Application of hydroxychloroquine sulfate in preparation of medicine for preventing and treating porcine reproductive and respiratory syndrome

Abstract

The invention discloses application of hydroxychloroquine sulfate in preparation of a medicine for preventing and treating porcine reproductive and respiratory syndrome. The hydroxychloroquine sulfate disclosed by the invention has a remarkable effect of resisting the porcine reproductive and respiratory syndrome virus. The maximum non-toxic dose of the compound is 100 mu M, and the compound can effectively inhibit infection and proliferation of porcine reproductive and respiratory syndrome virus and has a good treatment effect on piglet PRRSV infection. The compound can be used as a medicine for preventing and treating the porcine reproductive and respiratory syndrome, and has a very good application prospect in the aspect of preventing and treating the porcine reproductive and respiratory syndrome.

Description

Technical Field

[0001] The present invention relates to the technical field of antiviral, and more particularly to the use of hydroxychloroquine sulfate in the preparation of a medicament for preventing and treating porcine reproductive and respiratory syndrome. Background Art

[0002] Porcine reproductive and respiratory syndrome (PRRS), commonly known as blue ear disease, is an acute and highly contagious viral disease characterized by reproductive disorders and respiratory symptoms in pig herds. It is caused by Porcine Reproductive and Respiratory Syndrome Virus (PRRSV). In addition, PRRSV can suppress the immune system and increase the risk of secondary infections, such as Streptococcus suis and Haemophilus parasuis. PRRSV is a single-stranded positive-sense RNA virus belonging to the genus Arterivirus in the family Arteriviridae. The diameter of the inner nucleocapsid of the virus is about 25-30 nm. Most virus particles are spherical or oval, showing icosahedral symmetry and appearing smooth under an electron microscope. Genome sequencing results show that PRRSV can be divided into two types: PRRSV-I (European type, prototype strain Lelystad virus) and PRRSV-II (North American type, prototype strain VR-2332 virus). The PRRSV genome is about 15.4 kb and has more than 11 open reading frames (ORFs), encoding at least 16 non-structural proteins and 8 structural proteins. PRRSV causes a decrease in production efficiency, an increase in mortality, and an increase in treatment and prevention costs. Since the discovery of PRRSV more than 30 years ago, it has continuously threatened the global pig industry and caused huge economic losses.

[0004] At present, the prevention and control of PRRSV still mainly rely on vaccine research and development and biosecurity measures. There are few direct and effective antiviral drugs. Other antiviral drugs for livestock are basically ineffective for the treatment of PRRSV. Traditional antibiotics can only control secondary infections. The prevention and control of PRRSV mainly rely on vaccines and biosecurity measures. However, due to the genetic diversity of PRRSV and the complexity of immune responses, the effect of PRRSV vaccines is not ideal. Therefore, in the current situation, it is particularly important to develop effective anti-PRRSV drugs.

[0005] Hydroxychloroquine Sulphate, also known as HCQ Sulfate, is an antimalarial drug used in the treatment of autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis, inflammation, and skin diseases. It is also an inhibitor of autophagy and toll-like receptor (TLR) 7 / 9. Summary of the Invention

[0006] The object of the present invention is to overcome the deficiencies of the prior art and provide the use of hydroxychloroquine sulphate in the preparation of a drug for preventing and treating porcine reproductive and respiratory syndrome.

[0007] The first object of the present invention is to provide the use of hydroxychloroquine sulphate in the preparation of a drug for preventing and treating porcine reproductive and respiratory syndrome.

[0008] The second object of the present invention is to provide the use of hydroxychloroquine sulphate in the preparation of a drug for preventing and treating porcine reproductive and respiratory syndrome virus.

[0009] To achieve the above object, the present invention is realized through the following technical solutions: The present invention claims the following uses: The use of hydroxychloroquine sulphate in the preparation of a drug for preventing and treating porcine reproductive and respiratory syndrome.

[0010] The use of hydroxychloroquine sulphate in the preparation of a drug for preventing and treating porcine reproductive and respiratory syndrome virus.

