Cat infectious peritonitis virus isolate and application thereof

Abstract

The invention discloses a cat infectious peritonitis virus isolate and application thereof. The cat infectious peritonitis virus is separated from the peritoneal fluid of a 6-month-old female gradient silver cat, the preservation number of the cat infectious peritonitis virus is CGMCC (China General Microbiological Culture Collection Center) No.46420, and the name of the cat infectious peritonitis virus is SHAANXI-FIBV-II-2024. An animal model is successfully established by using the strain.

Description

Technical Field

[0001] The invention belongs to the field of prevention and control of feline infectious peritonitis and relates to the separation and identification of feline infectious peritonitis virus. Background Art

[0002] Feline infectious peritonitis (FIP) is a progressive, fatal infectious disease caused by the feline infectious peritonitis virus (FIPV). Wassamon Moyadee et al. analyzed 46 cats diagnosed with FIP and noted that the clinical manifestations of FIP, by systemic stratification, include abdominal distension, pallor or anemia, dyspnea, behavioral changes, and uveitis. Autopsy revealed that FIP ​​is typically characterized by fibrinoid and granulomatous serositis, protein-rich serous effusions, and pyogranulomatous lesions in multiple organs.

[0003] FIPV is a type of feline coronavirus (FCoV), belonging to the Coronaviridae family and the genus Alphacoronavirus. It is an enveloped, non-segmented, single-stranded, positive-sense RNA virus. The clinical signs of FIP in FIPV-infected cats can be categorized as exudative (wet or non-solid), non-exudative (dry or solid), or a mixture of the two, based on the presence of protein-rich effusions in the abdominal and pleural cavities. There is a clear distinction between the exudative and non-exudative forms of FIP. The exudative form is characterized by severe pleural and peritoneal effusions, abdominal masses on palpation suggestive of organ-omental adhesions, mesenteric lymphadenopathy, jaundice in the final stage, and, in male cats, scrotal swelling. The non-exudative form is characterized by granulomas, ocular lesions, and, in severe cases, central nervous system symptoms.

[0004] FCoV is divided into two different biotypes, FIPV and Feline Enteric coronavirus (FECV). Most cats can be infected with enteric coronavirus through the oronasal route, and once the virus mutates into FIPV, it can proliferate in the tonsil glands and the mucosal layer of the gastrointestinal tract. When the virus infects macrophages and begins to proliferate in macrophages, it enters the systemic infection stage, and the virus spreads more widely through macrophages to cause viremia. The main pathological feature of exudative FIP is that the virus will form purulent granulomas around the small veins of various target organs after infection. These inflammatory lesions begin on the surface of the organ and spread to the organ parenchyma, which can cause exudative FIP when cellular immunity is insufficient. When the cellular immune response is moderate, non-exudative FIP occurs. In the exudative state, purulent granulomatous vasculitis lesions occur on the serous surface of the body cavity, resulting in exudate in the body cavity. The lesions of non-exudative FIP are typical granulomas, and granulomatous lesions of non-exudative FIP can also be found in autopsy cases of exudative FIP.

[0005] With the prevalence of FIPV, FIP has become more and more common in recent years. For example, in the “2021-2022 Qingdao City Feline Infectious Peritonitis Epidemiological Analysis”, 149 suspected FIP cases of ascites samples were detected by fluorescent quantitative RT-PCR, and 125 cases were found to be FIPV positive samples; between 2022-2023, 543 cat anal swabs were randomly collected from pet hospitals in Changchun, Beijing, Hohhot and Baotou for FCoV identification, and the total positive rate was 33.7%. According to statistics between 2008-2023, the FIPV nucleic acid positive rate was 33.7%, and the overall prevalence rate of FIP was 2%.

[0006] The mortality rate of cats infected with FIPV is as high as 95%, basically all due to ineffective treatment and death, and the treatment time of affected cats is long and the cost is high, which has a great impact on the pet industry. China Patent CN116042538A discloses a strain of isolated type I feline coronavirus, but when constructing an attack model using the virus and using it to evaluate the antiviral efficacy of drugs, there are few reference signs (the signs are limited to body temperature) and the disease course is short (from infection to death for about 20 days), which affects the evaluation of drug efficacy (if the cat group has few typical symptoms and a short disease course, the test design will deviate from the real clinical scene and cannot represent the typical patient population, and the results will be difficult to generalize). SUMMARY

[0007] The purpose of the present application is to provide a feline infectious peritonitis virus isolate and its application.

