CN101100656A - Nephrotropic avian infectious bronchitis HN99 strain virus - Google Patents
Nephrotropic avian infectious bronchitis HN99 strain virus Download PDFInfo
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Abstract
A fowl-kidney-form infectious bronchitis HN99 virus has excellent immunogenicity. The diameter of coronal virus particle is 90-105 nm, it has membrane and is radiant with 18 nm vomer protrusion, it has no interference for NDV La Sota virus multiplying and no agglutination for 1% fowl red blood cell; regression flow experiment leads to respiratory tract symptom and renal boss. Ill fowl virus isolate has positive reaction with HN99 positive blood serum, has negative reaction with NDV La Sota strain, IBDV B87 strain and AIV H9N2 strains; HN99 positive blood serum has positive reaction with T strains, Hubei strain, Tianjin strain and Shandong strain and has negative reaction with other viruses. It has exogenous pathogenic pollution.
Description
Technical field
The present invention relates to a kind of poultry infective virus, relate in particular to a kind of avian nephropathogenic infectious bronchitis virus.
Background technology
1999, poulty house, Yingyang, Henan Province outburst was Clinical symptoms with the respiratory symptom, be the transmissible disease of anatomical features with kidney swelling, urate deposition, was separated to a strain virus from the kidney of chicken group model case and ureter, and code name is HN
99Strain.Go down to posterity by SPF chicken embryo, the SPF chick is measured its EID after returning rejuvenation
50Be 10
-6.40/ 0.1ml; We have carried out histological observation to the kidney of morbidity chicken, with electron microscopic observation the form and the size of coronavirus particle that should virus, carried out the biological property test as blood clotting, to the interference test of NDV, measured sensitivity and the test of other physics and chemistry heat, acid, chemical reagent (chloroform, ether etc.).Specificity test and serum neutralization test have been carried out with local strains such as international classic kidney C-type virus C strain such as Holte, Gray and Ausc-T and Henan, Hubei, Beijing, Tianjin, Harbin etc.Test-results confirms that this virus strain is a kidney type infective bronchitis virus, HN
99Virus strain and traditional respiratory infectious bronchitis virus M
41The serotype difference of strain, H120 strain, H52 strain is the new virus strain of a strain.With traditional M
41The vaccine of strain or H120 strain, H52 strain preparation can not prevent HN
99Kidney type infective bronchitis due to the strain virus has only the HN of using
99The vaccine of strain virus preparation could prevent kidney type infective bronchitis.
Summary of the invention
The object of the invention is to provide a kind of novel avian nephropathogenic infectious bronchitis HN
99Strain virus.
The invention discloses a kind of novel avian nephropathogenic infectious bronchitis HN
99Strain virus, this virus strain is deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center on May 26th, 2006, and deposit number is CGMCC No.1729.
This virus has following feature:
(1), see that under Electronic Speculum the coronavirus particle is arranged, diameter 90～105nm has cyst membrane, has the pears shape that is about 18nm outstanding on every side, is radial arrangement;
(2), morbidity chicken symptom is except that respiratory symptom, most dead chicken all has pathologies such as kidney enlargement, urate deposition, piebald kidney, with 10 age in days egg inoculation viruses, and through blind passage after 5 generations, between visible 24～72 hours dead chicken embryo all have congested or hemorrhage, hatch to the 17th day chicken embryo have 3/5 present growth little, curl, ossify, urate deposition;
(3), viral isolates produces interference to the breeding of NDV La Sota strain virus;
(4), viral isolates does not produce aggegation to 1% chicken red blood cell;
(5), return pathologies such as respiratory symptom can appear in the test of SPF chicken and kidney is swollen, piebald kidney;
(6), the specific serum neutralization results proves viral isolates of sick chicken and HN
99Positive serum is positive, and with NDV La Sota strain, IBDV B
87Strain, AIV H
9N
2The strain reaction that all is negative;
(7), with HN
99Strain positive serum and 100 EID
50M
41Viruses such as strain, Holte strain, Gray strain, T strain and Local Isolates Hubei strain, Harbin strain, Shandong strain, Beijing strain, Shanghai strain, Tianjin strain are done serum neutralization test, and the result shows, HN
99Strain positive serum and T strain, Hubei strain, Tianjin strain and Shandong strain are positive, with the reaction that all is negative of other strain virus.
