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Swine epidemic diarrhea virus and application thereof

A porcine epidemic diarrhea and virus technology, applied in the direction of antiviral agent, virus/phage, antiviral immunoglobulin, etc., can solve problems such as prevention difficulties, and achieve good serum titer, good immunogenicity, and strong pathogenicity Effect

Active Publication Date: 2015-02-25
GUANGDONG HAID ANIMAL HUSBANDRY & VETERINARY RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For most viral diarrhea, vaccine immunization is generally selected as the prevention and control measure. Although commercial vaccines for porcine epidemic diarrhea have been used in pig herds, the virus still appeared in immunized pig herds, giving the prevention of the disease It has brought great difficulties, so finding a new virus strain with better immunogenicity has great significance for the development of preventive measures and diagnostic methods for porcine epidemic diarrhea

Method used

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  • Swine epidemic diarrhea virus and application thereof
  • Swine epidemic diarrhea virus and application thereof
  • Swine epidemic diarrhea virus and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] The isolation culture and identification of embodiment 1 virus

[0026] (1) Virus isolation and culture

[0027] The RT-PCR-positive fecal samples of 5-day-old sick piglets were collected from a pig farm in Guangdong Province in 2013. After treatment, they were inoculated onto African green monkey kidney passage cells (Vero). Porcine epidemic diarrhea virus PEDV / CHGD01 was obtained.

[0028] (2) Identification of the virus

[0029] 1. Genome sequence analysis

[0030] The genome sequence of this virus is analyzed, and the whole gene sequence is the sequence shown in SEQ ID No.1 (see the sequence list), which is compared with the existing porcine epidemic diarrhea virus sequence in GenBank. The origin is above 99%.

[0031] 2. Electron microscope inspection

[0032] Observe porcine epidemic diarrhea virus PEDV / CHGD01 under the electron microscope, such as figure 1 As shown, the diameter of virus particles ranges from 90-190nm, and there is a capsule on the surface,...

Embodiment 2

[0039] The preparation of embodiment 2 vaccine

[0040] Porcine epidemic diarrhea virus PEDV / CHGD01 infected Vero cells, cultivated, harvested virus fluid, and determined TCID 50 After , inactivation and purification, the vaccine stock solution is obtained for further preparation of vaccines.

[0041] (1) Virus culture

[0042] Resuscitate Vero cells, after expansion, inoculate the virus PEDV / CHGD01 into the cell bottle at a ratio of 1:10-1:100 (v / v) after the cells grow to a thin monolayer, 37, ℃ 5% CO 2 Cultivate in an incubator, and observe the cytopathic changes every day. When the cytopathic changes reach more than 80%, the virus liquid is harvested and stored at -20°C for later use.

[0043] (2) Virus inactivation and purification

[0044] Determination of TCID before virus inactivation 50 . Inactivate the virus liquid prepared above with 1:1000-1:10000 (v / v) formaldehyde solution for 24-48h. Add 42.5g of ammonium sulfate to 100mL of the virus liquid, stir well, pu...

Embodiment 3

[0047] Embodiment 3 immunogenicity test

[0048] The virus PEDV / CHGD01 vaccine prepared in Example 2 was subjected to an immunogenicity test on piglets.

[0049] (1) Immunize the pregnant sows with the vaccine prepared above 40 days and 20 days before delivery, 4mL / head / time, observe and record the health status and litter condition of the sows during the whole experiment, and before immunization, The blood of the sows was collected at parturition and 7 days and 14 days after parturition, and the serum was collected for antibody neutralization determination.

[0050] The method is as follows: add the isolated sow serum to the 96-well cell culture plate, dilute to a certain concentration with the cell growth medium (4 replicates for each dilution hole), add the previously diluted to 200TCID 50 / mL of virus liquid, mixed evenly and placed in 37, ℃ 5% CO2 Neutralize in the incubator for 1h-2h, then add the cell suspension, the cell concentration is 2-3×10 5 Individual / mL, place...

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Abstract

The invention discloses an epidemic diarrhea virus with apreservation number of CCTCC NO:V201407. Through electron microscope observation, the virus has particle diameter in the range of 90-190 nm, envelope on the surface, and petaloid spikes on the envelope and radiating from the core to the periphery. The invention also relates to the application of the swine epidemic diarrhea virus. The virus In the invention can realize efficient proliferation in vero cells and reach virus concentration of 107TCID50 / mL, and has no exogenous virus contamination and strong pathogenicity to piglets; the virus titer of 10TCID50 can cause disease in piglets and has good immunogenicity; a vaccine prepared from the virus provided by the invention has good protective effect on swine epidemic diarrhea; and a high titer serum antibody prepared from the virus provided by the invention has god better serum titer.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a porcine epidemic diarrhea virus and its application. Background technique [0002] Porcine epidemic diarrhea (porcine epidemic diarrhea, PED) is an acute infectious disease caused by porcine epidemic diarrhea virus of the Coronaviridae family. It mainly causes diarrhea, vomiting, and dehydration in suckling piglets, and eventually leads to death, and the mortality rate is extremely high. The disease spreads widely in Europe and Asia, causing serious economic losses to some countries and regions every year, and bringing great harm to the pig industry. After April 2013, porcine epidemic diarrhea broke out in the United States and spread rapidly to more than 20 states in the United States, causing a serious blow to the American pig industry. For most viral diarrhea, vaccine immunization is generally selected as the prevention and control measure. Although commercial va...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N7/00C07K16/10A61K39/215A61P31/14C12R1/93
Inventor 贾爱卿冯晓声刘琪周如月王贵平
Owner GUANGDONG HAID ANIMAL HUSBANDRY & VETERINARY RES INST
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