CN1744915A - Escape mutants of newcastle disease virus as marker vaccines - Google Patents

Abstract

A vaccine against Newcastle Disease contains one or more mutant immunogens of the NDW strain. The mutant immunogen lacks the antigenic binding site on the F glycoprotein which is recognized by the monoclonal antibody mAb 54. Reagent kits and assay methods help to distinguish vaccinated members of a poultry flock from those that may have been infected with wild-type Newcastle Disease virus.

Description

The Avian pneumo-encephalitis virus escape mutants of vaccine as a token of

Invention field

The present invention relates to a kind of new anti-newcastle disease vaccine, and relate to the protection poultry and exempt to suffer from the method for newcastle.In more detail, the present invention relates to a kind of sign vaccine of safe and effective anti-newcastle, and the test kit and the detection method that are used to check poultry, support the fowl person and can will inoculate poultry among the fowl group and those poultry that may infect wild-type Newcastle disease virus make a distinction thereby make.The present invention also relates to the new mutant immunogen that is used for anti-newcastle disease vaccine.

Background of invention

For poultry, newcastle (ND) usually is a kind of serious mortality disease, thereby can cause great economic loss.This disease is caused that by Avian pneumo-encephalitis virus (NDV) NDV belongs to Paramyxoviridae (Paramyxovirdae) paramyxovirus genus (Paramyxovirus).In avian paramyxoviruses, 9 kinds of different serotypes had been discerned already, called after PMV-1 to PMV-9.NDV belongs to PMV-1 or serotype 1.According to U.S. Patent number 5,149,530, present called after of NDV strain " Wiltenberg " (also claiming " Wiltenburg ") or NDW strain, this patent is attached to herein by reference.

Two types protrusion of surface (furcella) is outstanding from the peplos of NDV.A kind ofly be made up of glycosylated protein, this albumen contains blood clotting (H) and two kinds of activity of neuraminidase (N) (HN glycoprotein) of virion.Another kind is made up of glycoprotein F, and glycoprotein F is responsible for cell fusion, haemolysis and virus and penetrates.Sugar-free stromatin M is incorporated into the inboard of film, as the binding site of nucleocapsid, when virus when plasma membrane sprouts, may participate in the aggreation of HN glycoprotein and F glycoprotein.

Produce anti-all these three kinds of proteic antibody of greatly mediation immunity.Those independent anti-HN or the proteic antibody of F are protectiveness.

Newcastle influences the poultry of many commercializations, comprises chicken, turkey, Phasiana, Guinea chicken, duck, goose and pigeon.In addition, it also may be brought disaster to and various captive birds and the birds of putting in a suitable place to breed of partly taming birds and free living, comprises the aquatic bird that migrates.The birds with in the aviary of raising in cages also may be affected.

Avian pneumo-encephalitis virus enters in the animal body by respiratory tract and intestinal.The granule less than 5 microns of air-borne transmission is covered with whole respiratory tract, comprises air bag.Enter conjunctiva, nasal cavity and trachea down to bronchus greater than 5 microns granules.In effect by cilium and the infection between the cell in the trachea and diffusion virus.After the invasion site began breeding, virulent virus entered spleen, liver, kidney and lung.The final brain of invading of virus, many thereupon birds begin death.

The symptom of newcastle mainly is respiratory symptom and nervous symptoms.Normally asthma.Nervous symptoms comprises wing and/or both legs are one-sided and diplegia, the circus movement of head and neck, swing/fluctuation, the tic of wing, neck or lower limb flesh.General Symptoms can comprise loss of appetite and the minimizing of laying eggs, and lays eggs usually and can reduce more than 40%.

The immune state that gets involved viral character and concrete fowl group is depended in the variation of mortality rate.Usually, the propagation of lethal those strains between infected poultry is slower than those slow lethal strains fast.In addition, there is long-term asymptomatic virus carrier in people's supposition in some poultry species such as chicken.Pathophorous the greatest danger is exactly the outbreak of disease that flows and cause owing to personnel or equipment.Because the intensification of many operations in the aviculture, therefore just there are personnel to flow in a large number and the resettlement of equipment from a fowl group to another fowl group.

Develop many vaccine protection birdss now and exempted to suffer from newcastle.Some active antigen vaccines and weak malicious antigen vaccine can give by eye drip, collunarium or spraying or drinking-water.Inactivated vaccine also can provide protection, does not all have postvaccinal respiratory reaction usually.This class vaccine is many to be oil emulsion vaccines, and wherein oil adjuvant is used for improving the immunogenicity of vaccine antigen.But vaccine also is inoculated into developmental Embryo Gallus domesticus in the ovum.Sometimes this mode may be more accurate.

U.S. Patent number 5,149,530,5,250,298,6,348,197,5,750,111 and 5,733,556 every kind of present situations that anti-new castle disease virus vaccine field all is described.Some document of quoting also further illustrates some combined vaccines, for example those contain one or more except that at the newcastle also at the antigenic combined vaccine of other poultry disease.

Unfortunately, can't be subjected to that pathogenicity is strong, the not inoculation fowl group of the newcastle disease virus infection of " wild type " makes a distinction with inoculating fowl group and those seemingly now.Under the both of these case, antibody all produces in animal body.Yet, the present antibody that induces of the anti-NDV immunogen of inoculation, usually with infect the antibody of being found behind the strong infectious NDV of pathogenicity and be difficult to difference usually.

Therefore, this area needs the novel vaccine of anti-new castle disease virus.When giving poultry vaccine in eye drip, collunarium or spraying or drinking-water or the ovum, this vaccine can safe and effective prevention newcastle.Also to support the fowl person vaccinated poultry member and the nonvaccinated poultry member who has infected newcastle can be made a distinction.Develop a kind of new vaccine and also should develop a kind of test kit judging whether suitably inoculation of animal, or infected virulent newcastle, perhaps perhaps inoculated multiple conventional NDV vaccine.

Summary of the invention

As a part of the present invention, a kind of anti-new castle disease virus vaccine is provided, described vaccine contains has an appointment 10 ⁰-10 ⁹EID ₅₀The mutant immunogen from Avian pneumo-encephalitis virus NDW strain, wherein said mutant immunogen lacks the antigen binding site on the F glycoprotein that the monoclonal antibody of mAb 54 by name discerns.

A kind of mutant immunogen of the Avian pneumo-encephalitis virus through identifying also is provided, and this Strain is preserved in CNCM, and preserving number is #I-2928.This mutant immunogen is suitable as the mother who is developed further into the ND vaccine and plants virus, and the present invention also provides a kind of immunogen of ND virus of the immunogenicity characteristics with above-mentioned preservation Strain.

A kind of method of protecting the poultry animal to exempt to suffer from newcastle also is provided, and described method comprises that giving described animal contains and have an appointment 10 ⁰-10 ⁹EID ₅₀The vaccine from the mutant immunogen of Avian pneumo-encephalitis virus NDW strain, wherein said mutant immunogen lacks the antigen binding site on the F glycoprotein that mAb 54 monoclonal antibodies discern.

The invention still further relates to a kind of test kit that detects the mutant immunogen of the anti-newcastle of inoculation, described test kit comprises standard NDV antigen and luminous antibody, wherein said luminous antibody combines with described antigen in inoculating birds serum, but not with do not inoculate birds serum in described antigen combine.

The method of the Avian pneumo-encephalitis virus mutant immunogen that a kind of production is used for anti-newcastle disease vaccine also is provided, cultivate Avian pneumo-encephalitis virus when described method is included in the monoclonal antibody of mAb 54 by name, cause described mutant immunogen when described monoclonal antibody is arranged, to grow and grow and do not neutralized by described monoclonal antibody.Binding site on the F glycoprotein of mutant immunogen shortage mAb 54 identifications, thereby also do not neutralized by this antibody.

