Application of jute nanocellulose or derivative thereof in preparation of medicine or feed for preventing and treating bee virus infection

Abstract

The invention discloses application of jute nanocellulose or derivatives thereof in preparation of drugs or feeds for preventing and treating bee virus infection, and belongs to the technical field of bee breeding. It is found that the jute nanocellulose can significantly improve the survival rate of Italian bees infected with chronic paralysis virus (CBPV) and reduce the death rate of the Italian bees. The jute nano-cellulose can inhibit proliferation of CBPV in bees and reduce the copy number of viruses. Meanwhile, the level of intestinal probiotics reduced due to CBPV infection can be recovered, and the congenital immune defense capability of bees is improved. The jute nanocellulose can be used for preparing drugs or feed additives for preventing and treating CBPV infection, has important significance on preventing and treating chronic paralysis viruses of Italian bees, and has good market application value.

Description

Technical Field

[0001] The present invention belongs to the technical field of bee breeding, and more specifically, relates to the application of jute nanocellulose or its derivatives in the preparation of drugs or feeds for preventing and treating bee virus infections. Background Art

[0002] Bees, as important pollinating insects, play an irreplaceable role in global agricultural production. According to research statistics, the economic value created by bee pollination accounts for approximately 9.5% of the total value of agricultural products consumed by humans globally. This pollination service not only significantly improves the yield and quality of crops but also makes important contributions to maintaining biodiversity and ecological balance. In addition, bees can produce numerous bee products, such as honey, royal jelly, bee pollen, and propolis. Therefore, the health of bees is directly related to food safety and ecological environment safety. Bee products produced by diseased bees may pose safety hazards, and drug residues used for preventing and treating bee diseases may also lead to quality and safety problems of bee products.

[0003] Bees are vulnerable to various aspects in nature, including viruses, pesticides, varroa mites, microplastics, and fungi. Among them, the majority of pathogens infecting bees are small RNA viruses, including Chronic Bee Paralysis Virus (CBPV). CBPV can infect individuals at all developmental stages. Infected individuals can spread horizontally in the hive through behaviors such as contact and food sharing, and can also be vertically transmitted through the queen bee through behaviors such as nursing and feeding. CBPV infection mainly causes bees to tremble, lose the ability to fly, crawl, and have bloated abdomens, so it is also called "big belly disease". CBPV disrupts the balance of the bee gut microbiota, affecting the growth of beneficial bacteria. This process weakens the intestinal barrier, thus exacerbating the symptoms of bee bloating and ultimately leading to colony collapse. Currently, the prevention and control of CBPV mainly adopt methods such as strengthening apiary management, replacing the queen bee and discontinuing brood rearing, and using broad-spectrum antibiotics. These methods are not only time-consuming and laborious, but also have unsatisfactory effects and the risk of contaminating bee products.

[0004] The gut microbiota of honeybees can provide nutrition for honeybees, assist in digestion, help resist toxins, resist pathogenic microorganisms such as bacteria and fungi, and participate in immune antiviral activities, which is an important defense line for honeybees to avoid pathogen invasion. Researchers found that Lactobacillus apis promotes the synthesis of host antimicrobial peptides (AMPs) by inducing the gene expression that regulates the Toll pathway, thereby inhibiting the proliferation of Hafnia alvei. The obligate honeybee symbionts Snodgrassella alvi and Gilliamella spp. colonized in the ileum form a biofilm to build a physical barrier, effectively reducing the risk of honeybees being infected with Crithidia bomb. Bombella apis can effectively protect larvae from Aspergillus flavus. In summary, the gut microbiota of honeybees plays an important role in the antiviral defense of honeybees.

[0005] Researchers found that the abundance of gut microbiota in honeybees can be significantly increased by adding dietary fiber. Corchorus capsularis L. leaves have a high cellulose content, and jute fiber is one of the main underutilized agricultural raw materials. Jute leaves have been used to treat fevers, constipation, dysentery, liver diseases, and indigestion in traditional applications due to their stimulating, analgesic, laxative, and appetizing properties. Chinese Patent CN 116376998A reported that jute nanocellulose can significantly improve the abundance of gut bacteria after being stressed by antibiotics, but did not report the effect of jute nanocellulose in antiviral infection and improving immunity after virus infection. Summary of the Invention

[0006] Aiming at the problems in the prior art, the purpose of the present invention is to provide the application of jute nanocellulose or its derivatives in the preparation of drugs or feeds for preventing and treating honeybee virus infections. Experiments have proved that administering jute nanocellulose can significantly increase the survival rate of worker bees of the Italian honeybee infected with chronic paralysis virus. On the one hand, jute nanocellulose can directly act on the virus to reduce the virus copy number. On the other hand, jute nanocellulose can also restore or increase the level of gut microbiota and enhance the innate immune defense of the body.

