CN114375896A

CN114375896A - Method for constructing influenza A virus and herpes simplex virus 1 double-infection mouse disease model

Info

Publication number: CN114375896A
Application number: CN202011125529.8A
Authority: CN
Inventors: 杨子峰; 吕元君; 钟南山; 周红霞
Original assignee: Guangzhou Institute Of Respiratory Health; First Affiliated Hospital of Zhengzhou University; First Affiliated Hospital of Guangzhou Medical University
Current assignee: Guangzhou Institute Of Respiratory Health; First Affiliated Hospital of Zhengzhou University; First Affiliated Hospital of Guangzhou Medical University
Priority date: 2020-10-20
Filing date: 2020-10-20
Publication date: 2022-04-22

Abstract

The invention discloses a method for constructing a disease model of a mouse doubly infected by influenza A virus and herpes simplex virus type 1, which comprises the following steps: 1) establishing a mouse disease model of herpes simplex virus 1-influenza A virus: the mice are infected with herpes simplex virus 1 firstly, and are infected with influenza A virus three days later; 2) establishing a herpes simplex virus 1-influenza A virus co-infected mouse disease model: the mice are infected with influenza A virus and herpes simplex virus type 1 at the same time; 3) establishing an influenza A virus-herpes simplex virus 1 mouse disease model: the mice are infected with influenza A virus firstly and infected with herpes simplex virus 1 three days later; the physiological indexes of the three mouse disease models are respectively detected. The disease model constructed by the method provided by the invention has high stability, is simple and convenient to operate, has clinical simulation, and can be used for exploring the immune response change of mice in different double-infection models.

Description

Method for constructing influenza A virus and herpes simplex virus 1 double-infection mouse disease model

Technical Field

The invention belongs to the field of biomedicine, and particularly relates to an influenza A virus and herpes simplex virus type 1 double-infection mouse disease model and a construction method thereof.

Background

Influenza Virus (IV) is a single-stranded negative-strand RNA enveloped virus belonging to the orthomyxoviridae family, and is classified into four types, i.e., a, b, c and d, according to Nucleoprotein (NP) and Matrix Protein (MP), among which Influenza A Virus (IAV) is the most pathogenic and can drain influenza pandemics. The airway mucosal epithelial cells are the main targets for the IAV to attack the body. After IAV infects organism, it depends on Hemagglutinin (HA) of virus to adhere to epithelial cell of respiratory tract mucosa, enter cytoplasm through pinocytosis, and is replicated and assembled, and finally released in bud form to infect other cells. The replication cycle of IAV is about 8 hours. IAV infection can cause degeneration, necrosis, exfoliation, release of inflammatory factors, congestion and edema of the mucous membrane and increased secretion of the mucous membrane, thereby causing a series of respiratory symptoms. Influenza a viruses are generally characterized by annual seasonal influenza behavior that, as estimated by the world health organization, results in approximately 10 million human infections each year, and although seasonal influenza is self-limiting, patients develop pneumonia and severe lung injury due to secondary lower respiratory tract infections that can result following influenza infection.

HSV-1 is a neurotropic DNA virus that infects the body through damaged skin or mucous membranes, causing localized lesions. Epidemiological studies have shown that worldwide infection rates of HSV-1 are 90%, which are common in infancy. HSV-1 can cause respiratory tract infection such as bronchitis, pneumonia and the like in addition to oral lip, genital herpes and herpes encephalitis. The study showed that ICU patient with about 1/3 positive for the lower respiratory secretion HSV-1; the detection rate of HSV-1 in the throat is 22% -54% and the detection rate of the lower respiratory tract is 16% -32% when a patient with mechanical ventilation is received; in patients with ARDS, the positivity of HSV-1 in alveolar lavage fluid (BALF) was about 57%.

Studies have shown that HSV-1 co-infection with RV can cause Acute Respiratory Distress Syndrome (ARDS). However, due to the limited number of clinical studies described above, it is unclear whether HSV-1 co-infection is the primary cause of exacerbation. Therefore, an IAV and HSV-1 double-infection mouse model is established, and the study on the pathogenicity of double infection is particularly important.

Viral superinfection is a multifactorial process mediated by interactions between viruses and host immune cells. After the virus infects the epithelial cells of the respiratory tract, the first defense system of the organism against pathogenic microorganisms, namely the innate immune response, is activated. The innate immune system is composed primarily of the body's physical barriers and innate immune cells (NK cells, macrophages, dendritic cells, etc.). The innate immune system recognizes pathogen-associated molecular patterns on the surface of viruses through pattern recognition receptors to induce the production of interferons, cytokines and chemokines, activate antiviral immunity, neutrophil recruitment, macrophage activation, dendritic cell maturation, and the like. Normally, the innate immune response of the body is maintained in a dynamic balance, an unbalanced or uncoordinated immune response, accompanied by a cytokine storm characterized by excessive cytokine production, which can accelerate disease progression and death. Activation of innate immune cells and adaptive immune response cells, as well as cytokine/chemokine production, are key factors in the body's resistance to viral invasion. Therefore, the study on the pathogenicity difference and the inherent immune response change causing the double infection can provide basis and reference for clinically treating the IAV and HSV-1 double infection.

Disclosure of Invention

The invention aims to solve the technical problems and provides a method for successfully constructing a mouse disease model with double infection of influenza A virus and herpes simplex virus type 1.

In order to achieve the above object, the present invention provides a method for constructing a disease model of a mouse doubly infected with influenza a virus and herpes simplex virus type 1, comprising the steps of:

1) establishing a mouse disease model of herpes simplex virus 1-influenza A virus: the mice are infected with herpes simplex virus 1 firstly, and are infected with influenza A virus three days later;

2) establishing a herpes simplex virus 1-influenza A virus co-infected mouse disease model: the mice are infected with influenza A virus and herpes simplex virus type 1 at the same time;

3) establishing an influenza A virus-herpes simplex virus 1 mouse disease model: the mice are infected with influenza A virus firstly and infected with herpes simplex virus 1 three days later;

the physiological indexes of the three mouse disease models are respectively detected.

As a preferred embodiment, the influenza a virus is H3N 2.

As a preferred embodiment, the detecting of the physiological index includes: measuring body weight change, observing survival rate, detecting lung index, detecting titer of influenza A virus and herpes simplex virus 1 virus, detecting pathological sections, and detecting cell factors in mouse alveolar lavage fluid; detecting changes of immune cells, natural killer cells, B cells, plasmacytoid dendritic cells, interferon-producing killer dendritic cells and macrophages in mouse alveolar lavage fluid, lung and spleen.

As a preferred embodiment, the mice were tested daily for body weight and death in all three models on day 0 of infection at the time of H3N2 infection; virus titers and lung indices were measured in lung homogenates and mouse alveolar lavage fluid on

days

4, 6, 8, and 10 post H3N2 infection; lung pathology was examined on day 8 post H3N2 infection.

As a preferred embodiment, the detection of lung pathology index comprises pathological section detection; detecting cytokines in mouse alveolar lavage fluid; detecting changes of immune cells, natural killer cells, B cells, plasmacytoid dendritic cells, interferon-producing killer dendritic cells and macrophages in mouse alveolar lavage fluid, lung and spleen.

As a preferred embodiment, the mouse is a C57BL/6 mouse.

As a preferred embodiment, the influenza a virus is cultured using MDCK cells.

In a preferred embodiment, the herpes simplex virus type 1 is cultured using Vero cells.

According to the construction method of the influenza A virus and herpes simplex virus type 1 double-infection mouse disease model, three double-infection mouse disease models of H3N2 and HSV-1 are established, and the modes of weight weighing, survival rate observation, virus titer detection, pathological section making and the like are adopted, so that whether the model is successfully established or not is determined, pathogenicity differences of double-infection simulation are discussed, and further, the immune response changes of mice in different double-infection models are researched by detecting the cell factors and the changes of immune cells of the mice. The establishment and research of three double infection models lays a further foundation for deeply exploring the pathogenesis of H3N2 and HSV-1 double infection hair dyeing and also lays a certain foundation for pharmacological experimental research. The disease model of the invention has high stability, simple operation and clinical simulation.

