Method for resisting viruses in mosquito insects and application of method

Abstract

The invention relates to a method for resisting viruses in mosquito insects and application of the method, and belongs to the technical field of gene symbiosis. The method for resisting viruses in the mosquito insects comprises the following steps: separating symbiotic bacteria in the mosquito insects, taking the symbiotic bacteria existing in a plurality of mosquito insect organs, screening the symbiotic bacteria to obtain candidate strains, and over-expressing antiviral protein in at least one candidate strain to obtain symbiotic engineering bacteria, and supplementing the symbiotic engineering bacteria into the bodies of the mosquitoidaceae insects. According to the invention, a brand new method for resisting viruses in the mosquitocidae insects is established, symbiotic bacteria capable of stably spreading in the mosquitocidae insects are screened, and the symbiotic bacteria are used for expressing antiviral proteins to realize inhibition of human pathogenic viruses in the mosquitoes, so that the spreading capacity of the mosquitoes is reduced.

Description

Technical Field

[0001] The invention belongs to the technical field of gene symbiosis and relates to a method for resisting viruses in mosquito insects and application thereof. Background Art

[0002] Research has shown that when mosquitoes feed on infected blood, the virus enters the mosquito's body through the bloodstream and multiplies in the mosquito's intestines, salivary glands, and other tissues. For example, during the infection and transmission cycle within mosquitoes, flaviviruses like dengue and Zika interact with key factors, such as the mosquito's intestinal microbiome, influencing the mosquito's ability to acquire and transmit the virus. Mosquito-borne viruses spread primarily through a host-to-mosquito transmission cycle in nature. After an infected host is bitten by a mosquito, the mosquito picks up the virus and then spreads it further by biting other healthy hosts.

[0003] Traditional methods for controlling mosquito-borne viral transmission include chemical control, environmental governance, and vaccine therapy. Chemical control involves the use of chemical insecticides, and long-term, heavy use leads to widespread and severe insecticide resistance in mosquitoes, reducing their effectiveness. These methods are harmful to non-target organisms (beneficial insects and aquatic organisms) and ecosystems, and may pose potential risks to human health. Environmental governance, including the removal of breeding grounds (stagnant water), is difficult to implement fully and sustainably in areas with high urbanization, dense populations, or weak health infrastructure. Vaccine therapy also presents challenges in the lack of effective vaccines or specific treatments for many mosquito-borne viral diseases. Meanwhile, biological control strategies have gradually become a research hotspot. The core idea of ​​biological control is to reduce mosquito populations or reduce their ability to transmit viruses by utilizing mosquitoes' natural enemies, pathogens, or symbiotic microorganisms, or by genetically modifying the mosquitoes themselves. This approach is expected to overcome the shortcomings of traditional methods to a certain extent. For example, pathogen blocking technology mediated by symbiotic microorganisms. Wolbachia is an intracellular symbiotic bacterium widely present in insects. Existing Wolbachia strains have been shown to significantly inhibit the replication of a variety of human pathogenic viruses (such as dengue, Zika, chikungunya, and yellow fever) in mosquitoes (mainly Aedes mosquitoes), reducing the mosquitoes' ability to spread (pathogen blocking).

[0004] In summary, the development of antiviral methods in mosquito insects is of great significance in the field of mosquito-borne virus transmission prevention and control. Summary of the Invention

[0005] In response to the deficiencies of the existing technology and actual needs, the present invention provides a method for antiviral treatment in mosquito-borne insects and its application, providing new methods and new ideas for the prevention and control of mosquito-borne virus transmission.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a method for antiviral treatment in mosquito insects, the method comprising:

[0008] Isolate the symbiotic bacteria in mosquito insects, take the symbiotic bacteria present in multiple organs of mosquito insects, screen the symbiotic bacteria to obtain candidate strains, overexpress antiviral proteins in at least one of the candidate strains to obtain symbiotic engineered bacteria, and replenish the symbiotic engineered bacteria into the body of the mosquito insect.

