Genetic engineering method for reducing pathogenicity of duck plague virus and application thereof

Abstract

The application discloses a gene modification method for reducing pathogenicity of duck plague virus and application. The method comprises the following steps: modifying UL45 gene in the genome of the duck plague virus in any one of the following ways: knocking out the UL45 gene, deleting the transmembrane region (73-95th amino acid) of the UL45 protein, or mutating the 59th, 67th, 89th, 107th, 118th, 135th, 180th, 193rd and 201st cysteine residues of the UL45 protein into serine to inhibit S-palmitoylation modification. The application also provides application of the UL45 protein as a target in preparation of a duck plague attenuated vaccine, and an attenuated vaccine prepared from recombinant duck plague viruses constructed by the above method. Experimental results show that the three modification methods can significantly reduce the proliferation ability of the duck plague virus in the duck body and the level of cloacal discharge of the duck plague virus, and reduce the histopathological damage, thereby providing a new candidate target and construction strategy for research and development of the duck plague attenuated vaccine.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, specifically to a genetic modification method for reducing the pathogenicity of duck plague virus, and the application of UL45 protein as a target in the preparation of attenuated duck plague vaccine. Background Technology

[0002] Duck plague (DP) is an acute, febrile, septicemic infectious disease caused by the duck plague virus (DPV), primarily affecting anseriformes birds such as ducks, geese, and swans. The disease spreads rapidly, with high morbidity and mortality rates. Clinically, it is characterized by vascular damage leading to lesions in parenchymal organs and severe damage to the digestive and immune systems. With the continuous increase in the scale and intensification of duck farming, duck plague seriously threatens the healthy development of the industry.

[0003] DPV is a linear double-stranded DNA virus belonging to the family Orthoherpesviridae, subfamily Alphaherpesvirinae, and genus Marek's virus. The highly virulent Chinese strain of duck plague virus, DPV-CHv (GenBank accession number: JQ647509.1), has a genome length of approximately 162 kb, exhibiting a typical D-type herpesvirus genome structure, containing 78 open reading frames (ORFs). Among these, the DPV UL45 gene (GenBank accession number: EU195107.1) encodes a structural protein associated with the viral envelope, which possesses a large transmembrane region. Currently, research on the biological function of the DPV UL45 protein and its post-translational modifications (such as S-palmitoylation) is insufficient. In other herpesviruses (such as HSV-1, EHV-1, and HCMV), the deletion of the UL45 homolog affects viral replication, release, and pathogenicity; however, in DPV, studies on the impact of the UL45 protein, its transmembrane region, and S-palmitoylation modifications on viral pathogenicity have not been reported.

[0004] Therefore, developing a method to reduce the pathogenicity of DPV by modifying the UL45 gene or its protein structure is of great significance for the prevention and control of duck plague and the development of attenuated vaccines.

[0005] It should be noted that the information disclosed in the background section above is only used to enhance the understanding of the background of this disclosure, and therefore may include information that does not constitute prior art known to those skilled in the art. Summary of the Invention

[0006] The purpose of this invention is to provide a genetic modification method to reduce the pathogenicity of duck plague virus, and the application of UL45 protein as a target in the preparation of duck plague attenuated vaccine, so as to overcome the lack of effective targets for constructing DPV attenuated strains in the prior art, and to help prepare DPV attenuated vaccine.

[0007] To achieve the above objectives, the present invention provides a genetic modification method for reducing the pathogenicity of duck plague virus, which reduces the pathogenicity of the virus by knocking out the UL45 gene, deleting the transmembrane region of the UL45 protein, or blocking its S-palmitoylation modification.

[0008] This invention provides a gene modification method for reducing the pathogenicity of duck plague virus, comprising modifying the UL45 gene in the duck plague virus genome in any one of the following ways to reduce viral pathogenicity; (a) Knock out the UL45 gene; (b) The transmembrane region of the protein encoded by the UL45 gene is missing; (c) Mutate the S-palmitoylation site of the protein encoded by the UL45 gene; The UL45 gene in (a) is derived from the Chinese virulent strain of duck plague virus (DPV-CHv) with GenBank accession number JQ647509.1, and its nucleotide sequence is shown in GenBank accession number EU195107.1.

[0009] Furthermore, the transmembrane region in step (b) of the method is composed of amino acids 73 to 95 of the UL45 protein, and its amino acid sequence is shown in SEQ ID NO.20.

[0010] Further, the S-palmitoylation site in step (c) of the method includes one or more of the cysteine ​​residues at positions 59, 67, 89, 107, 118, 135, 180, 193, and 201 of the UL45 protein.

[0011] Preferably, the mutation involves replacing all cysteine ​​residues at positions 59, 67, 89, 107, 118, 135, 180, 193, and 201 of the UL45 protein with serine residues.

[0012] The UL45 gene or its encoded protein provided by this invention can be used in the preparation of duck plague attenuated vaccine. By inhibiting the expression of UL45 protein, deleting its transmembrane region, or preventing its S-palmitoylation modification, the pathogenicity of duck plague virus can be reduced.

[0013] Furthermore, the encoded protein is derived from the UL45 gene, whose nucleotide sequence is shown in GenBank accession number EU195107.1.