The invention establishes a H3N2 and HSV-1 double-infection mouse disease model for the first time, and comprehensively shows the characteristics of H3N2 and HSV-1 double-infection diseases through three infection modes. As double or multiple respiratory virus infections increasingly become a main problem threatening human health, the earlier stage research of the invention shows that severe IAV infected patients have serious illness and poor prognosis after being combined with HSV-1 infection, and the evaluation of the pathogenicity of the double infection is beneficial to searching for treatment and intervention targets. Therefore, the establishment of a double-infection mouse model is necessary for pharmacological research and research on virus pathogenesis.

Drawings

FIG. 1 shows H3N2 and HSV-1 dual infection model detection index time nodes.

FIG. 2 shows the changes in weight, survival and lung index for the HSV-1-H1N1 double-infected mouse disease model: (A) change in mouse body weight, (B) change in survival rate, (C) change in lung index. P <0.05, P <0.01, P < 0.001.

FIG. 3 shows immunocytochemical assay of changes in BALF and lung tissue viral loads in mice model of HSV-1-H3N2 double infection: (A) H3N2 viral load; (B) HSV-1 viral load; p <0.05, P <0.01, P < 0.001;

FIG. 4 shows the body weight, survival rate and lung index changes in H3N2+ HSV-1 co-infected mice; (D) mouse weight change, (E) survival change, (F) lung index change; p <0.05, P <0.01, P < 0.001.

Fig. 5 shows: the immunochemical cell method is used for determining the BALF and lung tissue virus load change of the H3N2+ HSV-1 co-infection model mouse. (A) H3N2 viral load; (B) HSV-1 viral load; p <0.05, P <0.01, P < 0.001.

FIG. 6 shows the body weight, survival rate and lung index changes in H3N2-HSV-1 double-infected mice; (G) mouse weight change, (H) survival change, (I) lung index change; p <0.05, P <0.01, P < 0.001.

FIG. 7 shows immunocytochemistry assay of changes in BALF and lung tissue viral load in H3N2-HSV-1 double infection model mice. (A) H3N2 viral load; (B) HSV-1 viral load; p <0.05, P <0.01, P < 0.001.

Figure 8 shows the double infection group H3N2 viral load in the three models; p <0.05, P <0.01, P < 0.001.

FIG. 9 shows the H3N2 and the HE staining (200x) (A) HSV-1-H3N2 double infection model on day 6 post H3N2 infection in mice; (B) H3N2+ HSV-1 coinfection model; (C) H3N2-HSV-1 double infection model.

FIG. 10 shows the expression levels of cytokines and chemokines in the three models at day 6 after H3N2 infection. (A) HSV-1-H3N2 superinfection model; (B) H3N2+ HSV-1 coinfection model; (C) H3N2-HSV-1 superinfection model. And detecting the cell factors/chemotactic factors by adopting a Luminex liquid phase chip method. P <0.05, P <0.01, P < 0.001.

FIG. 11 shows the expression levels of cytokines and chemokines in the three models at day 8 after H3N2 infection. (A) HSV-1-H3N2 superinfection model; (B) H3N2+ HSV-1 coinfection model; (C) H3N2-HSV-1 superinfection model. And detecting the cell factors/chemotactic factors by adopting a Luminex liquid phase chip method. P <0.05, P <0.01, P < 0.001.

FIG. 12 shows the expression levels of cytokines and chemokines in the group of the double infection in the three models. (A) Expression level at day 6 after H3N2 infection; (B) expression level at day 8 after H3N2 infection; p <0.05, P <0.01, P < 0.001.

FIG. 13 shows NK, B, pDC, IKDC,

Flow cytometric analysis of cells was gated. (A) Setting gates for NK, B, pDC and IKDC cells; (B)

arranging a door on the cell; the NK cell is CD3-NK1.1 +; b cells are CD3-B220 +; pDC cells are CD3-B220+ CD11c +;

the cells are CD11b + F4/80 +; IKDC cells CD3-B220+ NK1.1+ CD11c +.

FIG. 14 shows flow cytometry analysis of three models of BALF, NK, B, pDC, IKDC, in lung and spleen, 6 and 8 days after H3N2 infection,

The expression level of the cells. (A) HSV-1-H3N2 superinfection model NK, B, pDC, IKDC, on day 6 post H3N2 infection,

The expression level of the cell; (B) H3N2+ HSV-1 Co-infection model NK, B, pDC, IKDC, on day 6 post H3N2 infection,

The expression level of the cell; (C) H3N2-HSV-1 superinfection model NK, B, pDC, IKDC, on day 6 post H3N2 infection,

The expression level of the cell; (D) HSV-1-H3N2 superinfection model NK, B, pDC, IKDC, on day 8 post H3N2 infection,

The expression level of the cell; (E) H3N2+ HSV-1 Co-infection model NK, B, pDC, IKDC, on day 8 post H3N2 infection,

The expression level of the cell; (F) H3N2-HSV-1 superinfection model NK, B, pDC, IKDC, on day 8 post H3N2 infection,

The expression level of the cells. P<0.05，**P<0.01，***P<0.001。

Detailed Description

The present invention will be further described with reference to the following examples. It should be understood that the following examples are illustrative of the present invention only, and are not intended to limit the scope of the present invention. Unless otherwise specified, the instruments and reagents used in the following examples are all common commercial products.

1. Experimental equipment

Optical microscope, paraffin embedding machine, paraffin slicer, cell tissue disruptor, Luminex TM 100/200 Magpix TM analyzer, BD FACSVerse flow cytometer, etc.

2. Primary reagent

Murine anti-NP antibody was purchased from Novus, Rabbit anti-HSV-1 antibody was purchased from Abcam, flow antibody anti-CD16/32(BD), APC anti-mouse CD3(Biolegend), APC/FireTM750 anti-mouse CD3(Biolegend), PE anti-mouse CD45R/B220(Biolegend), BV421 anti-mouse NK1.1(Biolegend), BV421 anti-mouse CD11c (Biolegend), Alexa

647 anti-mouse F4/80(Biolegend), FITC anti-mouse/human CD11b (Biolegend), cell fixation/rupture kit BD.

3. Strain and experimental animal

The study in this section relates to two virus strains, the virus type, source, amplification mode are shown in table 1 below.

Table 1: viral strain source and mode of amplification

Viral strains	Source	Amplification method
			Influenza A virus A/Aichi/2/1968(H3N2)	ATCC	9-11 day old SPF chick embryo
Herpes simplex virus type 1 (HSV-1)	Clinical isolates	Vero cell

Experimental animals:

female C57BL/6 mice, 6-8 weeks old, weighed approximately 17-19g, were purchased from the Guangdong provincial animal center for medical trials (animal Certification No. 44007200063361). Animals were housed in the SPF Biosafety class 2 laboratory animal center, Guangzhou university of medical science. All according to the guidelines and animal protection laws of the experimental animal center of Guangzhou university of medical science. The study was approved by the animal ethics committee of the Guangzhou university of medical science.

4. Viral amplification

4.1 chick embryo amplification of the H3N2 Virus Strain

(1) Selecting SPF (specific pathogen free) chick embryos of 9-11 days old, and observing the vitality, blood vessel distribution, health condition and the like of the chick embryos under an egg lighting lamp.

(2) The influenza A virus A/Aichi/2/1968(H3N2) strain (available from ATCC) was removed from a-80 ℃ freezer and placed in a freezer for thawing. Ultraviolet irradiating the biological safety cabinet for 30min, disinfecting the chick embryos by using 75% v/v alcohol, and transferring the chick embryos into the biological safety cabinet.

(3) The disposable aseptic protective clothing is worn, and the disposable powder-free latex gloves, the aseptic mask and the cap are worn.

(4) The air chamber of the chick embryo is upward, the range of the air chamber is marked under the irradiation of an egg inspection lamp, and the position 2-5mm away from the edge of the air chamber is marked, so that the blood vessel is avoided. The marked sites and their surroundings were wiped again with an alcohol cotton ball and a 10mm by 6mm rectangular fracture was drilled at the top of the chamber with a drill, taking care to avoid drilling the crust membrane.

(5) Diluting the unfrozen H3N2 virus by PBS, inoculating the virus into allantoic cavities of chick embryos by using a sterile syringe, and inoculating 0.2mL of virus into each chick embryo; melting paraffin candle, dripping paraffin candle at the drill hole, sealing, continuously placing in a constant temperature and humidity incubator at 37 ℃ for incubation, paying attention to the activity and growth condition of chick embryos every day by using an egg candler, and discarding chick embryos which die within 24h and are considered to be non-specific. Note that the strain name, inoculation time, and name of the experimental operator are marked on the chick embryo.

