Canine adenovirus type 1 and use thereof in constructing animal infection model

Abstract

This invention discloses a virulent strain of type I canine adenovirus and its application in constructing animal infection models, belonging to the field of biotechnology. The virulent strain of type I canine adenovirus is named canine adenovirus type 1 strain 0601, with accession number CCTCC NO: V202604. The virus titer reaches 10. 7.5 TCID 50 / mL. This virus can be used to construct an animal model of canine adenovirus type I infection, which exhibits typical clinical symptoms, such as hepatic hemorrhage, hepatocellular necrosis, and venous and sinusoidal congestion. The constructed animal model can be used for future canine adenovirus vaccine development, immune protection assessment, and efficacy evaluation of preventive and therapeutic drugs, providing excellent biological materials and theoretical basis for further control of canine adenovirus type I.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a virulent strain of type I canine adenovirus and its application in constructing animal infection models. Background Technology

[0002] Canine adenovirus type 1 (CAV-1) is a major pathogen causing infectious hepatitis in dogs, seriously endangering canine health. To further investigate the pathogenesis and immune response of CAV-1 and evaluate the efficacy of vaccines and drugs, it is crucial to construct a stable, reliable animal challenge model that simulates natural infection.

[0003] Currently, the common practice in establishing CAV-1 challenge models is to directly inoculate susceptible experimental animals with virus strains isolated in the wild or preserved in the laboratory. However, this existing technical approach has several significant drawbacks, limiting the accuracy and reproducibility of related studies: First, the pathogenicity of the viral strains used in existing models is unstable and heterogeneous. Traditional viral strains are often not fully cloned and purified, and their viral populations exhibit heterogeneity in genomic sequence and virulence. This directly leads to significant fluctuations in the clinical symptoms, pathological progression, and severity exhibited by animals in different laboratories, and even in different batches within the same laboratory, resulting in poor comparability of experimental data and making it difficult to draw reliable conclusions.

[0004] Second, there is a lack of standardized quantitative challenge benchmarks. Current technologies mostly use coarse viral titers to set challenge dosages, rather than establishing challenge dosage standards that are precisely correlated with specific clinical outcomes (such as specific pathological scores or serological transformation). This ambiguity in dosage setting makes it difficult to obtain sensitive and uniform benchmarks for evaluating vaccine immunogenicity or drug efficacy.

[0005] Third, there are issues of model reproducibility and animal welfare. Due to the aforementioned problems of heterogeneity in viral strains and inaccurate dosage, researchers often tend to use higher doses of virus for challenge in order to achieve the expected pathogenic effect. This not only exacerbates unnecessary suffering for experimental animals and violates the "3R" principle of animal ethics (reduce, optimize, replace), but also affects the reproducibility of the model due to excessive differences in individual animal responses.

[0006] Therefore, there is an urgent need in this field for a type I canine adenovirus challenge model that has stable and uniform pathogenic characteristics, precise and controllable challenge dosage, and can highly simulate the clinical process of natural infection, in order to overcome the randomness and unreliability of existing technologies and provide a standardized tool for basic and applied research related to CAV-1. Summary of the Invention

[0007] The purpose of this invention is to provide a virulent strain of type I canine adenovirus and its application in constructing animal infection models, thereby addressing the problems existing in the prior art. This invention isolates a type I canine adenovirus, named canine adenovirus type 1 0601 strain. The viral titer reaches 10. 7.5 TCID 50 / mL. It can be used to construct animal models of canine adenovirus type I infection, for the development of future canine adenovirus vaccines and the evaluation of immune protection, as well as the evaluation of the efficacy of preventive and therapeutic drugs, providing good biological materials and theoretical basis for further prevention and control of canine adenovirus type I.

[0008] To achieve the above objectives, the present invention provides the following solution: This invention provides a virulent strain of type I canine adenovirus (Canine adenovirus 1), named Canine adenovirus type 1 strain 0601, which has been deposited at the China Center for Type Culture Collection (CCTCC) on January 5, 2026, with accession number CCTCC NO: V202604, and classified as: Canine adenovirus type 1 strain 0601 (Canineadenovirus 1).