A kind of mutant immunogen that obtains from the nucleotide sequence shown in the Seq.ID No.1 also has been described.This partial nucleotide sequence coding lacks the immunogenic F glycoprotein of mAb 54 monoclonal antibody binding sites.Therefore, the aminoacid sequence shown in the Seq.ID No.2 is represented the appropriate section sequence of Avian pneumo-encephalitis virus F glycoprotein.

Therefore, the present invention also provides a kind of immunogen of Avian pneumo-encephalitis virus, and described immunogen has the immunogenicity feature of the aminoacid sequence mutant immunogen shown in the Seq.ID No.2.

The present invention also provides a kind of mutant immunogen of Avian pneumo-encephalitis virus, and the serine that this is viral 157 of wild type NDW strains, is replaced by arginine usually.

It is a kind of to judge that the poultry animal whether according to the present invention's inoculation as described below, perhaps uses other NDV vaccination that the present invention also provides in addition, perhaps whether infected the test kit and the method for wild type ND virus.

According to following detailed description and appended claims, the other objects and features of the invention will become apparent.

Detailed description of the preferred embodiments

Have found that now the mutant immunogen that derives from Avian pneumo-encephalitis virus Wiltenberg strain can be used to produce the sign vaccine of anti-newcastle.The immunogen of gained lacks the binding site that this area is called " mAb54 " monoclonal antibody, and all known existing ND Strain all have the binding site of mAb 54.MAb 54 is that M.S.Collins etc. has done further description in conjunction with the characteristic of the F fusion rotein of NDV, see " Evaluation of Mouse MonoclonalAntibodies Raised Against an Isolate of the Variant Avian ParamyxovirusType I Responsible for the Current Panzootic in Pigeons ", Arch.Virol. (1989) 104:53-61.

The production that lacks the mutant immunogen of mAb 54 binding sites can be by cultivating the live virus strain of Avian pneumo-encephalitis virus when monoclonal antibody mAb 54 is arranged.More preferably utilize the Wiltenberg or the NDW strain of this area.Suitable NDW strain can derive from the Institute Pasteur CNCM (state-run microbial preservation center (Collection Nationale de Cultures deMicroorganismes)) (25 of Paris, FRA, Rue de Docteur Roux, F-75724 PARISCEDEX), preserving number is 1-781, U.S. Patent number 5, done in 149,530 to further describe.At Russell, P.H., J.Gen.Virol.65 can find the multinomial technology of cultivating and selecting mutant immunogen among the 795-798 (1984).Say that briefly mAb 54 antibody combine with the viral binding site of antibody recognition, i.e. viral binding site combination on the F glycoprotein part, the Avian pneumo-encephalitis virus that neutralization is cultivated.Exception be the i.e. mutant immunogen of this binding site not of some escape mutants.Thereby these mutants thereby escape neutralization can be grown and breed.

These escape mutants are mutant immunogen just, and they further become the basis of vaccine development.Mutant immunogen can be in proper culture medium such as African green monkey kidney cell (VERO) be further cultivated, for example, and can be by immunoselection to gained mutant immunogen and mAb 54 antibody responses, and virulence is done further to screen.The wild-type virus back mutation can adopt heredity and antigen method to estimate.Some technology described in the top Russell are perhaps helpful, just as the more available technology in this area.

Suitable mutant immunogen, promptly mother plants the virus mutation immunogen, is deposited in the CNCM of above-mentioned address, and preservation day is on August 29th, 2002, and preserving number is I-2928.This particularly preferred mutant immunogen is a Mutagen, the further called after of Fort Dodge Animal Health " p.13. ".As described below, this p.13. mutant immunogen be the desirable object of further continuous passage.The present invention also comprises other preparation with the method that described method is identical basically and NDV mutant immunogen that have its substantially the same immunogenicity feature herein.

ND virus mutation of the present invention is immunogenic to be further characterized in that in the wild type NDW virus that 157 serine is replaced by arginine with mutant form.

Can preferably use the continuous passage of fowl embryo by using the fowl egg continuous passage, perhaps use embryo fibroblast or VERO (cercopithecus aethiops) cell to carry out tissue culture, use available technology, come the described mutant immunogen of large-scale production from Embryo Gallus domesticus.The present invention's expection is about 2 continuous passages, and verified useful about at the most 10 continuous passages.And preferably go down to posterity about 4 times to about 8 times through the technical staff.Embryo Gallus domesticus is ideal especially for the mutant immunogen that goes down to posterity.

Above-mentioned gained mutant immunogen is specially adapted to provide the immunoprotection of anti-newcastle.This mutant immunogen can be mixed with anti-newcastle disease vaccine.Suitable vaccine contains for every dose has an appointment 10 ⁰To about 10 ⁹EID ₅₀One or more sudden change NDW immunogen or its immunogenicity active components of (50% egg-infective dose).Preferred scope especially for spray inoculation, is about 10 ⁴To about 10 ⁹EID ₅₀Scope in.When through eye drip or collunarium, perhaps to drink water when giving, above-mentioned scope can be done further adjustment.If inoculation in the ovum, every dose of consumption is about 10 ⁰To about 10 ¹EID ₅₀Scope in.

Typical dosage is that about 0.01ml is to about 10ml, more preferably from about 0.01ml is to the vaccine (scope can be adjusted by the technical staff) of about 1.0ml, this vaccine contains the mutant immunogen of above-mentioned dosage, and any adjuvant, excipient and carrier, is described further below.

Vaccine of the present invention can contain one or more suitable vaccine adjuvants and pharmaceutically acceptable carrier, and suitable vaccine adjuvant can comprise available compatible oil in this area and oil emulsion.Pharmaceutically acceptable carrier can comprise for example water or normal saline.Also can comprise other excipient in the vaccine, for example, acceptable stabilizing agent of medicine and live vaccine field and antiseptic.These components can be mixed with sudden change ND immunogen, produce final vaccine.

Vaccine of the present invention is safe and effective to the poultry family member, comprising chicken, turkey, Phasiana, Guinea chicken, duck, goose, Bantam, pigeon or the like.

The present invention contains the immunogenic vaccine of sudden change ND also can provide immunoprotection to prevent the vaccine antigen of other poultry disease formulated together with other.The immunogen of the disease that for example can use anti-Bursal disease, Marek (Marek ' s disease) and cause by avian herpetoviruses.

The application method of vaccine of the present invention is preferably by spraying, but also can pass through eye drip or collunarium or drinking-water, the technical staff can adopt all usings method, also can use suitable ovum injection device to carry out injection inoculation, for example machine of North Carolina Embrex company in the ovum.Can give and hatch 1 day to inoculating up to about 21 days egg.Inoculate once especially preferably for the egg during about 10-18 days.Therefore, the protection poultry method of exempting to suffer from newcastle comprises described vaccine is inoculated into developmental poultry in the ovum as mentioned above.

Also comprise test kit and detection method as ingredient of the present invention, by this can the poultry of vaccine of the present invention and those not be inoculated and the poultry that infected strong poison, wild type or " field " Avian pneumo-encephalitis virus makes a distinction with inoculating, can also inoculate itself and those and utilize the poultry of the immunogenic conventional NDV vaccine that contains the F fusion glycoprotein to make a distinction.Example comprises any other suitable method that ELISA, immunoperoxidase staining procedure, western blotting or art technology person are used.

As unrestriced example, test kit can comprise " standard " NDV antigen (binding site with F fusion glycoprotein of mAb 54 identification), and also can with the bonded luminous antibody in this site on the standard antigen." luminous " antibody is by the available certain methods labelling in this area, thereby meeting " luminous " and when combining with standard antigen, can send the signal of color change.Luminous antibody also can be a kind of anti-mouse antibodies (because of mAb 54 derives from mice), for example is the enzyme labelling that produces the color chemical reaction with the catalysis of a kind of energy, just as the available method in this area.Anti-mouse anti physical ability combines with second antibody (as the part of test kit), combines (as in the sandwich assay of standard) with binding site on the standard antigen successively again.

