A duck reproductive disorder syndrome virus, its isolation method and application

Abstract

This invention discloses a duck reproductive disorder syndrome virus, its isolation method, and its applications. The isolation method yields a novel duck reproductive disorder syndrome virus. Further analysis of the virus's sequence and characteristics lays the foundation for the preparation of vaccines and drugs to prevent and treat duck reproductive disorder syndrome, and also provides necessary conditions for further detection of the virus.

Description

Technical Field

[0001] This invention relates to the field of virus isolation technology, and in particular to a duck reproductive disorder syndrome virus, its isolation method, and its application. Background Technology

[0002] Muscovy ducks belong to the genus Geese and are not closely related to common farmed animals such as chickens, ducks, and geese. Muscovy ducks have been farmed on a large scale in China for many years, and viral infectious diseases of Muscovy ducks have become increasingly complex. However, there are few research reports on Muscovy duck infectious diseases, and even fewer reports on the in vitro isolation of pathogens of Muscovy duck infectious diseases. In vitro isolation and purification of viruses is the foundation of infectious disease research, and in vitro isolation and culture methods for Muscovy duck viruses are extremely important.

[0003] Breeding stock is of paramount importance in aquaculture. In recent years, abnormal egg production drops in Muscovy ducks have become a frequent problem. However, there is currently no mature Muscovy duck cell line for the isolation of Muscovy duck viruses. Virus isolation mostly relies on primary duck embryo fibroblasts. Different viruses have different receptors and different tissue cell tropisms. The existing cells cannot meet the needs of all virus isolation. Obtaining viruses for isolation and identification is often the first step in infectious disease research. Therefore, in vitro culture research of different primary Muscovy duck cells is still irreplaceable in the fields of virology and vaccines.

[0004] Depending on the specific needs of virus research, primary Muscovy duck cells are mainly used for virus isolation and vaccine development. The isolation and culture of avian viruses using primary Muscovy duck cells provides a foundation for vaccine development. After obtaining viruses using primary cells, disease mechanism studies are conducted, employing primary cell models to investigate viral invasion mechanisms, host immune responses, and antiviral drug screening. In vitro isolation and culture of primary Muscovy duck cells enables rapid isolation and identification of duck-derived viruses, addressing the problem of delayed disease diagnosis in poultry farms. This accelerates the progress of prevention and control strategies such as vaccine development, provides stable host cells for the large-scale production of viral antigens (such as inactivated vaccines, viral vector vaccines, and diagnostic standard preparation), shortens vaccine development cycles, and reduces production costs. By simulating the replication dynamics of viruses in cells, highly effective antiviral drugs or interferon preparations can be screened to guide clinical medication regimens and optimize disease prevention and control strategies in poultry farms.

[0005] The core idea of ​​traditional virus isolation methods is to propagate the virus using live cells / living organisms, and then identify it. This is a common approach in virology. The methods typically involve processing clinical samples, inoculating them into cells, chicken embryos, or animals, culturing and incubating them, and often requiring multiple blind passages.

[0006] Traditional virus isolation methods are extremely difficult: First, many viruses replicate only on specific hosts and cells, and their tropism is unclear; second, some viruses have extremely short post-infection shedding periods, making it impossible to collect virus-bearing samples, or antibodies in the sample may have already neutralized the virus, leading to isolation failure, and there is also a risk of inactivation during sample processing; third, many viruses do not produce cytopathic effects (CPE), requiring multiple blind passages, which is time-consuming and labor-intensive; fourth, clinical samples often contain contaminants such as bacteria, fungi, and mycoplasma, making it impossible for cells to survive; fifth, the isolation, culture, and proliferation conditions are extremely demanding, and slight errors in temperature, pH, inoculum size, and incubation time can prevent replication, and some virus cultures rely on cooperating factors, requiring multiple blind passages and long isolation cycles, ranging from several days to a week per cycle, necessitating multiple rounds of culture and isolation.

[0007] In recent years, abnormal egg production in Muscovy ducks has been a frequent problem. In 2025, a new duck reproductive disorder syndrome virus was discovered. The clinical symptoms of this duck reproductive disorder syndrome virus are decreased egg production. Autopsy revealed endometrial hemorrhage. The tissue tropism is the reproductive system. However, the virus could not be isolated in vitro using known cell lines and existing duck embryo primary cells, duck embryos, chicken embryos and goose embryos.

[0008] Therefore, there is an urgent need to study a new method for isolating duck reproductive disorder syndrome virus. Summary of the Invention

[0009] The purpose of this invention is to provide a new method for isolating duck reproductive disorder syndrome virus and obtaining duck reproductive disorder syndrome virus, so as to lay a research foundation for the prevention and control of duck reproductive disorder syndrome.

