Construction method of mouse-adapted coxsackievirus a4 strain and infection model thereof

Abstract

The application discloses a mouse-adapted coxsackievirus A4 strain and a construction method of an infection model thereof, the virus strain has a preservation number of CCTCC NO:V202432, and an ICR mouse hand-foot-mouth disease infection model of 13 days old is established by using the virus strain, and the mouse hand-foot-mouth disease infection model can present similar pathological changes to human diseases, especially hand-foot-mouth disease. Compared with the previous mouse model of less than 7 days old, the mouse hand-foot-mouth disease infection model constructed by the application solves the defects of difficult drug administration, low drug administration volume and the like of the small-day-old mouse, so that the mouse model can be applied to long-time drug efficacy observation and vaccine evaluation, and the model has very important application value in researches on the pathogenesis of HFMD, the effectiveness evaluation of vaccines, the screening and drug efficacy evaluation of antiviral drugs and the like. The application solves the technical problems that the coxsackievirus A4 strain is used to establish a model by using 1 to 7 day-old mice at present at home and abroad, and the day-old mice are small in age, difficult to administer drugs, and the infection model is not easy to prepare successfully.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of virology, and in particular to a mouse-adapted Coxsackievirus A4 strain and a method for constructing an infection model thereof. BACKGROUND

[0002] Hand foot and mouth disease (HFMD) is an infectious disease caused by multiple enteroviruses, mainly infecting infants and children under 5 years old. Enterovirus is the pathogen causing HFMD, belonging to the Enterovirus genus of the Picornaviridae family, which has 116 serotypes, and more than 20 types of enterovirus can cause HFMD, with enterovirus 71 and Coxsackievirus A16 being the most common. Before 2009, enterovirus 71 and Coxsackievirus A16 were the dominant epidemic strains. However, after 2009, the prevalence of other types of enterovirus such as CVA4, CVA6, and CVA10 has been increasing year by year.

[0003] The CVA4 virus strain was first isolated in the United States in 1948, and CVA4 infection in children mainly causes herpangina, fever, rash, and other symptoms, which adversely affects the physical and mental health of children and increases the global public health burden.

[0004] Currently, the broad-spectrum antiviral drug ribavirin is commonly used in clinical practice for the treatment of HFMD, and there is no specific drug for HFMD. The 2018 edition of the "Hand foot and mouth disease diagnosis and treatment guidelines" recommends the early use of interferon alpha and ribavirin, but only plays a symptomatic treatment role, and long-term use of ribavirin can cause reproductive toxicity. There are more researches on treatment drugs related to EVA71 (enterovirus A71) and CVA16 infection, and as the vaccination rate of EVA71 vaccine increases, the number of children infected with EV71 causing HFMD has greatly decreased, but the vaccine has poor cross-protection for different subtypes. Therefore, the focus of vaccine and drug research should be shifted to other enteroviruses.

[0005] An ideal infectious disease animal model is an important carrier for studying the transmission route, pathogenesis, and pathological damage of infectious diseases, and is also an important supporting condition for evaluating the effectiveness and safety of vaccines, drugs, and other prevention and control measures. In recent years, domestic and foreign research on animal models of hand foot and mouth disease has become increasingly important, and currently, 1 to 7-day-old mice are mainly used for modeling, but 7-day-old mice have many uncontrollable factors due to their small size, incomplete immune system and organ development, and other issues, such as difficulty in administering drugs and difficulty in preparing the infection model. Therefore, a method for constructing a hand foot and mouth disease model by infecting a large-day CVA4 mouse is needed. SUMMARY

[0006] In order to overcome the defects of the prior art, the mouse-adapted Coxsackie virus A4 strain and the construction method of the infection model are provided to solve the problems of the small size, the limited dosage and mode of administration, the incomplete development of the immune system, the difficulty in preparing the infection model, and the like of the 1 to 7-day-old mouse model of the Coxsackie virus A4 strain.

[0007] In order to achieve the above-mentioned object, a mouse-adapted Coxsackie virus A4 strain is provided, and the preservation number of the virus strain is CCTCC NO: V202432.