In a further embodiment of detection kit and detection method, also there is state of conflict to detect, the wherein luminous detection kit antibody and the second detection kit antibody all can combine with standard N D detection kit antigen.These two kinds of antibody are all competed F glycoprotein binding site, and therefore different readings can show the positive and feminine gender.

The method of unrestriced check poultry is summarized as follows: investigated blood sampling among the poultry member certainly, then one by one inspection.The poultry that infects wild type, open-air NDV or inoculated another ND strain that still has F glycoprotein binding site all can produce inner antibody, this site (F fusion glycoprotein) on the virus of anti-these antibody recognition of inner antibody.When sample adds " standard " antigen of detection kit, these inner antibody also will combine with same binding site on the standard antigen.This will block or obviously reduce luminous antibodies this site (as in competitive assay) to the standard antigen, thereby will block or obviously reduce color change.On the other hand, the poultry member who inoculates by the present invention will lack the binding site on the F glycoprotein.Thereby can not produce the inside antibody (mAb 54) of anti-this binding site.When interpolation contained the reagent of standard antigen, luminous antibody capable combined with standard antigen, and color change takes place, and showed that specific animal is by the present invention's inoculation.Perhaps, the second detection kit antibody capable combines with standard antigen, thereby luminous antibody can combine with second antibody (as in sandwich assay) and color change takes place.

When the poultry member has inoculated conventional NDV vaccine, can also produce the antibody in anti-F glycoprotein site, but these poultry are usually infecting wild-type virus in varying degrees.In these cases, the difference of color change can be distinguished these animals.

Some work-around solutions of the embodiment of aforementioned agents box and detection method also are within the scope of the invention.

Following embodiment illustrates each preferred aspect of the present invention, but shall not be construed as limitation of the scope of the invention.

Embodiment

Embodiment 1-experimental program

The useful route of following 4 phases experimental program brief description exploitation anti-new castle disease virus sign vaccine.

1 phase

Whether induce the antibody of the antigen site that mAb 54 antibody discern when determining natural infection PMV1 (paramyxovirus serotype 1 is accredited as NDV).The escape mutants that produces the antigen site that shortage mAb 54 is discerned on the fusion rotein is done further to analyze.

2 phases

The escape mutants of the antigen site that shortage mAb 54 is discerned on the Genetic identification fusion rotein.

3 phases

Carry out the stability test of escape mutants, check wild type or virulence back mutation situation.

4 phases

Exploitation and affirmation are used to distinguish anti-wild type antibody and anti-vaccine strain detection of antibodies method.

Be described in more detail:

1 phase

Use competitive binding assay, the antibody of the antigen site that anti-mAb 54 is discerned in the serum of inspection natural infection PMV1 birds.

When being arranged, on chick embryo fibroblast (CEF) culture, cultivates mAb 54 the NDW strain of NDV.

The virus that is produced among the plaque purification CEF is produced viral stock solution (poison of pretending illness is stocked mother solution) through limited going down to posterity in the fowl embryo.

Adopt the escape mutants of immunoperoxidase (IPX) experiment sieving and mAb 54 responding property.

2 phases

Measure the nucleotide sequence of " wild type " and escape mutants fusion rotein.

Determine that according to the experimental program that is provided (when the competitive assay of 1 phase) virus stocks the research that mother solution is renderd a service in the chicken of the no-special pathogen (" SPF ") of 1 age in days.

Selection is carried out the test of 3 phases with the escape mutants in the former site of nonreactive/modified antigen site that mAb 54 determines.

3 phases

Go down to posterity the viral stock solution 2 times of selected escape mutants to produce female virus (going down to posterity for the first time) and work seed virus (second pass for) of planting with the fowl embryo.

Be used for the vaccine virus of stability test for 4 times with production with the fowl embryo work seed virus that goes down to posterity.

With the IPX test of mAb 54 and the feature of nucleotide sequencing affirmation vaccine virus.

With go down to posterity " confirmed " vaccine virus 10 times and confirm above-mentioned feature of fowl embryo.

With go down to posterity " confirmed " vaccine virus 10 times and confirm feature of CEF with IPX test.

With studying in the body that in 1 age in days SPF chicken, carries out virulence and wild type back mutation that goes down to posterity for 10 times altogether.

With the IPX of mAb 54 test and nucleotide sequencing affirmation feature from the 10th isolating again virus of interior generation (if be less than 10 times then from the last isolated viral again that once goes down to posterity).

Measure pathogenic index (ICPI) in the brain of female kind, vaccine, subculture in vitro separately (10x) vaccine and interior generation (10x, referring to above) vaccine virus with 1 age in days SPF chicken.

4 phases

Monospecific antiserum with 6 chicken productions in age in week anti-" wild type " and NDW escape mutants.Produce the latter's antiserum (referring to above) with vaccine virus.

The ELISA that exploitation detects NDV antibody tests to screen the fowl serum of anti-NDV " wild type " strain antibody and anti-NDV vaccine strain antibody as test kit.

If suitably (along with the progress of ELISA) improves the IPX test of existing NDV antibody, test to screen the fowl serum of anti-NDV " wild type " strain antibody and anti-NDV vaccine strain antibody as test kit.

The positive and negative serum with known anti-NDV wild type strain are confirmed the selection-breeding test.

The method of embodiment 2-selection-breeding escape mutants

Can adopt Russell, P.H., J.Gen.Virol. (1984) 65,795-798 institute describing method.These programs are summarized as follows.

With ' Technomouse ' preparation a collection of monoclonal antibody 617/54 (mAb54).

With 9 age in days fowl embryo culture NDW strains.

Measure the neutralization index of mAb54.Measure virus infection titer with the dilution hot deactivation mAb of difference by trace-neutralization test.The virus of equivalent (50 μ l) and mAb are adsorbed onto in the micropore of VERO cell then 37 ℃ of reactions 2 hours.Carry out the IIP test, the cell number that statistics infects.The expectation viral infection reduces by 10 ⁵More than.

The immune selection-breeding of virus plaque.The virus of equivalent and hot deactivation mAb were hatched 2 hours at 37 ℃.With the PBS/ culture medium this mixture is diluted by 1/10,1/100 and 1/1000.With every kind of solution, comprise that undiluted solution absorbs to 2 contains in the hole (100 μ l/ hole) of merging chick embryo fibroblast (CEF).37 ℃ after 1 hour, with containing dilution mAb of 1/100-1/500 and trypsin 2-5 μ g/ml) agar cover each hole.In 37 ℃/5%CO ₂Hatch flat board.

After 3-4 days, with the visible single plaque of dimethyl diaminophenazine chloride dyestuff.Pass agar to cellular layer " picking " plaque with pasteur pipet.The agar speckle that will contain mutant is put into PBS.The excessive mAb of each preparation reuse handled once then through CEF or fowl embryo repeated transmission generation.

With the VERO cell culture that contains and do not contain mAb 617/54 from each plaque through the repeated transmission virus in generation.Escape mutants with immunoperoxidase (IPX) experiment sieving and mAb 617/54 and polyclonal antiserum reaction.

Embodiment 3

The effect research of NDW escape mutants and ' POULVAC ' vaccine

Method

The group of two inoculations (mutant and ' Poulvac ' vaccine) is made up of 25 age in days chickens for every group.

Ten nonvaccinated chickens are matched group.

With the fowl embryo virus of taking out in the plaque is gone down to posterity six times, be used for all inoculations of mutant.