[0011] Preferably, the drug further comprises pharmaceutically acceptable excipients.

[0012] Preferably, the prevention and treatment of porcine reproductive and respiratory syndrome is to inhibit or avoid the occurrence of cytokine storm.

[0013] More preferably, the occurrence of cytokine storm is related to IL-1β and / or IL-6.

[0014] Preferably, the prevention and treatment of porcine reproductive and respiratory syndrome virus is to inhibit the infection of porcine reproductive and respiratory syndrome virus.

[0015] Preferably, the prevention and treatment of porcine reproductive and respiratory syndrome virus is to inhibit the proliferation of porcine reproductive and respiratory syndrome virus.

[0016] Preferably, the effective use concentration of hydroxychloroquine sulphate is 1 - 100 μΜ.

[0017] More preferably, the effective use concentration of hydroxychloroquine sulphate is 100 μΜ.

[0018] Preferably, the effective use dose of hydroxychloroquine sulphate is 6 - 7mg / kg.

[0019] More preferably, the effective dosage of hydroxychloroquine sulfate is 6.5 mg / kg.

[0020] Compared with the prior art, the present invention has the following beneficial effects: The present invention discloses that hydroxychloroquine sulfate has a significant effect against porcine reproductive and respiratory syndrome virus. Its maximum non-toxic dosage is 100 μM, which can effectively inhibit the infection and proliferation of porcine reproductive and respiratory syndrome virus, and has a good therapeutic effect on PRRSV infection in piglets. It can be used as a drug for preventing and treating porcine reproductive and respiratory syndrome, and has a good application prospect in the prevention and treatment of porcine reproductive and respiratory syndrome. Description of the Drawings

[0021] Figure 1 It is the experimental result of the toxicity of hydroxychloroquine sulfate to PAM cells and Marc-145 cells measured by using the CCK-8 kit.

[0022] Figure 2 It is the result of detecting the expression of PRRSV N protein after treating Marc-145 cells with different concentrations of hydroxychloroquine sulfate by using IFA.

[0023] Figure 3 It is the result of detecting the expression of PRRSV N protein after treating PAM and Marc-145 cells with different concentrations of hydroxychloroquine sulfate by using Western Blot.

[0024] Figure 4 It is the result of measuring the PRRSV Nsp9 mRNA level after treating PAM and Marc-145 cells with different concentrations of hydroxychloroquine sulfate by using real-time fluorescence quantitative PCR.

[0025] Figure 5 It is the result of measuring the expression of inflammatory factor IL-1β and IL-6 genes after treating PAM cells infected with PRRSV with different concentrations of hydroxychloroquine sulfate by using real-time fluorescence quantitative PCR.

[0026] Figure 6 It is the effect of hydroxychloroquine sulfate treatment on the survival rate, body temperature and body weight of piglets infected with PRRSV.

[0027] Figure 7 It is the ELISA detection result of PRRSV antibody in piglets infected with PRRSV after hydroxychloroquine sulfate treatment. The abscissa is the number of days after infecting with PRRSV.

[0028] Figure 8 It is the RT-qPCR detection result of Nsp9 in piglets infected with PRRSV after hydroxychloroquine sulfate treatment. The abscissa is the number of days after infecting with PRRSV.

[0029] Figure 9 Effect of hydroxychloroquine sulfate treatment on histopathology of lungs of PRRSV-infected piglets Specific implementation manners

[0030] The present invention will be further elaborated in detail below in conjunction with the accompanying drawings of the specification and specific embodiments. The embodiments are only used to explain the present invention and are not used to limit the scope of the present invention. The test methods used in the following embodiments are all conventional methods unless otherwise specified; the materials, reagents, etc. used are all reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0031] Hydroxychloroquine sulfate, CAS 747-36-4, was purchased from Chengdu Purigen Biotech Co., Ltd. and hereinafter referred to as HCQ in the examples and the accompanying drawings.

[0032] PRRSV: Highly pathogenic PRRSV strain XHGD (GenBank accession number EU624117), stored in the group of Zhang Guihong in the College of Veterinary Medicine, South China Agricultural University.

