Application of antiviral compound in prevention and treatment of lichen on lithiasis
By spraying oseltamivir solution to inhibit lichen viruses, the problem of erosion of stone cultural relics by lithotripsy was solved, an environmentally friendly and effective biological control effect was achieved, and stone cultural relics were protected.
Patent Information
- Application Number
- CN202410356946.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-03-27
- Publication Date
- 2025-09-30
AI Technical Summary
The erosion of stone cultural relics by lithotripsy lichens poses challenges to the protection of cultural heritage. Traditional chemical methods damage cultural relics, and it is necessary to explore environmentally friendly and long-lasting biological control methods.
Oseltamivir was used to inhibit lichen virus infection. Lichens were treated by spraying oseltamivir solution at a concentration of 5 μg/mL, affecting the symbiotic system in the lichen body to reduce its activity.
Significantly reduce lichen activity, reduce viral load, protect stone cultural relics, and avoid damage to cultural relics caused by chemical spraying.
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Abstract
Description
Technical Field
[0001] The invention belongs to the field of biotechnology, and particularly relates to application of antiviral compounds in preventing and treating lithotripsy. Background Art
[0002] Stone carvings and grottoes are a precious cultural and artistic legacy of humanity, but the erosion of these cultural heritage sites by lithotripsy (lichens) poses a serious challenge to cultural heritage conservation. While traditional chemical spraying methods can effectively remove lithotripsy, long-term use on stone artifacts can cause damage, such as flaking and cracking. Therefore, the development of environmentally friendly and long-lasting biological control methods for lithotripsy is crucial.
[0003] Lichens are a complex symbiotic system composed of lichen-type fungi and photosynthetic organisms (such as cyanobacteria or green algae). As the main component of the lichen tissue, lichen-type fungi rely on symbiotic algae for nutrients, while the symbiotic algae are protected and supported by the hyphal structure. Recent studies have revealed the presence of a rich microbial community in lichens, including fungi, photosynthetic organisms, bacteria and viruses. Mycoviruses (or Fungal viruses) are a class of viruses that can infect filamentous fungi and replicate and proliferate in them. There are many types of fungal viruses, among which the virus particles of the Partitiviridae family are spherical, with a diameter of approximately 25 to 43 nanometers, and all contain two dsRNA genome fragments of 1.4 to 2.4 kbp in length.
[0004] There are many types of antiviral compounds, with two main mechanisms of action: the first is to interfere with the virus's ability to enter host cells, and the second is to target the process of synthesizing viral components after the virus invades the cell. Among them, oseltamivir, a common anti-RNA virus compound, inhibits the spread of the virus by blocking the spread of replicated virus particles to neighboring cells.
[0005] Given the high adaptability of lichens as terrestrial pioneers, the unknown mechanisms of their infestation have hindered the development of effective biocontrol strategies. Lichens harbor diverse microbial communities, including viruses, which rely on living host cells for survival. These viruses may have formed a mutualistic symbiosis with lichens through long-term evolution and natural selection. Therefore, using exogenous techniques to inhibit viruses within lichens, thereby influencing the lichen's symbiotic system, offers a new approach to biocontrol of lithopelagic lichens and holds significant significance for the preservation of stone artifacts and cultural heritage. Summary of the Invention
[0006] The main problem to be solved by the present invention is how to inhibit the activity of lichens and reduce the erosion of stone cultural relics by lithotripsy.
[0007] In order to solve the above problems, the present invention provides the use of oseltamivir in any of the following:
[0008] 1. The application of oseltamivir in the prevention and treatment of lichenoid virus infections.
[0009] 2. The use of oseltamivir in the preparation of products for preventing and treating lichenoid virus infections.
[0010] 3. The application of oseltamivir in inhibiting the growth of lichenoid viruses.
[0011] 4. The use of oseltamivir in the preparation of products for inhibiting the growth of lichenoid viruses.
[0012] 5. Application of oseltamivir in inhibiting lichen activity.
[0013] 6. Use of oseltamivir in the preparation of products for inhibiting lichen activity.
[0014] Herein, the lichen is Pyxine petricola.
[0015] The lichenoid virus may be of the family Partitiviridae, and specifically may be of the genus Alphapartitivirus.
[0016] In a specific embodiment, the lichen-like virus described in the present invention may be the lichen bipartite virus Pyxinepetricola partitivirus 1 (abbreviated as PpPV1), and the PpPV1 virus has a conserved 5'-UTR and 3'-UTR structure, wherein the dsRNA1 is 1855nt in length (GenBank: MZ289897, updated on July 13, 2022), encoding the replicase RdRp; the dsRNA2 is 1781nt in length (GenBank: MZ289898, updated on July 13, 2022), encoding the capsid protein CP, and the two together complete the virus replication process.
[0017] The lichen viroids are distributed in the cytoplasm of the lichen fungal cells.
[0018] The present invention also provides a method for inhibiting lichen virus infection, which comprises spraying oseltamivir on lichens to prevent and control lichen virus infection.