[0014] Furthermore, the transmembrane region is composed of amino acids 73 to 95 of the UL45 protein, and its amino acid sequence is shown in SEQ ID NO. 20.

[0015] Furthermore, the inhibition of its S-palmitoylation modification is achieved by replacing the cysteine ​​residues at positions 59, 67, 89, 107, 118, 135, 180, 193, and 201 of the mutant UL45 protein with serine residues.

[0016] The present invention also provides a duck plague attenuated vaccine, wherein the duck plague attenuated vaccine uses recombinant duck plague virus constructed by the above method as the active ingredient.

[0017] The duck plague attenuated vaccine provided by this invention also includes a pharmaceutically acceptable adjuvant.

[0018] The present invention has the following advantages: This invention also first constructed a DPV UL45 gene deletion virus, a transmembrane region deletion virus, and an S-palmitoylation site mutant virus, and verified that all three modification methods can significantly reduce the pathogenicity of DPV to ducks.

[0019] The technical solution provided by this invention offers new candidate targets and construction strategies for the development of attenuated duck plague vaccines, and has important application value for the clinical prevention and control of duck plague. Attached Figure Description

[0020] Figure 1 Predicted results for the transmembrane region and S-palmitoylation modification sites of the DPV UL45 protein.

[0021] Figure 2 The results of the identification of S-palmitoylation modification of DPV UL45 protein.

[0022] Figure 3 Results of rescue and purification of different recombinant duck plague viruses.

[0023] Figure 4 The results of PCR identification of different recombinant duck plague viruses.

[0024] Figure 5 The results show the in vitro multi-step growth curve determination of different recombinant duck plague viruses.

[0025] Figure 6 Comparative analysis of the replication cycles of different recombinant duck plague viruses.

[0026] Figure 7 Results of erosion area determination for different recombinant duck plague viruses.

[0027] Figure 8 The appearance of ducks infected with different recombinant duck plague viruses.

[0028] Figure 9 The results of monitoring body temperature, weight and survival rate of ducks infected with different recombinant duck plague viruses.

[0029] Figure 10Gross pathological changes in various tissues and organs of ducks infected with different recombinant duck plague viruses.

[0030] Figure 11 Microscopic pathological changes in the liver, cecum, and thymus of ducks infected with different recombinant duck plague viruses.

[0031] Figure 12 Results of viral load determination in various tissues and organs of ducks infected with different recombinant duck plague viruses.

[0032] Figure 13 Results of cloacal viral shedding in ducks infected with different recombinant duck plague viruses.

[0033] Note: The significance level marked ns in the figure indicates no significant difference; * p <0.05;** p <0.01; *** p <0.001; **** p <0.0001 (with P <0.05 indicates a statistically significant difference. Detailed Implementation

[0034] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0035] Note: Unless otherwise specified, the experimental methods in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0036] Some of the experimental materials involved in this application: 1. Cells, strains, bacterial strains, and plasmids Duck embryo fibroblasts (DEF); Duck plague virus strain DPV-CHv, a highly virulent strain from China, was isolated and preserved by Sichuan Agricultural University. This virus strain was deposited on February 23, 2012, at the China Center for Type Culture Collection (CCTCC) located at Wuhan University, China, with accession number: CCTCC NO: V201209. DPV-CHv50 virus (obtained by uploading DPV-CHv 50 times to DEF). An infectious clone of the entire genome of DPV, pDPV-CHv50, constructed using a bacterial artificial chromosome as a vector; Commonly used plasmids such as pCAGGS, pCAGGS-3Flag, pCAGGS-3HA, and pEP-Kan-S were preserved and provided by Cheng Anchun et al. from the College of Animal Science and Technology, Guizhou University.

[0037] 2. Duck embryos, experimental animals 14-day-old ducklings and 9-11-day-old duck embryos were tested and found to be free of DPV and DPV antibodies.

[0038] 3. Some of the primers involved are shown in Table 1.

[0039] Table 1 Primer Sequences Experimental Example 1: Bioinformatics Prediction The transmembrane region and S-palmitoylation modification of the UL45 protein (GenBank accession number: AFC61837.1) were predicted using the online prediction website TMHMM 2.0 and the biological analysis software CSS-Palm 4.0, respectively. The prediction results from TMHMM 2.0 (see...) Figure 1 As shown in A), the UL45 protein contains a hydrophobic transmembrane region between amino acids 73 and 95, with the amino acid sequence (SEQ ID NO. 20) being: LITIGIVVGILIVGLVCFFVGVF. CSS-Palm 4.0 prediction results (see...) Figure 1 B) shows that the UL45 protein has 9 potential S-palmitoylation modification sites, namely cysteine ​​residues at positions 59, 67, 89, 107, 118, 135, 180, 193, and 201, with prediction scores of 0.572, 8.442, 0.947, 15.696, 1.158, 1.085, 1.404, 4.035, and 1.584, respectively.

[0040] Example 2: Identification of S-palmitoylation Modification of DPV UL45 Protein Using the DPV-CHv50 genome as a template, a 723 bp UL45 gene fragment was amplified by PCR using primers shown in SEQ ID NO. 1~2 (or the sequence could be synthesized directly based on the coding gene sequence). This fragment was then cloned into the pCAGGS-3Flag vector to obtain the recombinant plasmid pCAGGS-UL45-3Flag. Based on the predicted S-palmitoylation modification sites, nine cysteine ​​residues in the UL45 protein were site-directed to serine residues to obtain a mutant UL45 without S-palmitoylation capability. PN(P: Palmitoylation, N: Null), this mutant gene fragment was chemically synthesized by Kangwei Century Biotechnology Co., Ltd. and cloned into the pCAGGS-3HA vector to obtain pCAGGS-UL45. PN -3HA plasmid.

