CN115024278A - Construction method and application of CV-B1 infected rodent hand-foot-and-mouth disease model - Google Patents
Construction method and application of CV-B1 infected rodent hand-foot-and-mouth disease model Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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- A61K49/0004—Screening or testing of compounds for diagnosis of disorders, assessment of conditions, e.g. renal clearance, gastric emptying, testing for diabetes, allergy, rheuma, pancreas functions
- A61K49/0008—Screening agents using (non-human) animal models or transgenic animal models or chimeric hosts, e.g. Alzheimer disease animal model, transgenic model for heart failure
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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Abstract
The invention belongs to the technical field of animal model construction, and particularly provides a construction method and application of a CV-B1 infected rodent hand-foot-and-mouth disease model, wherein the construction method comprises the steps of infecting the rodent model with CV-B1 in a nasal cavity instillation mode, and establishing the CV-B1 infected rodent hand-foot-and-mouth disease model. The modeling method has the advantages of short time, simplicity, practicability, effectiveness, low mortality and stable model. Meanwhile, nasal drip is a real approach closer to human infection virus, better reflects the whole process of virus infection, and provides more optimized method and technical support for establishment of CV-B1 pathogen. According to the modeling method, after the syrian golden yellow hamster is infected by nasal drip, hand-foot-and-mouth disease symptoms similar to those of human beings appear, and multiple organ injuries such as meningitis, myocarditis and the like exist while virus replication conditions appear in various tissues and organs. Lays a foundation for the subsequent infection way and mechanism, the evaluation of pathophysiology drugs and the research and development evaluation of vaccines.
Description
Technical Field
The invention belongs to the technical field of animal model construction, and particularly relates to a construction method and application of a CV-B1 infected rodent hand-foot-and-mouth disease model.
Background
The hand-foot-and-mouth disease is a common viral infection disease caused by enteroviruses and causing self-healing herpes to appear in limbs, oral cavity, buttocks or genitals of a patient. At present, the hand-foot-and-mouth disease is mainly focused on research of EV-71, CV-A16, CV-A6 and CV-A10. However, in recent years, the occurrence of hand-foot-and-mouth disease and its complications caused by various global enteroviruses has been newly developed. The occurrence of hand-foot-and-mouth disease is often accompanied by diseases of nervous systems such as meningoencephalitis, brainstem encephalitis, spasm and convulsion; worldwide CVA6 infection rates are rising in uk, malaysia, spain, etc., and cases of respiratory distress syndrome with mixed EVD68 infection have occurred; CV-A10 is more susceptible in Asian young children and has a close relationship with demethylation; in addition, the infection rate of viruses such as CV-B1, CV-A2, CV-A4, CV-A9, CV-B3 and CV-B5 has also been increasing in recent years.
According to previous researches, CVA-16, EV-71, CV-A6 and CV-A10 have various animal models such as multi-line mice, transgenic mice and non-human primates, but the animal models of the rest pathogens are not well constructed and researched. CV-B1 establishes an intraperitoneal injection infected mouse myositis model, an pancreatitis model, an intraperitoneal injection infected guinea pig polymyositis model, an ear edge intravenous injection infected rabbit hepatitis model and the like. The CV-B1 animal model established above mostly adopts an intraperitoneal injection method to artificially infect animals, although the method can obtain relatively acute clinical symptom expression, the method has a certain difference with human infection virus approaches, a hand-foot-and-mouth disease animal model established by simulating human infection approaches is lacked, typical hand-foot-and-mouth herpes symptoms do not appear, and the hand-foot-and-mouth disease model caused by CV-B1 is not deeply researched. Therefore, the establishment of a hand-foot-and-mouth disease model simulating human infection paths is extremely necessary.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a construction method and application of a CV-B1 infected rodent hand-foot-and-mouth disease model.
In order to achieve the purpose of the invention, the invention adopts the technical scheme that:
the construction method of the CV-B1 infected rodent hand-foot-and-mouth disease model is provided, CV-B1 is infected to the rodent model in a nasal cavity instillation mode, and the CV-B1 infected rodent hand-foot-and-mouth disease model is established.
Further: the rodent is selected from 3-week-old Syrian golden hamster.
Further: CV-B1 virus titers were: 10 7.25 CCID 50 mL, 100 μ l of virus suspension was instilled through the nasal cavity each.
