Panax notoginseng MYB transcription factor gene PnMYB34 and application thereof

Abstract

This invention discloses a Panax notoginseng MYB transcription factor gene. PnMYB34 Its nucleotide sequence is as described in SEQ ID NO:1, encoding the MYB transcription factor. This invention confirms through molecular biology and functional genomics related technologies. PnMYB34 The gene has the ability to enhance the plant's resistance to viral infection, and the present invention will... PnMYB34 Genes were constructed into plant expression vectors and overexpressed in Panax notoginseng and tobacco, respectively. Experimental results showed that overexpression... PnMYB34 Genetically modified Panax notoginseng against Panax notoginseng A virus (GMO) Panax notoginseng virus A The resistance to ) was significantly enhanced, and overexpression PnMYB34 Genetically modified tobacco is susceptible to tobacco mosaic virus (BMP). Tobacco mosaic virus Its resistance was also significantly enhanced.

Description

Technical Field

[0001] This invention belongs to the fields of molecular biology and genetic engineering, and specifically relates to a Panax notoginseng MYB transcription factor gene with antiviral infection capabilities. PnMYB34 And its applications. Background Technology

[0002] Plant viral diseases are the second largest plant disease after fungal diseases, causing serious economic losses to agricultural production in countries around the world and becoming one of the most difficult plant diseases to control in agricultural production worldwide (Xiao Qinzhi et al. Research progress on biological control of plant viral diseases. Southern Agriculture, 2021, 15(34): 64-69). After long-term research, a relatively mature integrated control system for fungal diseases has been formed, including chemical fungicides, disease-resistant variety breeding, and cultivation management. However, plant viruses are obligate pathogens that parasitize living cells, and their replication and movement depend entirely on the metabolic system of the host plant. In addition, plants lack adaptive immune mechanisms similar to those of higher animals and cannot produce specific antibodies and immune memory, so once a virus invades, it can spread rapidly in the plant and cause systemic damage, making control extremely difficult (Anikina I, Kamarova A, Issayeva K, et al. Plant protection from virus: a review of different approaches. Frontiers in Plant Science, 2023, 14: 1163270).

[0003] Sanqi [ Panax notoginseng (Burk.) FH Chen] belongs to the genus Panax (Araliaceae). Panax This perennial herb is one of the most distinctive Chinese medicinal herbs in Yunnan Province. Its rhizome is used medicinally, possessing various effects such as relieving pain and swelling, stopping bleeding and dispersing blood stasis, enhancing immunity, and protecting the cardiovascular system (Xu Y, Zhu MJ, Feng YB, et al.). Panax notoginseng-microbiota interactions: From plant cultivation to medicinal application. Phytomedicine, 2023, 119: 154978). Modern phytochemical studies have shown that the main active components of Panax notoginseng are saponins, flavonoids, polysaccharides, etc. Among them, Panax notoginseng saponins have anti-inflammatory, antioxidant, and cardiovascular disease treatment effects (Guo Xi et al. Research progress on the components and clinical pharmacological effects of Panax notoginseng saponins. Journal of Nanjing University of Traditional Chinese Medicine, 2024, 40(09): 985-992). Panax notoginseng requires long-term shade cultivation during its growth process, and the resulting warm and humid environment creates favorable conditions for the infection and spread of pathogens, especially viral diseases, which have now become one of the main diseases of Panax notoginseng.

[0004] Viral infection can significantly reduce the height, stem diameter, leaf length, leaf width, and fresh weight of Panax notoginseng plants, thereby decreasing the yield and quality of Panax notoginseng medicinal materials. Viruses that infect Panax notoginseng include cucumber mosaic virus (Cucumber mosaic virus). Cucumber mosaic virus , CMV), Tomato Mosaic Virus (CMV), Tomato mosaic virus , ToMV), Tomato Mottle Virus ( Tomato spotted wilt virus TSWV), Panax notoginseng Y virus ( Panax virus Y PnVY), Chinese tomato yellow leaf curl virus ( Tomato yellow leaf curl China virus , TYLCCNV) and its satellites ( Tomato yellow leaf curl China betasatellite , TYLCCNB), Panax notoginseng A virus ( Panax notoginseng virus A , PnVA and tobacco torsion virus (PnVA) ... Tobacco vein distorting virus , TVDV and other viruses (Chen HJ, Li WY, Chen XH, et al. Viral infections inhibit saponin biosynthesis and photosynthesis in) Panax notoginseng . Plant Physiology and Biochemistry, 2023, 203: 108038).

