Construction method and application of monkey pox virus infected immunodeficiency mouse model

Abstract

The invention relates to a construction method and application of a monkey pox virus infected immunodeficiency mouse model, and belongs to the technical field of gene engineering and virus infection. The invention provides a construction method of a monkey pox virus infected immunodeficiency mouse model, which comprises the following steps of: knocking out interferon I, interferon II and interferon III passages by using a CRISPR / Cas9 technology, specifically knocking out a mouse I type interferon receptor IFNAR, knocking out a mouse II type interferon receptor IFNGR and knocking out a mouse III type interferon receptor IFNLR. The mouse constructed by the method disclosed by the invention has the severe condition of infection of the monkey pox virus IIb branch, and can be used for screening medicines for treating infection of the monkey pox virus IIb subtype branch.

Description

Technical Field

[0001] The invention relates to the technical field of genetic engineering and virus infection, and in particular to a method for constructing an immunodeficient mouse model infected with monkeypox virus and its application. Background Art

[0003] Monkeypox virus (MPXV) is an enveloped double-stranded DNA virus belonging to the genus Orthopoxvirus of the Poxviridae family. Its family members include human pathogenic viruses such as smallpox virus, cowpox virus, and vaccinia virus (Nat Microbiol 2024, supra). Smallpox virus was once an important virus that caused a pandemic, and the high mortality rate caused has always been a painful memory for mankind. In 2003, a local outbreak of monkeypox virus infection broke out in the United States. It was small in scale, with less than 100 cases, and was quickly quelled (J Infect Dis 2022, doi:10.1093 / infdis / jiaa559). However, this monkeypox virus outbreak has been going on for two years and is still spreading outside of Africa, posing a health threat to vulnerable populations. It can be seen that the monkeypox virus jumping out of Africa once again suggests that zoonotic poxviruses may once again affect public health security.

[0004] Monkeypox virus (MPXV) has existed in primates for a long time. From an evolutionary perspective, it has produced two major branches: I (locally transmitted in West Africa) and II (locally transmitted in Central Africa), of which branch II is further divided into IIa and IIb. The mortality rate caused by infection with branch I of monkeypox virus exceeds 10%, and the mortality rate caused by infection with branch IIa of monkeypox virus is about 3%. The mortality rate caused by infection with branch IIb is the lowest, but it still has a certain mortality rate. Fortunately, this round of global monkeypox virus epidemic was caused by infection with branch IIb, which ultimately did not cause a large number of deaths. However, on the African continent, monkeypox viruses I and IIa are still active (Nat Rev Immunol 2023, doi:10.1038 / s41577-022-00775-4), and their potential global threat cannot be underestimated as the process of global integration advances.

[0005] Currently, the prevention and treatment of monkeypox virus is extremely limited. The US FDA has only approved two vaccines, ACAM2000 and JYNNEOS (live attenuated vaccine), for pre-exposure prevention of orthopoxvirus (including varicella vaccine (VARV) and MPXV) infection, but only for the American population. In terms of drugs, only three drugs, Cidofovir, Brincidofovir and Tecovirimat, can be used to treat monkeypox (Signal Transduct Target Ther 2023, Mpox (formerly monkeypox): pathogenesis, prevention and treatment). Although new drugs and new vaccines are already under development, they still need to be clinically verified before they can be officially put into use.

[0006] An important link limiting the development of antiviral drugs and vaccines is the lack of pathogenic models of the monkeypox virus subclade IIb. Non-human primates support monkeypox virus infection, but do not show obvious clinical symptoms, especially the rate of severe illness is very low (Infect Med2023, doi:10.1016 / j.imj.2023.05.004); moreover, the high cost of non-human primate models also limits related exploration. Among rodents, CAST / EiJ mice can support monkeypox virus infection and are widely used. It should be emphasized that CAST / EiJ mice can show weight loss and death characteristics after infection with monkeypox virus subclade I and IIa; however, monkeypox virus subclade IIb infection of CAST / EiJ mice is not pathogenic, and cannot cause weight loss, death, etc., and the viral load is also very low (PNAS2022, / doi.org / 10.1073 / pnas.2220415120). However, the lack of an effective mouse model for monkeypox virus infection will seriously affect the development of antiviral drugs and vaccines for monkeypox virus. Summary of the invention

[0007] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a method for constructing an immunodeficient mouse model infected with monkeypox virus and its application. The immunodeficient mouse model infected with monkeypox virus is established through this scheme.

