NL7904021A - COMBINED VACCINE AND METHOD FOR PREPARING THIS COMBINED VACCINE AGAINST ADENO-LIKE VIRUSES DROP IN EGG PRODUCTION DROPS AND DISEASES CAUSED BY REO VIRUS. - Google Patents

Description

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Combined vaccine and method for preparing this combined vaccine against adeno-like viruses causing egg production decreases and reo virus-induced disease manifestations

The Applicants mention as inventors:

Peter Apontoweil »Johannes Anthonius Gerardus lok and Manfred Μ ·

Scratches,

The invention relates to a combined vaccine against infectious disease: in poultry and more in particular against egg production decreases caused by adeno-like viruses (hereinafter referred to as ALV) and disease phenomena in poultry caused by avian reo virus, and the preparation of this combined. vaccine and its application.

Reo viruses are known to cause various disease manifestations in poultry, such as arthritis / tenosynovitis, mycocarditis, hepatitis, hydropericardium, as well as digestive and / or respiratory disorders.

For example, Avian Pathology, 6, 271-284, 1977, lists several pathogenic microorganisms, which alone or in combination could cause viral arthritis / tenosynovitis 790 4 0 21 Λ s Ί j, such as Staphylococcus, Salmonella, " j | Pasteurella, Brysipelotrix, Mycobacterium avium, Bacterium arthro-! ! pyogenes, Escherichia venezuelensis, Streptobacillis monoliformis, I Mycoplasma gallisepticum, Mycoplasma synoviae and reo viruses, such as, for example, the reo virus isolate S 1133, the j L / z 671 virus, the SAM-1 virus, with reo viruses as the - main pathogens are considered. It is noted that • viral arthritis / tenosynovitis and other symptoms of t, g * v, j reo virus infection play an important role as a cause of aan-10 | significant economic losses in poultry farming in many parts of the world, [! 3 In addition, a method is proposed to establish a protection against viral arthritis by vaccination during! the growth period and subsequent transmission of maternal anti-i 15; bodies to offspring by means of vaccination with reo virus vaccines.

In this connection it has been found experimentally that imraunisa-! breeding animals only the first generation offspring ·; protected, but not. that of the second generation *: It was also known. from for example Avian Pathology no, 3, 7, 20: 407-419, that there would be a synergistic relationship between

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M »synoviae and reo virus, although not convincingly demonstrated, in the experimental cause of previously found disease manifestations in chickens, such as inflammation of the synovial membrane, the tendon sheath in particular in the tarsometatarsal region and myocarditis, 25 Also shown in previous publications, for example the one in

Neth.J. Fat.Sci., Vol. 3, No. 1 (1970) pp. 5-10, that reo viruses such as, for example, the L / Z 671 reo virus, are held primarily responsible for synovitis symptoms in broiler breeders.

30 Also, for example, from Poultry Digest, February 1978, p.

102, it appears that reo virus can cause growth inhibition and diarrhea in broilers and that oral infection with intestinal suspensions in chickens can result in growth inhibition. Although viral material propagated in eggs did not cause growth inhibition but oral pasting after oral administration, intraperitoneal injection of egg-cultured virus did produce growth inhibition. The 790 4021 - 2 -% τι '

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• observed signs of disease are associated with "a necessary apparent combination of infectious agents · | On the other hand, more recent publications show that the! the cause of rickets-like symptoms can primarily be sought in the occurrence of toxic substances formed by fungi such as Fusarium noniliforme Sheldon. 'For example, from Zbl.Vet.Med.B, 25 »29-44 (1978) it is known that during the last In recent years, recently observed rickets symptoms in broilers in primary f 10 i appear to be caused by toxic substances formed by the aforementioned fungus, which have been shown to be able to develop in poultry feeds from latent spores, preferably under the slightly humid conditions of the autumn and winter periods · 15 Although in previous years to · v. those in which the published symptoms occurred, the observed rickets symptoms could still be significantly combated by a therapy with vitamins, led the same therapy to the later found! the disease phenomena no more or much less to improve. It has therefore been attempted to reproduce the disease image identified later by administering specially selected feeds in this connection and by influencing and investigating more in depth the administration of vitamins and / or minerals, whereby by the application of suitable methods the occurrence of diseases caused by parasites or poultry pathogenic microorganisms such as reo viruses, Mycoplasma gallisepticum and Mycoplasma synoviae as well as other bacterial species has been excluded as far as possible »

It is concluded that, partly in view of the demonstrable harmful properties of the metabolic products, in particular from F · moniliforme, that have been found in the previous 30 years, there can no longer be any doubt that between the occurrence of fusari in feed and the diseases identified in broiler chickens. there must be a causal link without accurate and adequate identification of the actual metabolites responsible * 790 40 21 “3”

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For example, from Feedstuffs, May 8, 1978, biz. 95 and 96, J

shows that the real root cause of deza rickets appears-; in fact it has still not been found.

