JPS6220964B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a combination vaccine that can be immediately administered against poultry infections, and more particularly against diseases characterized by drastic drop in egg production caused by Newkatsur disease and adeno-like viruses. The present invention further relates to the production of this combination vaccine and to a method for producing vaccine components that prevent this reduction in egg production. Niukatsuru disease (hereinafter abbreviated as ND) is one of the most serious respiratory diseases in poultry.It is caused by a virus and causes high mortality in poultry of all ages. sell. Several vaccines have been developed for the control of P. elegans, and it has been found that these vaccines are preferably administered in their inactivated form, as this provides the highest degree of safety. Furthermore, the use of live vaccines sometimes results in the transmission of the inoculated virus from vaccinated poultry to unimmunized susceptible poultry. Various raw NDs that are relatively safe and have sufficient immunity
Vaccines are being developed (e.g. British patent no.
1510100), there is nevertheless a clear need for an inactivated ND vaccine. Because respiratory diseases in poultry are often the result of more than one source of infection, combination vaccines have been developed. For example, GB 2798835 consists of a combination of a vaccine against infectious bronchitis and an ND vaccine (both components derived from live viruses). Furthermore, published Dutch patent application no. Haemophilus gallinarium (Haemophilus gallium) in culture medium
A combination of a "killed vaccine against infectious rhinitis (coryza)" obtained by cultivating a strain of A. gallinarium and then inactivating the bacteria, a killed vaccine from infectious bronchitis virus, and a killed ND vaccine. have characteristics. Several documents [e.g. Avian Dis.7 (1963) 106
~122 pages, Am.J.Vet.Res.17 (1956) 294~298
Avian Dis. 11 (1967) pp. 399-406 and
Virology 33 (1967) pp. 598-608] discloses that the activity of each component can be reduced or completely eliminated by mixing live viruses due to their mutual inhibition. Adeno-like virus (hereinafter referred to as ALV)
The presence of medium heavy rays has been observed to increase over the past few years, especially medium heavy rays.
Some association between the rapid decline in egg production and/or increased production of soft-shelled and/or shell-less eggs observed in broiler mother hens and chicks of the Lay breed has already been reported [ Avian Pathology 5
(1976) pp. 261-272]. This literature sometimes refers to such symptoms as “low egg production syndrome” (egg production syndrome).
It is also referred to as "drop syndrome" in other literature [Avian Pathology 6 (1977) pp. 405-413].
The study reveals a relationship between the production of antibodies against a hemagglutinising virus called 127 virus and the economically disadvantageous symptoms described above. Other reports (Avian
Pathology 7 (1978) pp. 35-47] confirmed the postulated relationship between the presence of this adeno-like virus and the above-mentioned reduction in egg production. Furthermore, some properties of the virus types in question are described, such as the characteristic agglutination of chick red blood cells and the inability of antisera to neutralize known adenovirus types. Luxembourg Patent No. 79154 discloses a method for the production of an anti-"hypopeid syndrome" vaccine starting from ALV, which is characterized by specific clotting and antiserum reactions. In its detailed description and examples this virus is referred to as EDS-76 and BC-14. This vaccine can be administered in tissue culture, preferably in avian tissue culture such as chick embryonic fibroblasts and embryonal hepatocytes or kidney cells, or in embryonated duck eggs. It is produced by culturing in References [Avian Pathology 5 (1976) p. 262,
Section 2; Avian Pathology 6 (1977) p. 406; and Luxembourg Patent No. 79154, p. 7
