Separation method of bovine ephemeral fever virus, obtained bovine ephemeral fever virus and application of bovine ephemeral fever virus

Abstract

The invention discloses a separation method of a bovine ephemeral fever virus, the obtained bovine ephemeral fever virus and application of the bovine ephemeral fever virus. Belongs to the technical field of bovine ephemeral fever virus separation. The invention aims to provide an efficient and rapid method for separating the bovine ephemeral fever virus. The invention provides a method for separating bovine ephemeral fever virus, which comprises the following steps: inoculating a bovine anticoagulant sample to KC cells for culture, harvesting virus, inoculating to grow into monolayer BHK-21 cells, and carrying out passage to obtain passage cells with cytopathy of the first generation; the virus strain can also be used for screening, preparing and detecting a bovine ephemeral fever virus antibody and preparing a bovine ephemeral fever virus infected animal model.

Description

Technical Field

[0001] The present invention belongs to the technical field of bovine ephemeral fever virus isolation, and particularly relates to a method for isolating bovine ephemeral fever virus, the obtained bovine ephemeral fever virus and its application. Background Art

[0002] Bovine ephemeral fever (BEF), also known as three-day fever or temporary fever, is an acute and febrile infectious disease of cattle caused by bovine ephemeral fever virus. The virus is mainly transmitted by blood-sucking insects (such as mosquitoes, midges, horseflies, etc.), and has the characteristics of fast transmission speed and high incidence rate. During the epidemic season, the incidence rate of cattle herds can reach 50% - 80%, bringing huge economic losses to the cattle industry. Cattle infected with bovine ephemeral fever mainly show symptoms such as high fever, tearing, salivation, dyspnea, lameness, etc., which seriously affect the growth and development, reproductive performance and milk yield of cattle. For fattening cattle, the growth is retarded after getting sick, the feed-to-meat ratio increases, and the breeding economic benefit is greatly reduced; for dairy cows, the milk yield drops sharply, and even after some sick dairy cows recover, the milk production performance is difficult to fully recover.

[0003] At present, the isolation and identification technology of bovine ephemeral fever virus (BEFV) plays a key role in epidemic disease prevention and control. The traditional virus isolation method is mainly intracerebral inoculation of suckling mice and blind passage. This method has the disadvantages of high operation difficulty, long cycle, and great influence of animal individual differences on the results. In addition, there is also blind passage by inoculating BHK-21 or Vero cells, generally passing 3 - 5 generations, and some even passing 8 generations. This method has poor sensitivity, low isolation efficiency, and long experimental cycle. Due to the limitations of the current BEFV isolation method, the number of strains isolated in China is small at present, which seriously affects the epidemiological investigation of BEFV in China and the formulation of corresponding prevention and control measures. Summary of the Invention

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

[0005] The present invention provides a method for isolating bovine ephemeral fever virus (BEFV). The specific steps of the isolation method are as follows: inoculate the anticoagulant sample of cattle onto KC (Culicoides sonorensis) cells at a volume ratio of 1:10 - 1:200 and culture for 4 - 5 days, harvest the virus, and then inoculate the harvested virus onto monolayer BHK-21 cells at a volume ratio of 1:10 - 1:100 and culture for 4 - 5 days to obtain the virus solution with cytopathic effect in the first passage.

[0006] Further limitation: the volume ratio of the anticoagulant sample of cattle to KC cells is 1:10; the volume ratio of the harvested virus to monolayer BHK-21 cells is 1:10.

[0007] Further limitation: the volume ratio of the anticoagulant sample of cattle to KC cells is 1:200; the volume ratio of the harvested virus to monolayer BHK-21 cells is 1:100.

[0008] Further limitation: the virus solution showing cytopathic effect is identified as positive.

[0009] Further limitation: the identification methods are RT-PCR and IFA.