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It is also known, for example, from Avian Pathology 6,279, 5 that past attempts have been made to vaccination with reo virus vaccines against viral arthritis / tenosynovitis in poultry. It was certainly not obvious to an expert on the basis of # the literature discussed above to use reo virus vaccines for the benefit of 10! combating rickets-like symptoms or osteoperosis or * femur degeneration, which have become increasingly frequent in recent years and on all continents as a problem for which an effective and efficient solution is becoming increasingly urgent.

In Avian Pathology, 5, 261-272 (1976), connection 1 is already assumed: between the presence of adeno-like viruses: and part of the, in recent years increasingly observed, sharp decreases in the egg production and / or an increasing production of soft-shelled and / or scale-free eggs: mainly broiler breeders and chickens of semi-heavy laying-20 1 breeds; these drops are sometimes referred to in the literature as "egg drop syndrome". Also in Avian Pathology, jS, 405-413 (1977) a found relationship is described between the development of antibodies relative to a hemagglutinating virus, referred to as 127 virus and these previously described economically very damaging phenomena, while Avian Pathology 7, 35-47, 1978 confirms the previously suspected relationship between the occurrence of an adeno-like virus and the sudden drop in production described above; | in addition, a number of characteristic properties of the processing are described; Responsible virus type have been described, for example, the characteristic agglutination of chicken erythrocytes and the inability to be neutralized by antisera of the known adeno virus types.

! Luxembourg Patent No. 79154 describes a method of preparing a vaccine against egg drop syndrome * by starting from an AL virus, which is characterized by a specific hemagglutination and antisera. -reaction, In the description and the examples this virus, 790 4 0 21 _4_ I '-

j designated EDS-76 and BC-14 * I

This vaccine is prepared by propagating the new virus in cell cultures and preferably in an avian cell; culture, such as chicken embryo fibroblasts and embryonic 5 | liver or kidney cells or in embryonated duck eggs *: So many from Avian Pathology, 5, (ΐ97β) biz · 262, second; paragraph, as appears from, for example, p. 406 of Avian Pathology, (1977) "and p. *, lines 7-11 of Luxembourg Patent 79154; clearly, that a drop in egg production can be caused 1 0! by Nevcastle disease, infectious bronchitis, avian and cephalomyelitis, bird pox virus and combinations thereof *

In this connection it will be clear that for practical reasons poultry is often vaccinated simultaneously or briefly together with two or more vaccines against the aforementioned prevailing virus disease and that poultry is preferably vaccinated simultaneously, whereby the hitherto usual method arose from mixing on-site standard volumes of ready-to-use individual vaccines and administering the locally-formulated vaccine dose which, therefore, usually consisted of substantially double volume relative to that of the vaccine to be used separately. exact mixing is difficult or impossible to guarantee and problems can arise as a result of contamination with foreign contaminants *

An improved vaccine combination was therefore sought to adequately protect against the adeno-like viruses caused by egg production dams as well as against the symptoms caused by rheo viruses such as rickets-like symptoms, which would protect osteoperosis c * femur de-degeneration.

Surprisingly, it has now been found that the pervasive bone disorders of widespread prevalence, such as rickets-like symptoms, osteoperosis c * q * femur degeneration in poultry, and more particularly in broilers, often accompanied by growth inhibitions and diarrhea, can be controlled by vaccination of broiler dams and their offspring using inactivated vaccines derived from certain avian reo viruses. In particular, it has been found that combinations of rickets-like symptoms, osteoperosis c * q * femur degeneration and egg production declines can be particularly effectively controlled by vaccination of broiler breeders and their offspring using combined inactivated vaccines derived from certain avian reo viruses and adeno-like viruses.