[0009] The present invention establishes a new antiviral method in mosquito insects, screens symbiotic bacteria that can stably spread in mosquito insects, uses them to express antiviral proteins, and inhibits human pathogenic viruses in mosquitoes, thereby reducing the mosquitoes' ability to spread.

[0010] Preferably, the Culicidae insects include Aedes mosquitoes.

[0011] Preferably, the Aedes mosquito includes at least one of Aedes aegypti, Aedes albopictus or Aedes flavipictus.

[0012] Preferably, the candidate strains include at least one of Serratia marcescens, Asaia, Elizabethkingia or Flavobacterium.

[0013] It can be understood that the present invention found that Serratia marcescens can be stably vertically transmitted and express exogenous proteins in Aedes aegypti. In theory, strains with similar genetic traits within the same genus or species are all suitable for the technical solution of the present invention.

[0014] Preferably, the Serratia marcescens includes Serratia marcescens CGMCC 1.12941.

[0015] Preferably, the virus comprises at least one of dengue virus, Zika virus or chikungunya virus.

[0016] Preferably, the amino acid sequence of the anti-dengue virus protein includes the sequence shown in SEQ ID NO.1, and the nucleic acid sequence includes the sequence shown in SEQ ID NO.2.

[0017] SEQ ID NO.1:

[0018] QVQLVESGGGVVQPGRSLRLSCAASGFIFSNYGMHWVRQAPGKGLEWVAVISYDGSDKRYADSVRGRFTISRDNSKNTLFLQVTSLRAEDTAVYYCAKELSGYDPGFEYWGQGTPVTVSSGGGGSGGGGSGGGGSDIQMTQSPSTLSASVGDRVTITCRASQSINTWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYESYATFGQGTKVDIKLE。

[0019] SEQ ID NO.2:

[0020] .

[0021] Preferably, the screening includes overexpressing fluorescent protein in the symbiotic bacteria and replenishing them into the bodies of mosquito insects, using a fluorescence microscope to observe the colonization dynamics of various tissues and organs of the replenished mosquito insects, and tracking them to the next generation to select symbiotic bacteria that can be vertically transmitted in mosquito insects.

[0022] Preferably, the fluorescent protein includes EGFP and the like.

[0023] Preferably, the amino acid sequence of EGFP includes the sequence shown in SEQ ID NO.3, and the nucleic acid sequence includes the sequence shown in SEQ ID NO.4.

[0024] SEQ ID NO.3:

[0025] MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK。

[0026] SEQ ID NO.4:

[0027] .

[0028] It is understood that in the present invention, the common genetic modification methods in the art can be used to genetically modify Serratia marcescens to express the target protein, for example, by inserting the target protein encoding nucleic acid into a plasmid to construct a recombinant vector, or directly inserting the target protein encoding nucleic acid into the Serratia marcescens genome.

[0029] Preferably, the replenishment method comprises artificial sugar water feeding.

[0030] In a second aspect, the present invention provides the use of Serratia marcescens in preventing and controlling virus transmission in mosquito insects.

[0031] In a third aspect, the present invention provides an antiviral engineered bacterium, which is Serratia marcescens that overexpresses an antiviral protein.

[0032] Preferably, the virus comprises at least one of dengue virus, Zika virus or chikungunya virus.

[0033] In a fourth aspect, the present invention provides a composition comprising the antiviral engineered bacteria described in the third aspect.

[0034] Preferably, the composition further comprises a biologically acceptable carrier.

[0035] Preferably, the biologically acceptable carrier comprises at least one of a culture medium, a bacterial growth agent, a wetting agent, a penetrant, a dispersant, an emulsifier, a stabilizer, an adhesive or a filler.

[0036] In a fifth aspect, the present invention provides a method for preventing and controlling virus transmission in mosquito insects, the method comprising: replenishing the antiviral engineered bacteria described in the third aspect or the composition described in the fourth aspect into the body of the mosquito insect.