[0008] To achieve the above-mentioned purpose, the following technical solutions are adopted in the present application: In a first aspect, a feline infectious peritonitis virus (FIPV) isolate is provided. The isolate is named SHAANXI-FIPV-II-2024 (strain name) and has a deposit number of CGMCC No. 46420.

[0009] Preferably, sequencing analysis shows that the serotype of the isolate is feline coronavirus type II (FCoV-Ⅱ).

[0010] In a second aspect, a method for constructing an animal model is provided, comprising the following steps: The virus liquid of the above-mentioned isolate is prepared by blind transmission, and then the virus liquid is used to infect healthy animals. The symptoms (including changes in physical signs) and / or pathological indicators (including tissue pathological changes) of the animals are detected within a certain period of time after infection.

[0011] Preferably, the animal is a cat (eg, 3 to 9 months old).

[0012] Preferably, the infection is carried out by injecting the virus.

[0013] Preferably, the isolates are expressed as 1×10 5 TCID 50 / pcs~ 4×10 6 TCID 50 The virus was injected intraperitoneally at a dose of 100 mg / hice.

[0014] Preferably, the symptoms specifically include elevated body temperature and increased abdominal circumference starting 3 to 4 weeks after infection, and loss of appetite and jaundice at the end of the infection (one week before death, such as the fifth week).

[0015] A third aspect provides the use of the above-mentioned isolate in constructing an animal model or preparing a reagent (such as a modeling kit) for constructing an animal model.

[0016] Preferably, the animal model is a feline infectious peritonitis (FIP) model or a feline infectious peritonitis virus challenge model.

[0017] The beneficial effects of the present invention are embodied in: The present invention isolated a new FIPV virus from FIP-affected cats (its genetic evolution was confirmed by sequencing). The strain of this virus is named SHAANXI-FIPV-II-2024, which can be used to establish FIPV infection models and other related animal models. The resulting model has many typical symptoms of FIP (with changes in multiple physical signs such as fever and jaundice on the body surface) and a long course of disease (no animal died within five weeks after infection). BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1The results of PCR test of virus extracted from ascites (electrophoresis): Lane 1 is the negative control (referring to water as the template); Lane 2 is the ascites PCR product; Lane 3 is the DNA molecular weight standard.

[0019] Figure 2 Figure 3 shows the CPE produced by three generations of blindly propagated viruses in CRFK cells: A is uninfected CRFK cells; B is F1 generation infected CRFK cells; C is F2 generation infected CRFK cells; D is F3 generation infected CRFK cells.

[0020] Figure 3 This is the PCR test result of cell culture of the third generation virus (electrophoresis): Lane 1 is the DNA molecular weight standard; Lane 2 is the F3 generation virus PCR product; Lane 3 is the negative control (referring to water as the template).

[0021] Figure 4A The results of genome alignment for isolated FIPV (genomes ranked high in similarity were selected).

[0022] Figure 4B This is the genetic evolutionary tree of the S gene biotype of SHAANXI-FIPV-II-2024.

[0023] Figure 5 These are the gross autopsy changes of cats after infection: A is jaundice on the body surface (note: it can also be observed until the end of the infection); B is fluid accumulation in the abdominal cavity; C is a large number of nodules diffusely distributed in the intestinal serosa and enlarged mesenteric lymph nodes (indicated by arrows); D is a nodule attached to the surface of the spleen (indicated by arrows); E is a jelly-like nodule in the kidney capsule on both sides (indicated by arrows); F is a yellow needle-like nodule attached to the entire surface of the liver.

[0024] Figure 6 Pathological tissue morphological observations of cats after infection: A shows death of lymphocytes in the mesenteric lymph nodes (indicated by the arrow); B shows granulomas in the subcapsular layer of the mesenteric lymph nodes; C shows granulomas on the surface of the liver; D shows necrosis of hepatocytes (indicated by the arrow); E shows red blood cells in the renal tubules (indicated by the arrow), dilation of the tubular lumen, and vesicular degeneration of the renal tubular epithelial cells; F shows partial atrophy of the renal tubular epithelial cells; G shows obvious thickening of the splenic capsule with a large amount of fibrin exudate (indicated by the arrow); H shows organization of exudate from the surface of the spleen and the formation of granulomas. DETAILED DESCRIPTION

[0025] The present invention will be further described in detail below with reference to the accompanying drawings and examples. The examples are only used to explain the present invention but not to limit the scope of protection of the present invention.

