GI type Japanese encephalitis virus attenuated vaccine strain SD12-F120 and construction and application of infectious clone of GI type Japanese encephalitis virus attenuated vaccine strain SD12-F120

Abstract

The invention belongs to the field of molecular biology, and relates to a GI type Japanese encephalitis virus attenuated vaccine strain SD12-F120 and construction and application of infectious clone of the GI type Japanese encephalitis virus attenuated vaccine strain SD12-F120. In order to overcome the technical defects that an existing GIII type JEV vaccine is difficult to provide complete protection for GI type JEV, and the GIII type JEV vaccine is lacked in current practical application, the invention provides a GI type Japanese encephalitis virus attenuated vaccine strain SD12-F120 and an infectious clone construction method thereof, and the cDNA sequence of the attenuated vaccine strain SD12-F120 is shown as SEQ ID NO.1. The invention further provides a construction method of the infectious clone of the attenuated vaccine strain SD12-F120. The attenuated vaccine strain SD12-F120 provided by the invention has good immunogenicity and protection effect on the GI type Japanese encephalitis virus, and provides a new choice for development of vaccines for preventing the GI type Japanese encephalitis virus and screening of vaccine strains.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular biology and relates to a method for constructing an infectious clone of a GI-type Japanese encephalitis virus attenuated vaccine strain and an application thereof. Background Art

[0002] Japanese encephalitis (JE) is an important zoonosis caused by Japanese encephalitis virus (JEV). It is the most common viral encephalitis in the world and is widely spread in countries such as China, India, Indonesia, Bangladesh and Pakistan. JEV is prevalent in more than 24 countries worldwide, with an estimated 67,900 cases of JE reported each year, resulting in 10,000 deaths and approximately 15,000 neurological sequelae. JE is a natural epidemic disease. Many animals can be infected with this virus, but most of them are latent infections, especially cattle and sheep. Sick animals and the viremia stage during latent infection can become sources of infection, among which pigs are the main source of infection. JE is mainly transmitted through the skin through mosquito bites. Mosquitoes are not only vectors, but also reservoirs of the virus.

[0003] JEV is a single-stranded RNA virus belonging to the genus Flavivirus of the family Flaviviridae, with a genome length of approximately 11 kb. Based on the nucleotide sequence of the E gene or the complete viral genome, JEV is divided into five genotypes (GI, GII, GIII, GIV, and GV). JE studies have shown that JEV originated in the Indonesian-Malaysian region from a common ancestor, where it differentiated into five genotypes, of which two older, divergent genotypes (GIV and GV) are still circulating in the region, and three newly evolved genotypes (GI, GII, and GIII) have spread to other regions in recent decades. GIII was first discovered in Japan in 1935 and remained the most dominant genotype throughout Asia until the end of the 20th century. Previous surveillance data showed that GIII was replaced by GI in Japan in 1994, and since then, GI has become the main circulating JEV genotype in many Asian countries. Some recent studies have reported that GI replicates more efficiently than GIII in its JEV amplification hosts, which may be the reason why GI gradually replaced GIII as the new dominant genotype.

[0004] Vaccination against JE is the most effective control measure to prevent JEV infection. Currently, four different types of JE vaccines are available in different parts of the world: mouse brain-derived Zhongshan inactivated vaccine, cell culture-derived SA14-14-2 attenuated live vaccine (SA14-14-2 vaccine), culture-derived inactivated vaccine, and genetically engineered attenuated chimeric live vaccine, all of which are derived from the GIII strain. SA14-14-2 vaccine is a widely used vaccine in many Asian countries; however, the protective efficacy of GIII vaccine against G1 infection is not optimistic. In China and India, GI-type JEV viruses can still be isolated from patients vaccinated with SA14-14-2 vaccine. It can be seen that the current GIII-type JEV vaccine may not be able to provide complete protection against the GI type, so the development of a new GI-type JEV vaccine is imminent.

[0005] The Chinese journal article "Study on the Dynamic Distribution of Epidemic Japanese Encephalitis Virus GI Type Strain SD12 in C57BL / 6 Mice" used the qRT-PCR method to detect the dynamic distribution of the strain in various tissues of mice. The study showed that after the genotype I epidemic Japanese encephalitis virus infects C57BL / 6 mice with damaged blood-brain barrier, it can quickly invade various tissues and organs, mainly infecting the liver, lungs, kidneys, cecum and brain. Mice with a complete blood-brain barrier have a certain resistance to the virus, and the virus content in various tissues is relatively low. Summary of the invention

