CN115088674A

CN115088674A - Construction method and application of echovirus 30 type wild suckling mouse model

Info

Publication number: CN115088674A
Application number: CN202210656386.6A
Authority: CN
Inventors: 刘洪波; 瞿颖; 刘启亮; 王婧; 陈泳蓓; 朱丹东; 但汉亮; 章�宁; 何韵怡; 甘燕媚
Original assignee: Second Affiliated Hospital Of Guilin Medical University
Current assignee: Second Affiliated Hospital Of Guilin Medical University
Priority date: 2022-06-10
Filing date: 2022-06-10
Publication date: 2022-09-23

Abstract

The invention relates to a construction method and application of an Echovirus 30 (Echovrus 30, E30) wild suckling mouse model, relating to the field of biological medicine and comprising the following steps: step 1: animal inoculation: after the infection dosage, the day age, the route and the strain are screened, the E30 clinical isolate A538 virus liquid concentration is 3LD by intracranial injection ₅₀ ‑5LD ₅₀ Inoculating any one of ICR mouse, BALB/c mouse and KM mouse with age of 1-3 days; and 2, step: and (3) clinical observation: and (4) carrying out clinical observation and clinical scoring, and screening the infected mouse with the clinical score being more than or equal to 4, namely the E30 wild type suckling mouse model. The wild type suckling mouse model infected by E30A538 clinical isolate can be simulatedThe human natural infection characteristics and good repeatability can provide powerful tools for the research of E30 infection characteristics and pathogenic mechanisms, the research and development of vaccines and antiviral drugs, screening evaluation and the like.

Description

Construction method and application of echovirus 30 type wild suckling mouse model

Technical Field

The invention relates to the field of biomedicine, in particular to a construction method and application of an echovirus 30 type wild suckling mouse model.

Background

Echovirus type 30 (echoviruses 30, E30) is a member of the group B of the Enterovirus genus (enteroviruses, EVs) of the Picornaviridae family (Picornaviridae). E30 primarily infects children 0-4 years of age and adults 26-45 years of age; the Disease spectrum is wide, and besides being one of main pathogens of Viral Meningitis (VM), E30 infection can also cause various diseases such as Viral Encephalitis (VE), Hand-Foot-and-Mouth Disease (HFMD), Acute Flaccid Paralysis (AFP), Viral Myocarditis (VMC) and the like.

E30 has a wide range of popularity. In 1983 the first epidemic of E30 occurred in japan, and since then there was a small outbreak around the world in countries such as the united states, france, greece, italy, brazil, india, korea, etc. Statistics of data show that E30 is the second most common EV serotype in korea during 1999 to 2011 and is frequently monitored during 2010-2014; the prevalence of E30 in 2018 in europe was high compared to the data collected in 2015-oz 2017, and large-scale proliferation of VM associated with E30 was reported in denmark, germany, the netherlands, norway and sweden, with E30 being detected in 14.5% of all diagnosed EV cases and central nervous system damage in 75% of cases. In recent years, a plurality of E30 epidemic situations such as Shandong, Zhejiang, Fujian, Hebei, Gansu and Guangdong also appear in various parts of China. In 2004, VE caused by E30 outbreaks in various regions of zhejiang province, such as hangzhou, ningbo and wenzhou, from 4 months to 8 months in the same year, 414 VE cases were reported in cihai city in zhejiang province, wherein 19E 30 were separated from 67 cerebrospinal fluid specimens. 26 parts of E30 were isolated from 442 parts of cerebrospinal fluid of a child who was clinically diagnosed as VE and VM in the Children hospital in Hebei province from 2013 to 2015. In 6-8 months in 2015, the VE epidemic situation occurred in guzhou county, gansu, china, 132 samples of 74 cases were collected, 72 positive samples of EV nucleic acid detection were obtained, 71 molecular typing results were E30, which accounted for 98.61% of EV, and 46 samples of 29 cases were isolated as E30. E30 is found in epidemic situations around the world to be capable of recombining with other EV-B to form a new strain, so that the transmission or pathogenicity changes, and the E30 cases are the main reason of increasing. Therefore, the development of preventive vaccines and the screening of antiviral drugs is very slow.

The animal model with typical clinical symptoms can be used for discussing the pathophysiological mechanism of diseases, and is one of the important links of drug effect and vaccine evaluation. It has been reported that various EV animal models have been successfully established and used in drug, vaccine development and evaluation. The application of the suckling monkey model is limited to a certain extent due to the problems of high feeding cost, ethics and the like, and the suckling mouse model is most widely used due to the advantages of low price, easiness in feeding, short experimental period, establishment of various stable population systems, capability of well simulating central nervous system disease symptoms and the like. Similar to infant infections, EV models are often established using newborn suckling mice. However, due to the differences in murine and human receptors, only a few clinical isolates can directly construct good suckling mouse models. It has been reported that by serial passage of clinical isolates in animal tropic tissues, pathogenic or lethal models can be established by obtaining virulent strains with enhanced virulence, but with inevitable changes in the pathogenic mechanisms, pathological lesions, etc. characteristic of the viral strains. The clinical isolate can directly reflect the pathogenicity, the pathogenic process and the pathogenic mechanism of the human epidemic strain in a mouse body because the clinical isolate directly separates the patient with the disease, and has better application value for research and development and application of human vaccines and medicaments.

At present, ICR, BALB/C, KM and C57BL/6 suckling mice are often adopted to establish an EV infected mouse model. Among them, the C57BL/6 mouse Interferon (Interferon, IFN) yield is high, and the susceptibility to virus is poor. The I type IFN receptor gene IFNAR knock-out (IFNAR-/-) C57BL/6 milk mouse has weaker immunity compared with the wild type and is more susceptible to virus infection. Therefore, IFNAR-/-C57BL/6 suckling mice are used for the construction and characteristic study of an infection model of an E30 isolate. Unfortunately, knockout mice are expensive and the technical means are cumbersome. And IFNAR-/-suckling mice can not activate type I IFN related immune reaction pathways, and can not completely simulate the disease process of mice naturally infected by virus. In addition, IFNAR-/-suckling mice can not combine with I type IFN to generate antiviral protein, and are not suitable for the evaluation of I type IFN antiviral drugs such as IFN-alpha and IFN-beta, and have certain limitations.

