Isolation method of bovine epidemic fever virus, and bovine epidemic fever virus obtained and application thereof

Abstract

The application discloses an isolation method of bovine epidemic fever virus and the bovine epidemic fever virus obtained by the method and application of the bovine epidemic fever virus, and belongs to the technical field of bovine epidemic fever virus isolation. The application aims to provide an efficient and rapid method for isolating bovine epidemic fever virus. The application provides the isolation method of the bovine epidemic fever virus, wherein the anticoagulated blood sample of a bovine is inoculated on KC cells for culture, the virus is harvested, the BHK-21 cells are inoculated to grow into a monolayer, and the first generation of passage cells with cytopathic effect is obtained by passage. The virus strain can also be used for screening, preparation and detection of bovine epidemic fever virus antibodies, and for preparation of an animal model infected by the bovine epidemic fever virus.

Description

Technical Field

[0001] This invention belongs to the field of bovine ephemeral fever virus isolation technology, specifically relating to a method for isolating bovine ephemeral fever virus, the obtained bovine ephemeral fever virus, and its applications. Background Technology

[0002] Bovine ephemeral fever (BEF), also known as three-day fever or transient fever, is an acute, febrile infectious disease of cattle caused by the bovine ephemeral fever virus. This virus is primarily transmitted through blood-sucking insects (such as mosquitoes, midges, and horseflies), and is characterized by rapid spread and high morbidity. During epidemic seasons, the incidence rate in cattle herds can reach 50%-80%, causing significant economic losses to the cattle industry. Cattle infected with bovine ephemeral fever mainly exhibit symptoms such as high fever, lacrimation, salivation, difficulty breathing, and lameness, severely impacting their growth, reproductive performance, and milk production. For fattening cattle, the disease causes stunted growth and an increased feed conversion ratio, significantly reducing the economic benefits of cattle farming. For dairy cows, milk production drops sharply, and even after recovery, some infected dairy cows may not fully regain their milk production.

[0003] Currently, the isolation and identification technology of Bovine Ephemeral Fever Virus (BEFV) plays a crucial role in disease control. Traditional virus isolation methods primarily involve intracerebral inoculation in suckling mice followed by blind passage. This method is technically challenging, time-consuming, and susceptible to significant influence from individual animal variations. Another method involves blind passage via inoculation with BHK-21 or Vero cells, typically involving 3-5 passages, sometimes even 8. However, this method suffers from poor sensitivity, low isolation efficiency, and a long experimental cycle. Given the current limitations of BEFV isolation methods, the number of isolated strains in my country is relatively small, severely impacting BEFV epidemiological surveys and the development of corresponding control measures. Summary of the Invention

[0004] The purpose of this invention is to provide an efficient and rapid method for isolating bovine ephemeral fever virus.

[0005] This invention provides a method for isolating bovine ephemeral fever virus (BEFV). The specific steps of the isolation method are as follows: anticoagulated bovine blood samples are inoculated into KC (Culicoides sonorensis) cells at a volume ratio of 1:10 to 1:200 and cultured for 4-5 days. The virus is harvested, and the harvested virus is inoculated into a monolayer of BHK-21 cells at a volume ratio of 1:10 to 1:100 and cultured for 4-5 days. The cells are then passaged to obtain the first-generation virus solution exhibiting cytopathic effects.

[0006] Further, the volume ratio of bovine anticoagulated blood samples to KC cells was 1:10; the volume ratio of harvested virus to monolayer BHK-21 cells was 1:10.

[0007] Further, the volume ratio of bovine anticoagulated blood samples to KC cells was 1:200; and the volume ratio of harvested virus to monolayer BHK-21 cells was 1:100.

[0008] To further specify, viral fluid exhibiting cytopathic effects is identified as positive.

[0009] To further specify, the identification methods are RT-PCR and IFA.

[0010] Beneficial Effects: Addressing the technical deficiencies in existing technologies, this invention provides a highly efficient method for isolating bovine ephemeral fever virus (BEFV) inactivated virus, and isolates a candidate BEFV vaccine strain, BEFV / HN3 / 2024, and provides a method for preparing an inactivated vaccine using this strain. The virus strain of this invention can proliferate well on BHK-21 cells, exhibiting high viral titers, high immunogenicity, and good genetic stability. The inactivated vaccine prepared using this virus strain has high immunogenicity, is safe and reliable, and provides excellent immunoprotective effect against BEFV infection. Furthermore, the virus strain of this invention can also be used for screening, preparing, detecting, and / or using BEFV antibodies, as well as for preparing BEFV infection cell models or animal models, and subsequently for research on the pathogenesis of BEFV infection, screening antiviral drugs, developing new diagnostic and / or therapeutic strategies, and evaluating the immunoprotective effect of vaccines, demonstrating broad clinical application value.

