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Porcine epidemic diarrhea virus stain and application thereof

A porcine epidemic diarrhea and virus technology, applied in the direction of antiviral agents, viruses/bacteriophages, and medical preparations containing active ingredients, can solve problems such as acute onset, limited protective effect of porcine diarrhea, and economic losses of farmers, and achieve Good safety and good immune protection effect

Active Publication Date: 2014-04-16
兆丰华生物科技(南京)有限公司 +3
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no specific drug against porcine epidemic diarrhea virus. Due to the acute onset and rapid prevalence of the disease, once the pigs are infected, it will bring huge economic losses to the farmers.
[0003] At present, immunization is an important means to prevent and control the disease. There are reports on the use of vaccines at home and abroad, but the effect is not satisfactory.
Especially in recent years, the protective effect of the original vaccine on porcine diarrhea has become more and more limited, and cases of diarrhea continue to appear clinically. In some areas, the positive rate of porcine epidemic diarrhea antigen can be as high as 70%, which shows that the development of a vaccine that can Vaccines effective against porcine epidemic diarrhea are imminent

Method used

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  • Porcine epidemic diarrhea virus stain and application thereof
  • Porcine epidemic diarrhea virus stain and application thereof
  • Porcine epidemic diarrhea virus stain and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1 Isolation and identification of porcine epidemic diarrhea virus

[0024] 1. Isolation of porcine epidemic diarrhea virus

[0025] The small intestine of pigs with diarrhea was sent to a pig farm in Zhejiang, and the contents were collected for detection by RT-PCR. The result was positive for porcine epidemic diarrhea antigen.

[0026] Take an appropriate amount of the small intestine submitted for inspection, scrape the intestinal mucosa and contents, add PBS according to the ratio of 1:5 (weight: volume), freeze and thaw repeatedly 3 times, centrifuge to get the supernatant, filter with a 0.22 μm filter membrane, and add the final concentration to the filtrate 20 μg / ml trypsin, 37°C for 1.5 hours.

[0027] Inoculate Vero cells covered with a single layer according to the conventional method (wash three times with PBS of pH 7.4 before inoculation), inoculate the virus at a ratio of 10%, absorb at 37°C for 1 hour, and supplement the cell maintenance solution (...

Embodiment 2

[0040] Example 2 Attenuation of Porcine Epidemic Diarrhea Virus

[0041] 1. Attenuated cultivation of porcine epidemic diarrhea virus

[0042] The isolated porcine epidemic diarrhea virus was continuously passaged on Vero cells for 100 passages, cloned and purified during this process, and then continued to passage on Vero cells for 145 passages. The 145th generation virus was detected by RT-PCR, and the amplified S gene fragment was 484bp.

[0043]The PEDV ORF3 gene sequence was relatively stable in the 30th, 50th, 70th and 90th generations. There were changes in individual bases, but no impact on amino acids; and a sudden deletion of 49 nucleotides in the ORF3 gene from the 105th generation, and a stable deletion of 49 nucleotides in the 105th, 125th, and 145th generations (sequence See SEQ ID No. 6). The result is as Figure 5 . The primers for amplifying the ORF3 sequence are:

[0044] ORF3-F: TCCTAGACTTCAACCTTACG

[0045] ORF3-R: GGTGACAAGTGAAGCACAGA

[0046] The ...

Embodiment 3

[0072] Example 3 Safety Evaluation of Porcine Epidemic Diarrhea Virus ZJ08 Strain

[0073] Dilute passage 125 virus to 10 6.0 TCID 50 / ml (10 heads), 10 3-day-old piglets were selected, injected intramuscularly, 1ml each, and observed for 14 days, and 5 piglets in the control group were injected with the same dose of normal saline; in addition, pregnant sows one and a half months before delivery were selected. 10 pigs, each with 20 doses intramuscularly, and 5 sows in the control group were injected with the same dose of saline; the indicators of feed intake, body temperature, diarrhea and abortion of the sows were observed, and the litter size of the sows was counted.

[0074] The results proved that large-dose injection of the virus had no effect on piglets and sows, and no fever, diarrhea, and local inflammation occurred. The sows did not suffer from reproductive disorders such as abortion and stillbirth. There was no significant difference between the test group and the c...

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Abstract

The invention discloses a porcine epidemic diarrhea virus low-virulent stain ZJ08 and application thereof. The microbial collection number of the low-virulent stain is CGMCC No.7806. The PEDV low-virulent stain has high safety, and is safe for pregnant sows, baby pigs and pigs in all ages. The active protection ratio of the low-virulent stain for 3-day baby pigs reaches 100%, and the passive protective ratio reaches higher than 94.7%, which indicates that the low-virulent stain has favorable immunoprotection effect on porcine epidemic diarrhea.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an epidemic diarrhea virus and its application. Background technique [0002] Porcine epidemic diarrhea (porcine epidemic diarrhea, PED) is an important acute, contact viral intestinal infectious disease that causes diarrhea in pigs, characterized by watery diarrhea, vomiting, dehydration and loss of appetite. The disease first occurred in the United Kingdom and Belgium in the 1970s. In 1973, PEDV was isolated in Shanghai, Liaoning, Jilin and other places in my country. PEDV can persist in pig herds, and pigs of all ages are susceptible. The morbidity rate of suckling piglets, shelf pigs and fattening pigs can reach 100%, especially severe with suckling piglets, and the case fatality rate reaches 100%. At present, there is no specific drug against porcine epidemic diarrhea virus. Due to the acute onset and rapid prevalence of the disease, once the pigs are infected, it ...

Claims

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Application Information

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IPC IPC(8): C12N7/00A61K39/215A61P31/14C12R1/93
Inventor 侯艳红王贵华赵亚荣于萍萍刘明明卢会英满坤陈翠云
Owner 兆丰华生物科技(南京)有限公司
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