Chimeric togaviruses of the tambussu virus prm-e and methods of making and using the same

Abstract

The application discloses a chimeric Kunjin virus of tamabi virus prM-E and a preparation method and application thereof. On the basis of a reverse genetic manipulation system of the Kunjin virus KUNV, the KUNV prM / E gene is replaced by the DTMUV prM / E gene to construct a chimeric DTMUV virus infectious clone plasmid with the KUNV as a skeleton, which is named as pUC19-KUNV-DTMUV prM / E. Then, the chimeric tamabi virus prM-E Kunjin virus is obtained through virus rescue, and is named as rKUNV-CHv-prM / E (CHv). Analysis on the virus characteristics shows that the CHv can effectively proliferate in BHK-21 and DEF cells, and the duck embryo virulence is weaker than that of the KUNV and the DTMUV. The animal experiment results show that the CHv only detects obvious virus levels in the brain, and does not produce viremia; the CHv after immunization can produce similar IgG antibody levels to those after immunization of the KUNV and the DTMUV. Therefore, the recombinant virus of the chimeric DTMUV prM / E with the KUNV as a skeleton prepared by the application can induce the duckling to produce sufficient immune protective neutralizing antibody levels, and can be applied to preparation of a duck tamabi virus vaccine.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of molecular biology, and particularly relates to a chimeric tembusu virus prM-E Kunjin virus and a preparation method and application thereof. BACKGROUND

[0002] Duck tembusu virus disease, also known as duck hemorrhagic oophoritis and duck flavivirus disease, is a new acute infectious disease caused by duck tembusu virus (DTMUV). The virus mainly causes egg laying ducks to be depressed, to reduce food intake, and to suddenly reduce egg production; in the duckling or growing duck, the virus can cause neurological symptoms and hind limb paralysis. DTMUV infects various duck breeds, including but not limited to cherry valley ducks, muscovy ducks, Beijing ducks, etc. Ducks infected with the virus usually develop the disease at 20-40 days of age, and the main clinical symptoms are: elevated body temperature, significantly reduced food intake, limb weakness, paralysis, and significantly reduced egg production. After infection, the duckling is characterized by unsteady standing, paralysis, and head and neck tremors. The characteristic lesions of infected breeding ducks include follicle membrane hemorrhage, follicle deformation, and even rupture; after infection, the egg laying duck shows a sudden drop in egg production or even stops laying eggs, and severe infection can lead to death, with a mortality rate of 10%-30%. The lesions of infected laying ducks are mainly concentrated in the ovary and oviduct, and the ovary is hemorrhagic, atrophic or dysplastic, the oviduct is atrophic or necrotic, and the yolk of the severely infected egg is ruptured, causing yolk peritonitis.

[0003] Current prevention and control measures for TMUV are mainly through vaccination, although there are commercial attenuated live vaccines and inactivated vaccines, but ducks after immunization can still be infected with TMUV, and the weak strain obtained by isolating and passing the strain has potential safety risks. Therefore, the current DTMUV vaccine still has a lot of room for improvement, especially the development of a safe and effective DTMUV attenuated live vaccine SUMMARY

[0004] The purpose of the present application is to solve the above problems in the prior art, and to provide a chimeric tembusu virus prM-E Kunjin virus and a preparation method and application thereof.

[0005] To solve the above technical problems, the technical scheme adopted by the present application is as follows: a preparation method of a chimeric tembusu virus prM-E Kunjin virus, comprising the following steps:

[0006] S1, with pUC19-FL-KUNV-intron and pACYC-DTMUV plasmid as templates, respectively PCR amplification obtains SnaBI-C, prM-E and NS1-BamHI three fragments; then the three recovered fragments are assembled into linearized pUC19-FL-KUNV-intron vector by homologous recombination to obtain a chimeric infectious clone plasmid, named pUC19-KUNV-DTMUVprM / E;

[0007] S2, the chimeric infectious clone plasmid in S1 is transfected into cells, and the chimeric virus is rescued to obtain a chimeric Kunjin virus of Tamabi virus prM-E, named rKUNV-CHv-prM / E (CHv).

[0008] Preferably, in S1, the amplification primers are reCMV-F and CHv-C / prM-R, CHv-C / prM-F and CHv-E / NS1-R, and CHv-E / NS1-F and 2A-BamHⅠ-R, and the nucleotide sequences are shown in SEQ ID N0.1-6 in sequence.

[0009] Preferably, in S1, the linearized pUC19-FL-KUNV-intron vector is obtained by double digestion with SnaBI and BamHI restriction endonucleases.

[0010] The application also provides the chimeric Kunjin virus of Tamabi virus prM-E prepared by the preparation method.