Provides the application of a CV-B1 infected rodent hand-foot-and-mouth disease model, and the model is used for screening or identifying drugs capable of preventing, relieving or treating CV-B1 infection.
Provides the application of a CV-B1 infected rodent hand-foot-and-mouth disease model, and the model is used for screening or identifying a vaccine capable of preventing CV-B1 infection.
The beneficial effects of the invention are as follows:
the invention establishes the golden hamster hand-foot-and-mouth disease model infected by nasal drip, and the modeling method has the advantages of short time, simplicity, practicability, effectiveness, low mortality and stable model. Meanwhile, nasal drip is a real approach closer to human infection virus, better reflects the whole process of virus infection, and provides more optimized method and technical support for establishment of CV-B1 pathogen. The modeling method has the advantages that similar hand-foot-and-mouth disease symptoms to those of human beings appear after the syrian golden hamster is infected by nasal drip, and multiple organ injuries such as meningitis, myocarditis and the like exist while virus replication conditions appear in various tissues and organs. Lays a foundation for the subsequent infection way and mechanism, the evaluation of pathophysiology drugs and the research and development evaluation of vaccines.
Drawings
FIG. 1 is a graph of the clinical symptoms of infection of the syrian golden hamsters with CV-B1 in example 2; wherein, A) gingival bleeding and B) herpes are conditions of the experimental group; C) body temperature monitoring, D) body weight monitoring as conditions of the experimental group and the control group;
FIG. 2 is a graph of detoxification of the experimental group of EXAMPLE 2 from Shigella chinensis mice infected with CV-B1; wherein, A) pharyngeal swab viral load; B) nasal swab viral load; C) fecal viral load;
FIG. 3 is a graph showing the monitoring of biochemical indicators of infection of the golden hamster CV-B1 in the experimental group and the control group of example 2; wherein, A) liver function; B) (ii) a myocardial zymogram;
FIG. 4 is a graph of the viral load of tissues infected with CV-B1 from the experimental group of EXAMPLE 2; wherein, A) the nervous system; B) the genitourinary system; C) a digestive system; D) a circulatory system and a respiratory system;
FIG. 5 is a graph of the pathological changes in herpes HE of the experimental group of EXAMPLE 2, i.e., golden hamster infected with CV-B1; wherein, A) herpes; B) a lip portion;
FIG. 6 is a graph showing pathological changes in tissue-organ HE caused by infection of Philippine nude mice with CV-B1 in the experimental group of example 2 and the control panel; wherein, A) smells the ball; a brain; C) a heart; D) the lung; E) a liver; F) intestinal tract.
Detailed Description
The following description of the embodiments of the present invention is provided to facilitate the understanding of the present invention by those skilled in the art, but it should be understood that the present invention is not limited to the scope of the embodiments, and it will be apparent to those skilled in the art that various changes may be made without departing from the spirit and scope of the invention as defined and defined in the appended claims, and all matters produced by the invention using the inventive concept are protected.
Example 1
The construction method of the CV-B1 infected rodent hand-foot-and-mouth disease model is provided, and the rodent model is infected in a nasal drip mode to establish the CV-B1 infected rodent hand-foot-and-mouth disease model.
According to earlier studies, rodents are susceptible to CV-B1, and the syrian golden yellow hamster has clear genetic background, short growth cycle, larger size than mice and easier observation of lesions of smaller tissues and organs, and is convenient to operate, so that the rodent is selected as the model animal of the application.
In this example, 3-week-old Syria golden yellow hamster was used as the experimental animal, and the female animals had a body weight of 60g to 80 g. Feeding in a barrier environment, and feeding with the same feed. Sterilizing the required food every day at high pressure and high temperature, and eating within 72 h; the drinking water is sterilized at high pressure and high temperature and is drunk within 24 hours.
Coxsackievirus group B type 1 (CV-B1) virus titers were: 10 7.25 CCID 50 mL, 100 μ l of virus suspension was instilled into each nasal cavity of experimental group, 100 μ l of physiological saline was instilled into each nasal cavity of control group, and normal feeding was isolated from experimental group.
Example 2
Evaluation and analysis were performed on the CV-B1-infected rodent hand-foot-and-mouth disease model constructed in example 1:
1. sample collection
Collecting throat swabs, nasal lavage liquid 50 mu l, excrement samples and the like of experimental animals every day 0-7 days after animal challenge for monitoring virus load, measuring basic data of animal weight and body temperature, observing animal mental state, skin mucous membrane change, dynamic behavior, excrement state, appearance and respiration, observing whether nervous symptoms exist and the like, and recording and sorting observation results. Herpes tissues were harvested on the day of herpes emergence with sterile ophthalmic scissors and fixed with a neutral fixative for subsequent pathological examination.