[0006] In order to overcome the technical deficiency that the existing GIII-type JEV vaccine is difficult to provide complete protection against GI-type JEV and there is a lack of GIII-type JEV vaccines in practical applications, the present invention provides a GI-type Japanese encephalitis virus attenuated vaccine strain SD12-F120 and a method for constructing an infectious clone thereof, and a recombinant strain rSD12-F120 is obtained by performing reverse genetic operation on the GI-type Japanese encephalitis virus attenuated vaccine strain SD12-F120. The attenuated vaccine strain SD12-F120 of the present invention has good immunogenicity and protective effect against GI-type Japanese encephalitis virus, and provides a new choice for the development of vaccines for preventing GI-type Japanese encephalitis virus disease and the screening of vaccine strains.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] One of the purposes of the present invention is to provide an infectious clone of a GI type Japanese encephalitis virus attenuated vaccine strain. The attenuated vaccine strain is named SD12-F120, and its cDNA sequence is shown in SEQ ID NO.1.

[0009] The second object of the present invention is to provide a method for constructing the infectious clone of the attenuated vaccine strain SD12-F120 of the GI type Japanese encephalitis virus, which comprises the following steps:

[0010] (1) The nucleic acid fragments 1-2350, 2351-4470, 4471-7640 and 7641-10965 obtained by segmentally amplifying the cDNA sequence of the GI type Japanese encephalitis virus SD12 strain were cloned into vectors respectively, and the E gene in the nucleic acid fragments 1-2350 was subjected to PCR site-directed mutagenesis, and the mutated nucleic acid fragments and the nucleic acid fragments 2351-4470, 4471-7640 and 7641-10965 were amplified using specific primers, and the full-length cDNA of the attenuated vaccine strain containing the transcription promoter was obtained by fusion PCR;

[0011] (2) The obtained full-length cDNA was transcribed in vitro and transfected into cells to obtain the infectious clone strain rSD12-F120 of the attenuated vaccine strain of Japanese encephalitis virus SD12-F120.

[0012] Preferably, the sites for PCR site-directed mutagenesis of the E gene in step (1) are the 138th and 176th amino acid sites of the E gene, wherein the 138th amino acid mutates from glutamic acid to arginine, and the 176th amino acid mutates from isoleucine to phenylalanine.

[0013] Preferably, the nucleotide sequence of the mutated E gene is shown in SEQ ID NO.16.

[0014] Preferably, the sequence of the forward primer E-E138R-F for the mutation at the 138th amino acid site of the E gene is shown as SEQ ID NO.2, and the sequence of the reverse primer E-E138R-R is shown as SEQ ID NO.3; the sequence of the forward primer E-I176T-F for the mutation at the 176th amino acid site of the E gene is shown as SEQ ID NO.4, and the sequence of the reverse primer E-I176T-R is shown as SEQ ID NO.5.

[0015] Preferably, the forward-specific primer for the mutated nucleic acid fragment at positions 1-2350 is shown as SEQ ID NO.6, and the sequence of the reverse primer is shown as SEQ ID NO.7; the forward-specific primer for the nucleic acid fragment at positions 2351-4470 is shown as SEQ ID NO.8, and the sequence of the reverse primer is shown as SEQ ID NO.9; the forward-specific primer for the nucleic acid fragment at positions 4471-7640 is shown as SEQ ID NO.10, and the sequence of the reverse primer is shown as SEQ ID NO.11; the forward-specific primer for the nucleic acid fragment at positions 7641-10965 is shown as SEQ ID NO.12, and the sequence of the reverse primer is shown as SEQ ID NO.13.

[0016] Preferably, the transcription promoter in step 1) is a T7 promoter.

[0017] The present invention also verifies the GI type Japanese encephalitis attenuated vaccine strain SD12-F120, including drawing growth curves, observing plaque morphology, and conducting pathogenicity experiments on mice, and comparing it with the parent strain SD12. The experimental results show that 10 0 -10 5 PFU dose of SD12-F120 in mice, even at the highest dose of 10 5 PFU, no clinical signs of JEV infection were observed in mice inoculated with SD12 intraperitoneally from 10 1 Clinical symptoms of JEV infection began to appear at a dose of 1 PFU, and mice inoculated with SD12 via the intracerebral route began to develop clinical signs of JEV infection from a dose of 1 PFU. This indicates that the SD12-F120 strain has lost its neuroinvasiveness and neurotoxicity in mice, and the GI-type attenuated strain SD12-F120 has been successfully obtained.

