CN113521117A

CN113521117A - Application of cedar needle leaf extract in inhibiting avian H9N2 AIV proliferation

Info

Publication number: CN113521117A
Application number: CN202110671392.4A
Authority: CN
Inventors: 韩明政; 胡文锋; 陈彤; 胡学生; 朱剑锋
Original assignee: Bioforte Biotechnology Shenzhen Co ltd
Current assignee: Bioforte Biotechnology Shenzhen Co ltd
Priority date: 2021-06-17
Filing date: 2021-06-17
Publication date: 2021-10-22

Abstract

The application relates to the field of antiviral proliferation, in particular to application of cedar needle extract in inhibiting proliferation of H9N2 AIV of poultry, the cedar needle extract can effectively inhibit proliferation of H9N2 AIV of poultry in a concentration of 10-250 mg/mL, and can be prepared into medicines or feeds for treating the infection of the poultry with H9N2 AIV or medicines or feeds for preventing the infection of the poultry with H9N2 AIV, and active ingredients of the medicines or feeds contain the cedar needle extract. The cedar needle leaf extract can be prepared into medicines and feeds for treatment or prevention, can also be added into drinking water for feeding, plays a role in treating or preventing H9N2 AIV for poultry animals in the modes of daily feeding, feeding administration, spraying administration and the like, plays a role in preventing and treating virus infection by enhancing the immunity of the organism and has small side effect.

Description

Application of cedar needle leaf extract in inhibiting avian H9N2 AIV proliferation

Technical Field

The present application relates to the field of anti-viral proliferation, more specifically it relates to the use of cedar needle extract for inhibiting the proliferation of H9N2 AIV in avian animals.

Background

An H9N2 subtype avian influenza virus (H9N 2 subtype avian influenza virus, H9N2 AIV) belongs to LPAI (low pathogenic avian influenza), avian animals such as chickens, turkeys and ducks are easy to be infected, and bring great economic loss to feeders, especially when the situation is more serious along with secondary infection of bacteria and viruses.

In order to prevent poultry from being infected with H9N2 AIV, the national research and development of commercial vaccines are carried out continuously, and most of the H9N2 AIV vaccines are inactivated vaccines which are prepared by inactivating chick embryo allantoic fluid containing H9N2 AIV by formaldehyde and show good immune effect, but because the inactivated vaccines mainly depend on humoral immunity of organisms, the inactivated vaccines lack the functions of inducing effective mucosal immunity and cellular immunity and are easy to interfere immunological monitoring and epidemiological research of avian influenza viruses; in addition, there are many new vaccines such as recombinant vector vaccine, subunit vaccine, DNA vaccine and virus-like particle vaccine in the commercial vaccine, but because H9N2 AIV has antigen drift, the new vaccine does not protect the organism completely, resulting in the persistent epidemic of H9N2 AIV, so there is a defect in developing and producing anti-avian influenza virus drugs to supplement the vaccine, so as to effectively prevent and control the infection of H9N2 AIV.

The anti-avian influenza virus drugs found in current research and clinical application can be divided into three types: m2 ion channel blockers, neuraminidase inhibitors and viral polymerase inhibition. However, due to the high prevalence of resistant viruses in nature, the efficacy of the anti-avian influenza virus drugs for preventing or treating H9N2 AIV is reduced, and the anti-avian influenza virus drugs are all synthetic antiviral drugs, although the production is rapid, the side effects are high, the drug resistance rate is increased, and the drugs all have single target effect to play the drug effect, so that the use of the synthetic antiviral drugs is limited.

Disclosure of Invention

In order to solve the problem of insufficient prevention and control effects of H9N2 AIV due to limited use of vaccines and synthetic antiviral drugs, the application provides the application of the cedar needle leaf extract in inhibiting the proliferation of the H9N2 AIV of poultry animals.

In a first aspect, the application provides use of cedar needle extract for inhibiting the proliferation of H9N2 AIV in an avian animal.

Preferably, the cedar needle extract is effective in inhibiting H9N2 AIV proliferation in avian animals at a concentration of 10-250 mg/mL.

The cedar needle leaf extract is obtained by extracting cedar needle leaves serving as raw materials in a conventional extraction mode, contains active ingredients such as volatile oil, crude protein, amino acid, pigment, vitamin, polyphenol, fatty acid, flavone, glycosides and terpenes, and has excellent platelet aggregation resisting activity, blood sugar reducing, bacteriostatic, anti-aging, cell DNA oxidative damage resisting, anti-tumor activity, antioxidation, anti-fatigue, blood fat reducing and other effects.

The application provides an application of cedar needle extract in inhibiting H9N2 AIV proliferation, preferably adopts the cedar needle extract with the concentration of 10-250 mg/mL, and has better inhibition effect on the proliferation of H9N2 AIV, specifically, the concentration of the cedar needle extract can be 10-30 mg/mL, 30-60 mg/mL, 60-90 mg/mL, 90-120 mg/mL, 120-150 mg/mL, 150-180 mg/mL, 180-220 mg/mL or 220-250 mg/mL. The cedar needle extract is a natural medicine which has an antiviral mechanism and biological activity, can play a role in preventing and treating virus infection by enhancing the immunity of an organism, has small side effect and even no side effect compared with a synthetic antiviral medicine, and solves the problems of side effect and drug resistance of the synthetic antiviral medicine.

Preferably, said inhibition of H9N2 AIV proliferation in avians is achieved by inhibiting the expression of the NP protein of H9N2 AIV.