Avian infectious bronchitis virus strain of the present invention is deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center on May 26th, 2006, depositary institution is called for short: CGMCC, deposit number: CGMCC No.1729, classification name: avian nephropathogenic infectious bronchitis virus, English name is Avain nephropathogenic Infectious bronchitis virus HN99 Strain, the Latin name is called Tarpeianephrite pulli, depositary institution address: No. 13, North No.1 Row, Zhongguancun, Haidian District, Beijing City, postcode: 100080.
Embodiment
The present invention adopts following method to carry out separation, cultivation, the evaluation of virus:
(1), viral separation and Culture
The sick chicken lung of aseptic collection, kidney, the back of weighing add sterile saline by 1: 5 weight ratio and grind, and with 3000rpm centrifugal 15 minutes, get supernatant liquor, add penicillin 2000IU, Streptomycin sulphate 2000 μ g by every milliliter, 4～8 ℃ of effects 12 hours are standby.Get the supernatant liquor allantoic cavity and inoculate 10 of 10 age in days SPF embryos, every embryo 0.2ml rejects chicken embryo dead in 30 hours.From inoculating back 30 hours, observed 1 time every 4～6 hours, choose dead embryo, put 2～8 ℃ of cold depositing, took out all embryos to 72 hours, the aseptic allantoic fluid of collecting adds each 1000IU/ml of mycillin.All the other 5 pieces are continued hatching 5 days, observe and the record embryonic lesions.So continuously 3 generations of blind passage, preserve below-70 ℃, with this viral liquid in 1: the ratio of 2-3 (V/V) adds sucrose milk protective material, carry out freeze-drying with vacuum freeze drier after, be stored in preservation in-80 ℃ of cryogenic refrigerators.
(2), virus is identified
1. electron microscopic examination result
HN
99Strain is seen under Electronic Speculum the coronavirus particle, and diameter 90～105nm has cyst membrane, has the pears shape that is about 18nm outstanding on every side, is radial arrangement.The contrast allantoic fluid is not seen has above-mentioned virus particle to exist.
2.HN
99, HX viral hemoagglutination test EID
50Measure and to the interference test of NDV
HN
99Aggegation (HA test negative) does not take place to 1% chicken red blood cell in the chicken embryo isolate of handling without pancreatin, and the HX viral isolates is to 1% chicken red blood cell generation aggegation, and HA is 1: 4, proves the virus pollution of blood clotting, so gives up and no longer do other test.
HN through the pancreatin processing
99Chicken embryo viral isolates is to 1% chicken red blood cell generation aggegation, and average agglutination titer HA is 1: 2.
HN after phospho-esterase c is handled
99Chicken embryo viral isolates can make 1% chicken red blood cell generation aggegation, and average agglutination titer HA is 1: 4.
The EID of viral isolates
50Mensuration is with HN
99Chicken embryo virus allantoic fluid is measured its EID
50, the result is 10
-6.0～10
-7.0/ 0.1ml.
3. to the interference test of NDV
Interference test to NDV proves, adds and separates malicious HN
99HA be 1: 128, not adding the HA that separates poison is 1: 1024, and the malicious HN of separation be described
99Can suppress NDV La Sota in the internal breeding of chicken embryo.This test-results shows, separates malicious HN
99The biological characteristics that meets IBV.
4. physics and chemistry test
Viral isolates adds 20% ether, 2～8 ℃ handle 24 hours after inoculated into chick embryo, the chicken embryo has not been had infectivity.Prove that viral thing is to the ether sensitivity.
Add chloroform in viral isolates, making its ultimate density is 4.8%, and thorough mixing 30min under 4 ℃ of temperature inoculates responsive chicken embryo, to the EID of chicken embryo
50Only be 10
-3.5/ 0.1ml is than the control group (EID that does not add chloroform
50Be 10
-6.30/ 0.1ml) about 3 titres that descend prove that viral isolates is to the chloroform sensitivity.
