Colu virus attenuated strain and application thereof

Abstract

The invention provides a Garlu virus attenuated strain and application thereof, and relates to the field of virology and biological medicine. According to the invention, the screened Garlu virus attenuated strain is a natural attenuated strain derived from a tick sample, can grow in Vero cells, has the titer of more than or equal to 107 TCID50 / mL, does not have clinical pathogenicity to an immunodeficient host, and is safe; after the attenuated strain is used as an immunogen for pre-intervention, infection of a lethal virus strain can be avoided, the survival rate is increased to 100%, adverse effects on growth and life of experimental animals are avoided, particularly, a host can be stimulated to generate humoral immunity and cellular immunity at the same time, and the survival rate of the experimental animals is improved. And moreover, broad-spectrum cross-protection efficacy can be generated on Bandavirus lethal virus strains, and a foundation is laid for improvement of research on Bandavirus live vaccines such as the Colu virus and the like.

Description

Technical Field

[0001] This invention relates to the fields of virology and biomedicine, specifically to an attenuated strain of Gurtu virus and its applications, particularly its use in preventive vaccines and in immunocompromised individuals. Background Technology

[0002] Guertu virus (GTV) belongs to the class Bunyaviricetes, order Hareavirales, family Phenuviridae, and genus Bandavirus. Its primary host is the steppe tick (Tectus prairie tick). Dermacentor nuttalli The Gurtu virus (GTV) genome is a three-segment negative-sense RNA, with L, M, and S segments encoding RdRp, Gp, NP, and NSs proteins, respectively. GTV particles are spherical or nearly spherical, with a diameter of 80-120 nm, and surface glycoprotein protrusions of 5-10 nm, embedded in a lipid bilayer envelope approximately 5 nm thick. Although there are no reported cases of GTV infection, GTV seropositivity and the presence of neutralizing antibodies have been found in populations in Xinjiang Uyghur Autonomous Region of China and Pakistan, suggesting past infection with this virus in these regions and indicating its potential threat to human health.

[0003] Dabie bandavirus (formerly known as Severe Fever with thrombocytopenia syndrome virus, SFTSV), Heartland bandavirus (HRTV), and GTV all belong to the genus *bandavirus* and are tick-borne viruses. In 2010, it was discovered and subsequently named Dabie bandavirus (SFTSV) from samples of cases of Severe Fever with thrombocytopenia syndrome (SFTS) of unknown cause in Hubei and Henan provinces. Its primary host is the Haemaphysalis longhorn tick (*Haemaphysalis longhorn*). Haemaphysalis longicornis Currently, it is prevalent in Hubei, Henan, Zhejiang, Shandong, and other regions in central and eastern China, as well as in Japan and South Korea. Dabieban large virus (SFTSV) infection in humans causes clinical symptoms such as high fever, loss of appetite, muscle pain, chills, swollen lymph nodes, leukopenia, and thrombocytopenia, with a mortality rate of approximately 5% to 30%. In 2009, Heartland virus (HRTV) was isolated from blood samples of two male patients in the Heartland region of northwestern Missouri, USA. Its primary host is the American tick (Achnatherum sp.). Amblyomma americanumThe lone star tick (HRTV), commonly known as the "lone star tick," is currently prevalent in various states in the Midwestern and Eastern United States. Its pathogenicity and transmission mode are similar to those of the styrovirus tabeban (SFTSV), which causes SFTS. In severe cases, it can lead to multiple organ failure and even death, with a mortality rate of approximately 5%.

[0004] Gurtu virus (GTV), Dabie banyan virus (SFTSV), and Heartland virus (HRTV) are all newly emerging tick-borne viruses in the banyanvirus genus that are evolutionarily closely related. They are all segmented, single-stranded, negative-sense RNA viruses with a spherical envelope morphology. All are potential zoonotic pathogens, and infection with any of them can cause similar clinical symptoms such as fever, thrombocytopenia, and leukopenia. There are currently no vaccines for GTV, SFTSV, and HRTV, making research into specific therapeutic drugs and vaccines for these three viruses highly necessary. Although a broad-spectrum monoclonal antibody (CN116693667A) capable of simultaneously recognizing and neutralizing all three viruses has been developed, providing a possibility for the development of broad-spectrum therapies, there are still no approved vaccines for infection with any of these three viruses. Currently, vaccine research against SFTSV and HRTV mainly focuses on inactivated and subunit vaccines. However, these vaccines still have several problems, including short duration of immunity, insufficient immunogenicity, limited cross-protection, and their safety and efficacy in immunocompromised populations have not been fully verified. Furthermore, traditional vaccine development is time-consuming and costly, making it difficult to rapidly respond to the control of newly emerging or mutated viruses. At present, these vaccine research problems remain unresolved. Therefore, providing a vaccine against GTV, SFTSV, and HRTV would have significant scientific and practical value.

[0005] Vaccine research is central to long-term disease control. Currently, SFTSV vaccine development is progressing the fastest, particularly with live attenuated vaccines leading the way. The core advantage of live attenuated vaccines lies in their ability to mimic natural infection, eliciting comprehensive and durable immune protection. However, traditional vaccine development pathways face two major challenges: first, obtaining attenuated strains of a specific pathogenic virus through passage attenuation may not only carry the risk of virulence reversion but also pose a potential pathogenic threat to immunocompromised individuals; second, naturally selected attenuated strains are often comprehensively "weak" strains—weak in virulence, weak in vitro replication capacity, and weak immunogenicity—making them unsuitable for vaccine strain screening and development. The ideal live attenuated vaccine is based on obtaining a vaccine strain that perfectly balances "weak virulence" and "strong immunogenicity"—that is, screening a strain that stably maintains both "weak virulence" and "strong immunogenicity." Furthermore, it is necessary to improve safety studies for immunocompromised populations to ensure the safety and efficacy of the vaccine for a broad range of populations, including immunocompromised individuals, further expanding the applicable population for live vaccines and improving vaccine safety. Summary of the Invention

[0006] The present invention aims to solve the problems of existing live vaccines, such as reversion of vaccine strain virulence, poor stability of vaccine strain, insufficient immunogenicity, limited cross-protection efficacy, and inapplicability to immunocompromised populations.

[0007] The purpose of this invention is to provide a low-virulence, high-immunogenic attenuated strain of Gurtu virus, laying the foundation for the screening of Gurtu virus or Bandia virus vaccine strains and the preparation of live vaccines.

[0008] The purpose of this invention is to provide an attenuated Gurtu virus strain that can provide broad-spectrum cross-protection against lethal GTV, SFTSV, and HRTV strains.

[0009] To achieve the above objectives, the technical solution adopted by the present invention includes: In a first aspect, the present invention provides an attenuated strain of Gurtu virus, which is classified as GTV-Vac Guertu virus, deposited at the China Center for Type Culture Collection (CCTCC) at Wuhan University, Wuhan, China, with accession number CCTCC NO: V202592 and deposit date of December 1, 2025.

[0010] According to a preferred embodiment of the present invention, the attenuated Gurtu virus strain is a naturally attenuated strain derived from a tick sample and capable of growing in Vero cells.

[0011] According to a preferred embodiment of the present invention, the titer of the attenuated Gurtu virus strain is ≥10. 7 TCID 50 / mL.

[0012] According to a preferred embodiment of the present invention, the attenuated strain of Gurtu virus is non-pathogenic.

[0013] Preferably, the attenuated Gurtu virus strain is not clinically pathogenic to immunodeficient hosts.

[0014] In a second aspect, the present invention provides a vaccine composition comprising an immunizing dose of the attenuated Gurtu virus strain described in the first aspect.

[0015] According to a preferred embodiment of the present invention, the immunization dose in laboratory mice is 10. 4 TCID 50 / Only; Preferably, the experimental mice are C57BL / 6 mice with type I interferon receptor deficiency (IFNAR). - / - C57BL / 6 mice.