In a second aspect, the application provides a use of an extract of cedar needles for the manufacture of a medicament for the treatment or prevention of infection in an avian animal with H9N2 AIV.

Preferably, the active ingredient of the medicine comprises cedar needle extract.

The cedar needle extract is adopted for preparing the medicine, the H9N2 AIV infection of the poultry can be treated by a medicine feeding mode of administering the medicine to the poultry, the inflammatory cell and erythrocyte infiltration of the lung tissue and the tracheal tissue of the poultry can be reduced, the damage of the lung tissue and the tracheal tissue is reduced, the integrity of the lung tissue and the tracheal tissue is effectively protected, and a better treatment effect is achieved; the cedar needle leaf extract has an antiviral mechanism and biological activity, and is small in side effect, so that the influence of the medicine on poultry animals is small.

Preferably, the content of the cedar leaf extract in the medicine is 10-250 mg/mL.

The cedar needle extract with the content has a good inhibition effect on the proliferation of H9N2 AIV in poultry, has an antiviral mechanism and biological activity, is small in side effect, has little influence on poultry and has a good treatment effect.

Preferably, the administration mode of the medicine is feeding, injection or spraying.

Because the H9N2 AIV is mainly propagated in an air propagation mode, the therapeutic medicine taking the cedar needle leaf extract as the active ingredient can be fed, injected or sprayed, can be accurately fed or fed in a large range according to the required culture scale, and is convenient to operate.

In a third aspect, the application provides a use of cedar needle extract in the preparation of a feed for treating or preventing infection of H9N2 AIV in an avian animal.

Preferably, the active ingredient of the feed comprises cedar needle extract.

The cedar needle extract is added into the feed, such as the cedar needle extract is added into solid feed or fed drinking water, namely solid feed and liquid feed, the infection of H9N2 AIV is treated by daily feeding and drinking of poultry animals, the immunity of the poultry animals is enhanced to play a role in treating and preventing virus infection, inflammatory cells and erythrocyte infiltration of lung tissues and trachea tissues of the poultry animals can be reduced, the damage of the lung tissues and the trachea tissues is reduced, and the integrity of the lung tissues and the trachea tissues is effectively protected.

Preferably, the content of the cedar leaf extract in the feed is 10-250 mg/mL.

The cedar needle leaf extract with the content is added into the feed, so that the cedar needle leaf extract can play a role in treating virus infection by enhancing the body immunity of poultry in daily feeding and drinking water, has an antiviral mechanism and biological activity, is small in side effect, has a good inhibition effect on the proliferation of H9N2 AIV in the poultry, and further achieves the treatment and prevention effects.

In summary, the present application has the following beneficial effects:

1. the application discovers for the first time that the cedar needle extract can obviously inhibit the proliferation of H9N2 AIV in vitro, can reduce the infiltration of inflammatory cells and erythrocytes of lung tissues and trachea tissues when acting in vivo, reduces the damage of the lung tissues and the trachea tissues, effectively protects the integrity of the lung tissues and the trachea tissues, can treat or prevent the infection of H9N2 AIV by poultry animals, plays a role in preventing and treating virus infection by enhancing the immunity of organisms, has small side effect, and reduces the infection rate and death rate of the poultry animals.

2. The cedar needle leaf extract can be prepared into medicines and feeds for treatment or prevention, can also be added into drinking water for feeding, plays a role in treating or preventing H9N2 AIV for poultry animals in a mode of administration or daily feeding and the like, plays a role in preventing and treating virus infection by enhancing the immunity of the organism and has small side effect.

Drawings

FIG. 1 is a MDCK cell viability diagram of 8 concentration gradients of cedar needle extract on MDCK cytotoxicity test in Experimental example 1 of the present application;

FIG. 2 is a schematic view of 4 dosing modes in Experimental example 2 of the present application;

FIG. 3 is a graph showing the results of the efficacy tests of the experimental group, H9N2 group and the negative group in Experimental example 2 of the present application; wherein, the H9N2 group refers to the test group to which only virus was added without adding cedar needle extract; negative group refers to test group with neither virus nor cedar needle extract added;

FIG. 4 is a graph showing the results of IFA test in Experimental example 3 of the present application; wherein the test concentrations of the cedar needle leaf extract are 25 mg/mL, 12.5 mg/mL and 6.25 mg/mL; group H9N2 refers to addition of virus to cells incubated for 2H without addition of cedar needle extract; negative group refers to no virus or cedar needle extract added;

FIG. 5 is a graph showing the results of Western Blot assay in Experimental example 4 of the present application; wherein the test concentrations of the cedar needle leaf extract are 25 mg/mL, 12.5 mg/mL and 6.25 mg/mL; group H9N2 refers to addition of virus to cells incubated for 2H without addition of cedar needle extract; negative group refers to no virus or cedar needle extract added;

FIG. 6 is a lung histopathological section of SPF chickens administered and challenged on the third day in Experimental example 5 of the present application; wherein (a) is a negative group, which refers to the case where neither H9N2 virus was inoculated nor cedar needle extract solution was fed; (B) H9N2 group, which refers to inoculation of H9N2 virus only without feeding cedar needle extract solution; (C) feeding the cedar needle extract solution 3 days before challenge, feeding the cedar needle extract solution all the time, dripping the virus 3 days after feeding the cedar needle extract solution, and continuously feeding the cedar needle extract solution until the experiment is finished; (D) the group administered 1d after the challenge refers to first dripping the nose to inoculate the virus, starting to feed the cedar needle extract solution 1 day later, and continuously feeding the cedar needle extract solution until the experiment is finished;