At present, an E30 animal model is extremely lacking, and a stable E30 isolate suckling mouse model which is low in cost, can simulate the clinical characteristics of human natural infection and is urgently needed to be established so as to carry out research works of E30 infection or pathogenesis, vaccine and drug research and evaluation.

Disclosure of Invention

The invention aims to solve the technical problem of providing a construction method and application of an echovirus 30 type wild suckling mouse model. The method aims to establish a wild suckling mouse model which can simulate the natural infection symptoms of human beings, is stable and lethal and can be used for evaluating the infection of E30 clinical isolates of the vaccine or medicament effectiveness, provides powerful tools for infection characteristics, pathogenic mechanism research, E30 vaccine development and evaluation, antiviral medicament screening and evaluation, and has low model cost and simple and convenient operation.

In order to solve the above technical problems, a first object of the present invention is to provide a method for constructing an Echovirus 30 (Echovirus 30, E30) wild suckling mouse model, comprising the steps of:

step 1: animal inoculation: the infection route is IC injection, and the concentration of the E30A538 virus liquid is 3LD ₅₀ -5LD ₅₀ The infection dose of 10-30 mul, the infection product line with the inoculation infection day age of 1-3 days is any one of ICR mouse, BALB/c mouse and KM mouse;

and 2, step: and (3) clinical observation: and (2) carrying out isolated breeding on the inoculated mice in the step (1), continuously observing the infected mice, carrying out clinical scoring according to clinical observation results, and screening the inoculated mice with the clinical scoring more than or equal to 4, namely the echovirus 30 type wild suckling mouse model.

The clinical scoring criteria were: 0, health; 1, lethargy and reduced activity; 2, emaciation; 3, weakness of limbs; 4, hind limb paralysis; and 5, death.

The beneficial effects of the invention are: in the constructed E30 infected suckling mouse model, the suckling mouse is wild without any gene modification work and gene verification, so the cost is low, the suckling mouse model is easy to obtain, the feeding is convenient, and the operation is easy; the infected echovirus 30 is a clinical isolate model E30A538, can directly infect a mouse and is stably lethal without continuous passage in the mouse, and can directly reflect the pathogenicity, pathogenic process and pathogenic mechanism of a human epidemic strain in the mouse; the suckling mouse model can be used for researching systemic transmission of viruses and a human disease spectrum in an animal model; the E30A538 infection can cause the milk mouse to have typical clinical symptoms of brain, spinal cord, heart injury and the like and finally die, namely, the milk mouse model has the characteristics of human VM, VE, VMC and AFP, and the E30 infection is proved to be connected with the diseases.

On the basis of the technical scheme, the invention can be further improved as follows.

Further, the infection route of IC injection is adopted in the step 1, and the concentration of the E30A538 virus liquid is 3LD ₅₀ -5LD ₅₀ The infection product line inoculated with the infection of 1 day of age of day is any one of ICR mouse, BALB/c mouse and KM mouse.

The beneficial effect of adopting the further scheme is that: by selecting mice with 1 day old as ICR mice, BALB/c mice and KM mice, more inoculated mice with clinical scores of more than or equal to 4 are screened.

Further, the infection route using IC injection in the step 1, E30A538 the concentration of the virus solution is 5LD ₅₀ The ICR mouse was inoculated as an infected strain with an age of 1 day of infection.

The beneficial effect of adopting the further scheme is that: by adopting E30A538 virus liquid concentration 5LD ₅₀ The optimal echovirus 30 type wild suckling mouse model can be obtained by inoculating an infected strain with the age of 1 day of the infected day into ICR mice.

Further, the 3LD ₅₀ -5LD ₅₀ Middle 3LD ₅₀ Is 10 ^3.685 TCID ₅₀ ，5LD ₅₀ Is 10 ^3.907 TCID ₅₀ 。

Further, before the animal in the step 1 is inoculated, the selection of the infection dose, the selection of the day of infection, the selection of the infection route and the selection of the infection strain can be carried out, wherein the selection of the infection dose, the selection of the day of infection, the selection of the infection route or the selection of the infection strain comprises the following steps:

step 1-1: preparation of inoculated animals: setting corresponding experimental groups and negative control groups, wherein the sample amount of each group is 3-15, and isolating and feeding the samples in groups under the same environment;

step 1-2: preparing virus liquid: diluting the E30A538 isolate by using a diluent to prepare E30A538 virus solutions with different concentrations;

step 1-3: animal inoculation: inoculating the experimental group in the step 1-1 with E30A538 virus solution with corresponding concentration in the step 1-2; injecting a corresponding amount of the diluent of the step 1-2 into the negative control group of the step 1-1;

step 1-4: and (3) clinical animal observation: and (3) carrying out isolated breeding on the experimental group and the negative control group inoculated in the steps 1-3 in the same environment, continuously observing the clinical symptoms of the mice of the experimental group and the negative control group, and calculating the death rate.

Further, E30A538 was serially diluted 10-fold with sterile diluent 10-fold ^-2 -10 ^-8 I.e. 10 ^7.375 TCID ₅₀ -10 ^1.375 TCID ₅₀ The concentration is/mL, and then the 1-day-old ICR mice are inoculated with IC for screening the infection dose;

E30A538 as 5LD ₅₀ ：10 ^3.907 TCID ₅₀ Mice, IC-vaccinated 1, 3, 5 and 7 day-old ICR mice for screening for infection day-old;

inoculation of 5LD with 1 day old ICR mice by IC, IP or IM ₅₀ ：10 ^3.907 TCID ₅₀ E30A538 from mice, for screening of infection pathways;

by 5LD ₅₀ ：10 ^3.907 TCID ₅₀ E30A538 of mice was inoculated with IC into 1-day-old ICR mice, BALB/C mice, KM mice, and C57BL/6 mice, respectively, for infected strain screening.

The beneficial effect of adopting the further scheme is that: the severity of disease in E30A 538-infected mice was found to be dependent on viral dose, 5LD ₅₀ The lowest lethal dose for mice; finding an ICR suckling mouse at 1 day of age to be most susceptible; the mortality rate of the IC injection experimental group is the highest; ICR mice, BALB/C mice, KM mice were found to be susceptible to E30A538, with the exception of C57BL/6 mice.