[0018] The third purpose of the present invention is to request protection for the use of the JEV GI type attenuated vaccine strain SD12-F120 in preventing JEV infection in mammals. Preferably, the mammal is a boar or a sow. The present invention uses the JEV GI type attenuated vaccine strain SD12-F120 to prevent JEV infection in mouse models and boar models. The experimental results of Example 2 showed that SD12-F120 can provide complete protection for mice infected with GI SD12 and GIII NJ28 after immunization, while the immune control group SA14-14-2 cannot provide complete protection for mice infected with GI SD12 after low-dose immunization. At the same time, the Japanese encephalitis virus attenuated vaccine strain SD12-F120 has good immunogenicity and can induce mice to produce neutralizing antibodies that have a protective effect against GI type JEV SD12. The experimental results of Example 3 show that all clinical symptoms of all piglets in the immunized group are normal. All piglets in the challenge control group showed symptoms of Japanese encephalitis infection, including mental excitement and fever. Three boars showed varying degrees of testicular swelling and loss of appetite. Pathological sections showed that the normal structure of the seminiferous tubules in the testes of pigs in the challenge control group had been significantly destroyed, with a large number of inflammatory cells infiltrating, mainly lymphocytes, accompanied by plasma cells, neutrophils and monocytes, and obvious proliferation of interstitial fibrous tissue. The seminiferous tubules of the testes of pigs in the DMEM control group and the immunized challenge group were normal, indicating that the JEV attenuated strain SD12-P120 can effectively protect boars from orchitis. At the same time, SD12-F120 has good immunogenicity for boars, and the neutralizing antibody levels of the immunized piglets exceeded the protection level.

[0019] Compared with the prior art, the GI type Japanese encephalitis virus attenuated vaccine strain of the present invention has the following beneficial effects: the GI type Japanese encephalitis virus attenuated vaccine strain SD12-F120 and its infectious clone described in the present invention, the recombinant strain rSD12-F120 is obtained by reverse genetic operation of the GI type Japanese encephalitis virus attenuated vaccine strain SD12-F120, and the attenuated vaccine strain has a protective effect on both GI type and GIII type Japanese encephalitis viruses in a mouse model and has a good immune effect; at the same time, the GI type Japanese encephalitis virus attenuated vaccine strain SD12-F120 strain has good safety and immunogenicity in pigs. The GI type Japanese encephalitis virus attenuated vaccine strain SD12-F120 strain described in the present invention provides a candidate strain for the development and application of GI type JEV for gene type I Japanese encephalitis vaccine. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 is the agarose gel electrophoresis diagram of the full-length cDNA of the GI type SD12-F120 strain;

[0021] Figure 2 Growth curves of the rescued GI-type SD12-F120 strain and the GI-type parental strain SD12 strain on BHK-21 cells

[0022] Figure 3 The plaques of the rescued GI type SD12-F120 strain and the GI type parental strain SD12 strain on BHK-21 cells (A is the morphology of the plaques, and B is the diameter of the plaques)

[0023] Figure 4 The neurotoxicity and neuroinvasiveness of the rescued GI-type SD12-F120 strain and the GI-type parental strain SD12 strain in C57BL / 6 mice ( Figure 4 A is the pathogenicity result of SD12-F120, and B is the pathogenicity result of SD12)

[0024] Figure 5 The cross-protection results of the rescued GI type SD12-F120 strain and the commercialized SA14-14-2 against the GI and GIII type virulent strains ( Figure 5 A in the figure is the survival curve of SA14-14-2 after immunization and challenge with poison, and B is the survival curve of SD12-F120 after immunization and challenge with poison)

[0025] Figure 6 The level of neutralizing antibodies produced in mice after immunization with the rescued GI type SD12-F120 strain

[0026] Figure 7 Viremia and viral load in nasal swabs of young boars immunized with the rescued GI type SD12-F120 strain.

[0027] Figure 8 The neutralizing antibody levels in boars after immunization with the rescued GI type SD12-F120 strain.

[0028] Fig. 9 Antibody levels in boars after immunization with the rescued GI type SD12-F120 strain. DETAILED DESCRIPTION

[0029] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0030] Example 1 Construction and Identification of the Full-Length cDNA of the Genome of the Attenuated Vaccine Strain SD12-F120 of Type GI JEV (I) Construction of the Full-Length cDNA of the Attenuated Vaccine Strain SD12-F120

[0031] 1. Plasmid construction of attenuated vaccine strain SD12-F120

[0032] According to the sequence of GI type JEV SD12 strain (GenBank No.MH753127), its full-length gene was divided into 4 segments to construct cloning plasmids, which were constructed by Paisono Biotechnology Co., Ltd. The 4 nucleic acid fragments were the 1-2350 nucleic acid fragment (fragment 1), the 2351-4470 nucleic acid fragment (fragment 2), the 4471-7640 nucleic acid fragment (fragment 3) and the 7641-10965 nucleic acid fragment (fragment 4). The corresponding constructed plasmids were named JEV-SD12-1, JEV-SD12-2, JEV-SD12-3 and JEV-SD12-4.

