Canine adenovirus type 2 virulent strain, inactivated vaccine and application thereof

Abstract

This invention discloses a virulent strain of type II canine adenovirus, an inactivated vaccine, and their applications, belonging to the field of biotechnology. The virulent strain of type II canine adenovirus is named canine adenovirus type 2 Aa05 strain, with accession number CCTCC NO: V202605. The virus titer can reach 10. 6.0 TCID 50 / mL. This invention utilizes this strain to construct an animal model of canine adenovirus type II infection. This animal model exhibits typical clinical symptoms and can be used for future canine adenovirus vaccine development, immune protection assessment, and efficacy evaluation of preventive and therapeutic drugs. This invention also utilizes this strain to develop an inactivated vaccine with extremely high safety and immunogenicity. It can induce the body to produce high levels of specific neutralizing antibodies, which can protect immunized animals against infection with virulent CAV-2 strains and also against infection with highly pathogenic CAV-1 strains, demonstrating excellent cross-protection.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a virulent strain of type II canine adenovirus, an inactivated vaccine, and its application. Background Technology

[0002] Canine adenovirus (CAV) is a member of the genus Canine Adenovirus, mainly divided into type I (CAV-1) and type II (CAV-2) based on differences in antigenicity and pathogenicity. CAV-1 primarily causes infectious hepatitis in dogs, characterized by damage to hepatocytes and vascular endothelial cells, with a high mortality rate. CAV-2, on the other hand, is a core pathogen causing infectious laryngotracheitis (i.e., kennel cough syndrome). CAV-2 mainly affects puppies and immunocompromised dogs, causing upper respiratory symptoms such as fever, persistent dry cough, and nasal discharge. It can also lead to secondary bacterial infections such as Bordetella bronchiseptica due to impaired respiratory ciliary function, resulting in severe bronchopneumonia. This poses a continuous and serious threat to the global canine industry, pet health, and the combat effectiveness and service capacity of working dogs (such as police dogs and guide dogs). Therefore, research on the pathogenic mechanism of CAV-2, the development of novel vaccines, and the screening of antiviral drugs urgently require highly effective and safe vaccine candidate strains and stable and reliable animal experimental models.

[0003] Currently, the prevention and control of CAV-2 both domestically and internationally mainly relies on vaccination. Existing commercial vaccines are mostly attenuated live vaccines, which, while controlling outbreaks to some extent, still have potential safety issues, such as the risk of virulence reversion, safety concerns in pregnant dogs and immunodeficient dogs, and incomplete cross-protection between different serotypes (CAV-1 and CAV-2). Meanwhile, in the field of CAV-2 research, although there are occasional reports of challenge experiments using experimental dogs, the model construction and evaluation systems also have the following significant shortcomings: I. Crude challenge protocols and lack of clinical relevance: Existing challenge models mostly employ a single, high-dose viral inoculation method. This "all or nothing" challenge model, while inducing typical disease, often results in acute, severe systemic symptoms in animals, even death, failing to simulate the continuous disease spectrum from mild to severe common in natural infections. This severely limits the models' use in studying disease progression, host-differentiated responses, and evaluating the effectiveness of interventions for mild cases.

[0004] Second, the evaluation indicators are singular and subjective: the assessment of disease after viral challenge is usually limited to endpoint or qualitative indicators such as survival rate and rough clinical symptom observation (such as "depression" and "cough"). There is a lack of systematic recording of continuous, objective, and quantifiable core physiological indicators (such as dynamic body temperature and weight change curves) during the occurrence and development of the disease, resulting in extremely poor model reproducibility and data comparability between different laboratories.

[0005] Third, the pathological evaluation is unsystematic and lacks gradient correlation: the pathological analysis of the model endpoint often stops at a simple description of typical lesions, failing to establish a precise dose-response relationship between different challenge doses and the severity of multi-organ pathological damage. In particular, there is a lack of systematic semi-quantitative scoring of lesions in key target organs (such as the lungs and trachea), as well as detailed pathological examination of other potentially affected sites (eyes and nasal cavity). This makes it impossible for the model to accurately distinguish between different virulence strains or evaluate the subtle protective effects of interventions.

[0006] IV. Low Model Reproducibility and Standardization: Due to the non-standardization of the above-mentioned challenge protocols and evaluation systems, the CAV-2 challenge models constructed by different studies vary greatly in terms of disease severity, course, and outcome. Data are difficult to compare and integrate directly, which seriously hinders the accumulation and progress of research in this field. It also brings great uncertainty and operational difficulty to the evaluation of the immune protection effect of new vaccines.

