CN111297884A - Application of Adenosine in Prevention and Control of Honeybee Virus Infection - Google Patents

Abstract

The invention relates to the technical field of honeybee breeding, in particular to the application of adenosine in the prevention and control of honeybee virus infection. The present invention finds that adenosine can significantly improve the survival rate and reduce the mortality of Chinese honeybee larvae infected with CSBV. Adenosine can inhibit the proliferation of CSBV in honeybees and reduce the copy number of the virus; at the same time, it can also induce the expression of endogenous antimicrobial peptides and improve the innate immune defense of honeybees. Adenosine can be used to prepare medicines or feeds for preventing and controlling CSBV infection, which is of great significance for the prevention and control of Chinese honeycomb larvae, and has good market application value.

Description

Application of Adenosine in Prevention and Control of Honeybee Virus Infection

technical field

The invention relates to the technical field of honeybee breeding, in particular to the application of adenosine in the prevention and control of honeybee virus infection.

Background technique

Bees are important pollinators in nature. About one-third of the world's crops require bees to be pollinated to some extent, while some fruits and vegetables are completely dependent on bees for pollination. Bee pollination can significantly improve the yield and quality of crops, so it has important economic value in agricultural production. In addition, bees can also produce a wide variety of peak products such as honey, royal jelly, propolis, bee pollen, etc. The quality of bee products is closely related to the health status of bees. The bee products produced by infected bees have potential safety hazards, and the drugs used to treat bee diseases will also remain in bee products, resulting in safety problems for bee products.

Apis cerana, as a dominant bee species with strong anti-mite ability, low temperature resistance, and good at collecting scattered nectar sources, plays an important role in beekeeping and plays an extremely important role in maintaining the balance of the natural ecosystem. . However, Chinese honeybees are often harmed by a variety of pathogens, among which, Chinese honeybee virus (CSBV) is the most serious harm to Chinese honeybees. CSBV mainly infects 1 to 3-day-old larvae, resulting in the death of a large number of larvae in the colony. Even if they survive by chance, they cannot pupate, resulting in insufficient number of new worker bees leaving the house, which in turn leads to a weakened colony of middle bees and a decline in colony resistance. Invaded by other viruses, thus showing the phenomenon of multi-virus co-infection. CSBV can be transmitted in bee colonies through mutual feeding of food, fecal contamination, mating behavior and reproductive gametes. Drones can be used as viral vectors to transmit the virus to offspring through reproductive gametes. Due to the unswarming nature of drones, the genetic diversity of the colony can be directly determined, so it may have an important impact on the disease resistance of the colony. At present, Chinese honeybee cystic larvae disease is mainly controlled by comprehensive control measures such as changing the king's larvae, strict disinfection, strengthening management, and using antiviral drugs and antibiotics to prevent secondary infection. However, the control effect is not ideal, and the commonly used antiviral Drugs are difficult to effectively control the harm and spread of the virus, and also lead to drug residues in bee products.

As an important component of innate immunity, endogenous antimicrobial peptides play key roles in immune regulation and pathogen defense. Endogenous antimicrobial peptides have immunomodulatory activity and participate in the regulation of innate and adaptive immune responses. They are important mediators of the body's defense against foreign invasion, and are also immune active molecules produced by the body's adaptation to the environment.

When honeybees are infected by microorganisms or other exogenous substances, the hemolymph will produce a certain amount of antimicrobial peptides, including Hymenoptaecin, bee defensin, bee antimicrobial peptides (Apidaecin) and bee moths. Antibacterial peptide (Abaecin). These peptides are synthesized by the fat body and then secreted into the hemolymph. They are a fast and effective defense mechanism that can be used to rapidly kill or eliminate foreign microorganisms. This defense mechanism is different from the immune response of animals. Insects do not have an immune response mechanism in the strict sense. These antimicrobial peptides produced by insect hemolymph can be produced by one or more microbial invasions without specificity. The induced products are not only for Produced by specific invading microbes, often resistant to non-invading microbes. The molecular weight, chemical structure and antibacterial mechanism of these several antimicrobial peptides are different. Different antimicrobial peptides can resist different bacteria, fungi, viruses and tumor cells. Among them, honeybee antimicrobial peptides are the main antimicrobial peptides of honeybees, and Hymenoptera Antibacterial peptides are the most important supplementary peptides among honeybee antibacterial peptides, which mainly inhibit some gram-negative bacteria that are resistant to honeybee antibacterial peptides. Peptides and hymenopteran antimicrobial peptides only work when their bactericidal ability is lost. The expression of bee defensin is the least, and it is also the only antimicrobial peptide in bee hemolymph that inhibits Gram-positive bacteria. The changes of antimicrobial peptide content in honeybees were affected by different factors, including bacteria, fungi and microsporidium, viruses, bee mites and drugs. Antimicrobial peptides play a role in resisting bee viruses. The researchers found that: when bees were inoculated with acute bee paralysis virus (ABPV), the expression of antimicrobial peptide genes was significantly increased; in the midgut epithelium of bees, bee residual wing virus (DWV) infection The level was linearly correlated with the expression level of honeybee antimicrobial peptide genes; after being infected by virus, the expression levels of honeybee antimicrobial peptides, hymenopteran antimicrobial peptides, and bee moth antimicrobial peptides in honeybees were significantly reduced. Bee mites can also affect the expression of bee antimicrobial peptides. In terms of drugs, the researchers found that acaricides can significantly affect the expression of bee antimicrobial peptide genes. After bees were exposed to chlorfenthrin, the expression of hymenopteran antimicrobial peptides was significantly up-regulated, while myiacophos inhibited the expression of hymenoptera. The expression of antimicrobial peptides and the antibacterial peptide genes of bee moth were down-regulated. In conclusion, honeybee antimicrobial peptides are key components of honeybee humoral immunity, and they play a coordinated role together to resist the invasion of various pathogenic microorganisms.