[0008] Further, the virus strain is derived from a clinically isolated highly pathogenic Coxsackie virus strain.

[0009] Further, the mouse-adapted Coxsackie virus A4 strain with high lethality to 7-15-day-old mice is obtained by inoculating the virus strain into 11-day-old mice through muscle inoculation and repeatedly passing the virus strain in the 11-day-old mice and human RD cells for 10 times.

[0010] Further, the cell types infected by the virus strain include African green monkey kidney cells or human rhabdomyosarcoma cells.

[0011] Further, the virus strain is applied to the preparation of vaccines and diagnostic reagents for preventing or treating infectious diseases caused by the similar Coxsackie virus strain.

[0012] The present application provides a preparation method of a virus suspension of a mouse-adapted Coxsackie virus A4 strain, wherein the virus diluent prepared by diluting the virus strain with a buffer without inactivation of the virus is inoculated into cells capable of supporting replication of the Coxsackie virus to prepare the virus suspension.

[0013] The present application provides a preparation method of a virus suspension of a mouse-adapted Coxsackie virus A4 strain, wherein the virus diluent prepared by diluting the virus strain with a buffer without inactivation of the virus is inoculated into 11-day-old mice through muscle injection, and then the animal tissues of the mice are homogenized or ground to prepare the virus suspension, which is inoculated into 11-day-old mice again through muscle injection after being amplified on RD cells, and is amplified on RD cells, and the process is repeated for 10 times to obtain the in-vivo domesticated enhanced CVA4 strain.

[0014] The present application provides a method for establishing a Coxsackie virus A4 virus infection model by using a mouse-adapted Coxsackie virus A4 strain, which comprises the following steps:

[0015] The virus suspension of the mouse-adapted Coxsackie virus A4 strain is prepared into a virus suspension diluent by using a buffer without inactivation of the virus;

[0016] The Coxsackie virus A4 virus infection model is prepared by muscle injection of the virus suspension diluent into the mice.

[0017] Further, the mouse age is 7-15 day old mice.

[0018] Further, the virus titer of the 7-15 day old mice infected by intramuscular injection can range from 10-10 5 TCID 50 / each.

[0019] Further, the application also includes the use of the coxsackievirus A4 virus infection model for studying the pathogenesis of HFMD, evaluating the effectiveness of vaccines and screening of antiviral drugs.

[0020] The beneficial effects of the application are that the mouse adapted coxsackievirus A4 strain is a new coxsackievirus CVA4 mouse adapted strain with a titer of 10 8.8 TCID 50 , and the 7-day-old mouse hand-foot-mouth disease infection model established by the mouse can present similar pathological changes to human diseases, especially hand-foot-mouth disease. Compared with the previous 1, 3 or 7-day-old mouse model, the mouse hand-foot-mouth disease infection model constructed by the application solves the defects of small day-old mouse administration difficulty, low administration volume, etc., so that the mouse model can be suitable for long-term drug efficacy observation and vaccine evaluation. The model plays a very important role in studying the pathogenesis of HFMD, evaluating the effectiveness of vaccines and screening of antiviral drugs. BRIEF DESCRIPTION OF DRAWINGS

[0021] Other features, objects and advantages of the application will become more apparent from the following detailed description of non-limiting embodiments made with reference to the accompanying drawings:

[0022] Figure 1 CVA4 infection of RD cells caused cytopathic effect.

[0023] Figure 2 CVA4 mouse adapted strain YZ08 whole genome phylogenetic tree.

[0024] Figure 3 CVA4 mouse adapted strain YZ08 infection of mice to determine the best infection age.

[0025] Figure 4 CVA4 mouse adapted strain YZ08 infection of mice to determine the best infection dose.

[0026] Figure 5 CVA4 mouse adapted strain YZ08 infection of mice to determine the best infection route.

[0027] Figure 6 CVA4 mouse adapted strain YZ08 infection of mice of different ages for 3 days for analysis of viral load in each organ.