[0037] Compared with the prior art, the present invention has at least the following beneficial effects:

[0038] The present invention designs a precise and efficient prevention and control strategy, screens symbiotic bacteria that can stably spread in mosquito insects, utilizes the vertical transmission characteristics of heritable symbiotic bacteria, expresses antiviral proteins in them, and inhibits human pathogenic viruses in mosquitoes, thereby reducing the transmission ability of mosquitoes. It has long-lasting antiviral properties, strong targeting, and is eco-friendly, providing a new approach for the prevention and control of mosquito-borne diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 This figure shows the results of EGFP-labeled Serratia marcescens colonizing the intestine of Aedes aegypti after complementation.

[0040] Figure 2 Figure 1 shows the results of constructing the protein secretion system of Serratia marcescens. Figure A is a schematic diagram of plasmid construction, Figure B is the PCR molecular identification result of positive Serratia marcescens, Figure C is a schematic diagram of wild-type and fluorescent-expressing Serratia marcescens under a fluorescence microscope, and Figure D is the Western-blot identification result of the HasA protein secretion system expression.

[0041] Figure 3 A diagram showing the secretion results of the engineered Serratia marcescens antiviral protein.

[0042] Figure 4 This figure shows the results of in vitro virus neutralization by engineered Serratia marcescens.

[0043] Figure 5Virus neutralization results in the gut of Aedes aegypti after complementation with Serratia marcescens.

[0044] Figure 6 The figure shows the adaptive changes of Aedes aegypti after the engineered bacteria that secrete antiviral proteins and the wild-type bacteria are supplemented into the mosquito. Figure A shows the survival rate of Aedes aegypti after the supplementation, Figure B shows the blood-sucking rate of Aedes aegypti after the supplementation, Figure C shows the egg production of Aedes aegypti after the supplementation, and Figure D shows the hatching rate of Aedes aegypti after the supplementation. DETAILED DESCRIPTION

[0045] The technical solution of the present invention will be further described below with reference to the accompanying drawings and through specific embodiments. However, the following examples are merely simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

[0046] If no specific techniques or conditions are specified in the examples, the experiments were carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0047] In a specific embodiment of the present invention, the mosquito may be selected from Aedes aegypti (Liverpool), and the Serratia marcescens may be selected from Serratia marcescens CGMCC 1.12941.

[0048] Example 1

[0049] This example screens for symbiotic bacteria of Aedes aegypti.

[0050] Tissue samples from various organs of Aedes aegypti (intestines, ovaries, testes, etc.) were evenly spread on selective culture plates (LB medium: 5 g / L yeast extract, 10 g / L tryptone, 10 g / L sodium chloride, 20 g / L agar powder). Microbial pure culture technology was used to systematically isolate and purify the symbiotic bacterial strains in Aedes aegypti, and a symbiotic bacterial strain library of Aedes aegypti was constructed.

[0051] Based on the resources of isolated symbiotic strains of Aedes aegypti, by comparing and analyzing the composition of symbiotic bacteria from different organ sources, core symbiotic bacteria that are stably present in multiple organs were screened and identified, including Serratia marcescens, which was isolated in the intestine and other tissue parts of the insect body (excluding the intestine, ovaries, and testicles).

[0052] Example 2

[0053] This example constructs antiviral engineered bacteria.

[0054] The Serratia marcescens or Serratia marcescens CGMCC 1.12941 screened and identified in Example 1 was used as the starting strain, and the enhanced green fluorescent protein (EGFP) reporter gene was integrated into the genome of Serratia marcescens to construct a fluorescently labeled strain. The specific experimental process included:

[0055] 1. Construction of fluorescent plasmid

[0056] 1. Using pET28a-EGFP (EGFP gene sequence shown in SEQ ID NO. 4) as the plasmid backbone, the NPT II promoter was substituted for the T7 promoter in the pET28a-EGFP plasmid backbone to construct the plasmid pET28a-NPTII-EGFP. Seamless cloning was used for plasmid construction. Seamless cloning enzyme was purchased from Jinsha Biotechnology.