[0010] According to a first aspect of the present invention, a duck reproductive disorder syndrome virus is provided, the duck reproductive disorder syndrome virus having the accession number CCTCC NO:V202619. Thus, by isolating this new virus, further analysis of its sequence and characteristics can lay the foundation for the preparation of vaccines and drugs to prevent and treat duck reproductive disorder syndrome, and also provide the necessary conditions for further detection of the virus.

[0011] In some embodiments, a method for isolating the above-mentioned duck reproductive disorder syndrome virus is provided, the method comprising the following steps: S1: Separate the positive duck oviduct and obtain epithelial tissue of the uterine portion of the duck oviduct; S2: Digestion of duck oviduct uterine epithelial tissue: Add 0.25% trypsin + 0.02% EDTA to the duck oviduct uterine epithelial tissue from step S1 and let it stand at 37℃ to digest until the tissue becomes fluffy; S3: Centrifugation and resuspension of cells: Add FBS to the digested tissue in step S2 to remove enzymes, centrifuge to discard the FBS, add complete culture medium, shake well with magnetic beads, filter through a 100μm cell sieve, centrifuge to discard the supernatant, and resuspend the precipitated cells. S4: Cell counting and differential adhesion removal of fibroblasts: Count the cells obtained from resuspending in step S3, and seed them in 1.37x10 cm cell culture dishes. 7 The cells were aliquoted and plated, incubated in an incubator for 2 hours, and then the epithelial cells suspended in the cell culture medium were collected and the adherent fibroblasts were removed. S5: Cell plate coating: Coat cell plates with coating solution containing 100% FBS at 37°C; S6: Obtaining primary oviduct epithelial cells: Collect the cell clumps from step S4, resuspend them in complete culture medium, and incubate them statically in a 5% CO2 37℃ incubator using the cell plates coated in step S5 to obtain positive duck oviduct epithelial primary cells. S7: Collect cell supernatant: Culture the positive duck oviduct epithelial primary cells obtained in step S6 and collect the cell supernatant; S8: Isolation of Duck Reproductive Disorder Syndrome Virus: The cell supernatant from step S7 was subjected to sucrose density gradient ultracentrifugation. After centrifugation, the cells were separated into 7 layers. The precipitate layer with 35% sucrose was resuspended and centrifuged again to remove the sucrose. The resuspended precipitate was then used as the Duck Reproductive Disorder Syndrome Virus solution. The solution was then concentrated and purified to obtain the Duck Reproductive Disorder Syndrome Virus.

[0012] Therefore, this method can rapidly and efficiently isolate the duck reproductive disorder syndrome virus strain, laying the foundation for further detection of the virus and the preparation of vaccines or drugs for the prevention and treatment of the virus.

[0013] Traditional methods for avian viruses: (1) Grind and filter positive pathogens or add antibiotics to sterilize or kill them, and then use them as inoculation samples to inoculate chicken, duck, or goose embryos. The required embryo age varies depending on the breed and inoculation site; (2) Use existing passaged cell lines to inoculate the virus. First, culture the cells well, add the sample for incubation, and then culture them; (3) Prepare primary cells, first culture the cells well, add the sample for incubation, and then culture them. Generally, it is necessary to passage the virus three times in a row. Only if the virus is still detected after three passages can it be considered isolated, because the positive results detected within three passages are sometimes false positives due to sample residue. Generally, many viruses have one or more specific cells for reproduction. Not all cells can reproduce. As a new virus, it is uncertain which cells can be used to proliferate the duck reproductive disorder syndrome virus. Before this isolation method, all embryos and cells were tried, but the virus could not be proliferated and isolated. However, this method can efficiently isolate the duck reproductive disorder syndrome virus. Moreover, compared with the traditional virus isolation method, this method does not require inoculation and blind passage, and the isolation time can be saved by more than three weeks. The efficiency and success rate of virus isolation are higher.

[0014] In some embodiments, in step S1, 35-week-old positive ducks are selected. These positive ducks are those that have been excluded from other pathogen infections and exhibit clinical symptoms of duck reproductive disorder syndrome. Therefore, selecting 35-week-old positive ducks allows for the acquisition of high-density and morphologically uniform primary oviduct epithelial cells, which can further improve the isolation quality of duck reproductive disorder syndrome virus.

[0015] In some embodiments, the complete culture medium in step S3 consists of: 20% FBS, 2-3 mL of penicillin-streptomycin mixture, 2 mmol glutamine, 100 μg / mL heparin sodium, 100 μg epidermal growth factor, 100 μg insulin, and 100 μg IGF-1 recombinant transferrin added to every 500 mL of DMEM / F12 cell culture medium.

