Kumpu virus embedded with tembusu virus prM-E as well as preparation method and application of Kumpu virus

Abstract

The invention discloses a Kujin virus embedded with tembusu virus prM-E as well as a preparation method and application of the Kujin virus. According to the invention, on the basis of a reverse genetic operating system of a KUNV (Kumphu virus), a DTMUV prM / E gene is used for replacing a KUNVprM / E gene, and a chimeric DTMUV virus infectious clone plasmid taking the KUNV as a skeleton is constructed, and is named as pUC19-KUNV-DTMUV prM / E. Then, virus rescue is carried out, so that the kumpu virus embedded with the tembusu virus prM-E is obtained, and the kumpu virus is named as rKUNV-CHv-prM / E (CHv). The analysis of virus characteristics shows that CHv can effectively proliferate in BHK-21 and DEF cells, and the virulence of duck embryos is weaker than that of KUNV and DTMUV. Animal experiment results show that the CHv only detects an obvious virus level in the brain and does not generate viremia; after CHv immunization, an IgG antibody level similar to that after KUNV and DTMUV immunization can be generated. Therefore, the recombinant virus which takes the KUNV as the skeleton and is embedded with the DTMUVprM / E can induce the duckling to generate a neutralizing antibody level with enough immune protection, and can be applied to preparation of a duck tembusu virus vaccine.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular biology, and in particular to a chimeric Kunjin virus of Tembusu virus prM-E and a preparation method and application thereof. Background Art

[0002] Duck Tembusu virus disease, also known as duck hemorrhagic oophoritis and duck yellow virus disease, is a new acute infectious disease caused by duck Tembusu virus (DTMUV). The virus mainly causes depression, reduced feeding and a sudden drop in egg production in laying ducks; in ducklings or growing ducks, the virus can cause neurological symptoms and hind limb paralysis. DTMUV infects a variety of duck breeds, including but not limited to Cherry Valley ducks, Mallard ducks, and Beijing ducks. Ducks infected with the virus usually develop the disease at 20-40 days of age. The main clinical symptoms are: fever, a significant decrease in feed intake, weakness and paralysis of the limbs, and a significant decrease in egg production. After infection, ducklings are characterized by neurological symptoms such as unsteady standing, paralysis, and head and neck tremors. Characteristic lesions after infection in breeder ducks include bleeding of the follicle membrane, deformation and even rupture of the follicles; after infection in laying ducks, egg production drops sharply or even stops, and severe infection can lead to death, with a mortality rate of 10%-30%. After infection, the lesions in laying ducks are mainly concentrated in the ovaries and oviducts. Autopsy shows ovarian bleeding, atrophy or dysplasia, and oviduct atrophy or necrosis. In severe cases, the yolk ruptures and yolk peritonitis occurs.

[0003] At present, the prevention and control measures for TMUV are mainly carried out through vaccination. Although there are commercial attenuated live vaccines and inactivated vaccines, ducks can still be infected with TMUV after vaccination, and the weak strains obtained by separating and propagating the strains have potential safety risks. Therefore, there is still a lot of room for improvement in the current DTMUV vaccine, especially the need to develop a DTMUV live attenuated vaccine with high safety and good immune effect. Summary of the invention

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

[0005] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows: a method for preparing a chimeric Kunjin virus prM-E of Tembusu virus, comprising the following steps:

[0006] 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. The three fragments were then assembled into the 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 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 (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 NOs.1-6.

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

[0010] The present invention also provides the Kunjin virus chimeric with Tembusu virus prM-E prepared by the above preparation method.

[0011] The present invention also provides the use of the above-mentioned chimeric Tembusu virus prM-E Kunjin virus in the preparation of duck Tembusu virus vaccine, wherein the duck Tembusu virus vaccine is a live attenuated duck Tembusu virus vaccine.

[0012] The beneficial effects of the present invention are:

[0013] 1) Kunjin virus (KUNV) is a naturally attenuated strain of West Nile virus (WNV). Based on the reverse genetic operating system of KUNV, the present invention replaces the KUNVprM / E gene with the DTMUV prM / E gene to construct a chimeric DTMUV virus infectious cloning plasmid with KUNV as the backbone, named pUC19-KUNV-DTMUVprM / E.

