A duck Tembusu virus

Abstract

The present invention discloses a duck Tembusu virus, wherein the duck Tembusu virus DTMUV‑201909 has a deposit number of CCTCCNo: V202262, and was deposited in the China Center for Type Culture Collection on September 15, 2022. The duck Tembusu virus provided by the present invention has activation dependent on the infection time and infection multiplicity of p38MAPK in DEF cells. Activation of the p38MAPK pathway may achieve regulation of DTMUV replication, and then based on the in-depth analysis of DTMUV infection-induced host cell signal transduction in the later stage, it can help to clarify the molecular mechanism of viral pathogenicity and provide new targets for antiviral treatment.

Description

Technical Field

[0001] The invention relates to a duck Tembusu virus and an application field thereof, in particular to a duck Tembusu virus. Background Art

[0002] Duck tembusu virus (DTMUV) belongs to the Flavivtridae family and the Flavivirus genus. Its genome is a single-stranded positive-strand RNA with a total length of about 11 kb, encoding a polyprotein precursor containing 3425 amino acids. The virus mainly infects laying ducks, which can cause a rapid decline in egg production, ataxia, ovarian hemorrhage, inflammation, etc. It can also infect meat ducks, causing neurological symptoms and even death. DTMUV has a fast mutation rate, is highly contagious, and can be prevalent all year round, which brings great difficulties to the prevention and control of duck tembusu virus disease. Its commercial vaccines include attenuated vaccines, inactivated vaccines, and DNA vaccines. However, the newly emerged variants are highly pathogenic, and the existing vaccines cannot fully protect them. There is currently no specific preventive and control drug. Moreover, its host range is wide, and in addition to infecting poultry, it can also infect mammals. Epidemiological survey results show that DTMUV can be effectively transmitted between ducks through direct contact and aerosols. Literature reports that DTMUV neutralizing antibodies were detected in the serum of duck farm employees, which has not yet been shown to be pathogenic to humans, indicating that DTMUV can be transmitted between ducks and humans and has a potential zoonotic risk, which should be given sufficient attention.

[0003] p38 mitogen-activated protein kinase (MAPK) is a member of the MAPK family and is a serine / threonine protein kinase that can be activated by dual phosphorylation of the Thr and Tyr sites in its Thr-Gly-Tyr motif. Since viruses are completely dependent on host cells to complete their life cycle, they participate in regulating pre-existing intracellular signal transduction networks with the host for their own proliferation. At present, it is known that many viruses activate p38MAPK after binding, entry or replication, and use host pathways to regulate cellular or viral gene expression, or both, to achieve successful viral proliferation. For example, influenza A virus (IAV), hepatitis C virus (HCV), dengue virus (DENV), Zika virus (ZIKV), porcine epidemic diarrhea virus (PEDV), Newcastle disease virus (NDV), reovirus (ReoV), chikungunya virus (CHIKV), Ebola virus (EBOV), rotavirus (Rotavirus) and other viruses can activate p38 MAPK and promote viral replication during infection.

[0004] At present, Wang et al. have screened the HELIC domain of DTMUV NS3 protein through the yeast two-hybrid system and found that it interacts with PRDX1 protein in DEF cells. This interaction regulates p38 MAPK to avoid apoptosis caused by oxidative stress, making it possible for the virus to escape the host immune response. When screening inhibitors that may act on DTMUV infection, Tian et al. found that the JNK pathway inhibitor SP600125 can significantly inhibit viral replication. JNK is another important member of the MAPK family. These studies have shown that kinases play an important role in DTMUV infection. As an emerging virus, DTMUV is currently studied more in terms of etiology, epidemiology, vaccine development and diagnostic method establishment, while there are fewer applications that reveal the molecular mechanisms of key links in the commonality of viral replication. Summary of the invention

[0005] In view of the above-mentioned prior art, the purpose of the present invention is to provide a new duck Tembusu virus, and the virus can provide a new direction for in-depth analysis of host cell signal transduction induced by DTMUV infection based on the activation of the p38 MAPK pathway in cells, thereby helping to clarify the molecular mechanism of viral pathogenicity from the perspective of the commonality of viral replication and provide new targets for antiviral treatment in the later stage.

[0006] In order to achieve the above-mentioned purpose, the present invention provides a duck Tembusu virus, the duck Tembusu virus DTMUV-201909 has the accession number of CCTCC No: V202262, and was deposited in the China Center for Type Culture Collection on September 15, 2022.