FIG. 7 is a photograph of a pathological section of trachea tissue on the third day after administration and challenge to SPF chickens in Experimental example 4 of the present application; wherein (a) is a negative group, which refers to the case where neither H9N2 virus was inoculated nor cedar needle extract solution was fed; (B) H9N2 group, which refers to inoculation of H9N2 virus only without feeding cedar needle extract solution; (C) feeding the cedar needle extract solution 3 days before challenge, feeding the cedar needle extract solution all the time, dripping the virus 3 days after feeding the cedar needle extract solution, and continuously feeding the cedar needle extract solution until the experiment is finished; (D) the group administered 1d after the challenge refers to first dripping the nose to inoculate the virus, starting to feed the cedar needle extract solution 1 day later, and continuously feeding the cedar needle extract solution until the experiment is finished;

fig. 8 is a graph showing the viral load data of pharyngeal swabs, cloaca swabs, lungs, and trachea after administration and challenge to SPF chickens in experimental example 4 of the present application.

Detailed Description

The present application will be described in further detail with reference to fig. 1 to 8 and experimental examples.

The cedar needle extracts used in the following experimental examples and comparative examples were selected from cedar needle extracts produced by shanxi french natural products limited.

In addition, materials, reagents and the like used in the following experimental examples are commercially available unless otherwise specified.

Examples of the experiments

Experimental example 1: testing toxicity of cedar needle extract on MDCK cells

According to the experimental example, the cedar needle extract with different gradient concentrations is mixed with the MDCK cells and cultured for a certain period of time, the cell viability of the MDCK cells is tested, the toxicity of the cedar needle extract with corresponding concentration to the MDCK cells is indicated through cell viability indexes, the safe use concentration of the cedar needle extract is determined by the method, the related experiment that the cedar needle extract inhibits H9N2 AIV proliferation in vivo/in vitro is facilitated, toxic influence of the cedar needle extract with too high concentration on the cells is eliminated, and the cedar needle extract with certain concentration does not have toxic effect on the cells.

The specific detection operation is as follows:

inoculate 5X 10 per well in 96-well plates⁴The MDCK cells were seeded, and the 96-well plate was placed at 37 ℃ in 5% CO₂Culturing for 24 hours in a cell culture box; the assay can be started when the cells in each well are at a monolayer density of about 90%.

An experimental group and a negative group are set, the experimental group uses the cedar needle extract with 8 concentration gradients, the concentration is 50 mg/mL, 25 mg/mL, 12.5 mg/mL, 6.25 mg/mL, 3.12 mg/mL, 1.56 mg/mL, 0.78 mg/mL and 0.39 mg/mL in sequence, and the negative group uses the cedar needle extract with 0 mg/mL, namely the cedar needle extract is not added.

Before adding the cedar needle extract, 100 μ L of 1 × PBS solution is added into each well for washing, and the culture medium is washed clean for 3-5 times. Dissolving 8 concentration gradient cedar needle extract experimental groups by adopting a serum-free culture medium containing 5 mu g/mL trypsin, specifically dissolving 50 mg/mL cedar needle extract by adopting the serum-free culture medium to prepare a 50 mg/mL cedar needle extract solution, then starting to dilute the 50 mg/mL cedar needle extract solution by 2 times, designing each gradient for 3 times, diluting 7 gradients altogether, forming 8 gradient concentration cedar needle extract solutions as experimental groups, and adding 100 mu L of cedar needle extract with corresponding concentration into each experimental group. The negative group was added with 100. mu.L of serum-free medium containing 5. mu.g/mL trypsin only. The experimental group and the negative group were placed at 37 ℃ in 5% CO₂The cell culture box is used for culturing, and after 24 h and 48 h of culture, the CCK-8 kit is used for detecting the cell activity.

The results are shown in figure 1, when the cedar needle extract is used at a concentration of 50 mg/mL, the cell viability is significantly lower than that of the negative group, and at 25 mg/mL, the cell viability is not significantly different from that of the negative group, which indicates that the cedar needle extract is used at a maximum safe use concentration of 25 mg/mL.

Experimental example 2: detection of H9N2 AIV inhibitory effect of cedar needle extract on MDCK cell

In this experimental example, different drug-adding modes were set according to the virus propagation mode under the condition that the safe use concentration was 25 mg/mL, and the possible inhibition mechanism of the cedar needle extract at the cell level was explored.

The specific detection operation is as follows:

inoculation of 2X 10 per well in 24-well plates⁵The MDCK cells were seeded and the 24-well plates were then placed at 37 ℃ in 5% CO₂When the cells in each well are in a monolayer, the cells are cultured in the cell culture box for 24 hoursThe 90% density test of the inhibition effect of the cedar needle extract can be carried out.

Experimental groups, H9N2 groups and negative groups were set up, all added at the maximum safe concentration (25 mg/mL) of cedar needle extract, dosed in four different ways, and 3 experimental replicates were designed for each way. The cedar needle extract solution is prepared by dissolving 25 mg/mL cedar needle extract in a serum-free DMEM medium containing 5 mu g/mL trypsin to prepare a 25 mg/mL cedar needle extract solution; while the H9N2 group was added with 50. mu.L of 10⁶ EID₅₀Virus, without the addition of cedar needle extract solution; while the negative group was not added 50. mu.L 10⁶ EID₅₀The virus was also not supplemented with cedar needle extract solution.