[0016] According to a second aspect of the present invention, the use of duck reproductive disorder syndrome virus (DRSV) in the preparation of a vaccine against DRSV is provided. This provides a preventative effect against DRSV and reduces its harm to poultry farms.

[0017] According to a third aspect of the present invention, an application of duck reproductive disorder syndrome virus (DRSV) in the preparation of products for detecting DRSV is provided. This enables efficient detection of DRSV, facilitating subsequent development of prevention and control strategies and epidemiological investigations, thereby reducing its harm to farms.

[0018] According to a fourth aspect of the present invention, the use of duck reproductive disorder syndrome virus in the preparation of a medicament for treating duck reproductive disorder syndrome is provided. This allows for effective treatment of duck reproductive disorder syndrome virus and reduces its harm to poultry farms.

[0019] The beneficial effects of this invention are: 1. This invention isolates a duck reproductive disorder syndrome virus, with the preservation number CCTCC NO:V202619. This virus is a novel virus. Further analysis of its sequence and characteristics can lay the foundation for the preparation of vaccines and drugs to prevent and treat duck reproductive disorder syndrome, and also provide the necessary conditions for further detection of the virus.

[0020] 2. This invention also discloses a method for isolating duck reproductive disorder syndrome virus (DRS), which allows for rapid and efficient isolation of DRS virus strains. Furthermore, compared to traditional virus isolation methods, this method eliminates the need for inoculation and blind passage, resulting in higher virus isolation efficiency.

[0021] 3. The present invention also discloses the use of duck reproductive syndrome virus in the preparation of vaccines against duck reproductive syndrome virus, in the preparation of products for detecting duck reproductive syndrome virus, or in the preparation of drugs for treating duck reproductive syndrome. Attached Figure Description

[0022] Figure 1 Image of epithelial cells isolated from the uterine portion of the fallopian tube; Figure 2 A cellular diagram of the infundibulum of the fallopian tube isolated from cells; Figure 3 Cell image isolated from a 30-week-old Muscovy duck; Figure 4 Cell image isolated from a 35-week-old Muscovy duck; Figure 5 Image of cells isolated from a 38-week-old Muscovy duck; Figure 6 A cell diagram showing primary cells of Muscovy duck oviduct epithelial cells after optimized culture conditions; Figure 7 Figure showing the results of a passage experiment on primary cells of Muscovy duck oviduct epithelium; Figure 8 Image showing the cryopreservation and recovery results of primary epithelial cells from the oviduct of Muscovy ducks; Figure 9 Image showing the results of cell supernatant after sucrose density gradient ultracentrifugation; Figure 10 Image showing electron microscopic observation of duck reproductive disorder syndrome virus; Figure 11The results show the alignment of the 7 ORFs of DRDSV with the sequences in the NCBI virus database. Detailed Implementation

[0023] The present invention will now be described in detail with reference to specific embodiments. These embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that those skilled in the art can make various changes and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention.

[0024] Unless otherwise specified, all chemical substances, proteins, enzymes, or reagent kits used in this invention are commercially available.

[0025] Unless otherwise stated, the methods used in this invention, such as PCR amplification and RNA extraction, employ conventional experimental procedures in the field. These procedures can be performed with reference to commonly used experimental technical manuals or the operating instructions for commercially available reagent kits and instruments.

[0026] Example 1: Selection of positive Muscovy ducks.

[0027] A Muscovy duck farm experienced a decline in egg production. Autopsies of the affected ducks revealed endometrial hemorrhage. Tissue samples from the diseased ducks were collected and tested using existing methods for detecting known pathogens, but no pathogen was detected. The initial diagnosis was an unknown pathogen. Because the main clinical symptoms of this disease are decreased egg production and endometrial hemorrhage, and because the tissue has a strong affinity for the reproductive system, the disease was named "Duck Reproductive Disorder Syndrome." Duck Reproductive Disorder Syndrome, DRDS However, through the isolation of bacteria and mycoplasma from the diseased tissue, no bacteria or mycoplasma could be isolated. Therefore, it was inferred that the cause of the disease was a virus, and the unknown pathogen causing the disease was named "Duck Reproductive Disorder Syndrome Virus" (DRS). Duck Reproductive Disorder Syndrome Virus,DRDSV )".

[0028] Anal swabs were collected from ducks exhibiting Duck Reproductive Disorder Syndrome (DRDS) and then tested (using existing methods for detecting known pathogens). Ducks infected with other pathogens were removed, leaving only the positive ducks infected with DDS virus (positive ducks that have been completely excluded from infection with other pathogens) for subsequent research.