[0016] According to a preferred embodiment of the present invention, the vaccine composition further contains pharmaceutically acceptable excipients.

[0017] According to a preferred embodiment of the present invention, the vaccine composition may be an injection, a lyophilized powder, an aerosol, an emulsion, or other forms that are conducive to maintaining the immunogenic activity of the attenuated Gurtu virus strain.

[0018] Preferably, the immunization route of the injection is intraperitoneal injection.

[0019] More preferably, the target of the intraperitoneal injection is a mouse.

[0020] Thirdly, the present invention provides the use of the attenuated Gurtu virus strain described in the first aspect and the vaccine composition described in the second aspect in the preparation of a Gurtu virus vaccine.

[0021] Fourthly, the present invention provides the application of the attenuated Gurtu virus strain described in the first aspect and the vaccine composition described in the second aspect in the preparation of a broad-spectrum Bancrovirus vaccine.

[0022] The beneficial effects of this invention are: This invention provides an attenuated Gurtu virus strain and its applications. Firstly, this invention unexpectedly isolated an attenuated Gurtu virus strain (GTV-Vac) from a tick homogenate sample. This attenuated strain is a naturally attenuated strain, and its titer, sequence characteristics, and growth rate remained stable after 20 passages of culture. GTV-Vac can grow in Vero cells, and its titer value remained stable at 10⁻⁶ across different passages. 7 TCID 50 / mL~10 7.5 TCID 50 GTV-Vac, at a concentration of / mL, can be stably cultured in vitro in Vero cells and maintains a relatively high replication capacity. This allows GTV-Vac, as a vaccine strain, to provide sufficient antigen for immunization intervention while maintaining high replication capacity, effectively stimulating the body to generate an immune response. This attenuated strain is not clinically pathogenic to immunodeficient hosts (such as C57BL / 6 mice lacking type I interferon receptors), improving the safety of the vaccine strain and expanding its applicable population. This invention experimentally verifies that pre-intervention with GTV-Vac as an immunogen can prevent host infection with lethal viral strains, increasing the survival rate to 100%, without adversely affecting the growth and life of experimental animals. In particular, it can stimulate both humoral and cellular immunity in the host and provides broad-spectrum cross-protection against lethal Bancrovirus strains, laying the foundation for improving the research of live vaccines against Bancroviruses such as Gurtu virus.

[0023] The attenuated Gurtu virus strain (GTV-Vac) provided by this invention exhibits significantly reduced virulence, good immunogenicity (capable of simultaneously stimulating humoral and cellular immunity in the host), and maintains stability in terms of genetics (sequence identity ≥99.9%) and low virulence (GTV-Vac ≤15%) even after 20 consecutive passages in vitro. It achieves a perfect balance between "strong immunogenicity", "stability" and "low virulence", laying a valuable starting point for the development of live vaccines.

[0024] More importantly, the preferred attenuated Gurtu virus strain (GTV-Vac) of this invention further enhances its safety as a live vaccine due to its proven safety in immunodeficient hosts. Furthermore, this highly safe attenuated strain, GTV-Vac, when used as a vaccine strain, not only provides strong immune protection against lethal wild-type Gurtu virus (GTV-WT) but also offers broad-spectrum cross-immune protection against representative lethal SFTSV and HRTV strains. In other words, GTV-Vac provides positive protection against infection by all three lethal strains of the Dabie Bantamvirus genus: GTV, SFTSV, and HRTV, exhibiting broad-spectrum protection. This lays the foundation for the development of broad-spectrum vaccines against the Dabie Bantamvirus genus, enabling the achievement of trivalent immunization with a single vaccine. In particular, it provides a proactive control measure for the current prevalence of Dabie Bantamvirus (SFTSV) in central and eastern China.

[0025] The shortcomings of current vaccine development include: ① Insufficient immunogenicity: Although existing inactivated and subunit vaccines have high safety profiles, they often fail to produce a sufficiently strong immune response, resulting in limited protective efficacy. Therefore, multiple vaccinations are required to provide adequate protection, increasing the complexity and cost of vaccination. ② Limited cross-protection: Most existing live vaccines are designed for single-virus, one-to-one specific protection. The specificity of the protective site of a particular virus strain means they lack cross-protection against other lethal strains of the same genus. Furthermore, the protective efficacy of existing single-virus vaccines is insufficient against rapidly mutating virus types and the successive emergence of new strains. For example, influenza vaccines can only be designed and administered against a single possible circulating virus strain, and cannot provide protection against all serotypes and circulating strains of a particular virus. ③ Safety issues: Traditional attenuated live vaccines may carry the risk of strain reversal into pathogenicity, potentially leading to serious adverse reactions in immunocompromised individuals, thus limiting their applicability to certain populations. ④ Long R&D cycle and high cost: Traditional live vaccines undergo passage attenuation, resulting in complex R&D and production processes with long cycles. This makes them unable to quickly respond to emerging viral threats. Furthermore, the high production cost of live vaccines and the need for strict animal testing to control potential virulence reversion mutations further hinder rapid production and widespread application. ⑤ Short duration of immunity: Inactivated and subunit vaccines offer limited duration of immune protection, and some live vaccines require periodic booster vaccinations, posing challenges to the widespread implementation of public health prevention and control measures. ⑥ Insufficient research on immunocompromised populations: Most current vaccine research focuses on healthy individuals, with insufficient research on the safety and efficacy in immunocompromised populations. As can be seen, the Gurtu virus attenuated strain (GTV-Vac) provided by this invention, from the initial studies on its infectivity in experimental animals, has consistently been validated from different angles in a gene-deficient mouse model (C57BL / 6 mice lacking type I interferon receptor). The results show that GTV-Vac possesses good immunogenicity, cross-protection within the Bancrovirus genus, low virulence, stability as a live vaccine, relatively high replication and production capacity in Vero cells, and comprehensive immune protection against humoral and cellular immunity. In particular, it consistently demonstrates safety and efficacy data in immunodeficient individuals, meaning that GTV-Vac is effective against immunodeficient IFNAR. - / - C57BL / 6 mice were not lethal, so GTV-Vac addressed the urgent need for a safe attenuated vaccine strain that provides broad cross-protection in immunocompromised individuals. It is not only highly safe, but also provides broad cross-protection against lethal GTV strains and their related lethal SFTSV and HRTV strains, effectively compensating for the shortcomings and defects in existing vaccine development.

[0026] Furthermore, there are currently no approved specific or broad-spectrum vaccines for the genus *Pandaria*, and there is a lack of effective control measures against the potential spread of lethal GTV, SFTSV, and HRTV strains. Therefore, the attenuated Gurtu virus strain (GTV-Vac) provided by this invention has complete cross-protective efficacy against GTV and its lethal strains. Crucially, it has high safety performance and is suitable not only for healthy individuals but also potentially for immunocompromised individuals. It provides a proactive preventive measure for the spread of the three representative lethal strains of *Pandaria*: GTV, SFTSV, and HRTV. Early intervention with GTV-Vac can help improve biosafety defense capabilities against the spread of *Pandaria* viruses. Attached Figure Description