[0011] [Biological Preservation Information]: A strain of bovine ephemeral fever virus, named BEFV / HN3 / 2024, with accession number CGMCC No. 46379, deposited on February 19, 2025, at the China General Microbiological Culture Collection Center, Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Attached Figure Description

[0012] Figure 1 The results of RT-PCR detection of BEFV clinical samples in this invention are shown below; where 1~24: clinical anticoagulated blood samples 1~24; positive: BEFV positive control; negative: negative control (water); M is DL2000 Marker, from top to bottom: 2000 bp, 1000 bp, 750 bp, 500 bp, 250 bp;

[0013] Figure 2The study observed the cytopathic effect (CPE) induced by inoculating BHK-21 cells with the BEFV / HN3 / 2024 strain of this invention; wherein, Figure 2 In Figure A, BHK-21 cells were inoculated with the improved isolate BEFV / HN3 / 2024. Figure 2 B cells in the middle section are normal BHK-21 cells; Figure 2 In the middle section, the HN3 / 2024 strain, isolated using the traditional virus separation method, was inoculated into BHK-21 cells;

[0014] Figure 3 The observation of cytopathic effect (CPE) in cells of the BEFV / HN3 / 2024 strain after inoculation with BHK-21 was conducted for the present invention; wherein, Figure 3 In the case of BHK-21 cells, strain A (BEFV / HN3 / 2024) was inoculated and showed specific green fluorescence. Figure 3 In cell B, the cells are normal BHK-21 cells and do not exhibit green fluorescence. Figure 3 When HN3 / 2024 strain was inoculated into BHK-21 cells, it exhibited specific green fluorescence. Detailed Implementation

[0015] In the following examples, the BHK-21 cell culture medium was DMEM medium containing 10% fetal bovine serum, and the cell maintenance medium was DMEM cell culture medium without fetal bovine serum; the KC cell culture medium was Schneider's Drosophila medium (Gibco, 21720024) containing 10% fetal bovine serum; all the above culture media contained 200 μg / ml penicillin and 200 μg / ml streptomycin in DMEM medium.

[0016] Example 1: Isolation and identification of bovine ephemeral fever virus strain BEFV / HN3 / 2024

[0017] 1. Sample processing:

[0018] Twenty-four anticoagulated blood samples were collected from diseased cattle in Henan Province. After three freeze-thaw cycles, the supernatant was collected by centrifugation, filtered through a 0.22μm filter membrane for sterilization, and stored at -70℃ for later use.

[0019] 2. RT-PCR detection:

[0020] Based on the BEFV G gene sequence published in Genbank, two primers were designed in the conserved region using Oligo 6.0. The primer sequences are: upstream primer BEFV-F: 5`-AGAGCTTGGTGTGAATAC-3`, and downstream primer BEFV-L: 5`-CCAACCTACAACAGCAGATA-3`. The expected amplification length is 420 bp. The primers were synthesized by Harbin Ruiboxingke Biotechnology Co., Ltd.

[0021] RNA extraction was performed according to the kit instructions to obtain sample RNA. The reverse transcription system consisted of: 10 μL 5×PrimeScript Buffer, 6 μL dNTPs, 6 μL BEFV-L, 1 μL RRI, 1 μL PrimeScript, and 26 μL template RNA. The reverse transcription program was: 25℃ for 10 min, then 42℃ for 1 h. The obtained cDNA was used for subsequent PCR reactions. Using specific primers, the corresponding BEFV gene fragment was amplified by RT-PCR. The PCR system consisted of: 10 μL 5×PrimeSTAR Buffer, 4 μL dNTPs, 2 μL BEFV-L, 2 μL BEFV-F, 0.5 μL PrimeSTAR, 29.5 μL ddH2O, and 2 μL cDNA. The program was: 98℃ for 2 min, 98℃ for 10 s, 55℃ for 15 s, 72℃ for 1 min, and 72℃ for 10 min, for 35 cycles. The PCR products were detected by 1.0% agarose gel electrophoresis, and the results showed that ( Figure 1 Of the 24 samples, 17 samples amplified specific bands, consistent with the expected size of 420bp, indicating that these 17 samples were all BEFV positive.