[0008] To achieve the above object, the technical solution adopted by the present invention is:

[0009] In a first aspect, the present invention provides a method for constructing an immunodeficient mouse model infected with monkeypox virus, comprising the steps of knocking out interferon I, interferon II and interferon III pathways using CRISPR / Cas9 technology.

[0010] In the present invention, after we simultaneously knock out the three types of interferon pathways, I, II and III, the natural immunity of mice will be more severely defective, which will increase the disease sensitivity of mice and will better support the replication and pathogenicity of monkeypox virus.

[0011] As a preferred embodiment of the first aspect, the knockout of the three types of interferon pathways I, II, and III includes knocking out the mouse type I interferon receptor IFNAR gene, knocking out the type II interferon receptor IFNGR gene, and knocking out the type III interferon receptor IFNLR gene.

[0012] As a preferred embodiment of the first aspect, the mouse type I interferon receptor IFNAR includes two subunits, IFNAR1 and IFNAR2; the type II interferon receptor IFNGR is the IFNGR2 subunit; and the type III interferon receptor IFNLR is the IFNLR2 subunit.

[0013] The mouse interferon receptor genes are located on chromosomes 4, 7, and 16, respectively. Considering that mouse Ifnar2, Ifnlr2, Ifnar1, and Ifngr2 are arranged in tandem on chromosome 16, this 200kb region containing four genes can be knocked out through a single genetic operation, thereby obtaining mice with severe natural immunity deficiency in which the IFNAR, IFNGR, and IFNLR pathways are simultaneously knocked out in one step.

[0014] As a preferred embodiment of the first aspect, the method for knocking out IFNAR1, IFNAR2, IFNGR2 and IFNLR2 comprises the following steps:

[0015] (1) designing and in vitro transcribing sgRNA sequences for the 5'-specific target site and the 3'-specific target site; the sgRNA sequence for the 5'-specific target site includes the nucleotide sequences shown in SEQ ID NOs. 1 to 2; the sgRNA sequence for the 3'-specific target site includes the nucleotide sequences shown in SEQ ID NOs. 3 to 4;

[0016] (2) Cas9, sgRNA plasmids for the 5'-specific target site, and sgRNA plasmids for the 3'-specific target site were simultaneously injected into mouse fertilized eggs, and the fertilized eggs were transplanted into pseudo-pregnant female mice to obtain F0 generation mice. The F0 generation mice were further self-fertilized to obtain F1 generation mice in which the Ifnar2, Ifnlr2, Ifnar1, and Ifngr2 genes were simultaneously knocked out, thus obtaining an immunodeficient mouse model infected with monkeypox virus.

[0017] In a second aspect, the present invention provides an sgRNA sequence for knocking out mouse interferon I, interferon II and interferon III pathways, wherein the sequence is shown in any one of SEQ ID NOs. 1 to 4.

[0018] In a third aspect, the present invention provides the use of the sgRNA sequence in knocking out the Ifanr1 gene, Ifnar2 gene, Ifngr2 gene, and Ifnlr2 gene of mice.

[0019] In a fourth aspect, the present invention provides a PCR primer for identifying Ifanr1- / -Ifnar2- / -Ifngr2- / -Ifnlr2- / - genotype mice, wherein the primer comprises a nucleotide sequence as shown in SEQ ID NOs. 5 to 8.

[0020] In a fifth aspect, the present invention provides a method for identifying functional defects of three interferon pathways, I, II, and III, comprising the following steps:

[0021] Step 1: Isolating mouse fibroblasts constructed by the method described in the first aspect;

[0022] Step 2: Stimulate fibroblasts with IFNα4, IFNγ and IFNλ2 cytokines respectively;

[0023] Step 3: extract RNA from the cells in step 2, and detect the expression of Mx1, Ifit1 and Isg15 genes in the cells. If Mx1, Ifit1 and Isg15 genes are not expressed, it indicates that the functions of the three interferon pathways I, II and III are defective.