From the description in lines 11 to 11, it can be concluded that decreased egg production can be caused by avian and cefalomyelitis, avian spondylitis, avian and cefalomyelitis, birdpox viruses, and combinations thereof. It's obvious. Therefore, from a practical standpoint, poultry will often be vaccinated with two or more vaccines against the relatively commonly occurring viral diseases mentioned above, either simultaneously or at short intervals. In particular, poultry will be simultaneously vaccinated against snail disease and ALV-induced egg production loss. The conventional method for such co-administration consists of mixing ready-to-use standard doses of separate vaccines and administering a single dose of vaccine formulated on a spot, so that one The batch size is substantially two volumes and each viral load is diluted compared to a separately administered vaccine. Furthermore, accurate mixing cannot be guaranteed and on the other hand problems can arise due to foreign infectious agents. Therefore, the object of the present invention is to provide a ready-to-use compound which is effective against the disease of the snail and also against the reduction in egg production caused by ALV, and which meets the stringent requirements of veterinarians in many relevant countries today. This is the discovery of a vaccine. Surprisingly, it has been found that a combined ready-to-administer vaccine can be produced by mixing inactivated ND virus fluid and inactivated ALV fluid. As used herein, the terms "readily administrable,""immediatelyadministerable,""ready to use," and "immediately administrable""combined inactivated vaccine" all mean: . That is, the combination vaccine of the present invention is a vaccine in a completed form, that is, it is a combination vaccine that does not need to be mixed or added to each other when administered to a target animal. This is in contrast to prior art vaccines, where previously the two vaccines had to be injected separately or at least mixed to a standard volume immediately before use. It is. The invention also relates to an inactivated conjugate vaccine derived from at least one NDV fluid and at least one inactivated ALV fluid, optionally from two or more of the above-mentioned virus types. That will be understood. In the method according to the invention, the inactivated NDV-containing solution is produced in a manner known per se in principle, for example, by culturing the desired virus in fertilized chicken eggs and culturing the cultured virus with a titer typical for vaccines of this type. The virus is collected in a liquid, inactivated, suspended in an appropriate buffer solution, homogenized, and mixed with an inactivated virus solution derived from ALV. However, this
The inactivated virus solution derived from ALV is prepared by culturing the virus in a cell culture of fertilized duck eggs or duck cells, and collecting the resulting virus in a solution of a titer common to this type of vaccine. ,
It is produced by inactivation by methods known per se. This NDV-containing solution can be produced using various almost non-toxic or virtually non-toxic (slow-developing or rapid-developing (lentogenic)
ND viruses with excellent immunity such as LaSota or Hitchner virus (lentogenic) or Herts 33/56 (velogenic) virus can be used. Specific examples of virus strains that can be used are: Institut fuer Medizinische
Mikrobiologie, Infections und Seuchenmedizin
der Ludwig Maximilians Univorsitaet,
P/76/5, P/76/ obtained from Muenchen
4, and P/76/3 strain; American Type
Culture Collection, Rockville, Mayland, USA
VR-108, VR-107, VR-109, obtained from
VR-699, VR-623 strain; Centr.Vet.Labs.
Obtained from Weybridge, Great Britain
Queensland V4 strain or Collection d′ Institut
L/deposited in Pasteur, Paris as I.091
Z258P virus. For the production of this inactivated ALV liquid component, Collection
No. d′ Institut Pasteur, Paris. The passage virus 127 strain deposited as 090 may be preferably used, but other ALV virus types that cause reduced egg production due to antigenicity rather than morphology may also be fully utilized. This virus strain I.090 is preferably cultured in duck eggs. The production of this inactivated ALV solution is preferably carried out as described above.
Although starting from viruses derived from passage virus strain 127 as shown in the Avian Pathology literature, other ALV types that cause egg-laying diseases may also be fully utilized. The production of this conjugate vaccine involves inactivating and optionally homogenizing 150-250 ml of a buffered NDV-containing solution, along with 100-200 ml of a previously inactivated and optionally homogenized buffered ALV-containing solution.