Further, the TCID of the E30A538 virus fluid ₅₀ Measured by the Spearman-Karrer method.

The second purpose of the invention is to provide the application of the suckling mouse model constructed by the construction method of the echovirus 30 type wild suckling mouse model, wherein the suckling mouse model is used for screening and evaluating antiviral drugs or vaccines.

The beneficial effect of the scheme is that the suckling mouse model constructed by the method provides a powerful tool for research and development and evaluation of E30 vaccine, screening and evaluation of antiviral drug.

The third purpose of the invention is to provide the suckling mouse model for screening and evaluating the antiviral drugs, which specifically comprises the following steps:

step 21: preparation of inoculated animals: selecting an ICR mouse of 1 day old; dividing into a negative control group (1 XPBS), an E30 model group (E30+1 XPBS), a drug treatment group (E30+ drug +1 XPBS) and a drug control group (drug +1 XPBS); the sample volumes of the negative control group, the E30 model group, the drug treatment group and the drug control group are the same and are all 6-15, and the negative control group, the E30 model group, the drug treatment group and the drug control group are isolated and raised according to groups under the same environment;

step 22: preparing virus liquid and medicines: the preparation concentration is 5LD ₅₀ E30a538 virus solution of (1); respectively preparing medicines with different concentrations by using sterile buffer solutions;

step 23: animal inoculation: the E30 model group and the drug treatment group were injected with 20. mu.l of E30A538 virus solution 5LD by IC injection ₅₀ Mice, negative control group, drug control group were injected with IC with the same volume of 1 × PBS; after 1hpi (1hour post-infection,1hpi), the treated mice, drug control mice were IP injected with the corresponding therapeutic dose of drug, negative control mice, E30 model mice were IP injected with the same volume of 1 × PBS; subsequent days 1-6 (1-6days post-infection,1-6dpi), re-dose or 1 × PBS;

step 24: and (4) carrying out clinical observation on the mice in the step (23), comparing the weight change of the mice, the survival rate of the mice and the clinical scores of the mice, and screening out the drug with good curative effect of the antiviral drug.

Further, the drug treatment groups in step 21 are: interferon alpha 2a drug treatment group and interferon gamma drug treatment group; the therapeutic dose of the medicine in the step 23 is interferon alpha 2a10000U and interferon gamma 10000U in single administration dose.

The beneficial effect of adopting the further scheme is that: interferon gamma does not show the anti-E30 ability in vivo, and interferon alpha 2a can effectively inhibit the replication of E30A538 in vivo and improve the survival rate of newborn mice.

Drawings

FIG. 1 is a graph of infectious dose screening; survival of mice in other groups and negative control groups within 21dpi was analyzed using Mantel-Cox log rank test,. P <0.001,. P < 0.05;

FIG. 2 is a screening chart of day-old infected mice; survival of other groups and 1 day old groups of suckling mice within 21dpi was analyzed using Mantel-Cox log rank test,. P <0.001,. P < 0.05;

FIG. 3 is a screening chart of infection pathways; survival rates of other groups and IC groups of suckling mice in 21dpi are analyzed by adopting a Mantel-Cox logarithmic rank test, and ns represents no significant difference;

FIG. 4 is a graph of the sensitivity of different strains of mice to E30; the survival rates of other strain groups and ICR suckling mice within 21dpi are analyzed by adopting a Mantel-Cox logarithmic rank test; p <0.001, ns no significant difference;

FIG. 5 shows E30(5 LD) ₅₀ ) Body weight change profile of ICR suckling mice one day old infected with IC; unpaired t-test analysis of changes in body weight of mice from the E30 model group and the negative control group within 21dpi, error bars (error bars) representing SEM, P<0.001

FIG. 6 shows E30(5 LD) ₅₀ ) Survival plots of ICR suckling mice one day old infected with IC; differences in survival of negative control and E30 model groups of milk mice within 21dpi were analyzed using Mantel-Cox log rank test<0.001

FIG. 7 shows E30(5 LD) ₅₀ ) Mean clinical score plot of IC-infected one-day-old ICR suckling mice; unpaired t-test analyzed significant differences between the E30 model group and the negative control group of suckling mice within 21dpi, error bars (error bars) representing SEM, × P<0.0001

FIG. 8 shows E30(5 LD) ₅₀ ) A clinical symptom profile of one day old ICR suckling mice infected with IC;

FIG. 9 is a graph of the mean viral load of E30 infected suckling mouse tissues;

FIG. 10 is a graph showing the results of HE staining of mouse brains of negative control groups;

FIG. 11 is a graph showing the results of HE staining of the brains of mice in the E30 model group; black single arrows mark lesion sites: a. apoptosis of brain neuron cells, b. lymphocyte infiltration;

FIG. 12 is a graph showing the results of HE staining of the hearts of mice in the negative control group;

FIG. 13 is a graph showing the results of HE staining of mouse hearts of the E30 model group; black single arrow marks the lesion site: myocardial necrosis autolysis, rupture of muscle fibers;

FIG. 14 is a graph showing the results of HE staining of mouse livers of negative control groups;

FIG. 15 is a graph showing the results of HE staining of mouse livers of the E30 model group; black single arrows mark lesion sites: mild edema of hepatocytes;

FIG. 16 is a graph showing the results of HE staining of spinal cords of mice in the negative control group;

FIG. 17 is a graph showing the results of HE staining of spinal cords of mice in the E30 model group; black single arrow marks the lesion site: a, the spinal cord is scattered in lymphocytes, and b, the spinal cord is liquefied and necrotic;

FIG. 18 is a graph showing the results of HE staining of skeletal muscle in mice of the negative control group;

FIG. 19 is a graph showing the results of HE staining of skeletal muscle of mice in the E30 model group; black single arrows mark lesion sites: muscle is scattered in individual lymphocytes;

FIG. 20 is a graph showing HE staining results of back fat pads of negative control mice;

FIG. 21 is a graph showing HE staining results of back fat pads of mice of the E30 model group; black single arrows mark lesion sites: fat necrosis, calcium salt deposition;

FIG. 22 is a graph showing the result of IHC staining of the brain of a mouse in a negative control group;

FIG. 23 is a graph showing the results of IHC staining of the brains of mice in the E30 model group; black single arrows mark brown-yellow E30 antigen sites;

