CA1183473A - Infectious bronchitis vaccines for poultry and process for the preparation of such vaccines - Google Patents

Abstract

ABSTRACT

Infectious bronchitis virus strains of a novel sero-type deposited with the Czechoslovak National Collection of Type Cultures of the Institute of Hygiene and Epidemiology in Prague under the nos. CNCTC A 07/80, CNCTC A 08/80, CNCTC
A 09/80, CNCTC A 010/80, CNCTC A 011/80, CNCTC A 016/80, CNCTC A 014/80, CNCTC A 015/80 and CNCTC A 013/80 and with the Collection Nationale de Cultures de Microorganismes d'Institut Pasteur, Paris, under nos. I.111, I.112, I.113, I.109, I.110, I.132, I.134, I.135 and I.133. These strains are useful in the production of infectious bronchitis vac-cines for administration to poultry.

Description

a73 INFECTIOUS BRONCHITIS V~CCINES FOR P3ULTRY AND PRCCESS FOR THE
PREPARATION OF SUCH VPCCINES.

The invention is relating to infectious bronchitis (IB) vaccines for poultry and a process for the preparation of such vaccines and more particularly to improve infectious bronchitis vaccines, derived from at least one in~ectious bronchitis virus strain of a no~el serotype as to those of the virusses, which are known up to now for vaccination purposes.
The application of live infectious bronchitis vaccines for poultry is already known for many years.
Infectious bronchitis is an important affection of the respiratory system, the kidneys and oviduct of poultry. The cause of this syndrome is a corona virus. The poultry is severely affected by epizootics of this disease.
~he infectious bronchitis still causes a high mortality, especially in young poultry. Besides the mortality and more or less skrong respiratory symptoms, lesions to the oviducts and as a result thereof egg production drops occur caused by an IB
infection.
Moreover infections with IB virus may stimulate latent vir~ls-or bacterial infections and may give rise in ~is way to severe economical losses, especially in the broiler field.
~or the combat~ent of infectious hronchitis as well vaccines, derived from inactivated virus as tho9e ones der:ived ~rom live virus, are applied. However, it was ~ound that a loss of immunogenic pr.ope~ties o~urxed after inactivation of these viruses with e.g.
~.ormaline cmd ultra violet light (M.S. ~lo~stad, Diseases of Poultry, Biester and Schwarte, Iowa State University Press. Ames. (1565), 615).
As also normal, sound chickens can be killed or diseased by primary vaccination with a live, non or less attenuated virus vaccine, whereby an especial danger is existing for animals of less than

2 to 3 weeks old or for chickens short before the start of or during laying, pecple skilled in this art have a clear preference for the app.llc~lon of d~ad vaccin~ or of live vaccines, whereby ~a~ tried to ~ncre~e th~ h~rmlessness of such vaccines by means o~

~1 ,.