[0005] Transcription factors play a role in plant innate immunity by regulating pathogen-associated molecular patterns, effector-triggered immunity, hormone signaling pathways, and genes related to the synthesis of phytoalexins (Seo E, Choi D, Choi. Functional studies of transcription factors involved in plant defenses in the genomics era. Briefings in Functional Genomics, 2015, 14(4): 260-267). MYB is one of the largest transcription factor families in plants, and its identification and functions have been extensively studied in plants. Its roles include regulating secondary metabolism, controlling plant development, and participating in the physiological and biochemical processes of responses to biotic and abiotic stresses (Chen X, Mao YC, Chai WG, et al. Genome-wide identification and expression analysis of MYB gene family under nitrogen stress in Panax notoginseng . Protoplasma, 2023, 260(1): 189-205). MYB transcription factors are divided into four major subfamilies based on the number of adjacent repetitive sequences in the MYB domain: R1-MYB, R2R3-MYB, 3R-MYB, and 4R-MYB (Stracke R, Werber M, Weisshaar B. The R2R3-MYB gene family in Arabidopsis thaliana Current Opinion in Plant Biology, 2001, 4(5): 447-456). Among them, R2R3-MYB transcription factors are the most numerous subclass in plants and have been identified in many plant species. A total of 437 R2R3-MYB gene family members have been identified in blueberries (Wang HY, Zhai LL, Wang SW, et al. Identification of R2R3-MYBThe family in blueberry and its potential involvement of anthocyanin biosynthesis in fruits. BMCGenomics, 2023, 24(1): 505); 131 R2R3-MYB gene family members were identified in sweet potatoes (Li MX, Zhou YP, Li KF, et al. Genome-Wide comparative analysis of the R2R3-MYB gene family in six Ipomoea species and the identification of anthocyanin-related members in sweet potatoes. Plants (Basel), 2023, 12(8): 1731).

[0006] Transcription factors regulate the defense network in plant-pathogen interactions and are key targets for genetic engineering to enhance crop stress resistance. In terms of antifungal activity, in apples, MdMYB54 interacts with MdERF114 to synergistically activate cell wall synthesis-related genes, thereby enhancing resistance to Fusarium solani (Tomato rot fungus). Fusarium solani Physical defense resistance (Liu QW, Chen X, Li SJ, et al. MdMYB54 reduces disease severity caused by) Fusarium solani In apples, the transcription factor SmMYB44 modulates cell wall cellulose and pectate lyase-dependent defense. (The Plant Journal, 2025, 121(2): e17206). Regarding antibacterial activity, SmMYB44 activates the spermine synthase gene. SmSPDS The expression of [something] promotes spermidine accumulation, thereby enhancing the eggplant's resistance to Ralstonia solanacearum (…). Ralstonia solanacearumThe resistance to bacterial wilt (Qiu ZK, Yan SS, Xia B, et al. The eggplant transcription factor MYB44 enhances resistance to bacterial wilt by activating the expression of spermidine synthase. Journal of experimental botany, 2019, 70(19): 5343-5354). In terms of antiviral activity, the R2R3 type MYB transcription factor OsMYB4P in rice can activate the jasmonic acid signaling pathway, significantly enhancing the resistance of rice to Southern Rice Black-Streaked Dwarf Virus (SMR). Southern rice black-streaked dwarf virus, SRBSDV) and rice stripe virus ( Rice stripe virus , Broad-spectrum resistance to RSV (Lu MM, He QQ, Wang GD, et al. JA-responsive R2R3-type MYBtranscription factor OsMYB4P confers broad-spectrum antiviral immunity in rice. Plant Biotechnology Journal, 2025, 23(10): 4602-4617). Nicotiana benthamiana ( Nicotiana benthamiana NbMYB4L enhances the activity of superoxide dismutase and catalase through the ethylene signaling pathway, thereby inhibiting tobacco mosaic virus infection and enhancing the resistance of Nicotiana benthamiana to TMV (Zhu T, Zhou X, Zhang JL, et al. Ethylene-induced NbMYB4L is involved in resistance against tobacco mosaic virus in Nicotiana benthamiana . Molecular Plant Pathology, 2022, 23(1): 16-31). Summary of the Invention

[0007] This invention provides a Panax notoginseng MYB transcription factor gene. PnMYB34 Its application in improving the resistance of Panax notoginseng to Panax notoginseng A virus (PnVA) and tobacco to tobacco mosaic virus (TMV).