FIG. 24 is a graph showing the result of IHC staining of the heart of a mouse in a negative control group;

FIG. 25 is a graph showing the results of IHC staining of the hearts of mice in the E30 model group; black single arrows mark the brown-yellow E30 antigen particle positions;

FIG. 26 is a graph showing the result of IHC staining of spinal cords of mice in the negative control group;

FIG. 27 is a graph showing the results of IHC staining of spinal cords of mice in the E30 model group; the black single arrow marks the brown-yellow E30 antigen site;

FIG. 28 is a graph showing the result of IHC staining of skeletal muscle of a mouse in a negative control group;

FIG. 29 is a graph showing the result of IHC staining of skeletal muscle of mice in the E30 model group; black single arrows mark brown-yellow E30 antigen sites;

FIG. 30 is a graph showing the result of IHC staining of the fat pad on the back of a mouse in a negative control group;

FIG. 31 is a graph of the results of IHC staining of the small back fat pad of the E30 model group; black single arrows mark brown-yellow E30 particle positions;

FIG. 32 is a graph of the in vivo anti-E30 effect of interferon alpha 2a, interferon gamma vs. mouse body weight change; comparing the significant difference in weight change between the drug-treated mice and the model mice within 21dpi using Dunnett's m mu multiple complexes test; error bars represent SEM, # P <0.0001, ns is not significant;

FIG. 33 is a graph of in vivo anti-E30 effect of interferon alpha 2a, interferon gamma-mouse survival; the Mantel-Cox log rank test analyzes the significant difference of the survival rate of the mice in the drug treatment group and the mice in the model group, P <0.0001, and ns has no significance;

figure 34 is a graph of the in vivo anti-E30 effect of interferon alpha 2a, interferon gamma-mouse clinical scores; the significant difference in small clinical scores between drug-treated and model mice within 21dpi was compared using Dunnett's m μmultiple complexes test; error bars represent SEM, # P <0.0001, ns is not significant;

FIG. 35 is a graph of in vivo anti-E30 effects of interferon α 2a, interferon γ -mouse clinical symptoms.

Detailed Description

The principles and features of this invention are described below in conjunction with examples which are set forth to illustrate, but are not to be construed to limit the scope of the invention.

Description of experimental materials and methods employed in the present invention:

(1) experimental materials and sources

Virus strains: the E30a538 strain was isolated from clinical patient specimens with GenBank accession numbers: ON129560, stored at-80 ℃ until passaged for 3 cycles in RD cells before further use.

Cell line (b): the mammalian cell line used in the present invention is a Human rhabdomyomyoma cell (RD).

Experimental animals: the experimental animals are produced in Specific Pathogen Free (SPF) ICR, BALB/C, KM, C57BL/6 pregnant mice (purchased from Splakeda laboratory animals Co., Ltd., Hunan province, with the approved number of the experimental animals: SCXK 2019- .

Coli DH5 alpha (Producer, China) for expression of single clones.

Interferon alpha 2a was purchased from MCE corporation, usa, cat #: HY-P7022. Interferon gamma was purchased from MCE corporation, usa, cat #: HY-P7025.

(2) Cell culture and viral amplification

And (3) cell culture: RD cells were grown in Dulbecco's Modified Eagle's Medium (DMEM) (Invitrogen, USA) containing 10% special grade Fetal Bovine Serum (FBS) (Invitrogen, USA) and 1% penicillin-streptomycin double antibody, and grown at 37 ℃ and 5% CO ₂ Culturing in an incubator.

And (3) virus amplification: E30A538 infected RD cells, and at 37 ℃ and 5% CO ₂ Incubation in an incubator; at 24hpi, the cells may develop CPE such as cell rounding, shrinkage, rupture, dead float, etc. RD cells with a fusion rate of 70% -80% in T25 cell culture flasks were infected with 100. mu.l of E30A538 virus suspension, and 900. mu.l of DMEM was added and placed horizontally at 37 ℃ with 5% CO ₂ Incubate 1hpi in the incubator, discard unbound virus, wash infected RD cells twice in sterile phosphate buffered saline (1 × PBS, pH 7.4) solution, and then add maintenance medium (DMEM, 1% FBS). Cytopathic effect (CPE) of infected RD cells was observed daily.

(3) Determination of viral titre

Viral titers were counted using the Spearman-Karrer method. RD cells (1.5X 10) ⁴ Individual cells/mL) were seeded in 96-well plates and cultured for about 24 h. Frozen virus thawed to room temperature was diluted 10-fold in a gradient with maintenance medium (10) ^-1 ～10 ^-10 ) Washing RD cells, sequentially inoculating diluted virus diluent with each well being 100 μ L, each gradient having 8 multiple wells, simultaneously setting negative control wells which only adopt maintenance medium at 37 deg.C and 5% CO ₂ Culturing in an incubator, observing cell change continuously at 5-7dpi and recording the number of cytopathic holes. When the hole is drilledThe virus titer was calculated according to the Spearman-Karber method when 80% of the cells showed CPE and was recorded as + and calculated as logTCID ₅₀ Xm is the logarithm of the dilution at which the highest concentration of virus is used, d is the logarithm of the dilution factor (fold), and Σ pi is the sum of the percentages of cytopathic appearance at each dilution.

Viral titers of E30a538 in RD cells: according to TCID ₅₀ The detection method comprises observing and recording cytopathic condition every day, and marking the observation as an endpoint at the end of 5-7dpi after infection. The virus titer of E30A538 strain was 10 as calculated by the Spearman-Karber method ^9.375 TCID ₅₀ /mL。

Example 1 construction method of E30 clinical isolate suckling mouse model

Construction method of 1E 30 clinical isolate suckling mouse model

1.1 screening of infectious dose

1.1-1, preparation of vaccinated animals: selecting 1-day-old ICR suckling mice, dividing the ICR suckling mice into a negative control group (n is 6) and 9 dose experimental groups (n is 6 multiplied by 9), and carrying out isolation and breast milk feeding according to groups under the same environment;