attenuation of the original IB field virus isolate.
For exa~ple the H-strain, which is presently applied on world wide scale on account of his broad immunization spectre against am~ng others Massachusetts and Connecticut type IB-virus and which was isolated and attenuated by sijlenga, Hoekstra and Rispens, is disclosed in Tijdschr. Diergeneesk. 81:43, "Infectious bronchitis in ehicks in the Netherlands" (1956), Tijdschr. Diergeneesk, 85:320 (1960), Tijdschr. Diergeneesk. 85-279 (1960) and Tijdschr. Diergeneesk.
85:398 (1960).
E'or such m~dified vaccines, viruses, having undergone 25 or more embryo passages to reduce their pathogenicit~ and their disseminating ability, are applied up to ncw, such as e.g. the Massachusetts type and more particularly the IsV W 48, M 41, 82828 besides the H52 and H120 strains thereof, these two having been passaged approximately 52 and 120 times respectively on ehieken embryonated eggs, a Connectieut isolate, e.g. A 5968 or the Beaudette IBV type (I~V-42).
'I~e imm~nizing eapaeity of these viruses is very specifie against Massachusetts or Cbnnecticut types of IB virusses.
Although ~he use of vaeeines of these modified strains has presently appeared t~ be safe and effeetive, these vaeeines have appeared to be still unable to prevent outbreaks of infeetious bronehitis in a suffieient way under certain conditions, as appears from ~vian diseases vol. 20, no. 1, p. 42 and 177 and Avian Diseases vol. 19, no. 2, p. 323 and 583.
q~his shorteommg o the present IB vaeeines is attributed to c~eurrLng antlc~enie variations of the virus in an important degree, a~ al~o a~pp~ax~ e.g. from ~rehiv f~r die Ge.s,lmte Virusforschung 3~, p. 32 ~ 71) arld Cunningham C.EI., Develop. Biol. Standard, 33, 311 (1976).
Efforts were made therefore to reaeh an adequate vaeeination of poultry by means of preparation and application of co~bined vaeeines, derived from several IBV strains of different serotypes.
Elowever, hereby a elearly eneountered difficulty appeared to form the deerease of immunogenie properties of the respeet:ive start~lg viruse~, eaused by mutual ~Iterae-tion, as appears from ~n. J. Vet. Res.
36, ~, 52~ an~ 525 ~1965) a~ ~vian Diseases 12, 577 (1~68).
'rhexefore ~here :is ~ill existing a larqe need ~!

~!33~3 for IB vaccines with adequate immunizing properties.
It will be appreciated that the pursued improve-ment of these vaccines are still severely hampered, caused by changing immunogenic and other properties of the presently avai]able Is viruses after a large nuT~er of passages in embryonated chicken eggs, the appearance of new serotypes and the lack of sufficiently effectively a~plicable serological and immunological test procedures respecti~ely.
In this connection reference may be made to ~vian Diseases, 19, 2, 323 and 324 (1975).
As a result of extensive research and experimenta-tion, a numher of novel IB viruses could surprisingly be obtained, which can be regarded as deviating from the up to now most frequently applied IB viruses of the H type (e.g. IB H120 and IB H52) in cross neutralization tests (virus neutralization tests) according to e.g. the method as described in American Association of Avian Pathologist~, "Isolation and Iclentification of Avian Pathogens", page 184 (1975)~in the understanding that antisera diluted in a ratio of 1:5 are applied~and in challenge experiments with subse-quent vlrusreisolation tests. In other words at an 1nocula-tion with a virus o~ the H-type the concerning animals are not protected against virusreplication in the mucosa o~ the re~piratory sy~tem after a ch~llenge with the beforementioned cleviatlng novel IB vlru~cs.
Ilumoral antihodles against the IB H strain equally ~ppellrcd not to be able to neutralize ~ignificant amounts of IB vlru~ oE the mentioned devlating ~ypes.
O special irnportance ~or the practice is that the~ rlovel ~B vlru~e~ cause respiratory symp~om3 wLth ani-mal~ ~how:ln~ hL~h all~ibo(ly ~:iters a~ainst ihe ~B H sl:rain, and wi-th stlll l~ying anlmals, ec3cJ produ~tion clrons.
~ xamples of such novel viruses are those ones, identifi.~3d by tlle internal notation ~trecht.101 (U.101), Utrecht 102 (U.102), Drente.201 (Dv201), Lirnburg.501 (L.501), Llmburg.502 (L.502), Brabant.801 (B.801), I,;lmbu~c3.$86 ~L. 536), Overij~sel 728 ~0.728) ancl Utrecht.121 (U~ 12L) a~ CIQPO~:It~d at the Czeclloslovak National Collectlon o~ Typ~ Cul~uros o~ the Institute oE ~Iyg:Lene and ~p:Ldem:Lolo~Jy in Prague under the respective nos. CNCTC A07/80 ~ CNCTC A08t80, CNCTC A09/80, CNCTC A010/80, CNCTC A011~80, (on March 6, 1980), CNCTC A016/80, CNCTC A014/80, CNCTC A015/80 and CNCTC A013/80 (on September 9, 1980), and with the Col-5 lection Nationale de Cultures de11icroorganismes d'Institut Pasteur,Paris, under the resp.
nos. I-111, I- 112, I-113~ I-109, I-110 (on November 14, 1979) and I-132, I-134, I-135 and I-133 (on October 3, 1980) with 10 the Collection Nationale de Cultures de Microorganismes Institut Pasteur, Paris.
These viruses were isolated by means of the trachea swab method with flocks of laying chickens, which had been vaccinated two and three times respectively with IB H120 and 15 IB ~152 vaccine and which showed high humoral antibody titers as -to the Massachusetts type of the IB virus at the beginning of the occurrence of respiratory symptoms and/or egg production drops and with broilers, which sho~Jed in the second half of the ~attening period, respiratory symptoms a~ter previous vaccina-20 tlons with IB vaccine of the I1120 type, whereby both kindsof animals found themselves at the moment of isolation in districts, which have been indicated in the hereinbefore usecl internal notatlons.
There ~7a5 now found that by attenuation to SPF-~5 chitkt~n~mbryos, the isolrlted virusstrains have lost theirpakh~gqnicLk~ for SPF chlckens in a major degree, in spite o~ the fact that lheir immunising ability has rernained s-till present.
For example the viruss-traLn with lnternal notation 30 xr~v U~ c~ht.l01 ~howet~ these beEorementioned characterlst:lcs aEker 85 SPE' ~ype I chlckenelnhryo passages and the virus-strain IBV Limburg.536 ~howed these characteristics after 56 SPF type I chickenembryo passages.
There ~s surprisingly found during a comparing 35 test, using conven-tional ~1120 or H52 vaccines, ~hat the protectton against LB viru~;es of thc Massachusetts type ~ m~ur:~d a~ ~he amoullt oE virus neu~raliæ~ng antiboclles -h~cl n~t ~.o be dllninl.shet~ 1n a ~i~3ni~lcant d~yrce, i~ a l-l-7;~

