Duck-derived coronavirus attenuated strain IBV DCV35 and application thereof

A technology of coronavirus and attenuated strains, applied in the field of microorganisms, can solve the problems of low affinity and the inability of IBVZZ2004 to produce effective protection

Active Publication Date: 2018-02-02
HENAN INST OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The IBVZZ2004 strain has very low affinity with domestic popular IBV, and also has low affinity with domestic main vaccine strains, such as M41 strain, H120, Beaudette, and IBV4 / 91 have a homology of 85.2% to 87.9%.
The isolation and identification methods, pathogenicity, and gene variation of the virus isolate ZZ2004 have been demonstrated in the invention patent ZL201010225577.4. In production practice, the existing vaccines cannot produce effective vaccines against duck-derived IBVZZ2004. protection, making the disease more prevalent

Method used

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  • Duck-derived coronavirus attenuated strain IBV DCV35 and application thereof
  • Duck-derived coronavirus attenuated strain IBV DCV35 and application thereof
  • Duck-derived coronavirus attenuated strain IBV DCV35 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Embodiment 1 low temperature culture induces attenuated strain

[0018] ZZV6 seed poisoning (EID 50 10-4.88 / 0.2mL) first passed through a 56°C water bath for 20 minutes, filtered with a 0.22um bacterial filter, and inoculated with 9-day-old SPF chicken embryos, 5 eggs per generation, 0.2mL per embryo, and incubated at 31-32°C. Daily observation, chicken embryos that died within 24 hours were discarded, and the dead chicken embryos were temporarily placed in a refrigerator at 4°C, and the virus was aseptically collected for 72 hours. At the same time, the pathological changes of the chicken embryos were observed, and the allantoic fluid of typical diseased chicken embryos was selected for passage , each generation was treated and cultured in the same way, continuously passed to the 25th generation, cultured at 37°C from the 26th generation, and then continuously passed to the 35th generation to obtain DCV35.

Embodiment 2

[0019] Example 2 Passage Virus S1 Gene Sequence Determination

[0020] The 7th, 15th, and 20th to 35th generations of viruses were selected during the continuous passage, the S1 gene was cloned by RT-PCR, and its nucleic acid sequence was determined. S1 gene primers: upstream (P1): 5'-AGTTATTGGTTAGAGATGTTGGGGA-3', downstream (P2): 5'-CGTA TGG ACA GCT TGT GAC ATT TTC-3'.

[0021] Using the DNASTAR analysis software, the base sequence and deduced amino acid sequence of the sequenced 7th, 15th, 20th, 22nd to 35th generation poisons and the maternal virus's S1 gene are shown in Table 1, and the comparison and analysis results of the bases and amino acids of each generation See Appendix A and Appendix B. The results showed that 12 bases of the S1 gene mutated in the 22nd generation, and this variation remained stable in the 35th generation. The 12 mutation sites of the S1 gene are: 188ntT→C(Val→Ala), 191ntA→G(Asn→Ser), 228ntT→G(Asn→Lys), 232ntA→C(Ser→Arg), 347ntT→A (Phe→Try), 35...

Embodiment 3

[0024] The pathogenicity test of embodiment 3DCV35 to SPF chicks

[0025] (1) DCV35EID 50 Determination Take 1mL of DCV35 virus liquid and filter it with a 0.22um sterile filter, then make a 10-fold incremental dilution with normal saline, and inoculate five 9-day-old SPF chicken embryos at each dilution, 0.2mL / each embryo, and culture at 37°C. The number of dead embryos was observed and recorded every day, and the dead embryos within 24 hours were discarded. The remaining live embryos were dissected after incubation until 19 days old, and the dead and dwarf embryos were used as the criteria for judging infection. Nine-day-old SPF chicken embryos were inoculated with the same dose of normal saline as a control. Calculate EID by Reed-Muench method 50 .

[0026] The results showed that: with the increase of the number of passages of the virus, the adaptability of the virus to SPF chicken embryos became stronger and stronger, and EID 50 It also increased accordingly, and the ...

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Abstract

The invention relates to the field of microorganisms, and concretely provides a duck-derived coronavirus attenuated strain IBV DCV35 (with the preservation number being CGMCC NO.13852) and an application thereof. The duck-derived coronavirus attenuated strain IBV DCV35 is obtained through passage attenuation screening of a duck-derived coronavirus virulent strain IBV ZV2004 strain (with the preservation number being CGMCC NO.3842), the S1 gene sequence of the attenuated strain IBV DCV35 is significantly mutated, and the mutated sequence can be stably inherited in the 22-35th generations. SPF chickens are infected with the screened attenuated strain IBV DCV35 by artificial infection test and horizontal transmission test, and no virulence return phenomenon is observed. Experiments show thatthe attenuated strain can effectively activate the immune system in chickens and well prevent avian infectious bronchitis after being inoculated into the chickens, and can be used as a vaccine for preventing the avian infectious bronchitis.

Description

technical field [0001] The invention relates to the field of microorganisms, in particular to a duck-derived attenuated strain of coronavirus. Background technique [0002] Chicken infectious bronchitis virus (Infectious bronchitis virus, IBV) seriously endangers the breeding industry of our country, and the economic loss caused by chicken infectious bronchitis is very serious every year, and the prevention of this disease is mainly attenuated vaccine, which is the most widely used The most attenuated vaccines are H120 and H52. However, due to the large number of IBV serotypes and the easy mutation of the IBV genome, new IB mutant strains continue to emerge, making the existing vaccines unable to produce effective protection against the newly emerging mutant epidemic strains, making the prevention and treatment of IBV difficult. [0003] IBV ZZ2004 is a virus isolated from muscovy ducks that cause immunosuppression and growth suppression as the main symptoms. This virus can...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/00A61K39/215A61P31/14A61P11/00
CPCA61K39/12A61K2039/5252A61K2039/5254A61K2039/552C12N7/00C12N2770/20021C12N2770/20034
Inventor 刘兴友陈明艳姚四新欧长波刘明成
Owner HENAN INST OF SCI & TECH
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