Chinese patent CN103524602A discloses the treatment of bee follicle larvae disease by administering exogenous antimicrobial peptides, but the administration of exogenous antimicrobial peptides still faces potential safety risks that may exist in antimicrobial peptide drugs, the mass production of antimicrobial peptides is limited, and the production efficiency is relatively low. Low cost and high production cost (Lin Chengde, Peng Hongjuan, Wang Yanhai. Application and existing problems of antimicrobial peptides. Journal of Tropical Medicine. 2007, 1(7): 86-90.). Therefore, it is of great significance for the bee aquaculture industry to develop an efficient control method for the mesocystis larvae.

SUMMARY OF THE INVENTION

In order to solve the technical problems in the prior art, the purpose of the present invention is to provide the application of adenosine in the prevention and control of honeybee virus infection.

The adenosine described in the present invention is a compound known in the prior art, which is a compound formed by connecting N-9 of adenine and C-1 of D-ribose through a β-glycosidic bond, and its phosphate ester is adenosine acid. Adenosine is a metabolite of adenosine nucleotide, which plays an important physiological and biochemical effect in the body, and is an important intermediate for the synthesis of adenosine triphosphate (ATP), adenine, adenylate and adenosine arabinoside. The structural formula of adenosine is as follows:

The present invention provides the use of adenosine or its derivatives in the preparation of a medicament for treating or preventing viral infection of honeybees.

The present invention provides the use of adenosine or its derivatives in the preparation of feed for treating or preventing viral infection of honeybees.

The invention provides the application of adenosine or its derivatives in inhibiting the proliferation of honeybee virus.

The application of the adenosine or its derivative of the present invention in inhibiting the proliferation of honeybee viruses can be for inhibiting the proliferation of viruses in apparently healthy honeybees, or for inhibiting the proliferation of viruses in honeybees with virus diseases.

The virus described in the present invention is preferably Chinese bee vesicular virus. The present invention proves through experiments that the administration of adenosine can significantly improve the survival rate of Chinese honeybee larvae infected with bee sac virus. On the one hand, adenosine can directly act on the virus to reduce the number of virus copies; The expression of endogenous antimicrobial peptides enhances the body's innate immune defense against viruses and reduces the probability of disease in infected larvae.

According to the effect of adenosine on honeybee virus infection, substances containing adenosine structure that can metabolize adenosine in vivo (including adenosine, adenylate, etc.) or other adenosine derivatives should have the same effect.

The derivatives of adenosine according to the present invention include adenylate and adenylate.

The present invention proves through experiments that adenosine can induce the transcription and translation of endogenous antimicrobial peptide genes, and improve the expression of endogenous antimicrobial peptides (such as hymenopteran antimicrobial peptides, honeybee defensins, honeybee antimicrobial peptides and bee antibacterial peptides) level, thereby enhancing the body's immunity.

Therefore, the present invention provides the use of adenosine or its derivatives in the preparation of a medicament for improving the immunity of honeybees. The present invention also provides the application of adenosine or its derivatives in the preparation of feed for improving the immunity of honeybees.

The improvement of honeybee immunity according to the present invention is specifically realized by increasing the expression level of endogenous antimicrobial peptides of honeybees.

The endogenous antimicrobial peptides of the present invention can be any one or more selected from the group consisting of Hymenopteran antimicrobial peptides, honeybee defensins, honeybee antimicrobial peptides and bee moth antimicrobial peptides.

The active ingredients of the medicine or feed of the present invention include one or more selected from adenosine and its derivatives.

The present invention also provides a product for treating or preventing viral infection of honeybees, wherein the active ingredients of the product include one or more selected from adenosine and its derivatives. The product is a medicine, feed or feed additive.

The active ingredient of the medicament of the present invention may contain only adenosine or its derivatives; may also contain a variety of adenosine and its derivatives; may also contain other active ingredients other than adenosine and its derivatives ( For example: other active ingredients that can improve the immunity or antiviral ability of bees).

The medicament of the present invention may further comprise excipients allowed in the pharmaceutical field; the dosage form of the medicament may be any dosage form allowed in the pharmaceutical field.

The feed of the present invention may contain adenosine or its derivatives, or a variety of adenosine and its derivatives; and may also contain other active ingredients other than adenosine and its derivatives (for example: other In addition to the above-mentioned active ingredients, the feed of the present invention can also contain nutrients required by bees, such as pollen, royal jelly, fructose, glucose, sucrose, soybean meal, yeast Powder, skimmed milk powder, inorganic salts, vitamins, water, etc.

The feed according to the present invention can be solid feed, semi-solid feed or liquid feed.

In the above application of the adenosine provided by the present invention, it is preferable that the dose of adenosine administered to the honeybee is ≥15ng/bee/time. More preferably, it is 0.1 to 10 μg/piece/time. The time interval for each adenosine administration is preferably 22-24 hours.