[0022] 3. Virus isolation:

[0023] Samples identified as positive by RT-PCR in the above steps were inoculated into KC cells (Culicoides sonorensis KC, derived from Culicoides midge larvae) at a ratio of 1:100 for primary culture. The cells were then placed in a 30°C, 5% CO2 incubator for 4-5 days. The virus was harvested and inoculated into BHK-21 cells that had grown into a monolayer. The cells were passaged for another 4-5 days. When cytopathic effects appeared, the virus was harvested and identified. Cytopathic effects were observed in the first generation.

[0024] As a result, obvious CPE (congenital cerebral palsy) was observed in 13 samples in the first generation of BHK-21 cells. Figure 2 Because the viral load varies in different samples, the time lag before and after the appearance of CPE is 1-3 days. This improved method has a virus isolation cycle of approximately 8-10 days, with a virus isolation rate of 76.5%.

[0025] Meanwhile, following traditional cell isolation methods, positive samples were directly inoculated into BHK-21 and Vero cells at a 1:100 ratio for blind passage. Viruses were harvested and identified when cytopathic effects (CPE) appeared. If no CPE appeared, blind passage continued until CPE occurred, at which point identification was performed. Results showed that six virus strains were isolated from BHK-21 cells, with the earliest CPE appearing at passage 4 and the latest at passage 6, an experimental period of 16–24 days, and a virus isolation rate of 35.3%. Five virus strains were isolated from Vero cells, with the earliest CPE appearing at passage 5 and the longest at passage 8, an experimental period of 20–32 days, and virus isolation rates of 29.4%. Therefore, the traditional BHK-21 or Vero cell inoculation method has a long experimental period and a relatively low virus isolation rate.

[0026] 4. Identification by RT-PCR and IFA

[0027] Viruses exhibiting cytopathic effects were identified using RT-PCR and IFA. The RT-PCR method was the same as described above. Indirect immunofluorescence (IFA): Harvested viruses were inoculated at a 1% ratio into two wells of a well-grown BHK-21 cell monolayer (100 μl per well). The cells were incubated at 37°C in a 5% CO2 incubator for 36 hours. The cell culture medium was discarded, and 100 μl of cold acetone fixative was added to each well for 15 minutes. The fixative was discarded, and the cells were allowed to air dry. 100 μl of PBS containing 5% skim milk was added to each well, and the cells were placed on a shaker and gently shaken at 37°C for 1 hour to block the cell culture plate. The blocking solution was discarded, and the cells were washed three times with PBS and then dried. 100 μl of BEFV-positive serum (obtained by centrifugation of whole blood from bovines immunized with BEFV inactivated vaccine) diluted 100-fold with pH 7.4 PBS was added to both the BEFV-infected cell wells and the uninfected cell control wells. The wells were incubated at 37°C for 1 hour, followed by washing three times with PBS for 3 minutes each time. Then, 100 μl of FITC-labeled rabbit anti-bovine IgG diluted 100-fold with pH 7.4 PBS was added, and the wells were incubated at 37°C for 1 hour. The wells were washed three times with PBS for 3 minutes each time. Finally, 100 μl of PBS was added to each well, and the wells were inverted and observed under a fluorescence microscope. The results showed that the positive control wells exhibited specific green fluorescence (…). Figure 3 The negative control wells showed no green fluorescence. The test sample wells showed specific green fluorescence, indicating that all were BEFV positive. Based on these results, RT-PCR was performed using bovine ephemeral fever-specific primers, and IFA was performed using bovine ephemeral fever-positive serum. Therefore, both methods confirmed that the samples were bovine ephemeral fever virus.