[0033] Using plasmid JEV-SD12-1 as a template, site-directed mutagenesis primers were designed to mutate the 138th and 176th amino acid sites of the E gene. The resulting new plasmid was named JEV-SD120-E-1.

[0034] The specific PCR amplification system is as follows:

[0035]

[0036]

[0037] The specific PCR reaction procedure is as follows:

[0038]

[0039] All PCR products were digested with 1 μL DpnⅠ-HF endonuclease (purchased from NEB) at 37°C for 2 h, and the digested products were transformed with DH5α competent cells (purchased from TIANGEN). Monoclonal colonies were picked, and the plasmids were extracted (using the plasmid DNA mini-extraction kit, purchased from AXYGEN) and then sequenced for verification.

[0040] 2. Using JEV-SD120-E-1, JEV-SD12-2, JEV-SD12-3 and JEV-SD12-4 as templates, four pairs of specific primers were used to amplify four fragments of each, the concentration of the fragments was measured by gel recovery, and they were stored at -20°C. The forward specific primer of JEV-SD120-E-1 is shown in SEQ ID NO.6, and the sequence of the reverse primer is shown in SEQ ID NO.7; the forward specific primer of JEV-SD12-2 is shown in SEQ ID NO.8, and the sequence of the reverse primer is shown in SEQ ID NO.9; the forward specific primer of JEV-SD12-3 is shown in SEQ ID NO.10, and the sequence of the reverse primer is shown in SEQ ID NO.11; the forward specific primer of JEV-SD12-4 is shown in SEQ ID NO.12, and the sequence of the reverse primer is shown in SEQ ID NO.13.

[0041] 3. Use fusion PCR to obtain the full-length cDNA of the GI type attenuated vaccine strain SD12-F120 carrying the T7 promoter, the steps are as follows:

[0042] (1) The fusion PCR system is divided into two tubes, one of which contains fragments 1 and 2, and the other contains fragments 3 and 4, without adding primers.

[0043] The specific PCR amplification system is as follows:

[0044]

[0045] The specific PCR amplification reaction procedure is as follows:

[0046]

[0047] (2) Mix the two tubes of PCR products together, blow them evenly, and then divide them into two tubes again, and repeat the reaction conditions described above for 10 cycles;

[0048] (3) Add 2 μL of primers T7-SD120-1-F and SD120-JD-4R to two tubes respectively, mix well and perform amplification reaction; the specific PCR amplification reaction procedure is as follows:

[0049]

[0050] (4) Perform nucleic acid electrophoresis on the fusion PCR product using 0.8% agarose gel at 140V for 40 min, cut out the target fragment, and recover the fragment by gel (use RNase-free EP tube to avoid RNase contamination during operation). The recovered product is stored at -20°C. The results are as follows: Figure 1 shown.

[0051] (II) Rescue of the recombinant virus of the GI type JEV attenuated vaccine strain SD12-F120

[0052] 1. In vitro transcription

[0053] (1) The viral cDNA containing the T7 promoter was in vitro transcribed into 7-methylguanine-capped RNA using the mMESSAGE mMACHINETMT7 in vitro transcription kit (purchased from Thermo Fisher Scientific). The in vitro transcription reaction system was as follows;

[0054]

[0055] (2) After mixing, react in a 37°C water bath for 2 h, add 1 μL TURBO DNase I, continue the reaction at 37°C for 15 min, and store the RNA product at -80°C.

[0056] 2. RNA Transfection

[0057] (1) Using BHK-21 cells to rescue SD12-F120 virus, when BHK-21 cells grow to 70-80% in a 6-well plate, discard the culture medium and wash three times with PBS;

[0058] (2) Add 1 mL of Opti-MEM (purchased from Gibco) and 6 μL of DMRIE-C transfection reagent (purchased from Thermo Fisher Scientific) to the EP tube, vortex immediately and incubate at room temperature for 15 min, then add 1 μg of RNA product and vortex immediately;

[0059] (3) Add the mixed solution to a 6-well plate and prepare a blank control, and incubate in a 37°C incubator for 6 h;

[0060] (4) After discarding the liquid, wash three times with PBS, add 2 mL of 2% FBS and 1% SP DMEM maintenance solution (purchased from Gibco) to each well, and culture in a 37°C incubator until the cells show more than 80% lesions. Repeat the freezing and thawing three times, centrifuge at 3000 rpm at 4°C for 10 min, and then divide the supernatant into EP tubes and store at -80°C.