According to another feature of the invention, a method has been found for the preparation of a combined vaccine, wherein an inactivated ALV fluid is combined with an inactivated reo virus fluid.

It will be southern that the invention also relates to inactivated combination vaccines derived from at least one inactivated ALV fluid and at least from an inactivated reo virus fluid and also from vaccines derived from more than these two mentioned virus types. According to the invention, an inactivated ALV-containing liquid is substance prepared in a manner known per se, for example by breed the desired virus in fertilized duck eggs or on j. a cell culture of duck cells, collecting the virus formed in a suspension of titer customary for this vaccine type, and inactivate it, suspend the virus in a suitable buffer solution and homogenize it, and mix it with an inactivated virus liquid derived from a reo virus, which has been prepared, for example by further cultivating an appropriate reo virus in a fertilized chicken egg or in an I SPP chicken embryo fibroblast cell culture, collecting the cultured virus in a suspension of an appropriate titer and inactivating, suspending in an appropriate buffer solution and al or not to homogenize.

For the preparation of the inactivated ALV liquid, 1 is preferably started from a passaged virus strain 127 which was deposited with the Collection d'Institut Pasteur, Paris under no.

1,090, although other antigenically related ALV-inducing egg production decline may also be used successfully.

The raw virus-containing starting fluid is preferably prepared from an L / z 671 virus which is propagated in a fertilized chicken egg or in an SPF chicken embryo fibroblast cell culture. This L / z 671 virus has been deposited with the Collection d'Institut Pasteur, Paris under no. 1.092.

790 4 0 21. The £ ALV-containing starting fluid is preferably prepared by culturing an adeno-like virus in duck eggs or on a cell culture of duck embryofibroblasts, for example, in a ntonolayer roller bottle culture or culture of cells attached in 5 solid inert carriers · - To this end, the starting cells are suspended in a culture medium, consisting of at least 75-95 parts of Eagle medium with 5-15 parts of calf serum or otherwise, an aqueous sodium bicarbonate solution of 2-4% and 1-5 10 · parts of a solution or suspension of one or more antibiotics such as penicillin G, streptomycin and natamycin When the cell culture is almost closed, the culture fluid is separated and infection with the virus suspension takes place, incubation for 0.5-2 hours at 37 ° C t * b # v * the attachment of the virus and adding maintenance medium consisting of Eagle * s medium with 2-10% half serum, 4-6oto tryotose phosphate broth (30g / l) j and the above geno Emde additives · Harvesting of the virus-containing medium and cells is done 0.5-2 days after the appearance of the CPE.

The cell culture is infected with an ALV suspension of an activity of> 30 x 10δ TCID 50 / roller culture bottle. It should be noted in this regard that the use of cell suspension cultures of the aforementioned method has proved unsuitable.

According to a suitable embodiment of the method of preparing a suitable reo virus containing liquid, the virus is grown in an SPF1 chicken embryo fibroblast culture, for example in a monolayer roller bottle culture or a culture of cells adhered to solid inert carriers and preferably a monolayer roll-bottle culture, or in pre-hatched chicken eggs free of feathering 30 _ against avian reo viruses ·

Preferably, a cell culture is obtained, obtained by suspending the starting cells in a culture medium, consisting of at least 75-95 parts of Eagle medium with 5-1 or 5 parts of calf serum or not, an aqueous sodium bicarbonate solution of 2-4% and 35 1- 5 parts of a solution or suspension of one or more antibiotics such as penicillin G, streptomycin and natamycin, and separating the culture fluid when the cell culture monolayer is almost closed · 790 40 21 c -----: ”'i

The antibiotic solution contains eg 2-3 x 104 units penicillin, 20-30 mg streptomycin and 0.4-0.8 mg natamycin per ml »

After infection with the virus suspension, incubation is carried out for 0.5-3 hours at 37 ° C for the adhesion of the virus and maintenance medium consisting of Eagle's maintenance medium with 4-6 parts of calf serum, 3-6 parts of tryptose phosphate broth is added. (30 g / l) and the aforementioned additives, and the virus-containing medium and cells are harvested after 0.5-3 days after the appearance of the CPE.