[0033] PRRSV N antibody: JNT, China; no. JN0401.

[0034] The XH-GD plasmid is a plasmid containing the full-length cDNA of the highly pathogenic PRRSV strain XHGD, that is, pOKQ-XH. See the prior art: Wu Xinwei, Xiong Yongzhong, Qin Hongyang, etc. Construction of an infectious clone of porcine reproductive and respiratory syndrome virus strain XH [J]. Chinese Journal of Preventive Veterinary Medicine, 2011, 33(2): 97-100. DOI: 10.3969 / j.issn.1008-0589.2011.02.04, stored in the group of Zhang Guihong in the College of Veterinary Medicine, South China Agricultural University.

[0035] Example 1 Cytotoxicity of hydroxychloroquine sulfate on PAM and Marc-145 cells I. Experimental method 1. Preparation of hydroxychloroquine sulfate stock solution Prepare a 10 mM aqueous solution of hydroxychloroquine sulfate as the hydroxychloroquine sulfate stock solution. After aliquoting, wrap it with tin foil and store it at -20°C for later use.

[0036] 2. Treatment of PAM cells with hydroxychloroquine sulfate Dilute the stock solution of hydroxychloroquine sulfate with RPMI Medium 1640 medium containing 10% FBS (containing 100 U / mL penicillin, 50 μg / mL streptomycin, and 0.25 μg / mL amphotericin) so that the concentration of hydroxychloroquine sulfate reaches 1, 10, 50, 100, 150, 200, 250, 500, 1000, 1500 μM, and obtain RPMI Medium 1640 medium containing different concentrations of hydroxychloroquine sulfate.

[0037] Inoculate PAM cells into a 96-well plate. After the cells adhere to the wall, discard the culture medium in the plate, add RPMI Medium 1640 medium containing different concentrations of hydroxychloroquine sulfate, 100 μL per well, set up a blank cell control group (RPMI Medium 1640 medium), and make 6 replicates for each treatment concentration.

[0038] After culturing in a 37°C, 5% CO2 incubator for 48 h, discard the supernatant, wash twice with PBS, add fresh culture medium, then add CCK-8 reagent, 10 μL per well, continue to culture in the dark for 1 h, and then use an enzyme-linked immunosorbent assay (ELISA) reader to read the absorbance of each well at a reference wavelength of 450 nm.

[0039] 3. Treatment of Marc-145 cells with hydroxychloroquine sulfate Dilute the stock solution of hydroxychloroquine sulfate with DMEM medium containing 10% FBS (without antibiotics) so that the concentration of hydroxychloroquine sulfate reaches 1, 10, 50, 100, 150, 200, 250, 500, 1000, 1500 μM, and obtain DMEM medium containing different concentrations of hydroxychloroquine sulfate.

[0040] Inoculate Marc-145 cells into a 96-well plate. After the cells adhere to the wall, discard the culture medium in the plate, add DMEM medium containing different concentrations of hydroxychloroquine sulfate, 100 μL per well, set up a blank cell control group (DMEM medium without hydroxychloroquine sulfate), and make 6 replicates for each treatment.

[0041] After culturing in a 37°C, 5% CO2 incubator for 48 h, discard the supernatant, wash twice with PBS, add fresh culture medium, then add CCK-8 reagent, 10 μL per well, continue to culture in the dark for 1 h, and then use an enzyme-linked immunosorbent assay (ELISA) reader to read the absorbance of each well at a reference wavelength of 450 nm.

[0042] II. Experimental results The results are as Figure 1 shown. In PAM and Marc-145 cells, at a concentration of 100 μM, hydroxychloroquine sulfate has no toxicity to the cells at all.