Specifically, for 3-day-old bees, 15ng-1.5μg/bee/time; 4-day-old bees 22.5ng-2.25μg/bee/time; 5-day-old bees 37.5ng-3.75μg/bee/time; 6-day-old bees 60ng～6μg/only/time.

The beneficial effects of the present invention are as follows: the present invention finds for the first time that adenosine can significantly improve the survival rate of CSBV-infected Chinese honeybee larvae and reduce the mortality rate. On the one hand, adenosine can inhibit the proliferation of CSBV and reduce the copy number of the virus; on the other hand, adenosine can also induce the expression of endogenous antimicrobial peptides and improve the innate immune defense of bees. The experimental results of the present invention show that after adenosine intervention, the mortality rate of CSBV-infected honeybee larvae is greatly reduced, and the survival rate is significantly increased (up to 1.32 times), and the surviving larvae can pupate and emerge into adult bees; adenosine intervention After infection with CSBV, the number of virus copies in honeybee larvae decreased significantly (the maximum number of virus copies infected with CSBV was up to 121,000, no CSBV was detected after administration of adenosine, and there was a dose-effect relationship. At the same time, adenosine intervention could improve the appearance of the virus. The degree of virus infection in healthy larvae is conducive to the normal growth and development of larvae; the expression levels of hymenopteran antimicrobial peptides, bee defensins and bee antimicrobial peptides in CSBV-infected larvae were significantly up-regulated after adenosine intervention.

The new function of adenosine provided by the invention for virus infection of honeybees provides a basis for the research on the immune defense mechanism of honeybees. Adenosine can be used in the preparation of medicines or feeds for preventing and treating Chinese honeycomb larvae or improving the immunity of honeybees, which provides a new method for preventing and treating Chinese honeycomb larvae, and has a good market application prospect.

Description of drawings

Figure 1 shows the statistical results of the survival rate of 6-day-old larvae after adenosine intervention in Experimental Example 1 of the present invention; wherein, CK represents the control group, 0.75 represents the low concentration group of the preparation group, 7.5 represents the medium concentration group of the preparation group, and 75 represents the In the high concentration group of the preparation group, CSBV represents the infection group, CSBV+0.75 represents the low concentration group of the intervention group, CSBV+7.5 represents the medium concentration group of the intervention group, and CSBV+75 represents the high concentration group of the intervention group.

Figure 2 is the statistical result of mortality of 4-6 day old larvae after adenosine intervention in Experimental Example 1 of the present invention; wherein, CK represents the control group, 0.75 represents the low concentration group of the preparation group, 7.5 represents the medium concentration group of the preparation group, 75 represents the high concentration group of the preparation group, CSBV represents the infection group, CSBV+0.75 represents the low concentration group of the intervention group, CSBV+7.5 represents the medium concentration group of the intervention group, and CSBV+75 represents the high concentration group of the intervention group.

Figure 3 is the detection result of virus copy number of 4-6 day old larvae after adenosine intervention in Experimental Example 2 of the present invention; wherein, CK represents the control group, 0.75 represents the low concentration group of the preparation group, and 7.5 represents the medium concentration group of the preparation group , 75 represents the high concentration group of the preparation group, CSBV represents the infection group, CSBV+0.75 represents the low concentration group of the intervention group, CSBV+7.5 represents the medium concentration group of the intervention group, and CSBV+75 represents the high concentration group of the intervention group.

Figure 4 shows the pupation and eclosion of healthy larvae and adenosine-intervention larvae after exposure in Experimental Example 3 of the present invention; wherein, A is the adenosine intervention group (7.5 μg/ml); B is the control (CK) group.

Fig. 5 is the expression amount of honeybee antimicrobial peptide (Apidaecin) after exposure and adenosine intervention in Experimental Example 4 of the present invention; wherein, CK represents the control group, the low represents the low-concentration group of the preparation group, and the middle represents the medium-concentration group of the preparation group. , high represents the high concentration group of the preparation group, CSBV represents the infection group, CSBV+ low represents the low concentration group of the intervention group, CSBV+ medium represents the medium concentration group of the intervention group, and CSBV+ high represents the high concentration group of the intervention group.

Figure 6 is the expression of Hymenoptaecin after exposure and adenosine intervention in Experimental Example 4 of the present invention; wherein, CK represents the control group, low represents the low concentration group of the preparation group, and medium represents the medium of the preparation group. Concentration group, high represents the high concentration group of the preparation group, CSBV represents the infection group, CSBV+ low represents the low concentration group of the intervention group, CSBV+ medium represents the medium concentration group of the intervention group, and CSBV+ high represents the high concentration group of the intervention group.

Fig. 7 is the expression level of the antibacterial peptide (Abaecin) of the beetle after exposure and adenosine intervention in Experimental Example 4 of the present invention; wherein, CK represents the control group, the low represents the low concentration group of the preparation group, and the middle represents the medium concentration of the preparation group group, high represents the high concentration group of the preparation group, CSBV represents the infection group, CSBV+ low represents the low concentration group of the intervention group, CSBV+ medium represents the medium concentration group of the intervention group, and CSBV+ high represents the high concentration group of the intervention group.