[0007] Preferably, the cDNA sequence of the duck Tembusu virus DTMUV-201909 is shown in SEQ ID No: 1.

[0008] Preferably, the amino acid sequence of the duck Tembusu virus DTMUV-201909 is as shown in SEQ ID No: 2.

[0009] The duck Tembusu virus DTMUV-201909 provided by the present invention has an activation dependent on the infection time and infection multiplicity of p38MAPK in DEF cells, and the duck Tembusu virus C protein can activate p38MAPK. Based on the activation of the p38 MAPK pathway, it is possible to regulate the replication of DTMUV, and then based on the in-depth analysis of the host cell signal transduction induced by DTMUV infection in the later stage, it can help to clarify the molecular mechanism of viral pathogenicity and provide new targets for antiviral treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the following specific embodiments, they are used to explain the present invention but do not constitute a limitation of the present invention. In the accompanying drawings:

[0011] Figure 1A This is a graph showing the infection status of infected DEF cells detected by indirect immunofluorescence in detection example 1;

[0012] Figure 1B is a graph showing the expression results of DTMUV E protein detected by Western blot method in detection example 2;

[0013] Figure 1C This is a result diagram of detecting the RNA content of DTMUV using qRT-PCR in detection example 2;

[0014] Figure 2A This is the result diagram of the expression level of p38 and P-p38 proteins in DEFs cells detected by Western blot in Application Example 1;

[0015] Figure 2B This is a result diagram of the p38 RNA content in DEFs cells detected by qRT-PCR in Application Example 1;

[0016] Figure 3A This is a result diagram of the expression levels of p38 and P-p38 proteins in DEFs cells detected by Western blot in Application Example 2;

[0017] Figure 3B This is a result diagram of the p38 RNA content in DEFs cells detected by qRT-PCR in Application Example 2;

[0018] Figure 4 This is a graph showing the expression levels of p38 and P-p38 proteins in cells after incubation in Application Example 3. DETAILED DESCRIPTION

[0019] The specific embodiments of the present invention are described in detail below. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0020] In the present invention, duck embryos were purchased from Shandong Haotai Experimental Animal Breeding Co., Ltd.; DMEM cell culture medium, trypsin and sterile PBS solution were purchased from Gibco; the double antibody used for culturing cells was purchased from Shanghai Bio-Technology; fetal bovine serum was purchased from Biological Industries; ChamQ Universal SYBR qPCR Master Mix fluorescence quantitative kit, III RT SuperMix for qPCR (+gDNAwiper) reverse transcription kit and FastPure Cell / Tissue Total RNA Isolation Kit V2 total RNA extraction kit were purchased from Nanjing Novazon Biotechnology Co., Ltd.; protease inhibitors and phosphatase inhibitors were purchased from MCE; P-p38 MAPK (#4511) and p38 MAPK (#9212) antibodies were purchased from CST; GAPDH, peroxidase-labeled goat anti-rabbit IgG (H+L), and peroxidase-labeled goat anti-mouse IgG (H+L) were purchased from Yisheng Biotechnology (Shanghai) Co., Ltd.

[0021] The following is further described through specific examples.

[0022] Example 1. Obtaining duck Tembusu virus DTMUV-201909 in the present invention

[0023] Take the collected sick duck tissue, cut it into pieces under a sterile environment, add sterile PBS solution (containing 1000U double antibody) at a volume ratio of 1:5 between the sick duck tissue and the sterile PBS solution, freeze and thaw repeatedly for 3 times, centrifuge at 5000r / min for 10min, and take the supernatant. After filtering the supernatant with a 0.22μm filter, inoculate the treated supernatant into 9-11-day-old SPF duck embryos, incubate at 37℃, observe for 5 consecutive days, discard dead duck embryos within 24h, and store the dead duck embryos at 4℃ for the rest of the time. After the 5th day, all the alive duck embryos are placed at 4℃ overnight. Harvest all duck embryo allantoic fluid from the first to the fifth day (here refers to the mixture of duck embryo allantoic fluid collected within five days from all duck embryos that died and survived within five days, which is recorded as the primary allantoic fluid, and observe the duck embryo embryo lesions. The allantoic fluid collected above was blindly propagated continuously to the third generation, and the allantoic fluid blindly propagated to the third generation was harvested in the same way as the first generation allantoic fluid, and stored at -20°C for future use.

[0024] After RNA was extracted from the allantoic fluid harvested after three generations of blind transmission, it was reverse transcribed into cDNA, and the reverse transcribed cDNA was subjected to whole genome sequencing, and its sequence is shown in SEQ ID No:1.