1.1-2, preparing virus liquid: diluting the E30A538 isolate by using a diluent, and respectively preparing 9 groups of E30A538 virus solutions with different concentrations; the preparation method of the E30A538 virus liquid comprises the following steps: serial 10-fold dilution of E30A538 with sterile 1 XPBS ^-2 -10 ^-8 ，10 ^7.375 TCID50-10 ^1.375 TCID ₅₀ Per mL, 10 ^7.375 、10 ^6.375 、10 ^5.375 、10 ^4.375 、10 ^3.375 、10 ^2.375 、10 ^1.375 、10 ^3.907 (5LD ₅₀ )、10 ^3.685 (3LD ₅₀ )TCID ₅₀ A mouse;

1.1-3, animal inoculation: injecting 20 mu l of sterile 1 XPBS buffer solution into the negative control group mice IC in the steps 1.1-1, and injecting 20 mu l of E30A538 virus solution with different concentrations prepared in the steps 1.1-2 into the 9 dosage experimental groups respectively through IC;

1.1-4, clinical animal observations: and (3) carrying out isolated feeding on all groups of suckling mice in the steps 1.1-3 in the same environment, carrying out normal breast milk or water and feed feeding in the period, continuously observing the weight change and clinical symptoms of the suckling mice at 21dpi, carrying out clinical scoring, and calculating the death rate.

1.2 screening of best suckling mice for day-old of infection

1.2-1, preparation of inoculated animals: selecting ICR suckling mice, dividing the ICR suckling mice into a 1-day-old experimental group (n-6), a 3-day-old experimental group (n-6), a 5-day-old experimental group (n-6) and a 7-day-old experimental group (n-6), and a 1-day-old negative control group (n-6), wherein 5 groups are selected in total, and isolating and breast-feeding the ICR suckling mice according to groups under the same environment;

1.2-2, preparing virus liquid: the E30A538 isolate was diluted with sterile 1 XPBS buffer to prepare 5LD ₅₀ (5LD ₅₀ ：10 ^3.907 TCID ₅₀ Mouse) of E30A538 virus solution 1 mL;

1.2-3, animal inoculation and clinical observation: the 4 day-old group of mice IC from steps 1.2-1 was injected with 20. mu.l of E30A538 virus solution (5 LD) ₅₀ Mice), negative control group IC was injected with the same volume of 1 x PBS; and clinical observations were made on 1.2-1 groups of mice as per 1.1-4.

1.3 screening for optimal infection pathways

1.3-1, preparation of inoculated animals: selecting 1-day-old ICR suckling mice, dividing the ICR suckling mice into 4 groups of an IC injection experimental group (n-6), an Intraperitoneal (IP) injection experimental group (n-6), an Intramuscular (IM) experimental group (n-6) and an IC injection negative control group (n-6), and isolating and breast-feeding the ICR suckling mice according to groups under the same environment;

1.3-2, animal inoculation and clinical animal observation: injecting the group 3 mice in the step 1.3-1 with E30A538 virus solution (5 LD) prepared by the method of 1.2-2 by IC, IP and IM injection respectively ₅₀ Mice) 20. mu.l, negative control group IC was injected with the same volume of 1 XPBS; and clinical observations were made on groups of mice 1.3-1 by the method of 1.1-4.

1.4 comparison of sensitive mouse strains

1.4-1, preparation of vaccinated animals: selecting 1-day-old ICR suckling mice, BALB/C suckling mice, KM suckling mice and C57BL/6 suckling mice, dividing the ICR suckling mice into an ICR experimental group (n is 6), a BALB/C experimental group (n is 6), a KM experimental group (n is 6) and a C57BL/6 experimental group (n is 6), and isolating and breast-feeding the ICR suckling mice, the BALB/C suckling mice, the KM suckling mice and the C57BL/6 experimental mice according to groups under the same environment;

1.4-2, animal inoculation and clinical observation: groups 4 of mice from steps 1.4-1 were each IC-injected with 20 μ l of E30A538 virus (5 LD) ₅₀ A mouse); and clinical observations were made on 1.4-1 groups of mice according to 1.1-4 methods.

1.5 reproducibility of clinical isolate of E30 suckling mouse model

1.5-1, preparation of inoculated animals: selecting 1-day-old ICR suckling mice, dividing the ICR suckling mice into a negative control group (n-6) and an E30 model group (n-6), and carrying out isolation and breast milk feeding according to groups under the same environment;

1.5-2, animal inoculation and clinical observation: the negative control group in the 1.5-1 step was injected with 20. mu.l of sterile 1 XPBS buffer by IC, and the model group E30 ICR mice were injected with 20. mu.l of E30A538 virus solution (5 LD) ₅₀ A mouse); and carrying out clinical observation on 1.5-1 groups of mice according to a method of 1.1-4; and screening the model group mice with clinical scores more than or equal to 4.

2 results and discussion

2.1 screening of infectious dose

IC infection of 1 day old ICR mice after 10-fold serial dilution of E30a538 virus solution, negative control groups injected with the same volume of sterile 1 × PBS, n ═ 6, 21dpi survival rate was calculated, and it was found that disease severity in E30a538 infected mice was dependent on virus dose, 5LD ₅₀ The lowest lethal dose was given to mice (fig. 1).

2.2 screening of best suckling mice for day-old of infection

1. IC infection of 3, 5, 7 day old ICR suckling mice with E30A538 virus (5 LD) ₅₀ ) And n is 6, the survival rate of 21dpi is calculated, and the ICR suckling mouse at the age of 1 day is found to be most susceptible (figure 2).

2.3 screening for optimal infection pathways

1 day old ICR suckling mouse is infected by E30A538 virus (5 LD) through intracranial, abdominal cavity and muscle ₅₀ ) And n is 6, the survival rate of 21dpi is calculated, and the mortality rate of an IC injection experiment group is the highest (figure 3).

2.4 comparison of sensitive mouse strains

Infection of suckling mice of different strains with IC E30A538 Virus (10) ^3.907 TCID ₅₀ Mice), survival of each group of suckling mice (n ═ 6) was calculated, and ICR mice, BALB/C mice, and KM mice were all found to be susceptible to E30a538, except for C57BL/6 mice (fig. 4).