Fig. 8 is the expression of honeybee defensin (Defensin) after exposure and adenosine intervention in Experimental Example 4 of the present invention; wherein, CK represents the control group, the low represents the low-concentration group of the preparation group, and the middle represents the medium-concentration group of the preparation group , high represents the high concentration group of the preparation group, CSBV represents the infection group, CSBV+ low represents the low concentration group of the intervention group, CSBV+ medium represents the medium concentration group of the intervention group, and CSBV+ high represents the high concentration group of the intervention group.

Detailed ways

The preferred embodiments of the present invention will be described in detail below with reference to the examples. It should be understood that the following examples are given for illustrative purposes only, and are not intended to limit the scope of the present invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from the spirit and spirit of the present invention.

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

The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

Statistical analysis of data in the following examples was completed using SPSS 22.0 (SPSS Inc., Chicago, USA). The effect of adenosine on Chinese honeybee larvae was expressed as "Mean±SE". Analysis of variance and LSD multiple analysis of variance were used to compare the changes of CSBV virus copy number in A. chinensis larvae.

Example 1 Use of adenosine to interfere with healthy Chinese honeybee larvae and poisoned larvae

1. Sample for sample

The Chinese honey bee was obtained from the experimental bee colony of the Institute of Honey Bee, Chinese Academy of Agricultural Sciences. In order to obtain 2-day-old larvae, the queen bee was kept on the spleen to lay eggs for 96 hours, and the spleen containing 2-day-old larvae was taken out from the colony and placed in a 24-well plate, and placed at a temperature of 32±1°C and relative humidity. Perform pre-incubation in a constant temperature and humidity incubator at 75 ± 5% RH.

The reverse transcription-polymerase chain reaction (RT-PCR) method was used to detect whether the test larvae had CSBV and other viruses (including black queen bee virus BQCV, residual virus DWV, Israel paralysis virus IAPV, Kashmir virus KBV, acute paralysis virus ABPV, Chronic paralysis virus (CBPV) infection, healthy larvae without virus infection were used as samples in this example.

The detection primers of above-mentioned various viruses refer to the primer sequences provided by the literature, and are specifically as follows: CSBV detection primers refer to Grabensteiner, E., W.Ritter, M.J.Carter, S.Davison, H.Pechhacker, J.Kolodziejek, O.Boecking, I. . Derakshifar, R. Moosbeckhofer, E. Licek, and N. Nowotny. Sacbrood virus of the honeybee (Apis mellifera): rapid identification and phylogenetic analysis using reverse transcription-PCR. Clin. Diagn. Lab. Immunol. 2001, 8:93-104 .; BQCV detection primers refer to M, Benjeddou, N, Leat, M, Allsopp, S, Davison. Detection ofacute bee paralysis virus and black queen cell virus from honeybees byreverse transcriptase pcr.Applied and environmental microbiology.2001,67(5): 2384-2387.; DWV detection primers refer to Tentcheva, D., L.Gauthier, S.Jouve, L.Canabady-Rochelle, B.Dainat, F.Cousserans, M.E.Colin, B.V.Ball, and M.Bergoin.Polymerasechain reaction detection of deformed wing virus (DWV) in Apis mellifera and Varroa destructor.Apidologie.2004,35:431-439.; IAPV detection primers refer to Eyal, Maori, Shai, Lavi, Rita, Mozes-Koch, Yulia, Gantman, Yuv al, Peretz, Orit,Edelbaum,Edna,Tanne,Ilan,Sela.Isolation and characterization of Israeli acuteparalysis virus,a dicistrovirus affecting ho neybees in Israel: evidence fordiversity due to intra-and inter-species recombination. The Journal of generalvirology, 2007, 88(Pt 12): 3428-38.; KBV detection primers refer to Stoltz, D., X.R.Shen, C.Boggis, and G.Sisson.Molecular diagnosis of Kashmir bee virusinfection.J.Apic.Res.1995,34:153-165.; ABPV detection primers refer to Bakonyi,T.,R.Farkas,A.Szendroi,M.Dobos-Kovacs, and M.Rusvai.Detection of acute bee paralysis virus by RT-PCR in honey bee and Varroa destructor field samples:screening ofrepresentative Hungarian apiaries.Apidologie.2002,33:63-74.; CBPV detection primers refer to Ribie`re,M., C. Triboulot, L. Mathieu, C. Aurie'res, J. P. Faucon, and M. Pe' pin. Molecular diagnostic of chronic bee paralysis virus infection. Apidologie, 2002, 33:339-351.

The specific detection method is as follows: RNA was extracted using the RNeasy mini kit (Adelai), and RNA extraction was performed according to the product instructions (this method includes the step of removing DNA to ensure that only RNA is present in the final extract). Total RNA was eluted in 30 μL of elution buffer and used directly for RT-PCR. The extracted RNA was immediately used to generate first-strand cDNA using the Quantitec Reverse Transcription Kit (Takara) (according to the manufacturer's instructions), and the cDNA was stored at -20°C. PCR amplification reaction was 50 μL system; PCR reaction conditions included pre-denaturation at 94°C for 5 minutes; denaturation at 94°C for 30 seconds, annealing at 58°C for 30 seconds, extension at 72°C for 15 seconds, 35 cycles; and extension at 72°C for 5 minutes. Test results showed that CSBV and other viruses were not detected.