[0027] Figure 1 This is a one-step growth curve for the attenuated Gurtu virus strain (GTV-Vac) and the wild-type Gurtu virus strain (GTV-WT). The horizontal axis represents the TCID value after virus inoculation. 50 The collection time of the supernatant used for the assay is expressed in hours (h), and the vertical axis represents the corresponding viral titer value, expressed in TCID. 50 / mL; Figure 2 Infection of IFNAR with different doses of attenuated Gurtu virus strain (GTV-Vac) - / - Infection trend in C57BL / 6 mice; Figure 3 A graph showing the trend of infection changes in IFNAR- / -C57BL / 6 mice after infection with different doses of wild-type Gurtu virus (GTV-WT); exist Figure 2 and Figure 3 In the graph, A represents the trend of changes in the body weight of experimental animals after infection, and B represents the trend of changes in the survival rate of experimental animals after infection. The horizontal axis represents the time after infection, in days. The vertical axis represents the measurement standard of experimental animals after infection, in percentages (%). Specifically, in graph A, the vertical axis represents the percentage of mouse body weight recorded at different observation times after infection compared with the initial body weight before infection, and in graph B, the vertical axis represents the survival rate of each group of mice at different observation times after infection. Figure 4 The graph shows the ELISPOT detection results of splenic lymphocytes from mice after PBS intervention with attenuated Gurtu virus strain (GTV-Vac) and different stimuli; the vertical axis represents the number of spots detected by ELISPOT, in units of spots / 1×10⁻⁶. 5 Cells; Figure 5The graph shows the ELISA results of splenic lymphocytes from experimental mice after intervention with attenuated Gurtu virus strain (GTV-Vac) and PBS in response to different stimuli; the vertical axis represents the concentration values ​​detected by ELISA, in pg / μL. exist Figure 4 and Figure 5 In the diagram, the horizontal axis represents different stimuli, namely GTV-WT, SFTSV, HRTV, and PBS; Figure 6 The graph shows the trend of protective efficacy against wild-type Gurtu virus (GTV-WT) in mice after prior intervention with attenuated Gurtu virus strain (GTV-Vac) and PBS. In the graph, "-" indicates the level of protection after 10... 4 TICD 50 Mice were treated with a single dose of GTV-Vac and an equal volume of PBS. The text before the "-" indicates the use of 10... 4 TCID 50 / each, 10 5 TCID 50 / The experimental group challenged with only one dose of GTV-WT; Figure 7 The graph shows the trend of protective efficacy against SFTSV in mice after pre-intervention with attenuated Gurtu virus strain (GTV-Vac) and PBS. In the graph, "-" indicates the difference between 10 and 10% protection against SFTSV infection. 4 TICD 50 Mice were treated with a single dose of GTV-Vac and an equal volume of PBS. The text before the "-" indicates the use of 10... 3 TCID 50 / each, 10 4 TCID 50 / each, 10 5 TCID 50 / each, 10 6 TCID 50 / The experimental group challenged with only a single dose of SFTSV; Figure 8 The graph shows the trend of protective efficacy against Heartland virus (HRTV) infection in mice after pre-intervention with attenuated Gurtu virus strain (GTV-Vac) and PBS; in the graph, "-" indicates that the antibodies were administered at 10... 4 TICD 50 Mice were treated with a single dose of GTV-Vac and an equal volume of PBS. The text before the "-" indicates the use of 10... 3 TCID 50 / each, 10 4 TCID 50 / each, 105 TCID 50 / each, 10 6 TCID 50 / The experimental group challenged with only one dose of HRTV; exist Figures 6-8 In the figures, A represents the trend of changes in the body weight of experimental animals after challenge with different doses of lethal virus strains, and B represents the trend of changes in the survival rate of experimental animals after challenge with different doses of lethal virus strains; the horizontal axis represents the time after infection, in days; the vertical axis represents the measurement standard of experimental animals after infection, in percentages (%). Specifically, in figure A, the vertical axis represents the percentage of mouse body weight recorded at different observation times after challenge compared with the initial body weight before infection, and in figure B, the vertical axis represents the survival rate of each group of mice at different observation times after challenge. Figure 9 This is a structural diagram validating the safety of the attenuated Gurtu virus strain (GTV-Vac) and its cross-protective efficacy against SFTSV. The horizontal axis (1-5) represents the source, grouping, and sampling timing of blood samples used for routine blood tests and biochemical assays in the experimental groups, respectively. 1 represents sampling and testing on day 14 after intervention in the PBS intervention group; 2 represents sampling and testing on day 14 after intervention in the GTV-Vac intervention group; 3 represents sampling and testing on day 4 after challenge with SFTSV in the GTV-Vac intervention group (day 14 after intervention); 4 represents sampling and testing on day 4 after challenge with SFTSV in the GTV-Vac intervention group (day 14 after intervention); 5 represents sampling and testing on day 14 after challenge with SFTSV in the GTV-Vac intervention group (day 14 after intervention). The units corresponding to the blood routine indicators and the vertical axis in the graph are: WBC (×10⁻⁶). 9 / L), Lymph (×10 9 / L), Mon(×10 9 / L), Gran (×10 9 / L), Lymph(%), Mon(%), Gran(%), PLT(×10 9 / L); the units of blood biochemical indicators and the vertical axis in the graph are: ALT (U / L) and AST (U / L), respectively. Detailed Implementation

[0028] The exemplary embodiments of the present invention are described in detail below with reference to the accompanying drawings, wherein the same or similar refer to the same concepts, such as Gurtu virus attenuated strain = GTV-Vac, Gurtu virus wild strain = GTV-WT, Dabieban large virus = fever with thrombocytopenia syndrome virus = SFTSV, Heartland virus = HRTV, etc.

[0029] In the following detailed description, numerous specific details are set forth to provide a full understanding of the embodiments disclosed herein for ease of explanation. The following embodiments are for illustrative purposes only and should not be construed as limiting the scope of the invention.

[0030] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the materials and reagents used are commercially available. In this invention, the sequencing of the selected viral strains was commissioned to Sangon Biotech (Shanghai) Co., Ltd.; Vero cells (ATCC numbers: CCL-81) were purchased from ATCC in the United States; FBS (fetal bovine serum) was purchased from Gibco (Grand Island, NY, USA), a subsidiary of Thermo Fisher Scientific; DMEM culture medium was purchased from Hubei Xinzongke Viral Disease Engineering Technology Co., Ltd.; the preferred and claimed attenuated Gurtu virus strain (GTV-Vac) of this invention is deposited in [location missing]; the wild-type Gurtu virus strain (GTV-WT, strain DXM) obtained by screening in this invention is preserved in the National Virus Resource Bank of Wuhan Institute of Virology, Chinese Academy of Sciences (NVRC: CSTR:16533.06. IVCAS 6.6106); Dabieban large virus (SFTSV, C2 type HBGS13) is preserved and used in the National Virus Resource Bank of Wuhan Institute of Virology, Chinese Academy of Sciences (NVRC: CSTR:16533.06. IVCAS 6.6312); Heartland virus (HRTV, strain [missing information]) is also preserved and used in the National Virus Resource Bank of Wuhan Institute of Virology, Chinese Academy of Sciences (NVRC: CSTR:16533.06. IVCAS 6.6312); [missing information] MO-4 was derived from the World Reference Center for Emerging Viruses and Arboviruses at the University of Texas Medical Branch and is preserved and used by the National Virus Resource Bank of the Wuhan Institute of Virology, Chinese Academy of Sciences (NVRC: CSTR: 16533.06, IVCAS 6.6330). In this invention, all live bacterial operations involving GTV, SFTSV, and HRTV were performed in a biosafety level 3 (BSL-3) laboratory, while all experimental procedures involving the infection of experimental animals with these three types of viruses were performed in a biosafety level 2 (BSL-2) laboratory. Virus culture conditions included amplification and culture in Vero cells at 37°C in a 5% CO2 incubator, and virus storage at -80°C. Virus titer determination was performed using the endpoint method in Vero cells to determine the 50% tissue culture infectious dose (TCID). 50 The test was performed on C57BL / 6 mice (IFNAR) lacking type I interferon receptors. - / -The C57BL / 6 mice were purchased from Cyagen Biosciences Ltd.; the ELISPOT kit and ELISA kit for multiple factors (IFN-γ, IL2, IL4, IL10) were purchased from Dakota Biotechnology Ltd. However, it is clear that one or more examples may be implemented without these specific details, and where specific conditions are not specified in the examples, they should be performed under standard conditions or conditions recommended by the manufacturer.