[0057] 2. Using pBBR1MCS-2 as the plasmid backbone, insert the NPTII-EGFP expression cassette into the multiple cloning site of pBBR1MCS-2 to construct the plasmid pBBR1MCS-NPTII-EGFP. Seamless cloning was used for plasmid construction. Seamless cloning enzyme was purchased from Jinsha Biotechnology.

[0058] 2. Preparation of Serratia marcescens Competent Cells

[0059] 1. Take frozen glycerol culture of Serratia marcescens, streak it on LB plate, and culture at 30℃ until single colonies grow.

[0060] 2. Pick a single colony and place it in 3 mL of LB liquid medium. Culture overnight at 30°C and 200 rpm.

[0061] 3. Take 1 mL of activated bacterial solution and transfer it to a flask containing 100 mL of LB liquid medium. Cultivate at 30°C and 200 rpm for 2 to 3 hours until the OD 600 =0.5.

[0062] 4. Transfer the aliquots to 50 mL centrifuge tubes and centrifuge at 4000 rpm for 15 min at 4°C to collect the cells. Discard the supernatant.

[0063] 5. Add 5 mL of pre-chilled 10% glycerol to fully resuspend the cells. Add an additional 35 mL of 10% glycerol and mix thoroughly. Centrifuge at 4000 rpm for 15 minutes at 4°C to collect the cells and discard the supernatant.

[0064] 6. Add 5 mL of pre-chilled 10% glycerol to fully resuspend the cells. Add an additional 20 mL of 10% glycerol and mix thoroughly. Centrifuge at 4000 rpm for 15 minutes at 4°C to collect the cells and discard the supernatant.

[0065] 7. Add 12.5 mL of pre-chilled 10% glycerol to fully resuspend the cells. Centrifuge at 4000 rpm for 15 minutes at 4°C to collect the cells and discard the supernatant.

[0066] 8. Add 4 mL of pre-cooled 10% glycerol and fully resuspend the cells (quantitate to 10 using a hemocytometer). 9 ), aliquot into 1.5 mL EP tubes, 100 μL / tube. Quickly freeze in liquid nitrogen and store at -80°C.

[0067] 3. Electroporation of Plasmids into Serratia

[0068] 1. Remove the frozen competent cells and thaw on ice. Add 100 ng of plasmid and mix gently by pipetting. Transfer to a pre-chilled 1 mm electroporation cuvette.

[0069] 2. Use GenePulserⅡ electroporator, set the voltage to 2.5kV, time to 5ms, and perform electric shock.

[0070] 3. Immediately add 1 mL of pre-cooled antibody-free LB, mix well, transfer to a 1.5 mL EP tube, and incubate at 30°C and 200 rpm for 4 h.

[0071] 4. Centrifuge at 2000×g for 5 minutes, discard 900 μL of supernatant, and resuspend the bacteria in the remaining liquid.

[0072] 5. Spread on LB (Kana) plates and culture at 30°C overnight.

[0073] 4. Identification and preservation of positive strains

[0074] The strain emitting green fluorescence was observed under a fluorescence microscope, and the strain was picked and placed in LB liquid culture medium, 1% kana antibiotic was added overnight, and finally 50% glycerol was added and stored at -80°C.

[0075] 5. By artificial sugar water feeding, fluorescently labeled Serratia marcescens in the logarithmic growth phase was mixed with 10% sucrose solution to adjust the concentration to 1×10 8 / mL. The replenishment time was 24 hours. The colonization dynamics of the tissues and organs of the replenished Aedes aegypti mosquitoes were observed using a fluorescence microscope (such as Figure 1 Intestinal observation results), and collected eggs of female Aedes aegypti supplemented with fluorescent bacteria until they hatched into adult mosquitoes and tracked them to the next generation, systematically studied their vertical transmission characteristics in the host, and finally determined Serratia marcescens as the target strain for the transformation of engineered symbiotic bacteria.