[0025] Example 2: Cultivation of duck Tembusu virus DTMUV-201909 in duck embryo fibroblasts

[0026] A. Preparation of duck embryo fibroblasts: Take a 10-day-old SPF duck embryo in good condition and disinfect the eggshell with alcohol. Take the prepared sterile tweezers and gently break the eggshell at the air chamber, carefully remove the eggshell and expose the chorioallantoic membrane. Use new sterile elbow tweezers to gently open the chorioallantoic membrane to expose the duck embryo, carefully clamp the neck of the duck embryo with tweezers, take it out and put it in a sterile plate, and add sterile PBS solution to wash. Remove the limbs, head and internal organs, leaving the trunk part, change to a new plate and wash it again with sterile PBS solution until the blood and other residues in the trunk are removed. Put the washed duck embryo trunk into a 5mL beaker and cut the trunk with sterile small scissors until it is viscous and finely chopped. Transfer the cut tissue in the beaker to a 15mL sterile tube, add 10mL of 0.05% trypsin, and place it in a 37℃ incubator for digestion. Observe after about 10-15min, and when the tissue becomes round and blurred and cloudy, it indicates that the digestion is complete. Add DMEM culture medium containing 10% serum to terminate the reaction, centrifuge at 1200rpm for 5min, discard the supernatant, add 10mL DMEM culture medium with a concentration of 10% serum to the precipitate, transfer to a 50mL sterile tube, and blow with a pipette to disperse the cells into single cells. Filter the obtained cell fluid into a T25 cell bottle with a sterile cell filter, add an appropriate amount of DMEM culture medium containing serum, and continue to culture in a cell culture incubator to obtain cultured duck embryo fibroblasts.

[0027] B. Infection and amplification of duck embryo fibroblasts by duck Tembusu virus DTMUV-201909 of the present invention

[0028] Take the above-cultured duck embryo fibroblasts with good growth status (the judgment standard is that the density of 70%-80% confluence is reached on the second day, and the wall is well attached) and passaged into a T75 cell culture flask. The next day, the culture medium used for duck embryo fibroblast culture was replaced by serum-free DMEM culture medium from serum-containing DMEM culture medium, and the duck Tembusu virus DTMUV-201909 in the present invention was inoculated into the duck embryo fibroblasts in the above culture according to a multiplicity of infection (expressed as MOI) of 0.1. After 1 hour of virus infection, it was replaced with an appropriate amount of normal serum-containing DMEM culture medium and placed in a cell culture incubator for culture. After the cells began to fall off after lesions appeared, the cell bottle was frozen in a -80°C refrigerator, and the cells were repeatedly frozen and thawed 3 times to lyse the cells and release the duck Tembusu virus DTMUV-201909 therein. The cell lysate after repeated freezing and thawing was dispensed into sterile 10 mL tubes, centrifuged at 4000 rpm for 10 min at 4°C, the supernatant was collected, and the cell debris was removed with a 0.22 μm filter to obtain the duck Tembusu virus solution. After mixing, the solution was divided into devices and stored in a -80°C refrigerator for later use.

[0029] Test Example 1

[0030] After inoculating duck embryo fibroblasts (DEF cells) with 0.1 MOI of the duck Tembusu virus DTMUV-201909 of the present invention as described in step B of Example 2, the cells were cultured for 48 hours, fixed with formaldehyde, and blocked with 5% skim milk powder. Then, DTMUV E protein antibody (the DTMUV E protein antibody here is a polyclonal antibody obtained by inducing mice to be immunized using the duck Tembusu virus DTMUV-201909 prepared in Example 1 as an antigen, and the induction method here adopts a polyclonal antibody preparation method that can be understood and commonly used by technicians in the field, and no further details are given here) was diluted with a diluent at 1:10 as a primary antibody, incubated at room temperature for 4 hours, washed, added with 488 labeled goat anti-mouse antibody for 2 hours, washed, and observed under a fluorescence microscope. At the same time, duck embryo fibroblasts not inoculated with the virus were set up as negative controls. The results are as follows Figure 1A shown.

[0031] Test Example 2

[0032] The operation was performed in the manner of Detection Example 1, except that the duck Tembusu virus DTMUV-201909 of the present invention with different multiplicity of infection was used to inoculate DEF cells, wherein the multiplicity of infection gradient was set to 0 MOI (ie, negative control), 0.01 MOI, 0.1 MOI and 1 MOI, respectively, and the adherent cells were collected 48 hours after infection.