[0007] In summary, there is an urgent need in this field to isolate and obtain a highly pathogenic CAV-2 strain with good immunogenicity that covers currently circulating strains, and to develop a safe and effective inactivated vaccine based on this strain. Simultaneously, it is also necessary to establish a standardized challenge model that can simulate the natural infection spectrum of CAV-2, has a clear clinical gradient, and provides comprehensive and objective evaluation indicators, in order to provide a scientific and rigorous evaluation platform for the efficacy and cross-protective effects of novel vaccines. Summary of the Invention

[0008] The purpose of this invention is to provide a virulent strain of type II canine adenovirus, an inactivated vaccine, and their applications, thereby addressing the problems existing in the prior art. This invention isolates a type II canine adenovirus, named canine adenovirus type 2 Aa05 strain. The virus titer reaches 10. 6.0 TCID 50 / mL, can be used to construct animal infection models of type II canine adenovirus and to prepare inactivated vaccines, for future research and development of canine adenovirus vaccines and evaluation of immune protection, evaluation of the efficacy of preventive and therapeutic drugs, and prevention and control of canine adenovirus diseases.

[0009] To achieve the above objectives, the present invention provides the following solution: This invention provides a virulent strain of type II canine adenovirus (Canine adenovirus 2), named Canine Adenovirus Type 2 Aa05 strain, which has been deposited at the China Center for Type Culture Collection on January 5, 2026, with accession number CCTCC NO: V202605.

[0010] The present invention also provides the application of the above-mentioned virulent type II canine adenovirus strain in constructing a type II canine adenovirus infection model or in preparing a type II canine adenovirus inactivated vaccine.

[0011] The present invention also provides a method for constructing an animal infection model of type II canine adenovirus, including the step of challenging animals with the above-mentioned virulent strain of type II canine adenovirus.

[0012] Optionally, the animal may include a dog.

[0013] Optionally, the challenge dose is not less than 10. 3.0 TCID 50 / Only.

[0014] Further optionally, the attack dose is not less than 10. 4.0 TCID 50 / Only.

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

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

[0017] The present invention also provides a type II canine adenovirus inactivated vaccine, wherein the preparation method of the type II canine adenovirus inactivated vaccine includes the step of inactivating the above-mentioned type II canine adenovirus virulent strain to obtain an inactivated vaccine.

[0018] The present invention also provides the use of the above-mentioned type II canine adenovirus inactivated vaccine in the preparation of a drug for preventing canine adenovirus infection.

[0019] Optionally, the canine adenovirus includes type I canine adenovirus and type II canine adenovirus.

[0020] The present invention also provides a drug for preventing canine adenovirus infection, the drug comprising the above-mentioned canine adenovirus inactivated vaccine and a pharmaceutically acceptable carrier.

[0021] The present invention discloses the following technical effects: This invention successfully isolated a type II canine adenovirus from dogs infected with kennel cough in Wuhan, Hubei Province. Through genetic evolutionary analysis and genome comparison, it was found that this strain is the latest circulating strain in China, and the viral titer can reach 10. 6.0 TCID 50 / mL.

[0022] This invention successfully constructed a type II canine adenovirus infection model using this strain. In experimental dogs, 1 mL of 10... 4.0 TCID 50 After being challenged with a dose of / mL, dogs developed typical clinical symptoms. This model can be used for the future development of canine adenovirus vaccines, the evaluation of immune protection, and the assessment of the efficacy of preventive and therapeutic drugs, providing good biological materials and theoretical basis for further prevention and control of canine adenovirus type 2.

[0023] This invention also utilizes this strain to develop an inactivated vaccine. Regarding safety, experimental dogs administered an overdose of the inactivated vaccine showed no local or systemic adverse reactions; their mental state, appetite, and body temperature remained normal; absorption at the injection site was good, with no nodules, redness, swelling, or necrosis, confirming its good safety profile. In terms of immunogenicity, the vaccine induced high levels of specific neutralizing antibodies in the body after immunization. Regarding cross-protection, immunized dogs not only showed complete protection against high-dose challenge with the homologous virulent CAV-2 strain, but also effectively resisted high-dose challenge with the highly pathogenic CAV-1 wild-type strain, protecting them from infectious hepatitis caused by CAV-1, demonstrating excellent cross-protection. Attached Figure Description

[0024] 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.

