CN105296440B - One breeder c-type fowl metapneumovirus strain aMPV/C-JCZ and its application - Google Patents
One breeder c-type fowl metapneumovirus strain aMPV/C-JCZ and its application Download PDFInfo
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Abstract
The present invention provides a breeder c-type fowl metapneumovirus strain aMPV/C-JCZ and its application, and the deposit number of the virus stain is CGMCC No.10889.The virus is a breeder source fowl metapneumovirus C hypotype wild type strains.The virus of field separation carries out back chicken experiment on SPF chicken, causes severe respiratory infection clinical symptoms, and tracheae and lung pathologies is caused to damage, and being sequenced by M gene order confirms that the virus is fowl metapneumovirus C hypotype.Virus titer is measured after viral dilution in SPF chicken embryo, test result shows that EID50 is 10‑4.6/0.2ml.This is that the country is separated to chicken c-type fowl metapneumovirus for the first time, and there are biggish differences in genotype and antigen with fowl metapneumovirus A and subtype B.Direct support is provided for the pathogenic mechanism for further studying aMPV and the vaccine research for carrying out effective prevention and control disease.
Description
Technical field
The invention belongs to microorganism animal virus fields, and in particular to one plant of chicken c-type fowl metapneumovirus strain and its correlation
Using.
Background technique
Fowl metapneumovirus (Avian Metapneumovirus, aMPV) belongs to paramyxovirus section metapneumovirus category, is one
There are cyst membrane package, ameristic, the negative adopted RNA virus of sub-thread.The infection of fowl metapneumovirus is a kind of emerging infectious disease, can be in fire
The decline for causing acute, highly infective the infection of the upper respiratory tract in the birds such as chicken and chicken and laying eggs.The infection of fowl metapneumovirus
In worldwide distribution.The virus is divided into 4 hypotypes: A, B, C and D type, from world's most area such as Europe, Asia and South America point
From to be A or Type B, c-type infection has been reported that in the U.S., France and South Korea.The infection of D type is only found in France at present.Core
Nucleotide sequence analysis shows that, in the U.S., the c-type fowl metapneumovirus strain of discovery has 90% homology, but only has with A and Type B
The homology of 40-70%.Compared with other three kinds of hypotypes, c-type fowl metapneumovirus is developmentally inclined with the people that belongs in phyletic evolution
Pneumovirinae is more close.
We have collected difference successively from more than 70 a chicken houses in 8, the whole nation more than 20 areas of province (city) within 2009~2015 years
Age in days, the original seed chicken of different cultivars, breeder and commercial broiler blood serum sample, carry out the epidemiological survey of the disease, find institute
It is 60.45% that aMPV, which infects average total positives rate, in the chicken group of investigation, and individual chicken house positive rates are up to 100%, these survey datas
Show the white feather chicken of China's either local varieties or overseas introduction, aMPV infection is all generally existing.2012 in China east
South carries out discovery when epidemiological survey, and serious, acute respiratory tract clinical symptoms occurs in the diseased chicken for chicken house of falling ill, by returning
Chicken test and sequencing confirm that the disease is caused by the infection of chicken c-type fowl metapneumovirus.This is that we have found for the first time in the world
The infection of chicken c-type fowl metapneumovirus.Since the current country there is no the vaccine for the infection of fowl metapneumovirus, fowl metapneumovirus sense is caused
It contaminates and growth performance and the economic benefit generation of birds is seriously affected, this status has become generally existing in domestic breeding environment
Shape.
The infection of fowl metapneumovirus is a kind of emerging infectious disease, and it is tight that the infection of chicken c-type fowl metapneumovirus causes commercial generation chicken to occur
The respiratory symptom of weight, and the heredity of the virus and antigenicity with the A hypotype and subtype B that found originally in the presence of significantly poor
It is different, in order to preferably study pathogenic mechanism and effectively prevent and control the disease that virus must be separated.
Summary of the invention
The purpose of the present invention is to provide one plant of new fowl metapneumovirus, and the object of the invention is also to provide the fowl is inclined
The application of Pneumovirinae.