type vaccine combined with e.g. the novel IBV isolate Utrecht 101 was administered instead of the H-ty~e vaccine. During this experimentation one star-ted from intran~sal a~lication of the concerning vaccines.
For instance the following neutrali7ation indices were determined ~ weeks a.~ter adminlstration of the respective vacclnes and vaccine combinations.

~Table 1. I~nunisin~ ability of dif~erent vaccines measured on the humoral immune res~onse (neutralization-index) _ _ _ .. , ., . _ _ .. , _ _ _ _ .. _ _ _ _ _ .. ~ _ _ _ _ .. , .. .. _, .. _ _ _ _ . _ _ _ _ _ _ _ _ . _ .. _ _ . _ _ _ . _, _ _ vaccine testvirus/ IBV IBV
N.I.H (B222) U.101 L.536 1. H120 6.5 1.7 0.8 2. :[BV U.101 2.1 6.8 N.D.
15 3. H120 ~ IBV U.101 6.2 6.5 N.D.
(53~d ~gg pas~ags~) 0 9 N.D. 5.8 5. H120 ~ IBV L,536 6.7 N.D. 5.7 ... _~.~ .. ._. ,_ _____ ~ .. _ ._.. , ___,._._._ _ __ . ._ ___ ____ In addition -to the immuno response af-ter administra-t.i.on of different vaccines and vaccine cornbinations respec-20 tive:ly, the res.istance agains!t virusreplication and ~ersis-tence was also d~termi.1lesl in and on the mucosa of the trachea, cls3termlned by means Oe the v.irus rei.solation technique, as de~s~.lbed .in "Speci~:l.cat.ions ~or the pxoduct.ion and control O.e ~v:l.an li.ve v.lrus vaccines" of the Mini~try of Agriculture, 25 Fi.sherls~ ~nd Food of the United Kin~dom Central Veterlnary ~Jahoratory of F~ioloc~:i.c~l Products alld Stallsla:rds ~e~artm~ntr N~W ll~w, W~br.~dsJe, SuLxey KT 153 MB, 2nd Edlt.l~n (1977), p.12 Cxoss ncutralizati.on ~ests in SPF chicken embryos and cross inEection tests on SPF chickens were carried ou-t 30 accord.i.ng to the hereunder followiny tables:

