Super-high virulent chicken infectious bursal disease virus cell adapted strain and application thereof

A technology for infectious and bursal disease of chickens, which can be applied in the directions of viruses, antiviral agents, and viral antigen components, and can solve problems such as hidden safety hazards of chicken flocks, reduced protection effects of live medium virulence vaccines or virulent inactivated vaccines, etc. , to achieve the effect of stable immune efficacy, good immunogenicity and long duration

Active Publication Date: 2017-05-31
HENAN AGRICULTURAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Studies have shown that the highly virulent strain of chicken infectious bursal disease (vvIBDV) can break through the protection of the high titer antibody level of the original attenuated vaccine, causing damage to the bursa of chicks and becoming a safety hazard in chicken flocks
Moderately virulent live vaccines or highly virulent inactivated vaccines have a better protec...

Method used

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  • Super-high virulent chicken infectious bursal disease virus cell adapted strain and application thereof
  • Super-high virulent chicken infectious bursal disease virus cell adapted strain and application thereof
  • Super-high virulent chicken infectious bursal disease virus cell adapted strain and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Embodiment 1: Isolation and identification of chicken infectious bursal disease virus IBDV-CL strain

[0031] In March 2011, a suspected infectious bursal disease outbreak occurred in a large chicken farm in Henan. Chickens with typical clinical symptoms were selected to collect bursa, liver, spleen, lung, kidney and other tissues aseptically. Homogenize and grind, add penicillin 1,000U / mL, streptomycin 1,000U / mL sterilized saline at a volume ratio of 1:5 to make a tissue suspension, put in a refrigerator at 2-8°C, inhibit bacteria for 2 hours, and freeze repeatedly After thawing for 3 times, centrifuge at 4000rpm for 10min, take the supernatant, filter it with a sterile filter, and store it at -20°C for later use.

[0032] 1. Inoculation of virus in chicken embryos

[0033] The diseased material obtained from the above treatment was inoculated with 9-11 day-old SPF chicken embryos through the chorioallantoic membrane, 0.2 mL per embryo, hatched at 37°C, and the eggs w...

Embodiment 2

[0045] Example 2: Screening and identification of cell-adapted strains

[0046] 1. Cell-adapted virus seed preparation

[0047] Prepare chicken embryo fibroblasts (CEF) according to the method of "Chinese Veterinary Pharmacopoeia" 2010 edition. After the CEF cells grow into a single layer, discard the cell culture medium and wash with PBS solution for 3 times to remove non-adherent, aging and dead cells. and cell surface serum, the virus of the E5 generation was diluted with a sterile medium (DMEM culture fluid), inoculated on a single layer of cells, adsorbed in a 37°C incubator for 1h, and added to a virus maintenance solution (2% serum DMEM culture fluid) ), 37°C CO 2 Harvest in an incubator for 96 hours, or when the lesion reaches about 80%, harvest in a sterilized container after freezing and thawing three times, and store at -70°C for later use. According to this method, the cells were continuously blindly passed for 20 generations until the cells produced stable typic...

Embodiment 3

[0057] Embodiment 3: the biological characteristic determination of chicken infectious bursal disease virus strain

[0058] 1. Chicken embryo median lethal dose (ELD) of IBDV-CL strain 50 ) determination

[0059] The IBDV-CL strain was diluted 10 times, and 10-day-old SPF chicken embryos were inoculated through the CAM route. Five chicken embryos were inoculated for each dilution, and the inoculation volume was 0.1 mL. The dead embryos within 24 hours were observed until 144 hours, and the death of chicken embryos in each group was recorded. According to the Reed-Mueneh method, the chick embryo median lethal dose (ELD) of the virus in every 0.1 mL of cell culture fluid was calculated. 50 ), the results are shown in Table 2.

[0060] Table 2 Mortality of inoculated SPF chicken embryos and ELD of IBDV isolates 50

[0061]

[0062] 2. The cell half infection dose (TCID) of CL40 strain 50 ) determination

[0063] The virus that has been stably transmitted to the 8th pass...

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Abstract

The invention discloses a super-high virulent chicken infectious bursal disease virus cell adapted strain, the preservation number thereof is CCTCC NO: V201316, and application of the super-high virulent chicken infectious bursal disease virus cell adapted strain in preparation of a chicken infectious bursal disease virus vaccine, an antigen reagent for diagnosis, a positive serum reagent for diagnosis, an egg yolk antibody for therapy, and antiserum. The chicken infectious bursal disease virus CL40 strain of the invention has super-high epidemic strain character, has good immunogenicity, and is suitable for cell scale cultivation. The TCID50 of the chicken infectious bursal disease virus CL40 strain is 10<-8.0>/0.1ml, and has a 100% protection rate to the fetal attack of the super-high strain IBDV-CL, and is stable in immunity effect, long in duration time, and has important value for preventing and controlling chicken infectious bursal disease super-high virus prevalence.

Description

technical field [0001] The invention belongs to the technical field of biological vaccines, in particular to a cell-adapted strain of super-virulent chicken infectious bursal disease virus and its application. Background technique [0002] Infectious bursal disease (IBD) is an acute, highly contagious, viral infectious disease of chickens caused by infectious bursal disease virus (IBDV). . IBDV belongs to the family BiRNAvirus, and the genus Avian BiRNAvirus. There are two serotypes of bursal disease virus, type Ⅰ and type Ⅱ. Type Ⅰ chicken infectious bursal disease virus is pathogenic to chickens, type Ⅱ virus is not pathogenic to chickens, and type Ⅰ virus is divided into different types. subtypes, such as classic strains, mutant strains, super-virulent strains and attenuated strains. IBDV mainly infects the central immune organ, the bursa of Fabricius, in chickens, which not only causes disease and death in chickens, but also leads to immunosuppression, which makes chi...

Claims

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Application Information

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IPC IPC(8): C12N7/00A61K39/12A61P31/14C12R1/93
CPCA61K39/12A61K2039/5252A61K2039/552A61K2039/55566C12N7/00C12N2720/10021C12N2720/10034
Inventor 李新生崔保安黄宗梅周云飞常婧竹王洁琼王莉
Owner HENAN AGRICULTURAL UNIVERSITY
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