Insect specific virus NLBV3 and application of insect specific virus NLBV3 in reducing transmission of rice odontodwarf virus of brown planthopper

Abstract

The invention belongs to the field of biological prevention and control, and discloses an insect specific virus NLBV3 and application thereof in reducing transmission of rice odontodwarf virus of brown planthopper. The NLBV3 virus genome comprises three chains, the L chain encodes RNA dependent RNA polymerase (RdRP), the M chain encodes glycoprotein precursor (GP), the S chain encodes nucleoprotein (NP), and the nucleotide sequences are respectively shown as SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3; the application is realized by releasing the NLBV3-carrying brown planthopper population, and according to the screening and detection method of the NLBV3-carrying brown planthopper high-virus population provided by the invention, natural brown planthoppers can be effectively infected after the NLBV3-carrying brown planthopper high-virus population is released, the RRSV transmission capability of the brown planthoppers is reduced, and the virus is prevented from damaging rice production. The NLBV3 provided by the invention is an insect specific virus, is harmless to mammals, fishes and shrimps, natural enemy insects and pollination insects, and has the advantages of specific action object, environment friendliness and the like.

Description

Technical Field

[0001] This invention belongs to the field of biological control and relates to the identification of an insect-specific virus NLBV3 of the brown planthopper, the establishment of highly infected populations and its application. Specifically, it relates to an insect-specific virus NLBV3 and its application in reducing the transmission of rice tooth dwarf virus by the brown planthopper. Background Technology

[0002] Brown planthopper ( Nilaparvata lugens Brown planthopper (Rhizoctonia solani) is a piercing-sucking pest belonging to the family Planthopperidae in the order Hemiptera. It is one of the most important pests in rice-producing areas of Asia, exhibiting a distinct seasonal migratory pattern. It directly damages rice by sucking sap from the phloem, laying eggs, and secreting honeydew. It can also indirectly damage rice by transmitting plant viruses such as rice tooth-leaf dwarf virus (RRSV) and rice grass dwarf virus (RGSV). RRSV, belonging to the genus Ricevirus in the family Reoviridae, causes notched leaf margins, stunting, and increased tillering in rice plants, often preventing them from heading and producing grains. It can proliferate persistently within the brown planthopper, causing significant damage to rice. Currently, the control of rice tooth-leaf dwarf virus mainly relies on chemical control of the brown planthopper. However, the overuse of pesticides pollutes the environment and induces pest resistance, making this approach unsustainable. Therefore, there is an urgent need to develop new green control strategies.

[0003] Insect-specific viruses (ISVs) are a class of viruses that can only replicate within insects and their cell lines, and cannot multiply in vertebrates or plants. With the development of sequencing technology and vector-borne virus surveillance, a large number of ISVs have been identified in various insects, covering multiple groups such as Flaviviridae, Rhabdoviridae, Reoviridae, and Iflaviridae. Studies have shown that ISVs can affect the development, reproduction, lifespan, sex ratio, and immunity of host insects, with effects that can be beneficial or harmful depending on environmental regulation. Some ISVs can also inhibit the transmission of vector-borne viruses by competing for resources, activating immunity, or interacting with viral proteins; for example, PCV and NHUV significantly inhibit the replication and transmission of various mosquito-borne flaviviruses. In terms of application prospects, ISVs can be used as biocontrol tools, leveraging the Wolbachia inhibitory effect to directly or engineer the weakening of vector-borne virus transmission. Utilizing their host-limiting characteristics, safe recombinant vaccine vectors can be constructed (such as EILV chimeras protecting mice from CHIKV and VEEV infection) and highly efficient diagnostic reagents can be developed. Their unique biology and safety make ISVs of great potential in agricultural pest management, vaccine development and viral ecology research.