[0026] (1) Isolation and identification of feline infectious peritonitis virus A 6-month-old female silver tabby cat was admitted to a pet hospital in Yangling District, Shaanxi Province. Clinical examination revealed fever, loss of appetite, and abdominal effusion. Nucleic acid testing revealed only positive results for feline infectious peritonitis virus (FIPV). The cat died three days after treatment. A necropsy was performed, and the virus was isolated and identified from the peritoneal fluid. Details are as follows.

[0027] 1. Materials and Methods 1.1 Cells and main reagents CRFK cells (i.e., cat kidney cells) are maintained in our laboratory and are also commercially available. Fetal bovine serum (FBS) was purchased from Zhejiang Tianhang Biotechnology Co., Ltd.; DMEM cell culture medium was purchased from Gibco; reverse transcriptase and high-fidelity enzymes were purchased from Novigene Biotechnology (Beijing) Co., Ltd.; and the TA cloning kit was purchased from TAKRA. Histopathological staining was performed using conventional reagents.

[0028] 1.2 Gross autopsy and sampling The abdominal cavity was opened and the pathological conditions of the abdominal organs were observed; ascites was extracted and placed in a sterile centrifuge tube; the end of the large intestine and the pylorus were ligated; a section of the duodenum, jejunum, ileum, cecum, colon, and rectum was cut and placed in tissue fixative; the mesenteric lymph nodes, liver, kidneys, and spleen were removed and photographed, placed in tissue fixative, and recorded; the cat ascites samples collected in sterile centrifuge tubes were frozen at -80°C.

[0029] 1.3 Histopathological examination Small intestine, liver, and spleen tissue blocks of 1cm×1cm×0.3cm in size were fixed in 4% paraformaldehyde for 48h. The embedding surface was selected and cut into small pieces of 0.3~0.7cm in length with a blade. The pieces were placed in an embedding box and rinsed with running water for 12h. The specimens were dehydrated in different levels of ethanol in turn and transparentized in xylene. The transparent specimen blocks were placed in a wax immersion container for wax immersion and then embedded in paraffin. The temperature of the constant temperature spreader and the baking machine was adjusted to about 37℃ and kept in a constant temperature state. The trimmed paraffin-embedded blocks were placed on the microtome for sectioning. The wax strips were flattened onto the slides using the steam of hot water. The slides were baked on the baking machine for HE staining, sealed, and observed and photographed under an optical microscope.

[0030] 1.4 PCR detection Design of primers specific for the FIPV S gene: FIPV-S-Forward (ie SEQ.ID.NO.1): 5'-TACGACATAGCAGACTTGGTGTG-3' FIPV-S-Reverse (ie SEQ.ID.NO.2): 5'-GTCTGAGACACAAATGCATTAAGTGC-3' Ascites (1 mL) or viral RNA was extracted using the TRIzol method, reverse transcribed, and then PCR was performed using a high-fidelity enzyme and analyzed by electrophoresis.

[0031] 1.5 Virus isolation, culture and identification When CRFK cells reached 80% confluency, they were washed three times and added with serum-free 1× double-antibody DMEM. Ascites fluid was filtered through a 0.22 μm filter and added at a 5% ratio. The cells were adsorbed in a 37°C CO2 incubator for two hours. Maintenance medium (DMEM containing 2% FBS) was then added and the cells were incubated in a CO2 incubator for 24 hours. Observation and recording were performed during this period, and the virus was harvested after 48 hours. To harvest the virus, the cells were frozen at -80°C, frozen and thawed twice, and centrifuged at 10,000 rpm. The supernatant (viral fluid) was aliquoted into 1.5 mL EP tubes and labeled as the F1 generation virus isolated from the ascites. Several blind passages were performed, and the resulting third generation (F3 generation) virus was analyzed by PCR. The target fragment amplified with a high-fidelity enzyme was cloned using TA cloning. Positive colonies were identified and sent to a company (Sangon) for sequencing. Sequence analysis and homology comparison were performed using MEGA.