[0076] Using EGFP as a visual reporter gene (SEQ ID NO. 4), the HasA protein secretion system was selected as a vector, which was constructed into an expression plasmid vector through genetic engineering methods. The recombinant plasmid was introduced into Serratia marcescens using electroporation transformation technology. The specific experimental process included:

[0077] 1. Construction of protein secretion plasmid

[0078] Schematic diagram of plasmid construction Figure 2 As shown in Figure A, pBBR1MCS-NPTII-EGFP was used as the plasmid backbone, and the HasA nucleotide sequence (SEQ ID NO. 5) was inserted downstream of the pBBR1MCS-NPTII-EGFP plasmid to construct the plasmid pBBR1MCS-NPTII-EGFP-HasA. Seamless cloning was used for plasmid construction. Seamless cloning enzyme was purchased from Jinsha Biotechnology.

[0079] SEQ ID NO.5:

[0080] .

[0081] 2. Preparation of Serratia marcescens Competent Cells

[0082] Refer to the above method for preparing Serratia marcescens competent cells.

[0083] 3. Electroporation of Plasmids into Serratia

[0084] Refer to the above method for preparing Serratia marcescens competent cells.

[0085] 4. Identification and preservation of positive strains

[0086] The strain emitting green fluorescence was observed under a fluorescence microscope, and the strain was picked and placed in LB liquid culture medium, 1% kana antibiotic was added overnight, and finally 50% glycerol was added and stored at -80°C.

[0087] 5. Using Western-blot protein immunoblotting technology, the extracellular secretion of EGFP green fluorescent protein was qualitatively and quantitatively analyzed. The results are as follows: Figure 2 As shown, Figure B is the PCR molecular identification result of the engineered bacteria, wherein lane 1 is the plasmid PCR as a positive control, lane 2 is the engineered bacteria PCR as an experimental group, and lane 3 is the wild-type bacteria PCR as a negative control. The results show that the plasmid can enter the Serratia marcescens mixture through electroporation; Figure C shows the observation results of the engineered bacteria and wild-type bacteria expressing fluorescent protein under a fluorescence microscope, wherein Serratia marcescens (Sm) WL-2 is the engineered bacteria, and WT is the wild-type Serratia marcescens. The results show that the green fluorescent protein is successfully expressed in Serratia marcescens and shows green fluorescence under a fluorescence microscope; Figure D shows the detection result of the protein secretion system of Serratia marcescens, wherein Sm WL-2 is an engineered bacteria with a plasmid carrying a fluorescent protein sequence and a HasA protein secretion system, Sm WL-1 is an engineered bacteria with a plasmid carrying only a green fluorescent protein sequence, and Sm WL is the wild-type Serratia marcescens, Sup refers to the supernatant of the strain culture to verify the secreted protein, and the cell body after the strain is centrifuged. The results show that under the conditions of the HasA protein secretion system, green fluorescent protein (EGFP) can be secreted out of the cell as a secreted protein in Serratia marcescens, and Pre refers to the cell body sediment (intracellular protein) after the bacteria are centrifuged. The EGFP gene was replaced with the antiviral protein encoding gene (SEQ ID NO.2), and the electroporation technology was used again to obtain an engineered Serratia marcescens strain that can stably secrete antiviral proteins. The verification results are as follows Figure 3 As shown, Sup is the bacterial culture supernatant, and Pre is the cell body after centrifugation of the bacterial culture. The results show that antiviral proteins can be secreted extracellularly in Serratia marcescens.

[0088] Example 3

[0089] This example identifies the antiviral effect of antiviral engineered bacteria.

[0090] In vitro and in vivo antiviral activity evaluations were carried out respectively.

[0091] In vitro experiments used techniques such as cytopathic inhibition assay to detect the direct inhibitory effect of engineered bacteria on dengue virus. The specific experimental process included:

[0092] The frozen engineered bacteria were aspirated and placed in 5 mL of LB liquid medium. The culture was carried out at 37°C and 220 rpm overnight. Wild bacteria were used as a control.