The present invention has obtained one plant of new chicken c-type fowl metapneumovirus poison by separating identification from China southeast morbidity chicken house
Strain, is named as aMPV/C-JCZ, the virus virus is on June 24th, 2015 in Chinese microorganism strain preservation conservator
It can common micro-organisms center (address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode
100101) preservation, classification naming are fowl metapneumovirus (Avian metapneumovirus), deposit number CGMCC
No.10889。
By returning chicken test confirm individually to be infected by aMPV/C-JCZ that the serious breathing of commercial generation yellow-feather broiler can be caused
Road symptom；The discovery of the pathologic finding of lung and tracheae, virus infection cause serious inflammatory and venereal injury.It is expanded by RT-PCR
The M gene discovery of virus in tissue, which is chicken c-type fowl metapneumovirus, homologous with A hypotype and subtype B on viral genetic
Property only has 70.6%-71.7%.It is investigated and is shown according to the popularity of the virus, aMPV infection is generally existing, and C hypotype is excellent
Gesture strain.
Specifically, which has the following characteristics that
(1) morbidity diseased chicken shows serious, acute respiratory tract clinical symptoms: eye has secretion, rhinorrhea, conjunctiva
Inflammation, facial swelling, rale, sinus swelling, nasosinusitis and nasal sinus and tracheae have the dry of yellow or white around eye socket and under socket of the eye
Junket sample object.
(2) virus measurement is carried out using 10 age in days SPF chicken embryos find that 24-96 hours chick embryo developments are obstructed, liver and body
Body has blutpunkte.
(3) SPF chicken Orthogonal Rotational Regressive Tests are found, serious respiratory tract clinical symptoms, pathologic finding occur within 3-5 days after virus infection
It was found that tracheae and lung pathologies damage are serious: epithelial structure is destroyed, and epithelial cell and cilium fall off；The infiltration of inflammatory cell.Gas
There is bleeding, lymphocytic infiltration and hyperplasia in pipe epithelium and lamina propria；There are bronchial lymph cellular infiltration and submucosa in lung
Oedema thickens and lymphoid cell hyperplasia.Since monocyte infiltration and oedema cause alveolar spaces diffusivity mildly to be expanded.
In the visible epithelial cell to fall off of bronchial lumen, different phagocyte, macrophage and irregular fragment etc..
(4) amplification of virus M gene and sequencing are found using RT-PCR, M full length gene 765bp.Carry out systematic growth
It being found with Molecular Evolutionary Analysis, aMPV-JCZ and c-type fowl metapneumovirus homology are 96.0%-96.7%, and only with A and Type B
There is the homology of 70.6%-71.7%.
The present invention also provides the chicken c-type fowl metapneumovirus strain aMPV/C-JCZ to prevent and treat the sense of fowl metapneumovirus in preparation
Application in the vaccine of disease caused by contaminating.Disease caused by the fowl metapneumovirus infects can be the infection of fowl metapneumovirus and draw
Play acute upper respiratory tract infection egg drop reduction etc..
The beneficial effects of the invention are as follows one plant of new fowl metapneumovirus strain aMPV/C-JCZ is provided, which is domestic
Chicken c-type fowl metapneumovirus isolated for the first time, feature clinical symptoms are caused in SPF chicken, can be used for studying the inclined lung of c-type fowl
The foundation etc. of strong virus attack model in the pathogenic mechanism and vaccine preparation of virus.Production of vaccine seed culture of viruses in can be used for preparing, system
Standby vaccine, for preventing and treating disease caused by fowl metapneumovirus infects.
Detailed description of the invention
There is facial swelling A in Fig. 1 virus infection SPF chicken, 3-5 days diseased chickens of virus inoculation, and nasal cavity is full of dense nose liquid B, nose
There is cheesy object C in sinus and intratracheally has mucus D.
The pathologic finding of Fig. 2 virus infection SPF chicken, the normal tracheal cilia of A；The leaching of the inflammatory cell of B tracheae and submucosa
Profit；C normal lung tissue；The inflammatory cell infiltration of D lung.
The M gene order of Fig. 3 virus is compared with each subtype gene of virus.
Fig. 4 virus inoculation SPF chicken embryo, the normal SPF chicken embryo of A；Dead chicken embryo (B) and the chicken embryo of impaired development after inoculation
(C)。
Specific embodiment
Following embodiment further illustrates the contents of the present invention, but should not be construed as limiting the invention.Without departing substantially from
In the case where spirit of that invention and essence, to modifications or substitutions made by the method for the present invention, step or condition, the present invention is belonged to
Range.
Unless otherwise specified, the conventional means that technological means used in embodiment is well known to those skilled in the art.
The separation and identification of embodiment 1 aMPV/C-JCZ virus
1, the acquisition of pathological material of disease and the separation of virus:
From the morbidity of China southeast one chicken house, morbidity chicken serious respiratory tract clinical symptoms occur for pathological material of disease, seriously
Influence the growth performance of chicken.