- 6 ~ 73 Table 2 Cross neutrali2ation test in SPF chickenembryos Antiserum vlruslsolate against virustype H U.101 o. 728 L. 536 5 H ~,7,2 neg. neg. n~g.
U.101 neg. 5,3 neg. neg. x 0.728 neg. neg. 5,8 neg.N.I.(10 ) L.5~6 neg. neg. neg, ~,6,2 In this connection with the expression "neg." i~
10 meant, that no significant positive neutralization indexes (N.I.), i.e. N.I. ~ 102' are obtained.

Table 3.
Cross challenge tests car~ied out o~ SPF chickens;
results of virusresolationtests.
15 vacc~ne virus challenge virus "VOET" U.101 0.728 L.536 type Massachusetts __ ___ H neg. + + +
120th egg-passage U.101 + neg, 50th egg-passage 0,~.827 + ~ neg. +
pas sage 20 ~.S36 + + + neg.
80th ecJ~3, p~ ge ~
With the term "neg." is meant in this connection that none of the SPF eggs, injected wlth the tracheaswab material, ~howed symptorns which rnay be attributed to an infection with IB virus.

From the results of tables 2 and 3 (neutralization and cross-challenge tests) it appears that the four applied serotypes of the IB virus, namely those of the H-, U.101-, 0.728- and L.536-type, not only differ antigenitically from each other, but additionally - ha~7ing in mind the results of the challenge experiments with additional virusreisolation - show attractive im~nogenic properties as to the hom~logeous virus.
Field experiments shcwed that in sera of broilers, reproduction chickens and laying hens, antibodies were frequently occurring against the virustypes Utrecht.101, Limburg.536 and ~7erijssel.728.
It will be appreciated therefore that the novel IB virustypes are not only differing antigenitically in a significant degree from the usually applied H-virus, but moreover are shcwing significantly different properties as to each other.
The novel isolated virusstrains could be characteri~ed by the following tests:
Treat~ent of the infectious amnion-allantoic fluid obtained by cultivation of original virus containing samples from infected homDgenized organ and tracheaswabmaterial in the allantoic hole of 10 days prebrooded SPF eggs with chloroform according to Mayr, A. et al, Virologische Arbeitsmethoden G. Fischer Verlag, Jena, 1977, p. 2B5. resulted, in comparison with the non treated ~terial, un a reduction o the repeatedly measured viruscontent from 107 5 to 101'5 EID~o. ~his experience may point to the presence of a virus agent, which is containing in his envelope a lip:lde, which is nece~sary Eor the infectivity.
~ e in~ctious amnion-allantoic ~luid caused no agglutination with erythrocytes derived rom SPF chickens.
Addition of 5-fluordesoxyuridine (Ft~) to the culture medium of chicken kidney cell culturest serving for replication of the agent, did not influence the intracellular synthesis of the virus agent in a significank degree.
The EID50 content of the cellmaterial and culture medium appeared k~ xe~ide on comparable levels 2, ~ and 7 days after -the inoculation oE the vixu~ a~nt, l.e. the '73 nucleic acid to be replicated belonged to the group of the ribonucleic acid.
Exam mation with electron microscope showed, that the virus agent, present in the amnion allantoic fluid which was harvested within 30 hours after the artificial infection, possessed a diameter of about 100 nm. About 15 nm long projections were present on the surface of this virus. The virus has the size and shape of a corona virus, to which also the avian bronchitisviruses are regarded to belong.
It will be appreciated that the properties of the novel serotypes of IB-viruses as described hereinbefore make the novel virusstra~ns expecially suitable for the preparation of as well inactivated as live poultry vaccines on behalf of a more efficient protection against infectious bronchitis, especially in areas or countries, wherein the described deviating serotypes according to the present invention occur besides the IB viruses of the socalled H-tyee.
More particularly virusstrains of the serotypes of the hereinbefore mentioned novel virusstrains may successfully be applied for the preparation of mixed live and inactivated vaccines, derived as well ~rom the novel IB strains as from the H~strains with one or more o the novel IB-strains~