[0025] Figure 1 This is a PCR amplification diagram of the isolated type II canine adenovirus; where M is the DNA molecular standard quality standard-DL2000; 1 is the isolated type II canine adenovirus sample; 2 is the positive control; and 3 is the negative control. Figure 2 The genetic evolutionary tree of the isolated type II 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 Image of lung lesions in a 2-month-old experimental dog after viral challenge; Figure 5 A graph showing the changes in body temperature of 2-month-old experimental dogs after viral challenge; where 1-30 represent animal numbers. Figure 6 This is a graph showing the weight changes of 2-month-old experimental dogs after viral challenge; where 1-30 represent animal numbers. Figure 7 Clinical symptom scoring chart for 2-month-old experimental dogs after viral challenge; Figure 8 H&E staining images of lung pathological sections from 2-month-old experimental dogs after viral challenge; Figure 9 Immunohistochemical images of lung pathological sections from 2-month-old experimental dogs after viral challenge; Figure 10 Phenotypic images of the injection site in experimental dogs after receiving an overdose of inactivated vaccine; Figure 11 This is a phenotypic diagram of clinical symptoms in experimental dogs challenged with a secondary immune inactivated vaccine. Detailed Implementation

[0026] 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.

[0027] 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.

[0028] 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.

[0029] 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.

[0030] 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.

[0031] 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.

[0032] Example 1: Isolation and Identification of Type II Canine Adenovirus 1. Virus isolation Liver and spleen were collected from dogs with kennel cough 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 at 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 II canine adenovirus solution.

[0033] 2. Identification of the virus Take 200 μL of type II 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.

[0034] The primers used for PCR are: PCR CAV-F: 5'-CGCGCTGAACATTACTACCTTGTC- 3' (SEQ ID NO. 1); PCR CAV-R: 5'-CCTAGAGCACTTCGTGTCCGCTT-3' (SEQ ID NO. 2).

[0035] 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 genome of the Kewangfu canine quadrivalent vaccine (purchased from Keqian Biotechnology Co., Ltd.) 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. The results are shown below. Figure 1 As shown, a specific target band of approximately 1030 bp was amplified, indicating that a type II canine adenovirus was successfully isolated.

[0036] 3. Amplification and evolutionary analysis of the target gene Design specific primers to amplify the viral fiber gene by PCR.

[0037] The primers used for PCR are: PCR CAV2-Fiber-F: 5'-CTTACCTAATTTTTGCTAAGACGTCTGGGTCC- 3' (SEQ IDNO.3); PCR CAV2-Fiber-R: 5'-CGCGTGTATGAAAAATAAAGCTTTTAAACTTA-3' (SEQ ID NO. 4).

[0038] 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, 56℃ 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 a specific target band of 1629 bp. The target band was 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 to obtain the fiber target gene sequence. A phylogenetic tree was constructed, and the results are shown below. Figure 2 As shown. This virus belongs to type II canine adenovirus.

[0039] 4. Preservation of viruses The highly virulent strain of type II canine adenovirus was named Canine Adenovirus Type 2 Aa05 strain 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 adenovirus2 and has the accession number CCTCC NO: V202605.

[0040] 5. Culture and purification of type II canine adenovirus Type II 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.

[0041] 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 frozen and thawed three times and inoculated into 12-well MDCK cell culture plates. After complete cytopathic effect, the supernatant was collected, and the plates were frozen and thawed three times. The viral load of the clones was then measured. 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.

[0042] 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 .

[0043] The indirect immunofluorescence method is as follows: (1) Fixed Discard the culture medium, wash three times with 200 μL PBS, add 100 μL of pre-cooled methanol at 4 °C to each well, and fix at -15 °C for 10 min.

[0044] (2) Closed Discard the methanol, add 200 μL of PBS and wash 3 times. Add 100 μL of PBS containing 5% BSA to each well and incubate at 37°C for 1 hour.

[0045] (3) Add primary antibody Discard the blocking solution, wash three times with 200 μL PBS, add 100 μL of mouse monoclonal antibody (5E5) (diluted 1:2000 with PBS) to each well, and incubate at 37°C for 1 h.

[0046] (4) Add secondary antibody Discard the primary antibody, wash three times with 200 μL PBS, add fluorescently labeled goat anti-mouse IgG secondary antibody (diluted 1:2000 with PBS), and incubate at 37°C for 1 h.