[0009] This invention also provides the application of the above-mentioned virulent type I canine adenovirus strain in constructing an animal infection model of type I canine adenovirus.

[0010] The present invention also provides a method for constructing an animal infection model of type I canine adenovirus, including the step of challenging animals with the above-mentioned virulent strain of type I canine adenovirus.

[0011] Optionally, the animal may include a dog.

[0012] Furthermore, the dog in question is a 2-month-old puppy.

[0013] Optionally, the challenge dose is not less than 2 × 10⁻⁶. 4.5 TCID 50 / Only.

[0014] Optionally, the attack dose is not less than 2 × 10⁻⁶. 5.5 TCID 50 / Only.

[0015] The present invention also provides the application of the type I canine adenovirus infection model obtained by the above construction method in screening drugs for the prevention and / or treatment of type I canine adenovirus.

[0016] The present invention also provides the application of the type I canine adenovirus infection model obtained by the above construction method in evaluating the efficacy of drugs for the prevention and / or treatment of type I canine adenovirus.

[0017] This invention also provides the application of the type I canine adenovirus animal infection model obtained by the above construction method in screening type I canine adenovirus vaccines.

[0018] This invention also provides the application of the type I canine adenovirus infection model obtained by the above construction method in evaluating the immunization effect of type I canine adenovirus vaccines.

[0019] The present invention discloses the following technical effects: This invention successfully isolated a type I canine adenovirus from dogs infected with infectious hepatitis in Wuhan, Hubei Province, and named it canine adenovirus type 1 strain 0601. Through genetic evolutionary analysis and genome comparison, this strain was found to be the latest circulating strain, with a viral titer of 10. 7.5 TCID 50 / mL.

[0020] This invention successfully constructed a type I canine adenovirus infection model using this strain. The model was tested on experimental dogs using 2 mL of 10... 5.5 TCID 50 At a challenge dose of / mL, at least 80% of the experimental dogs developed the disease, exhibiting typical clinical symptoms such as hepatic hemorrhage, hepatocellular necrosis, and venous and sinusoidal congestion. This model can be used for future canine adenovirus vaccine development, immune protection assessment, and efficacy evaluation of preventive and therapeutic drugs, providing valuable biomaterials and theoretical basis for further control of type I canine adenovirus. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 The image shows the PCR amplification results of the isolated type I canine adenovirus; where M is the DNA molecular standard quality standard-DL2000; 1 is the isolated type I canine adenovirus sample; 2 is the positive control; and 3 is the negative control. Figure 2 The genetic evolution tree of the isolated type I canine adenovirus fiber gene; where red circles represent the strains isolated in this invention; Figure 3 Phenotypic images of clinical symptoms in 2-month-old experimental dogs after viral challenge; Figure 4 The graph shows the changes in body temperature of 2-month-old experimental dogs after viral challenge; where 1-25 are animal numbers. Figure 5This is a graph showing the weight changes of 2-month-old experimental dogs after viral challenge; where 1-25 are animal numbers. Figure 6 This is a graph showing the changes in aspartate aminotransferase levels in the plasma of 2-month-old experimental dogs after viral challenge; where 1-25 are animal numbers. Figure 7 This graph shows the changes in alanine aminotransferase levels in the plasma of 2-month-old experimental dogs after viral challenge; 1-25 are animal numbers. Figure 8 This image shows the change in whole blood lymphocyte count in 2-month-old experimental dogs after viral challenge; 1-25 are animal numbers. Figure 9 Clinical symptom scoring chart for 2-month-old experimental dogs after viral challenge; Figure 10 H&E staining images of liver pathological sections from 2-month-old experimental dogs after viral challenge; Figure 11 Immunohistochemical images of liver pathological sections from 2-month-old experimental dogs after viral challenge. Detailed Implementation

[0023] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0024] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0025] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0026] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0027] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0028] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the instruments and equipment used in the following examples are all conventional laboratory instruments and equipment; unless otherwise specified, the experimental materials used in the following examples were all purchased from conventional biochemical reagent stores.