[0061] 3. RNA Extraction

[0062] The obtained virus supernatant was used to extract SD12-F120 virus genomic RNA using the Trizol method, the steps are as follows:

[0063] (1) Take out the SD12-F120 virus solution from -80°C, place it on ice to melt, add 600 μL Trizol to 400 μL of the virus solution, vortex to mix, and let it stand at room temperature for 5 min;

[0064] (2) Add 200 μL of chloroform, vortex mix, let stand for 5 min, and centrifuge at 4°C and 12,000 rpm for 15 min;

[0065] (3) After centrifugation, the supernatant of the upper layer of the water sample was aspirated into a new 1.5 mL EP tube without RNA, 800 μL of isopropanol was added, and the tube was mixed by inversion. The tube was allowed to stand for 10 min, and then centrifuged at 4°C and 12,000 rpm for 10 min.

[0066] (4) After discarding the liquid, add 800 μL of 75% alcohol to gently rinse the precipitate, and centrifuge at 4°C and 12,000 rpm for 5 min;

[0067] (5) After discarding the liquid, the EP tube was inverted on absorbent paper and air-dried in a clean bench. After 10 minutes, 20 μL of RNase-free water was added to dissolve the RNA. After measuring the concentration, it was stored at -80°C. The RNA was reverse transcribed into cDNA using the TIANScriptⅡ cDNA first-strand synthesis kit (purchased from TIANGEN). The steps are as follows:

[0068] (1) Take 1-2 μg of viral RNA, add 2 μL of Random Primer and 1 μL of Super Pure dNTPs, add RNase-free water to 14.5 μL, incubate in a 65°C water bath for 5 min, and immediately put it in ice for 2 min;

[0069] (2) Add 4 μL 5× TIANScriptⅡRTase Buffer, 1 μL TIANScriptⅡRTase, and 0.5 μL RNasin to a total volume of 20 μL and mix well;

[0070] (3) The sample was placed in a PCR instrument and the program was 25°C for 10 min, 42°C for 60 min, and 85°C for 5 min to obtain the cDNA of SD12-F120, which was then stored at -20°C. The nucleic acid sequence of the cDNA of the SD12-F120 virus obtained by sequencing is shown in SEQ ID NO.1

[0071] 4. Amplification of E protein

[0072] The E protein was amplified by PCR using primers E-1-F and E-1500-R (Table 11).

[0073] The specific amplification system is as follows:

[0074]

[0075]

[0076] The specific reaction procedure is as follows:

[0077]

[0078] After PCR amplification, 1% agarose gel electrophoresis was performed, and the target fragments were respectively recovered by gel extraction using EZNA Gel Extraction Kit. The gel extraction products were sequenced by Shanghai Bioengineering Co., Ltd. The nucleic acid sequence of the E protein of the SD12-F120 virus obtained by sequencing is shown in SEQ ID NO.16.

[0079] (III) Identification of GI type attenuated vaccine strain SD12-F120 and GI type virulent strain SD12

[0080] 1. Multi-step growth curve

[0081] The multi-step growth curves of the SD12-F120 and SD12 strains of the virus on BHK-21 cells were determined as follows:

[0082] (1) When BHK-21 cells grew to 80% in a 6-well plate, the culture medium was discarded, the cells were washed three times with PBS, 0.01 MOI of virus solution was added, serum-free DMEM medium was supplemented to 1 mL, and the cells were incubated at 37°C for 2 h;

[0083] (2) Discard the virus solution, wash three times with PBS, add 2 mL of DMEM maintenance solution containing 2% FBS and 1% SP, take 200 μL of supernatant every 12 h, and add 200 μL of maintenance solution until 72 h of culture;

[0084] (3) The virus solution collected at each time point was centrifuged at 4°C and 3000 rpm for 10 min, and the supernatant was aspirated and stored at -80°C;

[0085] (4) The virus titer in the cell supernatant at different time points was measured, and the data were analyzed using GraphPad 8.0 software to draw a multi-step growth curve. The results are shown in Figure 2 shown.

[0086] 2. Plaque morphology

[0087] The plaque morphology of the virus strains SD12-F120 and SD12 on BHK-21 cells was determined as follows:

[0088] (1) After the BHK-21 cells have grown in a monolayer in a 6-well plate, discard the culture medium, wash three times with PBS, add 100 μL of 100 TCID50 SD12-F120 or SD12 virus solution, supplement with serum-free DMEM medium to 1 mL, and incubate at 37°C incubator for 2 h;

[0089] (2) Discard the virus solution, wash three times with PBS, and replace with 2×MEM medium containing 3% low-melting point agarose and 2% FBS. After solidification, place it upside down in a 37°C incubator;

[0090] (3) After 5 days, add 1 mL of 4% tissue cell fixative and fix at room temperature for 30 min;

[0091] (4) Rinse off the fixative and agar, add 1 mL of gentian violet stain, and stain at room temperature for 15 min;

[0092] (5) Rinse gently with water, dry, and use a vernier caliper to measure 20 plaques. Take the average to calculate the plaque size of the virus strains SD12-F120 and SD12. The results are as follows: Figure 3 shown.