2. Preparation of syrup

The preparation and feeding of syrup were adjusted according to the 2016 standard for feeding Italian honeybees (Karl Crailsheim, Robert Brodschneider, Pierrick Aupinel, Dieter Behrens, Elke Genersch, Jutta Vollmann & Ulrike Riessberger-Gallé. Standard methods forartificial rearing of Apis mellifera larvae. Journal of Apicultural Research, 2013, 52(1):1-15.). The formula, ratio and feeding dose of syrup fed to larvae of different ages are shown in Table 1.

Table 1 Syrup formula, ratio and feeding dose

3. Preparation of virus solution

From another Chinese honeybee breeding farm of the Institute of Honey Bee, Chinese Academy of Agricultural Sciences, we selected Chinese honeybee cysts with typical cystic larvae form, and confirmed the existence of CSBV in Chinese honeybees by RT-PCR. In order to obtain CSBV, 210 CSBV-infected larvae were taken, placed in a sterile grinder, and 2100 μL of sterile phosphate buffered solution (PBS) was added for grinding. After grinding, centrifuge for 30 min at 4°C, 8000 rpm, repeat twice, collect the supernatant, use the cDNA reverse transcribed after RNA extraction as a template, and perform RT-PCR detection. No other virus was detected, this supernatant was used. Named the original venom, and stored at -80°C for later use.

Virus detection using absolute quantitative PCR method (K.M.Hong, H.Najjar, M.Hawley, R.D.Press.Quantitative real-time PCR with automated sample preparation for diagnosis and monitoring of cytomegalovirus infection in bone marrowtransplant patients.Clinical Chemistry.2004,50(5 ):846-856;HU Zhi Gang,CHEN KePing,YAO Qin,GAO Gui Tian,XU Jia-Ping,CHEN Hui-Qing.Cloning andCharacterization of Bombyx mori PP-BP,a Gene Induced by Viral Infection.ActaEntomol.Sin. 2005, 48:871-875).

The PCR reaction was carried out in the BIOER LineGene9600 real-time PCR system, and the upstream and downstream primers were: 5'-ccttggagttttgctatttacg-3' and 5'-cctacatccttgggtcag-3', respectively. The real-time fluorescence quantitative PCR reaction system was 15 μL, including 0.3 μL of forward and reverse primers, 7.5 μL of SYBR reaction solution, 1 μL of template, and water to 15 μL. The qPCR reaction conditions adopt a two-step method: the first step includes a constant temperature section (denaturation at 95 °C for 3 min) and a cycle section (denaturation at 95 °C for 5 s; annealing at 60 °C for 30 s, 40 cycles); the second step is a melting section (denaturation at 95 °C for 15 s) ; annealing at 60°C for 5s; denaturation at 95°C for 15s.). The concentration of the original venom detected by qPCR was 6.74×10 ⁴ copies/μL, and the original venom was used for subsequent vaccination experiments.

Preparation of poisonous syrup: According to the IC ₅₀ detection results of pre-experimental virus infection, the ratio of feeding venom was selected as the original venom: syrup (see Table 1 for syrup composition) = 1:3, and 5 μL of virus stock solution and 15 μL of syrup were prepared. The mixture, a toxic syrup with a CSBV of 1.685×10 ⁴ copies/μL. The poisonous syrup was fed to 3-day-old test larvae.

4. Preparation of adenosine preparations

Purchase adenosine standard (source leaf), prepare a 1 mg/mL stock solution with high-purity water, filter and sterilize it for later use. During the test, the syrup of each age in Table 1 was used for doubling dilution to obtain adenosine preparations. The low concentration group (0.75 μg/mL), the medium concentration group (7.5 μg/mL) and the high concentration group (75 μg/mL) were set respectively. /mL).

Preparation of poisonous preparations: The original venom and adenosine preparations (see Table 1 for syrup composition) are prepared at a ratio of 1:3 (for example: to prepare 20μL of poisonous preparations, 5μL of virus stock solution is mixed with 15μL of adenosine preparations), that is, CSBV A toxic preparation of 1.685×10 ⁴ copies/μL. Toxic formulations were fed to 3-day-old test larvae.

5. Adenosine Intervention Trial

The larvae (3-day-old) after pre-cultivation for 1 day were taken out from the incubator, and the healthy larvae were put into 48-well plates and randomly divided into the control group, the infection group, the preparation group and the intervention group, with 24 larvae in each group. sample, repeated three times. The grouping and feeding conditions are shown in Table 2, and the composition of the syrup in Table 2 is shown in Table 1 (controlling each age).

Table 2 Grouping of Adenosine Intervention Trials

The best time for honeybee larvae to be infected with CSBV is 2-3 day old larvae. Therefore, CSBV infection and adenosine intervention treatment were carried out when the larvae were 3 days old to analyze the preventive and therapeutic effects of adenosine on honeybee CSBV infection. The infection group was fed with poisonous syrup (the preparation method was described in the above 3), and the intervention group was fed with the poisonous preparation (the preparation method of the poisonous preparation was as described in the above 4); The larvae were fed with syrup or adenosine preparation respectively, and only the larvae fed with syrup were used as CK control (refer to Table 1 for feeding amount). Place the above-fed food on the bottom side of the plate to avoid contact with the larvae. Feed every 24h, observe and record the feeding situation and mortality of larvae.

Use 75% ethanol (prepared with DEPC-treated water) to sterilize tweezers for 5 min and rinse three times with DEPC-treated water. Take out 5 surviving larvae from each treatment every day, freeze them with liquid nitrogen immediately, and store them in - 80°C refrigerator for subsequent virus copy number detection.