Wherein, the four different modes are respectively method 1-discarding the cell culture solution, adding 500. mu.L of 1 XPBS solution into each hole for cleaning, cleaning for 3-5 times, and then 50. mu.L of 10⁶ EID₅₀Premixing virus and 500 μ L25 mg/mL cedar needle extract solution at 4 deg.C for 30 min, adding cells, incubating for 2h, adding 500 μ L1 × PBS solution, cleaning for 3-5 times, cleaning the mixture, adding 500 μ L serum-free DMEM medium containing 5 μ g/mL trypsin, standing at 37 deg.C and 5% CO₂Culturing for 48 h in a cell culture box;

method 2-discard cell culture fluid, add 500. mu.L 1 XPBS solution per well for washing, wash 3-5 times, then 50. mu.L 10⁶ EID₅₀Adding virus and 500 μ L25 mg/mL cedar needle extract solution into cells at the same time, incubating for 2h, adding 500 μ L1 × PBS solution, washing for 3-5 times, cleaning the mixture, adding 500 μ L serum-free DMEM medium containing 5 μ g/mL trypsin, placing at 37 deg.C and 5% CO₂Culturing for 48 h in a cell culture box;

method 3-discard cell culture fluid, add 500. mu.L 1 XPBS solution per well for washing, wash 3-5 times, then 50. mu.L 10⁶ EID₅₀The virus infects cells and incubate for 2h, add 500 μ L1 XPBS solution to wash for 3-5 times,adding 500 μ L25 mg/mL cedar needle extract solution, incubating for 2h, adding 500 μ L1 × PBS solution, washing for 3-5 times, adding 500 μ L serum-free DMEM medium containing 5 μ g/mL trypsin, standing at 37 deg.C with 5% CO₂Culturing for 48 h in a cell culture box;

method 4-discard cell culture fluid, adding 500. mu.L 1 XPBS solution to each well for washing, washing 3-5 times, then adding 500. mu.L 25 mg/mL cedar needle extract solution to the cells for incubation for 2h, adding 500. mu.L 1 XPBS solution for washing 3-5 times, then inoculating 50. mu.L 10⁶ EID₅₀Incubating for 2 hr, washing with 500 μ L1 × PBS solution for 3-5 times, adding 500 μ L serum-free DMEM medium containing 5 μ g/mL trypsin, standing at 37 deg.C with 5% CO₂The cell culture box is used for culturing for 48 hours. The specific dosing manner is shown in figure 2.

After incubation in the four dosing regimes, viral copy number was determined by fluorescent quantitative PCR using QuantStudio 7 Flex (applied biosystems, thermo fisher Scientific, America).

The proliferation mode of the virus is usually that the nucleic acid is absorbed into the host cell, then the nucleic acid invades the interior of the cell, the nucleic acid of the virus utilizes the substances of the host cell to replicate and obtain the nucleic acid and the protein of the progeny virus (replication and proliferation), the nucleic acid and the protein are further assembled into the progeny virus, and then the progeny virus is released after the host cell is lysed. The method 1 is characterized in that viruses and cedar needle extract solution are premixed, whether the viruses can play an inhibiting role in vitro (outside cells) before invading host cells is mainly explored, and H9N2 AIV researched by the application is mainly infected through air transmission, so that the method 1 is set for exploring whether the cedar needle extract directly contacts the viruses to play an in-vitro inhibiting role on the viruses; adding the virus and the cedar needle extract solution into cells at the same time for incubation, and exploring whether the cedar needle extract can inhibit the virus or not when the virus and the cedar needle extract solution are added at the same time and the virus adsorbs and invades the cells; in the method 3, the virus firstly infects cells and incubates for 2 hours, the process that the virus finishes the processes of adsorbing and invading host cells is completed, and after the incubation for 2 hours, the cedar needle extract solution is added, and whether the cedar needle extract has inhibition effect on the replication and release of the virus in the host cells is mainly explored; in the method 4, the cedar needle extract solution is added into the cells and incubated for 2 hours, then the virus is inoculated and incubated for 2 hours, and whether the cedar needle extract can competitively inhibit the receptor of H9N2 AIV on MDCK cells is mainly explored, so that the adsorption and invasion processes of the virus on the cells are influenced, and the proliferation of the virus in the cells is inhibited.

The experimental results are shown in fig. 3, the experimental example performs the drug effect test on four drug adding modes, and screens the effective drug adding mode of the cedar needle extract solution, and the results show that compared with the H9N2 group, the dosage mode of the cedar needle extract solution in the method 1 has the obvious difference of the virus load compared with the H9N2 group, namely, the dosage mode of the method 1 has obvious in-vitro inhibition effect on the proliferation of H9N2 AIV (the in-vitro inhibition effect on the proliferation of H9N2 AIV) (the dosage mode of the method 1 has the obvious in-vitro inhibition effect on the proliferation of the H9N2 AIV)P<0.001) and the modes of administration of method 2, method 3 and method 4 do not effect in vitro inhibition; the reason may be that in the administration mode of the method 1, the cedar needle extract can destroy the structure of the virus in vitro, so that the virus is not easy to adhere to host cells or invade cells, and further plays a role in inhibiting the proliferation of the virus in the cells; the virus load of the method 2 is not significantly different from that of the H9N2 group, and probably under the scheme that the virus and the cedar needle extract solution are added and incubated at the same time, the cedar needle extract destroys part of the virus, so that part of the virus is not easy to adsorb and invade cells, but the destruction and inhibition of the cedar needle extract on the virus require a certain action time, the action time of the virus and the cedar needle extract solution is short, so that part of the virus still adsorbs and invades the cells to proliferate, the scheme of the inverse scheme 1 is that the virus and the cedar needle extract solution are premixed for 30 min, the drug has a certain time of action on the virus, the virus structure is destroyed, so that the virus cannot adsorb and invade the cells when the cells are added for incubation, and the proliferation is inhibited. The virus load of the method 3 is not significantly different from that of the H9N2 group, and probably the cedar needle extract has no inhibition effect on the release process of the virus; the virus load of method 4 was not significantly different from that of the H9N2 group, and it was probably cedar needle extractionThe compound has no obvious competitive inhibition effect on the receptor of H9N2 AIV, so that the compound has no obvious inhibition effect on the adsorption and invasion processes of H9N2 AIV.