[0014] 2) In the present invention, a Kunjin virus chimeric with Tembusu virus prM-E is obtained by virus rescue, and is named rKUNV-CHv-prM / E (CHv). Analysis of the rescued virus characteristics shows that CHv can effectively proliferate in BHK-21 and DEF cells, and its duck embryo toxicity is weaker than KUNV and DTMUV. Subsequently, CHv was inoculated into 3-day-old ducklings by intramuscular injection. After immunization, tissues and blood were collected to detect the viral tissue load and monitor the viremia, and serum was collected to detect antibody levels. The results showed that CHv only detected obvious virus levels in the brain, and did not produce viremia; after CHv immunization, IgG antibody levels similar to those after KUNV and DTMUV immunization were produced. Therefore, the recombinant virus prepared by the present invention with KUNV as the skeleton chimeric DTMUV prM / E can induce ducklings to produce neutralizing antibody levels with sufficient immune protection, and can be used to prepare duck Tembusu virus vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0016] Figure 2 The rescue of the recombinant KUNV with the chimeric DTMUVprM-E gene, including (A) CPE and IFA verification of CHv; (B) KUNV, DTMUV and CHv incubated with TMUV E protein polyclonal antiserum for verification; (C) KUNV, DTMUV and CHv incubated with KUNV E protein polyclonal antiserum for verification; (D) KUNV, DTMUV and CHv incubated with KUNV NS1 protein polyclonal antiserum for verification.

[0017] Figure 3 In vitro growth characteristics of CHv; (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) 1000TCID 50 CHv duck embryo toxicity; (G) 10000TCID 50 CHv duck embryo toxicity. "ns" indicates no significant difference, "*" indicates P < 0.1, "**" indicates P < 0.01, "***" indicates P < 0.001, and "****" indicates P < 0.0001.

[0018] Figure 4 This is a schematic diagram of the pathogenicity test operation for ducklings.

[0019] Figure 5Figure 2 shows the pathogenicity of the chimeric virus in ducklings, including (A) weight changes of ducklings; (B) mortality of ducklings; (C) viral load in lung tissue; (D) viral load in liver tissue; (E) viral load in brain tissue; (F) viral load in spleen tissue; (G) viral load in kidney tissue; (H) viral load in heart; (I) viral load in small intestine tissue; and (J) viral load in blood.

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

[0021] TMUV and KUNV are both deposited in the Poultry Disease Center of Sichuan Agricultural University. The public literature information is as follows:

[0022] TMUV: Chen S, HeY, et al. Establishment of areverse genetics system forduck Tembusu virus to study virulence and screen antiviralgenes. AntiviralRes.

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

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

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

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

[0027] Example 1

[0028] 1 Construction and rescue of recombinant KUNV carrying 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 endonucleases, and the linearized vector was recovered using Takara's DNA purification and recovery kit.

[0031] Using pUC19-FL-KUNV-intron and pACYC-DTMUV plasmids as templates, 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 as primers, PCR amplification obtained three fragments, SnaBI-C, prM-E, and NS1-BamHI. The recovered fragments were then assembled into the linearized pUC19-FL-KUNV-intron vector by homologous recombination to obtain a chimeric infectious clone plasmid named pUC19-KUNV-DTMUVprM / E. The primer names and sequences are shown in Table 1.

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

[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 during transfection. According to the instructions of Trans Liposomal transfection reagent, 2 μg of DTMUV, KUNV, and pUC19-KUNV-DTMUVprM / E plasmids were transfected into cells respectively. After 80%-90% of the cells showed cytopathic effect (CPE), the supernatant was collected and stored at -80°C for subsequent use. The cell samples were identified by IFA.

[0036] After KUNV, DTMUV and chimeric virus plasmids were transfected into BHK-21 cells, they were verified with 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 incubated with KUNV E protein polyclonal antibody produced a positive fluorescence signal, while the chimeric virus and DTMUV did not produce any specific fluorescence ( Figure 2 C); The chimeric virus and KUNV were incubated with KUNV NS1 protein polyclonal antibody to produce a strong specific fluorescent signal, while DTMUV did not produce any positive fluorescent signal ( Figure 2 D). The above results show 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. The viruses rescued by KUNV and DTMUV transfection were marked as rKUNV and rDTMUV.

[0037] (3) Analysis of in vitro characteristics of chimeric viruses

[0038] In order to verify the in vitro characteristics of CHv, in this study, it was infected with BHK-21 and DEF cells, and then the size of the plaques produced after the virus infected the 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 plaques produced by CHv were smaller than those of rKUNV ( Figure 3 A); Growth curve results showed that in BHK-21 cells ( Figure 3 B), after CHv infected cells, its proliferation ability was weaker than that of rKUNV, and the peak time of virus titer was advanced to 72h, and then showed a downward trend, while the rKUNV virus titer reached its peak at 96h; in DEF cells ( Figure 3 D), after CHv infected cells, the virus titer reached a peak at 48h and then showed a downward trend, while the rKUNV virus titer reached a peak at 96h, which was 493 times higher than 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 at 24-72h, the CHv genome copy number was 10 times, 4 times, and 3 times higher than that of rKUNV genome copy number, respectively; in DEF cells ( Figure 3 E), the CHv genome copy number was higher than the rKUNV genome copy number at all time points.