[0029] Example 2: Preparation of complete culture medium.

[0030] Complete culture medium components: Add 20% fetal bovine serum (FBS), 2-3 mL penicillin-streptomycin mixture, 2 mmol glutamine, 100 μg / mL heparin sodium, 100 μg epidermal growth factor, 100 μg insulin, and 100 μg IGF-1 recombinant transferrin to every 500 mL of DMEM / F12 cell culture medium. Then filter through a 0.22 μm filter membrane and dispense into sterile centrifuge tubes.

[0031] Example 3: Steps for isolating and culturing primary epithelial cells from the oviduct of positive Muscovy ducks.

[0032] In a clean bench, oviducts of positive Muscovy ducks (ducks thoroughly excluded from other pathogen infections) were rinsed in pre-cooled physiological saline. The oviducts were cut open, and the internal epithelial tissue was placed in a small beaker. The tissue was rinsed with PBS until clear, then minced and rinsed again with PBS until clear. The PBS was discarded. Then, 0.25% trypsin + 0.02% EDTA was added and incubated at 37°C until the tissue became fluffy. FBS was then added to remove the enzymes, and the FBS was discarded. Freshly prepared complete culture medium was added, and the mixture was shaken with magnetic beads and filtered through a 100μm cell sieve. Cells were counted, centrifuged at 1000 rpm for 3 minutes, the supernatant was discarded, and the cells were resuspended. The cells were then layered in 1.37 x 10⁻⁶ cells per 10cm cell culture dish. 7 Cells were aliquoted and plated, and incubated in an incubator for 2 h. Simultaneously, cell plates were coated with 100% FBS at 37℃ for later use. Non-adherent cells were centrifuged at 1000 rpm for 3 min. The resulting cell pellets were collected and resuspended in complete culture medium. The coated cell plates were then incubated statically in a 5% CO2 incubator at 37℃.

[0033] Example 4: Selection of oviduct location in Muscovy ducks.

[0034] Following the method in Example 3, epithelial tissue from the uterine and infundibulum portions of the oviducts of positive Muscovy ducks (ducks that have been thoroughly excluded from infection by other pathogens) was collected for epithelial cell isolation and culture. Cells isolated from the uterine portion were shown below. Figure 1 As shown: the cells have a uniform morphology, exhibiting typical epithelial cell cluster growth characteristics, good adhesion, few cell debris, occasional fibroblasts and other miscellaneous cells, and no bacterial contamination, indicating successful cell adhesion and proliferation. The funnel-shaped portion separates the cells as shown... Figure 2 As shown, the cells do not aggregate and, based on morphological and growth characteristics, are all fibroblasts; epithelial cells were not successfully isolated. Therefore, epithelial cell isolation and culture should be performed on the oviduct and uterine epithelial tissue of positive Muscovy ducks (positive ducks that have been fully excluded from infection by other pathogens).

[0035] Example 5: Selection of positive Muscovy duck age.

[0036] Following the method in Example 3, epithelial cells were isolated and cultured from the oviduct and uterine region of positive Muscovy ducks (completely excluding other pathogen infections) at 30, 35, and 38 weeks of age, respectively. The cell isolation results from the 30-week-old Muscovy ducks are as follows: Figure 3 As shown in the image: cells successfully adhered and proliferated, but proliferation was very slow; the cell density did not reach 90% after 120 hours, and cell morphology was inconsistent, with fibroblasts visible in the field of view. The results of cell isolation from 35-week-old Muscovy ducks are as follows. Figure 4 As shown: Cells successfully adhered and proliferated, reaching a density of 90% after 96 hours of culture. Cell morphology was uniform, and no fibroblasts were observed. The results of cell isolation from 38-week-old Muscovy ducks are as follows. Figure 5 As shown, the cells successfully adhered and proliferated, exhibiting uniform cell morphology, but their proliferation rate was lower than that of cells isolated from 35-week-old Muscovy ducks. Therefore, 35-week-old Muscovy ducks were ultimately selected as the target animal for primary cell isolation from the uterine epithelial region of the Muscovy duck oviduct.

[0037] Example 6: Optimization of culture conditions for primary epithelial cells from positive Muscovy duck oviducts.