[0008] This invention clones transcription factor genes from Panax notoginseng. PnMYB34Its nucleotide sequence is shown in SEQ ID NO:1. The CDS length of this gene is 900 bp, encoding a protein with the amino acid sequence shown in SEQ ID NO:2.

[0009] This invention clones the Panax notoginseng transcription factor gene. PnMYB34 CDS sequences, using Agrobacterium tumefaciens ( Agrobacterium tumefaciens This method mediates the transfer of the target gene into recipient plants and its overexpression. Experiments were conducted to verify whether the gene enhances plant antiviral activity. This lays the foundation for future applications of this gene to improve the resistance of Panax notoginseng to PnVA, tobacco to TMV, and other plants to viral diseases. The inventors named this gene... PnMYB34 .

[0010] The above PnMYB34 The gene was applied to improve the resistance of Panax notoginseng to PnVA and tobacco to TMV. The specific operation is as follows: (1) Amplification PnMYB34 Using specific primers, total RNA was extracted from Panax notoginseng and amplified by reverse transcription-polymerase chain reaction (RT-PCR). PnMYB34 The coding region was then ligated into the pGEM-T vector, and clones containing the target gene were obtained by sequencing. (2) Using restriction endonucleases Eco RI and Bam HI enzyme digestion of pGEM-T- PnMYB34 The target gene fragment was obtained by gel extraction from the vector. The plant expression vector pCAMBIA2300S was digested with the same restriction enzyme, and the desired large vector fragment was obtained by gel extraction. The obtained fragment was then... PnMYB34 The gene fragment was linked with the pCAMBIA2300S fragment to construct a plant overexpression vector. Finally, the constructed recombinant vector was transformed into Panax notoginseng and tobacco through Agrobacterium tumefaciens-mediated expression. (3) Transformants were screened using the resistance markers on the recombinant vector T-DNA. After obtaining the real transgenic Panax notoginseng and tobacco plants by PCR detection, they were inoculated with PnVA and TMV viruses respectively. The ability of transgenic plants to resist viral infection was analyzed, and transgenic plants with significantly enhanced resistance to viruses were screened out.

[0011] This invention provides a novel method for improving plant resistance to viral diseases. By using genetic engineering to cultivate disease-resistant plants, it overcomes the shortcomings of traditional breeding methods, shortening the breeding cycle, simplifying the operation, and making it easier to obtain highly resistant materials. In this invention, the resistant plants are derived from Panax notoginseng. PnMYB34The gene can enhance the resistance of Panax notoginseng to PnVA and tobacco to TMV. Introducing this gene into Panax notoginseng and tobacco can produce new varieties and materials with virus resistance. The use of genetic engineering technology to cultivate resistant plant varieties and materials has significant advantages and irreplaceable importance. It not only facilitates large-scale production of crops, medicinal herbs, and horticultural plants, significantly reducing the use of chemical pesticides, but also saves costs in agricultural production and reduces environmental pollution. Therefore, this invention has broad market application prospects. Attached Figure Description