[0007] The technical solution of the present invention to solve the above technical problems is as follows:

[0008] The present invention provides the application of jute nanocellulose or its derivatives in the preparation of drugs or feeds for preventing and treating honeybee virus infections.

[0009] Preferably, the virus is the chronic paralysis virus of honeybees.

[0010] The present invention also provides the application of jute nanocrystalline cellulose or its derivatives in the preparation of drugs or feeds for inhibiting the proliferation of bee viruses.

[0011] Preferably, the virus is Chronic bee paralysis virus.

[0012] The present invention also provides the application of jute nanocrystalline cellulose or its derivatives in the preparation of drugs or feeds for improving the immunity of bees after virus infection.

[0013] Preferably, the virus is Chronic bee paralysis virus.

[0014] Preferably, the immunity of bees is improved by improving the abundance of intestinal probiotic flora.

[0015] The jute nanocrystalline cellulose (abbreviated as NCC) described in the present invention is processed from jute leaves, and the processing method of jute dietary fiber is prepared with reference to "National Standard of the People's Republic of China - National Food Safety Standard - Determination of Dietary Fiber in Foods - GB5009.88—2014". The jute dietary fiber is dispersed in sterile deionized water at a ratio of 1:200 (w / v), and pulverized in an ice bath for 20 min with an ultrasonic cell disruptor with a power of 360 W, and finally a jute nanocrystalline cellulose suspension is prepared.

[0016] In the present invention, the jute nanocrystalline cellulose derivatives include soluble and insoluble jute dietary fiber.

[0017] In the present invention, the concentration of jute nanocrystalline cellulose in the drug or feed is 0.8 - 1.2 μg / mL, preferably 1 μg / mL. Specifically, the jute nanocrystalline cellulose is dissolved in the liquid food of bees and fed to the bees, and the final concentration of jute nanocrystalline cellulose is 1 μg / mL.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0019] The application of the jute nanocrystalline cellulose provided by the present invention provides a basis for the research on the mechanism of bee virus infection and the mechanism between bee viruses and intestinal microorganisms. Jute nanocrystalline cellulose can be applied to the preparation of drugs or feed additives for preventing and treating Chronic bee paralysis virus or improving the immunity of bees, providing a new method for preventing and treating Chronic bee paralysis virus, having good market application prospects, and also providing new ideas for the uses of nanocrystalline cellulose. Description of the Drawings

[0020] Figure 1 It is the statistical result of the survival curve of bees within 1 - 7 days after the intervention of jute nanocrystalline cellulose in Experimental Example 1 of the present invention;

[0021] Among them, Control represents the control group (CK group), NCC represents the group fed with jute nanofibrils alone, CBPV represents the group injected with chronic bee paralysis virus, and NCC + CBPV represents the group injected with chronic bee paralysis virus and fed with jute nanofibrils.

[0022] Figure 2 This is the detection result of the virus copy number after 2 days and 4 days of jute nanofibril intervention in Experimental Example 2 of the present invention;

[0023] Control represents the control group (CK group), CBPV represents the group injected with chronic bee paralysis virus, and NCC + CBPV represents the group injected with chronic bee paralysis virus and fed with jute nanofibrils.

[0024] Figure 3 This is the abundance statistics of 6 main probiotics in the intestines of bees after 5 days of jute nanofibril intervention in Experimental Example 3 of the present invention;

[0025] Control represents the control group (CK group), NCC represents the group fed with jute nanofibrils alone, CBPV represents the group injected with chronic bee paralysis virus, and NCC + CBPV represents the group injected with chronic bee paralysis virus and fed with jute nanofibrils. Detailed implementation mode

[0026] Many specific details are set forth in the following description in order to provide a thorough understanding of the present invention. However, the present invention can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without departing from the connotation of the present invention. Therefore, the present invention is not limited by the specific implementations disclosed below.

