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Attenuated strain for Muscovy duck aMPV (avian metapneumovirus)-C as well as preparation and application of attenuated strain

A technology of avian metapneumovirus and attenuated strains, applied in the direction of viruses, antiviral agents, virus antigen components, etc.

Active Publication Date: 2019-03-01
WENS FOOD GRP CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Duck-derived subtype C avian metapneumovirus has only been reported in France and Canada, and attenuated vaccines for the aMPV-C subtype derived from muscovy ducks have not yet been reported.

Method used

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  • Attenuated strain for Muscovy duck aMPV (avian metapneumovirus)-C as well as preparation and application of attenuated strain
  • Attenuated strain for Muscovy duck aMPV (avian metapneumovirus)-C as well as preparation and application of attenuated strain
  • Attenuated strain for Muscovy duck aMPV (avian metapneumovirus)-C as well as preparation and application of attenuated strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] The subculture of embodiment 1 duck C subtype metapneumovirus

[0024] Use the duck subtype C avian metapneumovirus S-01 strain isolated in the laboratory to cause Muscovy ducks to be morbid in nature, and inoculate the African green monkey kidney cell (Vero) T25 cell bottle covered with a single layer, and inoculate each bottle 100 times Dilute 500ul of the virus solution, after 2 hours of adsorption, discard the virus solution, add 5ml of DMEM culture solution containing 2% fetal bovine serum, and cultivate for 24 to 96 hours until cytopathic changes appear, freeze and thaw 3 times repeatedly, and absorb the virus solution Inoculate the next generation. Continuously subcultured to 40th generation, and then continuously purified 5 times by limiting dilution method, and passed to 62nd generation.

[0025] Subculture Results

[0026] F3 generation duck embryo yolk fluid was inoculated with Vero cells, F1-F3 generation had no obvious lesions, and F4 generation showed cy...

Embodiment 2

[0029] Example 2 Passage Pathogenicity Test of Subtype C Avian Metapneumovirus S-01 Strain

[0030] Select the above-mentioned S-01 strains for passage to generations S-01-5P, S-01-17P, S-01-28P, S-01-32P, S-01-40P, S-01-50P, S-01- 60P, Vero cell solution control group, a total of 9 groups, infected two-week-old ducklings with eye drops and nasal drops, and the infection dose was 10 4 TCID 50 / ml, observe the clinical symptoms every day after immunization and record the number and severity of diseased ducks in each group, collect the cloacal swabs and throat swabs of each group on the 5th day after immunization, and put the swabs of every 5 ducks in In a 15ml EP tube, carry out RT-PCR detection, a total of 6 samples to be tested in each group. By observing the clinical symptoms and lesions after necropsy, the pathogenicity of different generations to ducks was analyzed. (Refer to Ali A for the RT-PCR detection method, Reynolds DL: A reverse transcription-polymerase chain re...

Embodiment 3

[0034] Example 3 Efficacy experiment of subtype C avian metapneumovirus S-01 strain attenuated generation vaccine

[0035] The weakened passages in the above results were subjected to immunogenicity and different titer immune challenge tests. F40, F50, and F60 were selected for the test, and the specific implementation was 200 1-day-old young muscovy ducks (maybe aMPV and The detection of aMPV was negative) were randomly divided into 10 groups, 20 in each group. The 40th generation, 50th generation, and 60th generation were divided into three gradients (10 2.5 、10 3.5 , 10 4.5 TCID 50 / 0.1ml). Vero cell solution 0 is the blank group of the tenth group. The ducklings were immunized with eyedrops and nasal drops at 2w. Observe clinical symptoms every day after immunization, carry out the collection of cloaca swab and throat swab on the 5th day, every group collects 10 ducks at random, puts in the EP tube of 2ml respectively. Add 300ul of normal saline containing 2000U dou...

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Abstract

The invention belongs to the field of microbial animal viruses, and particularly relates to an attenuated strain for Muscovy duck aMPV (avian metapneumovirus)-C as well as preparation and applicationof the attenuated strain. The preservation number of the attenuated strain for Muscovy duck aMPV-C is CCTCC NO:V201823, the nucleotide sequence is shown as SEQ IDNO:1, and the amino acid sequences areshown as SEQ ID NO:2-SEQ ID NO:9. The attenuated strain for Muscovy duck aMPV-C is prepared from an aMPV-C S-01 strain by subculturing to the 50th generation. The attenuated strain for the Muscovy duck aMPV-C S-01 strain is obtained for the first time, and the foundation is laid for development of attenuated vaccines.

Description

technical field [0001] The invention belongs to the field of microbial animal viruses, and in particular relates to an attenuated strain of muscovy duck subtype C avian metapneumovirus and its preparation and application. Background technique [0002] Avian metapneumovirus is one of the acute and highly contagious diseases of upper respiratory tract infection caused by avian metapneumovirus (aMPV). Avian lung disease was first isolated in young turkeys with clinical signs of sneezing, tracheal rales, sticky discharge from the nose and eyes, and conjunctivitis. Therefore it is called turkey rhinotracheitis. In South Africa in 1978, aMPV was first isolated and subsequently reported in Europe, Asia and South America. In 1998, my country reported the isolation of subtype A of avian pneumovirus in chickens with swollen head syndrome for the first time. In 2012, subtypes B and C were successively isolated in chickens. In July 2010, in a duck farm in Guangdong Province, a strain...

Claims

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Application Information

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IPC IPC(8): C12N7/00C12N7/08A61K39/155A61P31/14C12R1/93
CPCA61K39/12A61K2039/5254A61K2039/552A61P31/14C12N7/00C12N2760/18321C12N2760/18334C12N2760/18364
Inventor 刘佳佳陈峰王占新鲁俊鹏覃健萍操胜
Owner WENS FOOD GRP CO LTD
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