[0011] The application also provides application of the chimeric Kunjin virus of Tamabi virus prM-E in preparation of a duck Tamabi virus vaccine, and the duck Tamabi virus vaccine is a duck Tamabi virus attenuated live vaccine.

[0012] The application has the following beneficial effects:

[0013] 1) Kunjin virus (Kunjin Virus, KUNV) is a natural attenuated strain of West Nile Virus (West Nile Virus, WNV), and the application replaces KUNV prM / E gene with DTMUV prM / E gene on the basis of reverse genetic manipulation system of KUNV, constructs a chimeric DTMUV virus infectious clone plasmid with KUNV as a skeleton, and is named pUC19-KUNV-DTMUVprM / E.

[0014] 2) The chimeric Kunjin virus with Tanapox virus prM-E in the present application is obtained by virus rescue, named rKUNV-CHv-prM / E (CHv). The analysis of the characteristics of the rescued virus shows that CHv can effectively proliferate in BHK-21 and DEF cells, and the virulence of duck embryo is weaker than that of KUNV and DTMUV. Subsequently, CHv is inoculated into 3-day-old ducklings through the muscle injection route, and the virus tissue load is detected and the viremia is monitored after immunization, and the serum is collected to detect the antibody level. The results show that CHv only detects obvious virus level in the brain, and does not produce viremia; CHv can produce similar IgG antibody level after immunization as KUNV and DTMUV. Therefore, the recombinant virus prepared in the present application, which is a chimeric DTMUV prM / E with KUNV as the skeleton, can induce ducklings to produce sufficient immune protective neutralizing antibody level, and can be applied to prepare duck Tanapox virus vaccine. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 Schematic diagram for construction of infectious clone of KUNV chimeric virus.

[0016] Figure 2 Schematic diagram for rescue of recombinant KUNV with chimeric DTMUV prM-E gene, wherein (A) CPE and IFA verification of CHv; (B) KUNV, DTMUV and CHv incubation with TMUV E protein polyclonal antibody verification; (C) KUNV, DTMUV and CHv incubation with KUNV E protein polyclonal antibody verification; (D) KUNV, DTMUV and CHv incubation with KUNV NS1 protein polyclonal antibody verification.

[0017] Figure 3 In vitro growth characteristics of CHv, wherein (A) plaque morphology; (B) growth curve of CHv in BHK-21 cells; (C) gene copy number of CHv in BHK-21 cells; (D) growth curve of CHv in DEF cells; (E) gene copy number of CHv in DEF cells; (F) 1000 TCID 50 CHv virulence in duck embryo; (G) 10000 TCID 50 CHv virulence in duck embryo. "ns" represents no significant difference, "*" represents P<0.1, "**" represents P<0.01, "***" represents P<0.001, and "****" represents P<0.0001.

[0018] Figure 4 Schematic diagram for operation of duckling pathogenicity test.

[0019] Figure 5Changes in serum IgG levels induced by rKUNV, rTMUV and CHv infection in 3-day-old ducklings.

[0020] Figure 6 Changes in serum IgG levels induced by rKUNV, rTMUV and CHv infection in 3-day-old ducklings.

[0021] TMUV and KUNV were preserved in the Avian Disease Center of Sichuan Agricultural University, and the public literature information is as follows:

[0022] TMUV: Chen S, He Y, et al. Establishment of a reverse genetics system for duck Tembusu virus to study virulence and screen antiviral genes. Antiviral Res.

[0023] 2018; 157: 120-127. doi: 10.1016 / j.antiviral.2018.06.016

[0024] KUNV: Cherkashchenko L, Merits, A, et al. Validation of flavivirus infectious clones carrying fluorescent markers for antiviral drug screening and replication studies. Front Microbiol. 2023; 14: 1201640. Published 2023 Sep 15.

[0025] doi: 10.3389 / fmicb.2023.1201640 DETAILED DESCRIPTION

[0026] The present application will be further described below in conjunction with the accompanying drawings and specific examples.

[0027] Example 1

[0028] 1 Construction and rescue of recombinant KUNV of chimeric duck Tembusu virus prM-E gene

[0029] (1) Construction of chimeric virus infectious clones

[0030] The strategy for constructing chimeric infectious clones is as follows Figure 1 The pUC19-FL-KUNV-intron vector plasmid was double-digested with SnaBI and BamHI restriction enzymes, and the linearized vector was recovered using Takara's DNA purification and recovery kit.