[0041] S-palmitoylation modification of the UL45 protein was detected using immunoprecipitation combined with acyl-biotin exchange (IP-ABE). pCAGGS-UL45-3Flag and pCAGGS-UL45 were compared. PN -3HA plasmids were transfected into DEF cells, and cell samples were collected and total protein was extracted after 36 h of culture. Samples were processed according to the instructions of the palmitoylated protein immunoblotting detection kit (catalog number: AM10314, AIMSMASS, China), and Western blotting analysis was performed. Biotinylated S-palmitoylated protein was detected by binding to horseradish peroxidase (HRP)-labeled streptavidin.

[0042] The experimental results showed that in the treated group (+hydroxylamine HAM), an S-palmitoylation modification band was detectable in wild-type UL45 protein, while this band was not observed in the untreated group (-HAM) (see [link to experimental results]). Figure 2 (A in the text); and UL45 PN Even after HAM treatment, the mutant did not show S-palmitoylation signaling (see Figure 2 (B in the text). The above results indicate that the DPV UL45 protein is S-palmitoylated.

[0043] Experiment Example 3: Construction of Infectious Clones This experiment will use the pDPV-CHv50 infectious clone as a backbone vector to construct UL45 gene deletion and reversion (pDPV-ΔUL45 and pDPV-ΔUL45-Rev) and transmembrane region deletion and reversion (pDPV-UL45) using Red homologous recombination technology. Δ73-95aa and pDPV-UL45 Δ73-95aa -Rev) and S-palmitoylation site mutations and reversions (pDPV-UL45) PN and pDPV-UL45 PN Infectious clones of -Rev).

[0044] 1. Amplification of the target fragment Using pEP-Kan-S plasmid as a template, and primers SEQ ID NO.3~4 and SEQ ID NO.9~10 in Table 1, ΔUL45-Kan (1086 bp) and UL45 were amplified by PCR. Δ73-95aa-Kan (1108 bp) targeting fragment; using primers SEQ ID NO.7~8 and SEQ ID NO.13~14 in Table 1, the UL45 / UL45-containing fragments were amplified by PCR. PN Gene homologous arm or UL45 73-95aa The Kan resistance gene fragment (1113 bp) of the transmembrane homologous arm.

[0045] Using the pDPV-CHv50 genome as a template, and with primers listed in Table 1 (SEQ ID NO. 5-6 and SEQ ID NO. 11-12), UL45 (760 bp) and UL45 were amplified by PCR. 73-95aa (119 bp) gene fragment. Using pCAGGS-UL45 PN Using the -3HA plasmid as a template, and primers SEQ ID NO.5~6 in Table 1, UL45 was amplified by PCR. PN Gene fragment (760 bp).

[0046] PCR amplification system: 5 μL PrimeSTAR ® Max DNA Polymerase, 0.2 μL each of forward and reverse primers, 0.2 μL of pEP-Kan-S, and 4.4 μL of ddH2O were used. The PCR amplification program was as follows: 98℃, 2 min; [98℃, 10 s; 55℃, 15 s; 72℃, 10 s], 30 cycles; 72℃, 10 min; 16℃, ∞.

[0047] Using overlap extension PCR, UL45 and UL45 were respectively... 73-95aa and UL45 PN Gene fragments were fused with their corresponding Kan resistance gene fragments containing homologous arms to obtain UL45-Kan-Rev / UL45. PN -Kan-Rev (1848 bp), UL45 73-95aa -Kan-Rev (1232 bp) and UL45 PN -Kan (1848 bp) targeting fragment. Overlap extension PCR system: 5 μL RimeSTAR ® Max DNA Polymerase, 0.5 μL each of forward and reverse primers, and 0.5 μL each of gene fragment were added, and ddH2O was added to a final volume of 10 μL. The PCR program was as follows: 98℃; 2 min; [98℃, 10 s; 55℃, 15 s; 72℃, 10 s], 32 cycles; 72℃, 10 min; 16℃.

[0048] 2. Preparation of electrocompetent states The frozen GS1783 bacteria containing the pDPV-CHv50 plasmid were revived on Cm / LB plates, and single colonies were picked and inoculated into Cm / LB medium, and cultured with shaking until OD. 600 The bacterial cell concentration reached approximately 0.5; after shaking in a 42℃ water bath for 15 min, the cells were placed in an ice bath for 20 min, centrifuged at 4℃ and 4500 r / min for 10 min to collect the cells. The precipitate was washed 5 times with pre-cooled ddH2O. Finally, the volume was adjusted to 500 µL of ddH2O, and the cells were dispensed in 100 µL tubes to obtain GS1783-pDPV-CHv50 electrocompetent cells, which were stored at -80℃ for later use. GS1783-pDPV-ΔUL45 and GS1783-pDPV-UL45 can be prepared using the same method. Δ73-95aa Electrocompetent state.