After 3 mice in the experimental group and 3 mice in the control group are euthanized on the 7 th day after infection, anticoagulation blood is collected by 500ul for routine blood detection and virus load measurement through heart blood collection, 3ml of non-anticoagulation blood is used for serum biochemical detection, after gross dissection, whether each organ is obviously diseased or not is observed, meanwhile, a tissue sample is collected for grinding, freezing and fixing, and then the virus load and pathological detection in the tissue are measured.
2. Clinical symptoms
Referring to FIG. 1, CV-B1 virus developed small red rash on the skin outside of the mouth on day 6 after nasal drip infection and subsided on day 7. Simultaneously with the gingival red swelling and bleeding condition, the clinical manifestation symptoms are not obvious or part of the initial stage of herpes occurrence is slightly red swelling, the intermediate stage of herpes occurrence is obvious erythema or herpes on the surface of the skin mucous membrane and the surrounding skin is red swelling, a small amount of pus is in the herpes, the herpes of the skin gradually regresses to pigmentation or herpes scab at the later stage of herpes development, the regressed skin texture is hard without pus, and finally the skin recovers to the original state.
After the three-week-old hamster is infected with CV-B1, the body temperature is irregularly reduced in a wave manner, the fever is low, and the body temperature reaches the lowest point on the 7 th day. In the initial stage of infection, the weight of two groups of mice regularly increases, the weight of the mice regularly decreases on day 6, the appetite of the animals is obviously reduced, and the mental is unconscious.
3. Throat-nose feces loading capacity
After throat swab, nose swab, feces, blood and tissue are collected, RNA in a sample is extracted by using a T method, and the virus load in the sample is determined by performing one-step method qRT-PCR by using the RNA as a template. The sequence of the upstream primer is as follows: (CV-B1-QF) 5'-GGTGTCTACACAAAAGACGGGTG-3', downstream primer sequence: (CV-B1-QR) 5'-TGGGTCTTGTGTGAAATCTTGCC-3' and Taqman probe sequence (CV-B1-QProbe)5 'FAM-CGCATGAGACTGGTTTGAGTGCCAGCG-BHQ 3' were synthesized by Biotechnology, Inc. The reaction system comprises CV-B1-QF 0.6 muL, CV-B1-QR 0.6 muL, CV-B1-QProbe 0.6 muL, RNA template 1 muL, 2 Xone Step RT-PCR Buffer III 10 muL, TaKaRa Ex Taq HS (5U/muL) 0.4 muL, PrimeScript RT Enzyme Mix II 0.4 muL, RNase Free dH2O 7.6.6 muL and 20 muL in total. The reaction conditions are as follows: 5min at 42 ℃ and 10s at 95 ℃; the temperature of 95 ℃ for 5s and the temperature of 60 ℃ for 30s are one cycle, and the detection is carried out for 40 cycles. And constructing a CV-B1 standard substance to draw a standard curve, and calculating the virus load in the sample according to the standard curve and the CT value of the sample.
Referring to fig. 2, the results show that viral load was detectable in pharyngeal swabs from day 1 to day 7, with the experimental group reaching a maximum on day 4 and then gradually decreasing to a minimum on day 6; the nasal lavage fluid samples exhibited two peaks on day 2 and day 4, respectively; the viral load was detectable in the stool samples on day 1 of infection, with the highest load in the stool samples on day 2 followed by a gradual decrease.
4. Physiology and biochemistry of blood
Serum from day 7 after infection was collected and subjected to biochemical index detection of liver function and myocardial zymogram, and referring to FIG. 3, the test group showed increased myocardial zymogram (LDH, CK-MB) and AST and ALT in liver function, and decreased GLB, compared with the control group.
5. Tissue viral load
Referring to FIG. 4, the results of nucleic acid detection on tissue samples showed that viral nucleic acid was not detected in the ovarian and gastrocnemius tissues, but in the other tissuesAll have virus load, and the virus load in the olfactory bulb can be as high as 10 on average 9.86 copies/g. Compared with the control group, the virus load of the visceral tissues of the challenge mice is obviously increased, which indicates that CV-B1 is successfully replicated and proliferated in the mice at the age of 3 weeks, and the CV-B1 strain infects the mice successfully.