[0031] Unless otherwise specified in the following examples, the molecular biology experimental methods were performed in accordance with the specific methods listed in J. Sambrook's *Molecular Cloning: A Laboratory Manual* (3rd Edition), or according to the kit and product instructions. The attenuated Gurtu virus strain (GTV-Vac) for which this invention is sought is a biological material that needs to be publicly disclosed and ensured to be accessible to the public. This biological material has been deposited with an international depository for biological materials recognized by the State Intellectual Property Office of China and has obtained the required certification documents.

[0032] This invention provides an attenuated strain of Gurtu virus and its screening method, immunogenicity verification method, broad-spectrum cross-protection effect and its application. Specific embodiments of this invention are described below.

[0033] Example 1: Isolation and Preliminary Verification of Gurtu Virus This invention discloses an attenuated strain of Gurtu virus, which is derived from steppe ticks (Dermacentor spp.) collected in the Xinjiang Uygur Autonomous Region. Dermacenter nuttalli Ticks were isolated from tick homogenate samples. The preparation process of the tick homogenate samples was as follows: approximately 50 frozen ticks were placed in a sterile culture dish and repeatedly rinsed with phosphate-buffered saline (PBS) or physiological saline containing antibiotics (such as penicillin or streptomycin) to remove surface contaminants. Ticks were grouped into three groups of 10-20 each to improve the detection rate. After mixing the ticks in each group, they were transferred to a centrifuge tube in a sterile homogenizer, and an appropriate amount of DMEM medium containing 5% FBS was added. The mixture was then homogenized thoroughly on ice until the tissue was completely homogenized. The tick homogenate was centrifuged at 8000-12000 rpm for 15 minutes in a low-temperature centrifuge. The supernatant was filtered through a 0.22 μm microporous membrane. The filtrate is the "tick homogenate supernatant", which can be used immediately for inoculation or aliquoted and stored at -80°C for long-term storage.

[0034] Virus isolation: Vero cells were cultured in 24-well cell culture plates. Once a dense monolayer (coverage approximately 80%-90%) was formed, the cell culture medium was discarded, and the aforementioned "tick homogenate supernatant" was added for inoculation at a volume of 200 μL / well. Uninoculated control wells were included. After adsorption at 37°C for 1-2 hours, maintenance medium was added, and the cells were incubated at 37°C in a 5% CO2 incubator. The growth and pathological changes of the "tick homogenate supernatant" in Vero cells were observed daily. Although typical CPE phenomena such as rounding, detachment, and aggregation did not occur during the screening process, the cell growth rate was slightly slowed, suggesting possible viral proliferation. After 5 days of culture, two GTV-positive virus strains were isolated from the culture supernatants of the three groups of "tick homogenate supernatant" inoculated with Vero cells using dual detection by qPCR (targeting the NP gene of GTV) and IFA (targeting the NP protein of GTV). Cell supernatants containing these two GTV-positive virus strains were then inoculated into newly prepared Vero T25 cells. The virus was amplified in cell culture flasks, and the culture medium was harvested 5-7 days after inoculation, aliquoted, and stored. A portion of the culture medium was retained for further passage to amplify and preserve the virus, while the remainder was used for qPCR, IFA, and whole-genome sequencing verification against GTV virus. The results confirmed that the two positive virus strains were GTV virus.

[0035] Take these two positive samples and continue to passage them in Vero cells for 20 generations. At each generation, perform dual verification using qPCR and IFA to confirm replication characteristics (TCID viral titer). 50 The two isolated Gurtu virus strains underwent whole-genome sequencing every five generations to verify their genetic stability (sequence identity ≥99.9%). Results: Both Gurtu virus-positive strains consistently belonged to the stably inherited Gurtu virus family, with viral titers reaching ≥10. 7 TCID 50 / mL, which proves that it can maintain a high replication capacity in Vero cells; the whole genome sequencing analysis results further prove that it is a stable genetic strain of Gurtu virus, and one of the strains always has a stable and unique mutation sequence in the UTR region.

[0036] One-step growth curve analysis: Two Gurtu virus strains (generation 10, P10) obtained above and verified by titer determination and sequencing were inoculated into Vero cells at an MOI of 5. Supernatants were collected at 0, 3, 6, 12, 18, 24, 30, 36, 48, 72, and 96 h post-inoculation, and the TCID values ​​of the virus titers were determined. 50 The detection was performed, and a one-step growth curve of the two isolated Gurtu viruses was plotted based on the viral titer values, as shown in the figure below. Figure 1As shown: Significant differences in viral titers were observed between the two virus strains 24 hours after inoculation. (The TCID value is then used to illustrate this.) 50 The strain with a relatively low viral titer was named the GTV attenuated strain for Vaccine (GTV-Vac), TCID. 50 The relatively high-yielding strain was named the Guthu virus wild-type strain (GTV-WT), and the results were as follows: Figure 1 As shown.

[0037] Preliminary results from virus isolation and validation revealed that 24 hours after viral infection of Vero cells, the viral load was significantly lower than that of the wild-type Gurtu virus (GTV-WT) with TCID levels. 50 Value ≥ 10 8 TCID 50 Despite high titers ( / mL), the viral titer of the GTV-Vac attenuated strain (GTV-Vac) remained stable at 10 from the primary (P1) to the 20th generation (P20). 7 TCID 50 / mL≤titer<10 7.5 TCID 50 / mL, however, due to the significant difference in viral titers between GTV-Vac and GTV-WT at different time points on the one-step growth curve, it is speculated that the weak virulence of GTV-Vac may be related to the stable and unique mutation sequence in the UTR region of GTV-Vac verified by whole-genome sequencing. It can be preliminarily determined that the GTV-Vac obtained by screening in this invention is a natural attenuated strain (= weak strain) with stable genetic performance isolated from tick samples. Given the low virulence of GTV-Vac, it has potential application value in vaccine strain research. Figure 1 The one-step growth curve shown further verifies that the cell infection and amplification of the two virus strains, GTV-Vac and GTV-WT, show similar trends. However, specific data on viral titer values ​​reveal that the viral titer value used to measure the amplification ability of GTV-Vac in Vero cells is significantly weaker than that of GTV-WT. This suggests that the virulence of the GTV-Vac strain is significantly weaker than that of GTV-WT, and it has the potential to be a candidate strain for a Gurtu virus vaccine.

[0038] Example 2: Infection of experimental animals with Gurtu virus Infectivity and virulence experiments in experimental animals: To screen for Gurtu virus vaccine strains suitable for immunocompromised populations, this invention uses gene-deficient mouse models, specifically C57BL / 6 mice lacking type I interferon receptors (IFNAR). - / -C57BL / 6 mice), IFNAR - / - C57BL / 6 mice were 6-8 weeks old female mice, with 6 mice in each group (n=6). The experimental groups underwent active infection intervention with different doses of Gurtu virus strains. The intervention method involved intraperitoneal injection of 100 µL of two different doses of Gurtu virus strains (GTV-Va and GTV-WT). The infection doses were: 10... 2 TCID 50 / each, 10 3 TCID 50 / each, 10 4 TCID 50 / each, 10 5 TCID 50 / each, 10 6 TCID 50 / mouse; the negative control group was injected intraperitoneally with an equal volume of PBS; after intraperitoneal injection, the experimental group and the negative control group were monitored for 14 consecutive days, and the changes in the physical status of mice in the experimental group with different doses of the two Gurtu viruses were recorded, and the weight change rate and survival rate of the mice were measured at the same time.