[0049] 3. Red homologous recombination 200 ng of the target fragments ΔUL45-Kan and UL45 were respectively used. Δ73-95aa -Kan was electroporated into GS1783-pDPV-CHv50 competent cells (2.5 kV). After electroporation, 800 µL of LB was added and the cells were incubated at 30℃ for 1 h. The supernatant was removed by centrifugation at 4500 r / min for 2 min. The bacterial pellet was resuspended in 200 µL of LB and plated on Cm / Kan / LB plates. The cells were incubated at 30℃ for 48 h, and positive clones containing the Kan resistance gene were screened.

[0050] Positive clones were inoculated into Cm / Kan LB medium and cultured at 30°C to the logarithmic phase. 10 µL of bacterial culture was added to 1 mL of Cm / Kan LB medium and cultured at 30°C for 2 h. Then, 1 mL of Cm / LB medium and 54 µL of 5 mol / L L-arabinose were added, and the mixture was induced in a water bath at 42°C and 180 r / min for 30 min, followed by shaking culture at 30°C for 2 h. 1 µL of bacterial culture was mixed with 200 µL of LLB medium and spread onto Cm / LB plates, then cultured at 30°C for 48 h. Single clones were picked and identified by PCR using the primers shown in Table 1 (SEQ ID NO. 15-16). The PCR reaction system was: 5 μL 2× Hieff ® PCR Master Mix, single colony, 0.2 μL each of forward and reverse primers, and 4.6 μL of ddH2O were used. The PCR reaction program was: 94℃, 5 min; [94℃, 10 s; 55℃, 20 s; 72℃, 30 s], 30 cycles; 72℃, 10 min; 16℃, ∞. Sequencing verification confirmed the presence of pDPV-ΔUL45 and pDPV-UL45. Δ73-95aa Infectious clones.

[0051] Using the same method described above, UL45-Kan-Rev and UL45 were respectively... PN -Kan, UL45 PN - The Kan-Rev firing fragment was imported into GS1783-pDPV-ΔUL45 competent cells for Red homologous recombination, yielding pDPV-ΔUL45-Rev and pDPV-UL45 sequentially. PN and pDPV-UL45 PN -Rev infectious clone; UL45 73-95aa -Kan-Rev shooting footage imported into GS1783-pDPV-UL45 Δ73-95aa The competent state, after two Red homologous recombinations, yielded pDPV-UL45. Δ73-95aa -Rev infectious clones.

[0052] Experimental results: After two rounds of Red homologous recombination, the colony PCR identification bands were consistent with expectations: pDPV-ΔUL45 was 536 bp, and pDPV-UL45... Δ73-95aa The value is 1142 bp, pDPV-ΔUL45-Rev, pDPV-UL45 Δ73-95aa -Rev、pDPV-UL45 PN and pDPV-UL45 PN -Rev values ​​were all 1211 bp, and sequencing verification was correct, indicating that all infectious clones had been successfully constructed.

[0053] Experiment Example 4: Rescue, purification and identification of recombinant duck plague virus 1. Rescue and purification of recombinant duck plague virus pDPV-ΔUL45, pDPV-ΔUL45-Rev, and pDPV-UL45 were respectively... Δ73-95aa pDPV-UL45 Δ73-95aa -Rev、pDPV-UL45 PN and pDPV-UL45 PN - The Rev infectious clone plasmid was transfected into DEF cells. Virus was harvested after widespread green fluorescent spots and cytopathic effects were observed in the cells under a fluorescence microscope. The rescued recombinant duck plague virus carrying the mini-F vector element was then used in DEF cells for homologous recombination to seamlessly excise the fluorescent protein expression cassette, ultimately yielding UL45 gene deletion virus and reversal virus (DPV-ΔUL45 and DPV-ΔUL45-Rev) that do not express fluorescent protein, and UL45 protein transmembrane region deletion virus and reversal virus (DPV-UL45). Δ73-95aa and DPV-UL45 Δ73-95aa -Rev) and UL45 protein S-palmitoylation site mutant viruses and reversion viruses (DPV-UL45) PNand DPV-UL45 PN -Rev).

[0054] Experimental results showed that 2-4 days after transfecting DEF cells with infectious clonal plasmids, green fluorescent spots appeared in the cells accompanied by cytopathic effects, indicating that the recombinant duck plague viruses were successfully rescued (see...). Figure 3 (A in the text); the virus was purified by homologous recombination in DEF cells to obtain various recombinant duck plague viruses that lost fluorescence but still caused cytopathic effects (see A in the text). Figure 3 (B in the middle).

[0055] 2. Identification of recombinant duck plague virus The rescued and purified recombinant duck plague viruses were inoculated into DEF cells. Viral samples were collected after 36 h, and viral DNA was extracted and used as a template. Gene fragments were amplified using primers shown in Table 1 (SEQ ID NO. 15-16). The PCR products were sent to Qingke Biotechnology Co., Ltd. for sequencing. The PCR identification system was: 5 μL 2×Hieff ® PCR Master Mix, 0.5 μL viral DNA, 0.3 μL each of forward and reverse primers, 4.1 μL ddH2O; PCR identification program: 94℃, 5 min; [94℃, 10 s; 55℃, 20 s; 72℃, 30 s], 30 cycles; 72℃, 10 min; 16℃, ∞.