[0031] Using the pUC19-FL-KUNV-intron and pACYC-DTMUV plasmids as templates, PCR amplification was performed using primers reCMV-F and CHv-C / prM-R, CHv-C / prM-F and CHv-E / NS1-R, and CHv-E / NS1-F and 2A-BamHI-R, respectively, to generate the SnaBI-C, prM-E, and NS1-BamHI fragments. The recovered fragments were then assembled into the linearized pUC19-FL-KUNV-intron vector by homologous recombination to generate a chimeric infectious clone plasmid named pUC19-KUNV-DTMUVprM / E. Primer names and sequences are shown in Table 1.

[0032] Table 1 Primers for constructing KUNV chimeric virus infectious clone plasmids

[0033]

[0034] (2) Rescue of chimeric viruses

[0035] BHK-21 cells were seeded in 6-well plates and cultured for 12-24 hours to allow the cell density to reach 80%-90% confluence at the time of transfection. Trans Liposomal transfection reagent instructions: 2 μg of DTMUV, KUNV, and pUC19-KUNV-DTMUVprM / E plasmids were transfected into cells. After 80%-90% of the cells showed cytopathic effect (CPE), the supernatant was collected and stored at -80°C for subsequent use. Cell samples were identified by IFA.

[0036] After KUNV, DTMUV and chimeric virus plasmids were transfected into BHK-21 cells, they were verified using DTMUV E protein polyclonal antibody, KUNV E protein polyclonal antibody and KUNV NS1 protein polyclonal antibody respectively ( Figure 2 AD), the results showed that the chimeric virus and DTMUV incubated with DTMUV E protein polyclonal antibody produced a strong positive fluorescence signal, while KUNV did not produce any specific fluorescence ( Figure 2B); KUNV incubation with KUNV E protein polyclonal antibody produced positive fluorescent signal, while chimeric virus and DTMUV did not produce any specific fluorescent signal Figure 2 C); chimeric virus and KUNV incubation with KUNV NS1 protein polyclonal antibody produced strong specific fluorescent signal, while DTMUV did not produce any positive fluorescent signal Figure 2 D). The above results showed that rKUNV-CHv-prM / E (CHv) was successfully rescued and could be specifically recognized by DTMUV E protein polyclonal antibody and KUNV NS1 protein polyclonal antibody. KUNV and DTMUV transfection rescued virus were labeled as rKUNV and rDTMUV.

[0037] (3) In vitro characterization of chimeric virus

[0038] To verify the in vitro characteristics of CHv, in this study, it was infected into BHK-21 and DEF cells respectively, and then the plaque size produced after virus infection of cells was observed, and the virus growth curve and gene copy number were detected. The results of comparing the plaque morphology of CHv and rKUNV in BHK-21 cells showed that the plaque produced by CHv was smaller than that of rKUNV Figure 3 A); the growth curve results showed that in BHK-21 cells Figure 3 B), the proliferation ability of CHv infected cells was weaker than that of rKUNV, and the time to reach the peak of virus titer was 72h, and then it showed a downward trend, while the virus titer of rKUNV reached the peak at 96h; in DEF cells Figure 3 D), after CHv infected cells, the virus titer reached the peak at 48h, and then it showed a downward trend, while the virus titer of rKUNV reached the peak at 96h, which was 493 times higher than that of CHv at the same time point. The results of gene copy number showed that in BHK-21 cells Figure 3 C), the proliferation trend and peak time point were consistent with the growth curve results, but the CHv genome copy number was 10 times, 4 times and 3 times higher than that of rKUNV genome copy number at 24-72h; in DEF cells Figure 3 E), the CHv genome copy number was higher than that of rKUNV genome copy number at the whole time point.

[0039] In 10-day-old duck embryos, 1000 TCID 50 and 10000 TCID 50 of CHv, rKUNV and rDTMUV were inoculated into the allantoic cavity respectively, and the virulence difference of the three viruses in duck embryos was compared. The results are shown in Figure 3 F-G: compared with rKUNV and CHv, 1000 TCID 50All duck embryos inoculated with TMUV died at 6 dpi, and duck embryos inoculated with 10 50 The death time of duck embryos inoculated with DTMUV was advanced to 4 dpi; the death time of duck embryos inoculated with rKUNV was later than that of duck embryos inoculated with rDTMUV, but whether inoculated with 10 50 TCID 50 and 10 50 TCID 50 All duck embryos inoculated with CHv were still alive at the end of the experiment.