[0038] Referring to the method in Example 3, and optimizing the culture conditions for primary Muscovy duck oviduct epithelial cells, an AF group was set up, namely: Group A (Optimized FBS content): The 20% FBS in the complete culture medium in Example 2 was changed to 10% FBS, the coating was 100% FBS, and the culture conditions were 37°C. Group B (Optimized coating solution FBS content): The complete culture medium (20% FBS) in Example 2 was used, and the coating was changed from 100% FBS to 20% FBS. The culture conditions were 37°C. Group C (Optimized culture medium and serum type): The 20% FBS in the complete culture medium in Example 2 was changed to 20% FBS + 5% Muscovy duck serum, and the coating was 100% FBS. The culture conditions were 37°C. Group D (Optimized FBS content): The 20% FBS in the complete culture medium in Example 2 was changed to 5% FBS, the coating was 100% FBS, and the culture conditions were 37°C. Group E (optimized FBS content): The complete culture medium formulation in Example 2 was used, with FBS content of 20%, and the coating was 100% FBS. The culture conditions were 37°C. Group F (optimized culture conditions): The complete culture medium (20% FBS) in Example 2 was used, the coating was 100% FBS, and the culture temperature was 39°C. Following the method described in Example 3, the AF group was cultured, and the results are as follows: Figure 6As shown in the figure, the results indicate that the cell density in groups A, B, C, D, and F is relatively low, while group E has the best cell adhesion and density. Therefore, group E was selected for subsequent cell culture in a complete medium supplemented with 20% FBS, 100% FBS serum coating solution, and a 5% CO2 37℃ incubator.

[0039] Example 7: Optimized method for isolating primary epithelial cells from positive Muscovy duck oviducts.

[0040] S1: Separate the oviduct of Muscovy ducks and obtain the epithelial tissue of the uterine part of the oviduct: Take the oviduct of a 35-week-old positive Muscovy duck (positive ducks that have been fully excluded from other pathogen infections) and wash it in pre-cooled physiological saline. Cut open the oviduct and take the epithelial tissue inside the uterine part. Put it into a small beaker and rinse it with PBS until it is clear. Cut it into small pieces and rinse it with PBS again until it is clear. Discard the PBS.

[0041] S2: Static digestion of the uterine epithelial tissue of the Muscovy duck oviduct: Add 0.25% trypsin + 0.02% EDTA to the shredded tissue sample from step S1 and let it sit at 37°C until the tissue becomes fluffy.

[0042] S3: Centrifugation and resuspending cells: Add FBS to the digested tissue from step S2 to remove enzymes, then discard the FBS. Add fresh, prepared complete culture medium (20% FBS, 2-3 mL penicillin-streptomycin mixture, 2 mmol glutamine, 100 μg / mL heparin sodium, 100 μg epidermal growth factor, 100 μg insulin, and 100 μg IGF-1 recombinant transferrin per 500 mL DMEM / F12 cell culture medium). Then, mix with magnetic beads, filter through a 100 μm cell sieve, centrifuge at 1000 r / min for 3 min, discard the supernatant, and resuspend the cells.

[0043] S4: Cell counting and differential adhesion removal of fibroblasts: Count the cells obtained from resuspending in step S3, and seed them in 1.37x10 cm cell culture dishes. 7 Cells were aliquoted and plated, incubated in an incubator for 2 hours, and then the epithelial cells suspended in the cell culture medium were collected, and the adherent fibroblasts were removed.

[0044] S5: Cell plate coating: Coat cell plates with coating solution containing 100% FBS at 37°C.

[0045] S6: Obtaining primary oviduct epithelial cells: Collect the cell clumps from step S4, suspend them in complete culture medium, and incubate them statically in a 5% CO2 37℃ incubator using the cell plates coated in step S5 to obtain positive Muscovy duck oviduct epithelial primary cells.

[0046] Example 8: Passage test of primary epithelial cells from positive Muscovy duck oviducts.

[0047] The primary Muscovy duck oviduct epithelial cells obtained in Example 7 were passaged: after each plating, when the cell density reached 90%, a 2:1 passage ratio was performed. The passage results are as follows: Figure 7 As shown: F1 primary cells aggregated and grew normally; F2 generation (cultured for 96 h after subculturing) showed morphological changes, with uneven cell morphology and fibrous formation; F3 generation (cultured for 48 h after subculturing) cells did not aggregate, but showed morphological changes, significant fibrosis, and accelerated growth rate. These results indicate that the morphology of these positive Muscovy duck oviduct epithelial primary cells changes after subculturing, therefore they cannot be subcultured and must be used immediately after preparation.

[0048] Example 9: Cryopreservation and thawing of primary epithelial cells from the oviduct of positive Muscovy ducks.