[0056] The amplified electrophoresis results are shown below Figure 4 ,in, Figure 4 In the diagram, A, B, C, D, E, and F represent DPV-ΔUL45, DPV-ΔUL45-Rev, and DPV-UL45, respectively. Δ73-95aa DPV-UL45 Δ73-95aa -Rev、DPV-UL45 PN and DPV-UL45 PN -Identification results of Rev virus. The results showed that the PCR identification results of each recombinant duck plague virus were as expected; the PCR amplification products were sent to a biotechnology company for sequencing, and the obtained sequences were compared online with NCBI-BLAST and were consistent with the theoretical reference sequences, indicating that the deletion / mutation and reversion of the UL45 gene, transmembrane region, and S-palmitoylation site were all accurate, namely DPV-ΔUL45, DPV-ΔUL45-Rev, and DPV-UL45. Δ73-95aa DPV-UL45 Δ73-95aa -Rev、DPV-UL45 PN and DPV-UL45 PN -Rev virus successfully built.

[0057] Experimental Example 5: Effects of UL45 protein, its transmembrane region, and S-palmitoylation on in vitro DPV proliferation. 1. Determination of in vitro multi-step growth curves of different recombinant duck plague viruses Viruses with an MOI of 0.01 (DPV-ΔUL45, DPV-UL45) Δ73-95aa DPV-UL45 PN DPV-ΔUL45-Rev, DPV-UL45 Δ73-95aa -Rev、DPV-UL45 PN DEF cells were infected with Rev and DPV-CHv50. Virus samples were collected at 24 h, 48 h, 72 h, and 96 h post-infection. After three freeze-thaw cycles at -80°C, 100 μL of each sample was serially diluted 10-fold and seeded into 96-well DEF cells at 100 µL / well. Each sample was tested in triplicate and cultured at 37°C in 5% CO2. The number of wells with cytopathic effects was observed and recorded daily, and the TCID of the virus was calculated using the Reed-Muench method. 50 Virus growth curves were plotted, and a two-way ANOVA was performed on the data using GraphPad Prism 9.0 software.

[0058] Experimental results: The viral titers showed an increasing trend from 24 h to 72 h, reaching their maximum values ​​at 72 h, and then began to decrease (see...). Figure 5 (A) Compared with the parent virus and the revertant virus, DPV-∆UL45, DPV-UL45 ∆73-95aa and DPV-UL45 PN The number of mature viral particles produced by viral replication in DEF is significantly reduced (see...) Figure 5 (B in the figure) indicates that the UL45 protein and its transmembrane region and S-palmitoylation modification affect the in vitro proliferation of DPV.

[0059] 2. Comparative analysis of replication cycles of different recombinant duck plague viruses (1) Virus adsorption The prepared DEF cells were pre-cooled at 4°C for 1–2 h and washed three times with pre-cooled PBS; then inoculated with 1 MOI of virus (DPV-ΔUL45, DPV-UL45) respectively. Δ73-95aa DPV-UL45 PN DPV-ΔUL45-Rev, DPV-UL45 Δ73-95aa -Rev、DPV-UL45 PN-Rev and DPV-CHv50), incubated at 4℃ for 2 h, discarded the virus solution and washed the cells 5 times with pre-cooled PBS; the viral genome copy number was determined using the Taq-Man MGB probe qPCR method established in our laboratory (Development of TaqMan MGBfluorescent real-time PCR assay for the detection of anatid herpesvirus1 [J]. Virology journal, 2009, 6(1): 71), using UL30-F / R primers and UL30-probe probes as shown in Table 1 as SEQ ID NO.17~19, and one-way ANOVA was performed using GraphPad Prism 9.0 software.

[0060] Virus adsorption detection results are shown below Figure 6 As shown in A, B, and C, it can be seen that compared with the parental toxin and the revertant toxin, DPV-ΔUL45 and DPV-UL45 Δ73-95aa The virus's ability to adsorb DEF was significantly weakened, while DPV-UL45 PN There was no significant difference between the groups, indicating that the UL45 protein and its transmembrane region affect the adsorption process of DPV.

[0061] (2) Virus invasion The prepared DEF cells were pre-cooled at 4°C for 1–2 h and washed three times with pre-cooled PBS; then inoculated with 1 MOI of virus (DPV-ΔUL45, DPV-UL45) respectively. Δ73-95aa DPV-UL45 PN DPV-ΔUL45-Rev, DPV-UL45 Δ73-95aa -Rev、DPV-UL45 PN -Rev and DPV-CHv50), incubated at 4℃ for 2 h, discarded the virus solution and replaced with maintenance solution containing 2% NBS for 1 h; washed 5 times with pre-cooled PBS, collected cell samples and extracted viral DNA, used the same qPCR method as (1) to detect the viral genome copy number, and performed one-way ANOVA using GraphPad Prism 9.0 software.

[0062] Intrusion detection results are shown Figure 6 As shown in D, E, and F, it can be seen that DPV-ΔUL45 and DPV-UL45 Δ73-95aa The virus's invasive ability is significantly lower than that of the parent virus and the revertant virus, while DPV-UL45 PN The virus showed no significant differences compared to its parental and revertant strains, indicating that the UL45 protein and its transmembrane region influence the invasion process of DPV.