[0004] Several ISVs have been identified in brown planthoppers, including the double-stranded RNA virus NLRV (Fijivirus, family Reoviridae), the single-stranded positive-strand RNA virus HiPV (bicistronicoviridae), NLHV1-3 (which can be horizontally transmitted via honeydew) from the Iflaviridae family, NlCV (which has both horizontal and vertical transmission potential) from the bicistronicoviridae family, and the newly discovered segmented positive-strand RNA virus NLQV1 (a Quenyavirus group, associated with various insect "dark viruses"). The presence and transmission mechanisms of these ISVs in brown planthopper populations suggest that they may regulate the accumulation and transmission of plant viruses in vector insects through competition for host resources, activation of immunity, or interviral interactions. This indicates the potential of using ISVs to construct biocontrol strategies, and further elucidation of their ecological functions and application prospects is warranted.

[0005] This patented invention, through high-throughput sequencing technology and transcriptome analysis, identified an insect-specific virus—Nilaparvata lugens Bunyavirus 3 (NLBV3)—that affects the transmission of RRSV in brown planthoppers. By vertically transmitting NLBV3 through infected females, a large number of progeny carrying NLBV3 are produced, thereby inhibiting the brown planthopper's ability to transmit RRSV and mitigating the damage caused by RRSV to rice. This provides a new approach for the green control of brown planthoppers and rice tooth dwarf virus, and offers a theoretical basis for better development and utilization of insect microbial resources. Summary of the Invention

[0006] This invention identifies NLBV3, an ISV of the Phaneroviridae family, in brown planthoppers through high-throughput sequencing and transcriptome analysis. The complete genome sequence of NLBV3 is cloned. By releasing brown planthopper populations carrying NLBV3, the natural population is infected through vertical transmission. The efficiency of brown planthoppers carrying NLBV3 in acquiring and spreading RRSV is significantly reduced, ultimately achieving the goal of controlling rice toothed leaf dwarf virus RRSV.

[0007] To achieve the above objectives, the present invention provides the following technical solution: In a first aspect, the present invention provides an insect-specific virus, Nilaparvata lugens Bunyavirus 3 (NLBV3), the genome of which comprises three strands: the L strand encodes an RNA-dependent RNA polymerase (RdRP), the M strand encodes a glycoprotein precursor (GP), and the S strand encodes a nucleoprotein (NP). The L-chain, whose nucleotide sequence is shown in SEQ ID NO:1; The M chain, whose nucleotide sequence is shown in SEQ ID NO:2; The S chain, with its nucleotide sequence, is shown in SEQ ID NO:3.

[0008] Preferably, the protein encoded by NLBV3 is: The L chain encodes an RNA-dependent RNA polymerase (RdRP), the amino acid sequence of which is shown in SEQ ID NO:4; The M chain encodes a glycoprotein precursor (GP), the amino acid sequence of which is shown in SEQ ID NO:5; The S chain encodes a nucleoprotein (NP), the amino acid sequence of which is shown in SEQ ID NO:6.

[0009] In a second aspect, the present invention provides an application of the insect-specific virus NLBV3 described in the first aspect in reducing the transmission of rice toothed leaf dwarf virus (RRSV) by brown planthoppers. The application is achieved by releasing brown planthopper populations carrying NLBV3 to inhibit the transmission of rice toothed leaf dwarf virus by brown planthoppers.

[0010] Preferably, the method for constructing the population includes: (a) Screening for female brown planthoppers carrying NLBV3 (♀+); (b) The female (♀+) was mated with the male and the offspring population with 100% NLBV3 infection rate was obtained through vertical transmission of eggs.

[0011] Preferably, the male insect is a male carrying NLBV3 (♂+) or a male not carrying NLBV3 (♂−).

[0012] In a third aspect, the present invention provides a primer set for specifically amplifying the NLBV3 viral genome described in the first aspect, comprising the primers shown in SEQ ID NO:7 to SEQ ID NO:24.

[0013] In a fourth aspect, the present invention provides a primer set for detecting the NLBV3 viral genome described in the first aspect, comprising the primers shown in SEQ ID NO:25 to SEQ ID NO:30.

[0014] Fifthly, the present invention provides a brown planthopper strain carrying NLBV3, wherein the NLBV3 genome has the nucleotide sequences shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3.