[0049] The positive Muscovy duck oviduct epithelial primary cells obtained in Example 7 were cryopreserved using 10% DMSO + 90% FBS. After thawing, the epithelial cells successfully adhered and proliferated, reaching a cell density of 90% after 96 hours of culture. This indicates that the isolated Muscovy duck oviduct epithelial primary cells can be cryopreserved and thawed (results are shown in Figure 7). Figure 8 (As shown).

[0050] Example 10: Harvesting, concentrating, and purifying duck reproductive disorder virus.

[0051] After the primary epithelial cells of the positive Muscovy duck oviduct obtained in Example 7 were successfully adhered to the culture medium, the cell supernatant was aspirated and fresh culture medium was added every day until the cells were fully harvested after 5 days.

[0052] The harvested cell supernatant was subjected to sucrose density gradient ultracentrifugation (4℃, 40,000 rpm, 3 h) with sucrose density gradients of 25%, 35%, and 45%. After centrifugation, the cells separated into 7 layers (see results). Figure 9 As shown in the figure, the precipitate of each layer was resuspended and diluted with PBS, and then centrifuged at 4°C and 50,000 rpm for 3 h to remove sucrose. The precipitate was then resuspended again with 100 μL of PBS.

[0053] Example 11: Identification of duck reproductive disorder syndrome virus.

[0054] The samples obtained from each resuspension in Example 10 were observed under a scanning electron microscope. Typical enveloped viral features were visible in the 35% sucrose precipitate layer (electron microscopy results are shown in Figure 10). Figure 10 As shown in the figure, no viral characteristics were found in other precipitation layers, proving that the virus clustered in the 35% sucrose precipitation layer was duck reproductive disorder syndrome virus.

[0055] Whole-genome sequencing was performed on the duck reproductive disorder syndrome virus isolated from a 35% sucrose precipitation layer. The complete genome sequence is shown in SEQ ID NO:1. Based on this whole genome sequence, open reading frame (ORF) prediction analysis was performed on the DRDSV whole genome using bioinformatics software, resulting in seven predicted ORFs (denoted as ORF1-ORF7). These seven ORFs (ORF1-ORF7) were then compared with the GenBank database. Only ORF3 showed a match with the viral rdrp (RNA-dependent RNA polymerase) protein; the other six ORFs did not show any match results. Furthermore, the alignment result of ORF3 is as follows: Figure 11 As shown: Figure 11 The first result is a metagenomically assembled genome (MAG), and the remaining results are all rdrp (RNA-dependent RNA polymerase) protein-related sequences of astroviruses, with the highest homology being only 52.13%. This indicates that the duck reproductive disorder syndrome virus is a novel virus.

[0056] The nucleotide sequences of the seven ORFs (denoted as ORF1-ORF7) are shown in SEQ ID NO:2-SEQ ID NO:8, and the amino acid sequences of the seven ORFs (denoted as ORF1-ORF7) are shown in SEQ ID NO:9-SEQ ID NO:15.

[0057] The isolated duck reproductive disorder syndrome virus was deposited under the accession number CCTCC NO:V202619, with the accession name DRDSV-01 and the classification name as follows: Duck Reproductive Disorder Syndrome Virus The depositary institution is the China Center for Type Culture Collection, the deposit address is Wuhan University, Wuhan, China, and the deposit date is March 6, 2026.

[0058] In a challenge experiment using this duck reproductive disorder syndrome virus on healthy Muscovy ducks, the infected ducks exhibited decreased egg production and endometrial hemorrhage. Furthermore, the tissue tropism was primarily in the reproductive system, consistent with duck reproductive disorder syndrome (DRS). Duck Reproductive Disorder Syndrome, DRDS The clinical symptoms were completely consistent, further indicating that the duck reproductive disorder syndrome virus was successfully isolated.

[0059] Example 12: Application of duck reproductive disorder syndrome virus.

[0060] By isolating and sequencing the genome of the duck reproductive disorder syndrome virus, primers or probes for its detection can be prepared for efficient detection. This facilitates the development of prevention and control strategies and epidemiological investigations, thereby reducing its harm to farms.

[0061] The virus obtained by isolating duck reproductive disorder syndrome can also be used to prepare corresponding vaccines or drugs for the prevention and treatment of the disease, thereby reducing its harm to farms.