[0024] In a sixth aspect, the present invention provides the use of mice constructed by the method in screening drugs for treating monkeypox virus subclade IIb viruses.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] The present invention knocks out the 200kb region including the mouse Ifnar2, Ifnlr2, Ifnar1 and Ifngr2 genes by one gene operation, and obtains mice with severe natural immunity deficiency in which the three pathways of IFNAR, IFNGR and IFNLR are simultaneously knocked out in one step. Compared with obtaining mice with simultaneous deficiency in the three pathways of IFNAR, IFNGR and IFNLR by conventional crossbreeding, which takes 3-4 years, the present invention has short production time and low cost.

[0027] The present invention improves the pathogenicity sensitivity of mice, solves the technical problem that the weak strain of monkeypox virus, sub-branch IIb virus, cannot cause disease and it is difficult to obtain a severe mouse model infected with the weak virus of this branch. The mouse model constructed by the method of the present invention can be used to screen drugs for treating sub-branch IIb virus infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1This is a schematic diagram of the principle of knocking out IFNAR- / -IFNGR- / -IFNLR- / - in AGL mice of the present invention;

[0029] Figure 2 Figure 1 is a schematic diagram of the genotype identification results of AGL mice. Figure A is a schematic diagram of the genotype identification results of the AGL mouse tail; Figure B is a schematic diagram of the sequencing results of the homozygous KO band PCR product;

[0030] Figure 3 Figure 3 is a schematic diagram of the functional validation results of AGL mice. Figure A is a schematic diagram of the functional validation experiment of AGL mice. Figures BD are schematic diagrams of the ISGs RT-qPCR results.

[0031] Figure 4 Schematic diagram of AGL mice supporting clade IIb monkeypox virus infection; Figure A is a schematic diagram of mice infected with monkeypox virus IIb; Figure B is a schematic diagram of mouse weight results; Figure C is a mouse survival curve; Figure D is a schematic diagram of AGL tissue viral load results; Figure E is a schematic diagram of tissue histochemistry results;

[0032] Figure 5 AGL mice can be used as a model for evaluating drugs for treating monkeypox virus; Figure A is a schematic diagram of the experiment of cidofovir treating monkeypox virus infection; Figure B is a schematic diagram of the body weight results; Figure C is a survival curve;

[0033] Figure 6 Schematic diagram of the NGS sequencing typing results of monkeypox patient samples. DETAILED DESCRIPTION

[0034] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0035] Example 1

[0036] 1. Mouse gene knockout strategy

[0037] 1. Based on the CRISPR-Cas9 technology, a large fragment of Ifnar1, Ifnar2, Ifngr2 and Ifnlr2 gene sequences were knocked out on chromosome 16 of mice to obtain a mouse model with severe natural immunity deficiency in which all three types of interferon pathways were blocked in the fastest way. Figure 1 As shown, CRISPR-Cas9 was used to knock out the Ifnar1, Ifnar2, Ifngr2 and Ifnlr2 genes in mice to obtain AGL mice in which the type I+II+III interferon pathways were completely blocked.

[0038] 2. Search the mouse genome (GRCm38 / mm10) chr16:91,372,783-91,565,623nt on the UCSC genome browser website, download the sequence, design the sgRNA sequence using the http: / / crispr.mit.edu / website, and clone it into the pUC57-sgRNA expression vector (addgene, Catalog#51132-DNA), identify it by PCR sequencing, and obtain the sgRNA expression plasmid. Use T7 RNA polymerase to transcribe in vitro to obtain sgRNA and Cas9 RNA.

[0039] 3. Microinjection: Microinject the CRISPR / Cas9 system sample into the fertilized eggs of mice with C57BL / 6JGpt background; transplant the surviving fertilized eggs after injection into pseudo-pregnant female mice and wait for them to become pregnant and give birth.