[0010] Beneficial effects: Aiming at the technical defects existing in the prior art, the present invention provides a method for highly efficient isolation of inactivated bovine ephemeral fever virus, and an inactivated vaccine candidate strain BEFV / HN3 / 2024 of bovine ephemeral fever virus is isolated, and a method for preparing an inactivated vaccine using this virus strain is provided. The virus strain of the present invention can proliferate well on BHK-21 cells, has a high virus titer, high immunogenicity and good genetic stability. The inactivated vaccine prepared using this virus strain has high immunogenicity, is safe and reliable, and has a good immune protection effect against bovine ephemeral fever virus infection. In addition, the virus strain of the present invention can also be used for screening, preparing, detecting and / or bovine ephemeral fever virus antibodies, as well as for preparing a bovine ephemeral fever virus-infected cell model or animal model, and further for the research on the pathogenic mechanism of BEFV infection, antiviral drug screening, development of new diagnostic strategies and / or treatment strategies, and evaluation of the immune protection effect of vaccines, etc., and has wide clinical application value.

[0011]

Biological deposit information

[0012] Figure 1 This is the RT-PCR detection result of BEFV clinical samples of the present invention; among them, 1-24: clinical anticoagulant samples 1-24; positive: BEFV positive control; negative: negative control (water), M is DL2000 Marker, from top to bottom are 2000 bp, 1000 bp, 750 bp, 500 bp, 250 bp in turn; Figure 2Observation of cytopathic effect (CPE) caused by inoculating BHK-21 cells with BEFV / HN3 / 2024 strain of the present invention; among them, Figure 2 In A, the BHK-21 cells were inoculated with the isolated strain BEFV / HN3 / 2024 by the improved method; Figure 2 In B, the normal BHK-21 cells; Figure 2 In C, the BHK-21 cells were inoculated with the isolated strain HN3 / 2024 by the traditional virus subculture method; Figure 3 Observation of cytopathic effect (CPE) of BHK-21 cells after inoculation with BEFV / HN3 / 2024 strain of the present invention; among them, Figure 3 In A, the BHK-21 cells were inoculated with BEFV / HN3 / 2024 strain, showing specific green fluorescence; Figure 3 In B, the normal BHK-21 cells, without green fluorescence; Figure 3 In C, the BHK-21 cells were inoculated with HN3 / 2024 strain, showing specific green fluorescence. Detailed implementation mode

[0013] The BHK-21 cell culture medium in the following examples 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; both of the above culture media contained 200 μg / ml penicillin and 200 μg / ml streptomycin in DMEM medium.

[0014] Example 1: Isolation and identification of bovine ephemeral fever virus BEFV / HN3 / 2024 strain 1. Treatment of samples: Twenty-four anticoagulant samples collected from diseased cattle in Henan were frozen and thawed 3 times, then centrifuged to take the supernatant, filtered through a 0.22 μm filter membrane to remove bacteria, and stored at -70°C in a refrigerator for later use.

[0015] 2. RT-PCR detection: According to the BEFV G gene sequence published on Genbank, two primers were designed in the conserved region using Oligo 6.0. The primer sequences were: upstream primer BEFV-F: 5`-AGAGCTTGGTGTGAATAC-3`, downstream primer BEFV-L: 5`- CCAACCTACAACAGCAGATA-3`. The expected amplified length was 420 bp, and the primers were synthesized by Harbin Ruibo Xingke Biotechnology Co., Ltd.

[0016] RNA extraction was carried out according to the kit instructions to obtain sample RNA. The reverse transcription system was as follows: 5×PrimeScript Buffer 10 μL, dNTP 6 μL, BEFV-L 6 μL, RRI 1 μL, PrimeScript 1 μL, template RNA 26 μL. The reverse transcription program was: 25°C for 10 min, 42°C for 1 h. The obtained cDNA was used for subsequent PCR reactions. Using specific primers, the corresponding gene fragment of BEFV was amplified by RT-PCR technology. The PCR system was: 5×PrimeSTAR Buffer 10 μL, dNTP 4 μL, BEFV-L 2 μL, BEFV-F 2 μL, PrimeSTAR 0.5 μL, ddH2O 29.5 μL, cDNA 2 μL. The program was: 98°C for 2 min, 98°C for 10 s, 55°C for 15 s, 72°C for 1 min, 72°C for 10 min, 35 cycles. The PCR products were detected by 1.0% agarose gel electrophoresis. The results showed ( Figure 1 ), specific bands were amplified from 17 out of 24 samples, which were consistent with the expected size of 420 bp, indicating that these 17 samples were all positive for BEFV.