[0029] Example 1: Isolation and Identification of Type I Canine Adenovirus 1. Virus isolation Liver and spleen were collected from dogs that died of canine infectious hepatitis in Wuhan, Hubei Province. The livers and spleens were cut into small pieces and transferred to 2 mL sterile EP tubes. Small steel balls were added to homogenize the mixture. The homogenized suspension was then mixed with penicillin (final concentration of 1000 IU / mL penicillin and 1000 μg / mL streptomycin), diluted, and centrifuged at 4°C and 10000 r / min for 10 min. The supernatant was collected, filtered through a 0.22 μm filter, and inoculated into adherent MDCK cells. The cells were blindly passaged for 3 generations to obtain type I canine adenovirus solution.

[0030] 2. Identification of the virus Take 200 μL of type I canine adenovirus solution and extract viral DNA / RNA according to the instructions of the viral DNA / RNA extraction kit. Identify the viral solution using PCR.

[0031] The primers used for PCR are as follows: PCR CAV-F: 5'-CGCGCTGAACATTACTACCTTGTC- 3' (SEQ ID NO. 1); PCR CAV-R: 5'-CCTAGAGCACTTCGTGTCCGCTT-3' (SEQ ID NO. 2).

[0032] The PCR reaction system consisted of 12.5 μL of 2×Taq Master Mix, 0.5 μL each of forward and reverse primers, 1 μL of DNA template, and ddH2O to a final volume of 25 μL. The reaction program was as follows: 95℃ for 5 min; 95℃ for 30 s, 62℃ for 30 s, 72℃ for 70 s, 35 cycles; 72℃ for 10 min; and storage at 4℃. The CAV-1 strain genome (NCBI ID: AC_000003) was used as a positive control, and ddH2O was used as a negative control. The PCR amplification results were detected by 1% agarose gel electrophoresis, and the results are shown below. Figure 1As shown, a specific target band of approximately 508 bp was amplified, indicating that a type I canine adenovirus was successfully isolated.

[0033] 3. Amplification and evolutionary analysis of the target gene Design specific primers to amplify the viral fiber gene by PCR.

[0034] The primers used for PCR are as follows: PCR CAV1-Fiber-F1: 5'-CAACACCAAAGCCATGAAGC- 3' (SEQ ID NO. 3); PCR CAV1-Fiber-R1: 5'-ATATCGGAAAGTCCAGGTCC- 3' (SEQ ID NO. 4); PCR CAV1-Fiber-F2: 5'-CGAGTCGCTTAAGAGTATCC- 3' (SEQ ID NO. 5); PCR CAV1-Fiber-R2: 5'-AGTGACTTTTCCTGAAGCCGC-3' (SEQ ID NO. 6).

[0035] The PCR reaction system consisted of: 25 μL of 2×PhantaMax Buffer, 1 μL of dNTP Mix (10 mM each), 1 μL each of forward and reverse primers, 5 μL of PhantaMax super-Fidelity DNA Polymerase, 5 μL of DNA template, and ddH2O to a final volume of 50 μL. The reaction program was: 95℃ for 5 min; 95℃ for 30 s, 52℃ for 30 s, 72℃ for 1 min, 35 cycles; 72℃ for 10 min; and storage at 4℃. 1% agarose gel electrophoresis detected the PCR amplification results, showing the amplification of specific target bands of 966 bp and 858 bp, respectively. The target bands were purified and recovered using the HiPure Gel Pure DNA Mini Kit (Guangzhou Meiji Biotechnology Co., Ltd.) and sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing and assembly to obtain the fiber target gene sequence. A phylogenetic tree was constructed, and the results are as follows. Figure 2 As shown. This virus belongs to type I canine adenovirus.

[0036] 4. Preservation of viruses The highly virulent strain of type I canine adenovirus was named Canine Adenovirus Type 1 0601 and was deposited on January 5, 2026, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China. It is classified as Canine adenovirus 1 and has the accession number CCTCC NO: V202604.

[0037] 5. Culture and purification of type I canine adenovirus Type I canine adenovirus solution was diluted 10-fold and inoculated into adherent MDCK cells using a synchronous inoculation method. After incubation at 37°C for 48 h, the cells were removed and subjected to three freeze-thaw cycles. The cells were then centrifuged at 4°C and 12,000 rpm for 10 min, and the supernatant was collected for subsequent virus proliferation and purification.

