Porcine parainfluenza virus type 2

The present invention relates to a viral agent, processes for the culture and replication of this agent and the utilization of this agent on its own or in combination with other bacterial or viral pathogens as a vaccine component for the protection of pigs from diseases of the respiratory and reproductive tract.
At the end of the eighties to the start of the nineties a new pig disease which spread like the plague and was accompanied by high economic losses occurred in North America and Europe. In the meantime, this has been officially called "Porcine Reproductive and Respiratory Syndrome" (PRRS).
The main clinical symptoms of these contagious diseases are fertility disorders in sows and respiratory tract diseases in piglets and fattening pigs.
Besides the irregularly occurring non-specific symptoms such as loss of appetite, apathy and fever, the disease is characterized in sows by late abortions, stillbirths and by the birth of mummified and weak piglets. As a result of the epidemic, symptoms of the mastitis-metritis-agalactia (MMA) complex and return to oestrus occur in large numbers.
In an endemic region are unweaned and weaner piglets in an endemic region are mainly affected at the start of the epidemic, in the further course fattening pigs increasingly fall ill. In this case, besides the mainly occurring diseases of the respiratory tract, other classical pig diseases can also be observed in association at an increased frequency. The epidemic causes considerable economic losses, which results besides the direct animal losses from the decrease in the characteristic production numbers (farrowing and weaning results, pregnancy rate, weight increase).
The primary infectious agent is assumed to be a new RNA virus replicating in pulmonary alveolar macrophages. On the other hand, epidemiological investigations indicate that respiratory and reproductive diseases can be additionally caused or intensified by secondary or multiple infections with other viruses or viruses and bacteria. It is therefore desirable to protect pigs not only against the main causative agent of PRRS, but also against the causative agents which are additionally responsible for respiratory and reproductive diseases.
The present invention relates to:
1. A vaccine against diseases of the respiratory and reproductive tract of pigs, in particular in connection with the disease complex called PRRS, characterized by containing as antigenic material, in whole form or in parts or submits parainfluenza viruses and their variants and mutants in modified live or inactivated form, prepared by conventional or recombinant techniques.
2. Antigens based on parainfluenza viruses which cause diseases of the respiratory and reproductive tract of pigs.
3. A process for the preparation of antigens based on parainfluenza viruses which cause diseases of the respiratory and reproductive tract of pigs, characterized in that parainfluenza viruses are replicated and the antigenic material is isolated in a manner known per se from the virus suspensions thus obtained
4. The use of antigens based on parainfluenza viruses which cause diseases of the respiratory and reproductive tract of pigs for the diagnosis and/or prevention of these diseases.
5. The use of antigens based on parainfluenza viruses which cause diseases of the respiratory and reproductive tract of pigs for the production of diagnostics for the detection of these diseases and for the production of vaccines for the prevention of these diseases.
Antigenic material which may be mentioned is:
1. Complete, live virus particles, obtained by replication of the virus in cell cultures or embryonated chicken eggs.
2. Complete, live, attenuated virus particles, obtained by continuous passages of the virus in primary cell cultures, permanent cell lines, embryonated poultry eggs or experimental animals with subsequent replication in cell cultures or embryonated chicken eggs.
3. Complete, killed virus particles which are prepared by means of conventional processes, such as chemical or physical inactivation.
4. Subunits of the virus particles prepared from virus which is replicated in cell cultures or embryonated eggs.
5. Subunits of the virus particles which are expressed by cell systems by the means of recombinant techniques and can optionally be separated from these or isolated from these.
6. Virus antigens which are expressed in vector systems, by inserting the genome of the virus or parts thereof employed by means of recombinant techniques in genome vectors such as vaccini viruses, herpes viruses, adenoviruses or other suitable vector systems.
Parainfluenza viruses type 2 (PIV-2) are preferably used. PIV-2 which have been isolated from the respiratory or reproductive tract of pigs which exhibit a PRRS-like symptomatology are particularly preferred. The PIV-2 strain having the notation SER, which was deposited according to the Budapest convention on 12.6.1993 in the Collection Nationale des Cultures et de Microorganismes (Institut Pasteur, Paris, France) under the number I-1331, is particularly suitable.
In the vaccines according to the invention, the antigenic material of the parainfluenza viruses can be present in a mixture with antigenic material of other viruses or bacteria. Those which may be mentioned are: Chlamydia, in particular Chlamydia psittaci and Chlamydia pecorum in concentrations of 10.sup.5 -10 .sup.10 FU.sup.1 /dose, Erysipelothrix rhusiopathiae in concentrations of 10.sup.7 -10.sup.12 CFU/dose, PRRS viruses in concentrations of 10.sup.4 -10.sup.9 TCID.sup.2.sub.50 /dose and porcines parvovirus in concentrations of 10.sup.4 -10.sup.9 TCID.sub.50 /dose.