(6) Harvested 48H after inoculation with H3N2 virus. Before harvesting, the chick embryos are placed in a refrigerator at 4 ℃ overnight or placed in a refrigerator at-20 ℃ for 1h to freeze the chick embryos and coagulate blood, so that blood vessels are prevented from being broken when the virus is harvested, erythrocyte flows out and the virus in allantoic fluid is prevented from being coagulated, and the virus titer is reduced.

(7) Wiping the eggshell of the air cell part with alcohol cotton ball, breaking the eggshell of the air cell part with aseptic forceps, tearing off the eggshell of the air cell part with another aseptic forceps, and breaking the eggshell at the position without large blood vessel of chorioallantoic membrane. Allantoic fluid is sucked by using a disposable sterile pasteur tube, and the allantoic fluid is put into a 50mL sterile centrifuge tube and is placed on ice for temporary storage.

(8) Cooling the low temperature centrifuge to 4 deg.C, centrifuging 50mL sterile centrifuge tube with 3000rpm for 10min to remove floccule in allantoic fluid.

(9) And (3) subpackaging the centrifuged supernatant into 1.5mL sterile EP tubes with the subpackage specification of 200 mu L per tube, labeling the strain name, the subpackage time and the name of an experimental operator on the EP tubes, and storing in a refrigerator at the temperature of-80 ℃.

4.2Vero cell expansion of HSV-1 Virus strains

(1) Vero cells grown in a T-75 culture flask, when the cells grow to 80%, the cells are passaged and inoculated in a 100mm cell culture dish.

(2) After 24h, when Vero cells in a 100mm cell culture dish are observed to grow to 80% -90% under a microscope, a cell growth solution is aspirated by a pipette, and the cells are washed for 3 times by PBS.

(3) Before cell inoculation, HSV-1 virus is taken out in a refrigerator at the temperature of-80 ℃ and unfrozen on ice. Diluting the thawed virus solution by 100 times by using serum-free DMEM-F12 medium, inoculating the virus solution into 100mm cell culture dishes, inoculating 1mL of virus solution into each cell culture dish, and slightly shaking to enable the virus solution to completely cover the cell surfaces.

(4) Placing the cell culture dish inoculated with the virus at 37 ℃ and 5% CO₂In the virus incubator, viruses are adsorbed for 1-2h, and the culture dish is shaken once at intervals of 15min, so that the viruses are uniformly distributed.

(5) After adsorption, the culture dish was removed, the virus solution was discarded, 10mL of DMEM-F12 medium containing 1% serum was added to each cell culture dish, and the mixture was incubated at 37 ℃ with 5% CO₂The virus incubator of (2) continues to culture. The cell culture dish is marked with the name of the virus strain, the inoculation time and the name of the operator.

(6) Observing Cytopathic condition every day, about 72-96 h, when all cells in the culture dish have Cytopathic effect (CPE), placing the culture dish in a refrigerator at minus 80 ℃, and repeatedly freezing and thawing for 2 times to improve the titer of harvested viruses.

(7) After freezing and thawing, a virus liquid is absorbed by a disposable sterile pasteur tube, collected in a 50mL sterile centrifuge tube, and centrifuged for 10min at 3000rpm in an ultra-low temperature centrifuge pre-cooled to 4 ℃ in advance to remove cell debris.

(8) And (3) respectively packaging the centrifuged virus liquid supernatant into 1.5mL sterile EP tubes with the packaging specification of 200 mu L per tube, labeling the strain name, the packaging time and the name of an experimental operator on the EP tubes, storing at-80 ℃ for later use, reserving one tube, and determining the HSV-1 virus titer by a plaque method.

4.3 determination of viral titre

Immunocytochemistry method for determining virus titer

(1) Vero cells and MDCK cells (purchased from ATCC) grown in a T-75 flask were passaged and seeded in a 96-well plate when the cells grew to around 80% (see cell passage for detailed steps). Wherein, the Vero cell is used for detecting the titer of HSV-1 virus, and the MDCK cell is used for detecting the titer of H3N2 virus.

(2) After 24h of cell passage, the cell state of the 96-well plate was observed under a microscope. When the cells grow to about 100%, the original culture medium is aspirated by a pipette, and 100 μ L of PBS is added to each well to wash the cells for 3 times.

(3) Lung homogenate and BALF supernatant were removed in advance and thawed on ice.

(4) Serum-free DMEM-F12 medium was used as 1: lung homogenate and BALF supernatant were diluted in 10-ratio gradient.

(5) Adding the diluted lung homogenate and BALF supernatant into cells of a 96-well plate respectively, wherein each well is 50 mu L; the 96-well plate is noted with cell type, type of inoculum, concentration, time and name of operator. Placing at 37 deg.C and 5% CO₂Adsorbing for 2h in a virus incubator, shaking the 96-well plate once at intervals of 15min to ensure that the virus is uniformly adsorbed in the cells.

(6) After incubation, the 96-well cell culture plate was removed and the diluted lung homogenate and BALF supernatant were aspirated by a micropipette. 2 × DMEM-F12 medium pre-warmed to 37 ℃ and 2% sodium carboxymethylcellulose 1:1, mixing uniformly. Adding a mixture of DMEM-F12 containing 1 mu g/mL TPCK pancreatin and 2% sodium carboxymethylcellulose into MDCK cells, adding a mixture of DMEM-F12 and 2% sodium carboxymethylcellulose into Vero cells, adding 100 mu L/well, placing at 37 ℃ and 5% CO₂The incubation was continued in the virus incubator.

(7) Cytopathic conditions in 96-well plates were observed daily for about 48-72 h, and when cells produced CPE, the cell culture plates were removed, the cell culture fluid was aspirated, and washed 3 times with PBS.

(8) Cells were fixed with 4% paraformaldehyde for 10min and washed 1 time with PBS.

(9) Adding 0.5% TritonX-100 to break membrane for 15min, and washing with PBST for 1 time.

(10) Incubating the primary antibody: primary antibody was diluted with 3% BSA, with murine anti-NP antibody diluted 1:4000, and added to MDCK cells; the dilution ratio of the anti-HSV-1 rabbit antibody is 1: 2500, adding to Vero cells; incubate at 37 ℃ for 1 h.

(11) After incubation, the cell plates were washed 3 times with PBST for 5min each time, and spun off.

(12) Incubation of secondary antibody: 3% BSA diluted secondary antibody, dilution ratio 1: 2000, HRP-anti-mouse secondary antibody was added to MDCK cells, and HRP-anti-rabbit secondary antibody was added to Vero cells, which were incubated for 1h at 37 ℃.

(13) After incubation, the cell plates were washed 3 times with PBST for 5min each time, and spun off.

(14) AEC mixtures were prepared according to the protocol and added to 96-well cell culture plates at 50. mu.L/well.

(15) Incubating in 37 deg.C incubator for 30min, removing AEC mixed solution, washing 96-well plate with deionized water for 3 times, spin-drying, and observing erythema number under microscope. Viral titer (PFU/mL) ═ average number of erythema × 20 × 1/corresponding dilution concentration.

5. Construction of a double infection model

5.1 mouse double infection model protocol design:

according to the purpose of experiments, the double infection of H3N2 and HSV-1 is divided into 3 models, each model is respectively provided with a corresponding double infection group, a single infection control group and a blank control group, the blank control group adopts PBSMock) to simulate infection, an HSV-1-H3N2 overlapping infection model and an H3N2+ HSV-1 co-infection model are established by selecting the H3N2 infection dose of 200PFU and the HSV-1 infection dose of 104PFU, and an H3N2-HSV-1 overlapping infection model is established by selecting the H3N2 of 10PFU and the HSV-1 infection dose of 104 PFU. The H3N2 or HSV-1 alone infected control and blank control were set in the three models with an infection time interval of 3 days. See table 2 for details.

Table 2: establishment and grouping of H3N2 and HSV-1 dual infection model

(1) HSV-1-H3N2 double infection model: the experimental group was infected with HSV-1 first, and was infected with H3N2 after 3 days; the control group was infected with PBS, HSV-1, PBS, respectively, and 3 days later, H3N2, PBS, respectively, were administered for 2 infections.

(2) H3N2 and HSV-1 co-infection model: the experimental group was simultaneously infected with HSV-1 and H3N2, and the control group was administered with H3N2, HSV-1, and PBS, respectively.