The viral isolates pH value is transferred to 3.0, measure EID with responsive chicken embryo
50Be 10
-5.5/ 0.1ml, comparison is according to only descending 10
-0.5About, virus still has certain infectivity to the chicken embryo, proves that viral isolates has tolerance to acid.
Through 30 minutes, responsive chicken embryo has not been had infectivity under 56 ℃ of the viral isolates, proved that viral isolates is thermo-labile.
5. animal returns test
With 10 of 7 age in days SPF chickens, with inoculation HN in eye droppings, collunarium and the tracheae
99Each 0.2ml of chicken embryo virus allantoic fluid, the inoculation back was observed 14.The inoculation back had 8 chickens' extract god not good enough since the 48th hour, and diet reduces, and phenomenons such as nose, wherein 1 chicken death appear coughing, get rid of in sick chicken.Symptomatic chicken is dissected, and visible tracheae, lungs have congested pathology.The chicken that dies of illness has kidney swollen, the piebald kidney, symptoms such as urate deposition are arranged in the ureter, the kidney of chicken that this is died of illness has been made tissue slice, carries out histological observation, and the major lesions of seeing kidney is renal tubules,convoluted generation granular degeneration, vacuolar degeneration and vitreous degeneration, interstitial edema, in infiltration such as lymphocyte is arranged, the kidney parenchymal tissue necrosis that has forms the focal zone that differs in size.
6. specific serum neutralization test
Use HN
99Infectivity resistant bronchitis positive serum respectively with viral isolates HN
99, NDV LaSota, IBDV B
87, AIV H
9N
2And M
41Strain virus carries out neutralization test with the chicken embryo, and the result only has viral isolates HN
99With HN
99Infectivity resistant bronchitis serum presents positive reaction, and its corrective is inoculated 10 age in days SPF chicken embryos, and hatching did not find that the chicken embryo had any pathology after 144 hours, and HN
99The chicken embryo of strain virus liquid positive control death is arranged or grow little, roll up, ossify, symptom such as urate deposition.NDV La Sota, IBDV B
87, AIV H
9N
2Present negative reaction with the infectivity resistant bronchitis, fetus all has symptoms such as hyperemia, hemorrhage, oedema, death behind its corrective inoculation SPF chicken embryo.The HA test shows NDV La Sota strain and AIV H
9N
2Chick embryo allantoic liquid 1: 128～512 blood clotting valency is all arranged, result such as following table:
The specific serum neutralization test
With HN
99Positive serum and 100 EID
50M
41Viral liquid balanced mix such as strain, Holte strain, Gray strain, T strain, Hubei strain, HB strain, BJ strain, TJ strain, SH strain, SD strain, in under 20～25 ℃ of conditions and 1 hour, inoculate 6 of 10 age in days SPF chicken embryos, every embryo 0.2ml, establish virus control group and normal control group simultaneously, continue hatching at 37 ℃, observe to 144 hours, with test group at least 5/6 strongly live, typical cytopathic appears in virus control group at least 50% or death, all strong work of normal control group are standard.Result such as following table:
Use M
41Strain, T strain, HO strain, G strain, HN
99Positive serum is done 1: 8 (V/V) dilution, with 100 EID
50HN
99Strain virus liquid balanced mix, in under 20～25 ℃ of conditions and 1 hour, 6 of inoculation SPF chicken embryos, every embryo 0.2ml, establish virus control and normal control simultaneously, continue hatching at 37 ℃, observe to 144 hours, with test group at least 5/6 strongly live, typical cytopathic appears in virus control group at least 50% or death, all strong work of normal control group are standard.Result such as following table:
The present invention adopts following method to virus HN
99Purifying and immunogenicity test are carried out in strain:
With chicken embryo kidney cell terminal point dilution method to IBV HN
99Carried out purification experiment, and it has been returned SPF chicken embryo, the basic no change of its malicious valency.Chicken embryo virus liquid with this strain is made deactivation vaccine, and immune SPF chicken carries out immunogenicity determining, proves IBV HN
99Strain has better immunogenicity.