[0039] The infection intervention experiments in the above-mentioned experimental animals were used to further verify the infection status of the two Gurtu virus positive strains (GTV-Va and GTV-WT) isolated above in immunodeficient experimental animal models. The infection trends of IFNAR- / -C57BL / 6 mice after infection with different doses of attenuated Gurtu virus strain (GTV-Vac) and wild-type Gurtu virus strain (GTV-WT) are as follows: Figure 2 and Figure 3 As shown.

[0040] Figure 2 It can be seen that, compared with the negative control group, when the infectious dose of Gurtu virus attenuated strain (GTV-Vac) is less than or equal to 10... 3 TCID 50 When the dose was 10 mg / mL, there was no significant difference in mouse body weight changes observed over 14 consecutive days, and it also had no effect on mouse survival. Therefore, it can be inferred that the intervention dose of the attenuated Gurtu virus strain (GTV-Vac) in experimental mice was less than 10 mg / mL. 3 TCID 50 At an infection concentration of 10-1, there were essentially no potential adverse effects on the growth of mice. The only effect was observed when the infection dose of the attenuated Gurtu virus strain (GTV-Vac) was 10-1. 6 TCID 50 / each, 10 5 TCID 50 / each, 10 4 TCID 50In the experimental groups of / mice, the lowest point of weight loss with a significant difference (greater than 10%) occurred on day 5 post-infection. Compared with the initial weight before infection, the rates of weight loss were 16.69%, 12.98%, and 10.86%, respectively. Encouragingly, if monitoring continued until day 14 post-infection, the differences between these three high-dose groups and the other three low-dose groups, even at the highest infection intervention dose of 10, remained significant. 6 TCID 50 In the experimental group, the ratio of mouse body weight to pre-infection body weight returned to normal, meaning the final monitored rate of weight change was not significantly different from the negative control group. This suggests that while high-dose GTV-Vac infection intervention in mice may initially have some adverse effects, in the long term, this GTV-Vac strain does not significantly negatively impact the growth of immunodeficient mice. Therefore, it can be considered a potentially safe viral strain for immunodeficient mouse immunization and vaccine research. Furthermore, according to monitoring records on day 14 post-infection, in all different GTV-Vac infection groups, even with the highest infection dose reaching 10... 6 TCID 50 / mouse, the survival rate of mice infected with this attenuated Gurtu virus strain (GTV-Vac) was 100%, indicating that: up to 10 6 TCID 50 The infection dose of 10n is also relatively safe for immunodeficient mice, and at least no lethal adverse effects have been found. The above experiments further verify that active intervention with the relatively "low-virulence" attenuated Gurtu virus strain (GTV-Vac) has virtually no adverse effects on the growth and well-being of immunodeficient mice, and that the maximum safe infection dose is at least 10n. 6 TCID 50 / Only.

[0041] However Figure 3 It is evident that the wild-type Gurtu virus strain (GTV-WT) except when the intervention dose is 10 0 TCID 50 / only=1TCID 50 At extremely low doses (e.g., 10 g / mL), there was no significant difference in the post-infection effects on experimental mice; that is, ultra-low intervention doses did not produce lethal infection effects on immunodeficient experimental mice, nor did they have significantly adverse effects on the weight changes of experimental mice. However, when the intervention dose was greater than or equal to 10 g / mL... 1 TCID 50 / each = 10TCID 50All four experimental groups posed a lethal risk of infection to the mice, and only the lower infectious dose group (10 mice) showed no significant difference in weight change as of day 14 post-infection. 1 TCID 50 / Only and 10 2 TCID 50 In these two experimental groups with relatively low intervention doses, it was observed that the wild-type Gurtu virus strain (GTV-WT) is a typical lethal Gurtu virus strain, with the lowest intervention dose being 10 TCID. 50 Even at a single dose, there is a risk of death in experimental mice, and higher intervention doses (greater than 10) pose a significant risk. 2 TCID 50 (per mouse) also has a significant adverse effect on the long-term growth and weight changes of experimental mice. Further determination of the median lethal dose (LD50) of GTV-WT was performed. 50 The TCID value is 31.6. 50 .

[0042] By comparing the effects of attenuated Gurtu virus strain (GTV-Vac) and wild-type Gurtu virus strain (GTV-WT) on the body weight and mortality rate of immunodeficient experimental mice, in stark contrast to the lethal effects of GTV-WT, GTV-Vac showed significant safety in immunodeficient IFNAR- / - C57BL / 6 experimental mice. The mice not only had a 100% survival rate, but also had virtually no impact on their growth and well-being. No adverse clinical effects such as disease onset were observed during the 14-day continuous monitoring period, further enhancing the possibility of GTV-Vac being used as a vaccine strain.

[0043] Example 3: Immunogenicity of GTV-Vac Intervention in immunogenicity studies: further validation of GTV-Vac for immunodeficient IFNAR. - / - The immune response in C57BL / 6 mice was investigated using an intervention method similar to the virulence experiment on experimental animals described above: the experimental animals were C57BL / 6 mice with type I interferon receptor gene knockout (IFNAR). - / - C57BL / 6), females, 6-8 weeks old, with 6 mice in each group (n=6); the intervention method was intraperitoneal injection of 100 µL of attenuated Gurtu virus strain (GTV-Vac), and the intervention dose in the experimental group was 10 µL. 4 TCID 50 / mouse; the negative control group received an intraperitoneal injection of an equal volume of PBS. On day 14 after intervention, the experimental mice were euthanized by cervical dislocation and immersed in 75% ethanol for 1-2 min. The spleen was dissected and separated in a biosafety cabinet. The aseptically obtained spleen tissue was prepared into a lymphocyte suspension for subsequent immunogenicity analysis.

[0044] Preparation of lymphocyte suspension: Add 4-5 mL of 1× lymphocyte separation medium to a 35 mm culture dish. Place the aseptically isolated spleen tissue in the dish and first cut it into small pieces with scissors. Then, use the flat end of a 2 mL syringe plunger to grind the chopped spleen until only white connective tissue remains in the culture dish. Since the separation medium is volatile, the entire grinding process should be controlled within 5 minutes to minimize its exposure time. Gently pipette the separation medium containing the suspended spleen cells to fully disperse the tissue cells, then immediately transfer it to a 15 mL centrifuge tube. Slowly cover the centrifuge tube with 200-500 μL of RPMI 1640 medium (serum-free). Place the centrifuge tube in a horizontal rotor centrifuge and centrifuge at 800×g for 30 minutes at room temperature, using a slow acceleration and deceleration to minimize damage to the lymphocytes. After centrifugation, the lymphocytes will float to the top and aggregate below the RPMI 1640 medium layer, forming a clear cell stratification. Carefully aspirate approximately 0.5–1 mL of the lymphocyte layer directly using a pipette, avoiding aspirating cells from other layers. Transfer the aspirated lymphocytes to a new centrifuge tube, add 10 mL of RPMI 1640 medium, and gently invert the centrifuge tube several times to thoroughly wash the cells. Place the centrifuge tube in a centrifuge and centrifuge at 250 × g for 10 min at room temperature. After centrifugation, the lymphocytes will precipitate at the bottom of the centrifuge tube. Discard the supernatant; the precipitate is the lymphocytes isolated from the tissue. Add 2 mL of erythrocyte lysis buffer to the centrifuge tube and gently mix to ensure the precipitated cells are in full contact with the lysis buffer. Lyse for 1–2 min, then add 3 mL of RPMI 1640 complete medium to terminate the reaction. Place the centrifuge tube in a centrifuge and centrifuge at 400–500 × g for 5 min. Discard the red supernatant. Depending on the condition of the lymphocyte precipitate, wash the tube 1–2 more times with RPMI 1640 complete medium to thoroughly remove the lysis buffer and other impurities. After washing and centrifugation, discard the red culture medium supernatant, resuspend the cells in 2 mL of RPMI 1640 complete culture medium, and dilute 20 times to obtain the isolated lymphocyte suspension. After live cell counting with trypan blue, it can be used for ELISPOT and ELISA detection, respectively.