[0047] (5) Observation and judgment Discard the fluorescent antibody, wash three times with 200 μL PBS, add 100 μL PBS to each well, and observe under a fluorescence microscope. Virus-infected cells show specific fluorescence and 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.

[0048] The TCID of the virus was calculated using the Reed-Muench formula. 50 10 6.0 TCID 50 / mL.

[0049] Example 2: Determination of type II canine adenovirus challenge dose 1. Grouping and virus attack Take a virus concentration of 10 6.0 TCID 50 The stock solution of canine adenovirus type 2 Aa05 strain was thawed at 4°C, vortexed, and serially diluted 10-fold with sterile PBS to 10⁻¹⁰ mL. 5.0 TCID 50 / mL, 10 4.0 TCID 50 / mL, 10 3.0 TCID 50 / mL and 10 2.0 TCID 50 Thirty 2-month-old Beagles, all 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 6 groups of 5 each. After anesthesia, the viral concentration in the nasal instillation for groups 1-5 was 10 mL. 6.0 TCID 50 / mL, 10 5.0 TCID 50 / mL, 10 4.0 TCID 50 / mL, 10 3.0 TCID 50 / mL and 10 2.0 TCID 50 / mL of virus solution, 1mL / dog. Group 6, anesthetized in the same manner, administered 1 mL of PBS intranasally as a blank control group. From -2 to 14 days post-challenge (day 1 of challenge as 0, negative numbers as before challenge), the experimental dogs' body temperature and weight were measured daily, and the disease progression was observed. Clinical symptom scores were recorded. At the end of the challenge period, the dogs were euthanized and necropsy performed. Lungs were harvested for pathological sections for 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 five aspects: coughing, vomiting, breathing pattern, eye and nasal discharge, and mental state and appetite. The cough scores were: normal (0 points), intermittent mild cough (1 point), and persistent severe cough (2 points). Vomiting was scored as follows: normal (0 points), intermittent mild vomiting (1 point), and persistent severe vomiting (2 points). The breathing pattern was scored as follows: normal (0 points), heavy breathing, mild dyspnea (1 point), and abdominal breathing (2 points). The scores for nasal secretions were: normal (0 points), serous nasal secretions (1 point), and purulent nasal secretions (2 points). The scores for mental and appetite aspects were: normal (0 points), depressed, decreased appetite (1 point), lethargic, and completely unable to eat (2 points).

[0050] 2. Results 2.1 Clinical symptoms Table 1 Results of virus challenge experiment on 2-month-old experimental dogs From Table 1 and Figures 3-7 It can be seen that, compared with the blank control group, 10 6.0 TCID 50 / mL challenge group dogs and 10 5.0 TCID 50 / mL Significant clinical symptoms began to appear on the second day after challenge, including fever, lethargy, loss of appetite, purulent nasal discharge, persistent severe cough, persistent severe vomiting, and abdominal breathing. Two dogs in each group died, and the remaining dogs continued to show symptoms for 9-10 days. Autopsy revealed enlarged lungs with large, diffuse areas of consolidation. The surviving dogs experienced a weight loss followed by a recovery during the challenge period; 10 4.0 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, purulent nasal discharge, persistent severe cough, persistent severe vomiting, and difficulty breathing. One dog died. All dogs continued to show symptoms until the end of the challenge period. Autopsy revealed mild lung swelling and small, localized areas of consolidation. The surviving dogs lost weight during the challenge period and then regained it. 3.0 TCID 50 In the / mL challenge group, only two dogs developed obvious clinical symptoms on the second day after challenge, including lethargy, loss of appetite, purulent nasal discharge, intermittent mild cough, and intermittent mild vomiting. No vomiting or respiratory distress occurred, and no deaths occurred. Symptoms persisted for up to 10 days. During the challenge period, the affected dogs experienced a weight loss followed by a recovery, while the remaining dogs showed a steady weight gain. 2.0 TCID 50 In the / mL challenge group, only one dog developed mild clinical symptoms on the second day after challenge, including fever, intermittent mild cough, and purulent nasal discharge. No vomiting or respiratory distress occurred, and no dogs died. All dogs showed an increasing weight trend during the challenge period.

[0051] 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 then 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℃.

[0052] 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, and then rinsed; stained with 0.5% eosin for 1 min, rinsed with distilled water, and then dehydrated sequentially in 80%, 90%, 95%, and 100% ethanol; immersed in xylene twice 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.