[0043] Example 2: Time-resolved immunofluorescence assay of the effect of hydroxychloroquine sulfate on PRRSV N protein I. Experimental method 1. Treatment of PRRSV-infected Marc-145 cells with hydroxychloroquine sulfate Marc-145 cells were seeded in 12-well plates. After the cells adhered, the culture medium was discarded. PRRSV (MOI = 0.1) was inoculated at 10 μL / well. After culturing at 37°C for 2 h, the virus supernatant was discarded, and the cells were washed twice with PBS. Then, DMEM medium containing 1 μM, 10 μM, and 100 μM hydroxychloroquine sulfate was added at 2 mL / well. Meanwhile, a cell group was set as a negative control (Mock: DMEM medium without inoculating PRRSV + hydroxychloroquine sulfate), and a virus-infected positive control group (PRRSV: DMEM medium inoculated with PRRSV + without hydroxychloroquine sulfate). The cells were cultured in an incubator at 37°C and 5% CO2 for 48 h.

[0044] 2. Time-resolved immunofluorescence assay (1) After 48 h, the culture medium was removed, and Marc-145 cells were fixed with 80% acetone at 37°C for 10 minutes, and then washed three times with PBS.

[0045] (2) 200 μl of 0.25% Triton X-100 was added to each well and incubated for 10 minutes to permeabilize the cell membrane, and then the cells were washed three times with PBS.

[0046] (3) 200 μl of 3% bovine serum albumin was added to each well and incubated at 37°C for 1 hour to block non-specific binding, and then the cells were washed three times with PBS.

[0047] (4) 200 μl of PRRSV-N antibody (dilution ratio 1:500) was added to each well and incubated at 4°C for 12 hours, and then the cells were washed three times with PBS.

[0048] (5) 200 μl of Alexa Fluor 488 (dilution ratio 1:1000) was added to each well and incubated in the dark at 37°C for 1 hour.

[0049] (6) Then, the fixed cells were stained with DAPI in the dark for 10 minutes, and the cells were observed using a Leica DMI 4000B fluorescence microscope (Leica, Wetzlar, Germany). II. Experimental results As Figure 2As shown, compared with the positive control group infected with the virus, the amount of PRRSV N protein was significantly reduced after treatment with hydroxychloroquine sulfate. When hydroxychloroquine sulfate was at 100 μM, it could inhibit more than 95% of the PRRSV N protein, and basically no PRRSV N protein appeared in the field of view.

[0050] Example 3 Western Blot Detection of the Effect of Hydroxychloroquine Sulfate on PRRSV N Protein I. Experimental Method 1. Treatment of PRRSV-infected PAM cells or Marc-145 cells with hydroxychloroquine sulfate PAM cells and Marc-145 cells were respectively inoculated into 6-well plates, and then according to the method of Example 2, "treatment of PRRSV-infected Marc-145 cells with hydroxychloroquine sulfate" was carried out; at the same time, Marc-145 cells were replaced with PAM cells to treat PRRSV-infected PAM cells with hydroxychloroquine sulfate.

[0051] 2. Western Blot Detection The treated cells on the above 6-well plate were scraped off with a cell scraper into a 1.5 mL centrifuge tube, centrifuged at 12000 g for 3 min at 4℃, and the supernatant was discarded. The precipitate was lysed thoroughly with NP40 lysis buffer on ice. After 30 min, it was centrifuged at 13000 g for 30 min at 4℃, and the supernatant was aspirated into a new 1.5 mL centrifuge tube. Protein loading was added, and after heating at 100℃ for 10 min, Western Blot detection was carried out.

[0052] First, SDS-PAGE was carried out on a Bio-Lab electrophoresis apparatus at a voltage of 80 V for 30 min, and then the voltage was adjusted to 120 V for 60 min; after transfer, it was blocked with 5% skim milk powder for 1 h, washed with TBST, 5 min / time, for a total of 3 times; Incubate the primary antibody: Use anti-N protein antibody and anti-GAPDH antibody, and incubate overnight at 4℃; recover the primary antibody, wash the membrane with TBST, 5 min / time, for a total of 3 times; Incubate the secondary antibody: The fluorescent secondary antibody of IRDye800CW Goat anti-mouse (diluted 1:10000), after incubating in the dark at 37℃ in an incubator for 1 h, Discard the secondary antibody, quickly wash the membrane with TBST in the dark on a shaker, 5 min / time, for a total of 3 times; Finally, use the Odyssey system of the two-color laser analysis instrument of LI-COR company to analyze the expression of the target protein.