2.5 reproducibility of clinical isolate of E30 suckling mouse model

Selecting 5LD ₅₀ Mice, IC-infected 1 day old ICR suckling mice, ICR suckling mice weight than the 4 th dpi begins to gradually decrease (figure 5); at 4-10dpi, symptoms such as lethargy, emaciation, arch and back, tremor, listlessness, quadriplegia, death, etc. appear (figure 8); and all died, with 100% mortality at 21dpi (FIG. 6); clinical score was 5 points (fig. 7).

Example 2 study of infection characteristics and pathogenic mechanisms of E30 clinical isolate in suckling mouse model

The infection characteristics and pathogenic mechanism of the E30 clinical isolate in example 1 were studied in a suckling mouse model.

Study on infection characteristics and pathogenic mechanism of 1E 30 clinical isolate suckling mouse model

The lowest lethal dose of 5LD ₅₀ The E30A538 of (A) was used as an E30 model group by IC infection of 18 1-day-old ICR suckling mice, and as a negative control group, 18 1-day-old ICR mice were IC-injected with the same volume of 1 XPBS. Infected animals were observed daily for disease manifestation. Mice with a clinical score of 4 or greater post-infection were euthanized and dissected model groups of mice with a clinical score of 4 or greater as described above, and their brain, intestine, skeletal muscle, heart, liver, lung, spinal cord, spleen, kidney, back fat pads were isolated and tissue viral load determinations were performed using qRT-PCR absolute quantitation. And collecting the tissues and organs of the negative control group mice at the same time, and detecting the pathological changes of the tissues by using Hematoxylin-eosin staining (HE) and detecting the distribution of specific virus antigens in the tissues by using Immunohistochemistry (IHC).

1.1 calculation of tissue viral load

The virus load detection steps are as follows: recombining and identifying standard plasmid → extracting tissue RNA → establishing a standard curve by a qRT-PCR method → calculating the tissue virus load.

1.1-1 recombination and characterization of a Standard plasmid (pMD19T-E30) encoding a partial cDNA of E30 VP 1: total RNA of E30 virus liquid is extracted by Trizol method, reverse transcription is carried out to cDNA by taking the total RNA as a template, RT-PCR is carried out by utilizing primer pair E30-A538-2378F (SEQ ID NO: 1)/3523R (SEQ ID NO: 2) (the primer is synthesized by Beijing Hua DageneCo., Ltd.), and DNA is purified for cloning after agarose electrophoresis identification. Using T-Vector pMD ^TM 19(Simple) DNA Ligation Kit Ver.2.1(Takara, China) clones the target DNA to pMD-19T vector, expresses it by Escherichia coli DH5 alpha expression system, picks up single clone PCR detection and sequences, namely, harvests positive standard plasmid (pMD19T-E30), the plasmid copy number is 6.73X 10 ¹⁰ copies/μL。

1.1-2 tissue RNA extraction: collecting fat pads of brain, intestine, skeletal muscle, heart, liver, lung, spinal cord, spleen, kidney and back, and extracting total RNA of the grinding fluid supernatant of each tissue and organ.

1.1-3 establishment of a standard curve by the qRT-PCR method: the method is completed by using a qRT-PCR absolute quantitative method. Taking 3 mice with clinical score more than or equal to 4, separating tissues, extracting total RNA of the supernatant of the grinding fluid of each tissue and organ, taking the standard plasmid gradient diluent containing the same gene segment as the tissue sample as a standard substance, and using a primer pair E30-A538-2389F (SEQ ID NO: 3)/2508R (SEQ ID NO: 4) (the primer is synthesized by Beijing Hua Dagong Co., Ltd.), Beyofast ^TM Performing qRT-PCR by SYBR Green One-Step qRT-PCR Kit (Biyunshi, China), and establishing a standard curve Y-4.0507X +47.929, R ² ＝0.999。

1.1-4 calculation of tissue viral load: based on the established standard curve, the average viral load of E30 in each tissue organ was detected and calculated.

1.2 processing of tissue sections

Processing of tissue sections (4% fixation of tissue cells → decalcification of bone tissue → dehydrated transparency → paraffin embedding → tissue sections): the collected tissue was fixed in 4% paraformaldehyde at room temperature for about 24 hours (24 hours), and the skeletal muscle and spinal cord tissues of mice were taken out from the fixation solution, washed with running water for 10 minutes (10min), and then decalcified by EDTA at room temperature for 12 hours. After decalcification, the tissue was washed with running water, dehydrated in 70%, 95% and 100% gradient ethanol, and cleared in xylene. And finally infiltrated with 4 changes of liquid paraffin. All incubations were performed at room temperature for 1h with gentle shaking at 100 rpm. Paraffin infiltration was performed in an oven set at 65 ℃. Paraffin-embedded tissue blocks (5 μm) were sectioned using a microtome, loaded onto poly-lysine-coated slides, and excess water was dried on a baking sheet machine to obtain tissue sections for later use.

1.3 histological section staining identification (HE):

staining of tissue sections: the pathological examination adopts hematoxylin (Biyun day, China) and eosin (Biyun day, China) for HE staining. The tissue sections were dewaxed and placed in 3 jars of xylene sequentially for 10min each jar. Rehydrating, sequentially passing through 100%, 95%, 85%, and 75% gradient ethanol for 2min each cylinder, and flushing with running water for 2 min. Staining cell nucleus, performing hematoxylin staining for 5min, and washing with running water for 10 min; 0.5% hydrochloric alcohol 6-10S; running water and turning blue for 10 min. Cytoplasmic staining, eosin staining for 1 min; flushing with running water for 10 min. Dehydrating, and transparent, and extracting with 95% ethanol for 5-10 s; 100% alcohol for 5 min; and 2 cylinders of dimethylbenzene I and dimethylbenzene II for 5min respectively, completely volatilizing ethanol in an oven, dripping 1 to 2 drops of neutral gum in the center of the tissue, and covering a cover glass to obtain a stained tissue section. Histopathological changes are judged and confirmed by clinical pathology specialists with extensive clinical experience and research experience.

1.4 immunohistochemical section (IHC):

after deparaffinization of the tissue sections, the tissue sections were subjected to heat-induced antigen recovery by incubation in a histological grade microwave oven and citrate buffer (pH 6.0) at 96 ℃ for 20 min. Use of ^TM HRP-Polymer Anti-Mouse IHC kit (New Biotechnology development, Inc., Fuzhou, China) was used for IHC analysis of the tissues. The primary antibody used was E30 polyclonal mouse antibody (1:500 dilution) prepared in this laboratory. After the tissue is developed, the tissue is counterstained by hematoxylin, differentiated by 1% hydrochloric acid alcohol, dehydrated and sealed to obtain the IHC tissue slice.