[0019] In the above method, the lichen may be a lithophilous lichen, and the virus may be Pyxinepetricola partiti virus 1.
[0020] In the above method, the spraying concentration of oseltamivir is 5 μg / mL, and the spraying amount each time is 5 μg / 4 cm 2 .
[0021] In the present invention, oseltamivir was purchased from Shanghai Jizhi Biochemical Technology Co., Ltd., with the product number O72521-20 mg. The oseltamivir stock solution had a concentration of 1 mg / mL and was diluted 200 times with double distilled water (ddH2O) to obtain the spray treatment concentration of 5 μg / mL.
[0022] The present invention proposes for the first time that the administration of antiviral compounds can have a lasting effect on the activity of lichen endosymbiotic systems by affecting them. BRIEF DESCRIPTION OF THE DRAWINGS
[0023] Figure 1 The Renshou Mountain Giant Buddha (left) and the petrified lichen (Pyxine petricola) (right).
[0024] Figure 2 Transmission electron microscopy was used to observe PpPV1 virus particles.
[0025] Figure 3 Spraying oseltamivir reduces the activity of lichens. A: Viral titer determination in lichen samples; B: Lichen photosynthetic activity assay; C: FDA staining to assess cell viability after oseltamivir treatment, observed under bright field and red fluorescence; D: Morphological characteristics of lichens in the treated area before and after oseltamivir spraying. DETAILED DESCRIPTION
[0026] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.
[0027] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.
[0028] The quantitative experiments in the following examples were performed in triplicate unless otherwise specified.
[0029] The 118 lichen samples used in the following examples are all stored in the Mycological Herbarium of the Institute of Microbiology, Chinese Academy of Sciences. Each sample has an HMAS number (see Table 2 for details). The public can obtain this biological material from the applicant for use only in repeating the experiments of the present invention and cannot be used for other purposes.
[0030] The sequence information of Pyxine petricola partitivirus 1 (PpPV1) described in the following examples has been registered with the NCBI database. The public can obtain this biological material from the applicant for use solely for replicating the experiments described herein and not for any other purpose.
[0031] The data in the following examples were processed using GraphPad Prism software. The values in the Student's t-test are mean ± standard deviation. The significance of the difference is indicated by an asterisk. One-way ANOVA was used for the test. P < 0.05 (*) indicates a significant difference, P < 0.01 (**) indicates a very significant difference, and P < 0.001 (***) indicates an extremely significant difference.
[0032] Example 1: Complete Sequence Cloning and Immunofluorescence Localization of a Novel Bipartite Virus Infecting Lichen
[0033] 1. Collection of Lichen Samples
[0034] Lichen samples were collected and isolated from the Giant Buddha at Renshou Mountain, Renshou County, Sichuan Province, China ( Figure 1 ).
[0035] 2. Viral genome sequence amplification and phylogenetic analysis
[0036] First, total RNA was extracted from the lichen and RNA metavirome analysis was performed. Based on the metagenomic data, sequencing fragments of dsRNA 1 and dsRNA 2 were amplified, and the full-length sequences of both viral strands were obtained using the 5' / 3' RACE technique.
[0037] Sequence analysis revealed a conserved 5'-UTR and 3'-UTR structure. The dsRNA1, 1855 nt in length (GenBank: MZ289897, updated July 13, 2022), encodes the replicase RdRp; the dsRNA2, 1781 nt in length (GenBank: MZ289898, updated July 13, 2022; MZ289898), encodes the coat protein CP, which together complete viral replication. Sequence alignment and phylogenetic analysis confirmed that the virus belongs to a new species of the genus Alphapartitivirus, family Partitiviridae, and was named Pyxine petricola partitivirus 1 (PpPV1).
[0038] 3. Localization and observation of virus PpPV1 in lichen
[0039] Transmission electron microscopy observation: Ultrathin sections were made under the electron microscope, and it was observed that PpPV1 virus particles were only distributed in the cytoplasm of lichen fungal cells ( Figure 2 ).
[0040] Example 2: Detection of the infection distribution of virus PpPV1 in different lichen hosts using RT-PCR
[0041] Based on the full sequence of the CP gene of the virus PpPV1 obtained in Example 1 (nucleotide sequence is GenBank: UTE99550.1), specific PCR primers were designed. The specific primer sequence information is shown in Table 1.
[0042] Table 1. Primer information in the present invention
[0043]
[0044] A total of 118 samples (including 47 lithophytic lichens) were collected from multiple provinces in China (including Sichuan, Anhui, Beijing, Fujian, Jiangxi, Shanxi, Gansu, Qinghai, and Tibet Autonomous Region), as well as Mongolia, Cambodia, Israel, and Antarctica, and total RNA was extracted from these lichen samples.