The chicken of every inoculation is predetermined to obtain 10 with the 0.5ml demineralized water ^6..5EID ₅₀

Give the virulent NDV of bird intramuscular injection (Herts/33), be used for counteracting toxic substances, every consumption 0.5ml10 ^5.0ELD ₅₀

During beginning, the titration mutant is measured the titre of virus.As shown in table 1.P.13, mutant is diluted to 10 ^6.5EID ₅₀/ 0.5ml and total amount 12.5ml isolate for every and feed 25 extensive spray deliveries of chicken.The titre of every bottle ' Poulvac ' vaccine is 10 ^10.02EID ₅₀(by FortDodge Animal Health Holland, The Netherlands provides) makes every chicken accept 0.5ml through preparation again and includes 10 ^6.5EID ₅₀The virus formulation that titration afterwards is used to inoculate chicken is to finish with the fowl embryo immediately after inoculation.These titres are used for calculating the meansigma methods (table 1) of the viral dosage that is calculated that every chicken accepts.The group of mixing at random between two rooms is used for counteracting toxic substances.The counteracting toxic substances dosage of every chicken acceptance contains 10 for 0.5ml Herts/33 ^4.6ELD ₅₀

Those chickens ill so that can not take food of experimental session can be dead.The sick chicken that can take food and drink water and the record of experiment remaining chicken after 15 days see table 2 and table 3 for details.

The result

Before the counteracting toxic substances and behind the counteracting toxic substances (15 days), gather serum, suppress (HI) reaction with the NDW viral hemoagglutination and detect from all remaining chickens.

The result of study of rendeing a service is summarized in table 2 and the table 3.

The virus group

Nonvaccinated

Ten nonvaccinated control chicks dead (mortality rate 100%) in three days of counteracting toxic substances.

The serum of these chickens all is that the HI test is negative before the counteracting toxic substances.

" Poulvac " vaccine

The chicken that has inoculated ' Poulvac ' vaccine is 4 days dead 1 (mortality rate 4%) behind counteracting toxic substances.

Reclaiming the strong ND virus of virulence from this dead chicken is by inoculating brain tissue homogenate to the fowl embryo.

In addition two chickens have shown clinical symptoms, a just for one day and another finishes up to experiment.

The HI antibody titer is at 2-16 before the counteracting toxic substances, and 24 chickens wherein are less than or equal to 8.The antibody titer of whole chickens all has increase (scope 16-512) behind the counteracting toxic substances.

Mutant p.13

There are 3 chickens to show clinical symptoms but none death (mortality rate 0%) in mutant 25 chickens p.13 though inoculated.

The HI titre is 8 or still less before the counteracting toxic substances, but the overwhelming majority obviously increases behind counteracting toxic substances, and scope is 32-1024.

Table 1.

The immunizing dose of the vaccine virus of every chicken

The mutant numbering	Production method CEF or Vero cell	Virus titer EID 50/0.1ml	Virus dosage a
				P13 ' Poulvac ' vaccine	Vero -- ?	10 7.83 10 10.02b?	10 5.95 10 6.6?

^aBy every chicken with acceptable dose that 0.5ml calculated

^bThe virus titer of every bottle

Table 2.

Use NDW escape mutants virus and ' POULVAC ' vaccine

The data summary of research vaccine potency

	Identify number	HI1	HI/D2	Estimate
					Do not inoculate matched group	888 889 890 891 892 893 894 895 896 897	2 1 2 1 2 1 ＜2 1 2 1 2 1 2 1 2 1 2 1 2 1	D D D D D D D D D D	Death in dead the 3rd day in the 3rd day dead the 2nd day dead the 2nd day dead the 3rd day dead the 3rd day dead the 2nd day dead the 2nd day dead the 3rd day dead the 3rd day

	Identify number	HI1	HI/D2	Estimate
					Inoculation ' Poulvac ' vaccine group	601 602 603 604 605 606 607 608 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623 624 625	2 3 2 2 2 3 2 2 2 2 2 3 2 3 2 3 2 2 2 2 2 3 2 2 2 3 2 2 2 3 2 1 2 3 2 3 2 4 2 3 2 3 2 3 2 2 2 2 2 3	2 5 2 4 2 7 2 5 2 8 2 5 2 9 2 7 2 5 2 8 2 8 2 8 2 7 2 6 2 8 2 6 2 5 2 6 2 6 2 6 2 5 D 2 8 2 6 2 5	Since morbidity in the 9th day *Only morbidity in the 5th day death in the 4th day

	Identify number	HI1	HI/D2	Estimate
					P.13, the inoculation mutant is organized	476 477 478 479 480 481 482 483 484 485 486	2 3 2 3 2 2 2 2 2 3 2 3 2 3 2 2 2 2 2 2 2 2	2 5 2 8 2 8 2 10 2 9 2 6 2 6 2 3 2 8 2 4 2 7	Morbidity in 7-11 days was since morbidity in the 5th day *

487 488 489 490 491 492 493 494 495 496 497 498 499 500

2 3 2 2 2 2 2 2 2 2 2 3 2 2 2 2 2 2 2 2 2 3 2 2 2 2 2 2

2 6 2 8 2 6 2 8 2 5 2 5 2 5 2 8 2 5 2 5 2 4 2 5 2 5 2 9

Morbidity in 7-10 days

Table 3.

Effect research-respectively organize per cent death loss

The group per cent death loss

Inoculation group 100

Poulvac ' vaccine group 4

P.13 organize 0

Note is applicable to table 2 and table 3.

¹Be expressed as the dilution inverse that serum suppresses 4 hemagglutination units of virus fully before the HI titre counteracting toxic substances.

²Remaining chicken number behind the HI titre counteracting toxic substances.D=is dead.

^*The chicken movement disorder, some lose weight, and only can move when being subjected to invading and harassing still and can take food and drink water.

Embodiment 4-is to NDW escape mutants and ' POULVAC ' vaccine

Fusion rotein (F _o) gene sequencing

Through go down to posterity 6 times mutant ABI Prism of Embryo Gallus domesticus ^TM310 Genetic Ahalyzer automatic sequencers are measured nucleotide sequence to detect its " wild type " back mutation.(partial sequence of F gene is shown in-Seq.ID No.1, and the amino acid sequence corresponding of enclosing is shown in Seq.IDNo.2).Measure the short sequence of mutant fusion protein F gene p.13, striden across the coding region that 157 amino acids change.Aminoacid sequence remains unchanged-and arginine is still at NDW ' wild type ' position of serine.Yet mutant arginine codon p.13 becomes CGC by AGA.

The discovery of order-checking is subjected to the support of this IPX test.Seemingly completely without mAb 54 and the bonded evidence of any mutant.Yet, NDW ' wild type ' obviously monoclonal antibody combination therewith.

To existing virus in ' Poulvac ' vaccine, measured the reading frame of complete genome sequence (comprising the F0 polypeptide precursor).NDW ' the wild type that this sequence and Fort Dodge provide ' mother of virus plants identical.Yet, these sequences and with Embryo Gallus domesticus further go down to posterity the female sequence of planting of NDW of gained have with.The silent mutation that Here it is gene 11 is 95.

Embodiment 5-further renders a service test

Used escape mutants is p.13 as follows: female virus (MSV) of planting, work seed virus (MSV+1 time go down to posterity), and experimental vaccine (MSV+5 time go down to posterity).Detect the virulence back mutation of MSV and detect hereditary stability after its tissue culture through egg and VREO cell goes down to posterity by regulation in the European Pharmacopoeia.The effectiveness of test experience vaccine (MSV+5 time go down to posterity) in commercial boiler.Described experiment shows that all escape mutants of the present invention (mutant immunogen) and vaccine all have efficient.

Embodiment 6-ND indicates test

General introduction

In the present embodiment, serum and anti-POULVAC  NDW vaccine (the Fort Dodge Animal Health of research difference inoculation ND sign strain chicken p.13, Fort Dodge, Iowa and Weesp, the Netherlands) feasibility of the serum of the chicken of the NDV antibody that serum of the chicken of antibody and antitoxin power are strong.The front points out, and p.13 ND sign strain lacks monoclonal antibody number is the binding site of 54 (Mab#54).