[0038] The virus was serially diluted 10-fold with DMEM cell maintenance medium (containing 2% newborn calf serum). Three wells of each diluted virus solution were inoculated into 12-well MDCK cell culture plates. The plates were incubated at 37°C with 5% CO2 for 1 hour for adsorption. The virus solution was then discarded, and the plates were covered with DMEM medium containing 2% newborn calf serum and 0.8% low-melting-point agarose and incubated at 37°C with 5% CO2 for 2-3 days. Once obvious vacuoles appeared under a microscope, the plates were stained with 0.1‰ neutral red at 37°C for 1 hour. The staining solution was discarded, and the vacuoles were picked and placed in 200 μL of DMEM cell maintenance medium. The plates were then subjected to three freeze-thaw cycles and inoculated into 12-well MDCK cell culture plates. The plates were incubated at 37°C with 5% CO2 until complete cytopathic effects were achieved. The supernatant was collected, and after three freeze-thaw cycles, the viral load of the clones was determined. Clones with high viral titers were selected and the above steps were repeated twice. Clones with high viral titers after three rounds of purification were selected for subsequent studies.

[0039] 6. TCID 50 Measurement The virus solution was serially diluted 10-fold with DMEM medium containing 2% newborn calf serum. Appropriate dilutions were seeded into 8 wells of 96-well cell culture plates, with 100 μL per well for each dilution. An 8-well control group was also included, with 100 μL of DMEM medium containing 2% newborn calf serum added to each well. The cell density was 5 × 10⁶ cells per well. 5 MDCK cell suspension was prepared at 100 cells / mL and cultured at 37°C with 5% CO2 for 3 days. TCID was determined by indirect immunofluorescence assay and calculated using the Reed-Muench method. 50 .

[0040] The procedure for indirect immunofluorescence assay is as follows: (1) Fixation: Discard the culture medium, wash 3 times with 200 μL PBS, add 100 μL of pre-cooled methanol at 4℃ to each well, and fix at -15℃ for 10 min.

[0041] (2) Blocking: Discard methanol, add 200 μL PBS and wash 3 times, add 100 μL PBS containing 5% BSA to each well, and incubate at 37℃ for 1 h.

[0042] (3) Add primary antibody: Discard the blocking solution, wash 3 times with 200 μL PBS, add 100 μL of mouse monoclonal antibody (5E5) to each well (diluted with PBS at 1:2000), and incubate at 37℃ for 1 h.

[0043] (4) Add secondary antibody: Discard the primary antibody, wash 3 times with 200 μL PBS, add fluorescently labeled goat anti-mouse IgG secondary antibody (diluted with PBS at 1:2000), and incubate at 37℃ for 1 h.

[0044] (5) Observation and judgment: Discard the fluorescent antibody, wash 3 times with 200μL PBS, add 100μL PBS to each well, and observe under a fluorescence microscope. Cells infected with the virus show specific fluorescence and have cell morphology; uninfected cells do not stain and the field of view is dark; cell culture wells with specific fluorescence are judged as positive, and cell culture wells without specific fluorescence are judged as negative.

[0045] The TCID of the virus was calculated using the Reed-Muench formula. 50 10 7.5 TCID 50 / mL.