[0063] (3) Virus replication With 1 MOI virus (DPV-UL45) PN DPV-UL45 PN -Rev and DPV-CHv50) infected monolayer DEF cells, incubated at 37°C for 6 h, then the virus solution was discarded and replaced with MEM maintenance medium containing 2% NBS for continued culture. Cell samples were collected and viral DNA was extracted at 6 h, 8 h, 10 h and 12 h. The viral genome copy number was detected using the same qPCR method as in (1), and the data were analyzed by two-way ANOVA using GraphPad Prism 9.0 software.

[0064] The experimental results are shown in Figure 6 As shown in G, DPV-UL45 PN The viral genome replication level in infected cells was not significantly different from that of the parent virus and the revertant virus, indicating that S-palmitoylation of the UL45 protein does not affect DPV genome replication.

[0065] (4) Virus release With 1 MOI virus (DPV-UL45) PN DPV-UL45 PN DEF monolayers were infected with Rev and DPV-CHv50, incubated at 37°C for 18 h, then the supernatant was discarded, and the cells were replaced with MEM maintenance medium containing 2% NBS for further culture. The supernatant was collected at 1 h, 2 h, 3 h, and 4 h to determine the viral TCID. 50 Two-way ANOVA was performed on the data using GraphPad Prism 9.0 software.

[0066] The experimental results are shown in Figure 6 As shown in the middle H, DPV-UL45 PN In virus-infected cells, the number of mature viral particles was significantly lower than that of the parent virus and revertant virus-infected groups, indicating that S-palmitoylation of the UL45 protein can affect the release of viral particles.

[0067] 3. Determination of the erosion area of ​​different recombinant duck plague viruses With 10 -3 MOI virus (DPV-ΔUL45, DPV-UL45) Δ73-95aa DPV-UL45 PN DPV-ΔUL45-Rev, DPV-UL45 Δ73-95aa -Rev、DPV-UL45 PNDEF monolayers were infected with Rev and DPV-CHv50, adsorbed at 37°C for 2 h, the virus solution was discarded, and the cells were washed three times with PBS. MEM maintenance medium containing 1.5% methylcellulose was added, and the cells were cultured for another 5–7 days. The culture medium was removed, the cells were washed with PBS, and then fixed with 4% paraformaldehyde at room temperature for 20 min. The cells were stained with 0.5% crystal violet for 30 min, washed off the stain, and photographed. Thirty plaques were randomly selected, and the plaque area was statistically analyzed using ImageJ and GraphPad Prism 9 software.

[0068] The experimental results are shown in Figure 7 As shown, where, Figure 7 In the image, A represents a photograph of the etched spot. Figure 7 B in the figure represents the quantitative result. It can be seen that, compared with the parental or reverted form of DPV-CHv50, DPV-ΔUL45 and DPV-UL45... Δ73-95aa and DPV-UL45 PN The area of ​​etched spots formed by viral infection of DEF was significantly reduced.

[0069] Example 6: Pathogenicity evaluation of UL45 deletion toxin, transmembrane deletion toxin, and S-palmitoylation mutant toxin. 1. Clinical symptoms 152 12-day-old ducks were randomly divided into groups of 19 each, numbered 1 to 19 within each group, according to Table 2. At 14 days of age, the ducks were infected with the virus via intramuscular injection. Ducks numbered 1 to 10 in each group were used for observation of clinical symptoms after infection and were continuously monitored for 10 days. The ducks' condition was observed and recorded daily, and their rectal temperature and weight were measured. The survival rate was also calculated.

[0070] Table 2 Grouping and Treatment of Laboratory Animals MEM 1 mL / each 19 Intramuscular injection DPV-ΔUL45 group <![CDATA[10 4 TCID 50 / mL 1 mL / only]]> 19 Intramuscular injection <![CDATA[DPV-UL45 Δ73-95aa Group <![CDATA[10 4 TCID 50 / mL 1 mL / only]]> 19 Intramuscular injection <![CDATA[DPV-UL45 PN Group <![CDATA[10 4 TCID 50 / mL 1 mL / only]]> 19 Intramuscular injection DPV-ΔUL45-Rev group <![CDATA[10 4 TCID 50 / mL 1 mL / only]]> 19 Intramuscular injection <![CDATA[DPV-UL45 Δ73-95aa -Rev Group]]> <![CDATA[10 4 TCID 50 / mL 1 mL / only]]> 19 Intramuscular injection <![CDATA[DPV-UL45 PN -Rev Group]]> <![CDATA[10 4 TCID 50 / mL 1 mL / only]]> 19 Intramuscular injection DPV-CHv50 group <![CDATA[10 4 TCID 50 / mL 1 mL / only]]> 19 Intramuscular injection The experimental results are as follows: (1) Ocular symptoms in each treatment group as follows Figure 8 As shown, it can be seen that DPV-ΔUL45 and DPV-UL45 Δ73-95aa and DPV-UL45 PN The ducks in the group with the same condition as those in the MEM group had clean feathers, no discharge from their eyes, and no lameness or neurological symptoms. The ducks in the DPV-CHv50 parental toxin group and the revertant toxin group showed symptoms such as ruffled feathers, eyelid adhesion, swelling of the head and neck, and paralysis of both legs. The most severe cases resulted in death.