[0045] Cell sample preparation and stimulation: Take cell culture plates (96-well plates for ELISPOT, 24-well plates for ELISA), adjust the concentration of the prepared lymphocyte suspension to the appropriate concentration, and add it to each well of the cell culture plate. The volume and cell seeding quantity per well are as follows: 100 μL / well for 96-well plates to maintain a cell seeding quantity of approximately 3 × 10⁶ cells / well. 5 Cell / well, add 500 μL / well to a 24-well plate to maintain a cell seeding density of approximately 1 × 10⁶ cells / well. 6 Cell / well; Stimulants are wild-type Gurtu virus (GTV-WT), Dabieban large virus (SFTSV), and Heartland virus (HRTV) with a MOI of 5. Positive and negative control wells are also set up. After adding the sample and stimulants, carefully close the cell culture plate lid and place the cell culture plate in a 37°C, 5% CO2 incubator for incubation.

[0046] Multifactor ELISPOT assay: After culturing the 96-well plates for 16-20 h, remove the plates and discard the suspended cells and culture medium. Add 200 μL / well of ice-cold deionized water and place the plates at 4°C to induce hypotonic lysis. After lysis for 10 min, discard the suspended cells and water. Add 200 μL / well of 1×Washing buffer and let the plate stand for 30-60 seconds before discarding. Repeat this washing process 5-7 times. After the last wash, blot the plate dry on absorbent paper. Add 100 μL / well of biotinylated antibody working solution (Biotin-IFN-γ, Biotin-IL2, Biotin-IL4, Biotin-IL10) according to the manufacturer's instructions. Block the plates with a sealing membrane and incubate at 37°C for 1 h. After incubation, discard the liquid in the wells and repeat the washing process 5-7 times with 1×Washing buffer, blotting the plate dry on the last wash. Add 100 μL of Streptavidin-HRP working solution to each well according to the instructions. After sealing with a sealing membrane, incubate at 37°C for 1 hour. After the second incubation, pour out the liquid from the wells and wash 5-7 times with 1× Washing buffer, following the same steps. For the last wash, firmly tap the plate to remove excess water. Add 100 μL of freshly prepared AEC chromogenic working solution to each well and incubate at room temperature in the dark for 15-45 minutes. After chromogenic development, pour out the liquid from the wells, remove the ELISPOT plate base, and wash both sides of the plate and the base 3-5 times with deionized water. After stopping the chromogenic process, place the ELISPOT plate in a cool, dark place at room temperature to air dry. Replace the base and use an ELISPOT spot counter to count the spots. Record the spot counts (unit: spots / 1×10⁻⁶) for different antibodies (four immune cytokines: IFN-γ, IL2, IL4, and IL10).5 Cells) and perform statistical analysis; ELISPOT detection results are as follows: Figure 4 As shown.

[0047] Figure 4 It is evident that after intervention with GTV-Vac and PBS in immunodeficient mice, the levels of four immune cytokines (IFN-γ, IL-2, IL-4, and IL-10) in the lymphocyte suspension isolated from the spleen showed significant differences compared to the negative control group (PBS). Since IFN-γ, IL-2, IL-4, and IL-10 represent Th1-type (IFN-γ, IL-2) and Th2-type (IL-4, IL-10) cytokines, respectively, they are key and relatively comprehensive immune detection indicators for assessing the type of immune response (e.g., cellular immunity vs. humoral immunity). The ELISPOT assay results, which showed significant differential expression of the four immune cytokines, indicate that GTV-Vac intervention elicited strong and relatively comprehensive immune responses in mice stimulated by GTV, SFTSV, and HRTV viruses.

[0048] ELISA assay: After 48 h of incubation in a 24-well plate, remove the plate and centrifuge under suitable conditions (room temperature, 200-300×g for 5-10 min) to collect the culture supernatant. Add 100 μL of the collected culture supernatant to each well of an ELISA plate pre-coated with the corresponding antigens (IFN-γ, IL2, IL4, IL10), seal with sealing film, and incubate at 37°C for 2 h. After incubation, carefully pour out the liquid from the ELISA plate wells, add 200 μL of 1×washing buffer per well, let stand for 30-60 seconds, then discard. Repeat this washing process 5-7 times. After the final wash, firmly blot dry on absorbent paper. Add 100 μL of diluted biotin-labeled antibody working solution (Biotin-IFN-γ, Biotin-IL2, Biotin-IL4, Biotin-IL10) to each well according to the instructions. After sealing with a membrane, incubate at 37°C for 1 h. After incubation, discard the liquid in the wells and wash 5-7 times with 1×Washing buffer, following the same steps as above. For the last wash, firmly tap the plate to remove excess liquid. Add 100 μL of diluted horseradish peroxidase (HRP)-labeled streptavidin working solution to each well according to the instructions. After sealing with a membrane, incubate at 37°C for 0.5 h. After another incubation, discard the liquid in the wells and wash 5-7 times with 1×Washing buffer, following the same steps as above. For the last wash, firmly tap the plate to remove excess liquid. Add 100 μL of TMB substrate to each well and incubate at 37°C in the dark for 20 min for color development. Stop the reaction by adding 50 μL of stop solution to each well. Immediately measure the OD values ​​at 450 nm and 630 nm using a microplate reader. Calculate the concentration values ​​of different antibodies (four immune cytokines: IFN-γ, IL2, IL4, and IL10) for ELISA detection based on the OD value results. Figure 5 The vertical axis is shown in the diagram.

[0049] Figure 5 It is evident that after GTV-Vac and PBS interventions in immunodeficient mice, the ELISA values ​​of IFN-γ, IL-2, and IL-10 in the lymphocyte suspension isolated from the spleen showed significant differences compared to the negative control group (PBS). Among these, the differences in IFN-γ and IL-2 were particularly pronounced. All three viral stimuli—GTV, SFTSV, and HRTV—showed significant responses in the lymphocytes after GTV-Vac intervention. This indicates that GTV-Vac intervention can elicit a strong immune response in experimental mice when stimulated by these three viral types.

[0050] Although the ELISA values ​​of IL-4 did not differ significantly between the experimental group and the negative control group, this may be due to two main reasons. Firstly, ELISPOT, which has a higher sensitivity, is capable of detecting cytokines secreted by individual cells and can sensitively capture cells that secrete the low-frequency cytokine IL-4. In contrast, ELISA detects the total amount of free cytokines in body fluids and is affected by factors such as half-life and metabolic degradation, which may underestimate the low-expression or transiently secreted cytokine IL-4. Secondly, because IL-4 belongs to the Th2 type of cytokine, compared to the Th1 type cytokines (IFN-γ, IL-2), the ELISA results at 48 hours of culture are affected by the sampling time point, which may indicate that IL-4 has been degraded or its detection peak has passed. Alternatively, it may simply indicate that IL-4 secretion occurs at a relatively low frequency. Hypothesis: After intervention with the attenuated Gurtu virus strain (GTV-Vac), regardless of the stimulus, although it can activate both Th1 and Th2 immune responses, there may be a shift in the immune response type, with the Th2 type gradually weakening and the Th1 type gradually strengthening. This result has important implications for studying the immunization strategy of GTV-Vac as a vaccine strain or optimizing the immunization mechanism.

[0051] Example 4: Protective efficacy of GTV-Vac immunization The above verification demonstrates the effectiveness of the attenuated Gurtu virus strain (GTV-Vac) in immunodeficient animal models (IFNAR). - / - The C57BL / 6 strain demonstrated excellent safety (non-lethal) and strong immunogenicity (eliciting a multifactorial immune response), meaning the balance between low virulence and strong immunogenicity lays the foundation for protection against challenge as a vaccine strain. Therefore, a thorough and systematic evaluation of the protective efficacy of GTV-Vac as a vaccine strain is needed.