[0028] Figure 7 Histopathological examination of the mouse model infected with the CVA4 mouse-adapted strain YZ08. DETAILED DESCRIPTION

[0029] The application will be further described below in conjunction with the drawings and examples. It can be understood that the specific examples described herein are only used to explain the related application, and not to limit the application. In addition, it should be noted that only the parts related to the application are shown in the drawings for ease of description.

[0030] It should be noted that the examples in the present application and the features in the examples can be combined with each other without conflict. The present application will be described in detail below with reference to the drawings and in conjunction with the examples.

[0031] The present application provides a mouse-adapted coxsackievirus strain A4, and the biological material preservation instruction thereof is as follows:

[0032] Strain number: V202432;

[0033] Classification name: Coxsackievirus strain A4 / Yangzhou / YZ08 / 2018;

[0034] Preservation unit name: China Center for Type Culture Collection;

[0035] Preservation unit abbreviation: China Center for Type Culture Collection (CCTCC);

[0036] Preservation unit address: China, Wuhan, Wuhan University Preservation Center, Postcode: 430072;

[0037] Preservation date: March 20, 2024;

[0038] Preservation number: CCTCC NO: V202432.

[0039] In the present example, the virus strain is derived from a clinically isolated highly pathogenic coxsackie strain.

[0040] Specifically, the virus strain is a mouse-adapted coxsackievirus strain A4 with high lethality to 7-15-day-old mice, which is obtained by repeatedly passing the virus strain through 11-day-old mice muscle inoculation in human RD cells for 10 times.

[0041] The cell types infected by the virus strain include African green monkey kidney cells or human rhabdomyosarcoma cells.

[0042] The virus strain of the present application is used in the preparation of vaccines and diagnostic reagents for preventing or treating infectious diseases caused by similar coxsackievirus strains.

[0043] The present application provides a method for preparing a virus suspension of a mouse-adapted Coxsackie virus A4 strain, wherein a virus diluent prepared by diluting the virus strain with a buffer without inactivation of the virus is inoculated into cells capable of supporting replication of the Coxsackie virus to prepare the virus suspension.

[0044] The present application provides a method for preparing a virus suspension of a mouse-adapted Coxsackie virus A4 strain, wherein a virus diluent prepared by diluting the virus strain with a buffer without inactivation of the virus is inoculated into cells capable of supporting replication of the Coxsackie virus to prepare the virus suspension.

[0045] The present application provides a method for establishing a Coxsackie virus A4 model of infection using a mouse-adapted Coxsackie virus A4 strain, comprising the following steps:

[0046] The virus suspension of the mouse-adapted Coxsackie virus A4 strain is prepared using a buffer without inactivation of the virus to obtain a virus diluent;

[0047] The Coxsackie virus A4 model of infection is prepared by intramuscular injection of the virus diluent into a mouse.

[0048] Preferably, the mouse is a 7-15 day old mouse. Preferably, the intramuscular injection of the virus into the 7-15 day old mouse has a virus titer ranging from 10-10 5 TCID 50 / mouse.

[0049] The method for establishing a Coxsackie virus A4 model of infection using a mouse-adapted Coxsackie virus A4 strain of the present application further comprises using the Coxsackie virus A4 model of infection to study the pathogenesis of HFMD, evaluate the effectiveness of vaccines, and screen antiviral drugs.

[0050] The experimental methods used in the following examples are conventional methods unless otherwise specified.

[0051] The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

[0052] The experimental animals used in the following examples are SPF grade ICR (Institute of Cancer Research) mice, and the mice are raised and handled in accordance with the animal welfare and ethical guidelines of the Jiangsu Province Experimental Animal Management Committee.

[0053] Statistical methods in the following examples were analyzed by GraphPad Prism 5 software. Reed-Muench method was used to calculate the tissue viral load and TCID 50 The quantitative variables were analyzed by mean and t-test, and P<0.05 was considered as significant difference.

[0054] TCID 50 in the following examples refers to the median tissue culture infectious dose, i.e. the amount of virus required to cause cytopathic effect or death (CPE) in half of the cells in a well of a culture plate.