[0033] The expression of DTMUV E protein was detected by Western blot. Figure 1B As shown, GAPDH is the internal reference protein expression band.

[0034] The RNA content of DTMUV was detected by qRT-PCR, and the results were as follows: Figure 1C shown.

[0035] pass Figure 1A-Figure 1C It can be seen that the DTMUV of the present invention can successfully infect DEF cells, and as the multiplicity of infection increases, the content of the virus in the cells increases significantly.

[0036] Application Example 1

[0037] DEF cells were plated on a 6-well plate for culture, and when the cell growth reached 60%-80%, the duck Tembusu virus DTMUV-201909 of the present invention was infected (the MOI of the duck Tembusu virus DTMUV-201909 used here was 1), and the medium was changed after 1 hour, and then the culture was continued. The cultured DEF cells were collected at 12h, 24h, 36h, and 48h.

[0038] 1. qRT-PCR detection: The total RNA in the cells was extracted using the FastPure Cell / Tissue Total RNA Isolation Kit V2, and the concentration of the extracted RNA was measured using Nanodrop. III RT SuperMix for qPCR (+gDNAwiper) reverse transcription kit was used to reverse transcribe 1 μg of RNA into cDNA.

[0039] Using the cDNA obtained by the reverse transcription as a template, the qRT-PCR method was used to perform fluorescence amplification on DTMUV, p38 and the internal reference gene GAPDH using the corresponding amplification primer pairs, and the Ct values ​​were detected accordingly, and the relative expression level of the target gene was calculated according to the corresponding Ct values ​​according to the 2^-ΔΔct method. Among them, the amplification primer pair of DTMUV is shown in SEQ ID No: 3 and SEQ ID No: 4, the amplification primer pair of p38 is shown in SEQ ID No: 5 and SEQ ID No: 6, and the amplification primer pair of the internal reference gene GAPDH is shown in SEQ ID No: 7 and SEQ ID No: 8.

[0040] 2. Western blot detection: The DEF cells collected above were lysed on ice for 30 minutes using RIPA cell lysis buffer with protease and phosphatase inhibitors, and the supernatant was centrifuged for SDS-PAGE electrophoresis, and then wet-transferred to a polyvinylidene fluoride (PVDF) membrane. Use 5% skim milk powder to block at room temperature for 2 hours, prepare the primary antibody with antibody diluent according to the antibody manual, incubate overnight at 4°C in a shaker, and wash; use HRP-labeled secondary antibodies of the corresponding species, dilute according to the book ratio, incubate at room temperature for 2 hours, wash, and finally add ECL super-sensitive luminescent liquid and expose in a chemiluminescence instrument.

[0041] Among them, the expression levels of p38 and P-p38 proteins in DEFs cells detected by Western blot were as follows: Figure 2A As shown, the p38 RNA content in DEFs cells detected by qRT-PCR is as follows Figure 2B shown.

[0042] pass Figure 2A and Figure 2B It can be seen that after DTMUV infected DEF for different time periods (12h, 24h, 36h, 48h), the expression level of phosphorylated p38 in 3 increased significantly after 6h, and the expression level of phosphorylated p38 increased significantly with the increase in infection time, which is consistent with the trend of DTMUV proliferation in DEF cells.

[0043] Application Example 2

[0044] The method of Application Example 1 was followed, except that DEF cells were infected with 0 MOI, 0.01 MOI, 0.1 MOI and 1 MOI, respectively, and then cultured for 48 h. The expression levels of p38 and P-p38 proteins in DEFs cells detected by Western blot were as follows: Figure 3A As shown, the p38 RNA content in DEFs cells detected by qRT-PCR is as follows Figure 3B shown.

[0045] pass Figure 3A and Figure 3B It can be seen that when DEF cells were infected with DTMUV at different MOIs (0, 0.01, 0.1, and 1), the total protein content of p38 in the cells did not change with the virus titer, but the expression level of phosphorylated p38 increased with the increase of virus titer, indicating that DTMUV activated p38MAPK after infection, and this activation was MOI-dependent.

[0046] Application Example 3

[0047] PCR amplification was performed using the cDNA obtained by reverse transcription in Application Example 1 as a template. The PCR amplification primer pair is shown in SEQ ID No: 9 and SEQ ID No: 10.

[0048] The PCR amplification system was 50 μL: 5 μL of 10×PCR Buffer for KOD-Plus-Neo, 5 μL of 2 mmol / L dNTPs, 3 μL of 25 mmol / L MgSO4, 1.5 μL of upstream and downstream primers (primer concentrations were 10 μmol / L each), 200 ng of template cDNA, 1 μL of 1 U / μL KOD-Plus-Neo, and made up to 50 μL with ddH2O.