Combining the four administration modes, for example, in the method 1, the virus and the cedar needle extract solution are premixed, and the virus can be acted in vitro within a certain premixed time, so that the structure of the virus is changed, the virus is not easy to adsorb cells and invade the cells, and further the effect of inhibiting the proliferation of the virus is achieved, in the

methods

2, 3 and 4, in the process of the virus invading the cells, the cedar needle extract can change the structure of the virus, and simultaneously part of the virus invades the cells, and the virus invading the cells is proliferated, even if the subsequent cedar needle extract enters the cells to destroy the structure of the virus, virus nucleic acid still exists, so that the fluorescent quantitative PCR can still detect a large amount of virus nucleic acid expression, and the differences among the

methods

2, 3, 4 and H9N2 in the fluorescent quantitative PCR detection result are not obvious. It follows that, before the virus is adsorbed to the cells, the cedar needle extract may have an effect of inactivating or destroying the structure of the virus, thereby affecting the proliferation of the virus so as to have an inhibitory effect.

Experimental example 3: concentration test for detecting H9N2 AIV proliferation inhibition of cedar needle extract

In this example, based on the mode 1 dosing method for inhibiting the in vitro proliferation of H9N2 AIV in example 2, the above mode 1 dosing method was used to search for the inhibitory effect of cedar needle extract at different concentrations on H9N2 AIV by immunofluorescence assay (IFA assay).

The specific detection operation is as follows:

(1) an experimental group, a H9N2 group and a negative group were set, and the experimental group was dosed according to the method 1 of Experimental example 2 (50. mu.L 10. mu.L)⁶ EID₅₀Premixing the virus and 500 mu L of cedar needle extract solution with corresponding concentration at 4 ℃ for 30 min, and then adding the mixture into cells for incubation for 2 h) to perform an experiment, wherein the cedar needle extract solution is prepared by dissolving cedar needle extract with corresponding concentration by adopting a serum-free DMEM medium containing 5 mu g/mL trypsin, and the experiment concentration of the cedar needle extract is 25 mg/mL, 12.5 mg/mL and 6.25 mg/mL; h9The N2 group was inoculated with 50. mu.L of 10⁶ EID₅₀Incubating the virus to the cells for 2h without adding cedar needle extract solution; the negative group was neither inoculated with 50. mu.L of 10⁶ EID₅₀The virus was also not supplemented with cedar needle extract solution; after the experimental group, the H9N2 group and the negative group were sampled, they were washed 3 times with 500. mu.L of 1 XPBS solution for 5min each.

(2) Fixing the sample washed in the step (1) with 4% paraformaldehyde for 15 min, and washing with PBST for 3 times, 5min each time.

(3) And (3) performing permeation treatment on the sample fixed in the step (2) by using a cell permeation agent of 0.4 percent TritonX-100, standing for 10-20 min, and washing for 5min by PBST (PBST).

(4) Blocking the permeable sample obtained in the step (3) by using 3% BSA (PBST dissolution) for 1h or overnight at 4 ℃; and then PBST is adopted for cleaning for 3 times, and each time, the shaking table is used for 5 min.

(5) Dissolve with 1% BSA (PBST) at 1: 50 dilution primary antibody (Mouse Anti-Influ A Virus Nucleoprotein antibody [ AA5H ]), adding into the sample washed in the step (4), and incubating for 1h at 37 ℃; and then PBST is adopted for cleaning for 3 times, and each time, the shaking table is used for 5 min.

(6) PBST is expressed as 1: and (3) adding a 300-diluted secondary antibody (Cy 3-laboratory coat anti-Mouse IgG (H + L) HSA) into the sample washed in the step (5), incubating for 1H at 37 ℃ in a dark place, and washing for 3 times by using PBST (shaking table) for 5 min.

(7) And (4) staining the sample cleaned in the step (6) with light for 7 min by using DAPI, cleaning the sample by PBST for 3 times, and cleaning the sample by shaking for 5min each time, wherein the result can be observed under a fluorescence microscope.

The above test procedure shows the NP protein of H9N2 AIV by the fluorescence effect of a secondary antibody (Cy 3-Labeled coat anti-Mouse IgG (H + L) HSA) with fluorescein, which is combined with the NP protein of H9N2 AIV by a primary antibody, and then combined with the primary antibody by a secondary antibody; if the fluorescence effect is obvious, the NP protein expression of H9N2 AIV is more, and the inhibition effect of the cedar needle leaf extract with the corresponding concentration in the test group on H9N2 AIV is weaker; if the fluorescence effect is not obvious, the NP protein expression of H9N2 AIV in the test group is less, and the fact that the cedar needle leaf extract with the corresponding concentration in the test group has the inhibition effect on H9N2 AIV is shown.