The serum of numbering " MV " is taken from the chicken of inoculation ND sign strain p13; The chicken that the serum of numbering " FS " is taken from actute infection NDV; The serum of numbering 9,10,11 and 12 is taken from the chicken of inoculation POULVAC  NDW vaccine.In contrast, comprise serum of SPF chicken and the serum of the chicken that has inoculated NDV B1 and usefulness NDV Herts 33 counteracting toxic substances in this research.Adopt immunoperoxidase staining test (IPX) to study.

The serum of numbering MV is blocked Mab#54 slightly, and SPF serum does not have.The reason of the blocking effect of the serum of numbering MV it be unclear that.If be not limited to any special theory, then may be because the NDV antibody and the spatial obstacle that is combined with near the binding site of Mab#54.This also may be caused by the active institute of non-specific binding.The combination of the medium blocking-up Mab#54 of serum of numbering FS, but some variation as a result.The serum of numbering FS shows the active blocking-up activity that is higher than the serum of numbering MV of its blocking-up.The blocking-up of the serum of numbering 9-12 is active fairly obvious and obviously be better than the blocking-up activity of the serum of numbering MV.Also there are notable difference between the two in serum and the NDV antiserum of numbering MV.

Can determine that the test of application IPx blocking-up Mab#54 might will be inoculated ND sign strain p13 from chicken serum chicken serum distinguishes:

-experimental infection NDV,

-inoculation NDW live vaccine.

Materials and methods

Material

96 hole tissue culturing plates of-fusion monolayer VERO cell.

-Mab#54

-NDV Ulster strain

-10% buffering formalin

-phosphate buffered saline(PBS) (PBS)

-SPF chicken serum

-antiserum that anti-POULVAC  NDW criticizes

-with the nontoxic B1 infected chicken of living, obtain the NDV positive serum with strong NDV Herts 33 counteracting toxic substances of virulence subsequently.

-from the chicken that suffers from acute newcastle, obtain open-air serum

-plant virus with the mother of ND sign strain p13 to give the chicken spray inoculation serum that obtained of intramuscular injection booster immunization subsequently

-goat anti-mouse immunoglobulin peroxidase conjugated antibody

0.2g carbamide H in the aseptic demineralized water of-substrate solution A:10ml ₂O ₂

Contain 0.1g 3-amino-9-ethyl carbazole in the-substrate solution B:25ml ethanol

Method, immunoperoxidase staining test (IPX)

-infect dull and stereotyped and standing over night with NDV Ulster-1/1000

-every hole adds 50 μ l 10% buffering formalin 20 minutes with fixed cell

-pour out formaldehyde, with twice in 0.1M Xian PBS hole of the temperature of 200 μ l pH7.2

-Jia 200 μ l PBS also leave standstill

-prepare serial dilution (1/2,1/10,1/25,1/50 and 1/100 P13, open-air serum and SPF serum) with PBS

-pour out PBS and plate is patted dry

-each dilution serum of 100 μ l is added in 10 holes of NDW virus, hatched 45 minutes for 37 ℃

-flick serum dilution, plate is patted dry and whole apertures are washed 3x with 200 μ l PBS.Plate is patted dry.

-preparing serial dilution 1/500-1/10 with PBS, 000 mAb 54 is added to every kind of diluent of 100 μ l in 1/2 hole, and the antigen/antibody of every kind of dilution test sera is contained in described hole.

Hatched dull and stereotyped 45 minutes for-37 ℃

-flick mAb and plate is patted dry

-clean each hole 3 times and plate is patted dry with 200 μ l PBS

-give to add 100 μ l goat anti-mouse immunoglobulin peroxidase conjugated antibody in each hole

Hatched 50 minutes for-37 ℃

-flick conjugate also with 200 μ l PBS hole flushing 3x

-be added among the 9.8ml PBS 0.1ml substrate solution A and 0.1ml substrate solution B and mixing, prepare final substrate solution

-Jia substrate, every hole 100 μ l put room temperature 15 minutes

Twice of-usefulness PBS hole flushing

-microscopically is distinguished and is read flat board

-brown cell is represented in conjunction with Mab#54

The result

The best dilution of Mab#54

SPF serum and NDV antiserum and different dilution antigen-reactives.Thereupon, Mab#54 also with different dilution antigen-reactives.This test is carried out twice.Result of the test sees Table 4 for the first time.(result of the test is very identical for the second time).

The result shows that the SPF antiserum can not block Mab#54 and combine with NDV is antigenic, though if can hang down slightly with the SPF seroreaction of 1/2 dilution.1/2, the combination of the positive antiserum blocking-up of 1/5 and 1/25 dilution NDV Mab#54.A little less than the 1/50 dilution serum blocking-up, and 1/100 dilution factor is blocked hardly.

Can think SPF serum and the NDV positive serum difference between the two, serum dilution be 1/5 and 25/1 o'clock the most obvious.

Further dilute Mab#54, the reactivity of 1/50 and 1/100 dilution positive serum descends.Decision is done further test with 1/2500 dilution factor Mab#54.

Behind the positive antiserum preincubate of table 4. and serial dilution degree SPF serum and NDV

Different dilution Mab#54 and the antigenic reactivity of NDV

The SPF antiserum

The antiserum dilution factor

The Mab dilution factor	1/2	1/2	1/5	1/5	1/25	1/25	1/50	1/100
									1/500	++	++	+++	+++	+++	+++	+++	+++
1/1000	++	++	+++	+++	+++	+++	+++	+++
									1/2500	++	++	+++	+++	+++	+++	+++	+++
1/5000	++	++	+++	+++	+++	+++	+++	+++

The NDV antiserum

The antiserum dilution factor

The Mab dilution factor	1/2	1/2	1/5	1/5	1/25	1/25	1/50	1/100
									1/500	-	-	-	-	-	-	++	+++
1/1000	-	-	-	-	-	-	+	++
									1/2500	-	-	-	-	-	-	+	++
1/5000	-	-	-	-	-	-	-	+

+ dyeing is obvious ++ strong dyeing ++ and the dyeing of+extra-heavy

-dye-free

The combination of different antiserum blocking-up Mab#54

What blocking-up was studied the results are shown in Table 5.The serum of numbering MV is taken from the chicken of inoculation ND sign strain p13.The serum of numbering FS is open-air serum, takes from the chicken of actute infection NDV.The serum of numbering 9,10,11 and 12 is taken from the chicken of inoculation POULVAC  NDW vaccine.

The result shows that SPF serum do not block the combination of Mab#54, and the combination of male NDV serum blocking-up Mab#54.The serum of numbering MV has blocking-up slightly active.When dilution factor is 1/2, in 8 parts of serum the combination of 2 parts of blocking-up Mab#54 is arranged, and when dilution factor is 1/10, detect the obvious combination of Mab#54.The blocking-up of the serum of numbering FS changes greatly; FS1 obviously blocks the combination of Mab#54, and FS6 shows that hardly blocking-up is active.

The serum of numbering 9-12 is all blocked the combination of Mab#54, and particularly 11 and 12.

1/2500 dilution Mab#54 behind the Different Chicken serum preincubate of table 5. and serial dilution degree

With the antigenic reactivity of NDV, A portion and B portion are separate detection

A portion.

The dilution factor of polyclonal antiserum

Antiserum	1/2		1/10	1/10	1/25	1/25	1/50	1/100
									MV1	-	-	+	+	++	++	+++	+++
MV2	-	-	+	+	++	++	+++
									MV3	+w	+w	++	++	+++	+++	+++	+++
MV5	+w	+	+++	+++	+++	+++	+++	+++
									MV6	+	+	+++	+++	+++	+++	+++	+++
MV7	+	+	+++	+++	+++	+++	+++	+++
									MV8	+？	+？	++	++	+++	+++	+++	+++
MV9	+w	+w	++	++	+++	+++	+++	+++
									FS1	-	-	-	-	+	+	+++	+++
FS3	-	-	+	+	++	++	+++	+++
									FS4	-？	-	++	++	+++	+++	+++	+++
FS5	+w	+w	++	++	+++	+++	+++	+++
									FS6	++	++	+++	+++	+++	+++	+++	+++
SPF	++	++	++	++	+++	+++	+++	+++
									NDV	-	-	-	-	-	-	-	+

B portion.