[0093] 3. Pathogenicity Experiments in Mice

[0094] The pathogenicity test of the virus strains SD12-F120 and SD12 was conducted on mice. The steps are as follows:

[0095] (1) 3-week-old SPF-grade C57BL / 6 female mice (purchased from Shanghai Sibeifu Animal Co., Ltd.) were divided into groups, with 8 mice in each group and blank control;

[0096] (2) The virus strains SD12-F120 and SD12 were injected intracerebrally (ic) at 30 μL / animal and intraperitoneally (ip) at 100 μL / animal, respectively. The challenge dose was 10 0 -10 5 PFU / 30μL and 10 0 -10 5 The control group was injected with an equal dose of PBS.

[0097] (3) The mice were observed for 21 days, and their health status and mortality were recorded every day. The LD50 was calculated using the Reed-Muench method. The results are as follows: Figure 4 shown.

[0098] The results showed that intraperitoneal or intracranial vaccination for 10 0 -10 5PFU dose of SD12-F120 in mice, even at the highest dose of 10 5 PFU, and no clinical signs of JEV infection were observed ( Figure 4 a). The LD50 of neuroinvasiveness and neurotoxicity of SD12-F120 is >10 5 In contrast, mice inoculated with SD12 via the intraperitoneal route showed a decrease in 1 PFU doses, clinical symptoms of JEV infection began to appear. The LD50 of SD12 neuroinvasiveness is 500 PFU ( Figure 4 b). In addition, mice inoculated with SD12 via the intracerebral route developed clinical signs of JEV infection starting from a dose of 1 PFU. The LD50 of neurotoxicity of SD12 is 2.5 PFU ( Figure 4 b). The above results showed that the SD12-F120 strain lost its neuroinvasiveness and neurotoxicity in mice, indicating that the GI-type attenuated strain SD12-F120 was successfully obtained.

[0099] Example 2 Evaluation of the immune protection of JEV GI attenuated vaccine strain SD12-F120 in a mouse model

[0100] 1. Protective immunity of attenuated vaccine strain SD12-F120

[0101] 150 three-week-old SPF-grade C57BL / 6 female mice were randomly divided into 15 groups, with 10 mice in each group. 4 -10 2 TCID50 / 100μL) virus content was intraperitoneally inoculated into mice, and 10 mice were inoculated at each dilution, with 100μL per mouse. Six groups of Japanese encephalitis attenuated live vaccine SA14-14-2 immunization control groups and one group of DMEM medium blank control group were set up, with 10 mice in each group. After 14 days of immunization, each group of mice was intraperitoneally challenged with GI JEVSD12 (20LD50) and GIII JEV NJ28 (50LD50), 100μL per mouse, and 30 mice were challenged with each genotype. After the challenge, the mice were observed for 25 consecutive days, the clinical symptoms of the mice were observed every day, the weight of the mice and the incidence and mortality of the mice were recorded, and the survival curve was drawn using GraphPad 8.0 software.

[0102] The results are as follows Figure 5As shown. After SPF mice were immunized with the SA14-14-2 weak strain for 14 days, the mice in the immunization group and the blank control group showed normal clinical manifestations, good mental state, normal appetite, and no local and systemic adverse reactions. All 60 mice in the immunization group and 10 mice in the blank control group survived, indicating that the SA14-14-2 weak strain is safe for mice. On the 14th day of immunization, mice were infected with lethal doses of the GI SD12 strain and the GIII NJ28 strong strain, respectively. During the entire observation period, all 30 mice in the three groups infected with GIII NJ28 survived. Among the three groups of mice infected with GI SD12, one mouse in the low-dose immunization group died on the 10th day after infection, and typical symptoms of Japanese encephalitis virus infection appeared before death. This shows that low-dose immunization with SA14-14-2 cannot provide complete protection for mice infected with GISD12 ( Figure 5 A)

[0103] Two weeks after SPF mice were immunized with the SD12-F120 attenuated strain, the mice in the immunization group and the blank control group showed normal clinical manifestations, good mental state, normal appetite, and no local and systemic adverse reactions. All 60 mice in the immunization group and 10 mice in the blank control group survived, indicating that the SA14-14-2 attenuated strain is safe for mice. After infection with lethal doses of the GI SD12 strain and the GIIINJ28 strong strain, all mice in all experimental groups survived during the observation period, and no typical clinical symptoms of JE appeared. This shows that SD12-F120 immunization can provide complete protection for mice infected with GI SD12 and GIII NJ28 ( Figure 5 B).