3. Results and discussion

3.1 viral load of E30A538 infected suckling mouse tissues (qPT-PCR):

using E30A538 (5 LD) ₅₀ ) Intracranial attacking 1-day-old ICR suckling mouse (n is 3), and measuring the virus load of each tissue organ of the mouse with the clinical score of more than or equal to 4 points by qRT-PCR; E30A538 was distributed in each tissue of ICR suckling mice infected with 1 day old with clinical score of 4 or more, but the average virus load in brain was the highest and reached 10 ⁴ copies/mg, higher than the mean viral load of other tissues and organs at the same time>0.15-1.4log 10. The level of E30A538 replication in the heart was slightly lower than that in the brain, with an average viral load of 9.55X 10 ³ copies/mg. (FIG. 9). Second, the viral load in the spinal cord and back fat pads, respectively, was 2.28X 10 ³ 、5.78×10 ³ copies/mg。

3.2 pathological lesions (HE) of tissues from mice infected with E30A 538:

pathological examination of stained tissue sections showed that no obvious pathological changes were observed in brain, heart, liver, spinal cord, skeletal muscle and back fat pad of the negative control group (fig. 10, 12, 14, 16, 18 and 20), and obvious pathological changes were observed in brain, heart, liver, spinal cord, skeletal muscle and back fat pad of the suckling mice after infection with E30a538, wherein the pathological changes of brain, heart and spinal cord were severe. The concrete expression is as follows: brain edema, apoptosis of brain neurons, and scattered lymphocyte infiltration occurred in the brain (fig. 11); severe necrolysis of myocardium, rupture of myocardial fibers (fig. 13); mild hepatocellular edema and sporadic lymphocytic infiltration in the liver occurred (fig. 15); the spinal cord was scattered throughout the spinal cord and infiltrated by individual lymphocytes and microglia (fig. 17); skeletal muscle scattered in single lymphocytes (fig. 19); necrosis and calcification of back fat (fig. 21).

3.3E 30A538 infection of suckling mice each tissue organ antigen distribution:

the tissue section IHC staining result is basically consistent with the tissue section HE staining result, no antigen is detected in the brain, heart, spinal cord, skeletal muscle, back fat pad (FIGS. 22, 24, 26, 28 and 30) and other tissues of the negative control group, a large amount of brown yellow E30 antigen is detected in the brain (FIG. 23) and heart (FIG. 25) of the E30A538 infected suckling mouse, positive antigen distribution is found in the spinal cord (FIG. 27), skeletal muscle (FIG. 29) and back fat pad (FIG. 31), and no positive antigen is found in the other tissues.

qPT-PCR, HE and IHC results are basically consistent, which indicates that E30A538 has strong neurotrophism, E30A538 infects a suckling mouse through IC, enters a central nerve through the brain, and then is propagated in the brain, the heart, the spinal cord, a back fat pad and skeletal muscle tissues to cause VM, VE, viral myelitis and necrotizing myocarditis of an experimental suckling mouse, and finally causes death, and the results are basically consistent with clinical characteristics of VE, VM, AFP, VMC and the like caused by human infection with E30A 538.

Example 3: application of E30 suckling mouse model in antiviral drug in-vivo evaluation

Screening application of E30 suckling mouse model in-vivo evaluation of antiviral drugs

1.1, preparation of inoculated animals: selecting ICR suckling mice of 1 day old; dividing into negative control group (1 × PBS, n-12); model group E30 (E30+1 × PBS, n ═ 12); interferon α 2a, interferon γ treatment group (E30+ drug +1 × PBS, n — 12 × 3); interferon alpha 2a and interferon gamma drug control groups (drug +1 × PBS, n is 12 × 3), which are isolated according to groups under the same environment and are breast-fed;

1.2, preparing virus liquid and medicine: the E30A538 isolate was diluted with sterile 1 XPBS buffer to prepare 5LD ₅₀ 2mL of E30A538 virus solution; respectively preparing medicines with different concentrations by using sterile 1 multiplied by PBS buffer solution, wherein interferon alpha 2a (10000U) and interferon gamma (10000U) are respectively administrated at single dose;

1.3, animal inoculation: at 0dpi, the E30 model and treatment groups were IC-injected with 20 μ l of E30A538 virus solution (5 LD) ₅₀ Mice), negative control group, drug control group IC injected with the same volume of 1 × PBS; injecting medicaments with corresponding treatment doses into the mice of the treatment group and the mice of the medicament control group at 1hpi through IP, and injecting 1 multiplied by PBS with the same volume into the mice of the negative control group and the mice of the E30 model group through IP; followed by 1-6dpi, and then again with 1 × PBS. And the groups of mice were clinically observed as described in 1.1-4 of example 1, and disease progression was monitored up to 21 dpi; disease progression in the treatment group was compared to the model group.

2. Results and discussion

1 day old ICR mice (n-12) were infected with E30a538 (5 LD) by IC ₅₀ ) 1hpi first time laterAdministering interferon alpha 2a or interferon gamma therapy; re-dosing at 24hpi with the same dose of drug, and continuing to dose 7 dpi; the negative control was injected with the same volume of sterile 1 × PBS, and the drug control group was not inoculated with virus and was given the corresponding dose of drug directly. It can be seen from fig. 32 that the body weight of the mice in the interferon α 2 a-treated group was significantly increased compared to the E30 model group (fig. 32). As can be seen in fig. 35, clinical symptoms were reduced compared to the E30 model group, the E30 model group showed moribund symptoms, the interferon α 2 a-treated group was lean, the interferon α 2a control group and the negative control group were in a healthy state (fig. 35), and the interferon γ -treated mice showed severe clinical symptoms, all died before 13 dpi; by 21dpi, the survival rates of the interferon alpha 2 a-treated mice and the interferon gamma-treated mice were 66.67% and 0, respectively (fig. 33), and the clinical scores were 2.17 and 5, respectively (fig. 34). Therefore, interferon gamma does not show the anti-E30 capability in vivo, and is possibly high expression of IFN-gamma after neuron infection virus, so that immunopathological reaction is aggravated, and interferon alpha 2a can effectively inhibit E30A538 from replicating in vivo, and the survival rate of newborn mice is improved.