The dilution factor of polyclonal antiserum

Antiserum	1/2		1/10	1/10	1/25	1/25	1/50	1/100
									9	-	-	+w	+w	++	++	+++	+++
10	-	-	ND	ND	ND	ND	ND	ND
									11	-	-	-	-	-？	-？	++	+++
12	-	-	-？	-？	+	+	+++	+++
									SPF	+++	+++	+++	+++	+++	+++	+++	+++
NDV	-	-	-	-	+	+	+++	+++

+ w=is weak positive

-?=very slight dyeing

+=dyeing

++=strong dyeing

++ +=extra-heavy dyeing

ND=serum deficiency is not done

Discuss

The serum outcome evaluation of numbering MV, FS and 9-12 is as follows.+ to be worth be 1, ++ value is 2, and +++to be worth be 3 ,-to be worth be 0.Calculate the meansigma methods of every part of serum according to this.The calculating of SPF serum and NDV positive serum is carried out equally.

The results are shown in Table 6.The serum of numbering MV is blocked Mab#54 slightly, and SPF serum is not blocked.The reason of the serum blocking effect of numbering MV it be unclear that.As be not limited to any special theory, then may be owing to the spatial obstacle that is combined with of NDV antibody with close Mab#54 binding site.This also may cause owing to the non-specific binding of serum component.

The blocking-up activity of the serum of numbering FS generally is better than the serum of numbering MV.The front points out that the result of the serum of numbering FS has some variations.The serum of numbering FS is taken from the chicken that actute infection NDV symptom is arranged.Because the time of infecting and collecting between serum is short, so has some may not have NDV antibody in these serum.There is individual variation for blocking-up is active in possible explanation between different serum.

The blocking-up of the serum of numbering 9-12 is active strong.Compare with it, the blocking-up of the serum of observed numbering MV is weak relatively.This species diversity serum dilution be 1/10 and 1/25 o'clock the most obvious.Serum and the NDV antiserum of numbering MV also have notable difference between the two.

Table 6. blocking-up result of study is summed up

Serum	Serum dilution
						1/2	1/10	1/25	1/50	1/100
	MV	0.75	2.13	2.75	3.00						3.00
FS						0.60	1.60	2.40	3.00	3.00
	9-12	0.00	0.33	1.00	2.67						3.00
SPF						2.50	2.50	3.00	3.00	3.00
	NDV	0.00	0.00	0.50	1.50						2.00

Conclusion

Might will inoculate ND sign strain chicken serum p.13 with the detection of IPX blocking-up Mab#54 from chicken serum distinguishes.

-experimental infection NDV,

-inoculated the NDV vaccine of living.

Although understand the present invention specifically with preferred embodiment, be expected under the situation without departing from the spirit and scope of the present invention, as given in description and the claims, the technical staff can make some modification to the present invention.

Sequence table

<110>Geerligs，Harmen?J.

Brown，Ian?H.

Alexander，Dennis?J.

Collins，Michael?S.

＜120〉the Avian pneumo-encephalitis virus escape mutants of vaccine as a token of

<130>AMl00044

<160>2

<170>PatentIn?version?3.2

<210>1

<211>1662

<212>DNA

＜213〉paramyxovirus/Avian pneumo-encephalitis virus

<220>

<221>CDS

<222>(1)..(1662)