[0028] 5. Improvement of method conditions optimization

[0029] (1) Proportion of first-generation drug recipients

[0030] Positive anticoagulated blood samples were inoculated into KC cells at volume ratios of 1:10, 1:50, 1:100, 1:200, and 1:300. Virus was harvested 6 days post-inoculation, and then inoculated into BHK-21 cells at a 1:100 ratio. The results showed that the 1:300 inoculation sample into KC cells did not exhibit CPE, and RT-PCR was negative. All other inoculation ratios showed CPE, and both RT-PCR and IFA confirmed BEFV. This indicates that virus isolation is possible at inoculation ratios from 1:10 to 1:200, but not at ratios less than 1:300. Therefore, the optimal inoculation ratio for KC cells was determined to be 1:10–1:200.

[0031] (2) Passage inoculation ratio

[0032] Virus fluid harvested after inoculation of KC cells was inoculated into BHK-21 cells at ratios of 1:10, 1:50, 1:100, 1:200, and 1:300. Virus was harvested 4 days after inoculation. The results showed that CPE occurred in the first generation of BHK-21 cells inoculated at ratios of 1:10 to 1:100, while CPE occurred in the second generation of BHK-21 cells inoculated at ratios of 1:200 and 1:300. Therefore, 1:10 to 1:100 was determined to be the appropriate inoculation ratio for passage of BHK-21 cells.

[0033] (3) Initial drug collection time

[0034] Positive anticoagulated blood samples were inoculated into KC cells at a volume ratio of 1:100. Viral fluid was harvested on days 3, 4, 5, and 6, and then inoculated into BHK-21 cells at a ratio of 1:100. The results showed that no CPE was observed in samples inoculated into KC cells on day 3, but CPE was observed in samples inoculated on days 4-5. This indicates that the optimal time for virus harvesting after inoculation into KC cells is 4-5 days.

[0035] (4) Time of drug collection through intergeneration

[0036] Virus fluid harvested from KC cells was inoculated into BHK-21 cells at a 1:100 ratio. CPE was observed daily, with the earliest appearance being 2 days and the latest being 4 days. Virus fluid was harvested on days 3, 4, and 5 for RT-PCR identification. All samples showing CPE were BEFV positive. To ensure sufficient viral replication in BHK-21 cells, the optimal harvest time was determined to be 4–5 days.

[0037] Example 2: Virus passage culture and virus titer determination

[0038] Thirteen BEFV strains that were positive by RT-PCR and IFA were stored at -70°C. One BEFV isolate (BEFV / HN3 / 2024) obtained by the modified method was selected as a candidate vaccine strain for subsequent passage culture and titer determination. At the same time, an isolate (HN3-BHK) isolated by conventional BHK-21 cells was selected as a parallel control. This virus came from the same anticoagulated blood sample as the BEFV / HN3 / 2024 strain.

[0039] 1. Virus propagation culture

[0040] Isolates BEFV / HN3 / 2024 and HN3-BHK were inoculated into BHK-21 monolayer cells at a 1:100 ratio and cultured for 48–72 hours. Cell cultures were then harvested. The cells were continuously passaged in BHK-21 to allow for cell adaptation and increase viral titer. After 10 passages, the harvested virus was stored at -70°C. RT-PCR was performed on the passaged virus.

[0041] The results showed that each generation of the virus could amplify a specific target band.

[0042] 2. Virus titer determination

[0043] Virus suspension was inoculated into BHK-21 cells for virus content detection. Once the cells reached a monolayer, the monolayer was digested with 0.1% trypsin. The cell suspension was then seeded into 96-well plates at 100 μl / well. After the cells reached a monolayer, the culture medium in the wells was discarded. 100 μl of virus suspension diluted 10-fold with cell maintenance medium was added to each well. -1 ~10 -8 Each dilution was replicated in 4 wells, with normal BHK-21 cells as a control. Cells were incubated at 37°C in a 5% CO2 incubator for 72 hours. Cytopathic effects were observed using an inverted microscope. The number of wells showing cytopathic effects and the number of wells without cytopathic effects were recorded. The Reed-Muench method was used to calculate the viral TCID. 50 .

[0044] The results showed that specific cytopathic effects were observed in BHK-21 cells inoculated with the virus, and the titer (TCID) of the BEFV / HN3 / 2024 strain was [not specified]. 50 ) is 1×10 -6.0 / ml~1×10 -7.0 / ml, titer of HN3-BHK strain (TCID) 50 1×10 -3.5 / ml~1×10 -4The titer of the two strains was significantly different, as shown in Table 1. Because the HN3-BHK strain virus (an isolate obtained by traditional methods after only five passages in BHK-21 cells, named HN3-BHK; both are viruses obtained from the same anticoagulated blood sample, only the isolation methods differ) had a low titer, it could not be used for vaccine production and application. Therefore, the BEFV / HN3 / 2024 strain was selected for subsequent vaccine research.