[0053] Depend on Figure 8 It can be seen that, compared with the blank control, 10 6.0 TCID 50 / mL challenge group, 10 5.0 TCID 50 / mL challenge group and 10 4.0 TCID 50 In the group challenged with / mL virus, the lung parenchyma showed significant destruction of bronchial structure, unclear epithelial cell structure, and the lumen filled with inflammatory cells; lung tissue consolidation was observed, alveolar wall structure was unclear, accompanied by mild fibrosis and extensive inflammatory cell infiltration, as well as widespread pulmonary edema. Powdery, frothy fluid was visible in the alveolar cavities. Meanwhile, in the 10 mL challenge group, the lung parenchyma showed significant bronchial structure destruction, unclear epithelial cell structure, and the lumen filled with inflammatory cells. 3.0 TCID 50 / mL challenge group and 10 2.0 TCID 50 The lung tissue surface of the / mL challenge group showed loose connective tissue with no obvious thickening; only a small area of ​​mild thickening of the alveolar walls was observed, and a small amount of inflammatory cell infiltration was visible in the alveolar walls, with no other obvious abnormalities.

[0054] 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 and alcohol, and turn blue with tap water; then immerse the sections in 70% alcohol, 85% alcohol, 95% alcohol, 100% alcohol I, and 100% alcohol II in a gradient dehydration process, with each immersion time being about 2-3 minutes; after dehydration, the sections need to be transferred to xylene I for 5-10 minutes; then transferred to xylene II for 5-10 minutes, and finally mounted with neutral resin for observation.

[0055] Depend on Figure 9 It can be seen that, compared with the blank control, 10 6.0 TCID 50 / mL challenge group, 10 5.0 TCID 50 / mL challenge group and 10 4.0 TCID 50 Lung tissue sections from dogs in the / mL challenge group showed abundant antigen-positive expression, manifested as numerous dark brown and yellowish-brown clumps of signal, mainly concentrated at the edges of severely lesion areas and within the nuclei and cytoplasm of alveolar epithelial cells, bronchial epithelial cells, and infiltrated inflammatory cells. Extensive signs of cell necrosis, including nuclear pyknosis and lysis, were observed in the core lesion areas with strong antigen positivity. Meanwhile, 10... 3.0 TCID 50 / mL challenge group and 10 2.0 TCID 50 Lung tissue sections from the / mL challenge group showed few positive signals, and the cell structure was not significantly abnormal.

[0056] Therefore, the challenge model was determined to be: 5 two-month-old experimental dogs were challenged with viral droplets via nasal administration after anesthesia, with a viral concentration of 10. 4.0 TCID 50 At least 4 / 5 of patients develop the disease after 1 mL of type II canine adenovirus ( / mL).

[0057] Example 3: Preparation and Safety Assessment of Type II Canine Adenovirus Inactivated Vaccine 1. Preparation of inactivated vaccines 1.1 Adherent MDCK cells were passaged, and canine adenovirus type II virus solution was added to the cell suspension simultaneously to make the multiplicity of infection (MOI) 1. The cells were cultured in square flasks at 37°C and the virus was harvested after 72 hours of sealed culture. 1.2 Repeat freeze-thaw cycles 3 times, centrifuge at 12000 r / min for 10 min, remove cell debris, collect the supernatant to determine the virus titer, and adjust the virus titer to 10. 6.5 TCID 50 / mL; 1.3 Virus inactivation: Add 1 μL of β-propiolactone to each 1 ml of virus solution, i.e., the final concentration of β-propiolactone is 0.1%; place on a rotary shaker at 4℃ for 48 h; 1.4 Hydrolysis of β-propiolactone: The prepared antigen was placed in a water bath at 37°C for 2 hours for hydrolysis; 1.5 Add adjuvant (MONTANIDE™ GEL02) slowly at a ratio of antigen:adjuvant = 9:1, and place on a rotating shaker at 4°C for 24 hours to obtain type II canine adenovirus inactivated vaccine.

[0058] 2. Safety evaluation of inactivated vaccines Ten 2-month-old beagle puppies were randomly divided into two groups of five each. In the first group, each puppy received a subcutaneous injection of 4 mL (4 doses) of the type II canine adenovirus inactivated vaccine prepared in step 1 into the neck. In the second group, an equal volume of physiological saline was injected subcutaneously into the neck. The puppies were observed for pain at the time of injection. For seven consecutive days after injection, their appetite, mental state, and body temperature were observed daily, as well as the appearance of obvious swelling or itching at the injection site. At the end of the observation period, all puppies were euthanized, and the injection sites were dissected to assess for adverse reactions such as nodules, redness, swelling, or necrosis.