[0062]

[0063] ORF1 nucleotide sequence (SEQ ID NO:2):

[0064] ORF2 nucleotide sequence (SEQ ID NO:3):

[0065] ORF3 nucleotide sequence (SEQ ID NO:4):

[0066] Nucleotide sequence of ORF4 (SEQ ID NO:5): ATGAATCCAAGTGCTCCAGTGTGGACACCGCAGAATAGTAATGGTAATAGTGTTGATGGTCCTCCGTATGGTTATACTAATCCACCGTTTCAGGCTTACCCTTGGGGTAATTTACCATCTAATCGGAATTGGTATCCCAGAAGAGGATGGTGTAGACCATGGCGTGGTCGTGGCAACTGGAATAATAGGAATCAAGACCGCGGGAGATCTCGTGATCGCGCATCATCAAGAGCTCGTTCGAGGAGTCGATCTGCTAGTCGTCGGCGTGGACATGGAGACGACTGGTACAGCCAGTGGGGACACAATGTCGTAACGTATGGTATGGTAACTGTGACACAGCAAGGGAAGTCTAATTTTAATCTTAGGAATCCCTGGAATTATAATAATAAGGACGTTGAACAGACAAAGTTGAGAGACTGGAGATTTCCAGCATCCACTGAAGTTGAAACAGTTGTCTTTGATGGTGATAACAATGGTGGTTTCTATCGTGTTGGGTTGAAAGTTGATATTGCTTCTCCTGATTCGAGGAAAGTAAGATTTGGTACACCTACTGATTTGGCAGATGGTGTTAAAAATTTAACTCTTGATTCTGAAGGCGATAAGAAGAAAAAGCGTGGTGGTATCTTTAGGAGATGGCGTTCCACGGAGCAGTTGTGA。

[0067] Nucleotide sequence of ORF5 (SEQ ID NO:6): ATGATGATCACCATCAATGCACACAACACACTTCGTTTGTTCCTCACAACACAACACCTTACAACCCATACGACCACGATTGCGATCAACAAGTGTTTTGCCAAACCAATCATCGATTTTATTATTACACTTCTTACAACATCTCTCTTTTGCATATTTACAGTCACGAACACTGTGACCAACAACGTTACACCAAATACAGGCTTGGCCAATACAAACATCACAATCACAATTCTTATCACACTGACACCTTGGACAATGACAATCACCATTCAAATTCGAATGTTTACTAATGCCACCACCAACAAACAATTGTTTTTTATCAAATTCACTACTAGATGGCTTAACCTTAGACTCCTGCCTTCTCCGCTGAGGCAAGGCTTCAACAACATCCTGCCTCCTCCGCTGTGGCAAAACTTCAACAACATTTTCCTCAACCGGTAA。

[0068] ORF6 nucleotide sequence (SEQ ID NO:7): ATGCATAACAGCATTATCCAATTCCAAAATGCTCCATCTCTCTTGACCTTAGCAACAAGAGGTCCACGAAAAATTTTAACAACCTTACCATCACAAAGACATGACAACAACCTCTCTCTAACCTCAACAAAACACAACAAAGCCGGATTAATTACTACCACCACCCACGACACAGGTGTTGCAATCCACGGAACAAAAAGCAAAACCAAAATCATAACACAAAACCAAATTCCATTAATAATACCAACATCCATAACACACCAAAACACAAAGACACCAACAAGTGATTTCACATCTCTCATGCCTCGCATATGA。

[0069] ORF7 nucleotide sequence (SEQ ID NO:8): ATGACAAGCCATGATGATCACCATCAATGCACACAACACACTTCGTTTGTTCCTCACAACACAACACCTTACAACCCATACGACCACGATTGCGATCAACAAGTGTTTTGCCAAACCAATCATCGATTTTATTATTACACTTCTTACAACATCTCTCTTTTGCATATTTACAGTCACGAACACTGTGACCAACAACGTTACACCAAATACAGGCTTGGCCAATACAAACATCACAATCACAATTCTTATCACACTGACACCTTGGACAATGACAATCACCATTCAAATTCGAATGTTTACTAA。

[0070] Amino acid sequence of ORF1 (SEQ ID NO:9):