[0093] 1. Inoculate hamster kidney cells (BHK-21 cells) into 96-well plates and culture with DMEM (10% FBS, 1% P / S) until the confluence reaches 70%-80%.

[0094] 2. Centrifuge the overnight culture at 4000 rpm for 10 minutes, filter the supernatant through a 0.22 filter membrane, and then mix according to the ratio in Table 1 to obtain a mixture.

[0095] Table 1

[0096] DMEM medium (supplemented with 2% FBS) 60μL Bacterial supernatant 9μL <![CDATA[Dengue virus DENV-II (1×10 8 PFU / mL)]]> 1 μL

[0097] 3. Add 70 μL of the mixture to each well containing BHK-21 cells, incubate at 37°C for 1.5 h, and then wash twice with PBS.

[0098] 4. Culture in DMEM (10% FBS, 1% P / S) for 1.5 days.

[0099] 5.1.5 days later, wash twice with PBS.

[0100] 6. Fix with methanol (5% acetone) for 20 min.

[0101] 7. Wash three times with PBS.

[0102] 8. Dilute the primary antibody (anti-flavivirus E protein) at 1:5000 and incubate for 2 h.

[0103] 9. Wash 3 times with PBS.

[0104] Dilute the secondary antibody at 1:10,000 and incubate for 1 hour.

[0105] 11. Wash 3 times with PBS.

[0106] 12. Add 100 μL of TMB.

[0107] 13. Detect OD with microplate reader 450 .

[0108] The results are as follows Figure 4 As shown, the intracellular OD of the engineered Serratia marcescens group was compared with that of the wild type group. 450 The lower values ​​indicated that the amount of intracellular DENV-II was reduced and the supernatant of engineered Serratia marcescens could neutralize dengue virus particles.

[0109] In vivo experiments artificially supplemented engineered bacteria into Aedes aegypti mosquitoes, using unmodified wild-type Serratia marcescens as a negative control and engineered Serratia marcescens that secreted antiviral proteins as an experimental group. Molecular biological techniques such as real-time fluorescence quantitative PCR were used to detect the level of viral replication and infection intensity in mosquitoes, comprehensively evaluating the antiviral effect of the engineered symbiotic bacteria. The specific experimental process includes:

[0110] In vivo testing:

[0111] 1. OD 600 = 1 LB bacterial solution: 10% sucrose solution in a ratio of 1:9, and placed in a small cup for feeding. The solution was changed once a day and fed to female mosquitoes that were 4-6 days old. The feeding lasted for 7 days.

[0112] 2.7 days later, goat blood containing DENV-II (virus titer of 1×10 7 PFU / mL) were used for blood feeding on the membrane, and then the blood-satiated mosquitoes were taken for further breeding.

[0113] 3. Dissect mosquito midguts 4 dpi after blood meal.

[0114] The viral copy number results in the mosquito midgut are as follows Figure 5 As shown, the number of virus particles was significantly reduced after the engineered bacteria were replenished, indicating that the engineered bacteria replenishment could play an antiviral role.

[0115] Example 4

[0116] This example tests the adaptive cost of antiviral engineered bacteria to Aedes aegypti.

[0117] Two experimental groups were set up: a blank control group (S.mWL-2(-)) fed sugar water without Serratia marcescens WL-2, and an experimental group (Sm WL-2(+)) fed sugar water containing Serratia marcescens WL-2. Through standardized artificial feeding methods, different strains were inoculated into Aedes aegypti mosquito populations. Multiple physiological indicators of Aedes aegypti were systematically monitored, including survival rate, blood-feeding rate, egg production, and hatching rate, to deeply evaluate the effects of the engineered symbionts on the host mosquito's adaptability.

