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Attenuated strain YN150 of variant porcine epidemic diarrhea virus and applications thereof

A technology for porcine epidemic diarrhea and attenuated strains, applied in the direction of viruses, antiviral agents, and virus antigen components, can solve the problems of inability to provide protection and ineffective prevention of diarrhea, etc., achieve infection prevention, simple preparation process, and easy storage and transportation Effect

Inactive Publication Date: 2016-08-03
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the PED vaccine immunized pig farms reported in my country in 2006 could not provide complete protection (ChenJFetal., 2010), and recent studies have found that the mutant strains in my country are dominant (GaoYetal., 2013). In China, it is used to prevent pig epidemics Live vaccine strains for acute diarrhea are mainly CV777 vaccine strains
In 2011, diarrhea caused by a variant strain of porcine epidemic diarrhea broke out in pig farms immunized with CV777 attenuated live vaccine, which indicated that the traditional CV777 attenuated vaccine immunization could no longer effectively prevent diarrhea caused by the variant strain, and PED caused by the current variant strain It is ravaging around the world, so it is an urgent need for the pig industry in the world to develop a safe, efficient and cheap PEDV attenuated vaccine derived from the mutant strains isolated from PEDV.

Method used

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  • Attenuated strain YN150 of variant porcine epidemic diarrhea virus and applications thereof
  • Attenuated strain YN150 of variant porcine epidemic diarrhea virus and applications thereof
  • Attenuated strain YN150 of variant porcine epidemic diarrhea virus and applications thereof

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Effect test

Embodiment 1

[0050] The collection and processing of embodiment 1 disease material

[0051] The disease samples were collected from the small intestine of a 3-day-old piglet that was immunized with porcine transmissible gastroenteritis-porcine epidemic diarrhea inactivated vaccine in Yunnan Province but died of an outbreak of PED. The porcine epidemic diarrhea virus was detected by RT-PCR. Add physiological saline to the small intestine of piglets at a ratio of 1:9 (volume ratio), mash, freeze and thaw twice, centrifuge at 2000r / min, 4°C for 15min, collect the supernatant, and add 1500ml of penicillin and streptomycin per ml. Unit, placed in a 4°C refrigerator overnight, and then centrifuged at 10,000r / min, 4°C, 15min, the supernatant was taken, filtered with a 0.22μm filter, and stored in a -80°C refrigerator.

Embodiment 2

[0052] Isolation and identification of embodiment 2 virus

[0053] Virus isolation Take out the sample used as infected cells from the -80°C refrigerator, add trypsin to the solution at 50 μg / mL, put it in a 37°C incubator for 30 minutes, and finally distribute 1mL per tube into different 1.5mL Store in sterilized EP tubes for later use. Take the Vero monolayer cells in good growth condition, discard the culture medium, and wash the monolayer cells twice with PBS solution. According to 25cm 2 Add 1 mL of the treated disease material sample for infection into the culture bottle, and add trypsin at a final concentration of 10 μg / mL, and incubate at 37°C CO 2 Adsorb in the incubator for 1 hour, during which it was shaken once, and normal cells were set up as a control. Discard the adsorption solution (adsorption solution after the second generation can not be discarded), and add 5 mL of DMEM maintenance solution (serum-free) containing 10 μg / mL final concentration of trypsin, ...

Embodiment 3

[0058] Passage weakening of embodiment 3 virus

[0059]The 80% confluent Vero cells grown for 24 hours were washed twice with serum-free DMEM, and inoculated with 0.1 MOI of YN1 according to the method of detoxification above, and the maintenance solution was DMEM containing trypsin at a concentration of 8 μg / mL. When about 36 hours, when the cytopathic effect reached 85%, the virus was harvested, and the virus was stored in a refrigerator at -80°C. The collected viruses were repeatedly frozen and thawed three times and then serially passaged.

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Abstract

The invention discloses an attenuated strain YN150 of variant porcine epidemic diarrhea virus and applications thereof. The attenuated strain is prepared by consecutively passing strain YN144 (microbial preservation number: CCTCC V201547) for six generations in Vero cells in the presence of pancreatin (10 [mu]g / mL). The PEDV attenuated strain is originated from variant porcine epidemic diarrhea virus, has good safety, and is safe to various pigs. The provided vaccine can stimulate the pigs to generate protective immune response so as to resist variant porcine epidemic diarrhea virus and effectively prevent the infection caused by variant porcine epidemic diarrhea virus.

Description

technical field [0001] The present invention relates to a mutant porcine epidemic diarrhea virus attenuated strain, in particular to a mutant porcine epidemic diarrhea virus attenuated strain YN150 and application thereof. Background technique [0002] Porcine epidemic diarrhea (PED) is an acute, highly contagious disease caused by porcine epidemic diarrhea virus (PEDV). After pigs are infected with PEDV, they often cause acute enteritis and fatal watery diarrhea in piglets until they die of dehydration. The clinical features are mainly vomiting, severe diarrhea and dehydration in piglets; in piglets infected with PEDV, the pathological changes of autopsy mainly manifest in the jejunum and Atrophy and shedding of intestinal villi in the ileal portion (Pensaert MB et al., 2006). Pigs of all ages are susceptible, but mainly affect suckling piglets, with a morbidity rate of 100% and a mortality rate of 80% to 100% (TimoneyJFetal., 1988), which brings significant economic losse...

Claims

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Application Information

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IPC IPC(8): C12N7/00A61K39/215A61P31/14A61P1/12C12R1/93
CPCC12N7/00A61K39/12A61K2039/542C12N2770/20021C12N2770/20034
Inventor 何启盖吴美洲陈芳洲李中华朱银杏刘洋徐凤琴库旭钢叶十一郭效珍
Owner HUAZHONG AGRI UNIV
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