[0017] 3. Virus isolation: The samples identified as positive by RT-PCR in the above steps were inoculated into KC cells (full name Culicoides sonorensis KC, sourced from Culicoides sonorensis larvae) at a ratio of 1:100 for primary culture. They were placed statically in an incubator at 30°C and 5% CO2. After 4 - 5 days, the virus was harvested and inoculated into confluent BHK-21 cells. Subculture continued for 4 - 5 days. When cytopathic effects appeared, the virus was harvested and identified. Cytopathic effects could be observed in the first generation.

[0018] Results, obvious CPE ( Figure 2 ) appeared in the first generation of BHK-21 cells inoculated with 13 samples. Due to different virus contents in different samples, the appearance of CPE differed by 1 - 3 days. The virus isolation cycle using this improved method was about 8 - 10 days, and the virus isolation rate was 76.5%.

[0019] Meanwhile, according to the traditional cell separation method, the positive samples were directly inoculated into BHK-21 and Vero cells at a ratio of 1:100 respectively for blind passage. When cytopathic effect (CPE) appeared, the virus was harvested and identified. If CPE did not appear, continuous blind passage on the cells was carried out until CPE appeared for identification. The results showed that 6 strains of virus were isolated from BHK-21 cells. The earliest appearance of CPE was in the 4th generation, and the latest was in the 6th generation. The experimental period was 16 - 24 days, and the virus isolation rate was 35.3%. 5 strains of virus were isolated from Vero cells. The earliest appearance of CPE was in the 5th generation, and the longest was in the 8th generation. The experimental period was 20 - 32 days, and the virus isolation rates were 29.4% respectively. Therefore, the traditional method of inoculating BHK-21 cells or Vero cells has a long experimental period and a relatively low virus isolation rate.

[0020] 4. Identification by RT-PCR and IFA The virus with CPE was identified by RT-PCR and IFA. The RT-PCR method was the same as above. Indirect immunofluorescence assay (IFA): The harvested virus was inoculated into 2 wells of a 96-well plate with a monolayer of well-grown BHK-21 cells at a ratio of 1%, 100 μl per well, and continued to be cultured in a cell incubator at 37°C with 5% CO2 for 36 hours. The cell culture medium was discarded, and 100 μl of cold acetone fixative was added to each well for 15 minutes of fixation. The fixative was discarded and air-dried naturally. 100 μl of PBS containing 5% skimmed milk was added to each well, placed on a shaker, and gently shaken at 37°C for 1 hour for blocking. The blocking solution in the cell culture plate was discarded, washed 3 times with PBS, and spun dry. 100 μl of BEFV positive serum diluted 100-fold with pH 7.4 PBS (serum obtained by centrifuging the whole blood collected from cattle immunized with BEFV inactivated vaccine) was added to the BEFV-infected cell wells and the non-infected cell control wells respectively, incubated in a 37°C incubator for 1 hour, then washed 3 times with PBS, 3 minutes each time; 100 μl of FITC-labeled rabbit anti-bovine IgG diluted 100-fold with pH 7.4 PBS was added, incubated in a 37°C incubator for 1 hour; washed 3 times with PBS, 3 minutes each time, and finally 100 μl of PBS was added to each well, and the results were observed under an inverted fluorescence microscope. The results showed that specific green fluorescence appeared in the positive control wells ( Figure 3 ), and no green fluorescence appeared in the negative control wells. Specific green fluorescence in the wells of the samples to be tested indicated that they were all positive for BEFV. Based on the above results, RT-PCR was performed using bovine epizootic fever-specific primers, and IFA was performed using bovine epizootic fever positive serum. Therefore, both identifications proved that it was bovine epizootic fever virus.

[0021] 5. Optimization of the improved method conditions (1) Initial inoculation ratio The positive anticoagulant samples were inoculated with KC cells at volume ratios of 1:10, 1:50, 1:100, 1:200, and 1:300. The virus was harvested 6 days after inoculation, and then inoculated with BHK-21 cells at a ratio of 1:100. As a result, no CPE was observed in the samples inoculated with KC cells at a ratio of 1:300, and RT-PCR identification was negative. CPE was observed in other inoculation ratios, and both RT-PCR and IFA identifications were BEFV. This indicates that the virus can be isolated at inoculation ratios of 1:10 to 1:200, and the virus cannot be isolated at ratios less than 1:300. Therefore, the inoculation ratio on KC cells was determined to be 1:10 to 1:200.