[0040] 4. Identification of F0 generation mice: The F0 generation pups born to recipient mice are numbered after their tails and toes are cut off at 5-7 days old, and genomic DNA is extracted for PCR and sequencing identification to confirm the genotype.

[0041] 5. Breeding of positive F0 generation mice After the positive F0 generation mice reach sexual maturity, they are self-fertilized; the newborn F1 generation mice are numbered after their tails and toes are cut off at 5-7 days old, and their genomic DNA is extracted for PCR and sequencing identification to confirm their genotypes.

[0042] 6. Preparation of heterozygous mice: Breeding of positive F0 generation mice After the positive F0 generation mice reach sexual maturity, they are mated with wild-type background mice; the newborn F1 generation mice are tail-cut and toe-cut on days 5-7, and are numbered, and genomic DNA is extracted for PCR and sequencing identification to confirm the genotype.

[0043] Table 1: 5' end sgRNA sequences

[0044]

[0045]

[0046] Table 2: 3' end sgRNA sequences

[0047]

[0048] (II. Mouse identification scheme

[0049] 1. Collect the mouse tail sample into a 1.5ml EP tube, add 180ul lysis buffer, and incubate at 95-100℃ for 10-15min until the mouse tail has a "fried hair" pattern;

[0050] 2. Add 20ul stop solution to stop the reaction, shake and mix, and centrifuge;

[0051] 3. Then dilute the sample 10 times with ddH2O and set aside;

[0052] 4. Perform PCR on the sample. The PCR primer information, system and procedure are shown in Tables 3 to 5:

[0053] Table 3: Genotype PCR identification primer sequences

[0054]

[0055] Table 4: PCR system

[0056]

[0057]

[0058] Table 5 PCR program

[0059]

[0060] 5. Prepare 1% agarose gel, spot the sample, and run electrophoresis at 120V for 20-25min, then take a photo under UV and make statistics of the results.

[0061] 3. Isolation of MEF cells (mouse embryonic fibroblasts) and functional verification in mice

[0062] 1. Isolate E10-14 day old mouse embryos in a sterile clean bench, remove the limbs, head and internal organs of the embryos, wash them three times with sterile PBS, and then transfer them to a 10 cm culture dish;

[0063] 2. Cut the embryonic tissue into pieces with surgical scissors, and then digest with 0.5% trypsin for 15 minutes;

[0064] 3. Terminate digestion with complete medium, transfer cells to a 50 ml centrifuge tube, and centrifuge at 200 g for 5 min.

[0065] 4. Remove the supernatant, resuspend the cells with MEF medium, and then seed the cells in a 10 cm culture dish, culture at 37°C, 5% CO2, and allow the cells to adhere to the wall for 3-5 days, then pass the cells, and seed the P3 cells in a 6-well plate at 200,000 cells per well;

[0066] 5. Culture the cells with culture medium containing 500 ng / ml IFNα4 / IFNγ / IFNλ2 for 8 h and 16 h, then collect the cell samples to extract RNA and detect the expression of IFN downstream pathway genes.

[0067] MPXV infection

[0068] 1. Patient sample collection: A portion of the samples were inactivated for NGS sequencing, and the samples that were confirmed to be monkeypox positive were sent to the P3 laboratory to isolate clade IIb monkeypox virus from monkeypox patient samples. The genome information of the clade IIb subbranch of the monkeypox virus is the GSA:CRA012029 sequence.

[0069] Cultivation and propagation of monkeypox virus: 0.5 ml of monkeypox patient samples or monkeypox virus seed virus was inoculated into T25 cell culture flasks, incubated at 37°C for 1 hour, washed with PBS, and supplemented with 5 ml of DMEM medium containing 2% double antibody and 10% FBS per flask, and cultured at 37°C and 5% CO2. Cytopathic effect (CPE) was observed on the second day after inoculation. After 80% CPE appeared, the cell culture was repeatedly frozen and thawed at -80°C and room temperature for 3 times, and the virus in the supernatant was harvested by centrifugation at 4°C and 4000 rpm for 10 minutes, and stored in a -80°C refrigerator in aliquots.