1, the preparation of SPF chicken embryo kidney cell (CEK)
SPF chicken embryo with 18 ages in days, it is dirty that the chicken embryonic kidney is taken out in aseptic technique, uses hank ' s liquid washing 3 times, is cut into small pieces with scissors, the pancreatin that adds volume percent 0.25%, in 37 ℃ of water-bath digestion 18～20 minutes, discard pancreatin, use hank ' s liquid to wash once again, add the RPMI-1640 that contains volume percent 8～10% calf serums, after blowing and beating cell repeatedly with suction pipe, be filtered to Tissue Culture Flask and Tissue Culture Plate with 4～6 layers of sterile gauze, in 37 ℃ of CO
2Incubator is cultivated, and connects poison after waiting to grow up to good individual layer.
2, go down to posterity and purifying
With HN
99Strain virus liquid is inoculated in the SPF chicken embryo kidney cell that grows up to individual layer, puts 37 ℃ of CO
2Incubator continue to be cultivated, and cell engenders pathology after 48 hours, contracts or draws in the net elongatedly as circle, receives poison after 96 hours, multigelation 3 times, with this method with HN
99Strain virus passed for 4 generations.With 10 times of serial dilutions of the 3rd generation cell toxicant work, get 10
-5, 10
-6, 10
-7, 10
-8Extent of dilution is inoculated in SPF embryo CEK individual layer respectively, 10 holes of every titre.Cultivate after 96 hours, high dilution is had the hole enlarged culturing of pathology, measure EID
50After carry out the terminal point dilution second time.Cytopathy liquid inoculation 9～10 age in days SPF embryos with obtaining after the terminal point dilution second time carry out viral rejuvenation and propagation.
3, HN
99The propagation of strain virus and evaluation
HN with purifying
99Virus liquid, inoculation 9～10 age in days SPF chicken embryos, the dead germ before 30 hours was rejected in the inoculation back, observes once every 4～6 hours after 30 hours, chooses dead embryo, puts 2～8 ℃ of preservations, chose all embryos to 72 hours.The aseptic allantoic fluid of collecting, and carry out every biological characteristics test, electron microscopic observation, the test of animal recurrence virus, physics and chemistry test, specificity test and serum neutralization test, steriling test, select asepsis growth, EID
50≤ 10
-6.0The HN that/0.1ml, every test meet the requirements
99The strain virus allantoic fluid, the packing freeze-drying is preserved, as seed culture of viruses.
Send U.S. SPAFA Jinan testing laboratory with the strain of purifying, carried out the external source cause of disease and detected, the result shows that this strain does not have the external source cause of disease and pollutes.
4, HN
99Immunogenicity test behind the strain virus purifying
With HN
99The strain purifying is selected qualified, the EID of steriling test
50≤ 10
-6.0The HN of/0.1ml
99The strain virus allantoic fluid, the tween-80 after the sterilization through adding 4% (V/V) after the deactivation is made water.Then in water: oil phase is the ratio of 1: 3 (V/V), is emulsified into oil-emulsion with high-speed tissue mashing machine.
With HN
99The strain oil-emulsion by 0.1,0.2,0.3,0.4,5 groups of different dosage immunity 28 age in days SPF chickens of 0.5ml, 8 every group.After 3 weeks, identical with condition 4 contrasts of immune chicken chickens are attacked HN together
99Strong poison, the HN of every chicken intratracheal injection 0.2ml
99Strain virus allantoic fluid 0.2ml observed result such as following table 14.