[0027] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present invention belongs. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention. It should be noted that the reagents used in this embodiment are all ordinary commercially available products.

[0028] The data statistical analysis in the following examples was all completed using Graph prism 8.0. The effects of jute nanofibrils on Apis mellifera ligustica were all expressed as "Mean±SE". The variance analysis and LSD multiple variance analysis methods were used to compare the changes in the virus copy number of Apis mellifera ligustica infected with CBPV and the abundances of 5 kinds of bacteria in the intestine.

[0029] Experimental Example 1 Intervention of healthy Apis mellifera ligustica and virus-injected bees with jute nanofibrils

[0030] 1. Test samples

[0031] Apis mellifera ligustica originated from the experimental bee colonies of the Institute of Zoology, Guangdong Academy of Sciences. The bee brood frames about to emerge were placed in a constant temperature and humidity incubator at a temperature of 30±1°C and a relative humidity of 75±5%RH for cultivation. Worker bees of Apis mellifera ligustica of the same day-old just emerged were obtained.

[0032] The reverse transcription-polymerase chain reaction (RT-PCR) method was used to detect whether the test larvae were infected with CBPV and other viruses (including black queen cell virus BQCV, deformed wing virus DWV, Israeli acute paralysis virus IAPV, Kashmir bee virus KBV, acute bee paralysis virus ABPV). Healthy worker bees without virus infection were used as samples in this example.

[0033] The detection primers for the above various viruses refer to the primer sequences provided by (Sguazza GH, Reynaldi Fy, Galosi CM, Simultaneous detection of bee viruses by multiplex PCR. Journal of Virological Methods, 2013, 194(1 / 2): 102-106.).

[0034] The specific detection method is as follows: RNA was extracted using the JianShi Biotechnology RNA extraction kit, RNA was reverse transcribed into cDNA using the abm company's 5X All-In-One MasterMix with AccuRT, and PCR amplification reaction was carried out using the JinSha Biotechnology 2*GS Taq PCR Mix. All the above steps were carried out according to the instructions. The detection results showed that CBPV and other viruses were not detected.

[0035] 2. Preparation of virus solution

[0036] The construction of the CBPV infectious clone and the synthesis of viral RNA refer to the previous research methods of this laboratory (Yang s, Zhao HX, Deng Yc, Deng s, Wang XL, Diao QY, Hou Cs, A reverse genetics system for the Israeli acute paralysis virus and chronic bee paralysis virus. Int. J. Mol. Sci. 2020, 21(5): 1742.).

[0037] 3. Preparation of sucrose syrup and jute nanocellulose preparation

[0038] Sucrose syrup: Sucrose was prepared into 50% sucrose syrup with sterile water for standby.

[0039] Jute nanocellulose preparation: Dissolve the jute nanocellulose solution in 50% sucrose syrup, and the final concentration of jute nanocellulose is 1 μg / mL.

[0040] 4. Jute nanocellulose intervention experiment

[0041] Select newly emerged bees of the same age within 24 hours and raise them in sterilized standard wooden cages (8 cm × 6 cm × 12 cm), with 30 bees in each cage and 3 cages in each group. Feed them with 50% sucrose syrup for subsequent experiments. CBPV group: Inject 2 μL of synthesized CBPV RNA (10 12 gene copies) into 30 newly emerged adult worker bees (3 groups) respectively, and infect them by thoracic injection. Feed them with 50% sucrose syrup from day 1 to day 7.

[0042] CK group: Inject 2 μL of 1×PBS into the thorax as a control, and feed them with 50% sucrose syrup from day 1 to day 7 after injection.

[0043] NCC group: Feed them with 50% sucrose syrup normally from day 1 to day 7.

[0044] CBPV+NCC group: Inject 2 μL of synthesized CBPV RNA (10 12 gene copies) into 30 newly emerged adult worker bees (3 groups) respectively, and infect them by thoracic injection. Feed them with jute nanocellulose preparation from day 1 to day 7.

[0045] Record the number of dead individuals in each group of bees every day from day 1 to day 7.