Add to 700ml of oil phase. The essential ingredients of this oil phase are at least one component selected from the group consisting of light paraffinic mineral oils (FDA quality), vegetable oils, and naphthenic mineral oils, and one or more suitable emulsifiers, such as alkylene oxide adducts and (or) hexahydric alcohol and (or) higher natural fatty acids (C ₁₀
~ _C20 ) and nonionic surfactant compounds such as esters and ester-ethers derived from C20). Examples of these include mannide monooleate (Span 80, Arlacel)
80, Aracel A] and polyoxyethylene (20) sorbitan monooleate (eg Tween 80). This buffered NDV-containing solution and the buffered
The volume ratio with the ALV-containing liquid may vary from 3:4 to 5:1, but is preferably about 3:2. The volume ratio of the aqueous phase and the oil phase consisting of both virus fluids can vary from 1:1 to 3:7, but is preferably 7:13. This aqueous phase is stirred vigorously to obtain the desired low viscosity, stable liquid finished emulsion.
It has been found that it must be added to the oil phase and/or while homogenizing. The oil phase contains 2 to 20% by weight (relative to the weight of the oil phase) of emulsifier, preferably 2 to 15% by weight of Aracel A or Aracel 80 or Span 80 and 0.2 to 4% of Towin 80. Each component of this oil phase is preferably heated separately in an autoclave to at least 110°C or sterilized as a mixture. Inactivation of both starting virus fluid components can be carried out using customary inactivating agents, for example β-propiolactone, optionally in combination with stabilizing agents or formaldehyde. Preferably, β-propiolactone is added to the buffered ND-virus-containing solution at a concentration of 0.05 to 0.25% by weight (based on the weight of the aqueous phase), and the solution is heated to about 37° C. for 1 to 2 hours. Preferably, the buffered ALV solution is incubated for 90 minutes while the buffered ALV solution is inactivated with formaldehyde, preferably at about 20° C. for about 20 hours at a concentration of 0.02-0.5% by weight. Next, if necessary, the virus solution is homogenized by a conventional method. A preservative such as thiomersal or formalin in a buffer solution may then optionally be added to the aqueous phase. In order to obtain a conjugate vaccine that gives the desired preferred titer after administration, an NDV starting solution with a titer of 10 ^9.8 to ^{10 10 .5} EID50/ml, preferably 10 ¹⁰ EID50/ml must be used ^. , and at least 10 ² HA units,
Preferably an ALV fluid with an activity of 10 ³ HA units should be used. It is clear that the preferred properties of the conjugate virus vaccine provided by the present invention could not have been foreseen at all by those skilled in the art. The reason is that there are several undesirable reactions that cannot be reliably excluded by those skilled in the art. The activity of one or both viral components in the final vaccine may be reduced to an unfavorable level due to unfavorable interactions between the viral fluid components or between these fluid components and one of the auxiliary chemicals utilized. can already decrease. For example, those skilled in the art know that added β-propiolactone acts for too long and excessively inactivates one viral component; It certainly cannot be excluded that it is necessary for the inactivation of the components of The use of a combination vaccine according to the invention requires a smaller volume for administration (e.g. 0.5 cm ³ instead of 1 cm ³ ) compared to prior art vaccines produced by mixing the constituent vaccines on a spot, and therefore It is advantageous in that it does not cause local irritation. Furthermore, less amount of chemical product foreign to the body is required for administration. Moreover, the above-mentioned problems caused by mixing and the introduction of foreign infectious agents are also avoided. Vaccines produced in this way are usually prepared 10 times before spawning.
Administered to chickens ~20 weeks of age. The present invention further relates to a method for producing a suitable ALV-containing stock solution, which involves injecting adeno-like viruses into duck eggs or cultures of duck embryo fibroblasts, such as monolayer rolling bottle cultures or cell cultures attached to solid inert supports. It consists of culturing in According to a preferred embodiment of the invention, the single-layer rolling bottle culture of duck embryo fibroblasts is carried out by submerging the raw material cells in at least 70 to 90 parts of Eagle's medium (Eagle's medium).