[0046] Example 2: Determination of type I canine adenovirus challenge dose 1. Grouping and virus attack Take a virus concentration of 10 7.5 TCID 50 The stock solution of canine adenovirus type 1 strain 0601 was thawed at 4°C, mixed by shaking, and serially diluted 10-fold with sterile PBS to 10⁻¹⁰. 6.5 TCID 50 / mL, 10 5.5 TCID 50 / mL and 10 4.5 TCID 50 / mL. Twenty-five 2-month-old Beagles, negative for canine influenza virus, canine respiratory coronavirus, canine parainfluenza virus, canine parvovirus, canine distemper virus, canine rotavirus, and *Bordetella leukemia* antigens, as well as canine adenovirus serum antibodies, and who had received one dose of canine parvovirus and canine distemper virus bivalent live vaccine, were randomly divided into 5 groups of 5 puppies each. Groups 1-4 received intramuscular injections of a viral concentration of 10 μL in their hind limbs. 7.5 TCID 50 / mL, 10 6.5 TCID 50 / mL, 105.5 TCID 50 / mL and 10 4.5 TCID 50 2 mL of virus solution per dog (2 mL / v) was administered. Group 5 received 2 mL of PBS as a blank control group. From day 1 to day 7 post-challenge (with day 1 as 0 and negative numbers representing pre-challenge), the dogs' body temperature and weight were measured daily to observe disease progression. Blood samples were collected for complete blood count and plasma biochemistry tests. Clinical symptom scores were recorded. At the end of the challenge period, the dogs were euthanized and necropsy performed. Liver samples were used for pathological sectioning, H&E staining, and immunohistochemistry. The clinical symptom scoring criteria are as follows: Clinical symptom score of experimental dogs: After viral challenge, the dogs were assessed in three aspects: eye discharge, liver damage, and mental state and appetite. The scores for eye discharge were: normal (0 points); serous eye discharge (1 point); purulent eye discharge (2 points). The liver damage score was: Normal (0 points); either aspartate aminotransferase or alanine aminotransferase in the plasma biochemistry test was higher than the normal value, i.e., aspartate aminotransferase > 55 U / L or alanine aminotransferase > 140 U / L (1 point); both aspartate aminotransferase and alanine aminotransferase in the plasma biochemistry test were higher than the normal value, i.e., aspartate aminotransferase > 55 U / L and alanine aminotransferase > 140 U / L (2 points). The scores for mental state and appetite were: normal (0 points); depressed and decreased appetite (1 point); lethargic and completely unable to eat (2 points).

[0047] 2. Results 2.1 Clinical symptoms Table 1 Results of virus challenge experiment on 2-month-old experimental dogs From Table 1 and Figures 3-9 It can be seen that, compared with the blank control group, 10 7.5 TCID 50 Dogs in the / mL challenge group began to show obvious clinical symptoms on the second day after challenge, including fever, lethargy, loss of appetite, and purulent eye discharge. Two dogs died on days 4 and 5, respectively, and the remaining dogs continued to show symptoms until day 7. During the challenge period, all dogs showed a continuous downward trend in weight, lymphocyte counts dropped below the normal reference range, and aspartate aminotransferase and alanine aminotransferase levels rose to abnormal values. 6.5 TCID 50Dogs in the / mL challenge group began to show obvious clinical symptoms on the second day after challenge, including fever, lethargy, loss of appetite, and purulent eye discharge. No deaths occurred. Symptoms persisted for 7 days in all dogs. Weight decreased and then recovered during the challenge period. Lymphocyte counts dropped below the normal reference range and then returned to the normal reference range. Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels were both elevated to abnormal values. 5.5 TCID 50 In the / mL challenge group, four dogs began to show obvious clinical symptoms on the second day after challenge, including fever, lethargy, loss of appetite, and purulent eye discharge. No deaths occurred. Symptoms persisted for up to 7 days. Weight decreased and then recovered during the challenge period. Two dogs' lymphocyte counts dropped below the normal reference range and then returned to the normal reference range. Aspartate aminotransferase (AST) levels were elevated in three dogs and alanine aminotransferase (ALT) levels were elevated in two dogs. Meanwhile, in the 10... 4.5 TCID 50 Dogs in the / mL challenge group began to show mild clinical symptoms on the 3rd day after challenge, including fever. Only 2 dogs were lethargic and had a decreased appetite. There was no eye discharge and no deaths occurred. Only 2 dogs lost weight and then recovered during the challenge period. The weight of the other dogs did not fluctuate. The lymphocyte count of all dogs changed within the normal reference range. Two dogs had abnormal values ​​for aspartate aminotransferase and alanine aminotransferase.