[0055] In the following examples, the standard for clinical score is:

[0056] 0 points, healthy;

[0057] 1 point, skin fold, spirit, limbs uncoordinated or single leg paralysis;

[0058] 2 points, both legs paralyzed;

[0059] 3 points, both legs completely paralyzed or have legs folded back;

[0060] 4 points, quadriplegia;

[0061] 5 points, on the verge of death or death.

[0062] The culture medium used in the following examples is DMEM (Dulbecco's modified eagle medium) culture medium containing 10% fetal bovine serum and 1% penicillin-streptomycin mixture.

[0063] Example 1, collection, isolation and identification of CVA4 virus strain.

[0064] The CVA4 virus strain of the present application is a highly pathogenic strain isolated from the clinic, which is obtained after being repeatedly passed through 11-day-old mice for 10 times, and does not belong to a natural strain, but a domesticated and modified strain. The CVA4 virus strain of the present application is a CAV4 virus strain obtained by plaque purification after being repeatedly passed through 11-day-old mice for 10 times, and the virus liquid obtained by RD cell amplification.

[0065] Specifically, the method for obtaining the CVA4 virus strain of the present application comprises the following steps:

[0066] S1, collection.

[0067] The throat swab samples of children with HFMD in hospitals in Jiangsu Province, China in 2018 were collected, transported at low temperature and stored in a -80℃ refrigerator.

[0068] S2, isolation and culture.

[0069] RD cells (human sarcoma cells) in logarithmic growth phase were plated into 24-well cell plates and cultured in DMEM medium containing 10% fetal bovine serum, and after the cells reached 80% confluence, the cell plates were washed twice with PBS, and then the sample collected in step S1 was centrifuged at 12000 rpm for 10 minutes, and the supernatant was inoculated into the RD cell culture plate, and incubated at 37°C, 5% CO2 for 1 hour, and then 2% fetal bovine serum-containing DMEM medium was added and cultured for 48-96 hours until 80-90% of the cells showed cytopathic effect (CPE), and the cell culture solution was collected. The aforementioned CVA4 virus solution was injected intramuscularly into 11-day-old mice, and then the animal tissues of the mice were homogenized or ground to prepare a virus suspension, which was amplified on RD cells and then injected intramuscularly into 11-day-old mice, and the RD cells were amplified, and this process was repeated 10 times to obtain a CVA4 mouse-adapted strain YZ08 with enhanced in vivo domestication, which was stored in a -80°C refrigerator.

[0070] S3, identification.

[0071] 200 μL of the CVA4 mouse-adapted strain YZ08 virus solution stored in step S2 was taken out, and the viral RNA was extracted by the Trizol method, and the cDNA was synthesized by RT-PCR method using Hiscript II first strand enzyme reverse transcription, and the CVA4 sequence primers were designed in sections, and the viral genome fragments were amplified in sections, and the obtained PCR products were sent to a sequencing company for sequencing, and after sequencing, the whole gene sequence was spliced according to the homologous alignment. See Figure 2 As shown in the figure, the neighbor-joining method was used to construct a genetic evolution tree based on the whole genome alignment of the CVA4 mouse-adapted strain YZ08 virus strain, and it was determined that the genotype of the CVA4 mouse-adapted strain YZ08 virus strain was type C.

[0072] S4, TCID of CVA4 mouse-adapted strain YZ08 50 determination.

[0073] See Figure 1 (Picture scale is 10 μm), and an appropriate amount of CVA4 mouse-adapted strain YZ08 virus solution was diluted with PBS to 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 , 10 -88 dilutions, then 50 μL of each dilution was inoculated into 96-well cell plates with RD cells, after 1 hour of incubation at 37℃, 5% CO2, cell maintenance solution was added, and the culture was continued for 72-96 hours, the cytopathic effect was observed daily, after more than 80% of the cells showed obvious cytopathic effect, the cell plate was inactivated by formalin fixation, then the cells were dyed with 0.5% crystal violet, and finally the virus titer of the CVA4 mouse-adapted strain YZ08 was quantified as 10 8.8 TCID 50 , and stored at -80℃ for use in subsequent examples.