The cell culture is preferably infected with a rheo; 6 δ virus suspension of an activity of 2x10 - 10 TCID 50 / roll culture bottle and preferably ^ 30 x 10 ^ TCID 50 / roll culture bottle ·

Inactivation of the starting virus liquid can be effected with the usual inactivators, such as, for example, using β-propiolactone, whether or not in combination with a stabilizer, or formaldehyde. For example, β-propiolactone can be added at a concentration of 0.05 to 0.25% by weight (calculated by weight of the aqueous phase) to the buffered virus-containing liquid and this liquid thereafter for 1-2 hours and preferably for 90 minutes are incubated at about 37 ° C. After this, the virus! liquids homogenized if necessary in the usual way · The water phase can subsequently also be used as a preservative! added, such as, for example, thiomersal or formalin in buffer · i For the preparation of the combined vaccines, "25 J 50 to 200 ml of an ALV-containing liquid is inactivated, whether or not" homogenized and together with 1 50-300 ml of a reo virus containing liquid, which has been pre-inactivated and whether or not homogenized is added to 500-700 ml of an oil phase of the above! said composition.

The volume ratio between the ALV-containing liquid: and the reo virus-containing liquid can vary from 4: 3 to 1; 6 and i! preferably about 1: 3 · 1 The volume ratio between the water phase formed by the. both virus fluids and the oil phase can vary from 1: 1 to 3: 7 35 1 and is preferably approximately 7: 13 · I- It was found that the water phase was stirred with vigorous stirring and / or 790 40 21 _ 8 ”! : 1 homogenization to the oil phase must be added to obtain the desired, stable and low viscosity final emulsion

The oil phase contains 2-20 wt% (calculated on weight of oil phase) emulsifier and preferably 2-15 wt% Arlacel A, Arlacel®30 or 5; Span ^ 8o and 0.2-4% Tween * 3 80 · The components of the oil phase are preferably individually autoclaved to at least 110 ° C; or sterile filtered as a mixture

To obtain a suitable combination of vaccines with * the desired favorable properties, it is necessary to start from I. 2 10; an ALV liquid with an activity of at least 10 KA units 3 and preferably 10 HA units and of a reo virus liquid with a titer of 10 ^ - 10 ^ ΞΙΰ ^ φ / πύ. and preferably> TOS, 0BID ^ 0 / ml.

The vaccines thus prepared are administered to slaughter-chick parent animals, preferably from 10 to 20 weeks old, before they are laid.

It will be clear that the immunological response occurring with the combination vaccines according to the invention for each of the virus components was found to be at least of the same order of magnitude as that caused by the individual virus vaccines, in no way by a One could predict or predict an expert, because this immunological response of the combination vaccine to one or both of the individual mono-vaccines contains a number of undesirable interactions between the virus fluid components which cannot be ruled out in advance. or with any of the auxiliary chemicals used, could drop to an unacceptable level · For example, it could be expected that applied 3-propiolactone would remain inactive for longer due to slower degradation in the mixture * 30 The use of the combined vaccine cf * the invention has the advantage that a smaller volume at a time, resulting in less space serious irritations, and a smaller amount of foreign chemicals need to be administered or fewer separate vaccinations are required, while moreover avoiding the aforementioned problems with regard to mixing and the occurrence of foreign contaminants * 790 4 0 2 1 - 9 - «✓ "1" '! "i

The invention will be elucidated on the basis of the following! examples

Example 1

Preparation combined ALV. ./reo virus vaccine.

5 a) Culture Adeno-like virus (passaged 127 strain)

Duck eggs incubated from 12/13 days are incubated in (Bellco) roller culture bottles of EBF in Eagle medium containing 10¾ calf serura medium, or 22% by weight NaHCO2 and 2% by volume anti-biotics solution consisting of penicillin G »streptomycin and 10% natamycin

The medium is aspirated when the cell cultures are almost closed. The cultures are then inoculated with the passaged 127 virus suspension incubated at 37 ° C for 1 hour and then Eagle maintenance medium containing 5¾ calf serum, 4 vol · ¾ tryptose phosphate 15 broth (30g / l), 0.22 wt * NaHCO 3 and 2 vol. * of the aforementioned anti biotics solution added again ·

The ALV containing medium containing the cells is harvested 1 day after the appearance of a CPE and the cells almost detach from the glass wall. The virus suspension is frozen at -20 ° C or below 20 and stored for vaccine production. · The HA titre and TCID are determined from this suspension. B) Culture reo virus (strain L / Z 671) I Chicken eggs incubated for 10 days in (Bellco) roll-culture bottles, cell cultures of KEF are applied in Eagle medium with 10 * calf {25: serum containing 0.22 wt * NaHCO 3 and 2 vol * antibiotic solution; tend penicillin G, streptomycin and natamycin.

j The medium is aspirated as the cell cultures (monolayers) | are almost closed · The cultures are then inoculated with L / z 671 virus suspension and then supplemented with Eagle maintenance medium, in which 4 vol. * calf serum, 4 vol. * tryptose phosphate broth (30g / l), 0.22 wt. * NaHCO3 and 3 vol. * Of the aforementioned antibiotic solution.