[0059] The results are shown in Table 2 and Figure 10 As shown, all experimental dogs exhibited normal appetite and mental state, did not struggle violently during vaccination, and showed no adverse reactions such as nodules, redness, swelling, or necrosis at the injection site. Some experimental dogs experienced a slight increase in body temperature on the first day after vaccination, but all remained within the normal range (38℃~39.5℃) and did not exceed the basal body temperature by more than 1℃. These temperatures returned to normal on the second and third days. All experimental dogs in the non-immunized control group had normal body temperatures. This indicates that the CAV inactivated vaccine prepared using the canine adenovirus type 2 Aa05 strain of this invention has good safety.

[0060] Table 2 Safety test results of experimental dogs Example 4: Immunogenicity and Cross-protection Validation of Type II Canine Adenovirus Inactivated Vaccine 1. Neutralizing antibody assay method (1) Serum dilution: Take a 96-well cell culture plate and add 0.05 ml of DMEM culture medium to each well using a multichannel pipette. Add 0.05 ml of serum to the first column and perform serial dilutions from the first column to the desired dilution ratio. Discard the 0.05 ml liquid in the last column (in sequence, 1:2, 1:4, 1:8, 1:16...).

[0061] (2) Virus dilution: Dilute the Aa05 strain virus solution with DMEM medium to a virus concentration of 100 TCID. 50 / 0.05ml.

[0062] (3) Virus neutralization Add 100 TCID to each well of the diluted 96-well plate. 50 0.05 ml of virus solution was placed in an incubator at 37°C for 60 min.

[0063] (4) Virus resurgence In a 96-well cell culture plate, four replicate infection wells were placed in each of columns 1 to 5, and the virus was diluted to 0.1, 1, 10, 100, and 1000 TCID, respectively. 50 For each dilution, prepare 4 wells and add 0.05 ml to each well.

[0064] (5) Cell infection Select MDCK cells in good growth condition, digest them with 0.25% trypsin, and then resuspend them in DMEM medium containing an appropriate amount of 2% new bovine serum to achieve a cell concentration of 5 × 10⁶ cells / year. 5 Cells / ml. Add 0.1 ml of cell suspension to each well. Incubate the cell culture plate at 37°C in an incubator containing 5% CO2 for 4 days.

[0065] (6) The antibody titer was determined by indirect immunofluorescence assay. The formula for calculating antibody titer was derived with reference to the Reed-Muench formula.

[0066] The calculation formula is: Distance ratio = (50% or higher - 50%) / (50% or higher - less than 50%) IgPD 50 = Logarithm of serum dilution with a protection rate of 50% or higher + distance ratio × logarithm of dilution factor.

[0067] Virus regression control at 0.1 TCID 50 It should have no specific fluorescence, and 100 TCID 50 Specific fluorescence is required; otherwise, the test cannot be performed.

[0068] 2. Grouping, immunization, and challenge Thirty-six healthy, susceptible dogs aged 6-8 weeks were divided into 12 groups of 3 dogs each. Groups 1-6 received 1 mL of Aa05 inactivated vaccine subcutaneously, while groups 7-12 served as the control group, each receiving an equal volume of DMEM culture medium subcutaneously. A second immunization was administered 21 days later using the same dose and method. Blood was collected from all dogs 21 days after the second immunization, serum was separated, and serum CAV neutralizing antibodies were measured. 21 days after the second immunization, each group of dogs was challenged with the highest amplifiable dose of each strain using CAV-1 wild-type virus (isolated from dogs with infectious hepatitis in Wuhan, Hubei) and CAV-2 wild-type virus (Aa05 strain isolated from Wuhan, Hubei; DY37 strain isolated from Chuzhou, Anhui; AS86 strain isolated from Anshan, Liaoning, from puppies with kennel cough and dogs with infectious bronchitis and laryngotracheitis). The group challenge details are shown in Tables 3 and 4.