(3) H3N2 and HSV-1 double infection model: the experimental group was infected with H3N2 first, and after 3 days, HSV-1 was infected; the control group was infected with H3N2, PBS, respectively, and 3 days later with PBS, HSV-1, PBS, respectively, for 2 infections.

The three models, 120 mice each, 30 mice each, were infected as described above.

The three models take the infection time point of H3N2 as the 0 th day of infection, and the weight and death condition of the mouse are detected every day; virus titers were measured in lung homogenates and BALF (lung bronchoalveolar lavage fluid) on

days

4, 6, 8, 10 post H3N2 infection; lung pathology was examined on day 8 post H3N2 infection, with time nodes shown in figure 1.

5.2 Experimental procedures for infection in mice

(1) In the SPF class biosafety class 2 animal center, experimental C57BL/6 female mice were weighed in biosafety cabinets, initial weights of the mice were recorded and labeled in groups.

(2) According to the experimental protocol design, the virus was diluted with PBS and placed on ice for use. Care should be taken to avoid cross-contamination when diluting the virus.

(3) Placing a cotton ball into an anesthesia container, adding 1mL of isoflurane anesthetic, slightly grabbing the mouse and placing the mouse into an anesthesia tank, taking attention to the good tightness of the anesthesia tank for about 30s, quickly taking out the mouse when the limbs of the mouse are observed to be immobile and the breath of the mouse becomes shallow, grabbing the mouse with the left hand to enable the mouse to be in a 45 ℃ supine position, sucking 50 mu L of virus liquid with a 100 mu L pipettor with the right hand, and slightly dropping the virus liquid into the nostril of the mouse. The mice were then placed in a well ventilated place to allow them to recover and the anesthetic was added in time when not sufficient. According to the above process, mice are infected with viruses of different doses, and the name, sex, infection time, infection dose, group and name of the experimental operator of the experimental animal are noted and marked.

(4) Establishing an overlapping infection model, infecting H3N2 virus with proper dose at the time point of IAV infection, dripping 50 mu L PBS solution before or 3 days after H3N2 infection to establish a co-infection model, and dripping the H1N1 or H3N2 virus and PBS to infect the mouse with nasal drip after the virus is uniformly mixed, wherein the total infection volume is 50 mu L.

(5) Daily body weight and mortality of the mice were monitored and recorded. In all models, the time of H3N2 infection was defined as day 0. Weight loss of more than 30% of the original body weight is considered the death limit of the study and mice are euthanized.

6 detection of physiological indexes

6.1 weight measurement and survival Change

The experimental mice were individually weighed before and after infection, and analyzed for variance and t-test using SPSS16.0 statistical software.

6.2 survival Rate Change:

the survival of experimental mice under different infection modes was recorded.

6.3 determination of pulmonary index:

(1) mouse weight: the mice were weighed and recorded daily.

(2) Lung weight of mice: in the experiment, the mice were dissected at different time points, lung tissues were cut off, blood was rinsed in precooled PBS, residual liquid was aspirated with sterile gauze, and the clear weight of lung tissues was weighed on an electronic analytical balance.

(3) Lung index: the lung index is calculated according to the formula "lung index is 100 × lung weight (g)/weight (g) of the corresponding mouse", and indirectly reflects the degree of lung inflammation and edema.

The method for obtaining the lung tissue of the mouse comprises the following steps:

(1) after 1% sodium pentobarbital was given to the anesthetized mice by intraperitoneal injection, the mice were sacrificed by eye bleed. The limbs are fixed on the dissection board in a supine position.

(2) The local skin of the mouse is disinfected by alcohol, and the skin of the chest is cut off in sequence along the median line by surgical scissors, so that the chest cavity is fully exposed, and the lung tissue is prevented from being damaged.

(3) Lung tissue for virus titer detection in lung homogenate was cut off lung lobes with scissors, blood was rinsed in precooled PBS, residual liquid was aspirated with sterile gauze, lung weight was weighed with an electronic balance, placed in a 1.5mL EP tube, experimental group, date and lung weight were marked, immediately poured into liquid nitrogen, snap frozen and stored in a-80 ℃ refrigerator for long-term storage.

(4) A lung tissue for lung pathology is prepared by cutting a small opening at left auricle of heart of mouse, extracting physiological saline with 20mL syringe, inserting into right ventricle, and flushing lung tissue until lung becomes white. A10 mL syringe was again used to inject 10% formalin from the right ventricular site for fixation in lung tissue. Lung tissue was cut from the trachea, placed in a 50mL centrifuge tube previously filled with 20mL 10% formalin, and fixed on a shaker for 24 h.

6.4 Virus titer detection: after anesthetizing the mice, the eyes bleed and the mice are sacrificed. Obtaining the mouse lung homogenate supernatant and the alveolar lavage fluid supernatant, and detecting the H3N2 and HSV-1 virus titer by adopting an immunocytochemistry method.

Among them, the lung tissue homogenate supernatant and alveolar lavage fluid (BALF) of the mouse were obtained as follows.

6.4.1 Lung tissue homogenate supernatant harvest

(3) Shearing off lung lobes with scissors, rinsing in precooled PBS to remove blood, sucking residual liquid with sterile gauze, weighing lung weight with an electronic balance, placing into a 1.5mL EP tube, marking experimental group, date and lung weight, immediately putting into a liquid nitrogen tank, quickly freezing, and storing in a refrigerator at-80 ℃.

(4) PBS containing 1% streptomycin double antibody was prepared: the volume ratio of the penicillin streptomycin double antibody to the PBS is 1: 100.

(5) Lung homogenization: frozen lung tissue was removed from the freezer at-80 ℃ and placed on ice, 300. mu.L of 1% penicillin bisanti PBS was added and the tissue samples were disrupted in a low temperature ultrasonication apparatus. Sonicate every 5s for a total of 3 sonications. After the sample is operated, the sample is quickly placed on ice, so that the virus titer degradation caused by excessive heat production is avoided.

(6) And (3) lung homogenate centrifugation: the homogenized sample was placed in a super low temperature high speed centrifuge precooled to 4 ℃ in advance, centrifuged at 12000rpm for 10 min.

(7) Subpackaging and storing: and (4) subpackaging the centrifuged sample supernatant into 600 mu L sterile centrifuge tubes, wherein the subpackaging specification is 100 mu L per tube, and storing in a refrigerator at the temperature of-80 ℃.

6.4.2 mouse BALF acquisition

(1) Mice were anesthetized with 1% sodium pentobarbital using intraperitoneal injection anesthesia. After anesthetizing the mice, the four limbs were fixed on the dissecting plate in the supine position.

(2) The local skin of the mouse is disinfected by alcohol, the skin of the neck is cut off along the median line by surgical scissors, and the trachea is fully exposed by blunt separation of surgical forceps.

(3) Inserting forceps under the trachea for supporting, inserting the remaining needle, after the needle head is inserted into the trachea, pulling out the inner needle core of the remaining needle, and keeping the outer layer soft needle head. 0.8mL of PBS buffer pre-cooled at 4 ℃ was extracted with a 1mL sterile syringe, an indwelling needle was connected and the alveolar lavage fluid was collected in a 5mL sterile centrifuge tube and repeated 2 times, and the recovery of alveolar lavage fluid was recorded (the recovery should be greater than 60%). Note that the operation should be slow to avoid damaging the lung tissue.

(4) The collected alveolar lavage fluid was centrifuged at 1000rpm in a 4 ℃ centrifuge for 10 min. Centrifuging, and subpackaging the supernatant with 1.5mL sterile EP tube with subpackage specification of 0.5 mL/tube and storing in a refrigerator at-80 deg.C for use; the cell pellet was used for subsequent immunocytology studies.

(5) BALF supernatant was used for virus titer and cytokine detection. Wherein, the virus titer is determined by an immune plaque method, and the cytokine is determined by a Luminex liquid phase chip technology.

6.5 lung pathology test: after anesthetizing the mice, the eyes bleed and the mice are sacrificed. Lungs were dissected, formalin fixed, HE stained and viewed under the mirror.

The preparation method of the pathological section comprises the following steps:

(1) material taking and fixing: after 1% sodium pentobarbital was given to the anesthetized mice by intraperitoneal injection, the mice were sacrificed by eye bleed. Fixing four limbs on an anatomical plate in a supine position, disinfecting local skin of a mouse with alcohol, and sequentially cutting off skin of a chest along a median line by using surgical scissors to fully expose the chest, so that lung tissues are prevented from being damaged. A small opening is cut at the left auricle of the heart of the mouse, a 20mL syringe is used for extracting the physiological saline, the physiological saline is inserted into the right ventricle, and the lung tissue is flushed until the lung is completely whitened. A10 mL syringe was again used to inject 10% formalin from the right ventricular site for fixation in lung tissue. Lung tissue was cut from the trachea, placed in a 50mL centrifuge tube previously filled with 20mL 10% formalin, and fixed on a shaker for 24 h.