The nov~l IBV vaccines o~ the present invention may be obtained by propagation of one or more novel virusstrains by methods kncwn in the ar~ in principle and optionally follc~ d by inaativation by methods known in the art in principle.
For instance the virus may b~ propayated in embryonated SPF chicken egg~ or in suitable cell cultures such as chicken lcidney cell cultures. However wit,h such a process there has to be checked whether the antigenic properties do not significantly change.
Hereafter the cultivatJed virus mat,erial is collected and puxi~ied. ~t last one or more stabilizers ancl possihly an~lbio~,ic~, ~uch as sodium penicillin Gl streptomycin or r - 9 ~
natamycin, may be added and the mix-ture is lyophilized.
~ore particularly, the concerning seed virus is lnoculated under sterile conditions into the allantoic cavity in 10-ll days prebrooded chicken egc3s of type I SPF
After incubation for 28 to 34 hours at 37C, the amnion-allantoic fluid of the then still living and of the specifically died (i.e. after 24 hours after the seed virus inoculation) embryos is harvested, purified and lyophilized after optional addition of stabili~ers and/or antibiotics.
According to this process, single vaccines could be prepared, which contained the virus after lyonhilisa~ion in an amount of ~lO4- ~ID50 per dose, while e.g. so prepared combined vaccines of a novel virusstrain and a known ~1-strain or of more novel virusstrains showed a virus 15 content of ~ 2 x 109- EID50 per dose and preerably a content of each of the virus cornponents of ~ 104 EID50 pcr dose.
It will ~e appreciated that the present invention .is also relating to novel, as well inactivated as live, ~o IBV vaccines, which have been at least derived fror~ one of the novel. IB virus~trains and to the application of such vaccines.
Preferably live vaccines, derived from one or more Oe t:he nove] vlruses or Erom the 11-tyne virus with one or ~ore of thr. novel IB-virllses, are applied.
More preferahly live vaccines, derived from ~120 or 1152 vlrusstrain and frorn one or rnore ~B viruses, selected Erom the Jrol1p cons:i6l:irlc3 of U.101, L.536 and ~728, are appL:Ied. ~rlle vclccl.ncs rnay also he applied with youny anim~ls an~ rnore preferab].y with broilers.
'rhe v~lccines may he ~dministered by the soccl1led eyerirop- or nose(1rop, t:l)e drLr1k;r)c3-water- or spraylneli1od ~or 1Ive vacc:lne~.
Vacc:ination hy means of novel live vaccines accord-ing to the present invention has preferably to be carried out 35 with poul~ry of an age of l day to 18 weeks. ~ovel inactivated vaccines are subcutaneously or intra1n1:lsc-1larly aclm:Lnil,tered to arlirnals. ., It wlll br3 a~ rec:i.cll.ed that a].so combLncd live (~r Lnacllvat:ed vacc:l1le~, r1erlvr~ rom at icast onr of the novel - 1 o - ~ 3 IB virustypes and one or more completelv other vlrusty~es, such as e.~. the Newcastle disease virus, adeno- or reo viruses, form a feature of the present invention too.
For the preparation of inactivated IBV vaccines according to the present invention there may be started from e.g. an amnion-allantoic fluid, to which a suitable carrier is added, after inactivation by methods known in the art, e.g. by means of beta-propiolactone or formaline.
Preferably the virus liquid of a suitable titer is processed to a water in oil emulsion vaccine, derived from a mineral or plant oil and one or more emulsifiers, such as non-ionic surface-active cornpounds derived from alkylene oxide and/or hexahydric alcohols and/or higher natural fatty acids (Clo-C20) such as esters or ester-ethers.
Examples of the lastmentioned emulsifiers are rn~nnide rnonooleate (Span ~ 80, Arlacel 80 ~ , Arlacel A ~
and polyoxyethylene (20) sorbita~ monooleate ~e.g. Tween 80 ~ ) The volurne ratio between the aqueous phase (virus 1uid) and the oily phase may vary from 3:7 to l:l and lies 20 preferably at a ratio of about 7:13.
~ rhe invention is illustrated by the following exam-plcs, however without any restriction o the object of the invention to these specific embodiments.