[0074] Example 2, determination of the optimal age of mice infected with the CVA4 mouse-adapted strain YZ08

[0075] Referring to Figure 3 , 7, 9, 11, 13, and 15-day-old mice (5 groups) were selected, each group had 8-10 mice of the same age, and the mice were injected intramuscularly with 100 TCID 50 / mouse of the CVA4 mouse-adapted strain YZ08 virus strain. After the mice were infected, the changes in body weight, clinical scores, and survival rates of the mice were observed and recorded daily.

[0076] Figure 3 In the table, A is the body weight change rate of 7, 9, 11, 13, and 15-day-old mice infected with CVA4, B is the clinical score of 7, 9, 11, 13, and 15-day-old mice infected with CVA4, C is the survival curve of 7, 9, 11, 13, and 15-day-old mice infected with CVA4, and D is the survival days of 7, 9, 11, 13, and 15-day-old mice infected with CVA4.

[0077] The death results of mice infected with the CVA4 mouse-adapted strain YZ08: all 7-day-old mice died within 5 days after inoculation, all 9-day-old and 11-day-old mice died on the 7th day after inoculation, all 13-day-old mice died on the 10th day after inoculation, and 15-day-old mice began to show symptoms such as typical hind limb paralysis after 3 days of infection, and no death was observed, indicating that the CAV4 mouse-adapted strain YZ08 described in the application is highly lethal to 7-13-day-old mice. Since the 9-day-old and 11-day-old mice are too small, the application preferably uses 13-day-old mice as a CAV4 disease infection model.

[0078] Example 3, determination of the optimal infection dose of the CVA4 mouse-adapted strain YZ08

[0079] Referring to Figure 4 , 13-day-old ICR mice were selected, each group had 8-10 mice, and the mice were inoculated with 10 TCID 50 , 102 TCID 50 , 10 3 TCID 50 , 10 4 TCID 50 , 10 5 TCID 50 The CVA4 mouse-adapted YZ08 strain virus dilution was used to infect the suckling mice, and the body weight changes, clinical scores and survival rates of the suckling mice were observed and recorded every day after infection.

[0080] Figure 4 In the table, A is the body weight change rate of 13-day-old suckling mice infected with different doses of CVA4 virus (10-10 5 TCID 50 ), B is the clinical score of 13-day-old suckling mice infected with different doses of CVA4 virus (10-10 5 TCID 50 ), C is the survival curve of 13-day-old suckling mice infected with different doses of CVA4 virus (10-10 5 TCID 50 ), and D is the survival days of 13-day-old suckling mice infected with different doses of CVA4 virus (10-10 5 TCID 50 ).

[0081] Death results of mice infected with different doses of CVA4 mouse-adapted YZ08 strain: 10 5 TCID 50 The 13-day-old ICR suckling mice inoculated with 10 4 TCID 50 and 10 3 TCID 50 also began to show symptoms at 2 days after infection, and reached the death peak at 6 days and 7 days after infection, respectively, and the suckling mice were all dead at 7 days and 8 days after infection, respectively. The 13-day-old ICR suckling mice inoculated with 100 TCID 50 began to show symptoms at 3 days after infection, hind limb paralysis began at 4 days after infection, forelimb paralysis began at 6 days after infection, and death began at 7 days after infection, with a final mortality rate of 83.3%. The 13-day-old ICR suckling mice inoculated with 10 TCID 50 began to show symptoms at 3 days after infection, death began at 8 days after infection, and clinical symptoms began to recover at 8 days after infection, with a final mortality rate of 50%. Since the onset time of the 13-day-old ICR suckling mice inoculated with 100 TCID 50 was close to the clinical incubation period, the present application preferably uses 100 TCID 50 to infect 13-day-old suckling mice as a CVA4 disease infection model for evaluating the protective efficacy of drugs or vaccines.

[0082] Example 4, Determination of the optimal inoculation route of CVA4 mouse-adapted strain YZ08

[0083] Referring to Figure 5 , 13-day-old ICR mice were selected, 8-10 in each group, and were inoculated with 100 TCID 50 of CVA4 mouse-adapted strain YZ08 virus strain diluent by intramuscular injection (IM), intraperitoneal injection (IP), and oral gavage (IG), respectively. After the mice were infected, the changes in body weight, clinical scores, and survival rates of the mice were observed and recorded daily.