[0071] (2) Changes in body temperature in each treatment group as follows Figure 9As shown in Figure A, it can be seen that ducks in the DPV-CHv50 parental virus group and each revertant virus group had body temperatures exceeding the normal range (>42.5°C) on days 3-4 post-infection, returning to normal levels on days 5-6; the body temperatures of ducks in the MEM and DPV-ΔUL45 groups fluctuated within the normal range throughout; the DPV-UL45 group... Δ73-95aa and DPV-UL45 PN The ducks in the group with the infection showed an increase in body temperature on days 4 to 6 after infection, but the time of the temperature rise was delayed and the magnitude of the rise was smaller compared with the parent virus group and the revertant virus group.

[0072] (3) Changes in body weight in each treatment group are as follows: Figure 9 As shown in B, it can be seen that DPV-ΔUL45 and DPV-UL45 Δ73-95aa and DPV-UL45 PN The ducks in the parent group showed a steady and continuous increase in weight. The ducks in the DPV-CHv50 parent virus group and the revertant virus group experienced decreased appetite due to high fever on days 2 to 5 after infection, resulting in a significant slowdown or even cessation of weight gain. Some ducks died acutely, while the surviving ducks gradually regained their weight after day 5.

[0073] (4) Survival rates of each treatment group are as follows: Figure 9 As shown in C, it can be seen that MEM, DPV-ΔUL45 and DPV-UL45 Δ73-95aa All 10 ducks in the group survived, achieving a 100% survival rate; DPV-UL45 PN The survival rate of the control group was 90%; the deaths of ducks in the DPV-CHv50 group and the relapsed virus group mostly occurred on the 4th to 6th day after infection, with a survival rate of only 50%-60%.

[0074] The above results indicate that, compared with the parental toxicant and the revertant toxicant, DPV-ΔUL45 and DPV-UL45... Δ73-95aa and DPV-UL45 PN The morbidity and mortality rates of ducks infected with the three recombinant viruses were significantly reduced.

[0075] 2. Pathological observation According to Table 2, ducks numbered 11-19 in each group were used for post-infection pathological observation. The period of significant clinical symptoms of duck plague was 3-6 days post-infection. On day 5 post-infection, three ducks from each group were euthanized to observe gross pathological changes in various tissues and organs. The collected tissues and organs (1 cm) were then examined. 3 (Size) were fixed in 4% paraformaldehyde solution, and pathological sections were prepared according to conventional methods (HE staining) to observe the microscopic pathological changes of each tissue and organ.

[0076] The experimental results are as follows: (1) Gross pathological changes in each treatment group are as follows: Figure 10As shown, no obvious lesions were observed in any tissues or organs of the ducks infected with DPV-ΔUL45; DPV-UL45 Δ73-95aa and DPV-UL45 PN In the infected group, the spleen of ducks was slightly swollen, and there were a few punctate hemorrhages in the thymus. The other organs were not significantly different from those in the MEM group. The DPV-CHv50 group and each revertant group showed systemic septicemia: the liver was fragile and diffusely congested; the proventricular mucosa was congested and hemorrhagic, and there was a clear hemorrhagic band at the junction of the esophageal dilatation and the proventriculus; the spleen was mottled with local grayish-white necrotic foci; the cecal wall was thinned and the mucosa was widely congested and hemorrhagic; the thymus was congested and severely atrophied.

[0077] (2) Microscopic pathological changes: The microscopic pathological changes of the liver, cecum, and thymus tissues of ducks in each treatment group are as follows: Figure 11 As shown. It can be seen that DPV-ΔUL45 and DPV-UL45 Δ73-95aa and DPV-UL45 PN The hepatocytes of the group of ducks were structurally intact and orderly arranged, with clearly defined nuclei of similar size and shape, except for DPV-UL45. PN A small amount of inflammatory cell infiltration was observed in the control group; the cecal villi structure was intact in all groups, and the cortex and medulla of the thymus were clearly demarcated without obvious lesions. In contrast, the hepatocyte structure of ducks in the DPV-CHv50 parent virus group and the revertant virus group was disordered, the central vein and hepatic sinusoids were filled with red blood cells and accompanied by fatty degeneration; the cecal villi were broken, and the submucosal capillaries were damaged and hemorrhagic; the thymic lobule structure was destroyed, inflammatory cell infiltration was observed, and the interstitial blood vessels were highly dilated and diffuse hemorrhage was observed.

[0078] The above results indicate that, compared with the parental toxicant and the revertant toxicant, DPV-ΔUL45 and DPV-UL45... Δ73-95aa and DPV-UL45 PN The pathological damage of the virus to various tissues and organs of ducks was significantly reduced.

[0079] 3. Viral load in tissues and organs Tissue samples were collected from each group on day 5 post-infection. 0.1 g of each sample was weighed, added to 1 mL of PBS solution, and homogenized thoroughly. 100 μL of the homogenate was used to extract viral DNA. The DPV genome copy number in each tissue and organ was detected using the qPCR method described in Experiment 5. Two-way ANOVA was performed on the data using GraphPad Prism 9.0 software.