It should be noted that: significant differences in clinical scores and mouse weight changes between drug-treated and model mice within 21dpi were compared using Dunnett's m mu multiple complexes test. Error bars represent SEM,. P <0.0001, ns indicates no significance. Mantel-Cox log rank test analysis of significant differences in survival rates between drug-treated and model group mice, P <0.0001, ns indicates no significance.

In conclusion, the invention establishes an ICR wild suckling mouse model infected by the E30 isolate, the model has good repeatability, simultaneously simulates the clinical characteristics of VM, VE, VMC and AFP, and is suitable for the pre-clinical research of E30 infection characteristics, pathogenesis and vaccines and antiviral drugs. When the model is applied, the interferon alpha 2a is proved to have effective anti-E30 effect in vivo and in vitro, and a basis is provided for clinical treatment of E30 related diseases.

Although embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are exemplary and not to be construed as limiting the present invention, and that changes, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.

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Claims

1. A method for constructing an echovirus 30 type wild suckling mouse model is characterized by comprising the following steps:

step 1: animal inoculation: the infection route of intracranial injection is adopted, and the concentration of E30A538 virus liquid is 3LD ₅₀ -5LD ₅₀ 10-30. mu.l of infectious dose, followed byThe infected strain with the age of 1-3 days is any one of ICR mouse, BALB/c mouse and KM mouse;

step 2: and (3) clinical observation: and (3) carrying out isolated breeding on the mice inoculated in the step (1), continuously carrying out clinical observation on the inoculated mice, carrying out clinical scoring according to clinical observation results, and screening the mice inoculated with hind limb paralysis and/or death, namely the echovirus 30 type wild suckling mouse model.

2. The method for constructing an echovirus 30 type wild suckling mouse model according to claim 1, comprising the steps of:

the infection route of IC injection is adopted in the step 1, and the concentration of the E30A538 virus liquid is 3LD ₅₀ -5LD ₅₀ The infection product line inoculated with the infection of 1 day of age of day is any one of ICR mouse, BALB/c mouse and KM mouse.

3. The method of claim 1, wherein the infection route of IC injection is adopted in step 1, and the E30A538 virus solution concentration is 5LD ₅₀ The ICR mouse was inoculated as an infected strain with an age of 1 day of infection.

4. The method of claim 1, wherein said 3LD is 3LD ₅₀ -5LD ₅₀ Middle 3LD ₅₀ Is 10 ^3.685 TCID ₅₀ ，5LD ₅₀ Is 10 ^3.907 TCID ₅₀ 。

5. The method for constructing the Eicovirus 30 type wild suckling mouse model according to any one of claims 1 to 4, wherein the step 1 of animal inoculation is performed with a selection of infection dose, a selection of day of infection, a selection of infection pathway and a selection of infection strain, wherein the selection of infection dose, the selection of day of infection, the selection of infection pathway or the selection of infection strain comprises the following steps:

step 1-1: preparation of inoculated animals: setting corresponding experimental groups and negative control groups, wherein the sample amount of each group is 3-15, and isolating and feeding the groups in the same environment;

6. The method for constructing the echovirus 30 type wild suckling mouse model according to claim 5, wherein in the animal inoculation of the step 1 to 3, the inoculation of an experimental group is specifically as follows:

serial 10-fold dilution of E30A538 with sterile diluent ^-2 -10 ^-8 I.e. 10 ^7.375 TCID ₅₀ -10 ^1.375 TCID ₅₀ The concentration is/mL, and then the 1-day-old ICR mice are inoculated with IC for screening the infection dose;

inoculation of 5LD with 1 day old ICR mice by IC, IP or IM ₅₀ ：10 ^3.907 TCID ₅₀ E30A538 from mice, for use in screening for infection pathways;

7. The method of claim 6, wherein the Eicovirus type 30 wild suckling mouse model is constructed byThe TCID of the E30A538 virus fluid ₅₀ Measured by the Spearman-Karrer method.

8. Use of a suckling mouse model constructed by the method for constructing an echovirus type 30 wild suckling mouse model according to any one of claims 1 to 7, wherein the suckling mouse model is used for screening and evaluating antiviral drugs or vaccines.

9. A method for screening and evaluating an antiviral drug, comprising the steps of:

step 21: preparation of inoculated animals: selecting an ICR mouse of 1 day old; dividing into a negative control group, an E30 model group consisting of suckling mouse models constructed by the construction method of the echovirus E30 type wild suckling mouse model as claimed in any one of claims 1 to 7, a drug treatment group and a drug control group; the sample volumes of the negative control group, the E30 model group, the drug treatment group and the drug control group are the same and are respectively 6-15, and the negative control group, the E30 model group, the drug treatment group and the drug control group are isolated and raised according to groups under the same environment;

step 22: preparing virus liquid and medicines: the preparation concentration is 5LD ₅₀ E30a538 virus solution of (a); respectively preparing medicines with different concentrations by using sterile buffer solution;

step 23: animal inoculation: the E30 model group and the drug treatment group were injected with 20. mu.l of E30A538 virus solution 5LD by IC injection ₅₀ Mice, negative control group, drug control group were injected with IC with the same volume of 1 × PBS; after 1hour of virus infection, injecting medicaments with corresponding treatment doses into the mice of the treatment group and the mice of the medicament control group through IP, and injecting buffer solution with the same volume into the mice of the negative control group and the mice of the E30 model group through IP; re-administration or buffer on days 1-6 post infection;

step 24: and (4) carrying out clinical observation on the mice in the step (23), comparing the weight change of the mice, the survival rate of the mice and the clinical scores of the mice, and screening out the medicine with good curative effect on the echovirus 30.

10. The method for screening and evaluating an antiviral drug according to claim 9, wherein the drug treatment groups in step 21 are: interferon alpha 2a drug treatment group and interferon gamma drug treatment group; the drug control group in step 21 is: an interferon alpha 2a drug control group and an interferon gamma drug control group; the single administration dose of the medicine with the therapeutic dose in the step 23 is interferon alpha 2a10000U and interferon gamma 10000U.