a segment-specific downstream primer with nucleotide sequences as shown in SEQ ID NO.16-22, the pCC1-DualPro vector according to claim 3, and the unidirectional expression plasmid pPolI-D / JY3002-PB1-240-GFP-240 according to claim 4.

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