The reo virus containing medium containing the cells is harvested: 1 day after the appearance of a CFE.

35 _ The virus suspension is frozen at -20 ° C or lower 790 40 21 -of temperature and stored for vaccine production · TCID ^ q of this suspension j is determined ·! c) Treatment of virus suspension

The frozen virus suspensions are thawed and placed in a water bath; inactivated with 0,05-0,25 β-propiolactone for 90 minutes at 37eC # The suspensions are stored overnight at + 4 ° C. The inactivation is checked by checking the formation of CPE on 3EF o £ ISF and subsequently the performance of a haem absorption test · In addition: pre-hatching SPF chicken eggs with the inactivated vi-10 | russel material incubated and subsequently checked

The virus suspensions are homogenized with an Ultra Turrax homogenizer. Optionally, the suspension is diluted with P3S + · 0.3¾ formalin, depending on TCID4 of the reo virus suspension and HA-titer of adeno virus suspension. Reo virus suspension and adeno— virus suspension. are then mixed in the ratio 6: 4 and used for the emulsion preparation * d) Preparation of emulsion

The virus suspensions are mixed with the oil phase in the ratio of 6.5 parts oil / 3.5 parts virus liquid and emulsified, so that the particle size of the water phase is approx. 0.05-0.5 µ, eg by injecting the virus suspension in the oil phase with simultaneous homogenization with an Ultra Turrax homogenizer or by passing the starting mixture through a colloid mill

The oil phase used has the following composition: 25 Harcol ^ 52 (white paraffinic Ssso oil) 93, δ vol.%

Arlacel®A or Arlacel® 30 or Spai ^ 80 (mannide monooleate) 6.0 vol%

Tweeri ^ 80 (polyoxyethylene 20 sorbitan monooleate) 0.4 vol »%

The oil phase components are autoclaved separately at 110 ° C or sterile filtered as a mixture ·

A stable emulsion was obtained, tested by: 1) pipetting drops of emulsion on a water surface immediately after it was filled, whereby the drops do not spread but remain intact.

2) Leave for 4 weeks at 37 ° C, with no water phase separating *

The vaccine obtained is used at a dose of -5 ml -5 ml per animal -intramuscularly-in-breast or -leg muscles -or -subcutaaa- 790 4021 / in the disease * From 8 days after inoculation, haemagglutination inhibitors ALV antibodies detectable. Precipitating reo virus antibodies can be detected from 10 days of inoculation.

Example 2 Preparation of combined ALV / reo virus vaccine * a) Culture Adeno-like virus (passaged 127 strain) Duck incubated eggs for 9 days are inoculated with the passaged 127 virus strain. After incubation at 37 ° C for 7 days, the AAV (amnion allantois liquid) is harvested. The AAV then contains approximately 1024 HA one to ten days. The virus suspension is then frozen at -20 ° C and saved for vaccine production.

| b) Culture reo virus (strain L / Z 671) | 10 days incubated SPF eggs are inoculated with LZ 671 seed virus. After incubation for 76 hours at 37 ° C, the AAV (amnion allantoic liquid

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15 'fabric). The AAV then contains approximately 10 * EID5Q / ml. The virus suspension is frozen at -20eC or lower temperatures and stored for vaccine production, c) Treatment of virus suspension

The frozen virus suspensions are thawed and inactivated with 20 I of formalin. (0.3%). The suspensions are stored overnight at + 4 ° C. The! inactivation is monitored by monitoring the formation of CPE

; or KEF or control of embryonated SPF chicken eggs after inoculation 1 with the inactivated virus material.

i The virus suspensions are homogenized with an Ultra

25! Turrax homogenizer. Optionally, the suspension is diluted with PBS

| + 0.3% formalin, depending on TCID ^ 0 or EID ^. j The virus suspensions of ALV and reo virus become afterwards! mixed in the ratio 1: 4 and used for emulsion preparation, j d) Preparation of emulsion The virus suspensions are mixed with the oil phase in the ratio! 6.5 parts of oil / 3.5 parts of virus fluid and emulsified so that the particle size of the water phase is about 0.05-0.5p. Emulsification can be carried out e.g. on an Ultra Turrax homogenizer or by means of passing the starting mixture through a colloid 3 mill.