HN
99The different immunizing dose test-results of strain
| Immunizing dose (ml) | The immunity chicken | The immunity number of elements | Attack toxic agent amount (ml) | Attack malicious immunoprotection | The result contrasts morbidity | |
| Kind | Age in days | |||||
| 0.1 | SPF | 21 | 8 | 0.2 | 6/8 | 4/4 |
| 0.2 | SPF | 21 | 8 | 0.2 | 8/8 | 4/4 |
| 0.3 | SPF | 21 | 8 | 0.2 | 8/8 | 4/4 |
| 0.4 | SPF | 21 | 8 | 0.2 | 7/7 | 4/4 |
| 0.5 | SPF | 21 | 8 | 0.2 | 8/8 | 4/4 |
HN
99The evidence of the different immunizing doses of strain can protect 6/8 after the immune chicken of this strain immunity 0.1ml is attacked poison, all can obtain full guard after the immune chicken of immunity 0.2～0.5ml is attacked poison.Contrast chicken morbidity 4/4, chicken cough occurs, gets rid of nose, has after the dissection that kidney is swollen, segmental bronchus, lungs are congested or different symptoms such as hemorrhage.Test shows, HN
99Strain has better immunogenicity, is the respond well virus strain of a strain.
Claims (1)
1, avian nephropathogenic infectious bronchitis HN
99Strain virus is characterized in that,
(1), see that under Electronic Speculum the coronavirus particle is arranged, diameter 90～105nm has cyst membrane, has the pears shape that is about 18nm outstanding on every side, is radial arrangement;
(2), viral isolates produces interference to the breeding of NDV La Sota strain virus;
(3), viral isolates does not produce aggegation to 1% chicken red blood cell;
(4), return pathologies such as respiratory symptom can appear in the test of SPF chicken and kidney is swollen, piebald kidney;
(5), the specific serum neutralization results proves viral isolates of sick chicken and HN
99Positive serum is positive, and with NDV La Sota strain, IBDV B
87Strain, AIV H
9N
2The strain reaction that all is negative;
(6), with HN
99Strain positive serum and EID
50M
41Viruses such as strain, Holt e strain, Gray strain, T strain and Local Isolates Hubei strain, Harbin strain, Shandong strain, Beijing strain, Shanghai strain, Tianjin strain are done serum neutralization test, and the result shows, HN
99Strain positive serum and T strain, Hubei strain, Tianjin strain and Shandong strain are positive, with the reaction that all is negative of other strain virus.
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|---|---|---|---|
| CNB2006100180654A CN100537753C (en) | 2006-07-03 | 2006-07-03 | Avian nephropathogenic infectious bronchitis HN 99Strain virus |
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|---|---|---|---|
| CNB2006100180654A CN100537753C (en) | 2006-07-03 | 2006-07-03 | Avian nephropathogenic infectious bronchitis HN 99Strain virus |
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| CN100537753C CN100537753C (en) | 2009-09-09 |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101906404A (en) * | 2010-07-14 | 2010-12-08 | 河南科技学院 | Avian infectious bronchitis virus (IBV) as well as culture method and application thereof |
| CN102978167A (en) * | 2011-11-30 | 2013-03-20 | 普莱柯生物工程股份有限公司 | Method for preparing nephropathogenic avian infectious bronchitis viruses and live vaccines by using cell lines |
| CN107287218A (en) * | 2012-12-06 | 2017-10-24 | 普莱柯生物工程股份有限公司 | Infectious bronchitis of chicken velogen strain S1 genes and its velogen strain and application |
-
2006
- 2006-07-03 CN CNB2006100180654A patent/CN100537753C/en not_active Expired - Fee Related
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101906404A (en) * | 2010-07-14 | 2010-12-08 | 河南科技学院 | Avian infectious bronchitis virus (IBV) as well as culture method and application thereof |
| CN101906404B (en) * | 2010-07-14 | 2012-09-05 | 河南科技学院 | Avian infectious bronchitis virus (IBV) as well as culture method and application thereof |
| CN102978167A (en) * | 2011-11-30 | 2013-03-20 | 普莱柯生物工程股份有限公司 | Method for preparing nephropathogenic avian infectious bronchitis viruses and live vaccines by using cell lines |
| CN107287218A (en) * | 2012-12-06 | 2017-10-24 | 普莱柯生物工程股份有限公司 | Infectious bronchitis of chicken velogen strain S1 genes and its velogen strain and application |
| CN107287218B (en) * | 2012-12-06 | 2021-08-27 | 普莱柯生物工程股份有限公司 | Avian infectious bronchitis virulent strain S1 gene and virulent strain and application thereof |
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|---|---|
| CN100537753C (en) | 2009-09-09 |
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