Experimental Example 1 Survival and mortality analysis of healthy larvae and infected larvae after adenosine intervention

1. Survival rate

The survival rate statistics of each group of honeybee larvae in Example 1 are carried out, and the results are shown in Figure 1. The results show that the survival rate of the larvae in the conventionally fed CK group and the preparation group can reach more than 86%, indicating that adenosine intervention is not effective. It will cause abnormal death of healthy larvae without affecting the growth and development of healthy larvae.

A large number of larvae in the infection group died, and the survival rate was only 43%. The survival rate of the larvae in the intervention group was greatly increased to more than 76%. Among them, all the larvae in the low concentration group (0.75μg/mL) survived at 6 days of age. Adenosine intervention can prevent about 50% of the infected larvae from dying and prevent the infected bee colony from collapsing, which plays an important role in the recovery and reproduction of the bee colony.

2. Mortality

Each group of honeybee larvae in embodiment 1 is carried out to mortality statistics, and the assay method of larval mortality is as follows: pre-cultivation (2-day-old) and the larvae death number on the day of inoculation (3-day-old) are not included in the analysis, to get rid of The effects of mechanical lethality of larvae during the experiment were counted every day from the next day of exposure (4-day-old), and the dead larvae were removed, and the above steps were repeated until the end of 6-day-old.

Daily larval mortality = number of dead larvae per day / number of larvae surviving per day × 100%;

The mortality of larvae in the whole process = the number of dead larvae / the total number of samples × 100%.

The statistical results of mortality in each group are shown in Figure 2. The results show that the daily mortality of 4-6 day-old larvae in the CK group fed routinely is less than 3%, and the daily mortality of 4-6 day-old larvae after each concentration of adenosine intervention The mortality rate did not increase significantly, indicating that adenosine intervention did not affect the growth and development of healthy larvae and could be used for assisted feeding of larvae.

While CSBV is a typical inducer, the mortality rate of the 3-day-old larvae (4-day-old) on the next day after exposure to the virus increased significantly, reaching as high as 25%, and the 5- and 6-day-old mortality decreased, but the daily mortality rate were more than 20%, and only 43% of the experimental larvae survived by 6 days of age (Fig. 1). Liang Qin et al. (Liang Qin, Chen Dafu. Bee Conservation [M]. China Agricultural Press, 2009.2-3.) found that CSBV was most susceptible to infecting 2-3 day old larvae, and the infected larvae could not pupate, and A large number of deaths occurred in the late larval stage, which is consistent with the experimental results of the present invention.

Compared with the infected group, the mortality rate of larvae at each age in the adenosine intervention group decreased significantly. The mortality rate of the 4-day-old larvae was about 15%, and then decreased day by day. At the 6-day age, the survival rate of the larvae in each concentration group reached more than 86%. Among them, the low-concentration group (0.75 μg/mL) intervened with the infected larvae all survived at 6 days of age (Figure 1). The results showed that the use of adenosine intervention can greatly reduce the fatality rate of the infected larvae and prevent the infected bee colony from collapsing , played an important role in the recovery and reproduction of bee colonies.

Experimental Example 2 Detection of virus copy number after adenosine intervention in healthy larvae and infected larvae

The CSBV detection was carried out on the apparently healthy 4-6 day-old bee larvae (CK group) in Example 1, and it was found that the virus copy number of each day-old larvae was 66-70, indicating that the apparently healthy larvae also had virus infection, but only No obvious symptoms were shown, indicating that CSBV generally has invisible infection in bee colonies, and larvae can survive with the virus within a certain concentration, which is similar to Liu Shan (Shan, L., Liuhao, W., Jun, G., Yujie ,T.,Yanping,C.,Jie,W.,Jilian,L.,ChineseSacbrood Virus Infection in Asian Honey Bees(Apis cerana cerana)and HostImmune Responses to the Virus Infection,Journal of InvertebratePathology.2017,doi:http:/ /dx.doi.org/10.1016/j.jip.2017.09.006) findings are consistent. After adenosine intervention, the virus copy number in apparently healthy 4-6-day-old larvae (preparation group) decreased to below 6, indicating that adenosine intervention can improve the virus infection degree of apparently healthy larvae and contribute to the normalization of larvae. growth and development.

The number of virus copies in the larvae of the infected group increased explosively. The number of virus copies in the 4-day-old larvae was as high as 12.1×10 ⁴ , which was more than 1700 times that of the CK group. %, with the growth and development, the number of virus copies in the 5-day and 6-day-old bodies decreased, but remained above 2.4×10 ⁴ , showing that the single-day larval mortality was still above 20% (Figure 3). The single-day virus copy number in the 4-6-day-old larvae of each concentration of adenosine intervention group decreased to less than 10, which can be regarded as no CSBV detection, indicating that after feeding adenosine, the infected larvae produced a strong antagonistic virus response, which was obvious. Reduced the degree of virus infection of larvae, thereby reducing the fatality rate of infected larvae.

Experimental Example 3 Morphological analysis of healthy larvae and infected larvae after adenosine intervention

On the 18th day of the experiment of Example 1 (the 22nd day from the egg stage), the state of each group of larvae in Example 1 was observed with a stereomicroscope, and photographed and recorded.