[0040] (4) Pathogenicity of chimeric virus to ducklings

[0041] To explore the influence of chimeric virus on the pathogenicity of ducklings and the difference from parent virus after replacing the prM / E gene of KUNV with the prM / E gene of DTMUV, 3-day-old ducklings were infected with 10 4.6 TCID 50 / 200 μL of rKUNV parent virus or 10 4.3 TCID 50 / 200 μL of chimeric virus by intramuscular injection, the body weight change, viremia and mortality were recorded every day, and the tissues of ducklings at 3 dpi, 5 dpi, 7 dpi, 14 dpi and 28 dpi were collected for determination of tissue load of virus. Figure 4

[0042] The results of body weight change showed that the body weight change trend of ducklings in CHv and rDTMUV infection groups was the same, close to the body weight change of DMEM group; compared with CHv and rDTMUV infection groups, the body weight gain of ducklings in rKUNV infection group decreased from 9 dpi Figure 5 A). The results of mortality of ducklings were similar to the results of body weight change, the ducklings in rKUNV infection group appeared death at 2 dpi, while the ducklings in CHv and rDTMUV infection groups appeared death at 4 dpi; the mortality of ducklings in rKUNV and rDTMUV infection groups was 20% (2 / 10); in contrast, the mortality of ducklings in CHv infection group was only 10% (1 / 10) Figure 5 ​B) Viral tissue load detection results showed that in the rKUNV infected group of ducklings, virus distribution could be detected in each tissue at 28 dpi, among which brain, lung, kidney, heart and small intestine tissues were more obvious, especially the heart tissue could detect virus at 3 dpi and 5 dpi; in the rDTMUV infected group of ducklings, virus could only be detected in the early infection, and the virus load in each tissue was similar except that the virus load in the spleen tissue was higher; in the CHv infected group of ducklings, only brain tissue could detect virus distribution from 3 dpi to 28 dpi, and the virus load in the rest of the tissues was similar to that of the DMEM control group; compared with the rKUNV or rDTMUV infected group, the virus load in each tissue of the CHv infected group was lower Figure 5 C-I).

[0043] Viremia detection results showed that only the rDTMUV infected group of ducklings detected low viremia from 3 dpi to 28 dpi, and the ducklings in the other two infected groups did not detect viremia, which may be due to the low level of viremia produced after rKUNV and CHv infection of ducklings, which did not reach the minimum detection baseline Figure 5 J) The above shows that after replacing the prM / E gene of KUNV with the prM / E gene of DTMUV, the pathogenicity of CHv to ducklings is weakened.

[0044] (5) Antibody levels induced by chimeric viruses

[0045] This study monitored the changes of serum IgG levels against rKUNV, rDTMUV and CHv in duck serum within 4 weeks after the ducklings were infected with rKUNV, rDTMUV and CHv. The detection results are shown in Table 5. Figure 6 As shown in Table 5, antibodies could be detected in two weeks after infection of the three viruses, and the serum collected after immunization of duck with the commercialized vaccine of duck Tembusu virus was used as a positive control. Antibodies could be detected throughout the detection period. The above data show that after the duck is inoculated with CHv, it can induce and infect similar serum IgG antibody levels as rKUNV, rDTMUV under the condition of relatively lower pathogenicity in the body.

[0046] The specification and drawings of the present application are considered to be illustrative rather than restrictive, and based on the present application, those skilled in the art can make some substitutions and modifications to some technical features according to the disclosed technical content without creative labor, which are all within the protection scope of the present application.

Claims

1. A method for preparing a chimeric Kunjin virus prM-E of Tembusu virus, characterized in that: The following steps are involved: S1. Using pUC19-FL-KUNV-intron and pACYC-DTMUV plasmids as templates, PCR amplification was performed to obtain three fragments, SnaBI-C, prM-E, and NS1-BamHI, respectively; the amplification primers were reCMV-F and CHv-C / prM-R, CHv-C / prM-F and CHv-E / NS1-R, and CHv-E / NS1-F and 2A-BamHI-R, and the nucleotide sequences are shown in SEQ ID NOs. 1-6, respectively; the three recovered fragments were then homologously reassembled into the linearized pUC19-FL-KUNV-intron vector to construct a chimeric infectious clone plasmid, named pUC19-KUNV-DTMUV prM / E; S2. The chimeric infectious clone plasmid in S1 was transfected into cells to rescue the chimeric virus, and the chimeric Kunjin virus with Tembusu virus prM-E was obtained, which was named rKUNV-CHv-prM / E.

2. The preparation method according to claim 1, characterized in that In S1, the linearized pUC19-FL-KUNV-intron vector is obtained by double digestion with SnaBI and BamHI restriction enzymes.

3. A chimeric Kunjin virus containing Tembusu virus prM-E obtained by the preparation method according to claim 1 or 2.

4. Use of the chimeric Tembusu virus prM-E Kunjin virus as claimed in claim 3 in the preparation of duck Tembusu virus vaccine, characterized in that: The duck Tembusu virus vaccine is a live attenuated duck Tembusu virus vaccine.

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