[0053] II. Experimental Results As Figure 3 shown, hydroxychloroquine sulfate inhibits the expression of PRRSV N protein in a dose-dependent manner, and hydroxychloroquine sulfate can inhibit the expression of more than 95% of PRRSV N protein at 100 μM.

[0054] PRRSV N protein is a structural protein of porcine reproductive and respiratory syndrome. This protein is expressed early in virus infection and is present in relatively large amounts, serving as a marker for detecting porcine reproductive and respiratory syndrome virus. If the expression of N protein is inhibited, the virus cannot complete genome packaging, resulting in the inability of virus particles to be normally assembled or released. Considering the results of Examples 2 and 3, hydroxychloroquine sulfate can inhibit the expression of PRRSV N protein, indicating that hydroxychloroquine sulfate can inhibit PRRSV infection and replication.

[0055] Example 4 RT-qPCR Detection of the Effect of Hydroxychloroquine Sulfate on Nsp9 mRNA I. Experimental Method 1. Treatment of PRRSV-infected PAM cells or Marc-145 cells with hydroxychloroquine sulfate PAM cells and Marc-145 cells were respectively seeded into 6-well plates, and "treatment of PRRSV-infected PAM cells or Marc-145 cells with hydroxychloroquine sulfate" was performed according to the method of Example 3.

[0056] 2. RT-qPCR Detection Cell supernatants were collected, and the genomic DNA of PRRSV was extracted using the AxyPrep Body fluid Viral DNA / RNA Miniprep Kit (Axygen), and then the RNA was reverse-transcribed into cDNA.

[0057] Using the upstream and downstream primers of Nsp9 mRNA and the internal reference gene GAPDH in Table 1, SYBR Green I was used as the fluorescent dye for RT-qPCR detection. The Bio-Rad CFX96 qRT-PCR detection system was used to detect the relative expression of Nsp9 mRNA. The reaction conditions were as follows: activation of the hot start enzyme at 95°C for 2 min; denaturation at 95°C for 15 s, annealing at 60°C for 1 min, extension at 72°C for 30 s, and 40 cycles of reaction. Fluorescent signals were collected in this step.

[0058] Table 1

[0059] II. Experimental Results As Figure 4As shown, compared with the positive control group infected with the virus, hydroxychloroquine sulfate can inhibit the synthesis of DNA of more than 95% of PRRSV Nsp9 mRNA at 100 μM, and the difference is very significant (P<0.001). The gene PRRSV Nsp9 is highly conserved and is a necessary gene for virus detection. The results indicate that HCQ can inhibit the synthesis of PRRSV mRNA.

[0060] Example 5 Effect of hydroxychloroquine sulfate treatment on inflammatory factors in PAM cells infected with PRRSV I. Experimental method 1. Treatment of PAM cells infected with PRRSV with hydroxychloroquine sulfate Inoculate PAM cells into 24-well plates and treat the PAM cells infected with PRRSV with hydroxychloroquine sulfate according to the method of Example 3.

[0061] 2. RT-qPCR detection Detect the relative expression of IL-1β, IL-2, and IL-6 mRNA genes. The specific method is the same as that in Example 4, and the primers are shown in Table 2.

[0062] Table 2

[0063] II. Experimental results As Figure 5 shown, hydroxychloroquine sulfate inhibits the upregulation of IL-1β and IL-6 induced by PAM cell infection with PRRSV in a dose-dependent manner within the concentration range of 1-100 μM. Compared with the control group, hydroxychloroquine sulfate significantly inhibits IL-1β and IL-6 at 100 μM, suppressing the occurrence of inflammation.

[0064] After PRRSV infection, the levels of pro-inflammatory factors IL-1β and IL-6 increase significantly, which is one of the core markers of cytokine storm. The results show that hydroxychloroquine sulfate can limitedly inhibit the cytokine storm caused by PRRSV infection.