[0015] The strain was obtained by microinjecting NLBV3 into female brown planthoppers to obtain females carrying NLBV3 (♀+), and then mating the females carrying NLBV3 (♀+) with males to obtain offspring with a 100% NLBV3 virulence rate.

[0016] Compared with the prior art, the beneficial effects of the present invention are: This invention discloses an insect-specific virus NLBV3 of the brown planthopper. By releasing brown planthopper populations carrying NLBV3 into natural populations, the ability of brown planthoppers to transmit rice toothed dwarf virus RRSV can be significantly reduced, thus decreasing the susceptibility rate of rice. This indicates that NLBV3 can effectively inhibit the transmission of rice toothed dwarf virus RRSV by brown planthoppers, and has important value and good application prospects in the biological control of brown planthoppers and rice toothed dwarf virus RRSV. Attached Figure Description

[0017] Figure 1 Phylogenetic analysis of NLBV3 in brown planthopper; Figure 2 For the identification of NLBV3 in the brown planthopper; Figure 3 NLBV3 is mainly transmitted vertically through infected females in brown planthopper populations. Figure 4 The effect of NLBV3 on the acquisition and spread of RRSV by brown planthoppers is shown in (A) for the efficiency of brown planthoppers in acquiring RRSV and (B) for the efficiency of brown planthoppers in spreading RRSV. Detailed Implementation

[0018] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that, unless otherwise specified, the following embodiments and features described therein can be combined with each other.

[0019] Example 1: Amplification of the whole genome sequence of NLBV3 of the brown planthopper

[0020] (1) After stunning several brown planthoppers by freezing, add 1 ml of RNAiso plus and grind thoroughly; let stand at room temperature for 5 min, centrifuge at 12000 rpm and 4℃ for 5 min, and take the supernatant; add 200 μL of chloroform, shake vigorously for 30 s, let stand for 10 min, centrifuge again for 10 min, and take the upper aqueous phase; add an equal volume of isopropanol, mix well, let stand for 20 min, centrifuge at 12000 rpm and 4℃ for 10 min, and discard the supernatant; wash twice with 75% ethanol, centrifuge at 12000 rpm and 4℃ for 5 min each, centrifuge empty for 2 min, and discard the supernatant; open the lid and dry for 20 min, dissolve in DEPC water, detect with NanoDrop, and store at −80℃ for later use.

[0021] (2) Using total RNA extracted from brown planthoppers as a template, first-strand cDNA was synthesized according to HiScript II Q RT SuperMix for qPCR (+gDNA). In an RNase-free centrifuge tube, 2 μL of 4×gDNA wiper mix and 1 μg of total RNA were prepared, and RNase-free ddH2O was added to a final volume of 8 μL. After mixing, the tube was placed in a PCR instrument and the reaction conditions were set at 42℃ for 2 min to remove gDNA. Subsequently, 2 μL of 5×HiScript II qRT SuperMixII was added to the RNase-free centrifuge tube, gently mixed with a pipette, and collected by brief centrifugation. The tube was then placed in a PCR instrument and the reaction conditions were set as follows: 50℃ for 15 min; 85℃ for 5 s for reverse transcription. The first-strand cDNA product could be directly used for RT-PCR.

[0022] (3) Primers were designed based on the three sequences (L, M, S) of Bunyavirus NLBV3 obtained from high-throughput sequencing and transcriptome analysis of brown planthopper (Table 1). The primers were synthesized by Hangzhou Youkang Biotechnology Co., Ltd.