[0048] 2.2 H&E staining and immunohistochemistry of tissue sections 2.2.1 Tissue embedding Fresh tissue samples were collected and fixed in formalin for 72 hours. The fixed tissue blocks were then removed, trimmed, and rinsed with tap water overnight. The tissue blocks were then treated sequentially with 70% ethanol for 50 minutes, 85% ethanol for 50 minutes, 95% ethanol for 50 minutes, and 100% ethanol for 50 minutes. Xylene was then used, with the treatment time varying depending on the tissue texture and size; the process of removing strands was observed, stopping when the tissue block gradually stopped exhibiting stranding in xylene. Low-melting-point paraffin was used: paraffin I (58℃) for 1 hour, and paraffin II (58℃) for 2 hours. The tissue blocks were embedded in a mold filled with paraffin. The embedded paraffin blocks were cut into thin slices of approximately 5–8 μm, dipped in hot water, and then mounted onto glass slides and dried at 55℃.

[0049] 2.2.2 H&E staining observation The fixed sections were immersed twice in xylene, 5 min each time. Then, they were immersed in 100%, 95%, 90%, and 80% ethanol for 5 min each time, sequentially; rinsed twice with distilled water for 3 min each time; stained with hematoxylin for 1-2 min; rinsed twice with distilled water for 5 min each time. Differentiated with 1% hydrochloric acid ethanol for 1-3 seconds, rinsed with water, immersed in ammonia water until partially turned blue, then rinsed; stained with 0.5% eosin for 1 min, rinsed with distilled water, then dehydrated sequentially in 80%, 90%, 95%, and 100% ethanol; immersed twice in xylene for 3-5 min each time, and air-dried at room temperature; neutral resin was added, a coverslip was placed on top, and the slide was mounted for observation. Results are as follows: Figure 10 .

[0050] Depend on Figure 10 It can be seen that, compared with the blank control, 10 7.5 TCID 50 / mL challenge group, 10 6.5 TCID 50 / mL challenge group and 10 5.5 TCID 50 In the group challenged with 10 mL of the virus, hemorrhagic foci were frequently observed in the liver tissue, with necrosis of hepatocytes around the hemorrhage, loss of nuclei, increased eosinophilicity, and a small number of fatty degenerations of hepatocytes. Tiny round vacuoles were present in the cytoplasm. Venous and sinusoidal congestion was common, and the arrangement of hepatocyte cords was disordered, with frequent punctate infiltration of inflammatory cells. 4.5 TCID 50 In the / mL challenge group, only a small amount of hepatocyte steatosis was observed, with few focal infiltrations of inflammatory cells, and no other obvious abnormalities were seen.

[0051] 2.2.3 Immunohistochemistry Paraffin sections were baked at 60°C for 2 hours, then immersed twice in xylene for 5 minutes each time. They were then immersed sequentially in 100%, 95%, 90%, and 80% ethanol for 5 minutes each time; washed twice with distilled water for 3 minutes each time; for heat antigen retrieval, the sections were immersed in EDTA antigen retrieval buffer (pH 9.0), microwaved on high heat to boiling, then on medium-low heat for 15 minutes, and then allowed to cool naturally to room temperature; the sections were then rinsed with PBS (pH 9.0). 7.4) Wash the slides three times, 5 minutes each time; add 3% hydrogen peroxide solution and incubate at room temperature in the dark for 25 minutes to block endogenous peroxidase; wash three more times with PBS; add normal goat serum blocking solution and block at room temperature for 30 minutes; discard the blocking solution, and directly add 1:4000 diluted canine adenovirus monoclonal antibody 5E5 (prepared and provided by Wuhan Keqian Biotechnology Co., Ltd.), and incubate overnight in a humidified chamber at 4°C; after warming up the next day, wash three times with PBS; add HRP-labeled goat anti-mouse IgG (purchased from Wuhan Boster Biological Engineering Co., Ltd.), and incubate at room temperature for 50 minutes. Wash three times with PBS; add freshly prepared DAB chromogenic solution, control the chromogenic time under a microscope, and rinse thoroughly with tap water after chromogenic development to stop the reaction; counterstain cell nuclei with hematoxylin for 30 seconds, differentiate with hydrochloric acid-ethanol, and return to blue with tap water; then, immerse the sections sequentially in 70% ethanol, 85% ethanol, 95% ethanol, 100% ethanol I, and 100% ethanol II for gradient dehydration, with each immersion time being approximately 2-3 minutes; after dehydration, transfer the sections to xylene I for 5-10 minutes, then to xylene II for 5-10 minutes, and finally mount with neutral resin for observation. Results are as follows: Figure 11 .