medium), optionally 5-15 parts calf serum, 2-4
% sodium bicarbonate solution and 1 to 5 parts of a solution or suspension of one or more antibiotics such as penicillin G, streptomycin, and natamycin, the culture medium being suspended in a cell culture medium. is separated when it is almost finished. The cell culture was infected with virus suspension and incubated at 37°C for 0.5-2 hours to allow virus attachment, followed by 2-10% calf serum, 2-10 parts tryptose phosphate broth (30 g/ ), a maintenance medium consisting of Eagle's medium containing 10-20 parts of bovine amniotic fluid (BAF) and the additives mentioned above is added, and the virus-containing medium and cells are harvested 0.5-2 days after the appearance of CPE. The cell culture is inoculated with 30×10 ⁶ TCID ₅₀ /rolling culture bottle of active ALV suspension. It should be noted in this connection that cell suspension culture has been found to be unsuitable for the manufacturing methods discussed so far. Collection d′ Institut is involved in the production of this vaccine.
Passage virus strain 127, deposited with Pasteur, Paris under no.I.0.90, is preferably used, and this strain is preferably cultured in duck eggs. While it is true that Luxembourg Patent No. 79154 generally discloses the production of vaccines against "decreased egg production syndrome", it is true that the production of vaccines with acceptable high titers according to the present invention is not possible on a commercial scale. No suitable manufacturing method is mentioned or even suggested in the specification. In this regard, the Luxembourg patent specification, page 2, lines 28-30 clearly states that the virus (i, a.)
It is pointed out that it has been described that the method can be cultured on chick embryonic fibroblasts. However, it has been found that such a method does not quite give the desired economically demanding results. 6th page 29~
Although page 35 discloses the use of culture of immature chick hepatocytes, it is still extremely difficult and therefore an expensive manufacturing method to cultivate immature hepatocytes on a commercial scale. It is believed that it will never yield the desired economically required performance. The invention is illustrated by the following examples. Example 1 a Culture of adeno-like virus (passage 127) in rolling culture bottles from Bellco containing 0.22% by weight NaHCO ₃ and 2% by volume of an antibiotic solution containing penicillin G, streptomycin and natamycin. Cell cultures from duck eggs incubated for 12-13 days were produced on DEF (Duck Egg Fibroblasts) in Eagle's 10% calf serum medium. Aspirate this medium when the cell culture is almost complete. Therefore, the culture was inoculated with a suspension of 127 subcultured viruses, kept constant at 37°C for 1 hour, and supplemented with 5% calf serum and 10% tryptose by volume.
Phosphate broth (30g/), 10% by volume
of bovine amniotic fluid (BAF), 0.22% by weight NaHCO ₃ and 2% by volume of the above-mentioned antibiotic solution. When CPE appears and the cells almost reach the glass wall, the ALV-containing medium and cells are harvested the next day. This virus suspension is frozen at -20°C and stored for vaccine production. HA titer of this suspension and
Measure TCID ₅₀ . b ₁ Culture of NDV SPF eggs after 11 days of incubation were inoculated with L/Z258P virus. This culture was kept constant at 37°C for 72 hours, and the AAF (Amnion Allantonic Fluid) was harvested. AAF now contains approximately 10 ¹⁰ EID ₅₀ /ml. This virus suspension is frozen at -20°C or below and stored for vaccine production. b ₂ Herts in SPF eggs incubated for 11 days
33/56 types of viruses were inoculated. The AAF is harvested after incubation at 37°C for 24-48 hours, at which point it contains approximately 10 ¹⁰ EID ₅₀ /ml. The virus suspension is frozen at -20°C or below and stored for vaccine production. c. Treatment of virus suspension Thaw the frozen virus suspension and inactivate it with 0.05-0.25% β-propiolactone in a water bath at 37° C. for 90 minutes. Store the suspension overnight at +4°C. This inactivation is performed by DEF (duck egg fibroblast) or CEF.