[0065] Example 6 Effect of hydroxychloroquine sulfate treatment on survival rate, body temperature and body weight of PRRSV-infected piglets I. Experimental method Randomly divide 15 four-week-old piglets into 3 groups, with 5 piglets in each group, namely the negative group (Ctrl), the positive group (PRRSV), and the treatment group (PRRSV+HCQ).

[0066] The negative group is fed normally, the positive group is inoculated with 2×10 5 / mL TCID50 of PRRSV through trachea, and the treatment group is also inoculated with 2×10 5PRRSV with TCID50 / mL.

[0067] After inoculating with PRRSV for 24 h, the treatment group was continuously medicated for 12 days. Each time, hydroxychloroquine sulfate (diluted with normal saline) was injected intramuscularly at a dose of 6.5 mg / kg.

[0068] After inoculating with PRRSV, the body weight, survival rate, body temperature of piglets were monitored and recorded every day, and whether there were clinical symptoms such as dyspnea and cough was observed. And blood was collected every day to collect serum for subsequent analysis.

[0069] II. Experimental Results As Figure 6 shown, the survival rate of the treatment group was significantly higher than that of the positive group. After inoculating with PRRSV, the average body weight of the treatment group gradually increased compared with that of the positive control group, and the body weight growth trend was basically the same as that of the control group and remained at a relatively stable level. The average body temperatures of the treatment group and the negative control group were at the same level and remained relatively stable. The phenomenon of increased body temperature occurred in the positive control group.

[0070] The results showed that hydroxychloroquine sulfate treatment effectively improved the survival rate of piglets infected with PRRSV and reduced the clinical symptoms of weight loss and increased body temperature caused by PRRSV infection.

[0071] Example 7 Effect of Hydroxychloroquine Sulfate Treatment on the Virus Titer of PRRSV-Infected Piglets I. Experimental Methods 1. Detection by Enzyme-Linked Immunosorbent Assay (ELISA) INGEZIM PRRS AMERICA kit from Ingnia Company was used to detect the PRRSV antibodies in the sera collected from the piglets in each group in Example 6 every day after inoculating with PRRSV.

[0072] 2. RT-qPCR Detection (1) Construction of the Standard Curve for the Viral Copy Number of PRRSV Preparation of the standard product: The standard product used was XH-GD plasmid. The concentration of the standard product was measured by Nanodrop, and the copy number of the standard product was calculated by the formula The calculation formula is: ; Dilution of the standard product: The standard product was serially diluted 10-fold for 8 consecutive dilution steps. The standard products at each dilution step and the blank control were subjected to qRT-PCR reaction. The qRT-PCR reaction used the upstream and downstream primers of the internal reference genes Nsp9 and GAPDH in Table 1 according to the method of Example 4, with SYBR Green I as the fluorescent dye. Three replicates were performed for each dilution step. Drawing of the standard curve: Based on the copy numbers and corresponding Ct values of each dilution, the standard curve can be made using [specific method].

[0073] (2)Detection of the viral copy number of PRRSV Use the AxyPrep Body fluid Viral DNA / RNA Miniprep Kit (Axygen) to extract the genome of PRRSV from the whole blood collected in Example 6, and then reverse transcribe the RNA into cDNA.

[0074] According to the method of Example 4, use the upstream and downstream primers of the Nsp9 and GAPDH internal reference genes in Table 1, and use SYBRGreen I as the fluorescent dye to perform RT-qPCR. Substitute the corresponding CT values into the standard curve formula to calculate the viral copy number of PRRSV in the serum of each group of piglets.

[0075] II. Experimental results Figure 7 This is the ELISA test result for PRRSV antibodies, where S: absorbance value of the Sample, P: absorbance value of the Positive control. S / P = (Sample value - Negative control value) / (Positive control value - Negative control value). When S / P ≥ 0.4, the result is positive. The larger the S / P value, the higher the concentration of PRRSV antibodies. The results show that the treatment group can inhibit the replication of PRRSV in piglets compared with the positive group.