[0022] (2)Subculture inoculation ratio The virus solution harvested after inoculating KC cells was inoculated with BHK-21 cells at ratios of 1:10, 1:50, 1:100, 1:200, and 1:300. The virus was harvested 4 days after inoculation. As a result, CPE appeared in the first generation when inoculating BHK-21 cells at ratios of 1:10 to 1:100, while CPE appeared in the second generation when inoculating BHK-21 cells at ratios of 1:200 and 1:300. Therefore, the subculture inoculation ratio for BHK-21 cells was determined to be 1:10 to 1:100.

[0023] (3)Primary harvest time of virus The positive anticoagulant samples were inoculated with KC cells at a volume ratio of 1:100. The virus solution was harvested on the 3rd, 4th, 5th, and 6th days respectively, and then inoculated with BHK-21 cells at a ratio of 1:100. As a result, no CPE was observed in the samples inoculated with KC cells for 3 days, and CPE was observed in the samples from 4 to 5 days. This indicates that the optimal virus harvest time after inoculating KC cells is 4 - 5 days.

[0024] (4)Subculture harvest time The virus solution harvested after inoculating KC cells was inoculated with BHK-21 cells at a ratio of 1:100. The CPE situation was observed every day. CPE appeared as early as 2 days and as late as 4 days. The virus solution was harvested on the 3rd, 4th, and 5th days respectively for RT-PCR identification. As a result, all the samples with CPE were positive for BEFV. In order to allow the virus to fully replicate on BHK-21 cells, the optimal harvest time was determined to be 4 - 5 days.

[0025] Example 2: Virus subculture and virus titer determination Thirteen BEFV strains identified as positive by RT-PCR and IFA were stored in a -70°C refrigerator. A BEFV isolate (BEFV / HN3 / 2024 strain) obtained by the improved method of isolation was selected as the candidate vaccine strain for subsequent subculture and titer determination. At the same time, an isolate (HN3-BHK) isolated from traditional BHK-21 cells was selected for parallel control. This virus and the BEFV / HN3 / 2024 strain were from the same anticoagulant sample.

[0026] 1. Proliferation and culture of virus Inoculate the isolate BEFV / HN3 / 2024 strain and HN3-BHK into BHK-21 monolayer cells at an inoculation ratio of 1:100 and culture for 48 - 72 hours, then harvest the cell culture. Continuously passage in BHK-21 cells to adapt the cells and increase the virus titer. Continuously passage until the 10th generation, and store the harvested virus at -70 °C. At the same time, perform RT-PCR detection on the virus after continuous passage.

[0027] The results showed that specific target bands could be amplified for each generation of virus.

[0028] 2. Determination of virus titer Inoculate the virus solution into BHK-21 cells for virus content detection. When the cells grow to a monolayer, digest the BHK-21 cells grown into a monolayer with 0.1% trypsin, inoculate the cell suspension into a 96-well plate, 100 μl / well. After the cells grow to a monolayer, discard the culture medium in the wells; add 100 μl of virus solution diluted 10-fold serially with cell maintenance medium (10 -1 ~10 -8 ) to each well, repeat 4 wells for each dilution, set normal BHK-21 cells as the control, culture in an incubator at 37 °C and 5% CO2 for 72 hours, observe the cytopathic effect through an inverted microscope, record the number of wells with cytopathic effect and the number of wells without cytopathic effect, and calculate the virus TCID 50 using the Reed-Muench method.

[0029] As shown by the results, specific cytopathic effects were visible in the BHK-21 cells inoculated with the virus. The titer (TCID 50 ) of the BEFV / HN3 / 2024 strain was 1×10 -6.0 / ml - 1×10 -7.0 / ml, and the titer (TCID 50 ) of the HN3-BHK strain was 1×10 -3.5 / ml - 1×10 -4 / ml. There was a significant difference between the two, and the results are shown in Table 1. Since the titer of the HN3-BHK strain virus (this is an isolate obtained by passing through BHK-21 cells 5 times using the traditional method, named HN3-BHK. The two are viruses obtained from the same anticoagulated blood, only the isolation methods are different) was relatively low and could not be used for vaccine production and application, the BEFV / HN3 / 2024 strain was selected for subsequent vaccine research.