(Chicken Egg Fibroblasts)
observed by the formation of CPE'S, followed by a hemabsorption test. Furthermore, pre-incubated chicken eggs were inoculated with an inactivated virus solution, kept at constant temperature, and used as a control. This virus suspension is homogenized using an Ultra Turrax homogenizer. If desired, this suspension may be mixed with PBS + 0.3% depending on the EID ₅₀ of the AAF and HA titers of the adeno-like virus suspension.
dilute with formalin. After that, this NDV-
AAF and adeno-like virus suspension are mixed in a ratio of 6:4 and used for emulsion production. d Manufacture of the emulsion by mixing the virus suspension with an oil phase in a ratio of 6.5 parts oil to 3.5 parts virus liquid, for example by injecting the virus suspension into the oil phase and simultaneously homogenizing it in an Ultra Teurax homogenizer. The aqueous phase is emulsified so that the average droplet size is about 0.05 to 0.5μ. The oil phase is composed of: Marcol 52 (white paraffinic Esso oil) 93.6% by volume Aracel A or Aracel 80 or Span 80 (mannide monooleate) 6.0% by volume Toin 80 (Polyoxyethylene-20-sorbitan monooleate) 0.4% by volume Each component of this oil phase was separately placed in an autoclave.
Either heated to 110°C or sterilized as a mixture. Stable emulsions were obtained and tested as follows: 1 Immediately after emulsification, a few drops of the emulsion were transferred onto the surface of the aqueous phase, during which time the drops did not spread and remained intact; 2 37°C. The aqueous phase does not separate even after being left for 4 weeks. The final concentrations of the emulsions thus produced were 21% NDV containing buffered solution and 14% NDV.
A buffered solution containing ALV127. The resulting vaccine was administered intramuscularly into the pectoral or leg muscles or subcutaneously into the neck at a dose of 0.5 ml per animal. On the 8th day after vaccination, hemagglutination inhibiting compound (hemagglutination inhibiting compound) was administered.
anticompounds) were recognized. Although anticoagulation is an indicator of immunocompetence, the effectiveness of vaccination in the above test was determined by the fact that the presence of relevant antibodies (i.e. anticoagulant antagonist compounds) could be observed 8 days after the vaccination. It was recognized by. Example 2 The method of Example 1 was repeated, but with the following changes: a.
An ALV-containing solution is produced by inoculating duck eggs that have been incubated for days with the passage 127 virus strain. 7 at 37℃
AAF (Amnion
Harvest allantoic fluid). At that point
AAF has a HA titer of approximately 1:1024. This virus suspension is subsequently frozen at -20°C and stored for vaccine production. b Treat this NDV suspension as described in Example 1, section c, at 0.02-0.5% for 20 hours at 22°C.
After inactivating this NDV suspension with formaldehyde, the NDV-liquid and ALV-
The liquids are mixed in a 4:1 ratio and used for emulsion preparation as described in Example 1, section d. Example 3 a An ALV-containing liquid was prepared according to the corresponding steps of Example 2. b An NDV-containing liquid was prepared according to the corresponding steps of Example 1. c This NDV suspension is processed in the same manner as in Example 1, and the ALV suspension is processed as in Example 2. This processed NDV-AAF and
Mix ALV-AAF at a ratio of 5:1. d This mixed virus suspension was added to the oil phase in a ratio of 6.5 parts of oil phase:3.5 parts of virus-containing liquid, mixed therewith, and passed through a colloid mill to produce an emulsion. The applied oil phase has the following composition: Marcol 52 91.4% by volume Aracel A or Aracel 80 or Span 80 8.0% by volume Towin 80 0.6% by volume Example 4 ALV-containing liquid and NDV-containing liquid corresponding to Example 1 respectively It was manufactured according to the following steps. this virus
