Avian infectious bronchitis virus (IBV) as well as culture method and application thereof

A coronavirus and duck-derived technology, applied in the field of bioengineering, can solve the problems of chicken immune failure and lack of cross-protection

Inactive Publication Date: 2012-09-05
HENAN INST OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are more than 26 serotypes of IBV that have been reported at home and abroad. Due to the large number of serotypes and new variants, there is a lac

Method used

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  • Avian infectious bronchitis virus (IBV) as well as culture method and application thereof
  • Avian infectious bronchitis virus (IBV) as well as culture method and application thereof
  • Avian infectious bronchitis virus (IBV) as well as culture method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1 Virus Isolation and Culture

[0024] In May 2004, a suspected IB disease with diarrhea and growth retardation as the main symptoms occurred in a farm in Zhengzhou, Henan Province. The infected chickens mainly showed depression, diarrhea, slow growth and decreased resistance. The morbidity rate was 100%, and the mortality rate was 100%. Reaching more than 10%, the production performance of infected flocks is significantly reduced. According to the investigation, there was a group of 1700 meat ducks only about 150m away from the chicken coop. About 10 days before the first batch of chickens became ill, the duck group had a disease with similar symptoms, but did not cause a large number of deaths. The lesions of dead chickens are mainly small intestinal bleeding, intestinal wall thinning, some have ulceration, small intestinal villi shedding, glandular stomach papilla swelling, mucosal shedding, some have bleeding at the junction of gizzard and glandular stomach,...

Embodiment 2

[0025] Embodiment 2 virus identification

[0026] (1) Chicken embryo median lethal dose (ELD) 50 ) determination

[0027] The above-mentioned gained seed poison (duck-derived coronavirus ZZ2004) is diluted by 10 times, and 10 -3 ~10 -7 Chicken embryos were inoculated with each dilution, and five 9-11-day-old SPF chicken embryos were inoculated for each dilution, cultured at 37°C, and dead chicken embryos within 24 hours were discarded, and the number of dead embryos was observed and recorded every day until the age of 18 embryos. Calculation of ELD by Reed-Muench method 50 (Reed and Muench, 1938).

[0028] (2) RT-PCR identification of virus ZZ2004 strain

[0029] The primer sequences were designed as follows: upstream primer 5'-GACCGCTTGTCAAAGCAAATT-3', downstream primer 5'-TGAGTACTAAGAGTGCAATT-3'.

[0030] Concentration method of ZZ2004 strain

[0031] Dilute the allantoic fluid infected by the ZZ2004 strain 1:10 with sterilized PBS, inoculate 0.2ml into the chorioalla...

Embodiment 3

[0053] The physicochemical test of embodiment 3 virus

[0054] (1) Sensitivity test of virus to ether and chloroform

[0055] ① Sensitivity test of the virus to 20% ether: Take 800ul of ZZ2004 virus in a sterile ep tube, add 200ul of ether, put it in a refrigerator at 4°C for 18-24h, shake it well from time to time, centrifuge at 2500rpm at 4°C for 20min, and take The liquid in the lower layer was repeatedly blown to evaporate the ether. Determination of its EID by diluting with sterile saline 50 , and a virus control group was set up at the same time. The test results are shown in Table 4.

[0056] ② Sensitivity test of virus to 0.5% chloroform: Take 995ul of CSISV virus in a sterile EP tube, add 5ul of chloroform, put it in a refrigerator at 4°C for 20min, shake it well from time to time, centrifuge at 2500rpm at 4°C for 20min, and take the upper liquid Determination of its EID by diluting with sterile saline 50 , and a virus control group was set up at the same time. ...

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Abstract

The invention relates to an avian infectious bronchitis virus (IBV) ZZ2004 as well as a separating culture method and application thereof. The avian IBV ZZ2004 has the microorganism preservation number of CGMCC No.3842. The separating culture method comprises the following steps of: collecting infectious attacked poultry kidney under the aseptic condition; after adding sterilizing PBS (Phosphate Buffer Saline) for repeatedly washing, grinding to emulsion; after adding sterilizing PBS diluent, separately charging into a freezing storage tube; repeatedly freezing and melting for many times, filtering and sterilizing; vaccinating filter liquid to an SPF (Specific Pathogen Free) chick embryo at ages of 10 days by an allantoic cavity; culturing at a temperature of 37 DEG C for 72 hours, collecting allantoic fluid and carrying out continuous blind pass for more than five generations; and taking chick embryo allantoic fluid which is negative by a hemagglutination test. Compared with coronaviruses of other poultries, the virus separating strain derived from a culturing farm with chick and duck mixed culture has high variation degree and wider infection spectrum on animals; and the discovery and research of the virus have significance for disclosing an IBV variation rule, distinguishing different serotypes and preparing a novel IBV vaccine and a medicament for preventing and treating avian infectious bronchitis.

Description

technical field [0001] The invention relates to the field of bioengineering, in particular to a duck-derived coronavirus (Avian infectious bronchitis virus, IBV) ZZ2004 and its isolation and cultivation method and application. Background technique [0002] In 1937, coronaviruses (Coronaviruses) were first isolated from chickens. In 1965, the first human coronavirus was isolated. It is named "coronavirus" because it can be observed under an electron microscope that there are obvious rod-shaped particle protrusions on its outer membrane, making it look like the crown of a medieval European emperor. According to the serological characteristics of the virus and the differences in nucleotide sequences, the Coronaviridae family is currently divided into two genera, coronavirus and torovirus. The representative strain of Coronaviridae is Avian infectious bronchitis virus (IBV). The virus is an enveloped, moderately pleomorphic spherical or oval virion with rod-shaped protrusions...

Claims

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Application Information

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IPC IPC(8): C12N7/00A61K39/215A61K48/00A61P31/14A61P11/00C12R1/93
Inventor 刘兴友姚四新赵淑秋刘金华王宪文王丽荣王自良赵坤陈仕钧赵朴
Owner HENAN INST OF SCI & TECH
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