Concha, tracheae, larynx and the lung of aseptic collection morbidity chicken, are added according to 1:4 weight ratio containing 1% blueness after weighing
After grinding, with 3500rpm centrifugation 15 minutes, supernatant is stored in -80 DEG C of refrigerators by the PBS of the dual anti-sterilizing of streptomysin.
2, animal returns experiment
2 week old SPF chickens of random grouping, collunarium eye droppings be inoculated with 200 microlitres of tissue-derived virus, day by day observation experiment group
Clinical symptoms, it is wet to start to occur nose for second day individual chicken after inoculation, is collected nose liquid within the 3rd day in inoculation and pharynx is wiped
Son extracts RNA, RT-PCR reaction detection virus.It is depressed all to there is within the 5th day after inoculation obvious spirit, partially occurs getting rid of head and plays spray
It sneezes, audible apparent snore, chicken has translucent mucilage secretion to come out in nose, individual facial swellings (Figure 1A).It connects
It cuts open within 5 days and kills after kind, it is seen that intratracheal mucus has cheesy object (Figure 1B, C, D) in nasal sinus.In progress dissect on the 5th of inoculation,
The sensitive organization of morbidity chicken is taken to carry out check pathological section, it is seen that the inflammatory cell infiltration and lung of tracheae and submucosa
The inflammatory cell infiltration (Fig. 2) in portion.
3, the M gene of RT-PCR method amplification virus, is sequenced and is compared:
Diseased chicken tissue or viral cultures are collected, total serum IgE is extracted using Qiagen RNeasy Mini Kit, uses
III Kit and M gene-specific primer of SuperScriptTM, the fowl metapneumovirus cDNA of synthesis are that template carries out RT-PCR expansion
Increase, amplified production is detected with agarose gel electrophoresis, is connected in carrier T after recycling, by the raw work biology in Shanghai after cloning successfully
Engineering technology Co., Ltd is sequenced.The inclined tuberculosis of A, B, C hypotype fowl registered on M gene (SEQ IDNo.1) and GenBank
Poison, according to MEGA 5.10 and DNAstar software progress sequence homology compares and phylogenetic tree construction, finds aMPV-JCZ
It is 96.0%-96.7% with c-type fowl metapneumovirus homology, and there was only the homology (figure of 70.6%-71.7% with A and Type B
3)。
Virocyte adaptation strain is named as aMPV/C-JCZ, the virus is micro- in China on June 24th, 2015
Biological inoculum preservation administration committee common micro-organisms center (address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Chinese science
Institute of microbiology of institute, postcode 100101) preservation, classification naming is fowl metapneumovirus (Avian metapneumovirus), is protected
Hiding number is CGMCC No.10889.
2 SPF chicken embryo of embodiment measures virus titer
Saving virus directly can be inoculated with SPF chicken embryo detection virus titer by allantoic cavity.Firstly, connecting virus is saved
Continuous 10 times of dilutions, are inoculated with 5 pieces of SPF chicken embryos, 200 microlitres of every embryonic breeding kind respectively.It is incubated in 38 DEG C, the incubator that humidity is 40%
It educates, observation in 24 hours discards dead germ, then observes chicken embryo death situation for every eight hours.Chicken embryo is placed in 4 DEG C of refrigerators after a week by inoculation
Cooling, tweezers break up chorion after disinfection, tear chorioallantoic membrane after removing shell membrane, and allantoic fluid is sucked out, interior to contain a large amount of viruses,
Freeze after collection and is saved in -80 DEG C；Chicken embryo is taken out simultaneously, observes chicken embryo death or die down (chicken embryo stopping development, bleeding or hair
It is sparse).The EID50, lgEID50=10 of virus are calculated according to Reed-Muench method-4.6/0.2ml.A is control group in Fig. 4,
It is inoculated with PBS.B and C is that chick embryo development is caused after various concentration virus inoculation to be obstructed and dead.