790 4 0 2 1 ~ 12 "

The oil phase used has the following composition: Marcol ^ 52 (white paraffinic Ssso oil) 91 »63»; Arlacel Ji ^ f'Arlace ^ ^ 80 or Spaxi ^ O (mannide monooleate) 6f0 * * Tveen ^ O (polyoxyethylene 20 sorbitan monooleate) 2 »4% 5 The components of the oil phase are autoclaved separately at 110®C or the mixture sterile filtered.

i i 790 4 0 21 -13 -

Claims (2)

1 · Combined inactivated vaccine against at least adeno-like virus-induced egg production decreases and roe virus-induced rickets-like phenomena, osteoperosis c * q * 5 femur degeneration * 2 * Combined inactivated vaccine according to claim 1, characterized in that it is derived of at least one adeno-like virus-containing liquid, obtained by propagating a passaged virus strain 127 * 10 3 * Combined inactivated vaccine according to claim 1, characterized in that it is derived from at least one reo virus; The liquid obtained by propagating the L / z 671 virus * 4 * Combined inactivated vaccine according to claims 1-3, characterized in that it contains an oil phase which as essential constituents selected at least one of the components from the group consisting of light mineral oils (FDA quality), * vegetable oils and naphthenic mineral oils, and one or more suitable emulsifiers of alkylene oxide and / or of hexahydric 20. alcohols and / or of higher natural fatty acids (c 0 -C2j) derived, non-ionic surfactants, such as esters and ester ethers, contains * 5 * Combined inactivated vaccine cf * Claims 1-4, characterized in that the volume ratio between the adeno-like virus-containing liquid and the reo virus-containing liquid can vary from 4: 3 to 1: 6 and preferably about 1: 3 * 6 * Combined inactivated vaccine cf * claims 1-5, characterized in that the volume ratio between the aqueous phase formed by the two virus fluids and the oil phase can vary from 30 1: 1 to 3: 7 and is preferably 7: 13 * 7 * Combined inactivated vaccine according to claims 1-6, characterized in that the oil phase contains 2-20 wt.% emulsifier and preferably 2-1 5 wt.% Arlacel. or Arlace ^ or O or Span 8 (i®en 0.2-4% Tween 35 8 * Process for the preparation of a combined inactivated vaccine against adeno-like virus-induced egg production 790 4 0 2 1 - 14- «V drops and by reo virus caused rickets-like symptoms, osteoperosis c * q * femur degeneration, in which a reo virus containing fluid was inactivated, whether or not homogenized and together with an adeno-like virus containing fluid * which was inactivated and whether or not homogenized added to an oil phase, the essential constituents of which are at least one of the components selected from the group consisting of light mineral oils (PDA grade), vegetable oils and naphthenic mineral oils, and one or more suitable emulsifiers based on alkylene oxide and / or contains hexahydric alcohols and / or higher natural fatty acids () derived nonionic surfactants, such as esters and ester ethers, with or without addition to the aqueous phase a preservative may be added * 9. method according to claim 3, characterized in that β-propiolactone (whether or not in combination with a stabilizer) or formaldehyde is used as inactivator for both virus liquids * 10 * method according to * claim 9, characterized in that β-propiolactone is used at a concentration of 0.05-0.25 v. 2 or 20 formaldehyde at a concentration of 0.02 to 0.5 g ^ sw * ^ (based on weight of the water phase) 11. Method according to claim 9 and 10, characterized in that incubation is effected for 1-2 hours at 37 ° 0 * 12 * Method according to * claim 8, characterized in that 5 5 7 5 25 is started from a reo virus liquid with a titer of 10 * -10 1 13 * Method cf * conclusion 8, characterized in that of an adeno .... 2-like virus fluid with an activity of at least 10 HA units is added. 790 4 0 21 -is-