[0032] 1.6 Determination of virus titer After CRFK cells were digested and resuspended, the cells were counted and the cell concentration was adjusted to 1×10 5 Cells were plated in 96-well cell culture plates (100 μL / well) and cultured in a 37°C, 5% CO2 incubator. After the cells were fully plated, the virus was diluted 10-fold to 10 -10 8 replicates were set for each dilution, and uninfected CRFK cells were used as the control group. The cells were observed for 5 consecutive days, and the number of lesion wells at each dilution was recorded. The virus titer (TCID) was calculated using the Reed-Muench method. 50 ).

[0033] 1.7 Strain Storage The confirmed virus was divided into 1.5 mL centrifuge tubes and frozen at 1 mL / tube in a -80℃ refrigerator.

[0034] 2. Results 2.1 Autopsy, histopathological examination, and PCR testing of ascites Gross autopsy revealed ascites, which was brown-red in color; a large number of beige nodules ranging in size from a needle tip to an elm seed were diffusely distributed throughout the intestinal serosa, mesentery, and the membrane surface of both lungs; in addition, similar nodules were scattered on the membrane surface of the kidneys and spleen.

[0035] Light microscopy revealed purulent granulomatous vasculitis in the intestines, liver, and spleen.

[0036] The specific primers of FIPV S gene were designed, and the product of reverse transcription of RNA extracted from ascites was used as a template for PCR. The results of electrophoresis of the PCR product are shown in Figure 1 , and the target fragment of 536 bp was amplified, which was identified as FIPV positive.

[0037] 2.2 Virus isolation and identification The collected ascites was inoculated into CRFK cells, and the blind passage was performed for three generations. The results are shown in Figure 2 , and the virus of the first, second and third generations appeared cytopathic effect (CPE) 24 hours after inoculation. The cell necrosis, deformation and detachment from the bottle wall were observed under a microscope. The virus was passed to the tenth generation on CRFK cells, and the CPE phenomenon still appeared in the cells. The virus was detected and not lost, indicating that the virus could stably proliferate when blind passage in CRFK cells.

[0038] 2.3 PCR detection and genetic evolution analysis of the virus The specific primers of FIPV S gene were designed, and the product of reverse transcription of RNA extracted from ascites was used as a template for PCR. The results of electrophoresis of the PCR product are shown in Figure 3 , and the target fragment of 536 bp was amplified, which was identified as FIPV positive.

[0039] The amplified target fragment (S gene fragment) was cloned by TA, and the sequence of the identified positive colonies was compared. The comparison results are shown in Figure 4A , and the similarity with the reference strain DF-2 is 99.66%, which confirms that the cell culture (i.e. the virus liquid prepared after blind passage) contains a new feline infectious peritonitis virus isolated from ascites samples, and is named SHAANXI-FIPV-Ⅱ-2024. According to the results of the genetic evolution tree based on S gene, the isolated strain SHAANXI-FIPV-Ⅱ-2024 is FCoV-Ⅱ serotype, which is in the same branch as DF-2, WSU-79-1146 and other strains Figure 4B .

[0040] The full length of S gene on the genome of the strain SHAANXI-FIPV-Ⅱ-2024 is as follows (i.e. SEQ.ID.NO.3): The strain SHAANXI-FIPV-Ⅱ-2024 was deposited in the General Microbiology Center of the China Culture Collection Administration (CGMCC for short, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing) on ​​May 15, 2025, with the deposit number CGMCC No. 46420 and the classification name as feline infectious peritonitis virus.

[0041] 2.4 Virus titer determination When the virus was blindly propagated to the third generation, the virus titer was detected and the TCID of the virus (specifically the strain SHAANXI-FIPV-Ⅱ-2024) was calculated. 50 is 10 -7.875 / mL.

[0042] (II) Establishment of pathological model 1. Materials and Methods 1.1 Animal exposure Three 9-month-old cats were selected and tested negative for FIPV, feline panleukopenia, feline herpes virus, feline calicivirus, and feline coronavirus nucleic acid. The virus (i.e., the virus liquid of strain SHAANXI-FIPV-Ⅱ-2024) stored at -80°C was placed in a 4°C refrigerator to thaw at low temperature, then taken out and placed at room temperature for about 30 minutes, and then the virus was tested according to 4×10 6 TCID 50 / cat was aspirated into a sterile syringe and the virus was infected into the cats by intraperitoneal injection.