[0039] In 10-day-old duck embryos, 1000 TCID 50 and 10000TCID 50 The virulence of the three viruses in duck embryos was compared. Figure 3 FG shows: Compared with rKUNV and CHv, the inoculation of 1000 TCID 50All duck embryos inoculated with TMUV died at 6 dpi and were inoculated with 10,000 TCID 50 The death time of duck embryos inoculated with DTMUV was advanced to 4 dpi; although the death time of duck embryos inoculated with rKUNV was later than that of duck embryos inoculated with rDTMUV, 50 and 10000TCID 50 , duck embryos died at 7dpi and 5dpi, respectively; compared with rKUNV and rDTMUV, inoculation with 1000TCID 50 and 10000TCID 50 The duck embryos of CHv were still alive at the end of the experimental observation.

[0040] (4) Pathogenicity of chimeric viruses in ducklings

[0041] In order to explore the effect of replacing the prM / E gene of KUNV with the prM / E gene of DTMUV on the pathogenicity of the chimeric virus to ducklings and the difference with the parental virus, in this example, 3-day-old ducklings were infected with 10 4.6 TCID 50 / 200 μL of rKUNV parent virus or 10 4.3 TCID 50 / 200μL chimeric virus, record daily weight changes, viremia and mortality, and collect tissues of ducklings at 3dpi, 5dpi, 7dpi, 14dpi and 28dpi to determine the tissue load of the virus ( Figure 4 ).

[0042] The results of weight changes showed that the weight changes of ducklings in the CHv and rDTMUV infection groups had the same trend, close to that of the DMEM group; however, compared with the CHv and rDTMUV infection groups, the weight gain of ducklings in the rKUNV infection group decreased from 9 dpi ( Figure 5 A). The results of duckling mortality were similar to those of weight change. The ducklings in the rKUNV-infected group died first at 2 dpi, while the ducklings in the CHv- and rDTMUV-infected groups died at 4 dpi. The mortality of ducklings in the rKUNV- and rDTMUV-infected groups was 20% (2 / 10). In contrast, the mortality of ducklings in the CHv-infected group was only 10% (1 / 10) ( Figure 5B). The results of viral tissue load detection showed that in all tissues of ducklings in the rKUNV infection group, the virus distribution could be detected at 28 dpi, especially in brain, lung, kidney, heart and small intestine tissues, especially in heart tissues where the virus could be detected at 3 dpi and 5 dpi; in all tissues of ducklings in the rDTMUV infection group, the virus could only be detected in the early stage of infection, and except for the spleen tissue with a higher viral load, the viral loads in all tissues were similar; in all tissues of ducklings in the CHv infection group, the virus distribution was only detected in brain tissue from 3 dpi to 28 dpi, and the viral loads in other tissues were similar to those in the DMEM control group; compared with the rKUNV or rDTMUV infection groups, the viral loads in all tissues of the CHv infection group were lower ( Figure 5 CI).

[0043] The results of viremia test showed that only the ducklings in the rDTMUV infection group had low viremia from 3 dpi to 28 dpi, while no viremia was detected in the ducklings in the other two infection groups. This may be because the viremia level produced by rKUNV and CHv infection in ducklings was extremely low and did not reach the minimum detection baseline ( Figure 5 J). The above results showed that the pathogenicity of CHv to ducklings was weakened after the prM / E gene of KUNV was replaced by the prM / E gene of DTMUV.

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

[0045] This study monitored the changes in serum IgG levels against rKUNV, rDTMUV, and CHv in ducklings within 4 weeks after infection with rKUNV, rDTMUV, and CHv. Figure 6 As shown, antibodies can be detected in all three viruses two weeks after infection, and the serum collected after immunization with the duck Tembusu virus commercial vaccine was used as a positive control. Antibodies can be detected throughout the entire detection period. The above data show that after ducks are vaccinated with CHv, they can induce serum IgG antibody levels similar to those of rKUNV and rDTMUV when the body shows relatively lower pathogenicity.

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

Claims

1. A method for preparing a Kunjin virus chimeric with Tembusu virus prM-E, 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. The three fragments were then assembled into the linearized pUC19-FL-KUNV-intron vector by homologous recombination to obtain a chimeric infectious clone plasmid named pUC19-KUNV-DTMUVprM / 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 (CHv).

2. The preparation method according to claim 1, characterized in that: In the 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 NOs.1-6.

3. 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.

4. A Kunjin virus chimeric with Tembusu virus prM-E obtained by the preparation method according to any one of claims 1 to 3.

5. Use of the chimeric Tembusu virus prM-E Kunjin virus as claimed in claim 4 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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