3 aMPV/C of embodiment attacks the foundation and the minimum determination for attacking toxic dose of malicious model
2 week old SPF chickens of random grouping, every group 10, collunarium eye droppings be inoculated with 100EID50,200EID50,500EID50,
The aMPV/C-JCZ of 1000EID50,5000EID50,10000EID50, clinical symptoms of observation experiment group day by day, wherein
100EID50 connects poison group chicken and has no morbidity, and 200EID50 group only has 2 presentation mild respiratory symptoms.500EID50 inoculation group
There are within 3rd 4 chickens that apparent respiratory symptom occurs after inoculation.Whole chicken morbidities (being shown in Table 1) on the 5th.It is developing vaccine
During, it needs to establish experimental animal and attacks malicious model, chicken c-type fowl metapneumovirus belongs to emerging infectious disease, and the current country there is no
Specified attacks malicious model evaluation vaccine effect.SPF chicken clinical incidence is inoculated with using 500EID50aMPV/C-JCZ of the invention
Up to 90% or more, the validity and protecting effect of evaluation vaccine may be used as.
Table 1: collunarium eye droppings approach is inoculated with different EID50aMPV/C-JCZ SPF chicken morbidity statistics
The validity and protecting effect of 4 aMPV/C of the embodiment vaccine that judges
Vaccine is fowl metapneumovirus aMPV/C-JCX, which protects on June 23rd, 2015 in Chinese microorganism strain
Hiding administration committee's common micro-organisms center, (address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Chinese Academy of Sciences microorganism are ground
Study carefully institute, postcode 100101) preservation, classification naming is fowl metapneumovirus (Avian metapneumovirus), and deposit number is
CGMCC No.10900。
The attenuated live vaccines that aMPV/C-JCX is aMPV/C-JCZ are screened after passing on several times by aMPV/C-JCZ and are obtained
?.The TCID50, lgTCID50=10 of virus are calculated according to Reed-Muench method-4.2/0.1ml。
21 age in days SPF chickens are randomly divided into 5 groups, every group 10.It is set to: healthy control group, aMPV-
JCX2000TCID50 immune group, 5000TCID50 immune group, 10000TCID50 immune group, and non-Immunization group.Immune 3
Zhou Hou, in addition to healthy control group, remaining four groups pass through collunarium eye droppings approach inoculation 500EID50aMPV-JCZ and are attacked, by
Day observation clinical symptoms.As a result, it has been found that attacking all morbidities of poison group, there is apparent respiratory symptom.2000TCID50 and
5000TCID50 group can play part protection, and protective rate is 50% and 70% respectively.10000TCID50 immune group is protected completely
(protective rate 100%).Illustrate, respiratory tract disease caused by capable of being infected chicken c-type fowl metapneumovirus using aMPV-JCX cell toxicant
Shape plays good protecting effect, and aMPV/C-JCZ may be used as the validity and protecting effect of evaluation vaccine.
Table 2. evaluates aMPV/C-JCX protective rate with aMPV-JCZ
Claims (4)
1. chicken c-type fowl metapneumovirus strain aMPV/C-JCZ, deposit number is CGMCC No.10889.
2. chicken c-type fowl metapneumovirus strain aMPV/C-JCZ described in claim 1 draws in preparation prevention and treatment fowl metapneumovirus infection
Application in the vaccine of the disease risen.
3. application according to claim 2, which is characterized in that disease caused by the fowl metapneumovirus infects is the inclined lung of fowl
Virus infection causes upper respiratory disease, egg drop reduction.
4. application of the chicken c-type fowl metapneumovirus strain aMPV/C-JCZ described in claim 1 in preparation production of vaccine seed culture of viruses.
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| WO2012140032A1 (en) * | 2011-04-12 | 2012-10-18 | Intervet International B.V. | Avian metapneumovirus in oncolysis |
| CN103360472A (en) * | 2013-07-30 | 2013-10-23 | 北京市农林科学院 | Acian Metapneumovirus antibody ELISA detection kit |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012140032A1 (en) * | 2011-04-12 | 2012-10-18 | Intervet International B.V. | Avian metapneumovirus in oncolysis |
| CN103360472A (en) * | 2013-07-30 | 2013-10-23 | 北京市农林科学院 | Acian Metapneumovirus antibody ELISA detection kit |
Non-Patent Citations (2)
| Title |
|---|
| AVIAN METAPNEUMOVIRUS SUBGROUP C INFECTION IN CHICKEN,CHINA;WEI L，et al.;《EMERG INFECT DIS》;20130731;第19卷(第7期);第1092页左栏第2段-地1093页右栏第3段 * |
| 鸡C亚型禽偏肺病毒的分离和鉴定;韦莉 等;《中国畜牧兽医学会畜牧兽医生物技术学分会暨中国免疫学会兽医免疫学分会第十次学术研讨会论文集》;20141222;摘要、第85页左栏第2段-第86页右栏第1段 * |
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