[0045] 1) RT-PCR amplification system and reaction conditions
[0046] The reverse transcription PCR system was as follows: 2 μg RNA; 1 μL Olig D18 Primer; ddH2O to 7 μL. The reaction conditions were set in a PCR instrument as follows: 65°C for 5 minutes; 4°C for 2 minutes. The following reaction solutions were then added: 1 μL E-mix; 1 μL gDNA remover; and 10 μL R-mix. The reaction conditions were set in a PCR instrument as follows: 42°C for 30 minutes; 85°C for 5 seconds; and 4°C for ∞.
[0047] The PCR amplification system (50 μL) contained 2 μL template DNA; 2 μL each of upstream and downstream primers (10 μM); 1 μL 10 μM dNTPs; 1 μL Phanta Super-Fidelity DNA Polymerase; 10 μL 5× SF Buffer; and 32 μL ddH₂O. The total reaction volume was 50 μL. The reaction conditions were: 95°C for 2 min; 30 cycles of 95°C for 30 sec, 55°C for 30 sec, and 72°C for 2 min; and 72°C for 10 min.
[0048] 2) RT-PCR detection
[0049] RT-PCR tests were performed on the collected lichen samples. The test results showed that the virus infection rate in the lithophytic lichen samples was relatively high, reaching 70.2% (Tables 2 and 3).
[0050] Table 2. Growth and virus infection of 118 lichens
[0051]
[0052]
[0053]
[0054]
[0055]
[0056] Table 3. Statistics of lichen toxin rates on different growth substrates
[0057]
[0058]
[0059] Example 3: Oseltamivir spraying treatment reduces the activity of Lichen lichen
[0060] Oseltamivir was purchased from Shanghai Jizhi Biochemical Technology Co., Ltd., catalog number O72521-20 mg. The oseltamivir stock solution had a concentration of 1 mg / mL and was diluted 200-fold with double-distilled water (ddH2O) to obtain a spray treatment concentration of 5 μg / mL oseltamivir solution.
[0061] The experimental site was Fenghuangling Scenic Area in Beijing. The lichen samples (part of the lithophytic lichens grown in the Fenghuangling Natural Scenic Area Park in Beijing) were divided into six equal-sized areas (each area was about 4 cm 2 , the total area is about 24cm 2 ), divided them into two groups (experimental group and control group), and sprayed the same concentration of oseltamivir solution (5μg / 4cm 2 ) and an equal amount of water (control treatment) were sprayed continuously for three weeks (once a week, 1 mL each time, for a total of three weeks), and samples were taken regularly during this period.
[0062] The treated samples were subjected to real-time quantitative PCR analysis to detect the virus titer. The specific steps are as follows: Next qPCR Mix series kit (Toyobo (Shanghai) Biotechnology Co., Ltd., Cat. No. QPX-201) instructions.
[0063] Cell viability staining and photosynthetic activity assays were performed on lichens. The specific steps were as follows: cell viability was assessed using fluorescein diacetate (FDA) staining, and images were captured using bright-field and red fluorescence imaging.
[0064] The experimental results showed that compared with the control group, the spraying of oseltamivir significantly reduced the titer of PpPV1 virus carried by the experimental group ( Figure 3 Middle A), lichen activity was significantly reduced compared with the control group ( Figure 3 B), and the number of lichen algae cells decreased ( Figure 3 In addition, the lichen area in the treated area was measured before and after oseltamivir spraying, and the ratio of the lichen area before and after treatment was 1:0.89, indicating that oseltamivir treatment had a significant effect on the shape and structure of the lichen ( Figure 3 Middle D).
[0065] Statistical analysis was performed using Graphpad Prism version 8.0, and significant differences were observed at **P < 0.01 and ***P < 0.001. The results suggest that topical oseltamivir can significantly affect viral load and, in turn, alter the activity of Lichen spp.
[0066] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that further improvements can be made to the present invention. In short, according to the principles of the present invention, this application is intended to include any changes, uses or improvements to the present invention, including changes that depart from the disclosed scope in this application and are made using conventional techniques known in the art.
Claims
1. The application of oseltamivir in the prevention and treatment of lichenoid virus infection.
2. The use of oseltamivir in the preparation of products for preventing and treating lichenoid virus infections.
3. The application of oseltamivir in inhibiting the growth of lichenoid viruses.
4. The use of oseltamivir in the preparation of products for inhibiting the growth of lichenoid viruses.
5. Application of oseltamivir in inhibiting lichen activity.
6. Use of oseltamivir in the preparation of products for inhibiting lichen activity.
7. The use according to any one of claims 1 to 6, characterized in that: The lichen is a lithophytic lichen.
8. A method for inhibiting lichenoid virus infection, characterized in that: The method comprises spraying oseltamivir on lichens to prevent and treat lichenoid virus infection.
9. The method according to any one of claim 8, characterized in that: The lichen is a lithophytic lichen, and the virus is a lichen-like virus Pyxine petricola partitivirus 1.
10. The method according to any one of claims 8 or 9, characterized in that: The spraying concentration of oseltamivir is 5 μg / 4cm 2 lichen.