[0084] Figure 5 In the table, A is the body weight change rate of 13-day-old mice infected by intramuscular, intraperitoneal, and gavage, B is the clinical score of 13-day-old mice infected by intramuscular, intraperitoneal, and gavage, C is the survival curve of 13-day-old mice infected by intramuscular, intraperitoneal, and gavage, and D is the survival days of 13-day-old mice infected by intramuscular, intraperitoneal, and gavage.

[0085] Death results of mice infected by different inoculation routes of CVA4 mouse-adapted strain YZ08:

[0086] The IM and IP groups showed paralysis on the 2nd day after infection, and the IG group of mice did not show obvious clinical symptoms after infection, and showed a transient decrease in body weight on the 7th day after infection, and then steadily increased. The IM group died completely on the 8th day after infection, and the IP group died completely on the 9th day after infection. The body weight reduction rate and clinical score of the IM group were slightly higher than those of the IP group, but there was no significant difference, which indicated that both IM and IP methods could successfully infect mice, but mice were more sensitive to intramuscular injection, so the intramuscular injection was finally selected as the best infection route.

[0087] For the above reasons, the present application preferably uses 100 TCID 50 of the infection dose to infect 13-day-old mice by intramuscular injection as a CVA4 disease infection model for evaluating the protective efficacy of drugs or vaccines.

[0088] Virus infection model is an extremely important experimental method in the field of medical research, and is a basic condition for studying the pathogenesis of virus and screening vaccines and antiviral drugs. The reason for selecting ICR mice as the infected animals is that the ICR mice are larger in weight than BALB / c and C57 mice of the same age, more sensitive to coxsackie group A virus, and the CVA4 mouse-adapted strain YZ08 infection of the suckling mice is selective and dependent on three factors of inoculation dose, infection route and age of the suckling mice. The CVA4 mouse-adapted strain YZ08 can make the suckling mice get sick fastest through the IM route, the muscle tissue can quickly absorb the CVA4 mouse-adapted strain YZ08 and make the virus spread throughout the body along the circulatory system. The small age suckling mice (≤13) are most susceptible to the CVA4 mouse-adapted strain YZ08, showing a high mortality rate, and the immune system of the large age suckling mice (≥15) has gradually developed, and the symptoms are mild or even asymptomatic after inoculation of the CVA4 mouse-adapted strain YZ08, which is not suitable for establishing a virus infection model. Through the optimal combination of infection conditions, the CVA4 mouse-adapted strain YZ08 virus strain suckling mouse infection model can realize high repeatability and stability of subsequent experiments.

[0089] Example 5, Detection of virus load in different organs of CVA4 mouse-adapted strain YZ08 virus strain infected suckling mouse animal model

[0090] Infection group: 7, 9, 11, 13, 15-day-old suckling mice were infected with a lethal dose of CVA4 mouse-adapted strain YZ08 virus strain (100 TCID 50 / suckling mouse) by IM.

[0091] Negative control group: 7-day-old suckling mice were inoculated with PBS buffer containing no CVA4 mouse-adapted strain YZ08 virus by IM.

[0092] On 3 dpi after infection of different age suckling mice with CVA4 mouse-adapted strain YZ08 virus, the heart, lung, hind limb muscle, liver, spleen, kidney, small intestine and brain of the infection group and the negative control group suckling mice (3 suckling mice in each group) were taken, and after tissue grinding, the supernatant was obtained by centrifugation, and the virus load in each organ was determined, and the change trend of the virus load in different organs with time was monitored, and the specific operation method was as follows: an appropriate amount of tissue grinding supernatant was diluted by 10 times gradient (10 -1 , 10 -2 ... 10 -8 ), 50 μL of tissue supernatant diluent was added to a 96-well cell culture plate of RD cells, 3 parallel holes were set for each dose, and a normal control group was also set, and after adsorption at 37℃, 5% CO2 for 1 h, DMEM maintenance solution was used for continuous culture for 72 h. The degree of pathological change and the number of holes were observed and recorded under an inverted microscope every day, and after 72 h, the cell plate was fixed with 10% formaldehyde aqueous solution, stained with crystal violet, and finally the virus load in the tissue supernatant was calculated according to the Reed-Muench method.