[0030] Table 1. Virus titers of isolated virus strains

[0031] Example 3: Preparation of Inactivated Vaccine and Immunization Effect 1. Propagation of Virus The 10th generation virus seed of BEFV / HN3 / 2024 was diluted with DMEM cell maintenance solution, and then inoculated into well-grown BHK-21 cells at 0.1 MOI. The cells were cultured in a 37°C cell incubator containing 5% CO2 for 3 days, and the cell culture was harvested. The virus titer (TCID 50 ) was 1×10 -7.0 / ml, which was used as the antigen for vaccine preparation.

[0032] 2. Virus Inactivation The above cell culture was centrifuged at 8000 rpm for 10 min to remove cell debris, and inactivated with beta-propiolactone (BEI), that is, 1% of 0.05 mol / L BEI solution was added while stirring to make it fully mixed. It was inactivated at 37°C for 24 hours, and then 1 mol / L sodium thiosulfate solution filtered and sterilized was added to the inactivated virus solution to make the final volume of sodium thiosulfate 30% of the volume of the added BEI solution. After thorough mixing, the inactivation was terminated to obtain the BEFV virus inactivated solution (i.e., the inactivated antigen of bovine ephemeral fever virus), which was stored at 2 - 8°C.

[0033] 3. Vaccine Preparation and Evaluation of Immunization Effect Immunization experiment: Take 2 ml of inactivated antigen of bovine ephemeral fever virus (virus content was 2×10 7 TCID 50 ) and emulsify it with 2 ml of ISA 206 adjuvant at a volume ratio of 1:1. Five to twelve-month-old healthy cattle (n = 16) were inoculated, and each cattle was intramuscularly injected with 4 mL, and boosted with the same dose once at an interval of 21 days. Blood was collected 21 days after the second immunization, and the neutralizing antibody titer was detected to be 1:1024 - 1:4096.

[0034] It showed that the immunogenicity of the inactivated virus of BEFV / HN3 / 2024 was good.

[0035] Challenge experiment: Five immunized cattle (n = 5) were selected for the challenge protection experiment. Each cattle was intravenously injected with 2 ml of blood virus. After challenge, they were continuously observed for 14 days. The body temperature was measured every day, and the clinical symptoms were observed and recorded. Blood was collected on the 7th, 10th, and 14th days after challenge for comprehensive index evaluation of body temperature (body temperature increased by more than 40.5°C and lasted for at least 2 days), clinical symptoms (symptoms such as listlessness and loss of appetite) and virus excretion detection.

[0036] As a result, no clinical symptoms of BEFV were observed in 4 cattle, and 1 cattle showed transient mild fever symptoms. All cattle in the blank control group developed the disease. The protection rate against challenge could reach over 4 / 5, that is, more than 80% protection against virulent challenge. The above results indicate that the inactivated vaccine can induce strong neutralizing antibodies and can resist virulent infection. Therefore, it can be used as a candidate strain for BEFV vaccine.

Claims

1. A method for isolating Bovine Ephemeral Fever Virus (BEFV), characterized in that, The specific steps of the separation method are as follows: inoculate the anticoagulant sample of cattle onto KC (Culicoides sonorensis) cells at a volume ratio of 1:10 - 1:200 and culture for 4 - 5 days, harvest the virus, and then inoculate the harvested virus onto monolayer BHK-21 cells at a volume ratio of 1:10 - 1:100 and culture for 4 - 5 days to obtain the virus solution with cytopathic effect in the first passage.

2. The separation method according to claim 1, wherein The volume ratio of the anticoagulant sample of cattle to KC cells is 1:10; the volume ratio of the harvested virus to monolayer BHK-21 cells is 1:

10.

3. The separation method according to claim 1, wherein The volume ratio of the anticoagulant sample of cattle to KC cells is 1:200; the volume ratio of the harvested virus to monolayer BHK-21 cells is 1:

100.

4. The separation method according to claim 1, characterized in that, The virus solution with cytopathic effect is identified as positive.

5. The separation method according to claim 1, wherein The identification methods are RT-PCR and IFA.

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