[0046] The statistical results are as follows Figure 1 shown: A large number of bees in the CBPV-infected group died. There was no difference in the mortality rate of bees between the CK group and the NCC group. The survival rate of bees in the CBPV+NCC group was significantly higher than that in the CBPV-infected group. The results show that jute nanocellulose does not affect the mortality rate of normal bees and can greatly increase the survival rate of bees infected with CBPV.

[0047] Experimental example 2 Detection of virus gene copies after jute nanocellulose intervention in healthy Italian bees and bees injected with venom

[0048] On the 2nd and 4th days of experimental example 1 respectively, select 9 - 12 bees from each group for CBPV detection.

[0049] Figure 2The results showed that on the 2nd day after injecting CBPV, the CBPV virus copy number in the bees of the CBPV-infected group increased explosively compared with the CK group; the virus copy number in the bees of the CBPV+NCC group showed a significant decreasing trend compared with the CBPV group (P<0.01). On the 4th day after injecting CBPV, the CBPV copy numbers in the bees of the three groups showed a significant decreasing trend compared with the 2nd day; the CBPV+NCC group still showed a significant decreasing trend compared with the CBPV group (P<0.05). This indicates that feeding with jute nanofiber preparation significantly enhanced the antiviral ability of bees, significantly reduced the degree of virus infection, and thus improved the survival rate.

[0050] Experimental Example 3 Effects of CBPV infection and jute nanofiber intervention on the intestinal microbiota of bees

[0051] On the 5th day of Experimental Example 1, 5 bees were taken from each replicate in each group (3 replicates in total). Under a stereomicroscope, the bee samples were fixed on the wax top of the dissection tray with insect pins, and then the intestines were gently pulled out with sterilized forceps. The intestinal tissues were washed 3 times in 1×PBS buffer (10 mmol / L, pH 7.4) and then stored in a -80 °C refrigerator. DNA extraction was performed according to the instructions of the bacterial genomic DNA extraction kit (Tiangen Biochemical Technology Co., Ltd.). Then the samples were sent to Novogene Bioinformatics Technology Co., Ltd. in Beijing by dry ice for metagenomic sequencing on the Illumina HiSeq 2500 platform to construct a library. After pretreatment, Clean Data was obtained, and the MEGAHIT assembly software was used for assembly analysis and gene annotation. Species annotation was performed based on the gene information to obtain the corresponding species information and the abundance distribution of species.

[0052] Figure 3The results showed that the abundances of Lactobacillus apis, Lactobacillus Firm4, Snodgrassella alvi, Bifidobacterium asteroides, and Bartonella apis in the intestines of bees infected with CBPV were significantly lower than those in the CK group and the NCC group. The abundances of the five bacteria in the CBPV+NCC group showed a significant upward trend compared with those in the CBPV group. This indicates that jute nanocellulose can not only restore the abundances of probiotics in the bee intestine but also increase the abundances of probiotics in the bee intestine, thereby providing immunity. The abundance of Bifidobacterium asteroides in the intestine of the NCC group was significantly higher than that in the CK group, indicating that jute nanocellulose also has a significant protective and promoting effect on some intestinal microorganisms of normal bees.

[0053] Combined Figure 2 with Figure 3 , in the present invention, jute nanocellulose can inhibit the proliferation of CBPV virus and improve the abundances of probiotic flora in the bee intestine after CBPV virus stress. It can be seen that jute nanocellulose can be used for the prevention and treatment of CBPV virus in bees and can also be used to improve the immunity of bees after CBPV virus infection. Jute nanocellulose can enter the bee body by injection (drug) or ingestion (feed) to achieve its function.

[0054] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. Application of jute nanocellulose or its derivatives in the preparation of drugs or feed for the prevention and treatment of bee virus infections.

2. The use according to claim 1, characterized in that: The virus is bee chronic paralysis virus.

3. Application of jute nanocellulose or its derivatives in the preparation of drugs or feeds for inhibiting the proliferation of honey bee viruses.

4. The use according to claim 3, characterized in that: The virus is bee chronic paralysis virus.

5. Application of jute nanocellulose or its derivatives in the preparation of drugs or feeds for improving immunity of honey bees after virus infection.

6. The use according to claim 5, characterized in that: The virus is bee chronic paralysis virus.

7. The use according to claim 5, characterized in that: Improving honey bee immunity by improving the abundance of intestinal probiotic flora.

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