(2) Baking slices: and (3) putting the paraffin tissue slices into a 60 ℃ baking machine for baking for 5 min.

(3) Dewaxing: TO I (10min) → TO II (10 min).

(4) Hydration: absolute ethanol (2min) → 95% ethanol (2min) → 85% ethanol (2min) → 75% ethanol (2min) → water washing (1min)

(5) Hematoxylin staining: 5min, removing oxidized particles before each use

(6) Flushing hematoxylin staining solution with running water: 1min

(7) 1% hydrochloric acid alcohol differentiation solution: 1-5s

(8) Returning the flowing water to blue: for 10min

(9) Eosin staining: 20s

(10) Flushing eosin dye solution with running water: 10s

(11) And (3) dehydrating: 75% ethanol (10s) → 85% ethanol (10s) → 95% ethanol (30s) → absolute ethanol (1min)

(12) And (3) transparency: phenol TO (1min) → TO II (1min) → TO III (1min)

6.6H3N2 and HSV-1 double infection mouse model establishment and immune index detection time point

(1) Three models of HSV-1-H3N2 superinfection model, H3N2+ HSV-1 co-infection model and H3N2-HSV-1 superinfection are established according to the above, each model is provided with an independent infection control group and a blank group, and 30 mice in each group.

(2) And (3) detecting cytokines: IFN-gamma, TNF-alpha, IP-10, MCP-1, IL-10, IL-6 levels were measured on

days

6 and 8 after H3N2 infection.

(3) And (3) detecting immune cells: detection of NK on

days

6 and 8 after H3N2 infection,

pDC, IKDC cellular changes.

(4) Detection of adaptive immune cells: detecting changes of B cells, CD 4T cells and CD8T cells at 6 and 8 days after H3N2 infection; virus-specific CD8T cell changes were detected on day 10 post H3N2 infection. Specific time points are shown in fig. 1.

6.7 mouse spleen tissue harvesting

(2) The local skin of the mouse is disinfected by alcohol, and the abdominal skin is cut off in sequence along the median line by surgical scissors, so that the abdominal cavity is fully exposed.

(3) Finding a dark red strip spleen on the left abdomen of the mouse, quickly picking the spleen, putting the spleen into a PRMI-1640 culture medium which is added with 1% double antibody in advance, and storing the spleen on ice for later use.

6.8 preparation of Single cell suspensions of Lung tissue and spleen tissue of mice

(1) The lung tissue and spleen tissue were cut into pieces using sterile ophthalmic scissors.

(2) The lung tissue is cut into pieces and then put into a sterile centrifuge tube containing 3mL of digestive juice, digested in water bath at 37 ℃ for 30min, shaken once every 10min, and then added with C-RPMI culture medium to stop digestion.

(3) Placing a nylon filter screen with the diameter of 70 mu m in a 50mL sterile centrifuge tube, respectively pouring digested lung tissue or chopped spleen tissue on the nylon filter screen, grinding the fragments of the lung tissue or the spleen tissue by using a piston head of a syringe, fully grinding, and washing the nylon filter screen by using an RPMI-1640 culture medium containing 1% double antibody.

(4) Centrifuging the centrifuge tube containing the tissue grinding fluid in a low temperature centrifuge at 4 deg.C and 1500rpm for 5 min.

(5) And (4) after the centrifugation is finished, discarding the supernatant, adding erythrocyte lysate into the cell sediment to lyse the erythrocytes for 5-10min, and observing the state of the cells.

(6) After the lysis is finished, adding 1% double-antibody RPMI-1640 culture medium to stop the lysis, and centrifuging at 4 ℃ and at 1500rpm for 5 min.

(7) The supernatant was discarded, 1mL of FACS buffer was added to resuspend the cells, the cells were counted, the cell density was adjusted to 1X 106/mL, and the cells were stored on ice until use.

6.9Luminex cytokine/chemokine assay

(1) Taking out the BALF supernatant to be tested, and unfreezing the BALF supernatant on ice for later use.

(2) Resuspending and diluting standards: the standards were removed and centrifuged at 2000g for 10s in a high speed centrifuge, 250. mu.L of Universal Assay Buffer (1X) resuspended standard, and centrifuged at 2000g for 10 s. Diluting the standard substance by 4 times of concentration gradient, taking out an 8-connection tube, and marking as 1-8; 200. mu.L of the resuspended standards were added to

tube

1, and 150. mu.L of Universal Assay Buffer (1X) was added to tubes 2-7, respectively. Taking 50 μ L from the 1 st tube, adding into the 2 nd tube, mixing, taking 50 μ L from the 2 nd tube, adding into the 3 rd tube, and so on, and gradually diluting. Only 200. mu.L of Universal Assay Buffer (1X) was added to tube 8; and diluting the standard substance for later use.

(3) And taking out the 96-well plate in the kit, and marking the standard well, the sample well and the blank well.

(4) Adding magnetic beads, wherein each well is 50 mu L, placing a 96-well flat-bottom plate on a handheld magnetic plate washer, standing for 2min, and waiting for the magnetic beads to gather at the bottom of the wells; quickly throwing off liquid in the 96-pore plate; and adding 150 mu L of washing solution into each hole, standing for 30s, waiting for the magnetic beads to gather at the bottom of the 96 holes, quickly throwing off the washing solution, and taking down the 96 hole plate from the handheld magnetic plate washer.

(5) According to the design of the pore plate, a standard substance, a blank control and a sample to be detected are sequentially added into a 96 pore plate, and each pore is 50 mu L. Shaking at 500rpm for 60-120min in dark at room temperature; and (5) washing the plate for 3 times according to the step (4) after the oscillation is finished.

(6) Adding 25 mu L of antibody to be detected into each hole, and shaking at 500rpm for 30min in a dark place at room temperature; and (5) washing the plate for 3 times according to the step (4) after the oscillation is finished.

(7) Adding 50 μ L SAPE into each well, and shaking at 500rpm for 30 min; and (5) washing the plate for 3 times according to the step (4) after the oscillation is finished.

(8) Add 120. mu.L of Reading buffer to each well, shake at 500rpm for 5min in the dark at room temperature, and then detect on Luminex 100/200 Magpix.

6.10 molecular markers of immune cells on day 6 and day 8 after H3N2 infection

(1) After centrifugation of single cell suspensions of BALF, lung or spleen, 1:200 dilutions of anti-CD16/32 antibody were incubated on ice for 30min to block Fc receptors, 50. mu.L per sample.

(2) After the blocking is finished, adding 1mL of FACS buffer to wash the cells, and centrifuging at 4 ℃ for 500g for 5 min; the supernatant was discarded, and 1mL of FACS buffer was added again to wash the cells 1 time, and the cells were centrifuged at 4 ℃ at 500g for 5 min.

(3) After centrifugation, adding a flow cell surface antibody diluted by a FACS buffer, wherein the specific antibody is as follows: anti-mouse/human CD44 (FITC; BioLegend), anti-mouse CD62L (PE/Cy 7; BioLegend), anti-mouse CD3(APC/FireTM 750; BioLegend), anti-mouse CD45R/B220 (PE; BioLegend), anti-mouse NK1.1(BV 421; BioLegend), anti-mouse CD11c (BV 421; BioLegend), anti-mouse F4/80(Alexa A)

647; BioLegend), anti-mouse/human CD11b (FITC; BioLegend) antibody dilution ratio 1:200, 50 μ L/tube. After incubation for 30min in the dark on ice, the cells were washed 2 times according to step (2).

6.11 statistical analysis

The experimental data were analyzed using SPSS 21.0 statistical software, plotted using GraphPad Prism 5.0, and all experiments were repeated in parallel at least three times, keeping the experimental results stable. The experimental data are presented as mean ± standard deviation, the differences between the two groups using either t-test or non-parametric test, and the multiple group comparisons using one-way analysis of variance (ANOVA) or Kruskal-Wallis test. P <0.05 was considered to have statistical differences, # P <0.05, # P < 0.01; p < 0.001.