[0012] Figure 1 This is the present invention. PnMYB34 Figure 1 shows the PCR detection results of genomic DNA from transgenic Panax notoginseng and transgenic tobacco. Figure A shows the PCR detection results of genomic DNA from transgenic Panax notoginseng, and Figure B shows the PCR detection results of genomic DNA from transgenic tobacco. The marker in the figures is the DL2000 DNA Marker (Takara Bio Engineering (Dalian) Co., Ltd., China), which consists of six DNA fragments of 2,000 bp, 1,000 bp, 750 bp, 500 bp, 250 bp, and 100 bp. The positive control is plasmid pCAMBIA2300S- PnMYB34 The PCR product is a template; WT (negative control) is a PCR product using non-GMO Panax notoginseng or tobacco as a template. Figure 2 This invention is based on the overexpression of transgenic Panax notoginseng. PnMYB34 The results of transcriptional expression analysis and plant disease resistance identification are shown in Figure A, where Figure A shows the results of transgenic Panax notoginseng overexpression. PnMYB34 Transcriptional expression analysis results; in the figure, WT represents non-transgenic Panax notoginseng (wild type), and Pn1-Pn6 represent... PnMYB34 Transgenic Panax notoginseng; Figure B shows wild-type Panax notoginseng plants (WT) and those overexpressing it. PnMYB34 Symptoms of transgenic Panax notoginseng plants (Pn2) after inoculation with PnVA are shown in the figures. 0 dpi represents the initial state before virus inoculation, and 14 dpi represents the state 14 days after virus inoculation (including overall plant symptom images and magnified views of leaf parts). Figure C shows wild-type Panax notoginseng and overexpressing PnVA. PnMYB34 Comparison of PnVA loading in transgenic Panax notoginseng plants; Figure 3 This invention relates to the overexpression of transgenic tobacco. PnMYB34 Figure A shows the results of transcriptional expression analysis and plant disease resistance identification, where Figure A shows the results of transgenic tobacco overexpression. PnMYB34 Transcriptional expression analysis results, where WT represents non-transgenic tobacco (wild type), and Nb1-Nb6 represent... PnMYB34 Transgenic tobacco; Figure B shows wild-type tobacco plants (WT) and those overexpressing [transgenic tobacco]. PnMYB34Symptoms of transgenic tobacco plants (Nb6) after TMV inoculation are shown in the figures. 0 dpi represents the initial state before inoculation, and 14 dpi represents the state 14 days after inoculation (including overall plant symptom images and magnified leaf details). Figure C shows wild-type tobacco and overexpressing plants. PnMYB34 A comparison of TMV loading in transgenic tobacco plants. Detailed Implementation

[0013] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. However, the scope of protection of the present invention is not limited to the contents described. Unless otherwise specified, the methods in the embodiments are conventional methods, and the reagents used are conventional commercial reagents or reagents prepared according to conventional methods unless otherwise specified.

[0014] Example 1: PnMYB34 Gene cloning, sequence analysis, and construction of plant overexpression vectors After grinding the Panax notoginseng tissue with liquid nitrogen, total RNA was extracted using the guanidine isothiocyanate method, and then processed using GoScript. TM The first strand of cDNA was synthesized using the Reverse Transcriptase System. Using this cDNA as a template, it was amplified using primers 5'-ATGAGAGAAGATGAGTCAAATTGGTTTT-3' and 5'-GAGTCATCCCCTGTTGGAAACAG-3'. PnMYB34 Gene. The PCR reaction system (50 μL) included: 2 μL cDNA, 5 μL 10×Ex Taq Buffer (containing 20 mM Mg). 2+ The reaction mixture consisted of 4 μL dNTPMix (2.5 mM each), 1 μL each of forward and reverse primers (5 μM), 0.25 μL TaKaRa Ex Taq (5 U / μL), and 36.75 μL ddH2O. The reaction program was: 94℃ for 5 min, 94℃ for 30 s, 57℃ for 30 s, 72℃ for 40 s, for 32 cycles; 72℃ for 5 min. The PCR product was detected by 1.2% agarose gel electrophoresis, showing a single target band.

[0015] The target fragment was recovered using the SanPrep column-based PCR product purification kit and cloned into a pGEM-T vector. The ligation system consisted of 4 μL PCR product, 0.7 μL pGEM-T vector, 0.9 μL T4 DNA ligase, and 5 μL 2×RapidLigation Buffer, and ligated overnight at 16°C. The ligation product was transformed into *E. coli* DH5α using the heat shock method, and positive clones were screened on LB agar plates containing ampicillin. Colony PCR and sequencing confirmed the presence of the ligation product. PnMYB34The full-length cDNA sequence (900 bp) encodes a protein containing 299 amino acids, with a predicted molecular weight of approximately 32.30 kDa and an isoelectric point of approximately 6.33. SignalP 4.1 analysis indicates that the protein does not contain a signal peptide; subcellular localization predicts its location in the cell nucleus.