[0043] 1.2 Symptom examination and autopsy Daily temperature and abdominal circumference measurements were taken, along with observations of changes in food intake and body appearance, and mental status scores were performed. Blood samples were drawn weekly for routine blood tests and biochemical analysis until death, with the time of death recorded. Following death, the animals were autopsied, lesion findings recorded, and lesion sites were sampled and fixed.

[0044] 1.3 Histopathological examination Tissues were fixed in 4% paraformaldehyde for four days. Extruded, uneven, or unneeded sections were trimmed. The trimmed tissue blocks were rinsed for 12 hours. The specimens were dehydrated in various levels of ethanol, cleared in xylene, and then embedded in paraffin solution for fixation, sectioning, and drying. Four slices were cut from each tissue block, two of which were stained with hematoxylin and eosin for histopathological examination.

[0045] 2. Results 2.1 Construction of animal model After intraperitoneal injection of the virus (strain SHAANXI-FIPV-II-2024) into healthy animals (negative for FIPV, feline panleukopenia, feline herpesvirus, feline calicivirus, or feline coronavirus), the cats developed fever in the third week (specifically, day 21 after infection). Their mental state became increasingly depressed, and their abdominal circumference increased. After developing jaundice, they died in the fifth week (specifically, between days 37 and 40 after infection). Results showed that all three infected cats were successfully modeled, with a 100% success rate.

[0046] 2.2 Autopsy After the cat died, a gross autopsy was performed, which showed severe icterus on the skin, a large amount of fluid in the abdominal cavity, and diffuse yellow nodules in the mesentery and peritoneal organs (see Figure 5 ).

[0047] 2.3 Histopathological examination After the cat died, a histopathological examination was performed, and the results were as follows: Figure 6 shown.

[0048] In summary, the present invention isolated and identified the virus from cats diagnosed with FIP by gross autopsy, histopathological examination, and PCR detection, and successfully isolated a FIPV strain named SHAANXI-FIPV-II-2024. The TCID 50 is 10 -7.875 / mL. Homology analysis and genetic evolution analysis were performed on part of the S gene sequence of the isolated FIPV strain SHAANXI-FIPV-II-2024. The results showed that SHAANXI-FIPV-II-2024 was closely related to FCoV-Ⅱ serotypes such as the American isolate WSU-79-1146 and the Hungarian isolate DF-2, but was distantly related to the isolates of the FCoV-Ⅰ serotype, that is, SHAANXI-FIPV-II-2024 belonged to the FCoV-Ⅱ serotype and FIPV biotype. An animal model was constructed using SHAANXI-FIPV-II-2024. Gross anatomy revealed typical granulomatous nodules distributed on the serous or capsular surfaces of the intestines, mesentery, lungs, kidneys, etc., and a large amount of peritoneal fluid. Therefore, the strain SHAANXI-FIPV-II-2024 isolated by the present invention can provide basic data references for the clinical prevention and control of feline infectious peritonitis, and provide stable and reliable guarantees for the research and development of related drugs and biological products.

Claims

1. A feline infectious peritonitis virus isolate, characterized in that: The deposit number of this isolate is CGMCC No.46420.

2. A feline infectious peritonitis virus isolate according to claim 1, characterized in that: The serotype of the isolate is feline coronavirus type II.

3. A method for constructing an animal model, characterized in that: The construction method includes the following steps: Animals are infected with the feline infectious peritonitis virus isolate as described in claim 1.

4. The method for constructing an animal model according to claim 3, characterized in that: The construction method further comprises the following steps: detecting symptoms and / or pathological indicators of the infected animals.

5. The method for constructing an animal model according to claim 3 or 4, characterized in that: The animal is a cat.

6. The method for constructing an animal model according to claim 3 or 4, characterized in that: The infection is carried out by injecting the virus.

7. The method for constructing an animal model according to claim 6, characterized in that: The isolates were expressed as 1×10 5 TCID 50 / pcs~ 4×10 6 TCID 50 The virus was injected intraperitoneally at a dose of 100 mg / hice.

8. The method for constructing an animal model according to claim 4, characterized in that: The symptoms include one or more of increased body temperature, increased abdominal circumference, loss of appetite, and jaundice on the body surface.

9. Application of the feline infectious peritonitis virus isolated strain as claimed in claim 1 in building an animal model or preparing a reagent for building an animal model.

10. The use according to claim 9, characterized in that: The animal model is a feline infectious peritonitis model or a feline infectious peritonitis virus challenge model.

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