7. Results and analysis

7.1 double infection of HSV-1 and H3N2 three models mice weight, survival rate and lung index change and mice BALF, H3N2 in lung homogenate, HSV-1 virus load change

(1) Mouse weight, survival rate and lung index changes in HSV-1-H3N2 superinfection model

The model is that the mice are infected with HSV-1 firstly (day-3), are infected with H3N2 (day 0) on day 3 after infection, and start to lose weight on day 3 after H3N2 infection, the virus-infected mice begin to lose weight, wherein the weight loss degree of the HSV-1-H3N2 overlapping infected mice is higher than that of the HSV mice infected with the H3N2 alone (HSV-1-Mock group) but is obviously lower than that of the H3N2 virus alone (Mock-H3N2 group), the weight loss degree is higher than 20%, and the weight loss degree is lower than that of the HSV-1-H3N2 double-infected groups, and the weight loss degree is the HSV-1 mice infected with the virus alone (see figure 2 and A). None of the four groups of mice died (see figure 2, B; P < 0.05; four lines coincide).

The lung index is an index reflecting lung inflammation and edema, and the lung index is increased after mice are infected with virus. On day 4 after H3N2 infection, the lung index of the HSV-1-Mock group was significantly higher than that of the HSV-1-H3N2 group and Mock-H3N2 group; on day 6 post H3N2 infection, the lung index was higher in the HSV-1-H3N2 group than in the HSV-1-Mock group; the lung index of the HSV-1-H3N2 group at

day

8, 10 post H3N2 infection was lower than that of the Mock-H3N2 group but higher than that of the HSV-1-Mock group (FIG. 2, C; P < 0.05). The above results suggest: the previous infection of HSV-1 can reduce the weight loss and lung injury caused by the reinfection with H3N 2.② the inflammatory response caused by HSV-1 infection is earlier than H3N2, which leads the lung index of the early HSV-1 group to be higher than that of the other groups, while the inflammatory response caused by H3N2 infection is taken as the main factor in the later infection period, which leads the lung injury degree of the Mock-H3N2 group to be higher than that of the HSV-1-H3N2 group.

The H3N2 viral load was significantly higher in the BALF and lung homogenate of the Mock-H3N2 group than in the HSV-1-H3N2 group on

days

4, 6, and 10 after H3N2 infection, and was highest on day 6 and largely cleared on day 10 after H3N2 infection (FIG. 3, A; P < 0.05). Whereas the HSV-1-H3N2 group showed lower HSV-1 viral loads in lung homogenate at day 4 and BALF at day 6 after H3N2 infection (FIG. 3, B; P <0.05) compared to the HSV-1-Mock group, but there was no significant difference at other times. The above results show that: firstly, HSV-1 can inhibit the replication of H3N2 by early infection and promote virus elimination; infection with H3N2 can also slightly inhibit replication of HSV-1; ③ the advanced infection of HSV-1 has a certain protective effect on the secondary infection of H3N 2.

(2) Mouse weight, survival rate and lung index changes in H3N2+ HSV-1 co-infection model

The model mainly comprises an H3N2+ HSV-1 co-infection group, an H3N2 group, an HSV-1 group and a Mock group. The weight reduction degree of the H3N2+ HSV-1 co-infected mice in the 4 th to 10 th days after the H3N2 infection is not obviously statistically different from that in the H3N2 group, but is obviously higher than that in the HSV-1 group (figure 4, D; P < 0.05). On day 10 after H3N2 infection, the mortality rate in the H3N2+ HSV-1 co-infected group was 41%, significantly higher than the 8.5% mortality rate in the H3N2 group, while no mortality occurred in the HSV-1 group (FIG. 4, E; P < 0.05; HSV-1 line coincides with blank line). The pulmonary index of the H3N2+ HSV-1 co-infected group was higher on day 6 post-H3N 2 infection than the H3N2 group, and higher on

days

4, 8, 10 than the HSV-1 group (FIG. 4, F; P < 0.05). The results indicate that the H3N2+ HSV-1 co-infected group has heavier lung injury and higher mortality than the H3N2 infected group alone.

In the H3N2+ HSV-1 group, compared to the H3N2 group, the lung homogenate H3N2 virus titer was significantly increased at

days

4, 6, and 10 after H3N2 infection (FIG. 5, A), but there was no significant difference in BALF; similarly, the H3N2+ HSV-1 group had the highest H3N2 viral titer at day 6 post H3N2 infection and was gradually cleared at day 10. The H3N2+ HSV-1 group showed a significant reduction in lung homogenate HSV-1 virus titer compared to the HSV-1 group, whereas the HSV-1 virus titer in both groups of BALF was statistically significantly different only on day 8 after H3N2 infection. The above results show that: H3N2 and HSV-1 are infected simultaneously, so that the clearance speed of H3N2 is reduced, and the virus load is high. ② the co-infection can inhibit the replication of HSV-1 virus and accelerate the clearance of HSV-1 (figure 5, B).

(3) Mouse weight, survival rate and lung index changes in H3N2-HSV-1 double infection model

The model is mainly divided into an H3N2-HSV-1 group, an H3N2-Mock group, a Mock-HSV-1 group and a Mock-Mock group. The results showed that the mice in the H3N2-HSV-1 group all had significantly higher weight loss, mortality, and pulmonary index than the H3N2-Mock and Mock-HSV-1 groups (FIG. 6; P < 0.05). The body weight of mice in the H3N2-HSV-1 and H3N2-Mock groups decreased gradually from day 4 to day 9 after H3N2 infection, with a significant statistical difference from day 6 to day 8 (FIG. 6, G; P < 0.05). The mortality rate for the H3N2-HSV-1 group was 28.5%, significantly higher than 7.6% for the H3N2-Mock group (FIG. 6, H; P < 0.05; Mock-HSV-1 group overlaps with Mock-Mock group black line). The lung index of H3N2-HSV-1 mice was significantly higher on

day

8 and 10 after H3N2 infection than that of the H3N2-Mock group, and on day 6-10 than that of the Mock-HSV-1 group (FIG. 6, I; P < 0.05). The results indicate that the pathogenicity is obviously increased and the death rate is high when HSV-1 is infected again after the H3N2 is infected.

Compared with the H3N2-Mock group, the H3N2-HSV-1 group has obviously increased virus loads of H3N2 in BALF and lung homogenate at 4 th, 6 th, 8 th and 10 th days after H3N2 infection, wherein the virus loads are highest at 4 th to 6 th days and the virus loads are reduced slowly; at the same time point, the viral load in BALF was slightly higher than in lung tissue (fig. 7, a). The H3N2-HSV-1 group showed a significant reduction in HSV-1 virus titer in BALF compared to Mock-HSV-1 group, whereas the lung homogenate HSV-1 virus titer in both groups was only statistically different at day 6 and day 10 after H3N2 infection (fig. 7, B). The above results show that: H3N2 delays virus clearance speed after HSV-1 infection, which leads to the increase of H3N2 virus load; secondly, H3N2-HSV-1 double infection can effectively inhibit the replication of HSV-1 and reduce the virus load of HSV-1.

(4) The weight, survival rate and lung index change of mice in a double infection group and the difference of H3N2 and HSV-1 virus loads in the three models

Transverse comparison of the double-infection group in the three models shows that the maximum weight loss degree of the H3N2+ HSV-1 and the H3N2-HSV-1 is similar to 23%, and the weight loss degree is statistically different from 12% of the maximum weight loss degree of the HSV-1-H3N 2. The highest mortality rate was H3N2+ HSV-1 and H3N2-HSV-1, with no significant statistical difference between the two groups, and no mortality in the HSV-1-H3N2 mice. The lung indices of the three groups were not statistically significantly different. The synthesis of the following steps: firstly, HSV-1 is infected in advance, so that the weight loss degree and the lung index caused by reinfection with H3N2 can be reduced, and the death rate is reduced; ② the pathogenicity of H3N2 can be obviously increased by infecting HSV-1 and H3N2 simultaneously or infecting HSV-1 again after H3N2 is infected.