Example 1 25 Pre~Axation of CB virus vaccine of the strain U.101.
A. Cultiv~tion of virus~
'rype ~ SE'F chicken eyyci, which were preincubal:ed 10 to 11 days, are inoculated with 103- to 104~ E~D50 C~V U.101 seed vixus ~0.2 rnl pro eyy) in the allantoic vlt:y.
The ecJy~ ~re inspected for the first t;me after 20 to 24 hollrs from the virusinoculAtion and all a3pecific died embryos are removed.
After an incubationperiod of in total 28 hours 35 at-~37C, the arnnion-al]antoic fluid (AAF) i3 harves~ed.

B. Treat.men~ of virus su.~pension.
Aeter purl~ic~ion of the A~E' b~ rnean.s of centr1~u-gation at 2000 r.p.m. in a cooling oe ntrifuge and/or filtration,5 x 105 units of sodium penicillin G and 800 mg of streptomycin per liter are added to this AAF.
The virusmaterial is subsequently stabilized by a~dition of at least 3% by weight of albumin and/or mannitol.
The stabilized buLk virusmaterial is frozen to at least -35C and stored at such temperature until the further processing phase.
Samples of this material are meanwhile tested on their virus content by means of the EID50 (Egg Infection Dose 50%) assay method.
After the test results have become available, the virus material is thawn up again and filled out into lyophilization flasks.
~ he virus content (volume) is adjusted hereby in such a way, that at the end of the subsequent lyophilization there are still at least 10 5 EID50 of the concerning virus per dose present in the vaccine.
m e flasks are sealed under vacuurn at the end of the lyophilization.
With the preparation of the multivalent (mixed) vaccine care ha to be taken that the minimal virus contents for all virus-components are reached.

Example 2 Preparation of IB virus vaccine of the strain L.536.
~. Cultivation of virus.
Type I SPF chicken eggs, which have been preincubated during 10 to 11 day~, are inoculated with 103- to 104- EID50 IgV L.536 seed virus (0.2 ml pro egg) into the allantoic cavity.
The egy~ are inspected for the firsk time and all aspecifically died ~nb~y~ a~ remov~d after 20 to 24 hours ~rom the virus inoculation. A~ter an incubation period of in total 32 ho~s at ~37C, the A~F is harvested.
B. Treatment of the virus suspension.
After purification of the AAF by means of centrifugation at 2000 r.p.m. in a cooling centrifuge and/or -- 12 -- 131.~?9L73 filtration, 6 x 105 units of sodium penicillin G and 900 mg of streptomycin per liter are added to this AAF.
The virus material is subsequently stabilized by addition of about 5~ by ~eight of albumin and/or mannitol.
As albumin e.g. bovine albumin may be applied. The stabilized bulk virus material is subsequently f~ozen to at least -35C
and kept at such temperatu~e until the further processing ,_ phase.
Meanwhile samples of this material are tested on their virus content by means of the EID50 (Egg Infectious Dose 50%) assay method. The virus material is thawn u~
again after the testresults have become available and filled out into lyophilisationflasks.
The virus content (volume) is adjusted hereby in ~uch a way that at the end of the lyophilisation process at least 10 '5EID50 of the concerning virus per dose are ~resent in the vaccine. The flasks are sealed under vacuum at the end o~ the lyoph:ilisation.
During the preparation of the multivalent (mixed) vaccine care has to be taken that the minimum virus content for all vLrus components is reached.

