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Preparation method and application of replication-defective West Nile virus

A West Nile virus and replication-deficient technology, which is applied in the field of preparation of replication-deficient West Nile virus, can solve the problems of high difficulty, low virus yield, and long time, and achieve good immune protection, good genetic stability, and high safety sexual effect

Active Publication Date: 2019-08-23
WUHAN INST OF VIROLOGY CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the influence of transfection reagents, transfection conditions, etc., the virus titer is usually low, and the virus needs to be re-transfected every time the virus is amplified, which is time-consuming and laborious
To screen a cell line that stably expresses the missing gene, you only need to transfect the RNA that lacks the gene into the cell line to complement and rescue the defective virus, and to re-amplify the virus, you only need to infect the cell line with the rescued defective virus. Continuously obtain a large number of defective viruses, but it takes a long time and is difficult to screen a stable and efficient cell line
The applicant successfully screened and obtained 293T that provided WNV NS1 protein in trans in previous research NS1 cell line ( Zhang HL , Ye HQ , Deng CL , Liu SQ , Shi PY , Qin CF , yuan Z M , Zhang B .Generation and characterization of West Nile pseudo-infectious reporter virus for antiviral screening.Antiviral Res. 2017May; 141:38-47.), although this cell line can complement the replication defect of WNV NS1 and obtain replication defective virus, the highest titer of WNV-ΔNS1 replication defective virus on this cell line can only reach about 10 5 FFU / mL, the virus yield is low, and the cell source is not Vero cells suitable for production, which restricts its research on vaccines

Method used

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  • Preparation method and application of replication-defective West Nile virus
  • Preparation method and application of replication-defective West Nile virus
  • Preparation method and application of replication-defective West Nile virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Selection of WNV NS1 expressing cell line Vero by puromycin resistance gene using retrovirus system NS1

[0056] 1. The construction method of the eukaryotic expression plasmid pBabe-WNV-NS1 for screening NS1 expression cell lines is as follows: use the pACYC-WNV-WT wild-type virus clone already in our laboratory as a template, and use pBabe-WNV-NS1- BamHI-F: 5'-cgcGGATCCACCATGGATAGGTCCATAGCTCTCACG-3', pBabe-WNV-NS1-EcoRI-HA-R: 5'-ccgGAATTCTTAAGCGTAATCTGGAACATCGTATGGGTAAGCATTCACTTGTGACTGC-3' as primers, PCR amplification with PrimeSTAR HS enzyme, recovery of PCR products, PCR reaction system is : 94°C for 2min, 94°C for 20s, 55°C for 10s, 68°C for 2min, 68°C for 10min, 30 cycles. The recovered fragments were double digested with BamHI and EcoRI, ligated with pBabe-puro treated with the same enzymes, and then transformed into E. coli competent DH5α; the plasmids were confirmed to be correct by DNA sequencing, and named WNV NS1 eukaryotic expression plasmid pBabe -WNV-N...

Embodiment 2

[0065] A preparation method for replication defective West Nile virus, comprising the steps of:

[0066] 1. Rescue of WNV-ΔNS1 replication deficient virus: WNV-ΔNS1 RNA transcribed in vitro was transfected into NS1 expressing cell line Vero by lipofection method NS1 and 293T NS1 (Control): On the day before transfection, inoculate 2×10 5 Vero NS1 and 5×10 5 293T NS1 Cells were placed in a 35mm cell culture dish, so that the cells reached about 80% on the day of transfection; when transfecting, first discard the culture medium in the culture dish, wash once with 1mL Opti-MEM, and then add 1mL Opti-MEM (to make the cells infiltrate state); add 1mL Opti-MEM to a 1.5mL EP tube, add 4μl DMRIE-C (Invitrogen) (mix well before using DMRIE-C), first mix up and down, and then add 1μg WNV-ΔNS1 obtained by in vitro transcription Mix the RNA upside down; quickly discard the Opti-MEM in the culture dish, and add the mixture to the dish (the movement should be gentle, do not blow agains...

Embodiment 3

[0079] Comparison of the WNV-ΔNS1 replication-deficient virus rescued by the present invention and the WNV wild-type virus WT in growth curve, immune plaque morphology, structural protein size and immunogenicity:

[0080] 1. WNV-ΔNS1 replication-deficient virus and WNV wild-type virus WT growth curve comparison: with embodiment 1, inoculate 2 * 10 respectively in two 35mm cell culture dishes 5 Vero NS1Cells, according to the MOI of 0.1, add WNV-ΔNS1-deficient virus and WNV wild-type virus WT to each dish respectively, after 2 hours of adsorption, discard the virus liquid, add 2mL DMEM medium containing 2% serum to each dish 24 / 36 / 48 / 72h after infection, receive the virus supernatant respectively, measure the virus titer according to the method in Example 1, and draw the growth curve ( image 3 Middle A). The results showed that the growth trend of WNV-ΔNS1 replication-deficient virus was similar to that of wild-type virus WT.

[0081] 2. Comparison of WNV-ΔNS1 replication-d...

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Abstract

The invention belongs to the field of biotechnologies and particularly discloses a preparation method and application of a replication-defective West Nile virus. A WNV-Delta NS1 replication-defectivevirus generated by trans-complementary rescue of an NS1 cell line is cloned by utilizing the West Nile virus lacking a nonstructural protein NS1 gene, is similar to a wild-type WNV virus in growth tendency and is same as the wild-type WNV virus in immunogenicity, has high genetic stability, can only be replicated and amplified in a screened cell line expressing WNV NS1, and can be used for carrying out vaccine-related research on the virus in a biosafety secondary laboratory. The virus can be used as a safe and efficient candidate vaccine to prevent WNV virus infection, and has a good application prospect and important strategic significance for coping with potential threats of WNV in China.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a preparation method and application of a replication-deficient West Nile virus. Background technique [0002] West Nile virus (WNV) belongs to the Flavivirus genus of the Flaviviridae family, and other viruses in the same family include dengue virus (DENV), yellow fever virus (YFV), Zika virus (ZIKV) and so on. The transmission cycle of WNV in nature is bird-mosquito-bird. Birds are the storage hosts of the virus, mosquitoes are the main transmission medium, and humans and horses can be the occasional hosts of the virus. Human infection with WNV can lead to West Nile fever, and can be secondary to fatal encephalitis, meningitis and other central nervous system diseases. WNV was first discovered in the West Nile area of ​​Uganda in 1937. At first, it spread only in a small area and did not cause serious diseases. After the outbreak in New York, USA in 1999, it spread rap...

Claims

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Application Information

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IPC IPC(8): C12N7/00C12N7/02A61K39/12A61P31/14C12R1/93
CPCC12N7/00A61K39/12A61P31/14C12N2770/24151C12N2770/24134Y02A50/30
Inventor 张波李娜张亚南邓成林史佩勇袁志明
Owner WUHAN INST OF VIROLOGY CHINESE ACADEMY OF SCI
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