After the suspension was treated as in Section c of Example 1, the NDV-AAF and the ALV-containing liquid were mixed in a ratio of 4:3 to produce an emulsion as described in Section d of Example 1. However, here the oil phase and the mixed virus-containing liquid were mixed in a ratio of 6:4, and the oil phase used therein was that used in section d of Example 1. Example 5 A combination vaccine produced according to Example 4 was tested as follows. Eight-week-old SPF chicks that had not been previously vaccinated were immunized with this combination vaccine. In this case, chicks were inoculated with 0.5 ml of the combined vaccine per chick by intramuscular (thigh) injection. Injections may be made into the chest muscle or even subcutaneously. The HAI titer, expressed as the number of HAI (anticoagulant) units in the serum from this immunized animal, is the antibody level seen in animals immunized with the corresponding single vaccine of the composition being compared under identical conditions. There was no significant deviation from the The attached figure shows a progressive curve obtained by plotting the found HAI units and HAI levels against time in a 2 ^× logarithmic scale. Significantly higher titers of this NDV-HAI unit were observed for animals that had been pre-immunized before being treated with live NDV vaccine (as is usually done in the field). In this animal, for example, approximately 10 ^16.0 to ^{10 17.0 NDV} -HAI
4-8 after immunization with combined NDV-ALV vaccine
Detected in weeks. Comparative Example A comparative example will be described below to demonstrate the technical basis that the combined inactivated vaccine of the present invention is effective against decreased egg production caused by adeno-like viruses. Antibody production in each test group was compared after vaccination of roosters with the combined NCD/ALV vaccine and after vaccination of different groups of roosters with the NCD vaccine alone and the ALV vaccine alone. Four groups of 4 to 5 roosters, 10 weeks old, were given 0.5 ml of each of the above vaccines (oil/water ratio was 65/35 for each group) per bird. ) was injected intravenously. Group A: NCD single vaccination Group B: ALV single vaccination Group C: NCD/ALV combined vaccine: Group D Control: 2, 4, 7, 9, and 14 weeks after vaccination, the above 20 roosters were tested for Hemagglutination Inhibition. test) (HI) was conducted. In this case (the amount of NCD virus suspension alone and the amount of ALV virus suspension were known).
In this test, sequential dilutions (1:2, 1:
4, 1:8...1:2048) was first contacted with a virus suspension of a known concentration. When the virus is not inactivated by the serum (no effective antibodies are present for this serum dilution), subsequent contact with chick red blood cells will cause clotting. The antibody activity of the serum is thus determined and expressed as titer (the maximum dilution ratio at which anticoagulation occurs). The results of the above HI test are shown in Table 1.

【table】

[Table] From the results in Table 1, when the combined vaccine of the present invention is used for vaccination, the titer against both NCD and ALV viruses is achieved by vaccination using NCD vaccine alone and ALV vaccine alone. It was concluded that the titer was not different from that obtained.

[Brief explanation of the drawing]

Figure 1 of the attached drawings shows the detected HAI units and
This is a progressive curve showing the HAI level (vertical axis) versus time (weeks) (horizontal axis).

Claims (1)

[Scope of Claims] 1. A combined inactivated vaccine that can be administered immediately against Newkatsurus disease and against decreased egg production caused by adeno-like viruses, wherein the vaccine comprises an aqueous phase and an oil phase, and the aqueous phase At least ^109.8 EID ₅₀ ^/
ml of an inactivated, optionally homogenized, Nyukatsuru disease virus-containing solution and an inactivated, optionally homogenized adeno-like virus-containing solution having a titer of at least 10 ² HA units/ml; , the adeno-like virus-containing solution is obtained by culturing subcultured virus strain 127 in duck eggs or duck embryo fibroblasts in cell culture, and is previously inactivated and optionally It is homogenized into
The volume ratio of the liquid containing the Newkatsurus disease virus to the liquid containing the adeno-like virus can vary from 3:4 to 5:1, and the oil phase is comprised of paraffinic light mineral oil, vegetable oil, and naphthenic mineral oil. Contains at least one component selected as an essential component from the group consisting of esters and esters/ethers derived from alkylene oxide adducts and/or hexahydric alcohols and fatty acids (C ₁₀ - C ₂₀ ) The "aqueous phase formed from both virus liquids" containing at least one emulsifier selected from the group