6. Pathological HE staining
Referring to fig. 5, lip muscle cells appearing in the herpes group became necrotic and rounded, cytoplasmic eosinophilia was increased, cell nucleus was pycnotic, fragmented and disappeared, necrotic muscle cells were disintegrated and disappeared, and interstitial little fibrous tissue was proliferated. Referring to fig. 6, in the olfactory bulb, the tissue nerve fiber mass becomes small in volume, the structure is loosened and loose, interstitial neurons are reduced and are accompanied by more microglial cell infiltration, inflammatory cell infiltration and a small amount of bleeding are generated around a small amount of blood vessels, granular cells of a granular layer are reduced, the arrangement is disordered and the nucleus is fixed and contracted; the cerebral cortex has loose structure, the meninges and parenchyma can be infiltrated by neutrophils and a small amount of inflammatory cells, local vascular inflammatory cell surrounding infiltration is performed, and the increase of glial cells is increased; the microscopic examination of heart tissue shows that a small amount of myocardial cells are necrotic, the nuclei are fixedly contracted and disintegrated, and the eosinophilia of cytoplasm is enhanced; epithelial cell proliferation and a small amount of neutrophil infiltration in the lung tissue of the experimental group; the hepatic cells around the central vein of the hepatic tissue are subjected to water sample degeneration in different degrees, are changed like balloons, vacuolate, cell body swelling, nucleus centering and hepatic sinus stenosis; intestinal villus edema, epithelial rupture and desquamation of intestinal tissues, blood capillary congestion, separation of inherent layer edema and mucosal epithelium, enlargement of gland interval, gland atrophy and disappearance, and lymphocyte infiltration.
The invention uses SPF level syrian golden yellow hamster of 3 weeks old, infect the animal through nasal cavity instillation, the skin mucous membrane of experimental group appears erythema or herpes within 7 days, herpes and herpes position have obvious pathological change, meanwhile animal body temperature reduces, serum enzyme changes, pharynx, nose and excrement continue to detect virus nucleic acid, each tissue virus load is obviously raised compared with the contrast group, multiple tissue organs have obvious pathological change, the above shows that the virus has wide tissue tropism in the organism, can successfully construct the hand-foot-and-mouth disease model through respiratory infection. Meanwhile, compared with the traditional intraperitoneal injection, the nasal drip is more close to the real way of human infection virus, and the hand-foot-mouth symptoms also appear, and meanwhile, the complications such as viral encephalitis, pneumonia, hepatitis and the like exist.
The Coxsackie group B virus type 1 infection Sjogren golden hamster hand-foot-and-mouth disease model is successfully constructed by nasal instillation, and a foundation is provided for the subsequent development of virus-related research, medicine and vaccine evaluation and pathogenic mechanism research.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned.
Furthermore, it should be understood that although the present specification describes embodiments, not every embodiment includes only a single embodiment, and such description is for clarity purposes only, and it is to be understood that all embodiments may be combined as appropriate by one of ordinary skill in the art to form other embodiments as will be apparent to those of skill in the art from the description herein.
Claims (5)
1. A construction method of a CV-B1 infected rodent hand-foot-and-mouth disease model is characterized by comprising the following steps: CV-B1 is infected to the rodent model in a nasal cavity instillation mode, and a CV-B1 infected rodent hand-foot-and-mouth disease model is established.
2. The method for constructing a CV-B1 infected rodent hand-foot-and-mouth disease model according to claim 1, wherein: the rodent is selected from 3-week-old syrian golden hamster.
3. According toThe method of constructing a CV-B1 infected rodent hand-foot-and-mouth disease model of claim 1, wherein: the CV-B1 virus titers were: 10 7.25 CCID 50 mL, 100 μ l of virus suspension was instilled through the nasal cavity each.
4. Use of the CV-B1 infected rodent hand-foot-and-mouth disease model prepared by the construction method according to any one of claims 1 to 3, for screening or identifying drugs capable of preventing, alleviating or treating CV-B1 infection.
5. Use of the CV-B1 infected rodent hand-foot-and-mouth disease model prepared by the construction method according to any one of claims 1 to 3, for screening or identifying a vaccine capable of preventing CV-B1 infection.
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