[0052] The protective efficacy experiment included intervention and challenge. The intervention method was similar to that described above: the experimental animals were C57BL / 6 mice with type I interferon receptor gene knockout (IFNAR). - / - C57BL / 6), females, 6-8 weeks old, with 6 mice in each group (n=6); the intervention method was intraperitoneal injection of 100 µL of attenuated Gurtu virus strain (GTV-Vac), with the experimental group receiving 10 µL of pre-intervention dose. 4 TCID 50 / mouse; the negative control group received an equal volume of PBS via intraperitoneal injection. The challenge method was as follows: on day 14 post-intervention, mice pre-treated with GTV-Vac were challenged via intraperitoneal injection with three different lethal viral strains: wild-type Gurtu virus (GTV-WT), Dabieban large virus (SFTSV), and Heartland virus (HRTV) at different lethal doses. The challenge dose for GTV-WT was 10... 4 TCID 50 / each, 10 5 TCID 50 / each, the challenge dose for both SFTSV and HRTV was 10 3 TCID 50 / each, 10 4 TCID 50 / each, 10 5 TCID 50 / each, 10 6 TCID 50 / mouse, after challenge injection, the protective effect of the experimental group that had undergone prior GTV-Vac immunization was monitored and recorded for 14 consecutive days against different types and doses of lethal virus strains. The criteria for evaluating the protective effect were still the changes in mouse body weight and survival rate. The protective efficacy of GTV-Vac against GTV-WT, SFTSV, and HRTV infections were as follows. Figures 6-8 As shown.

[0053] Regarding the rate of weight change Figures 6-8As can be seen in Figure A: Immunodeficient mice pre-intervened with an equal volume of PBS, when challenged with relatively high doses of lethal GTV, SFTSV, and HRTV, showed a clearly discernible trend of weight loss as early as the first day after infection. The mice died before the end date of continuous monitoring, with their weight dropping to about 70% of the pre-infection weight. Since weight loss is a sensitive indicator of systemic disease and metabolic disorders caused by viral infection, if the weight loss rate of mice in the PBS negative control group reaches 30%, it can indicate to some extent that the mice are in an extremely serious disease state, meaning that multiple organ failure has occurred, which is a typical precursor to death. In contrast, immunodeficient mice pre-intervened with 104 TCID50 / mouse GTV-Vac showed no significant weight loss or even a slight weight gain when exposed to lethal doses of GTV-WT, SFTSV, and HRTV. Furthermore, during 14 consecutive days of monitoring after challenge, the mice in the pre-protected group with GTV-Vac, which received lethal viral strains, showed no significant differences in fur condition, activity level, diet, and water intake compared to normal unchallenged mice. This indicates that pre-immunization with GTV-Vac as a vaccine strain almost completely blocked the occurrence of the disease, and the mice remained in a relatively healthy growth state.

[0054] Regarding survival rate Figures 6-8 As shown in Figure B, immunodeficient mice pre-intervened with an equal volume of PBS, regardless of the lethal dose of any lethal virus strain, experienced a premature drop in survival rate to 0 before the endpoint of continuous monitoring. This indicates that the PBS in the negative control group did not provide protection against any lethal virus, whereas the PBS in the negative control group (10...) provided protection against the lethal virus. 4 TCID 50 Immunodeficient mice pre-treated with GTV-Vac showed a 100% survival rate against lethal doses of GTV-WT, SFTSV, and HRTV infection. This demonstrates that using GTV-Vac as a vaccine strain for pre-immunization significantly improves survival rates against subsequent infections with these three lethal GTV, SFTSV, and HRTV strains (all belonging to the same genus *Panthera* virus), with a maximum survival rate potentially reaching 100%. Figures 6-8The study demonstrated significant differences in weight change rate (stable weight vs. rapid weight loss) and survival rate (100% survival vs. 0% survival) after GTV-Vac and PBS intervention, further validating that GTV-Vac can protect against infection by the genus *Pandoravirus*. It not only prevents lethal infection by GTV, SFTSV, and HRTV, but also almost completely blocks the occurrence of the disease. Therefore, GTV-Vac provides very positive and relatively complete protection, exhibiting broad-spectrum protective efficacy against lethal *Pandoravirus* strains with extremely strong protective efficacy. This further validates the potential of Gurtu virus attenuated strain (GTV-Vac) as a vaccine candidate for further research.

[0055] Example 5: Security of GTV-Vac and its cross-protection effectiveness against SFTSV Given the prevalence, mortality rate, and urgent need for vaccines and other immunizations against SFTSV, we aim to further evaluate the in vivo safety of GTV-Vac as a vaccine strain in immunodeficient animal models after early intervention immunization and its protective efficacy against lethal SFTSV infection.

[0056] To verify the safety of GTV-Vac in immunocompromised individuals and its cross-protective efficacy against SFTSV infection, the vaccine intervention method using GTV-Vac as the preferred attenuated strain of this invention was similar to that described above: the experimental animals were still C57BL / 6 mice with type I interferon receptor gene knockout (IFNAR). - / - C57BL / 6), females, 6-8 weeks old, were first divided into a GTV-Vac intervention group and a PBS intervention group, with 12 animals in each group (n=12). The intervention method was intraperitoneal injection of 100 µL of attenuated Gurtu virus strain (GTV-Vac). 4 TCID 50 / each; the PBS intervention group served as a negative control group to verify the safety of GTV-Vac, with the intervention method being an intraperitoneal injection of an equal volume of PBS. Secondly, the GTV-Vac intervention group and the PBS intervention group were further divided into a simple intervention group and an SFTSV challenge group. Specifically, the grouping and testing verification method was as follows: in the above-mentioned GTV-Vac intervention group (10 4 TCID 50 On day 14 after intraperitoneal injection of GTV-Vac into immunodeficient mice and in the PBS intervention group (equal volume of PBS), six mice were randomly selected from each group as the intervention group to compare and verify the safety of GTV-Vac intervention in immunization, i.e., without challenge treatment (e.g., no GTV-Vac per mouse). Figure 9As shown in Figures 1 and 2), after fixing 6 mice in the simple intervention group, blood was collected from the posterior orbital venous plexus: approximately 0.5 mL of whole blood was collected using heparin-free EDTA-K2 anticoagulant tubes, and at least 0.5 mL of serum was collected using procoagulant tubes; the collected whole blood and serum samples were subjected to complete blood count and biochemical analysis of the serum samples, respectively, within 2 hours. On the same day (i.e., day 14 after intervention), the remaining 6 mice in the GTV-Vac intervention group and the PBS control group were used as the SFTSV challenge group ( Figure 9 As shown in Figures 3-5), 10 mg was injected intraperitoneally. 4 TCID 50 / dose of SFTSV was used in a challenge experiment. The protective effect of PBS intervention group and GTV-Vac intervention group against SFTSV was continuously monitored after challenge injection until day 4 of challenge. Figure 9 As shown in Figure 3-4), whole blood and serum were collected according to the above blood sample collection methods for routine blood tests and biochemical tests of serum samples. If the experimental mice in the GTV-Vac intervention group maintained normal growth after SFTSV challenge, they were continued to be fed until day 14 after SFTSV challenge. Figure 9 As shown in Figure 5), blood samples were collected again using the above method, and complete blood count (CBC) and serum biochemical tests were performed. The CBC included eight tests: WBC (white blood cell count), Lymph (lymphocyte count), Mon (monocyte count), Gran (granulocyte count), Lymph% (lymphocyte percentage), Mon% (monocyte percentage), Gran% (granulocyte percentage), and PLT (platelet count). The two representative serum biochemical tests were: ALT (alanine aminotransferase) and AST (aspartate aminotransferase). The results of the eight CBC tests and two biochemical tests are shown below. Figure 9 As shown, the units for the above blood routine test and biochemical test items are as follows: Figure 9 The vertical axis is shown in the diagram.