[0071] Amino acid sequence of ORF2 (SEQ ID NO: 10): MAFHGAVVICLGFLNLCYCDPFLNFGFSLGWFNDTKVNGSNDSNVTTTSLSTAFGSIQGSVWGVVFGNQDSSGVVYVDWAGRTGGFVGADQIFNFTGNHSALGCNWSSNDTLVAGGPRPPGTRVPQFDNVTVDCGDLAVRLDFGGGSKLICGIDSKEDRHRGQVIGGITFGCDDSGCSSTSYYFRCYGNRSMEIDSNCTNGGGNNTNGTRCGKPVVKRWSFVRPVEEVSDKWNFTDKIVVCSYDCWGFSCADFNNCTCTGGPGAICCSTGNNMTSCRVLGDGTETWWSHHFEVIGLGLIVFLILVICVEIVLFCCGFKRKKKRRWLITNGETTIEAKGWFFVFLLWYCGFRSATAISIGKNDLNRWFSGPEDFGFKVTSLGFVANGSKFEERCCQWGHSTAGGISCAVDTPDNDNDPCYKWSQDPTQWDCRSYENGVAGWNDGCFLENGNTVSWCCKCRCRSSWTGWRFKFTGKPVGSIMLRSANHTLVVNISGDGTYYTAFANVTVSGCTGPMALDAWVSRGHWLDAHSCGLSIGDRPLHGAVHCSGRSNWGSVVTSCYCPQGGELGPISVDVDLGFPYKQKNVPAARVSGTCDVNFEWVEGKQNLGNCSLWCGDWSDGWCQVHIMNDTCIFQNVTQTKGITWIYCGNDMIIGKSKCNGTVWRPTVPGPYNPDEKHDSEHDNGFAHLWKEFLEFWQKWFYWIVIVIIVFVFWLIVRLLFCCNRK。

[0072] Amino acid sequence of ORF3 (SEQ ID NO: 11): MIFEWDKDFVKNFYKYLEDKMVKCCGMVNNFPHSIFNRDVVHGLRYLIDNDCNERVVDEFKPGNGFEWLSTGKYVRESGYVYGEYTTRDYIEDMFLKRFYESPFYPELRFNKLSKDFGVKYCRGLISVGMMPSRIRDVCKVNYPFEAEVPSVDLEAERFFKNYDWCNKIIGAYSWPKICLKSEMESVARFCPDERLCSGLEGLKFNSILIAFNYCVEDWNMGRSSVGLSDEDFVVTNRSWTNSPGYPYNKNFCRQAREAFDVYFGLLKHYEYSSMNDWMPTVFNVFCKKEILKAEKVLANDIRTIIAPALCQQLVMQKLTLAISMRVSSNWRFSHTSIGRTRWKDDVDFTGCRIGRFDFICEYDISKWDRSIKSFLLKLFWFYCWFVIDTTKLEHFWQLSNGFESVIYSFMLHKNGEVIRKHYGVPSGFTLTSYVNSWVHTFLNVLCFWELCPVECDINYFKKHCDFVCYGDDGLMGISKEAAEWFTIDARSEWLKTKWGITMDPKKCKMVDRYYFTFDGVDVDGIMFLGDVMKVDEDMGRIVPVFKISKIINQFILGNAVKNYMPSEKILICFGHYVECFFHPNCEVIREYLCFLMNKYKYSYIMKTMFKSEYVEYLAMTNNDLICKIRSLCFDRDKFRAFIMNMFYSVKDESKCSSVDTAE。

[0073] Amino acid sequence of ORF4 (SEQ ID NO:12): MNPSAPVWTPQNSNGNSVDGPPYGYTNPPFQAYPWGNLPSNRNWYPRRGWCRPWRGRGNWNNRNQDRGRSRDRASSRARSRSRSASRRRGHGDDWYSQWGHNVVTYGMVTVTQQGKSNFNLRNPWNYNNKDVEQTKLRDWRFPASTEVETVVFDGDNNGGFYRVGLKVDIASPDSRKVRFGTPTDLADGVKNLTLDSEGDKKKKRGGIFRRWRSTEQL。

[0074] Amino acid sequence of ORF5 (SEQ ID NO:13): MMITINAHNTLRLFLTTQHLTTHTTTIAINKCFAKPIIDFIITLLTTSLFCIFTVTNTVTNNVTPNTGLANTNITITILITLTPWTMTITIQIRMFTNATTNKQLFFIKFTTRWLNLRLLPSPLRQGFNNILPPPLWQNFNNIFLNR。

[0075] Amino acid sequence of ORF6 (SEQ ID NO:14): MHNSIIQFQNAPSLLTLATRGPRKILTTLPSQRHDNNLSLTSTKHNKAGLITTTTHDTGVAIHGTKSKTKIITQNQIPLIIPTSITHQNTKTPTSDFTSLMPRI。

[0076] Amino acid sequence of ORF7 (SEQ ID NO:15): MTSHDDHHQCTQHTSFVPHNTTPYNPYDHDCDQQVFCQTNHRFYYYTSYNISLLHIYSHEHCDQQRYTKYRLGQYKHHNHNSYHTDTLDNDNHHSNSNVY。

Claims

1. A duck reproductive disorder syndrome virus, wherein, The preservation number of the duck reproductive disorder syndrome virus is CCTCCNO:V202619.

2. The use of the duck reproductive syndrome virus of claim 1 in the preparation of a vaccine against duck reproductive syndrome virus.

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