[0070] 2. In the P3 laboratory, anesthetized mice with severe natural immunity deficiency were given 105 PFU of clade IIb monkeypox virus by nasal drops;

[0071] 3. Monitor the weight changes and clinical symptoms of mice every day;

[0072] 4. After the body weight of mice dropped below 80%, the mice were euthanized, and then blood, skin, muscle, testis, spleen, kidney, intestine, liver, heart, lung, brain, nasal concha and other tissue samples were collected;

[0073] 5. Blood samples were divided into anticoagulated blood and non-anticoagulated blood. For anticoagulated blood, 50-100ul was taken into a 2ml spiral cryotube, and 20ul proteinase K, 200ul PBS and 200ul AL were added. After inactivation at 56°C, the samples were transferred out of the P3 laboratory, and viral DNA was extracted from the blood. The viral copy number was determined by RT-qPCR.

[0074] 6. For non-anticoagulated blood, after standing for 30 minutes, centrifuge at 4000 rpm for 10 minutes, take the upper serum, inactivate at 60°C for 30 minutes, and then send it out of the P3 laboratory for subsequent detection of virus-binding antibody production by ELISA method;

[0075] 7. Tissue samples are divided into samples for DNA extraction, RNA extraction, samples for live virus titer determination, and samples for paraformaldehyde solution (PFA) fixation. First, the tissue samples are ground and broken, and then packaged;

[0076] 8. DNA extraction: For tissue samples, add 180ul lysis buffer and 20ul proteinase K, lyse at 56°C for 10min, and after complete lysis, send out to P3 laboratory, extract DNA, and determine the viral copy number by qPCR method;

[0077] 9. RNA extraction tissue samples, add 500ul trizol, mix well to fully lyse, and then transfer to the P3 laboratory to extract RNA and detect viral replication by RT-qPCR method;

[0078] 10. Determination of live virus titer: For tissue samples, grind and homogenize the samples, then centrifuge them, take the supernatant, and serially dilute them to infect Vero cells, and determine the virus TCID50.

[0079] 11. Tissue fixed samples were fixed and inactivated by adding 10 ml of 4% PFA, and then transferred out of the P3 laboratory for subsequent pathological sectioning.

[0080] 5. Results

[0081] like Figure 2 As shown, the electrophoresis results Figure 2 A shows that the wild-type (WT) mouse has a single band of 380 bp, the heterozygote (Hetero) has double bands of 380 bp and 619 bp, and the homozygote (Homo) has a single band of 619 bp. Figure 2 B is the sequencing result of the PCR product of the homozygous KO band, which shows that the knockout 5' and 3' homologous sequences are connected.

[0082] like Figure 3 As shown, Figure 3 A shows that MEF cells from WT mice and AGL mice were isolated and then stimulated with 500 ng / ml mouse type I interferon IFNα4, type II interferon IFNγ, and type III interferon IFNλ2 for 8 h and 16 h, respectively; Figure 3 The results in B showed that the expression of the type I interferon downstream gene Mx1, type II interferon downstream gene Ifit1, and type III interferon downstream gene Isg15 in AGL mice was not stimulated, indicating that the Ifnar1, Ifnar2, Ifngr2, and Ifnlr2 genes were successfully knocked out in AGL mice, and the type I+II+III interferon pathway was completely blocked ( Figure 3 In B, WT-MOCK represents the wild-type untreated group, WT represents the wild-type treated group, KO-MOCK represents the AGL knockout untreated group, and KO represents the AGL gene knockout treated group).

[0083] like Figure 4 As shown, Figure 4 B shows that AGL mice suffered from weight loss after infection with monkeypox virus IIb; Figure 4C shows the death of AGL mice after infection with monkeypox virus IIb; Figure 4 D shows that AGL mice developed systemic infection in various tissues after being infected with monkeypox virus IIb; Figure 4 E shows that the lung tissues of AGL mice developed necrotizing bronchopneumonia, necrotizing vasculitis, fibrinous pleurisy, and necrotizing pleurisy, and the bronchial epithelium, alveolar epithelium, vascular endothelium, and pleural tissues were all positive for monkeypox virus.