The morphological observation results are shown in Figure 4. The larvae in the infection group could not pupate even if they did not die, but the larvae in the CK group and the low concentration group (0.75 μg/mL) intervention group had very active life activities, and all of them could pupate and survive. Emerges into adult bees. CSBV infection is characterized by infecting 1-3 day old larvae, causing a large number of larvae in the colony to die and unable to pupate. Healthy larvae pupate and emerge into adult bees, which is a normal development process of honey, while CSBV-infected larvae can pupate and emerge into adult bees. The adult bees showed that proline intervention played a role in antagonizing the virus, so that the infected larvae could develop normally.

Experimental Example 4 Effects of CSBV infection and adenosine intervention on the expression of immune-related genes in larvae

In order to analyze the impact of CSBV infection and adenosine intervention on the immune system of honeybee larvae, real-time fluorescence quantitative PCR was used to detect the expression levels of antimicrobial peptide genes in each group of honeybee larvae in Example 1, and β-actin was used as an internal reference gene (Shan, L., Liuhao, W., Jun, G., Yujie, T., Yanping, C., Jie, W., Jilian, L., ChineseSacbrood Virus Infection in Asian Honey Bees(Apis cerana cerana) and HostImmune Responses to the Virus Infection, Journal of InvertebratePathology. 2017, doi: http://dx.doi.org/10.1016/j.jip.2017.09.006). The test results are as follows:

1. Expression detection of bee antimicrobial peptide (Apidaecin) gene

Honeybee antimicrobial peptide (Apidaecin) is the main antimicrobial peptide of bees. The detection results are shown in Figure 5. The relative expression of bee antimicrobial peptides in control (CK) larvae is only 1.24 to 1.55. After 24 hours of infection with CSBV, bee antimicrobial peptides The relative expression level of peptides increased in larvae, and the relative expression level of honeybee antimicrobial peptides in 4-6-day-old larvae was maintained at about 20.

Adenosine intervention to healthy larvae (preparation group) caused a significant increase in the expression of bee antimicrobial peptides, with the highest at 6 days of age, and the relative expression of the low concentration group (0.75μg/ml) was up-regulated to about 1000; Adenosine intervention in poisonous larvae (intervention group) also significantly increased the expression of honeybee antimicrobial peptides, with the highest at 6 days of age, and the relative expression reached about 650, which was 32 times that of the infected group.

The results showed that adenosine intervention strongly induced the ability of the infected larvae to express antimicrobial peptides, enhanced the larvae's innate immune defense against CSBV, and reduced the fatality rate. It indicated that adenosine has the ability to strongly induce the expression of antimicrobial peptides in healthy larvae and CSBV-infected larvae.

2. Hymenoptaecin

Hymenopteran antimicrobial peptides are the most important supplementary peptides in honeybee antimicrobial peptides. The detection results are shown in Figure 6. The relative expression levels of hymenopteran antimicrobial peptides in control (CK) larvae are only 1.31-1.56. After 24 hours, the expression level of hymenopteran antimicrobial peptides in larvae increased, and the relative expression level of 4-6-day-old larvae did not change significantly and remained at about 20.

Adenosine intervention to healthy larvae (preparation group) significantly increased the expression of hymenopteran antimicrobial peptides, and the relative expression was up-regulated to 97.24; adenosine intervention to poisoned larvae (intervention group) significantly increased Hymenopteran antimicrobial peptides The expression level of the peptide, the highest relative expression level in a single day can be as high as 342.14, which is 17 times that of the infection group.

The results showed that adenosine intervention strongly induced the expression of hymenopteran antimicrobial peptides, enhanced the larval innate immune defense against CSBV, and reduced the mortality. It indicated that adenosine had the ability to strongly induce the expression of Hymenopteran antimicrobial peptides in healthy larvae and CSBV-infected larvae.

3. Abaecin

As the backup peptide of honeybee antimicrobial peptide, the antibacterial peptide of bee moth can only play a role when the bactericidal ability of honeybee antibacterial peptide and hymenopteran antibacterial peptide is lost. The detection results of the antibacterial peptides of the bee moth are shown in Figure 7. The relative expression of the antibacterial peptides of the larvae of the control (CK) group was only 1.52 to 3.68. After 24 hours of infection with CSBV, the expression of the antibacterial peptides of the bee moths in the larvae was only 1.52 to 3.68. Significantly increased, increased to 73.25, but the expression level of 5-6 days old dropped to 25.4-26.97.

Adenosine intervention to healthy larvae (preparation group) can cause the expression of antibacterial peptides of the bee moth to increase to about 35, and the expression of antibacterial peptides of bee moth in the low concentration group (0.75 μg/ml) on a single day increased to about 100; Giving adenosine intervention (intervention group) can significantly up-regulate the expression of antibacterial peptides of 6-day-old larvae, up to 50.81, which is 88.4% higher than that of the infected group. effect.

The results showed that adenosine intervention strongly induced the expression of antibacterial peptides in the larvae, enhanced the innate immune defense of larvae against CSBV, and reduced the fatality rate. It indicated that adenosine had the ability to strongly induce the expression of antimicrobial peptides in healthy larvae and CSBV-infected larvae.

4. Bee Defensin

The detection results of bee defensins are shown in Figure 8. The relative expression of bee defensins in control (CK) larvae was only 1.32 to 1.62. After 24 hours of infection with CSBV, the expression of bee defensins in larvae increased to some extent. , reached 46.26, the highest expression at 5-day-old was 52.59, and dropped to 22.28 at 6-day-old.