Example _ Pre~arati,on of IB-virus vacc~ne of th~? strain 0.728.
A. Cultivation of viru.s.
Type I SPF chicken eggs, Which have been preincubated 2~ durin~ 10 to 11 day~, are inoculated with 103- to 104'0EID50 I~V 0.728 seed viru~ (0,2 ml per egg).
'rhe c?gcJs are inspec-ted or the first tirne and all aspe?ciflcally 11ed embryos are rernoved after 20 t:o 24 hours ~m ~he ViX'll~ inoc~ .ation. ~t~?r an 1ncuhation period of

3~ ln l,otal 3~ hours at ~37C, I:h~ ~AF is haL-vested.

B. Treatment of lhe virus suspension.
After purification of the ~AF by means of centrifu~
yation at 2000 r.p.m in a cooling cen-trifuc3e and/or iltratlon, 8 x 105 wnits of sodiurrl penicillln G and 1100 mg of strepto-35 rrly~Ln~? I1~?r lit~?r a~? ac~cled to th~? ~F.
'rh? viru~ malerial ls ~ub~ec,~u~?ntly stabilizecl by ~3~73 the addition of about 7% by weiyht of albumine and/or manni-tol.
The stabilized hulk virus material is subsequently frozen to at least -35C and kept at this temperature until the further processing phase.
Meanwhile samples of this material are tested on their virus content by means of the EID50 assay method.
The virus material is thawn un again and filled out into lyophilisationflasks after the testresults have be-come available.
The virus content (volume) is adjusted hereby in such a way tha-t at the end of the lyophilisation process at least 10 EID50 of the concerning virus per dosis are present ln the vaccine. The flasks are sealed under vacuurn at the end of the lyophilisation.
During the preparation of the multivalent (mixed) vaccine care has to be taken that the minimum virus content for all virus components is reached.

~ le 4 Preparation of a combined IB-virus vaccine of the strains U.lOl, L.536 and 0.728.
.. . .. . _ _ ... . , . . . . _ . . . ............ . _ A. Cultivation of virus.
Type I SPF chicke~ eggs, which have been preincubated during 10 to ll days, are inoculated wi-th 103- to 104-0EID50 LBV U.lOl, 1..536 and 0.728 seed virus (0,2 ml in total per egg) inko the allantoi.c cavLty.
T}le egc3~ are in~pected for the fJrst tlrne and all aspeci~lcally died embryos are removed after 20 to 24 hours ~rom the virus inoculation. After an incubatLon period of in total 32 hours a~ 37C, the ~AF :Ls harve~tecl.

B. rrreatment oE the virus suspension.
After purification of the A~F by rneans of centrifu-gation at 2000 r.p.m in a cooling centrifuge and/or filtration, 8 x 105 units of sodium penicillin G and 1000 rng of strepto-lnycine per liter are aclded to this AAF.
3S '['he vi~us Inater:ial is subs4quently stabilizecl by ~clclLt-Lon o~ at ~e~ 3~ by weicJht o;E albllnlLne and/or mannltol.