At the same time point after H3N2 infection, the H3N2 viral load differences between the double-infected groups in the three models were further compared laterally. The results show that the H3N2 viral loads in the BALF have obvious statistical differences among the HSV-1-H3N2 group, the H3N2+ HSV-1 group and the H3N2-HSV-1 group, wherein the H3N2-HSV-1 group has the highest viral load, and the HSV-1-H3N2 group has the lowest viral load; the H3N2 virus titers in the three groups of lung homogenates were statistically different at

days

6 and 10 after H3N2 infection, with H3N2+ HSV-1 having the highest viral load at day 6 after H3N2 infection and H3N2-HSV-1 having the highest viral load at day 10 after H3N2 infection. Secondly, the clearance rate of H3N2 virus is different among the three groups, the clearance rate of the virus is fastest among the HSV-1-H3N2 group, the clearance rate of the virus is second among the H3N2+ HSV-1 group, and the clearance rate of the virus is slowest among the H3N2-HSV-1 group (figure 8). This again demonstrates that premature HSV-1 infection can facilitate rapid clearance of H3N2, thereby reducing H3N2 viral titers; in contrast, infection of HSV-1 with H3N2, either simultaneously or after H3N2, slows the clearance of H3N2, resulting in high H3N2 viral titers.

7.2H 3N2 Lung Pathology HE staining following double infection with HSV-1

Lung organization pathology is the gold standard reflecting the extent of lung injury. After viral infection, the severity of tissue inflammation correlates with the magnitude of viral load in lung tissue. Normal mice have uniform bronchial wall thickness, regular shapes and uniform sizes of bronchial cells, bronchiole cells and alveolar epithelial cells, and no red blood cells and inflammatory cells are in the alveolar cavities. In the three models of the study, lung tissue was taken for HE staining on day 8 after H3N2 infection to assess the degree of lung injury.

In the HSV-1-H3N2 superinfection model, Mock-H3N2 group has the advantages of bronchiolitis cilia epithelial cell cilia shedding, interstitial fibrosis, alveolar space widening and filling with a large amount of mononuclear lymphocytes and erythrocytes; the lung interstitial fibrosis and alveolar septal broadening of the HSV-1-H3N2 group are not obvious, and inflammatory cell infiltration is less than that of the Mock-H3N2 group; the HSV-1-Mock group showed only a small lymphocyte infiltration with no apparent lung injury (FIG. 9, A).

In the H3N2+ HSV-1 co-infection model, the bronchioles epithelium of the H3N2+ HSV-1 group is completely damaged, the structure disappears, a large number of lymphocytes around the airway are gathered, the interstitium is changed, the alveolar space is broken and obviously widened, and the alveolar space is filled with red blood cells; the H3N2 group has a clear local tracheal epithelial structure, mononuclear lymphocyte aggregation is seen locally, alveolar space is slightly widened, and a small amount of red blood cells are seen in a lumen; HSV-1 group has clear bronchioles and alveolar spaces, local inflammatory cells aggregate into clumps and local fibrosis (FIG. 9, B).

In the H3N2-HSV-1 superinfection model, the thin bronchial walls of the H3N2-HSV-1 group are widened, the basal cells are subjected to myxopoiesis, inflammatory cells around the walls of the thin bronchial walls are aggregated, the interstitium is seriously fibrillated and changed, the alveolar space is widened and broken, the cilia of local thin bronchial cilia epithelial cells of the H3N2-Mock group collapse are fallen off, the local fibrosis and the alveolar space are broken, but the alveolar space has a proper structure, and the infiltration of lymphocytes is less than that of the H3N2-HSV-1 group; the Mock-HSV-1 group had no significant lesions (FIG. 9, C). Comparing lung pathology HE staining of a double infection group in three models, the results show that inflammatory cells around mouse trachea are aggregated, lung tissue fibrosis and degree of compaction are H3N2-HSV-1 group > H3N2+ HSV-1 group > HSV-1-H3N2 according to the severity degree, and the results are consistent with the expression condition of H3N2 virus load. In conclusion, HSV-1 preinfection can reduce the degree of lung injury caused by H3N 2; however, infection of HSV-1 and H3N2 at the same time or after H3N2 infection can obviously increase the degree of lung inflammation, fibrosis and inflammatory cell infiltration, and the lung injury caused by subsequent HSV-1 infection after H3N2 infection is the most serious.

7.3 expression differences of cytokines and chemokines in mouse alveolar lavage fluid (BALF) in three models of double infection of HSV-1 and H3N2s

HSV-1-H3N2 double infection model

The expression level of the cytokines and chemokines in HSV-1-H3N2 groups at 6 days and 8 days after H3N2 infection is mostly between that in Mock-H3N2 group and HSV-1-Mock group. On the 6 th day after H3N2 infection, the expression levels of IFN-gamma, TNF-alpha, IP-10 and IL-10 in the HSV-1-H3N2 group are lower than those in the Mock-H3N2 group, while the expression levels of IFN-gamma, TNF-alpha, IP-10, MCP-1, IL-10 and IL-6 are higher than those in the HSV-1-Mock group, and the statistical difference is achieved (figure 10, A; P < 0.05). On day 8 after H3N2 infection, the expression levels of IFN-gamma, TNF-alpha, MCP-1 and IL-10 in the HSV-1-H3N2 groups were still lower than that in the Mock-H3N2 group, and the expression levels of IFN-gamma, TNF-alpha, IP-10, MCP-1, IL-10 and IL-6 were still higher than that in the HSV-1-Mock group, with statistical differences (FIG. 11, A; P < 0.05). As shown above, the advanced infection of HSV-1 can reduce the level rise of inflammatory factors caused by the infection of H3N 2; infection with H3N2 promotes the levels of inflammatory factors caused by HSV-1 infection.

H3N2+ HSV-1 coinfection model

On day 6 after H3N2 infection, the level of TNF-alpha in the H3N2+ HSV-1 co-infected group was higher than that in the H3N2 group, while the expression level of IP-10 and IL-10 was lower than that in the H3N2 group (FIG. 10, B; P < 0.05). On day 8 after H3N2 infection, the expression levels of IFN-. gamma.TNF-. alpha.IP-10 and IL-6 in the H3N2+ HSV-1 co-infected group were higher than in the H3N2 group (FIG. 11, B; P < 0.05). Compared with the HSV-1 group, the H3N2+ HSV-1 co-infection group has significantly higher expression levels of IFN-gamma, TNF-alpha, IP-10, MCP-1, IL-10 and IL-6 at the 6 th and 8 th days after the H3N2 infection (figure 10, B; figure 11, B; P < 0.05). As shown above, the co-infection of H3N2 and HSV-1 can promote the release of a large amount of cytokines and chemokines in the later stage of infection, and trigger inflammatory reaction.

H3N2-HSV-1 double infection model

On day 6 after H3N2 infection, there was no significant difference in cytokine expression levels between the H3N2-HSV-1 and H3N2-Mock groups, except that IL-6 was significantly elevated; compared with the Mock-HSV-1 group, the H3N2-HSV-1 group has obviously reduced expression levels of IFN-gamma, TNF-alpha and IP-10 and increased IL-6 (figure 10, C; P < 0.05). On day 8 after H3N2 infection, IFN-gamma, MCP-1 and IL-6 in the H3N2-HSV-1 group were significantly higher than that in the H3N2-Mock group, and the expression levels of IFN-gamma, TNF-alpha, IP-10, MCP-1, IL-10 and IL-6 were all higher than that in the Mock-HSV-1 group (FIG. 11, C; P < 0.05). The results show that after HSV-1 infection is secondarily caused by H3N2, the body can be promoted to generate a large amount of inflammatory factors, a persistent inflammatory reaction is caused, and lung injury is aggravated.

The expression difference of cytokines and chemokines of the double infection group in the three models

The expression difference of cytokines and chemokines of the double infection group in the three models is transversely compared, and the result shows that: on day 6 after H3N2 infection, the expression levels of IFN-gamma, TNF-alpha and IL-10 in the H3N2+ HSV-1 group were higher than those in the H3N2-HSV-1 group and the HSV-1-H3N2 group, the expression level of IP-10 was lower than that in the H3N2-HSV-1 group, and the expression levels of other factors were not significantly different in the three groups (FIG. 12, A). On the 8 th day after H3N2 infection, the expression levels of IFN-gamma, IP-10, MCP-1, IL-10 and IL-6 are obviously higher in the H3N2-HSV-1 group than in the H3N2+ HSV-1 group and the HSV-1-H3N2 group, and the statistical difference is realized (figure 12, B; P <0.05), wherein the abnormal increase of IFN-gamma, IP-10, IL-10 and IL-6 is mainly used; the expression level of TNF-alpha in the H3N2+ HSV-1 group is higher than that in the H3N2-HSV-1 group and the HSV-1-H3N2 group (P < 0.05). The above results again demonstrate that premature infection with HSV-1 reduces the inflammatory response to H3N2 infection with low levels of inflammatory factors, whereas infection with HSV-1, or subsequent infection with HSV-1, with H3N2, produces a persistent inflammatory response characterised by a large number of cytokines.