[0016] Extract pGEM-T- PnMYB34 And the plasmid of the plant expression vector pCAMBIA2300S, after Eco RI and Bam After double digestion with HI, the enzymes were recovered separately. PnMYB34 The fragment and the linearized vector fragment were ligated using T4 DNA ligase to construct the recombinant plasmid pCAMBIA2300S- PnMYB34 The recombinant plasmid was transformed into Escherichia coli DH5α, and positive clones were screened on LB agar plates containing 50 mg / L kanamycin. After PCR verification, the recombinant plasmid was extracted and purified.

[0017] pCAMBIA2300S- was prepared by liquid nitrogen freeze-thaw method. PnMYB34 The bacteria were transformed into *Agrobacterium tumefaciens* LBA4404 competent cells. The transformed bacterial culture was plated on LB agar plates containing 50 mg / L kanamycin and incubated at 28°C. Single clones were then picked for PCR verification. Positive clones were added to glycerol and stored at -80°C for later use.

[0018] Example 2: Agrobacterium-mediated genetic transformation of Panax notoginseng and functional analysis of transgenic Panax notoginseng against viral infection Using annual Panax notoginseng as the genetic transformation recipient, the experiment was conducted during its dormant period, when the above-ground stems and leaves had fallen off but the new buds at the cut ends had not yet sprouted. The sample contained pCAMBIA2300- PnMYB34 Agrobacterium tumefaciens was streaked onto LB solid medium containing 20 mg / L rifampin and 50 mg / L kanamycin and incubated at 28°C for 48 h. Single colonies were scraped and inoculated into MGL liquid medium supplemented with 30 μM acetylsyleugenol and cultured at 28°C with shaking at 200 rpm until OD reached. 600Approximately 0.8 μg was used for infection. The Agrobacterium tumefaciens bacterial suspension was injected into the bud anode using a 1 mL sterile syringe until a distinct infiltration area was formed. After infection, the plants were cultured for 40 days, and newly formed plants were harvested for further analysis. Genomic DNA was extracted from plant leaves using the CTAB method. After agarose gel electrophoresis to check its integrity and concentration, PCR amplification was performed using the genomic DNA as a template with 35s promoter-specific primers: upstream primer: 5'-TGCCCAGTCATAGCCGAATAG-3', downstream primer: 5'-CGAGGAGCATCGTGGAAAAA-3'. The PCR reaction system (20 μL) included: 1 μL DNA, 10 μL 2×GS Taq PCR Mix, 1 μL each of the upstream and downstream primers, and 7 μL ddH2O. The reaction program was: 94℃ for 5 min, 94℃ for 30 s, 57℃ for 30 s, 72℃ for 40 s, 32 cycles; 72℃ for 5 min. The PCR products were subjected to electrophoresis detection. The amplification results of some transgenic Panax notoginseng plants are as follows: Figure 1 As shown in A; Total RNA was extracted from transgenic Panax notoginseng leaves and reverse transcribed into cDNA, which was then used as a template for qRT-PCR analysis. PnMYB34 Amplification was performed using specific primers (upstream: 5'-ATTCTGACCATTTCTTGCCGTT-3'; downstream: 5'-CTGCTGTGATTGCCTTGCC-3'). The reaction mixture (20 μL) consisted of 100 ng cDNA, 10 μL 2×GoTaq® qPCR Master Mix, 1 μL each of upstream and downstream primers (2 μM), and PCR-Grade water to a final volume of 20 μL. The amplification program was as follows: 95℃ pre-denaturation for 2 min; followed by 39 cycles (95℃ denaturation for 5 s, 60℃ annealing / extension for 30 s, fluorescence signal acquisition at 60℃); finally, melting curve analysis (60-95℃) was performed. A single peak in the melting curve indicated good primer specificity and suitability for a wide range of applications. PnMYB34 Specific detection. Using 2 -ΔΔCt Method calculation PnMYB34 The relative expression levels, and the detection results of partially overexpressed transgenic Panax notoginseng are as follows: Figure 2 As shown in Figure A.