The above-mentioned composite inactivated vaccine, characterized in that the volume ratio of the and oil phase can vary from 7:13 to 3:7. 2. The composite inactivated vaccine according to claim 1, wherein the volume ratio of the buffered Newkatsu virus-containing solution to the buffered adeno-like virus-containing solution is 3:2. 3 Collection d' Institut Pasteur, Paris
1. Combined inactivated vaccine according to claim 1, characterized in that the passaged virus strain 127 deposited on 1.090 is used. 4. The combined inactivated vaccine according to claim 1, which uses serotype adeno-like virus strain 127. 5 Collection d' Institut Pasteur, Paris
The combined inactivated vaccine according to claim 1, characterized in that the New Katsuru disease virus strain L/Z258P deposited under No. 1.091 is used. 6 Serotype Newkatsuru disease virus strain L/
The combined inactivated vaccine according to claim 1, characterized in that Z158P is used. 7 Emulsifier in the oil phase of 2 to 20% by weight, preferably 2 to 20% by weight
The composite inactivation according to claim 1, characterized in that it contains 15% by weight of Arlacel A or Arlacel 80 or Span 80 and 0.2-4% of Tween 80. vaccine. 8. In a method for producing a combination inactivated vaccine that is effective and immediately administrable against Newkatsurus disease and reduced egg production caused by adeno-like viruses, NDV ₍ ^NDV) having a titer of at least 10 ⁹ . Inactivate and optionally homogenize the solution containing Niukatsuru disease virus), and optionally homogenize the previously inactivated and optionally homogenized solution with a titer of at least 10 ² HA units/ml.
ALV (adeno-like virus)-containing liquid is added to the oil phase with the proviso that the ALV-containing liquid is passaged in duck eggs or in a cell culture of duck embryo fibroblasts. It is obtained by culturing in blast cells, inactivated in advance, and optionally homogenized, and the volume ratio of the liquid containing the New Katsuru disease virus and the liquid containing the adeno-like virus varies from 3:4 to 5:1. The above oil phase contains at least one component selected as an essential component from the group consisting of light paraffinic mineral oil, vegetable oil, and naphthenic mineral oil, an alkylene oxide adduct, and/or a hexahydric alcohol and a fatty acid. (C ₁₀ - _{C 20} ) and at least one selected from the group consisting of esters and esters/ethers derived from
containing a seed emulsifier, and that the volume ratio of the "aqueous phase formed from both viral fluids" and the oil phase can vary from 7:13 to 3:7, optionally containing the aqueous phase. The above manufacturing method is characterized in that a preservative is added to. 9. Process according to claim 8, characterized in that formaldehyde is used as an inactivating agent for the ALV liquid component. NDV with a titer of 10 10 ^{9.8 to 10 10 .5} EID ₅₀ /ml
9. Process according to claim 8, characterized in that starting from a liquid. 9. Process according to claim 8, characterized in that starting from an NDV liquid with a titer of 11 10 ¹⁰ EID ₅₀ /ml. 12. A method according to claim 8, characterized in that the adeno-like virus is cultured in a monolayer rolling bottle culture or in a cell culture of duck embryo fibroblasts attached to an inert solid support. 13. Monolayer cell culture with starting cells of at least 70 ~
characterized in that it is produced in suspension in a culture medium consisting of 90 parts of Eagle's medium, optionally containing 5 to 15 parts of calf serum, 0.22% by weight sodium bicarbonate solution and 1 to 5 parts of A solution or suspension of one or more antibiotics such as penicillin G, streptomycin, and natamycin is added, and when the cell culture is almost complete, the culture medium is separated and inoculated with a virus suspension and incubated at 37°C. The virus was kept at constant temperature for 0.5 to 2 hours to attach the virus, and the mixture was incubated with 2 to 10% calf serum and 2 to 10 parts of tryptose phosphate broth (30%).
g/) 10-20 parts bovine amnion
claim 12, wherein the virus-containing medium and cells are harvested 0.5 to 2 days after the appearance of CPE. The method described in. 14 Claim 13, characterized in that the cell culture is inoculated with an ALV suspension having the activity of 30×10 ⁶ TCID ₅₀ /rolling culture bottle.
The method described in section.