[0118] The results are as follows Figure 6Figure 1 shows the effects of Serratia marcescens WL-2 on mosquito lifespan. Mosquito survival was monitored daily. Percentages of survival are the means of three biological replicates (100 mosquitoes per replicate, mean ± standard deviation). Statistical significance of survival curves was analyzed using the log-rank test (Mantel-Cox test). Figure 1 shows the effects of Serratia marcescens WL-2 on mosquito blood-feeding behavior. Percentages are the means of three biological replicates (50 mosquitoes per replicate, mean ± standard deviation). No significant differences were detected between groups (Student's t-test). Figures 1 and 2 show the effects of Serratia marcescens WL-2 on mosquito fecundity and reproductive behavior. Figure 2 shows the number of eggs laid by each female Aedes aegypti mosquito collected three days after treatment (mean ± standard deviation); Figure 3 shows the egg hatching rate (mean ± standard deviation). Fertility data are the means of three biological replicates (20 mosquitoes per replicate). No significant differences were detected between groups (Mann-Whitney test). This indicates that the antiviral engineered bacteria designed by the present invention have no significant effect on the adaptability of the host mosquito.

[0119] In summary, the present invention achieves breakthroughs in visual tracking and mechanism research through genetic engineering of symbiotic bacteria and incorporation of reporter genes, designs precise and efficient prevention and control strategies, utilizes the vertical transmission characteristics of heritable symbiotic bacteria, achieves long-lasting antiviral effects, improves targeting and eco-friendliness, conducts in vitro and in vivo testing and physiological indicator monitoring, and ensures application from multiple dimensions to ensure the feasibility and safety of the solution, opening up new avenues for mosquito-borne disease prevention and control.

[0120] The applicant declares that the above is only a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention fall within the scope of protection and disclosure of the present invention.

Claims

1. A method for antiviral treatment in mosquito insects, characterized in that: The antiviral method in mosquito insects comprises: Isolate the symbiotic bacteria in mosquito insects, take the symbiotic bacteria present in multiple organs of mosquito insects, screen the symbiotic bacteria to obtain candidate strains, overexpress antiviral proteins in at least one of the candidate strains to obtain symbiotic engineered bacteria, and replenish the symbiotic engineered bacteria into the body of the mosquito insect.

2. The antiviral method in mosquito insects according to claim 1, characterized in that: The mosquito family includes Aedes; Preferably, the Aedes mosquito includes at least one of Aedes aegypti, Aedes albopictus or Aedes flavipictus.

3. The antiviral method in mosquito insects according to claim 1 or 2, characterized in that: The candidate strains include at least one of Serratia marcescens, Asiaticus, Elizabethella or Flavobacterium.

4. The antiviral method in mosquito insects according to any one of claims 1 to 3, characterized in that: The virus includes at least one of dengue virus, Zika virus or chikungunya virus.

5. The antiviral method in mosquito insects according to any one of claims 1 to 4, characterized in that: The screening includes overexpressing fluorescent protein in the symbiotic bacteria and replenishing them into the bodies of mosquito insects, using a fluorescence microscope to observe the colonization dynamics of various tissues and organs of the replenished mosquito insects, and tracking them to the next generation to select symbiotic bacteria that can be vertically transmitted in mosquito insects.

6. Application of Serratia marcescens in preventing and controlling virus transmission in mosquito insects.

7. An antiviral engineered bacterium, characterized in that: The antiviral engineered bacteria is Serratia marcescens that overexpresses antiviral proteins; Preferably, the virus comprises at least one of dengue virus, Zika virus or chikungunya virus.

8. A composition, characterized in that The composition comprises the antiviral engineered bacteria according to claim 7.

9. The composition according to claim 8, characterized in that The composition further comprises a biologically acceptable carrier; Preferably, the biologically acceptable carrier comprises at least one of a culture medium, a bacterial growth agent, a wetting agent, a penetrant, a dispersant, an emulsifier, a stabilizer, an adhesive or a filler.

10. A method for preventing and controlling virus transmission in mosquito insects, characterized in that: The method comprises: replenishing the antiviral engineered bacteria according to claim 7 or the composition according to claim 8 into the body of mosquito insects.

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