[0104] 2. Immunogenicity determination of attenuated vaccine strain SD12-F120

[0105] The immunogenicity testing process of the attenuated vaccine strain SD12-F120 is as follows:

[0106] (1) The GI type Japanese encephalitis virus attenuated vaccine strain SD12-F120 was added to 10 4 PFU doses were used for immunization, and blood was collected from the mouse orbits on days 14 and 21;

[0107] (2) determine the titer of neutralizing antibodies in serum samples collected from vaccinated mice using the plaque reduction neutralization test (PRNT);

[0108] (3) After the blood was allowed to stand for 2 h, it was centrifuged at 3000 rpm for 10 min at room temperature to separate the serum, inactivated in a 56°C water bath for 30 min, and then serially diluted;

[0109] (4) The diluted serum sample was mixed with an equal volume of virus SD12 (20 LD50) and incubated at 37°C for 1 h. The mixture was then distributed into BHK-21 cells and incubated at 37°C for 2 h.

[0110] (5) After 2 h of adsorption, cells were covered with 1.2% methylcellulose (ThermoFisher Scientific) in DMEM containing 2% FBS and incubated at 37°C for 3–5 days;

[0111] (6) Plaques were stained with crystal violet and counted. The formula for calculating the neutralizing antibody (PRNT50) titer was antibody titer = final dilution × titer. The positive cutoff value of the neutralizing antibody titer was defined as PRNT50 ≥ 10.

[0112] Serum samples collected from pre-vaccinated mice showed background levels of PRNT50 titers <10 for all JEV strains examined. Figure 6 As shown, the PRNT50 titer of the serum samples of mice vaccinated with SD12-F120 vaccine against the lethal virus SD12 strain was greater than 50. This indicates that the attenuated vaccine strain of Japanese encephalitis virus SD12-F120 has good immunogenicity and can induce mice to produce neutralizing antibodies that have a protective effect against GI type JEV SD12.

[0113] Example 3 Evaluation of the immune protection of JEV GI attenuated strain SD12-F120 in a boar model

[0114] 1. Safety of attenuated vaccine strain SD12-F120 in pigs

[0115] (1) Clinical symptoms after immunization of young boars

[0116] Ten healthy boars were randomly divided into two groups, one group was immunized with SD-F120 attenuated strain, and the other group was injected with an equal volume of DMEM as a negative control. During the 14 days of continuous observation, the boars in the immunized group and the blank control group showed normal clinical manifestations, normal body temperature, appetite, good mental state, no neurological symptoms, no local or systemic adverse reactions, and all 5 piglets in the immunized group and the DMEM blank control group survived healthily. This shows that the GI JEV SD-F120 virus strain is safe for boars. The results are shown in Table 1.

[0117] Table 1 Safety results of GI JEV SD12-F120 strain immunized boars

[0118]

[0119] (2) Detection of viral load in blood and nasal swabs of young boars after immunization

[0120] The viremia and viral load in nasal swabs of young boars were tested on the 7th and 14th days after immunization. The results showed that the viremia and viral load in nasal swabs of young boars after immunization remained at very low levels or undetectable on the 7th and 14th days. Figure 7 ).

[0121] 2. Protective effect of attenuated vaccine strain SD12-F120 in pigs

[0122] (1) Clinical symptoms of young boars infected with virulent strains two weeks after immunization

[0123] All piglets in the immunized group showed normal clinical symptoms. All piglets in the challenge control group showed symptoms of Japanese encephalitis infection, including mental excitement and fever. The highest detected temperature was 40.9℃ and 40.5℃ for two piglets on the third day after the infection. Three of the piglets showed varying degrees of testicular swelling and loss of appetite. The blank control group showed no other symptoms except that two piglets had a slightly elevated body temperature on the seventh day. The results are shown in Table 2.

[0124] Table 2 GI JEV SD12-F120 strain immune protection results against boars

[0125]

[0126]

[0127] (2) Pathological anatomy and section observation

[0128] The autopsy revealed that the testicles were swollen to varying degrees, the testicular parenchyma was congested, hemorrhagic and had necrotic foci, there was a large amount of mucus in the tunica vaginalis of the testis, fibrous thickening appeared on the edge of the epididymis and the visceral layer of the tunica vaginalis, and one young boar had severe unilateral testicular lesions.