[0076] Figure 8 This is the RT-qPCR test result of Nsp9 in infected piglets. The results show that the treatment group can inhibit the replication of PRRSV in piglets compared with the positive group.

[0077] The results show that hydroxychloroquine sulfate treatment effectively reduces the viremia caused by PRRSV infection in pigs and inhibits the replication and proliferation of PRRSV in pigs.

[0078] Example 8 Effect of hydroxychloroquine sulfate treatment on the histopathology of the lungs of PRRSV-infected piglets I. Experimental method For each group of piglets in Example 6, on the 14th day after inoculation with PRRSV (14 dpi, D14), after recording the body weight and measuring the body temperature, euthanasia was performed on each group of piglets and autopsy was carried out.

[0079] After rinsing the lungs clean, pathological observation was carried out and photographed. Subsequently, after collecting the lung tissue, it was fixed with 10% Neutral Buffered Formalin (NBF) as the fixative for 24 - 48 h for subsequent histopathological analysis.

[0080] After fixation, the samples are subjected to a series of dehydration steps using ethanol with increasing concentrations, and then cleared in xylene. The dehydrated tissues are embedded in paraffin for easy sectioning. Thin sections approximately 4 μm thick are cut from the paraffin-embedded tissues using a microtome. These sections are mounted on glass slides, then dewaxed with xylene and rehydrated through a series of ethanol grades. For histological evaluation, the slides are stained with hematoxylin and eosin (HE) to allow detailed observation of cell and tissue structures. After staining, the slides are examined under an optical microscope.

[0081] II. Experimental Results As Figure 9 shown, in the negative control group, the lungs of piglets had normal morphology and no obvious lesions; in the positive control group, the piglets showed enlarged lungs with diffuse swelling, increased weight, a dark red appearance on the lung surface, congestion in the lung lobes, a hardened texture of the lungs, reduced elasticity, and certain pulmonary adhesions.

[0082] Compared with the positive control group, the treatment group showed less severe lung lesions. In the treatment group, except for the grayish-white pathological changes in the visceral layer of the lung mucosa, there were no obvious lesions.

[0083] Histopathological analysis of the lungs showed that the positive group exhibited interstitial pneumonia with thickened alveolar septa, a large number of inflammatory cell infiltrations, interstitial edema and fibrosis in the interstitium, and destruction of the lung structure; the treatment group only showed mild inflammatory cell infiltration in the lungs. The results indicate that treatment with hydroxychloroquine sulfate can reduce the lung lesions in piglets caused by PRRSV infection.

[0084] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. For those of ordinary skill in the art, other different forms of changes can be made based on the above description and ideas. It is not necessary and impossible to enumerate all implementation manners here. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention shall be included in the protection scope of the claims of the present invention.

Claims

1. Application of hydroxychloroquine sulfate in the preparation of drugs for preventing and treating porcine reproductive and respiratory syndrome.

2. Application of hydroxychloroquine sulfate in the preparation of drugs for preventing and treating porcine reproductive and respiratory syndrome virus.

3. The use according to claim 1 or 2, characterized in that: The medicine also includes pharmaceutically acceptable auxiliary materials.

4. The use according to claim 1, characterized in that: The method of preventing and treating porcine reproductive and respiratory syndrome is to inhibit or avoid the occurrence of inflammatory storm.

5. The use according to claim 4, characterized in that: The occurrence of the inflammatory storm is related to IL-1β.

6. The use according to claim 4, characterized in that: The occurrence of the inflammatory storm is related to IL-6.

7. The use according to claim 2, characterized in that: The method of preventing and controlling porcine reproductive and respiratory syndrome virus is to inhibit porcine reproductive and respiratory syndrome virus infection.

8. The use according to claim 2, characterized in that: The method of preventing and controlling porcine reproductive and respiratory syndrome virus is to inhibit the proliferation of porcine reproductive and respiratory syndrome virus.

9. The use according to claim 1 or 2, characterized in that: The effective concentration of hydroxychloroquine sulfate is 1-100 μM.

10. The use according to claim 9, characterized in that: The effective concentration of hydroxychloroquine sulfate is 100 μM.

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