<400>1

atg?ggc?tcc?aga?tct?tct?acc?agg?atc?cca?gta?cct?ctg?atg?ctg?acc 48

Met?Gly?Ser?Arg?Ser?Ser?Thr?Arg?Ile?Pro?Val?Pro?Leu?Met?Leu?Thr

1 5 10 15

gtc?cgg?gtc?gcg?ctg?gca?ctg?agt?tgc?gtc?tgt?ccg?aca?agc?tcc?ctt 96

Val?Arg?Val?Ala?Leu?Ala?Leu?Ser?Cys?Val?Cys?Pro?Thr?Ser?Ser?Leu

20 25 30

gat?ggc?agg?cct?ctt?gca?gct?gca?ggg?att?gtg?gtg?aca?gga?gac?aaa 144

Asp?Gly?Arg?Pro?Leu?Ala?Ala?Ala?Gly?Ile?Val?Val?Thr?Gly?Asp?Lys

35 40 45

gca?gtc?aac?ata?tac?acc?tca?tct?cag?aca?ggg?tca?atc?ata?gtc?aag 192

Ala?Val?Asn?Ile?Tyr?Thr?Ser?Ser?Gln?Thr?Gly?Ser?Ile?Ile?Val?Lys

50 55 60

tta?ctc?cca?aat?atg?ccc?aaa?gat?aaa?gag?gcg?tgt?gca?aaa?gcc?ccg 240

Leu?Leu?Pro?Asn?Met?Pro?Lys?Asp?Lys?Glu?Ala?Cys?Ala?Lys?Ala?Pro

65 70 75 80

ttg?gag?gcg?tac?aac?agg?aca?ttg?act?act?ttg?ctc?acc?ccc?ctt?ggt 288

Leu?Glu?Ala?Tyr?Asn?Arg?Thr?Leu?Thr?Thr?Leu?Leu?Thr?Pro?Leu?Gly

85 90 95

gat?tct?att?cgt?agg?ata?caa?gag?tct?gtg?act?aca?tct?gga?gga?ggg 336

Asp?Ser?Ile?Arg?Arg?Ile?Gln?Glu?Ser?Val?Thr?Thr?Ser?Gly?Gly?Gly

100 105 110

aaa?cag?gga?cgc?ctt?ata?ggc?gcc?att?atc?ggc?ggt?gca?gct?ctc?ggg 384

Lys?Gln?Gly?Arg?Leu?Ile?Gly?Ala?Ile?Ile?Gly?Gly?Ala?Ala?Leu?Gly

115 120 125

gtt?gca?acc?gct?gca?cag?ata?aca?gca?gct?tcg?gct?ctg?ata?caa?gcc 432

Val?Ala?Thr?Ala?Ala?Gln?Ile?Thr?Ala?Ala?Ser?Ala?Leu?Ile?Gln?Ala

130 135 140

aac?caa?aat?gct?gcc?aac?atc?ctc?cgg?ctt?aaa?gag?aga?att?gct?gca 480

Asn?Gln?Asn?Ala?Ala?Asn?Ile?Leu?Arg?Leu?Lys?Glu?Arg?Ile?Ala?Ala

145 150 155 160

acc?aat?gag?gct?gtg?cac?gag?gtc?act?gat?gga?tta?tca?caa?cta?gca 528

Thr?Asn?Glu?Ala?Val?His?Glu?Val?Thr?Asp?Gly?Leu?Set?Gln?Leu?Ala

165 170 175

gtg?gca?gtt?ggg?aag?atg?cag?caa?ttt?gtt?aat?gac?cag?ttt?aat?aaa 576

Val?Ala?Val?Gly?Lys?Met?Gln?Gln?Phe?Val?Asn?Asp?Gln?Phe?Asn?Lys

180 185 190

aca?gct?cag?gaa?ttg?gac?tgt?ata?aaa?att?acc?cag?cag?gtt?ggt?gta 624

Thr?Ala?Gln?Glu?Leu?Asp?Cys?Ile?Lys?Ile?Thr?Gln?Gln?Val?Gly?Val

195 200 205

gaa?ctc?aac?ctg?tat?cta?act?gaa?ttg?act?aca?gta?ttc?ggg?cca?caa 672

Glu?Leu?Asn?Leu?Tyr?Leu?Thr?Glu?Leu?Thr?Thr?Val?Phe?Gly?Pro?Gln

210 215 220

atc?act?tcc?cct?gcc?tta?acc?cag?ctg?act?atc?cag?gcg?ctt?tac?aat 720

Ile?Thr?Ser?Pro?Ala?Leu?Thr?Gln?Leu?Thr?Ile?Gln?Ala?Leu?Tyr?Asn

225 230 235 240

cta?gct?ggt?ggg?aat?atg?gat?tac?ttg?ttg?act?aag?tta?ggt?gta?ggg 768

Leu?Ala?Gly?Gly?Asn?Met?Asp?Tyr?Leu?Leu?Thr?Lys?Leu?Gly?Val?Gly

245 250 255

aac?aac?caa?ctc?agc?tca?tta?att?ggt?agc?ggc?ctg?atc?acc?ggc?aac 816

Asn?Asn?Gln?Leu?Ser?Ser?Leu?Ile?Gly?Ser?Gly?Leu?Ile?Thr?Gly?Asn

260 265 270

cct?att?ctg?tac?gac?tca?cag?act?cag?ctc?ttg?ggt?ata?cag?gta?acc 864

Pro?Ile?Leu?Tyr?Asp?Ser?Gln?Thr?Gln?Leu?Leu?Gly?Ile?Gln?Val?Thr

275 280 285

cta?ccc?tca?gtc?ggg?aac?ctg?aat?aat?atg?cgt?gcc?acc?tac?ttg?gaa 912

Leu?Pro?Ser?Val?Gly?Asn?Leu?Asn?Asn?Met?Arg?Ala?Thr?Tyr?Leu?Glu

290 295 300

acc?ttg?tct?gta?agt?aca?acc?aaa?gga?ttt?gcc?tca?gca?ctc?gtc?cca 960

Thr?Leu?Ser?Val?Ser?Thr?Thr?Lys?Gly?Phe?Ala?Ser?Ala?Leu?Val?Pro

305 310 315 320

aag?gtg?gtg?atg?aag?gtc?ggt?tcc?gtg?ata?gaa?gaa?ctt?gac?acc?tca 1008

Lys?Val?Val?Met?Lys?Val?Gly?Ser?Val?Ile?Glu?Glu?Leu?Asp?Thr?Ser

325 330 335

tac?tgt?ata?gag?acc?gat?ttg?gat?cta?tat?tgt?aca?aga?ata?gtg?aca 1056

Tyr?Cys?Ile?Glu?Thr?Asp?Leu?Asp?Leu?Tyr?Cys?Thr?Arg?Ile?Val?Thr

340 345 350

ttc?cct?atg?tct?cct?ggt?att?tat?tcc?tgt?ttg?agc?ggc?aat?aca?tcg 1104

Phe?Pro?Met?Ser?Pro?Gly?Ile?Tyr?Ser?Cys?Leu?Ser?Gly?Asn?Thr?Ser

355 360 365

gct?tgc?atg?tac?tcg?aag?act?gaa?ggc?gca?ctc?act?acg?ccg?tac?atg 1152

Ala?Cys?Met?Tyr?Ser?Lys?Thr?Glu?Gly?Ala?Leu?Thr?Thr?Pro?Tyr?Met

370 375 380

act?ctc?aaa?ggc?tca?gtt?att?gcc?aac?tgt?aag?atg?aca?aca?tgt?aga 1200

Thr?Leu?Lys?Gly?Ser?Val?Ile?Ala?Asn?Cys?Lys?Met?Thr?Thr?Cys?Arg

385 390 395 400

tgt?gca?gac?ccc?ccg?ggt?atc?ata?tcg?caa?aat?tat?gga?gaa?gct?gtg 1248

Cys?Ala?Asp?Pro?Pro?Gly?Ile?Ile?Ser?Gln?Asn?Tyr?Gly?Glu?Ala?Val

405 410 415

tct?cta?ata?gat?agg?caa?tca?tgc?aat?gtc?cta?tcc?tta?gac?gga?ata 1296

Ser?Leu?Ile?Asp?Arg?Gln?Ser?Cys?Asn?Val?Leu?Ser?Leu?Asp?Gly?Ile

420 425 430

act?ttg?agg?ctc?agt?ggg?gaa?ttt?gat?gca?act?tat?caa?aag?aat?atc 1344

Thr?Leu?Arg?Leu?Ser?Gly?Glu?Phe?Asp?Ala?Thr?Tyr?Gln?Lys?Ash?Ile

435 440 445

tca?ata?caa?gat?tct?caa?gta?atc?gtg?aca?ggc?aat?ctc?gat?atc?tcg 1392

Ser?Ile?Gln?Asp?Ser?Gln?Val?Ile?Val?Thr?Gly?Asn?Leu?Asp?Ile?Ser

450 455 460

act?gag?ctt?ggg?aat?gtc?aac?aac?tcg?ata?agt?aat?gct?tta?gat?aag 1440

Thr?Glu?Leu?Gly?Asn?Val?Asn?Asn?Ser?Ile?Ser?Asn?Ala?Leu?Asp?Lys

465 470 475 480

tta?gag?gaa?agc?aac?agc?aaa?cta?gac?aag?gtc?aat?gtc?aaa?ctg?acc 1488

Leu?Glu?Glu?Ser?Asn?Ser?Lys?Leu?Asp?Lys?Val?Asn?Val?Lys?Leu?Thr

485 490 495

agc?aca?tcc?gct?ctc?atc?acc?tat?atc?gtt?tta?act?gtc?ata?tct?ctt 1536

Ser?Thr?Ser?Ala?Leu?Ile?Thr?Tyr?Ile?Val?Leu?Thr?Val?Ile?Ser?Leu

500 505 510

gtt?tgt?ggt?ata?ctt?agc?ctg?gtt?cta?gca?tgc?tac?ctg?atg?tac?aag 1584

Val?Cys?Gly?Ile?Leu?Ser?Leu?Val?Leu?Ala?Cys?Tyr?Leu?Met?Tyr?Lys

515 520 525

caa?aag?gcg?caa?cag?aag?acc?ttg?tta?tgg?ctt?ggg?aat?aat?acc?ctg 1632

Gln?Lys?Ala?Gln?Gln?Lys?Thr?Leu?Leu?Trp?Leu?Gly?Asn?Asn?Thr?Leu

530 535 540

gat?cag?atg?aga?gcc?act?acg?aaa?atg?tga 1662

Asp?Gln?Met?Arg?Ala?Thr?Thr?Lys?Met

545 550

<210>2

<211>553

<212>PRT

＜213〉paramyxovirus/Avian pneumo-encephalitis virus

<400>2

Met?Gly?Ser?Arg?Ser?Ser?Thr?Arg?Ile?Pro?Val?Pro?Leu?Met?Leu?Thr

1 5 10 15

Val?Arg?Val?Ala?Leu?Ala?Leu?Ser?Cys?Val?Cys?Pro?Thr?Ser?Ser?Leu

20 25 30

Asp?Gly?Arg?Pro?Leu?Ala?Ala?Ala?Gly?Ile?Val?Val?Thr?Gly?Asp?Lys

35 40 45

Ala?Val?Asn?Ile?Tyr?Thr?Ser?Ser?Gln?Thr?Gly?Ser?Ile?Ile?Val?Lys

50 55 60

Leu?Leu?Pro?Asn?Met?Pro?Lys?Asp?Lys?Glu?Ala?Cys?Ala?Lys?Ala?Pro

65 70 75 80

Leu?Glu?Ala?Tyr?Asn?Arg?Thr?Leu?Thr?Thr?Leu?Leu?Thr?Pro?Leu?Gly

85 90 95

Asp?Ser?Ile?Arg?Arg?Ile?Gln?Glu?Ser?Val?Thr?Thr?Ser?Gly?Gly?Gly

100 105 110

Lys?Gln?Gly?Arg?Leu?Ile?Gly?Ala?Ile?Ile?Gly?Gly?Ala?Ala?Leu?Gly