[0129] The results of pathological sections showed that the normal structure of the seminiferous tubules in the testes of pigs in the challenge control group had been significantly destroyed, with a large number of inflammatory cells infiltrating, mainly lymphocytes, accompanied by plasma cells, neutrophils and monocytes, and obvious proliferation of interstitial fibrous tissue. The seminiferous tubules of the testes of pigs in the DMEM control group and the immunized challenge group were normal, and supporting cells, sperm, primary spermatocytes, interstitial cells, etc. could be clearly distinguished. It can be seen that the JEV attenuated strain SD12-P120 can effectively protect boars from orchitis.

[0130] 3. Immunogenicity of attenuated vaccine strain SD12-F120 in pigs

[0131] After immunization, the neutralizing antibody levels of the piglets exceeded the protective level, reaching a maximum of 1:113 ( Figure 8 ), the antibody results showed the same trend ( Fig. 9). This indicates that SD12-F120 has good immunogenicity in young boars.

[0132] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A GI type Japanese encephalitis virus attenuated vaccine strain SD12-F120, characterized in that: Its cDNA sequence is shown as SEQ ID NO.

1.

2. A method for constructing an infectious clone of the attenuated vaccine strain of GI-type Japanese encephalitis virus according to claim 1, characterized in that: The steps include: (1) The cDNA sequence of the attenuated vaccine strain SD12-F120 of the GI type Japanese encephalitis virus was segmentally amplified to obtain the nucleic acid fragments 1-2350, 2351-4470, 4471-7640 and 7641-10965, respectively cloned into a vector, and the E gene in the nucleic acid fragments 1-2350 was subjected to PCR site-directed mutagenesis, and the mutated nucleic acid fragments and the nucleic acid fragments 2351-4470, 4471-7640 and 7641-10965 were amplified using specific primers, and the full-length cDNA of the attenuated vaccine strain containing the transcription promoter was obtained by fusion PCR; (2) The obtained full-length cDNA is transcribed in vitro and transfected into cells to obtain an infectious clone strain of the attenuated vaccine strain SD12-F120 of Japanese encephalitis virus.

3. The method for constructing an infectious clone of the attenuated vaccine strain of GI type Japanese encephalitis virus according to claim 2, characterized in that: The sites for PCR site-directed mutagenesis of the E gene in step (1) are the 138th and 176th amino acid sites of the E gene.

4. The method for constructing an infectious clone of an attenuated vaccine strain of GI-type Japanese encephalitis virus according to claim 3, characterized in that: The 138th amino acid mutated from glutamic acid to arginine, and the 176th amino acid mutated from isoleucine to phenylalanine.

5. The method for constructing an infectious clone of an attenuated vaccine strain of GI-type Japanese encephalitis virus according to claim 4, characterized in that: The nucleotide sequence of the mutated E gene is shown in SEQ ID NO.

16.

6. The method for constructing an infectious clone of an attenuated vaccine strain of GI-type Japanese encephalitis virus according to claim 2, characterized in that: The sequence of the forward primer E-E138R-F for the mutation at the 138th amino acid site of the E gene is shown as SEQ ID NO.2, and the sequence of the reverse primer E-E138R-R is shown as SEQ ID NO.3; the sequence of the forward primer E-I176T-F for the mutation at the 176th amino acid site of the E gene is shown as SEQ ID NO.4, and the sequence of the reverse primer E-I176T-R is shown as SEQ ID NO.

5.

7. The method for constructing an infectious clone of an attenuated vaccine strain of GI-type Japanese encephalitis virus according to claim 2, characterized in that: The forward specific primer for the mutated nucleic acid fragment at positions 1-2350 is shown as SEQ ID NO.6, and the sequence of the reverse primer is shown as SEQ ID NO.7; the forward specific primer for the nucleic acid fragment at positions 2351-4470 is shown as SEQ ID NO.8, and the sequence of the reverse primer is shown as SEQ ID NO.9; the forward specific primer for the nucleic acid fragment at positions 4471-7640 is shown as SEQ ID NO.10, and the sequence of the reverse primer is shown as SEQ ID NO.11; the forward specific primer for the nucleic acid fragment at positions 7641-10965 is shown as SEQ ID NO.12, and the sequence of the reverse primer is shown as SEQ ID NO.

13.

8. The method for constructing an infectious clone of an attenuated vaccine strain of GI-type Japanese encephalitis virus according to claim 2, characterized in that: The transcription promoter in step (1) is T7 promoter.

9. Use of the attenuated vaccine strain of GI type Japanese encephalitis virus according to claim 1 in preventing JEV infection in mammals.

10. The use of the attenuated vaccine strain of GI type Japanese encephalitis virus according to claim 9, characterized in that: The mammal is a boar or a sow.

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