[0045] Table 1. Virus titers of isolated virus strains

[0046]

[0047] Example 3: Preparation and Immunization Effect of Inactivated Vaccine

[0048] 1. Viral reproduction

[0049] The 10th generation of BEFV / HN3 / 2024 virus seed was diluted with DMEM cell maintenance medium and then inoculated into well-grown BHK-21 cells at 0.1 MOI. The cells were cultured at 37°C in a cell incubator containing 5% CO2 for 3 days. The cell culture was then harvested, and the virus titer (TCID) was determined. 50 ) is 1×10 -7.0 / ml, used as an antigen for vaccine preparation.

[0050] 2. Virus inactivation

[0051] Centrifuge the above cell culture at 8000 rpm for 10 min to remove cell debris, and inactivate it with diethyleneimine (BEI). Add 1% of 0.05 mol / L BEI solution while stirring to ensure thorough mixing. Inactivate at 37°C for 24 hours. Then add filtered sterile 1 mol / L sodium thiosulfate solution to the inactivated virus solution, making the final volume of sodium thiosulfate 30% of the volume of BEI solution added. Mix thoroughly to terminate the inactivation process and obtain the BEFV virus inactivated solution (i.e., bovine ephemeral fever virus inactivated antigen). Store at 2–8°C.

[0052] 3. Vaccine preparation and evaluation of immunization efficacy

[0053] Immunization experiment: Take 2 ml of bovine ephemeral fever virus inactivated antigen (virus content 2×10⁻⁶). 7 TCID 50 Emulsify 2 ml of ISA 206 adjuvant at a 1:1 volume ratio. Vaccinate healthy cattle aged 5-12 months (n=16) with 4 ml intramuscularly per head, followed by a booster immunization with the same dose 21 days later. Blood samples were collected 21 days after the second immunization, and the neutralizing antibody titer was measured to be 1:1024~1:4096.

[0054] This indicates that the BEFV / HN3 / 2024 inactivated virus has good immunogenicity.

[0055] Challenge experiment: Immunized cattle (n=5) were selected for the challenge protection experiment. Each cow was injected with 2ml of blood virus intravenously. After challenge, the cattle were observed for 14 consecutive days. The body temperature was measured daily, and the clinical symptoms were observed and recorded. Blood was collected on days 7, 10 and 14 after challenge. The comprehensive evaluation of the body temperature (body temperature rises above 40.5℃ and lasts for at least 2 days), clinical symptoms (symptoms such as lethargy and loss of appetite) and virus excretion detection was carried out.

[0056] The results showed that four cows did not exhibit clinical symptoms of BEFV, one cow developed transient mild fever, and all cows in the blank control group developed the disease. The protection rate against challenge with the virus reached over 4 / 5, meaning it provided over 80% protection against virulent infection. These results indicate that the inactivated vaccine can induce strong neutralizing antibodies and resist virulent infection, thus it can be considered a candidate strain for BEFV vaccine.

Claims

1. An isolation method of Bovine Ephemeral Fever Virus (BEFV) characterized in that, The specific steps of the separation method are as follows: Bovine anticoagulated blood samples are inoculated into Culicoides sonorensis KC cells at a volume ratio of 1:10-1:200 and cultured for 4-5 days. The virus is harvested, and the harvested virus is inoculated into a monolayer of BHK-21 cells at a volume ratio of 1:10-1:100 and cultured for 4-5 days. The cells are then passaged to obtain the first generation of virus solution exhibiting cytopathic effects.

2. The separation method of claim 1, wherein, The volume ratio of bovine anticoagulated blood samples to KC cells was 1:10; the volume ratio of harvested virus to monolayer BHK-21 cells was 1:

10.

3. The separation method of claim 1, wherein, The volume ratio of bovine anticoagulated blood samples to KC cells was 1:200; the volume ratio of harvested virus to monolayer BHK-21 cells was 1:

100.

4. The separation method of claim 1, wherein, Viral fluid exhibiting cytopathic effects tested positive.

5. The separation method according to claim 4, characterized in that, The identification methods were RT-PCR and IFA.

Images