[0049] PCR reaction conditions: 94°C for 2 min, 98°C for 10 s, 68°C for 1 min, 30 cycles, 68°C for 5 min. PCR products were obtained.

[0050] The pET-30a empty vector was double-digested by EcoR I and Xho I to obtain the digestion product. The PCR product and the digestion product obtained above were identified by agarose gel electrophoresis, purified and recovered by a gel recovery kit, and then the two products were connected by homologous recombination enzyme. The homologous recombination product was transformed into competent cells DH5α, and coated on a kanamycin-resistant LB plate, inverted and cultured overnight at 37°C for 12 hours, and the plasmid with correct digestion identification was selected for gene sequencing to construct the prokaryotic expression plasmid of duck Tembusu virus C protein of the present invention, and named pET-30a-C.

[0051] The pET-30a-C obtained above was transformed into BL21 (DE3) competent cells, and a single colony was picked and inoculated into 10 mL of kanamycin-resistant LB medium, and cultured at 37°C and 220 r / min for 4 hours. When the OD600 of the bacterial solution was about 0.6-0.9, IPTG with a final concentration of 1.0 mmol / L was added, and the culture was induced at 37°C and 220 r / min for 4 hours, and then centrifuged at 12000 r / min to obtain the bacteria, washed 2-3 times with PBS solution and resuspended, ultrasonically broken and lysed under ice bath conditions until the solution was clear, and the supernatant and precipitate were obtained by centrifugation, and the expression of protein C was analyzed by SDS-PAGE. The correctly expressed pET-30a-C plasmid was induced in large quantities, the precipitate was collected, washed with PBS solvent, and resuspended with inclusion body lysis solution, purified by Ni affinity chromatography pre-packed column, and the target protein was eluted with different concentration gradient imidazole buffer. SDS-PAGE analysis and Western-blot analysis were performed, and the protein concentration of the purified protein was determined using the BCA method and stored at -80°C for future use. The purified protein was a prokaryotic expression protein, named His-DTMUV C.

[0052] Western blot detection: DEF cells were plated on 6-well plates. When the cell growth reached 60% to 80%, duck Tembusu virus C protein (the above-mentioned His-DTMUV C) was added at a concentration of 10 μmol / L and incubated for 12 hours. The incubated cells were collected and lysed on ice for 30 minutes using RIPA cell lysis buffer with protease and phosphatase inhibitors. The supernatant was centrifuged and subjected to SDS-PAGE electrophoresis, and then wet-transferred to a polyvinylidene fluoride (PVDF) membrane. 5% skim milk powder was used for blocking at room temperature for 2 hours. The primary antibody was prepared with antibody diluent according to the antibody manual, incubated overnight at 4°C in a shaker, and washed. The HRP-labeled secondary antibody of the corresponding species was used, diluted according to the book ratio, incubated at room temperature for 2 hours, washed, and finally ECL supersensitive luminescent solution was added and exposed in a chemiluminometer. The expression levels of p38 and P-p38 proteins in the detected cells were as follows: Figure 4 shown.

[0053] pass Figure 4It can be seen that after the prokaryotically expressed His-DTMUV C protein was co-incubated with DEF cells for 12 hours, the phosphorylated p38 content in the cells increased significantly, indicating that the duck Tembusu virus C protein of the present invention can activate the p38MAPK signaling pathway of DEF cells.

[0054] It can be seen from the above embodiments, test examples and application examples that the duck Tembusu virus DTMUV-201909 provided by the present invention has an activation dependent on the infection time and infection multiplicity of p38 MAPK in DEF cells, and the duck Tembusu virus C protein can activate p38 MAPK. The activation of the p38 MAPK pathway may regulate the replication of DTMUV. The in-depth analysis of host cell signal transduction caused by DTMUV infection of all genotypes in the later stage will help to clarify the molecular mechanism of viral pathogenicity and provide new targets for antiviral treatment.

[0055] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0056] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

[0057] In addition, various embodiments of the present invention may be arbitrarily combined, and as long as they do not violate the concept of the present invention, they should also be regarded as the contents disclosed by the present invention.

Claims

1. A duck Tembusu virus, characterized in that The deposit number of the duck Tembusu virus is CCTCC No: V202262, and it was deposited in the China Center for Type Culture Collection on September 15, 2022.

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