[0052] Depend on Figure 11 It can be seen that, compared with the blank control, 10 7.5 TCID 50 / mL challenge group, 10 6.5 TCID 50 / mL challenge group dogs showed significant antigen deposition in liver tissue sections, with multifocal to diffuse distribution. In areas of strong antigen positivity, cell structure disintegrated, forming homogeneous clumps. Numerous hepatocytes showed perinuclear halos, and cells with pyknosis and dense staining of the nucleus were also observed; 10 5.5 TCID 50 The liver tissue sections of the / mL challenge group showed slightly weaker positive signals, with diffuse pale staining mainly in the cytoplasm, and numerous hepatocytes that were extremely swollen with clear, net-like cytoplasm, forming typical balloon-like cells; while the 10 4.5 TCID 50 Liver tissue sections from the group challenged with / mL virus showed few positive signals, and the hepatocyte structure was not significantly abnormal.

[0053] Therefore, the challenge model was determined to be: 5 two-month-old experimental dogs challenged with the virus via intramuscular injection at a viral concentration of 10.5.5 TCID 50 At least 4 / 5 of patients develop the disease after consuming 2 mL of type I canine adenovirus at a concentration of 1 mL / mL.

[0054] In summary, the canine adenovirus type 1 strain 0601 isolated in this invention can stably replicate under specific challenge doses and induce typical clinical symptoms and characteristic pathological changes in infected dogs. It can effectively construct an animal model of canine adenovirus infection and has good application prospects and practical value. This indicates that this viral strain can be used as a standardized challenge strain for evaluating the immunoprotective efficacy and challenge protection of different immunization programs and vaccine strains against canine adenovirus type 1. By setting gradient challenge doses, it can accurately simulate the pathogenesis and pathological damage of natural infection, providing a reliable experimental system for evaluating the protection rate, duration of immunity, and critical challenge protection dose of candidate vaccines. Simultaneously, this viral infection model can reproduce the core pathological features of canine adenovirus infection, such as liver hemorrhage, hepatocellular necrosis, and venous and sinusoidal congestion. 5.5 TCID 50 At a viral load of / mL, at least 4 / 5 of the experimental dogs developed the disease. This ensures the stability and reproducibility of the model while avoiding excessive mortality caused by excessively high doses, which could affect experimental observation. It can be widely used in the study of the pathological mechanism of canine adenovirus infection, the screening of antiviral drugs and the evaluation of efficacy, and provides key experimental support for the research and optimization of related prevention and control technologies.

[0055] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A highly virulent strain of type I canine adenovirus (Canine adenovirus 1), characterized in that, The highly virulent strain of type I canine adenovirus was named canine adenovirus type 1 0601 strain and has been deposited at the China Center for Type Culture Collection on January 5, 2026, with accession number CCTCC NO: V202604.

2. The application of the virulent type I canine adenovirus strain described in claim 1 in constructing an animal infection model of type I canine adenovirus.

3. A method for constructing an animal model of canine adenovirus type I infection, characterized in that, The method includes the step of challenging animals with the virulent strain of type I canine adenovirus as described in claim 1.

4. The construction method according to claim 3, characterized in that, The animals mentioned include dogs.

5. The construction method according to claim 4, characterized in that, The dog in question is 2 months old.

6. The construction method according to claim 3, characterized in that, The dose of the virus used for the attack was no less than 2 × 10⁻⁶. 4.5 TCID 50 / Only.

7. The use of the type I canine adenovirus infection model obtained by the construction method according to any one of claims 3-6 in screening drugs for the prevention and / or treatment of type I canine adenovirus.

8. The use of the type I canine adenovirus infection model obtained by the construction method according to any one of claims 3-6 in evaluating the efficacy of drugs for the prevention and / or treatment of type I canine adenovirus.

9. The application of the type I canine adenovirus animal infection model obtained by the construction method according to any one of claims 3-6 in screening type I canine adenovirus vaccines.

10. The application of the type I canine adenovirus animal infection model obtained by the construction method according to any one of claims 3-6 in evaluating the immunization effect of type I canine adenovirus vaccine.

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