The IFA test result is shown in fig. 4, while the 25 mg/mL cedar needle extract test group does not observe fluorescence, the 12.5 mg/mL cedar needle extract test group observes little fluorescence, the fluorescence of the 6.25 mg/mL cedar needle extract test group is not significantly different from that of the H9N2 group, which indicates that the use of 25 mg/mL cedar needle extracts and 12.5 mg/mL cedar needle extracts can effectively inhibit the proliferation of H9N2 AIV in vitro, the specific inhibition mechanism may be to destroy the NP protein structure of the virus, so that the NP protein of H9N2 AIV cannot be expressed, and simultaneously the virus with damaged protein structure is not easy to adsorb cells and incorporate into invading cells, thereby playing a role in inhibition, and the virus load detected by the test group in the fluorescence quantitative PCR test result may be the virus nucleic acid of H9N2 AIV with damaged structure, and not structurally complete H9N2 AIV.

Experimental example 4: concentration test for detecting H9N2 AIV proliferation inhibition of cedar needle extract

In this example, the inhibition effect of the cedar needle extract at different concentrations on H9N2 AIV was investigated by immunoblotting (Western Blot test) using the dosing method of the above-described mode 1, based on the dosing method of the mode 1 for inhibiting the in vitro proliferation of H9N2 AIV in the experimental example 2.

In this experimental example, the concentration of the extract of the needle leaf of cedar capable of inhibiting the proliferation of H9N2 AIV in vitro was measured by detecting the expression of the protein, as in the above experimental example 3.

The specific detection operation is as follows:

(1) an experimental group, a H9N2 group and a negative group were set, and the experimental group was dosed according to the method 1 of Experimental example 2 (50. mu.L 10. mu.L)⁶ EID₅₀Premixing the virus and 500 mu L of cedar needle extract solution with corresponding concentration at 4 ℃ for 30 min, and then adding the mixture into cells for incubation for 2 h) to perform an experiment, wherein the cedar needle extract solution is prepared by dissolving cedar needle extract with corresponding concentration by adopting a serum-free DMEM medium containing 5 mu g/mL trypsin, and the experiment concentration of the cedar needle extract is 25 mg/mL, 12.5 mg/mL and 6.25 mg/mL; H9N2 group was inoculated with 50. mu.L of 10⁶ EID₅₀VirusIncubating the cells for 2h without adding cedar needle extract solution; the negative group was neither inoculated with 50. mu.L of 10⁶ EID₅₀The virus is not added with cedar needle extract solution, and the cells are incubated for 2 hours; after the experimental group, the H9N2 group and the negative group were sampled, the supernatant was discarded, 50. mu.L of 2% TritonX-100 was added for lysis, mixed and ice-cooled for 30 min, 40. mu.L was taken out to a new centrifuge tube, and 10. mu.L of 5 × Loading Buffer was added to each tube to obtain each test sample.

(2) Carrying out boiling water bath on the test sample obtained in the step (1) for 5min, then loading the sample for SDS-PAGE gel electrophoresis, wherein the program is 80V and the program is 30 min; 120V, 40 min.

(3) Film transfer: cutting filter paper and a membrane with proper sizes, wherein the filter paper is common filter paper in the industry, the membrane is a PVDF membrane, the membrane is firstly soaked in methanol for 5min and then soaked in membrane transferring liquid for 15 min, and the filter paper is soaked in the membrane transferring liquid. The procedure is as follows: constant pressure 100V, 40 min or constant flow 300A, 40 min. The preparation of the membrane transferring liquid is as follows: 6.04 g Tris base and 28.8 g glycine were weighed and dissolved in 1500 mL distilled water, and then 500 mL ethanol was added to make 2L volume.

(4) And (3) sealing: shaking-sealing with 5% skimmed milk powder for 1 hr or overnight at 4 deg.C, and washing with PBST for 3 times (5 min each time).

(5) Primary antibodies (Mouse Anti-Influenza A Virus nucleic acid antibody [ AA5H ] and GAPDH Mouse Monoclonal antibody) were added to the washed samples from step (4), incubated with shaking at room temperature for 1h, and the primary antibodies were washed with PBST 3 times for 5min each.

(6) Adding a secondary antibody (HRP-conjugated affinity Goat Anti-Mouse IgG (H + L)) into the washed sample obtained in the step (5), performing shaking table incubation at room temperature for 1H, and washing the secondary antibody with PBST for 3 times, wherein each shaking table time is 5 min.

(7) According to the following steps: preparing ECL developing solution according to a proportion of 1, paving the cleaned PVEF film on a panel of an imager, dripping the ECL developing solution in a dark place, and adjusting appropriate parameters to photograph and observe results.

The test step comprises the steps that a primary antibody is combined with NP protein of H9N2 AIV, a secondary antibody is combined with the primary antibody, an ECL color development solution is combined with the secondary antibody, and the condition of the NP protein of H9N2 AIV is presented through the color development condition of a protein band of the ECL color development solution; if the H9N2 protein band can be observed, the corresponding concentration of the cedar needle extract in the test group has weaker inhibition effect on NP protein of H9N2 AIV; if no H9N2 protein band is observed, it indicates that the corresponding concentration of cedar needle extract in the test group has inhibitory effect on NP protein of H9N2 AIV.