[0093] With reference to Figure 6 (TCID 50 The results showed that muscle tissue was the main site of CVA4 mouse-adapted strain YZ08 virus replication, and the rapid replication of the virus was consistent with the clinical manifestations of muscle bundle rupture and hind limb paralysis in the suckling mice.

[0094] Example 6, Histopathological examination of CVA4 mouse-adapted strain YZ08 virus infection in a suckling mouse animal model

[0095] A 13-day-old suckling mouse model was injected with 100 TCID 50 of CVA4 mouse-adapted strain YZ08 virus solution, and on the 3rd day after infection, the skeletal muscle tissue of the suckling mouse was collected. The pathological changes of skeletal muscle, lung, small intestine and brain were observed by HE staining to study the histopathological changes caused by CVA4 mouse-adapted strain YZ08 infection.

[0096] CVA4 mouse-adapted strain YZ08 infection histopathology results:

[0097] With reference to Figure 7 (Picture scale is 200 μm), after the suckling mouse was infected with CVA4 mouse-adapted strain YZ08, skeletal muscle tissue showed skeletal muscle fiber rupture and a large number of inflammatory cell infiltration; the lungs showed uneven alveolar size, alveolar wall damage, thickened alveolar septum, vascular dilation and hyperemia with a large number of inflammatory cell infiltration; the small intestine villi showed obvious hyperemia, small intestine mucosa ulceration with inflammatory cell infiltration; a large number of neuron cells degeneration, necrosis, inflammatory cell infiltration and reduced number of neurons were observed in the brain.

[0098] The above description is merely preferred embodiments of the present application and a description of the principles of the technology used. It will be understood by those skilled in the art that the scope of the application involved in the present application is not limited to the technical solutions formed by the specific combinations of the above technical features, and also covers other technical solutions formed by any combination of the above technical features or their equivalent features without departing from the inventive concept. For example, the above features are replaced with each other to form technical solutions with similar functions disclosed in the present application (but not limited to).

Claims

1. A mouse-adapted Coxsackie A4 virus strain, characterized in that The deposit number of the virus strain is CCTCC NO: V202432.

2. The mouse-adapted Coxsackie A4 virus strain according to claim 1, characterized in that The virus strain is derived from a highly pathogenic Coxsackie virus strain isolated from a clinic.

3. The mouse-adapted Coxsackie A4 virus strain according to claim 2, characterized in that The highly pathogenic Coxsackie virus strain isolated from the clinic was inoculated into 11-day-old suckling mice through mouse muscle, and then repeatedly passaged with human sarcoma cells 10 times to obtain a mouse-adapted Coxsackie A4 virus strain that is highly lethal to 7-15-day-old suckling mice.

4. A method for preparing a virus suspension of a mouse-adapted Coxsackie A4 virus strain as claimed in claim 1, characterized in that: The virus suspension is prepared by diluting the virus strain with a buffer solution without virus inactivation to obtain a virus dilution, and inoculating cells capable of supporting the replication of Coxsackie virus.

5. A method for establishing a Coxsackievirus A4 infection model using a mouse-adapted Coxsackievirus A4 strain, characterized in that: The following steps are involved: The virus suspension prepared by the preparation method of claim 4 is prepared with a buffer solution having no virus inactivation effect to obtain a virus suspension dilution solution; The virus suspension dilution was injected intramuscularly into suckling mice to prepare a Coxsackievirus A4 infection model.

6. The method according to claim 5, characterized in that The suckling mice are 7-15 days old.

7. The method according to claim 5, characterized in that The virus titer range of the intramuscular infection of 7-15 day old suckling mice was 10-10 5 TCID 50 / Only.

8. The method according to claim 5, characterized in that It also includes using the Coxsackievirus A4 virus infection model to study the pathogenesis of hand, foot and mouth disease, evaluate the effectiveness of vaccines and screen antiviral drugs.

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