[0080] Experimental results are as follows Figure 12 As shown, the DPV-ΔUL45 group of ducks had the lowest viral load in tissues and organs (10). 4.80 ~10 7.72(copies / 0.1 g), DPV-UL45 Δ73-95aa and DPV-UL45 PN The viral load in the tissues and organs of the group of ducks was 10. 5.17 ~10 8.68 copies / 0.1 g and 10 5.75 ~10 8.75 The viral copy number range in various tissues and organs of the DPV-CHv50 parental virus and revertant group ducks was 10 copies / 0.1 g. 6.68 ~10 10.34 The viral load in the proventricular tissue was significantly different from that in the deletion and mutant groups, with the viral load being DPV-UL45, respectively. PN The results show that the DPV-ΔUL45 group was 420 times stronger than the DPV-ΔUL45 group and 2900 times stronger than the DPV-ΔUL45 group. Δ73-95aa and DPV-UL45 PN The virus's ability to multiply in ducks is weakened.

[0081] 4. Cloaca excretion volume Cloacal secretions from ducks in each group were collected on days 1, 3, 5, 7, and 9 post-infection using disposable sampling swabs. The swabs were placed in 1 mL of PBS solution and shaken to mix. Viral DNA was extracted from 100 μL of the sample. The DPV genome copy number was detected using the qPCR method described in Experiment 5. Two-way ANOVA was performed on the data using GraphPad Prism 9.0 software.

[0082] Experimental results are as follows Figure 13 As shown, 1–3 days post-infection, the deletion virus and mutant virus (DPV-ΔUL45, DPV-UL45) showed significant differences. Δ73-95aa and DPV-UL45 PN The viral shedding in the first group of ducks remained at a low level, significantly lower than that in the DPV-CHv50 parental virus and revertant virus groups; on day 5 post-infection, the viral shedding in all groups rose to its peak, with the viral shedding in the deletion virus and mutant virus groups ranging from 10... 5.53 ~10 7.20 The viral load was 0.1 mL / copies, while the viral loads of the DPV-CHv50 parental and revertant groups were as high as 10 copies / mL. 8.23 Copies / 0.1 mL; After 7 days of infection, the viral shedding in ducks in all groups showed a decreasing trend, but the difference in cloacal viral shedding between the parental virus or revertant virus group and the deletion virus and mutant virus group persisted. These results indicate that DPV-ΔUL45 and DPV-UL45... Δ73-95aa and DPV-UL45 PN The viral shedding level in the cloaca of ducks decreases after viral infection.

[0083] In summary, this invention has been verified to show that the absence of the UL45 protein or its transmembrane region, as well as mutations in its S-palmitoylation site, weaken the proliferation of DPV in ducks and the excretion of the virus from the cloaca of infected ducks, thereby reducing the pathogenicity of the virus.

[0084] Although the present invention has been described in detail through the preferred embodiments above, it should be understood that the above description should not be considered as a limitation of the present invention. Various modifications and substitutions to the present invention will be apparent to those skilled in the art after reading the above description. Therefore, the scope of protection of the present invention should be defined by the appended claims.

Claims

1. A genetic modification method for reducing the pathogenicity of duck plague virus, characterized in that, This includes modifying the UL45 gene in the duck plague virus genome in any one of the following ways to reduce viral pathogenicity; (a) Knock out the UL45 gene; (b) The transmembrane region of the protein encoded by the UL45 gene is missing; (c) Mutate the S-palmitoylation site of the protein encoded by the UL45 gene; The UL45 gene in (a) is derived from the Chinese virulent strain of duck plague virus (DPV-CHv) with GenBank accession number JQ647509.1, and its nucleotide sequence is shown in GenBank accession number EU195107.

1.

2. The method according to claim 1, characterized in that, The transmembrane region in (b) is composed of amino acids 73 to 95 of the UL45 protein, and its amino acid sequence is shown in SEQ ID NO.

20.

3. The method according to claim 1, characterized in that: The S-palmitoylation site in (c) includes one or more of the cysteine ​​residues at positions 59, 67, 89, 107, 118, 135, 180, 193, and 201 of the UL45 protein.

4. The method according to claim 3, characterized in that, The mutation involves replacing all cysteine ​​residues at positions 59, 67, 89, 107, 118, 135, 180, 193, and 201 of the UL45 protein with serine residues.

5. The application of the UL45 gene or its encoded protein in the preparation of duck plague attenuated vaccine, characterized in that: The pathogenicity of duck plague virus can be reduced by inhibiting the expression of UL45 protein, deleting its transmembrane region, or preventing its S-palmitoylation modification.

6. The application according to claim 5, characterized in that, The encoded protein is derived from the UL45 gene, whose nucleotide sequence is shown in GenBank accession number EU195107.

1.

7. The application according to claim 5, characterized in that, The transmembrane region is composed of amino acids 73 to 95 of the UL45 protein, and its amino acid sequence is shown in SEQ ID NO.

20.

8. The application according to claim 5, characterized in that, The method to prevent its S-palmitoylation modification is to replace the cysteine ​​residues at positions 59, 67, 89, 107, 118, 135, 180, 193, and 201 of the mutant UL45 protein with serine.

9. A duck plague attenuated vaccine, characterized in that, The attenuated duck plague vaccine uses recombinant duck plague virus constructed by the method described in any one of claims 1 to 4 as its active ingredient.

10. The duck plague attenuated vaccine according to claim 9, characterized in that, It also includes pharmaceutically acceptable adjuvants.

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