[0023] Table 1. Primers for NLBV3 gene amplification

[0024] (4) Using brown planthopper cDNA as a template, the whole genome of NLBV3 was amplified by PCR using the primers shown in Table 1. The specific reaction system consisted of 2 × Phanta Max Buffer 25 μL, dNTP Mix 1 μL, Forward primer (10 μM) 2 μL, Reverse primer (10 μM) 2 μL, Phanta Max Super-Fidelity DNA Polymerase 1 μL, Template cDNA 2 μL and ddH2O 17 μL. The PCR reaction program was as follows: pre-denaturation 95℃, 3 min; denaturation 95℃, 15 sec; annealing 60℃, 15 sec; extension 72℃, 1 kb / min; 32 cycles; and final extension 72℃, 5 min. The gel-recovered products were sent to Hangzhou Youkang Biotechnology Co., Ltd. for sequencing. The sequencing products were sequenced and assembled using software such as NCBI BLAST, SnapGene, and DNAStar. Finally, the complete genome sequence of NLBV3 virus containing three strands, L (6701bp), M (2582bp), and S (3079bp), was obtained. The obtained sequences are shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively.

[0025] Example 2: Phylogenetic analysis of the brown planthopper NLBV3

[0026] (1) Predict the ORFs of three sequences (L, M, S) using NCBI ORFfinder (https: / / www.ncbi.nlm.nih.gov / orffinder / ).

[0027] (2) In order to assess the taxonomic status of NLBV3, a phylogenetic tree was constructed using the maximum likelihood (ML) method based on the RdRp amino acid sequences of some viruses of Phasmaviridae, Phenuiviridae, Hantaviridae and Tospoviridae.

[0028] (3) The RdRP sequences were aligned using MAFFT (7.450) (Katoh and Standley, 2013), and gaps were pruned using Gblock. The alternative model was evaluated using Model Test-NG. Then, an ML tree was constructed using RAxML-NG (0.9.0), validated using bootstrap, and iterated 1000 times. The results are as follows: Figure 1 As shown, NLBV3 belongs to the genus Orthophanivirus in the family Phaneroviridae.

[0029] Example 3: Detection method and determination of vertical transmission of brown planthopper NLBV3 1. Detection of NLBV3 virus Using the NLBV3 virus sequence as a reference, suitable primers (Table 2) were designed to amplify the target band for NLBV3 identification. NLBV3 was identified in 24 brown planthoppers. The identification results showed that the sequence lengths of the L (RdRP), M (GP), and S (NP) chains were 1187 bp, 324 bp, and 1611 bp, respectively. Figure 2 Primers SEQ ID NO.25—SEQ ID NO.30 were synthesized by Hangzhou Youkang Biotechnology Co., Ltd.

[0030] Table 2. Primers for NLBV3 virus detection

[0031] 2. Determination of vertical transmission of NLBV3 in brown planthoppers (1) Collect brown planthoppers at the end of the 5th instar and carrying NLBV3. Feed the males and females separately in different glass tubes to avoid premature mating.

[0032] (2) After the initial emergence, single female and male insects are placed in a clean glass tube and mated and laid eggs for 10 days.

[0033] (3) Two combinations were set: ♀+ / ♂− and ♀− / ♂+; “+” indicates brown planthoppers carrying NLBV3, and “-” indicates brown planthoppers not carrying NLBV3. The offspring were fed to the 3rd instar and then tested. The test primers are shown in Table 2.

[0034] (4) Five biological replicates were set up for each combination, and approximately 10 progeny brown planthoppers were randomly selected from each replicate, meaning approximately 50 progeny plantshoppers were tested for each combination. The test results are as follows: Figure 3 As shown, 100% of the offspring of females carrying NLBV3 carried the virus. This indicates that NLBV3 is mainly transmitted vertically within the brown planthopper population through infected females.

[0035] Example 4: Determination of the ability of brown planthoppers carrying NLBV3 to acquire and transmit the virus. (1) Select about 30 second-instar nymphs of brown planthopper carrying NLBV3. Place the insects in a cage planted with RRSV virus for 5 days to complete the acquisition process of RRSV virus.

[0036] (2) After feeding, the brown planthoppers were transferred to fresh, non-toxic rice seedlings and raised for 10 days. After the cycle period, single-tube, single-seedling, and single-insect virus transmission experiments were conducted (each insect was in contact with a single rice seedling for 5 days to transmit the virus), and the finger tubes and rice plants were marked with numbers.