[0084] like Figure 5 As shown, Figure 5 A is a schematic diagram of the experiment of using cidofovir to treat monkeypox virus infection, in which cidofovir was administered by intraperitoneal injection at 10 mg / kg per day on day 0, day 1, and day 2, and then 5 mg / kg per day. Figure 5 B shows that after cidofovir treatment, AGL mice delayed weight loss. Figure 5 C shows that the mortality rate of AGL mice was reduced after cidofovir treatment.

[0085] like Figure 6 As shown, NGS sequencing of monkeypox patient samples determined that the virus was monkeypox virus IIb, B.1.3.

[0086] The present invention knocks out the Ifnar1, Ifnar2, Ifngr2 and Ifnlr2 genes of mice, obtains AGL mice with all I+II+III type interferon pathways blocked, provides a mouse model infected with the attenuated strain IIb branch of monkeypox virus and solves the technical problem that the attenuated strain IIb branch of monkeypox virus cannot cause disease or death.

[0087] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. A method for constructing an immunodeficient mouse model infected with monkeypox virus, characterized in that: It includes: Using CRISPR / Cas9 technology, the steps of the interferon I, interferon II and interferon III pathways were knocked out.

2. The construction method according to claim 1, characterized in that: The step of knocking out the interferon pathways I, II and III includes knocking out the mouse type I interferon receptor IFNAR gene, knocking out the type II interferon receptor IFNGR gene, and knocking out the type III interferon receptor IFNLR gene.

3. The construction method according to claim 2, characterized in that: The mouse type I interferon receptor IFNAR includes two subunits, IFNAR1 and IFNAR2; the type II interferon receptor IFNGR is the IFNGR2 subunit; and the type III interferon receptor IFNLR is the IFNLR2 subunit.

4. The construction method according to claim 3, characterized in that: The method for knocking out IFNAR1, IFNAR2, IFNGR2 and IFNLR2 comprises the following steps: (1) designing and in vitro transcribing sgRNA sequences for the 5'-specific target site and the 3'-specific target site; the sgRNA sequence for the 5'-specific target site includes the nucleotide sequences shown in SEQ ID NOs. 1 to 2; the sgRNA sequence for the 3'-specific target site includes the nucleotide sequences shown in SEQ ID NOs. 3 to 4; (2) Cas9, sgRNA plasmids for the 5'-specific target site, and sgRNA plasmids for the 3'-specific target site were simultaneously injected into mouse fertilized eggs, and the fertilized eggs were transplanted into pseudo-pregnant female mice to obtain F0 generation mice. The F0 generation mice were further self-fertilized to obtain F1 generation mice in which the Ifnar2, Ifnlr2, Ifnar1, and Ifngr2 genes were simultaneously knocked out, thus obtaining an immunodeficient mouse model infected with monkeypox virus.

5. A sgRNA sequence for simultaneously knocking out mouse interferon I, interferon II and interferon III pathways, characterized in that: The sequence is shown in any one of SEQ ID NOs. 1 to 4.

6. Use of the sgRNA sequence as described in claim 5 in knocking out the Ifanr1 gene, Ifnar2 gene, Ifngr2 gene and Ifnlr2 gene of mice.

7. A PCR primer for identifying Ifanr1- / -Ifnar2- / -Ifngr2- / -Ifnlr2- / - genotype mice, characterized in that: The primers include nucleotide sequences as shown in SEQ ID NOs. 5-8.

8. A method for identifying functional defects of three interferon pathways I, II, and III, characterized in that: The method comprises the following steps: Step 1: Isolating mouse fibroblasts constructed by the method of any one of claims 1 to 4; Step 2: Stimulate fibroblasts with IFNα4, IFNγ and IFNλ2 cytokines respectively; Step 3: extract RNA from the cells in step 2, and detect the expression of Mx1, Ifit1 and Isg15 genes in the cells; if Mx1, Ifit1 and Isg15 genes are not expressed, it indicates that the functions of the three interferon pathways I, II and III are defective.

9. Use of the mouse constructed by the method according to any one of claims 1 to 4 in screening drugs for treating viruses infected with monkeypox virus subclade IIb.

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