[0069] Table 3 Grouping of CAV-1 wild-type virulent strains for challenge Table 4 Grouping of CAV-2 wild-type virulent strains for challenge The results are shown in Tables 5 and 6. Figure 11 As shown: 21 days after the second immunization, the type II adenovirus inactivated vaccine stably induced high levels of adenovirus neutralizing antibodies in dogs. After high-dose challenge with the CAV-1 wild-type virulent strain, dogs in the control group exhibited symptoms such as persistent high fever, lethargy, loss of appetite, purulent eye discharge, elevated plasma aspartate aminotransferase or alanine aminotransferase levels, and death. The immunized group showed no symptoms and achieved complete protection. After high-dose challenge with the CAV-2 wild-type virulent strain, dogs in the control group exhibited symptoms such as persistent high fever, difficulty breathing, coughing, eye and nasal discharge, vomiting, and death. The immunized group showed no symptoms and achieved complete protection. This indicates that the type II canine adenovirus inactivated vaccine prepared in this invention has good immunogenicity and cross-protection.

[0070] Table 5. Antibody levels and challenge results of each challenge group for the highly virulent wild-type CAV-1 strain. Note: "T" indicates a body temperature exceeding 39.5℃ for ≥2 days; "V" indicates lethargy and loss of appetite; "Y" indicates purulent eye discharge; "A" indicates elevated plasma aspartate aminotransferase (AST) or alanine aminotransferase (ALT) levels (AST > 55 U / L; ALT > 140 U / L); "D" indicates death; "-" indicates no obvious clinical symptoms. Table 6. Antibody levels and challenge results of each challenge group for highly virulent wild-type CAV-2 strain. Note: "T" indicates a body temperature exceeding 39.5℃ for ≥2 days; "B" indicates difficulty breathing; "C" indicates cough; "Y" indicates nasal and ocular discharge; "M" indicates vomiting; "D" indicates death; "-" indicates no obvious clinical symptoms.

[0071] In summary, the canine adenovirus type 2 Aa05 strain isolated in this invention is a highly virulent type II canine adenovirus strain, with a purified titer reaching 10. 6.0 TCID 50 / mL, with stable pathogenicity, excellent immunogenicity, and strong cross-protection. At 10 4.0 TCID 50 Intranasal administration of 1 mL or 1 mL / dog can induce disease in 100% of experimental dogs, replicating typical clinical and pathological features. This can serve as a standardized strain for constructing a robust infection model, applicable to pathological research, drug screening, and vaccine validation. The inactivated vaccine prepared from this strain demonstrates good safety, with a neutralizing antibody titer ≥1:512 after a second immunization, providing complete protection against challenges from multiple type I and type II canine adenovirus wild-type strains. This strain can also be used for epidemiological surveillance, development of diagnostic kits, and provides materials for innovation in veterinary biological products. It has significant application value and broad prospects in canine disease prevention and control, biological product development, and basic research.

[0072] 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 virulent strain of type II canine adenovirus 2, characterized in that, The virulent strain of type II canine adenovirus was named canine adenovirus type 2 Aa05 strain and has been deposited at the China Center for Type Culture Collection on January 5, 2026, with accession number CCTCC NO: V202605.

2. The use of the virulent type II canine adenovirus strain described in claim 1 in constructing an animal model of type II canine adenovirus infection or in preparing an inactivated type II canine adenovirus vaccine.

3. A method for constructing an animal model of canine adenovirus type II infection, characterized in that, The method includes the step of challenging animals with the virulent strain of type II 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 3, characterized in that, The dose of the virus used for the attack was not less than 10. 3.0 TCID 50 / Only.

6. The use of the type II canine adenovirus animal infection model obtained by the construction method according to any one of claims 3-5 in screening canine adenovirus vaccines or drugs for the prevention and treatment of canine adenovirus.

7. The use of the type II canine adenovirus animal infection model obtained by the construction method according to any one of claims 3-5 in evaluating the efficacy of canine adenovirus vaccines or drugs for the prevention and treatment of canine adenovirus.

8. A type II canine adenovirus inactivated vaccine, characterized in that, The method for preparing the type II canine adenovirus inactivated vaccine includes the step of inactivating the virulent strain of type II canine adenovirus as described in claim 1 to prepare an inactivated vaccine.

9. The use of the type II canine adenovirus inactivated vaccine according to claim 8 in the preparation of a medicament for the prevention of canine adenovirus infection.

10. A drug for preventing canine adenovirus infection, characterized in that, The drug comprises the type II canine adenovirus inactivated vaccine of claim 8 and a pharmaceutically acceptable vector.

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