[0037] (3) After the virus transmission was completed, all brown planthoppers were collected, RNA was extracted from single brown planthoppers, and the first strand of cDNA was synthesized. Then, the RRSV target gene was amplified using cDNA as a template. The primers used, SEQ ID NO.31 and SEQ ID NO.32, are shown in Table 3. The primers were synthesized by Hangzhou Youkang Biotechnology Co., Ltd.

[0038] Table 3. Primers for RRSV virus identification

[0039] (4) Continue to cultivate rice that has been fed on by brown planthoppers, keep the numbering, and collect rice seedling leaves about 14 days later to test and confirm the RRSV infection status of each seedling.

[0040] (5) Calculate the efficiency of brown planthoppers in acquiring RRSV and the efficiency of RRSV transmission: Acquisition rate (%) = number of brown planthoppers carrying RRSV / total number of brown planthoppers detected × 100; Transmission rate (%) = number of rice plants infected with RRSV / number of rice plants consumed by brown planthoppers × 100. The experimental results are as follows: Figure 4 As shown.

[0041] The results showed that the infection rate of brown planthoppers carrying NLBV3 was 24%, lower than that of brown planthoppers without NLBV3 (50%). Furthermore, the efficiency of brown planthoppers carrying NLBV3 in transmitting RRSV was 0%, significantly lower than that of brown planthoppers without NLBV3 (23.1%).

[0042] Overall, NLBV3 reduced the efficiency of brown planthoppers in acquiring and spreading RRSV, with RRSV transmission being significantly inhibited.

[0043] The embodiments described above are merely illustrative of specific implementations of the present invention, and while the descriptions are detailed, they should not be construed as limiting the scope of protection of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. An insect-specific virus NLBV3, characterized in that, The genome of the NLBV3 comprises: an L chain, the nucleotide sequence of which is shown as SEQ ID NO: 1; an M chain, the nucleotide sequence of which is shown as SEQ ID NO: 2; an S chain, the nucleotide sequence of which is shown as SEQ ID NO:

3.

2. The insect-specific virus NLBV3 according to claim 1, characterized in that: The NLBV3 encodes proteins as follows: the L chain encodes an RNA-dependent RNA polymerase, the amino acid sequence of which is shown as SEQ ID NO: 4; the M chain encodes a glycoprotein precursor, the amino acid sequence of which is shown as SEQ ID NO: 5; the S chain encodes a nucleoprotein, the amino acid sequence of which is shown as SEQ ID NO:

6.

3. Use of the insect-specific virus NLBV3 according to claim 1 for reducing the transmission of Rice Leaf Beetle Dwarf Virus by Nilaparvata lugens, characterized in that, The application is achieved by releasing a NLBV3-carrying population of the brown planthopper.

4. Use according to claim 3, characterized in that, The method for constructing the population comprises: (a) screening NLBV3-carrying female brown planthoppers (♀+); (b) mating the female brown planthoppers (♀+) with male brown planthoppers to obtain a NLBV3-carrying population with a NLBV3 infection rate of 100% through vertical transmission of eggs.

5. Use according to claim 4, characterized in that, The male brown planthoppers in step (b) are NLBV3-carrying male brown planthoppers (♂+) or NLBV3-non-carrying male brown planthoppers (♂-).

6. A primer set for specifically amplifying the NLBV3 viral genome according to claim 1, characterized in that, The primers comprise SEQ ID NO: 7 to SEQ ID NO:

24.

7. A primer set for detecting the NLBV3 virus genome according to claim 1, characterized by, The primers comprise SEQ ID NO: 25 to SEQ ID NO:

30.

8. A brown planthopper strain carrying the virus NLBV3, characterized in that, The genome of the NLBV3 is the nucleotide sequence shown as SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO:

3.

9. The NLBV3-carrying brown planthopper strain according to claim 8, wherein: the strain is obtained by microinjecting NLBV3 into a female brown planthopper to obtain a NLBV3-carrying female brown planthopper (♀+), and mating the NLBV3-carrying female brown planthopper (♀+) with a male brown planthopper to obtain a NLBV3-carrying population with a NLBV3 infection rate of 100%.

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