~ he stabilized bulk v,irus material is subsequently frozen to at least -35C and kept at this temperature until the further processing phase.
Meanwhile samples of this material are tested on their virus content by means of the EID50 assay method.
The virus material is thawn u~' again and filled c,ut into lyophilisationflasks after the teqtresults have be-come available.
The virus content (volume) is adjusted hereby in 10' such a way that at the end of the lyophilisation Process at least 10 ' EID50 of each concernincJ virus per dosis is present in the vaccine. The flasks are sealed under vacuo at the end of the lyophilisation.
During the preparation of the multivalent (mi~ed) vaccine care has to be taken that the minimum virus content for all virus components is reached.

Preparation of inactivated combined IB-virus vaccine of the strains H.120 U.101, I,.'536 and 0.7~8 In a similar way as described in example 4.A., the ' 20 virus was cultiva-ted in SPF eggs and the obtained virus sus-pension was treated in a similar way as in example 4.s., un-til the frozen phase is rea~hed, however, without addition of antibiotics and stabilizers.
The ~rozen ~AF is defrosted and inactivated in a water halh by 0.l~ o~ propiolactone during 90 minutes at 37~C.
The virus suspension Ls kept overnight a-t ~4C, The inactlvation ls checked by ino~ulation of prebrooded embryo-nakecl SPF chicken e~s wi-th thc lnactlvated vlru~ material and ~0 ~uhsQc,luQnk Lncuba~Lon.
rrhe Lnactivated A~F is diluted if necessary, w,ith PBS t 0,3% of formaline, dependent on t,he E:LD50 of each virus t~rpe, determined in the non-inacti~atecl A~F (at least 107' for all virusstrains).
To the virus suspension of the four st:rains 3.5% of Tween 80 are ac1dcd.

The inactivated virus suspension is mixed with an oil phase in the ratio of 6.5 parts of oil / 3.5 parts of virus fluid and emulsified, so that the average particle size of the aqueous phase is about 0.5 u.
The emulsifying .is carried out by means of an Ultra Turrax homogeniser or by means of passing the starting mixture through a colloid+mill.
The applied oily phase has the following composi-tion:
10 Marcol 5 2~ (white paraffinic Esso oil) 93,5 Arlacel A~ Arlacel 8 ~ or Span 8 ~
(mannide monooleate) 6,5 ~, The components of the oily phase are separately heated to l10C in an autoclave or the mixture is sterily filtrated.

Claims (4)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows: -

1. Infectious bronchitis virus strains of a novel sero-type indicated by means of the internal notation Utrecht.101, Utrecht.102, Drente.201, Limburg.501, Limburg.502, Brabant.801, Limburg.536, Overijssel.728 and Utrecht.121 and deposited with the Czechoslovak National Collection of Type Cultures of the Institute of Hygiene and Epidemiology in Prague under the nos.
CNCTC A 07/80, CNCTC A 08/80, CNCTC A 09/80, CNCTC A 010/80, CNCTC A 011/80, CNCTC A 016/80, CNCTC A 014/80, CNCTC A 015/80 and CNCTC A 013/80 and with the Collection Nationale de Cul-tures de Microorganismes d'Institut Pasteur, Paris, under nos. I.111, I.112, I.113, I.109, I.110, I.132, I.134, I.135 and I.133.

2. Infectious bronchitis virus strain, indicated by means of Utrecht.101 and deposited with the Czechoslovak National Collection of Type Cultures of the Institute of Hygiene and Epidemiology in Prague under the no. CNCTC A 07/80 and with the Collection d'Institut Pasteur, Paris, under no.
I.111.

3. Infectious bronchitis virus strain, indicated by means of Limbur.536 and deposited with the Czechoslovak National Collection of Type Cultures of the Institute of Hygiene and Epidemiology in Prague under the no. CNCTC A 014/80 and with the Collection d'Institut Pasteur, Paris, under no.
I.134.

4. Infectious bronchitis virus strain, indicated by means of Overjssel.728 and deposited with the Czechoslovak National Collection of Type Cultures of the Institute of Hygiene and Epidemiology in Prague under the no. CNCTC A
015/80 and with the Collection d'Institut Pasteur, Paris, under no. I.135.