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Porcine deltacoronavirus and application thereof

A coronavirus and virus strain technology, applied in the field of isolated and purified porcine delta coronavirus, can solve the problems of reduced body resistance of piglets, no commercial vaccine, harm to the breeding industry, etc., and achieves easy clinical application, low pathogenicity, Good immunogenicity

Active Publication Date: 2020-02-04
CHINA ANIMAL HUSBANDRY IND
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

With the continuous occurrence of various porcine enteroviral diarrhea diseases, the complexity of the external environment and the decline of piglets' body resistance, more and more pig farms have successively broken out PDCoV, which has caused certain harm and economic losses to the breeding industry. There are currently no commercial vaccines against PDCoV
There are few studies on how PDCoV viral proteins regulate host functions, use host mechanisms to evade immune responses, and facilitate the survival and proliferation of the virus itself

Method used

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  • Porcine deltacoronavirus and application thereof
  • Porcine deltacoronavirus and application thereof
  • Porcine deltacoronavirus and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] The isolation of embodiment 1 porcine delta coronavirus

[0027] 1. Collection and processing of intestinal tissue

[0028] Collect porcine intestinal tissue, cut it into pieces, add 10 times the volume of DMEM medium containing 500 units of double antibody to grind, centrifuge at 12,000 rpm for 5 minutes at 4°C, take the supernatant of the grinding liquid, and freeze it at -80°C for later use.

[0029] 2. Pathogen detection in intestinal tissue

[0030] Take 200 μl of the supernatant of the above grinding liquid, and use the DNA / RNA Extraction Kit of Beijing Quanshijin Biological Company to extract DNA and RNA according to the instructions; then, use the Quanshijin Reverse Transcription Kit to reverse transcribe the RNA into cDNA. Using the extracted DNA and reverse-transcribed cDNA as templates, respectively, swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine pseudorabies virus (PRV), porcine circovirus type 2 / 3 ( PCV2 / PCV...

Embodiment 2

[0047] Example 2 Pathogenicity Experiment of Porcine Delta Coronavirus PDC-SX19

[0048] Nine 40-day-old piglets negative for porcine δcoronavirus pathogen and antibody were screened and randomly divided into 3 groups with 3 piglets in each group. The test group was nasal cavity and muscle challenge, and each pig was inoculated with 2ml of 10 7.5 TCID 50 F9 passage virus liquid, intramuscular injection 1ml10 7.5 TCID 50 F9 subcultured virus liquid; each pig in the control group was inoculated with an equal volume of sterile PBS solution in the nasal cavity. The status of the pigs was observed daily, and the rectal temperature was measured and recorded.

[0049] After continuous observation for 10 days, the body temperature of the test group and the control group fluctuated within the normal range. The observation of clinical symptoms showed that the pigs in the test group and the control group had normal appetite, no diarrhea occurred, and no pigs died, and all survived. ...

Embodiment 3

[0056] Application of Example 3 Isolated Strain PDC-SX19 in the Preparation of Porcine Delta Coronavirus Inactivated Vaccine

[0057] 1. Preparation of porcine deltacoronavirus inactivated vaccine

[0058] The PDC-SX19 isolate was inoculated in ST cells or LLC-PK cell monolayers or suspension cells according to 1% of the culture volume for continuous passage, and the virus liquid was harvested. After repeated freezing and thawing for 3 times, the supernatant was collected by centrifugation, and the virus was determined. Titer. Virus titer 10 7.5 After TCID50, sterility and exogenous virus inspection are qualified, formaldehyde with a final concentration of 0.1% is added for inactivation. Add an adjuvant with the virus liquid at a volume ratio of 1:1 (there are two adjuvants A and B: the A adjuvant is a colloidal water adjuvant made by Zhongmu (formula: 90% aqueous solution for injection, 2% polyvinylpyrrolidone, polyvinylpyrrolidone Lactic-glycolic acid (PLGA) 3%, Macrogol ...

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Abstract

The invention provides a porcine deltacoronavirus and application thereof. The porcine deltacoronavirus is isolated from pig intestinal tissues, and a porcine deltacoronavirus PDC-SX19 is obtained bypassage and purification, with a preservation number of CGMCC No. 18332. The isolate can realize stable proliferation on the passage cells and produce typical cytopathy. The porcine deltacoronavirus isolate of the invention has an excellent immunogenicity; a vaccine prepared by the isolate can induce piglets to produce a high-level neutralizing antibody; and an ELISA kit prepared by the isolate can better detect the porcine deltacoronavirus antibody in serum.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an isolated and purified porcine delta coronavirus and its application. Background technique [0002] Porcine deltacoronavirus (Porcine deltacoronavirus, PDCoV) is a newly discovered coronavirus that infects pigs in 2012 and belongs to the genus Deltacoronavirus of the Coronaviridae family. PDCoV is a single-stranded RNA virus with a diameter of 120-180nm, an envelope and fibers, and a genome length of 25.4kb, including 5' and 3' non-coding regions and 7 open reading frames, which encode polymerase proteins 1a / 1b, Spike protein (S), small membrane protein (E), membrane protein (M), nonstructural protein 6 (NS6), nucleocapsid protein (N) and nonstructural protein 7 (NS7). PDCoV can cause diarrhea symptoms in sows and piglets, which are characterized by vomiting, watery diarrhea, dehydration and loss of appetite. Pigs of all ages are susceptible, and the mortality rate of ...

Claims

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Application Information

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IPC IPC(8): C12N7/00A61K39/215A61P31/14G01N33/68G01N33/569C12R1/93
CPCA61K39/12A61K2039/5252A61K2039/552A61P31/14C12N7/00C12N2770/20021C12N2770/20034G01N33/56983G01N33/6854G01N2333/165G01N2469/20
Inventor 杨小蓉马良许磊王天恺常昊天李晓冉潘京学
Owner CHINA ANIMAL HUSBANDRY IND
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