[0057] Figure 9It is evident that: Firstly, in both the eight routine blood tests and the two biochemical tests, all ten tests were within the normal range in the simple intervention group (including the PBS intervention group and the GTV-Vac intervention group) and the GTV-Vac intervention + SFTSV challenge group (including days 4 and 14). It is also evident that in the PBS intervention group (represented by "1"), the GTV-Vac intervention group (represented by "2"), and the two SFTSV challenge groups after GTV-Vac intervention (days 4 and 14), all tests of the experimental mice were within the normal standard range, indicating that the experimental mice were generally in good health. Secondly, in the simple intervention group, only the measured values ​​of PLT (platelet count) and ALT (alanine aminotransferase) showed discernible differences between the PBS intervention group and the GTV-Vac intervention group. This difference, without affecting the growth of mice, may mean that the GTV-Vac intervention may have stimulated a certain degree of immune response in mice. This positive immune response interfered with the physiological indicators (PLT and ALT), but this interference did not have an adverse effect on the growth and safety of mice. Third, among the SFTSV challenge groups (3-5), the changes in the PBS intervention + SFTSV challenge group (represented by "3") were the most significant and could reflect the adverse effects on the normal growth of mice: among the 10 physiological indicators, except for Mon which showed no significant change, the values ​​of the other 9 physiological indicators all showed significant differences, and the changes in 6 of the values ​​were very large or even abnormal (Lymph↓, Lymph%↓, Gran%↑, PLT↓, ALT↑, AST↑), indicating that SFTSV challenge had caused serious damage to the immune system, blood system and liver of mice; in contrast, on the 4th day (represented by "4") and the 14th day (represented by "5") of the GTV-Vac intervention + SFTSV challenge group, all 10 physiological indicators were within the normal range, indicating that the early intervention of GTV-Vac had a positive cross-protective effect on SFTSV challenge, avoiding damage to the tissues and organs of mice and avoiding large-scale abnormal changes in physiological indicators. Fourth, further comparison of the GTV-Vac intervention + SFTSV challenge group on days 4 and 14 showed that WBC, Lymph, Gran, and ALT showed significantly increasing trends, with the increase in ALT being extremely significant ("****", P value less than 0.001). This suggests that the mice, due to GTV-Vac intervention, rapidly induced a positive protective immune response when challenged by SFTSV, thereby effectively inhibiting the pathogenicity of SFTSV.

[0058] The attenuated Gurtu virus strain (GTV-Vac) obtained by screening in this invention has been verified as a genetically stable natural attenuated strain through passage culture and sequencing. This virus strain has been deposited at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, Wuhan, China, with accession number CCTCC NO: V202592, and deposited on December 1, 2025. The deposited material is classified and named Gurtu virus GTV-Vac. Cell passage culture has verified that GTV-Vac has weak virulence (10-10). 7 TCID 50 / mL~10 7.5 TCID 50 The titer of GTV-Vac (≥10 mL / mL) remained stable, ensuring biosafety for experimental or clinical use; in vitro experiments confirmed that although the toxicity was weak, the titer value of GTV-Vac was relatively high (≥10 mL / mL). 7 TCID 50The fact that GTV-Vac can be continuously and stably passaged in Vero cells and maintain a relatively high replication capacity ( / mL) means that GTV-Vac can not only replicate at high rates in vitro as a vaccine strain, but also provide sufficient antigen in pre-interventioned immunodeficient experimental animals to effectively stimulate the body to produce an immune response. In addition, pre-intervention immunization with GTV-Vac can not only ensure that the experimental animals after intervention are resistant to infection with lethal doses of wild-type Gurtu virus (GTV-WT), but also effectively promote cross-resistance to infection with lethal doses of Dabieban large virus (SFTSV) and Heartland virus (HRTV). That is, GTV-Vac can provide broad-spectrum and complete protection against challenge with GTV-WT, SFTSV and HRTV, which are all lethal infections (cellular immunity and humoral immunity, with survival rate increased to 100%). Further safety and cross-immunoprotection evaluation experiments of GTV-Vac further validated that GTV-Vac can induce a strong immune response in immunodeficient animal hosts against the invasion and infection of SFTSV, without affecting the normal growth of immunodeficient animals (weight change rate, blood routine and biochemical test indicators were all normal). These results validate that GTV-Vac, as a natural attenuated strain of Gurtu virus, possesses a perfect balance of safety, immunogenicity, and broad-spectrum protective efficacy, supporting its selection as a candidate live attenuated vaccine for subsequent clinical trials. Therefore, it can be inferred that if GTV-Vac is used as the active ingredient in vaccine strains and vaccine products, and pharmaceutically acceptable excipients are added, such as stabilizers, buffers, surfactants, lyophilization protectants, or nano-delivery carriers, it can be prepared into a preventative vaccine for pre-intervention, which can be an injection, lyophilized powder, aerosol, emulsion, or other forms that help maintain the immunogenic activity of GTV-Vac. Prophylactic vaccines prepared from GTV-Vac, if their immunization schedule is optimized and their safety, efficacy, and quality control are comprehensively evaluated in accordance with drug development standards, can produce significant and broad-spectrum cross-protection against infections caused by viruses of the Bancrovirus genus. In particular, they provide a vaccine strain that can still be used in immunocompromised individuals. This not only provides an important basis for the development of vaccines that can provide broad-spectrum immunization against GTV, SFTSV, and HRTV, but also effectively broadens the applicable population for vaccine immunization, providing a potential, usable, safe, and effective vaccine strain for the elderly, children, and other immunocompromised individuals.

[0059] Specific embodiments of the present invention have been described in detail, making them readily understandable to those skilled in the art. However, based on all the disclosed descriptions, various modifications or substitutions may be made to those details, and all such changes are within the scope of protection of the present invention. The full scope of the present invention is given by the appended claims and any equivalents thereof.

Claims

1. A live attenuated strain of Goulbourn virus, characterized in that: The taxonomic designation of the attenuated strain is Guertu virus GTV-Vac Guertu virus, which is preserved in the China Center for Type Culture Collection, Wuhan University, Wuhan, China, with the preservation number CCTCC NO: V202592, and the preservation date is December 1, 2025.

2. The attenuated strain of Chikungunya virus according to claim 1, characterized in that: The attenuated strain is a natural attenuated strain derived from a tick sample and is capable of growing in Vero cells.

3. The attenuated strain of Chikungunya virus according to claim 1, characterized in that: The titer of said attenuated strain of Getah virus is > 10 7 TCID 50 / mL.

4. The attenuated strain of Chikungunya virus according to claim 1, characterized in that: The Guertu virus attenuated strain is non-pathogenic.

5. The attenuated strain of Chikungunya virus according to claim 4, characterized in that: The Guertu virus attenuated strain is non-clinically pathogenic to immunodeficient hosts.

6. A vaccine composition, characterized by: The vaccine composition comprises an immunizing dose of the Guertu virus attenuated strain of claim 1.

7. The vaccine composition of claim 6, wherein: It also contains pharmaceutically acceptable adjuvants.

8. The vaccine composition of claim 6, wherein: It can be an injection solution, a lyophilized powder, an aerosol, an emulsion, or other forms that are conducive to maintaining the immunogenic activity of the Guertu virus attenuated strain.

9. The Guertu virus attenuated strain of any one of claims 1-5, or the vaccine composition of any one of claims 6-8, for use in the preparation of a Guertu virus vaccine.

10. The Guertu virus attenuated strain of any one of claims 1-5, or the vaccine composition of any one of claims 6-8, for use in the preparation of a broad-spectrum Tetrovirus vaccine.

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