115 120 125

Val?Ala?Thr?Ala?Ala?Gln?Ile?Thr?Ala?Ala?Ser?Ala?Leu?Ile?Gln?Ala

130 135 140

Asn?Gln?Asn?Ala?Ala?Asn?Ile?Leu?Arg?Leu?Lys?Glu?Arg?Ile?Ala?Ala

145 150 155 160

Thr?Asn?Glu?Ala?Val?His?Glu?Val?Thr?Asp?Gly?Leu?Ser?Gln?Leu?Ala

165 170 175

Val?Ala?Val?Gly?Lys?Met?Gln?Gln?Phe?Val?Asn?Asp?Gln?Phe?Asn?Lys

180 185 190

Thr?Ala?Gln?Glu?Leu?Asp?Cys?Ile?Lys?Ile?Thr?Gln?Gln?Val?Gly?Val

195 200 205

Glu?Leu?Asn?Leu?Tyr?Leu?Thr?Glu?Leu?Thr?Thr?Val?Phe?Gly?Pro?Gln

210 215 220

Ile?Thr?Ser?Pro?Ala?Leu?Thr?Gln?Leu?Thr?Ile?Gln?Ala?Leu?Tyr?Asn

225 230 235 240

Leu?Ala?Gly?Gly?Asn?Met?Asp?Tyr?Leu?Leu?Thr?Lys?Leu?Gly?Val?Gly

245 250 255

Asn?Asn?Gln?Leu?Ser?Ser?Leu?Ile?Gly?Ser?Gly?Leu?Ile?Thr?Gly?Asn

260 265 270

Pro?Ile?Leu?Tyr?Asp?Ser?Gln?Thr?Gln?Leu?Leu?Gly?Ile?Gln?Val?Thr

275 280 285

Leu?Pro?Ser?Val?Gly?Asn?Leu?Asn?Asn?Met?Arg?Ala?Thr?Tyr?Leu?Glu

290 295 300

Thr?Leu?Ser?Val?Ser?Thr?Thr?Lys?Gly?Phe?Ala?Ser?Ala?Leu?Val?Pro

305 310 315 320

Lys?Val?Val?Met?Lys?Val?Gly?Ser?Val?Ile?Glu?Glu?Leu?Asp?Thr?Ser

325 330 335

Tyr?Cys?Ile?Glu?Thr?Asp?Leu?Asp?Leu?Tyr?Cys?Thr?Arg?Ile?Val?Thr

340 345 350

Phe?Pro?Met?Ser?Pro?Gly?Ile?Tyr?Ser?Cys?Leu?Ser?Gly?Asn?Thr?Ser

355 360 365

Ala?Cys?Met?Tyr?Ser?Lys?Thr?Glu?Gly?Ala?Leu?Thr?Thr?Pro?Tyr?Met

370 375 380

Thr?Leu?Lys?Gly?Ser?Val?Ile?Ala?Asn?Cys?Lys?Met?Thr?Thr?Cys?Arg

385 390 395 400

Cys?Ala?Asp?Pro?Pro?Gly?Ile?Ile?Ser?Gln?Asn?Tyr?Gly?Glu?Ala?Val

405 410 415

Ser?Leu?Ile?Asp?Arg?Gln?Ser?Cys?Asn?Val?Leu?Ser?Leu?Asp?Gly?Ile

420 425 430

Thr?Leu?Arg?Leu?Ser?Gly?Glu?Phe?Asp?Ala?Thr?Tyr?Gln?Lys?Asn?Ile

435 440 445

Ser?Ile?Gln?Asp?Ser?Gln?Val?Ile?Val?Thr?Gly?Asn?Leu?Asp?Ile?Ser

450 455 460

Thr?Glu?Leu?Gly?Asn?Val?Asn?Asn?Ser?Ile?Ser?Asn?Ala?Leu?Asp?Lys

465 470 475 480

Leu?Glu?Glu?Ser?Asn?Ser?Lys?Leu?Asp?Lys?Val?Asn?Val?Lys?Leu?Thr

485 490 495

Ser?Thr?Ser?Ala?Leu?Ile?Thr?Tyr?Ile?Val?Leu?Thr?Val?Ile?Ser?Leu

500 505 510

Val?Cys?Gly?Ile?Leu?Ser?Leu?Val?Leu?Ala?Cys?Tyr?Leu?Met?Tyr?Lys

515 520 525

Gln?Lys?Ala?Gln?Gln?Lys?Thr?Leu?Leu?Trp?Leu?Gly?Asn?Asn?Thr?Leu

530 535 540

Asp?Gln?Met?Arg?Ala?Thr?Thr?Lys?Met

545 550

Claims (19)

1. the vaccine of an anti-new castle disease virus, described vaccine comprises the mutant immunogen from Avian pneumo-encephalitis virus NDW strain of about 100-109EID50, the antigen binding site on the F glycoprotein that the monoclonal antibody of wherein said mutant immunogen shortage mAb 54 by name is discerned.

2. the vaccine of claim 1, wherein said mutant immunogen is continuous passage.

3. the vaccine of claim 2, wherein said continuous passage is carried out in fowl egg.

4. the vaccine of claim 3, wherein said continuous passage comprises at least about 2 times and about at the most 10 times continuous passage.

5. the vaccine of claim 4, wherein said continuous passage comprises about 4 to about 6 times continuous passage.

6. the vaccine of claim 1, p.13 wherein said mutant immunogen is accredited as, and is preserved in the CNCM of Paris, FRA, and preserving number is I-2928.

7. the immunogen of an Avian pneumo-encephalitis virus, the immunogen of described Avian pneumo-encephalitis virus has the immunogenicity feature that the preserving number that is preserved in CNCM is the strain of I-2928.

8. method of protecting the poultry animal to exempt to suffer from newcastle; described method comprises the mutant immunogen from Avian pneumo-encephalitis virus NDW strain that gives the about 100-109EID50 of described animal, the antigen binding site on the F glycoprotein that wherein said mutant immunogen shortage mAb 54 monoclonal antibodies are discerned.

9. the method for claim 8, the wherein mutant immunogen of about 104-109EID50.

10. production method that is used for the Avian pneumo-encephalitis virus mutant immunogen of anti-new castle disease virus vaccine, cultivate Avian pneumo-encephalitis virus when described method is included in the monoclonal antibody of mAb 54 by name, cause described mutant immunogen when described monoclonal antibody is arranged, to grow and grow and do not neutralized by described monoclonal antibody.

11. the method for claim 11, wherein said Avian pneumo-encephalitis virus are the NDW strains.

12. the method for claim 12, wherein said virus is cultivated in the fowl embryo.

13. the mutant immunogen of an Avian pneumo-encephalitis virus, the F glycoprotein of wherein said virus derives from the partial nucleotide sequence shown in the Seq.ID No.1, and wherein said glycoprotein lacks the binding site of the monoclonal antibody of mAb 54 by name.

14. a mutant immunogen, described immunogen has the aminoacid sequence shown in the Seq.ID No.2.

15. the immunogen of an Avian pneumo-encephalitis virus, described immunogen have the immunogenicity feature of the mutant immunogen of the aminoacid sequence shown in the Seq.ID No.2.

16. the mutant immunogen of an Avian pneumo-encephalitis virus, wherein 157 serines are replaced by arginine.

17. detection kit that is used for detecting inoculation by anti-newcastle mutant immunogen, described detection kit comprises standard NDV antigen and luminous antibody, wherein said luminous antibody combines with the described antigen of inoculation in the serum, but not with do not inoculate serum in described antigen combine.

18. the detection kit of claim 17, wherein said standard NDV antigen contains F glycoprotein binding site, and the site combination therewith of wherein said luminous antibody.

19. the detection kit of claim 18 wherein in described inoculation serum, does not produce the antibody of anti-F glycoprotein binding site, causes when adding the standard antigen of the described F of containing glycoprotein binding site, described luminous antibody freely combines with described site.