GAPDH is widely distributed in cells in various tissues and is used for parallel comparison with H9N2, and a GAPDH band is still shown in FIG. 5, which indicates that the Western Blot test of the experimental example can normally develop color.

The results of the Western Blot are shown in FIG. 5, and show that 25 mg/mL and 12.5 mg/mL cedar needle extract solution groups have no H9N2 protein band, while the histone band of the cedar needle extract solution of 6.25 mg/mL has no obvious difference with the H9N2 group, which shows that the cedar needle extract with the concentration of 25 mg/mL and 12.5 mg/mL can effectively inhibit the proliferation of H9N2 AIV in vitro, and the specific inhibition mechanism is probably to destroy the NP protein structure of the virus, so that the NP protein of the H9N2 AIV cannot be expressed, simultaneously, the virus with damaged protein structure is not easy to adsorb cells and invade the cells, plays a role in inhibiting, meanwhile, corresponding to the IFA result, it can also be shown that the viral load detected by the extract group in the fluorescence quantitative PCR detection result may be virus nucleic acid of H9N2 AIV with a damaged structure, but not H9N2 AIV with a complete structure.

Experimental example 5: detecting whether the action of cedar needle extract on animal has the effect of inhibiting H9N2 AIV proliferation

In the experimental example, the cedar needle extract (25 mg/mL) with a specific concentration is adopted to clinically test the treatment and prevention effects of the cedar needle extract on the infection of the animals with H9N2 AIV.

The specific operation is as follows:

80 SPF (specific pathogen free) chickens of 21 days old are randomly and averagely divided into 4 groups, namely a pre-challenge 3d administration group, a post-challenge 1d administration group, a positive control group and a negative control group, wherein each group comprises 20 SPF chickens, the SPF chickens are respectively raised in 4 negative pressure isolators, and all the chickens are fed with free drinking water and fed with food.

The administration group before 3 days of challenge is administered by nasal drip (100 μ L for each nostril) of 200 μ L and oral administration of 1 mL from 22 days old, wherein the administration is to a cedar needle extract solution of 25 mg dissolved in 1 mL of ultrapure water, the concentration of the cedar needle extract is 25 mg/mL, and the administration is continued after challenge.

The administration group after 1d of the offensive toxin is administered every day from 1d after the offensive toxin, the administration concentration is consistent with that of the administration group before the offensive toxin, 1 mL of ultrapure water is adopted to dissolve 25 mg of cedar needle extract solution, the concentration of the cedar needle extract is 25 mg/mL, the administration mode is also consistent with that of the administration group before the offensive toxin, 200 muL of nasal drops (100 muL of each of the left and right nostrils) and 1 mL of oral administration are administered every day, and the administration is continuously performed all the time after the offensive toxin.

At 25 days of age, 200. mu.L 106 EID was inoculated by eye drop nasal drip to all groups except the negative control group₅₀H9N2 virus, negative control group was replaced with 200. mu.L of 1 XPBS. (since H9N2 virus is airborne virus, the mode of nasal drop inoculation of virus is adopted)

The specific groups, administration modes, toxicity counteracting modes and sampling are shown in the following table 1.

TABLE 1 animal protocol

。

The administration method of this example is different from the administration method 1 of example 2, but mainly in example 2, the experiment for inhibiting in vitro proliferation mainly acts on MDCK cells (a cell model cell line known in the art as H9N2 AIV-infected cells), in addition, the experimental example is clinically applied to animal experiments, the H9N2 AIV infection is started by the poultry inhaling or feeding infectious H9N2 AIV virus particles, the hemagglutinin protein on the surface of the virus can be cracked by enzyme similar to pancreatin in the endothelial cells of the respiratory tract and the intestinal tract, therefore, viruses can replicate in respiratory tracts and intestinal tracts and release infectious virus particles, so that the embodiment of inhibiting in vitro proliferation on cells is different from the embodiment of inhibiting proliferation on animals, the inhibition of proliferation on cells is mainly to screen out extracts capable of inhibiting virus proliferation, and the fact that whether the extracts have an inhibiting effect in clinical application needs to be clinically embodied on animals.

Tissues such as throat swabs, cloaca swabs, lung, trachea and the like are collected for detection in each test group.

Wherein, the pathological section of lung tissue of SPF chicken administered and infected by virus at the third day is shown in figure 6. The lung histopathological section results showed that H9N2 group (group B) had a large number of inflammatory cells (indicated by the left arrow in fig. 6B) and red blood cell infiltration (indicated by the right arrow in fig. 6B). The pre-challenge 3d dosing group (group C) had only a small inflammatory cell infiltration (indicated by the left arrow in fig. 6C) and a small bleeding (indicated by the right arrow in fig. 6C). The group 1D after challenge (group D) had only a small inflammatory cell infiltration (indicated by the right arrow in the right panel of fig. 6D) and local bronchiectasis (indicated by the left arrow in the right panel of fig. 6D), and the lesions in the cedar needle extract group were reduced compared to the H9N2 group. The cedar needle extract can reduce inflammatory cells and red blood cell infiltration of lung tissues by both preventive administration and therapeutic administration, thereby reducing the damage of the lung tissues.