Adenosine intervention to healthy larvae (preparation group) caused a significant increase in the expression of bee defensin, with the highest expression at 5-day-old, and the low concentration group (0.75μg/ml) was up-regulated to more than 160; Adenosine was administered to infected larvae The intervention (intervention group) significantly up-regulated the expression of bee defensin, with the highest expression at 5-day-old, and the highest relative expression could reach more than 210, which was 4 times that of the infection group.

Honeybee defensin is the only antimicrobial peptide in honeybee hemolymph that inhibits Gram-positive bacteria, but the expression level is the least. The invention strongly induces the ability of endogenous honeybee defensin expression through adenosine intervening in this exogenous pathway, enhances the innate immune defense of larvae to resist CSBV intrusion, and reduces the fatality rate. It indicated that adenosine had the ability to strongly induce the expression of endogenous bee defensins in healthy larvae and infected larvae.

According to the present invention, by analyzing the dynamic changes of the immune-related gene expression in the 4-6-day-old larvae infected with CSBV, it can be known that the gene expression of antimicrobial peptides in the infected larvae is generally higher than that of the healthy larvae, indicating that infection with CSBV can promote the in vivo immunity of the honeybee larvae The expression of related genes, this result is consistent with Liu Shan (Shan, L., Liuhao, W., Jun, G., Yujie, T., Yanping, C., Jie, W., Jilian, L., Chinese Sacbrood Virus Infection in AsianHoney Bees (Apis cerana cerana) and Host Immune Responses to the VirusInfection, Journal of Invertebrate Pathology. 2017, doi: http://dx.doi.org/10.1016/j.jip.2017.09.006) research results are consistent.

The invention takes CSBV-infected Chinese honeybees and healthy Chinese honeybees as research objects, and uses the method of fluorescence quantitative PCR to quantitatively detect and compare the expression conditions of four antibacterial peptide genes in the developmental stages of susceptible and healthy bee larvae. The immune response of Chinese bees after infection with CSBV was investigated; and the expression of four antimicrobial peptide genes after adenosine was administered to susceptible and healthy bee larvae was quantitatively detected and compared:

Regardless of whether it was healthy larvae or infected larvae, adenosine intervention could up-regulate the expression of Hymenoptaecin, an antimicrobial peptide Hymenoptaecin, in 4-6-day-old larvae. Expression levels of the antibacterial peptides Apidaecin and Abaecin in instar larvae of honeybees. In conclusion, intervention with adenosine strongly induced the ability of infected larvae to express the endogenous antimicrobial peptides Hymenoptaecin, Defensin, Apidaecin, and Apidaecin. The high expression of these four antimicrobial peptides in one or more days successfully resisted the intrusion of CSBV on larvae, reduced the mortality rate of larvae, and promoted larvae to pupate and emerge into adult bees. At the same time, adenosine intervention can also up-regulate the expression level of antimicrobial peptides in healthy Chinese bee larvae, forming a fast and effective defense mechanism to quickly kill or remove exogenous pathogenic microorganisms and protect the growth and development of larvae.

In summary, adenosine can inhibit the proliferation of CSBV and reduce the copy number of CSBV in honeybee larvae; it can also induce the expression of endogenous antimicrobial peptides in honeybee larvae, improve the innate immune defense of honeybees, and resist the damage of CSBV to larvae. And prevent further infection by CSBV and other pathogenic microorganisms, so it can be used to prevent and treat honeycomb larvae in honeybees.

Although the present invention has been described in detail above with general description, specific embodiments and tests, some modifications or improvements can be made on the basis of the present invention, which is obvious to those skilled in the art . Therefore, these modifications or improvements made without departing from the spirit of the present invention fall within the scope of the claimed protection of the present invention.

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<120> Application of adenosine in the control of honeybee virus infection

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Claims (10)

1. Use of adenosine or a derivative thereof in the manufacture of a medicament for the treatment or prevention of a bee viral infection.

2. Use of adenosine or a derivative thereof in the manufacture of a feed for the treatment or prevention of viral infection in bees.

3. Use of adenosine or a derivative thereof for inhibiting the proliferation of bee virus.

4. The use of any one of claims 1 to 3, wherein the virus is a Chinese bee sacbrood virus.

5. Use of adenosine or a derivative thereof in the preparation of a medicament for enhancing the immunity of bees.

6. Use of adenosine or a derivative thereof in the preparation of a feed for enhancing the immunity of bees.

7. The use of claim 5 or 6, wherein the improvement of bee immunity is achieved by increasing the expression level of endogenous antimicrobial peptides of bees;

preferably, the endogenous antimicrobial peptide is any one or more selected from the group consisting of hymenoptera antimicrobial peptide, bee defensin, bee antimicrobial peptide and bee moth antimicrobial peptide.

8. The use according to any one of claims 1 to 7, wherein the adenosine derivative comprises adenosine, an adenylate.

9. Use according to any one of claims 1 to 8, wherein the active ingredient of the medicament or feed comprises one or more selected from adenosine and derivatives thereof.

10. A product for use in the treatment or prevention of a viral infection of honey bees, characterized in that the active ingredient of the product comprises one or more selected from adenosine and derivatives thereof; the product is a medicament, feed or feed additive.

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