[0019] Naturally PnVA-infected plants were collected from a Panax notoginseng cultivation base in Wenshan, Yunnan. Leaves infected with PnVA were ground in liquid nitrogen to prepare a virus suspension. Using a 1 mL sterile syringe, the PnVA-infected Panax notoginseng leaf suspension was injected separately into wild-type (WT) and... PnMYB34 Leaves of the transgenic Panax notoginseng (Pn2) plant with the highest expression level were inoculated with approximately 0.1 mL per leaf and cultured in a greenhouse for 2 weeks. Wild-type Panax notoginseng (WT) and... PnMYB34Disease incidence in genetically modified Panax notoginseng (Pn2). Results are as follows: Figure 2 As shown in Figure B, before PnVA inoculation (0 dpi), wild-type Panax notoginseng plants (WT) and those overexpressing PnVA... PnMYB34 Transgenic Panax notoginseng plants all exhibited uniform, dark green leaf color, smooth and flat leaf surfaces, and no abnormal phenotypes such as yellowing, chlorosis, or malformation. The plants grew vigorously and had a consistent overall morphology. Fourteen days after PnVA inoculation, wild-type Panax notoginseng leaves were covered with irregular yellow-green mosaic patterns, and scattered small brown necrotic spots were visible on the leaf margins; while... PnMYB34 Genetically modified Panax notoginseng exhibits milder symptoms, with only small chlorotic spots appearing on the leaf edges. The leaves are well-shaped, uniformly colored, and smooth and glossy, without any obvious deformities, necrosis, or curling.

[0020] Wild-type Panax notoginseng (WT) and PnMYB34 cDNA samples from transgenic Panax notoginseng leaves were analyzed by RT-qPCR under the same conditions. The content of PnVA in the leaves was quantitatively calculated based on the PnVA recombinant plasmid standard. Figure 2 As shown in Figure C, the PnVA loading in the leaves of transgenic Panax notoginseng was significantly lower than that in WT. Clearly, PnMYB34 Transgenic Panax notoginseng exhibits significant resistance to PnVA.

[0021] Example 3: Analysis of Agrobacterium-mediated genetic transformation of tobacco and its functional resistance to viral infection in transgenic tobacco. This study used Agrobacterium-mediated leaf disc transformation to obtain differentiated Nicotiana benthamiana seedlings. Genomic DNA was extracted from plant leaves using the CTAB method, and its integrity and concentration were detected by agarose gel electrophoresis. PnMYB34 PCR amplification was performed using specific primers. 8 μL of the product was then subjected to electrophoresis. The amplification results for some transgenic tobacco plants are shown below. Figure 1 As shown in B. Six strains were analyzed using the qRT-PCR method described in Example 2. PnMYB34 Genetically modified tobacco, using 2 -ΔΔCt Method calculation PnMYB34 The relative expression level, the detection results are as follows Figure 3 As shown in Figure A. Tobacco plants infected with TMV were cultured in our laboratory greenhouse, including wild-type tobacco (WT) and... PnMYB34 The transgenic tobacco plant with the highest expression level (Nb6) was inoculated with TMV suspension for 14 days, and then compared with wild-type tobacco (WT) and... PnMYB34 Disease incidence in genetically modified tobacco (Nb6). Results are as follows: Figure 3 As shown in B, before TMV inoculation (0 dpi), wild-type tobacco plants (WT) and those overexpressing TMV... PnMYB34The transgenic tobacco plants all exhibited uniform, dark green leaves with smooth, flat surfaces, free from abnormal phenotypes such as yellowing, chlorosis, or deformity. The plants were vigorous and showed consistent overall morphology. Fourteen days after TMV inoculation, wild-type tobacco leaves showed typical yellowing and curling symptoms; while... PnMYB34 Although the leaves of the genetically modified tobacco showed yellowing symptoms, the overall disease performance was significantly less severe than that of the wild type.

[0022] Wild tobacco (WT) and PnMYB34 cDNA samples from transgenic tobacco (Nb6) leaves and TMV recombinant plasmid standards were analyzed by RT-qPCR under the same conditions, and the TMV content was determined by Ct value. Figure 3 As shown in Figure C, the TMV loading in transgenic tobacco leaves was significantly lower than that in WT. Clearly, PnMYB34 Genetically modified tobacco exhibits significant resistance to TMV infection.

Claims

1. A Panax notoginseng MYB transcription factor gene PnMYB34 Its nucleotide sequence is shown in SEQ ID NO:

1.

2. The Panax notoginseng MYB transcription factor gene as described in claim 1 PnMYB34 Application in improving Panax notoginseng's resistance to Panax notoginseng A virus.

3. The Panax notoginseng MYB transcription factor gene as described in claim 1 PnMYB34 Application in improving tobacco resistance to tobacco mosaic virus.

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