Wherein, the pathological section of trachea tissue of SPF chicken at the third day after administration and challenge is shown in figure 7. Pathological section results of the trachea tissue show that H9N2 group (group B) had a loss of local mucosal epithelial cells (indicated by the lower arrow in the right panel of fig. 7B), more inflammatory cells infiltrated, the epithelial cells were irregularly arranged (indicated by the arrow in the left panel of fig. 7B and the arrow in the upper left side of the right panel of fig. 7B), and capillary congestion was visible (indicated by the upper right arrow in the right panel of fig. 7B). Only a small inflammatory cell infiltration (shown by the left arrow in fig. 7C) was observed in the pre-challenge 3d dose group (group C). Only a small inflammatory cell infiltration was observed in the post-challenge 1D-dose group (group D) (shown by the left arrow in fig. 7D and the right arrow in fig. 7D). The cedar needle leaf extract can effectively protect the tissue integrity of tracheal tissues and reduce inflammatory cell infiltration when being used for both prevention administration and treatment administration, thereby reducing tissue damage.

A graph of the viral load of throat swabs after SPF chicken administration and challenge is shown in FIG. 8A. The results of the virus load detection of the throat swabs show that the virus load detection of the throat swabs ranges from 1 day to 5 days (1 d) after the virus challengepi to 5 dpi), the virus load of the pharyngeal swabs administered 3d before challenge was significantly lower than that of the H9N2 group (respectivelyP<0.01、P<0.05、P<0.05、P<0.005、P<0.01) (FIG. 8A), whereas at 1dpi, 4dpi, and 5dpi, the viral load of pharyngeal swabs administered 1d after challenge was significantly lower than that of H9N2 (respectivelyP<0.01、P<0.005、P<0.01) (fig. 8A), indicating that cedar needle extract is effective in reducing the expulsion of toxins from the body's respiratory tract both by prophylactic and therapeutic administration.

Additionally, a graph of cloacal swab viral load data after SPF chicken administration and challenge is shown in FIG. 8B. The virus load detection results of the pharyngeal cloaca swabs show that the virus load of the cloaca swabs in the 3d administration group before challenge is significantly lower than that in the H9N2 group (respectively at 1dpi, 4dpi and 5 dpi)P<0.05、P<0.05、P<0.01) (FIG. 8B), whereas from 3 to 5dpi, the cloaca swab viral loads were significantly lower in the post-challenge 1d dose groups than in the H9N2 group (each oneP<0.05、P<0.05、P<0.05) (fig. 8B), indicating that the cedar needle extract is effective in reducing the expulsion of the cloaca of the body both by prophylactic and therapeutic administration.

In addition, a data graph of the tracheal viral load after administration and challenge to SPF chickens is shown in figure 8B. The tracheal viral load detection results show that the viral load of the trachea of the 3d administration group before challenge is remarkably lower than that of the H9N2 group (respectively, the viral load of the trachea is 1dpi to 5 dpi)P<0.05、P<0.005、P<0.01、P<0.01、P<0.01) (FIG. 8C), whereas at 4dpi and 5dpi, the tracheal viral load was significantly lower in the 1d post challenge group than in H9N2 (respectivelyP<0.01、P<0.05) (fig. 8C), indicating that the cedar needle extract was effective in inhibiting the proliferation of H9N2 AIV in the trachea both by prophylactic and therapeutic administration.

In addition, a graph of the lung viral load data after SPF chicken administration and challenge is shown in figure 8B. The lung viral load test results showed that at 4dpi, the viral loads of the lungs of both the pre-challenge 3d dose group and post-challenge 1d dose group were significantly lower than those of the H9N2 group (each of which is a group with a lower viral load than that of the other groups)P<0.01、P<0.01) (FIG. 8D), and at 5dpi, no viral load was detected in the lungs of both the pre-challenge 3D and post-challenge 1D groups (FIG. 8D), indicating that the cedar needle extract was effective in inhibiting the proliferation of H9N2 AIV in the lungs both when administered prophylactically and therapeutically.

The specific experimental examples are only for explaining the present application and are not limiting to the present application, and those skilled in the art can make modifications without inventive contribution to the experimental examples as required after reading the present specification, but all of them are protected by patent laws within the scope of the claims of the present application.

Claims

1. Use of cedar needle extract for inhibiting the proliferation of H9N2 AIV in avian animals.

2. The use of cedar needle extract according to claim 1 for inhibiting the proliferation of H9N2 AIV in avians, characterized in that: the cedar needle extract can effectively inhibit the proliferation of H9N2 AIV in the poultry at a concentration of 10-250 mg/mL.

3. The use of cedar needle extract according to claim 1 for inhibiting the proliferation of H9N2 AIV in avians, characterized in that: the inhibition of H9N2 AIV proliferation in avians is achieved by inhibiting the expression of the NP protein of H9N2 AIV.

4. Use of cedar needle extract for the manufacture of a medicament for the treatment or prevention of infection in avian animals with H9N2 AIV.

5. Use of cedar needle extract according to claim 4 for the preparation of a medicament for the treatment or prevention of infection in avians with H9N2 AIV, characterized in that: the active ingredient of the medicine comprises cedar needle leaf extract.

6. Use of cedar needle extract according to claim 4 for the preparation of a medicament for the treatment or prevention of infection in avians with H9N2 AIV, characterized in that: the administration mode of the medicine is feeding, injection or spraying.

7. Use of cedar needle extract for the preparation of a feed for the treatment or prevention of H9N2 AIV in an avian animal.

8. Use of cedar needle extract according to claim 7 for the preparation of feed for the treatment or prevention of H9N2 AIV in avian animals, characterized in that: the active ingredients of the feed comprise cedar needle extract.