7.4HSV-1 and H3N2 double infection of three models mice NK, B, pDC, IKDC,

Immunocytic changes FIG. 13 shows NK, B, pDC, IKDC,

Flow cytometric analysis of cells was gated.

(1) HSV-1-H3N2 double infection model

On day 6 after H3N2 infection, the expression levels of NK, B, pDC, IKDC cells in BALF and lung tissues and B cells in spleen tissues were significantly reduced in HSV-1-H3N2 group compared with Mock-H3N2 group. While the group HSV-1-H3N2 compares with the group HSV-1-Mock in BALF

Cells, NK cells in lung tissue, pDC cells, IKDC cells,

Increased expression levels in cells (FIG. 14, A, P)<0.05). On day 8 after H3N2 infection, the expression levels of NK cells in BALF, NK cells in pDC, IKDC and IKDC in lung tissues and NK cells in spleen tissues are obviously reduced in the HSV-1-H3N2 group compared with the Mock-H3N2 group. HSpDC and pDC in BALF compared with HSV-1-Mock in group V-1-H3N2

pDC cell expression level was elevated in cells and lung tissue, while NK cells, lung tissue NK and B cells were significantly reduced in BALF (FIG. 14, D, P)<0.05). The above results show that the expression levels of NK, pDC and IKDC of the HSV-1-H3N2 group are between the Mock-H3N2 group and the HSV-1-Mock group.

The reasons are mainly that: the innate immunity peaks caused by HSV-1 and H3N2 infections are different. The peak of innate immunity for HSV-1 was on day 3 post HSV-1 infection, while the peak of innate immunity for H3N2 was on days 4-6. When HSV-1 is infected for 3 days, H3N2 is infected again, the immune response which is enhanced in vivo can generate effective inhibition effect aiming at H3N2, the virus clearance of HSV-1-H3N2 group is promoted, and the inflammatory response and the injury degree of lung are reduced. With effective virus clearance, there was a gradual decrease in the level of innate immune cells in vivo, so on

days

6 and 8, the level of innate immunity was higher in the HSV-1-H3N2 group than in the Mock-H3N2 group.

(2) H3N2+ HSV-1 coinfection model

NK, B, pDC, IKDC, in BALF, compared with H3N2, on day 6 after infection with H3N2 in H3N2+ HSV-1 group,

The cell percentage is lower, while the expression levels of lung NK cells and spleen IKDC cells are higher. H3N2+ HSV-1 in BALF compared to HSV-1

Cells, lung NK, pDC, IKDC,

The expression level of IKDC in cells and spleen is obviously increased (FIG. 14, B, P)<0.05). On day 8 after H3N2 infection, NK, B, pDC, IKDC, B, C, B, C, B, C, B, C, B, C, B, C, B, C, B, C, B, C, B, C, B, C, B, C, B, C, B, C, B, C, B, C, B, C,

the cell expression level is still low, no obvious difference exists in lung tissues, and spleen pDC is fineThe cell expression is higher. NK, B, pDC, IKDC, in BALF, H3N2+ HSV-1, compared with HSV-1,

Cells, lung

The cells and splenic B, pDC cells were significantly increased, while the expression levels of lung NK and B cells were relatively decreased (FIG. 14, E, P)<0.05). The above results suggest that the innate immune response in the group co-infected with H3N2 and HSV-1 was weaker than in the group H3N2, NK, pDC, IKDC,

And the innate immune cells can not provide effective antiviral action, so that the clearance rate of the H3N2 virus in the co-infection group is low, the virus is greatly replicated in vivo, the viral load and inflammatory factors are increased, and the virus attacks the epithelial cells of the respiratory mucosa, thereby causing serious lung injury.

H3N2-HSV-1 superinfection model

On day 6 after H3N2 infection, BALF and lung B, H3N2-HSV-1 compared with H3N2-Mock,

The expression level of the cells is obviously increased, and other cells have no obvious difference; H3N2-HSV-1 group in BALF compared to Mock-HSV-1 group

Cells, lung and spleen pDC, IKDC cells were also significantly elevated (FIG. 14, C, P)<0.05). On day 8 after H3N2 infection, NK, pDC and IKDC cells, lung IKDC, in BALF in H3N2-HSV-1 group compared to H3N2-Mock group,

Cells and splenic pDC, IKDC cells were still significantly elevated. H3N2-HSV-1 group compared to Mock-HSV-1 group, B, pDC, IKDC, B, C,

cells, lung IKDC,

Cells and splenic pDC cells are obviously increased, while NK cells, lung NK, B cells and splenic NK in BALF,

Then significantly decreases (FIG. 14, F, P)<0.05). The above results indicate that the H3N2-HSV-1 group elicits the highest innate immune response in the middle and late stages of infection. The reason is that H3N2-HSV-1 group has high virus load and serious lung injury, promotes an organism to generate strong innate immune response and secretes high-level inflammatory cytokines, and is shown in that the levels of immune cells such as NK, pDC, IKDC and the like are increased, and the immune cells migrate to infected parts such as lung tissues, air passages and the like to resist virus action.

In three models, the double infection groups NK, B, pDC, IKDC,

Comparing the cell expression levels with those of NK, B, pDC, IKDC of the double infection group in three models,

The expression difference of the cells shows that the BALF and lung tissues B, pDC, IKDC, H3N2-HSV-1 group on 6 days and 8 days after H3N2 infection,

The expression level of cells such as cells is higher than that of H3N2+ HSV-1 group and HSV-1-H3N2 group. This again highlights that the innate immunity elicited by the H3N2-HSV-1 group was more intense in the 3-group double-infected group. This is related to the highest viral load and the most severe degree of lung injury in the H3N2-HSV-1 group among three groups, and attracts the body's innate immune cells to reach the local resistance to viral invasion.

Taken together, innate immune cells play an important role in regulating the difference in pathogenicity of H3N2 and HSV-1 superinfection. After virus infection, the immune cells are stimulated to gather from peripheral immune organs to lung tissues and air passages of infected parts, and the expression level of the immune cells is related to the virus load and the lung injury degree. Because the intrinsic immune response peaks caused by the H3N2 and HSV-1 are different, the immune response is different in strength and strength according to different infection sequences. The inherent immune cells effectively limit the replication and diffusion of viruses and reduce lung injury in the early stage of virus infection; the increase of the tissue virus load and the serious lung injury can further stimulate the organism to generate strong innate immune response, mobilize innate immune cells to reach lung tissues and air passages, and play an antiviral role.

Claims

1. A method for constructing a disease model of a mouse doubly infected by influenza A virus and herpes simplex virus type 1 comprises the following steps:

2. The method of claim 1, wherein the influenza a virus is H3N 2.

3. The construction method according to claim 1 or 2, wherein the detection of the physiological index comprises: measuring body weight change, observing survival rate, detecting lung index, detecting titer of influenza A virus and herpes simplex virus 1 virus, detecting pathological sections, and detecting cell factors in mouse alveolar lavage fluid; detecting changes of immune cells, natural killer cells, B cells, plasmacytoid dendritic cells, interferon-producing killer dendritic cells and macrophages in mouse alveolar lavage fluid, lung and spleen.

4. The construction method of claim 1, wherein the time point of H3N2 infection is taken as the 0 th day of infection, and the weight and death condition of the mouse are detected every day in all three models; virus titers and lung indices were measured in lung homogenates and mouse alveolar lavage fluid on days 4, 6, 8, and 10 post H3N2 infection; lung pathology was examined on day 8 post H3N2 infection.

5. The construction method according to claim 4, wherein the detection of lung pathology index comprises pathological section detection; detecting cytokines in mouse alveolar lavage fluid; detecting changes of immune cells, natural killer cells, B cells, plasmacytoid dendritic cells, interferon-producing killer dendritic cells and macrophages in mouse alveolar lavage fluid, lung and spleen.

6. The method of constructing according to claim 1, wherein the mouse is a C57BL/6 mouse.

7. The method of claim 1, wherein the influenza a virus is cultured using MDCK cells.

8. The method according to claim 1, wherein the herpes simplex virus type 1 is cultured using Vero cells.