A mixture of PIV-2 and Chlamydia, in particular Chlamydia psittaci or Ch. pecorum, is particularly preferred.
.sup.1 IFU=Inclusion Forming Units
.sup.2 TCID=Tissue Culture Infective Dose
The following terms are used in the following details:
______________________________________Cotransfection Simultaneous introduction of two different DNA sequences into cells in which viruses can be replicated, with the aim of inducing virus recombinants which contain foreign DNA sequences. The different DNA sequences are (1) foreign DNA which can be inserted in shuttle vectors and (2) the purified genome of the vector virus.Genome vector Live causative agents, in particular viruses, which are suitable for the in- sertion of foreign DNA and infect cells or organisms with the foreign DNA inserted in their genome and express the foreign DNA therein.Immunogens Peptides or proteins which elicit an immunological reaction in a higher or- ganism and can be expressed in vectors by means of foreign DNA sequences.Cloning Insertion of foreign DNA sequences in vectors.Plasmid Extrachromosomal, cyclic DNA sequences which are replicated in procaryotic or eucaryotic cells.Shuttle vector Bacteriophages or plasmids, in particu- lar bacterial plasmids, which contain inserted foreign DNA which is flanked by DNA sequences of the vector virus.Transfection Transfer of DNA sequences to procaryotic or eucaryotic cells with the aim of in- ducing recombinants of the cell genome with the DNA sequences introduced.Vectors Plasmids, bacteriophages or viruses which carry foreign DNA sequences in their genetic information.______________________________________
The replication of the viruses for the production of complete live virus particles is carried out in a manner known per se, on the one hand in tissue cultures of animal cells as primary cells or permanent cell lines, e.g. in porcine cells, monkey cells or bovine cells, preferably in porcine kidney cells such as e.g. the cloned, permanent porcine kidney cell PK15 (ATCC CCL33 or its derivatives) or the primary porcine kidney cell EPK or monkey kidney cells such as the permanent monkey kidney cells BGM (Flow 03-240 or its derivatives) or Vero (ATCC CCL81 or its derivatives) or bovine kidney cells such as the permanent bovine kidney cell MDBK (ATCC CCL22 or its derivatives) and on the other hand in embryonated chicken eggs (e.g. Valo hatching eggs, Lohmann).
Replication in cell cultures is carried out in a manner known per se in stationary roller or carrier cultures in the form of monolayers or in suspension cultures. The growing media employed for the cells are all cell culture media known per se e.g. described in the product catalogue of Gibco BRL GmbH, Dieselstra.beta.e 5, 76344 Eggenstein, such as, in particular, Minimal Essential Medium (MEM), which as essential constituents contains amino acids, vitamins, salts and carbohydrates, completed with buffer substances such as e.g. sodium bicarbonate (NaHCO.sub.3) or hydroxyethylpiperazine-N-2-ethanesulphonic acid (Hepes) and optionally animal sera, such as e.g. sera from cattle, horses or their foetuses. Eagles MEM having a content of NaHCO.sub.3 of 0.1-5 g/l, preferably 0.5-3 g/l, and foetal calf serum in a concentration of 1-30% by volume, preferably 2-10% by volume, is particularly preferably employed.
The cells and cell lawns used for the replication of the viruses are grown in conventional manner almost to confluence or to the optimal cell density. Before their infection with viruses, the cell growth medium is preferably removed and the cells are preferably washed with virus replication medium. The virus replication medium employed are all cell culture media known per se, such as, in particular, the abovementioned MEM. Infection is then carried out using a virus suspension. In the virus suspension, the virus is diluted in the virus replication medium to a MOI (=multiplicity of infection, corresponds to the ratio of the number of infectious virus particles to the number of cells present) of 0.01-50, preferably 0.1-10.
The replication of the viruses is carried out with or without addition of animal sera. In the case where serum is employed, it is added to the replication medium in a concentration of 1-30% by volume, preferably 2-10% by volume.
Infection and virus replication are carried out at temperatures between room temperature and 40.degree. C., preferably between 32 and 39.degree. C., particularly preferably at 37.degree. C. for several days, preferably up to complete destruction of the infected cells.
The virus-containing medium of the infected cells is worked up further, e.g. by removing the cells and cell debris by means of filtration using pore sizes of e.g. 0.1-0.45 .mu.m and/or centrifugation up to 10,000.times.g.
Replication in embryonated chicken eggs is carried out in a manner known per se in the allantoic cavity of chicken hatching eggs which have been preincubated for 9-12 days, preferably 10 days, at a temperature of 37-39.degree. C., preferably 38.5.degree. C., and a relative humidity of 30-90%, preferably 50-60%, in a commercially available incubator, preferably a power-driven incubator.
Before inoculation, the hatching eggs used for replication of the viruses are placed standing vertically in the incubator on the pointed end of the egg for 1-3 hours, preferably 2 hours, and then, after preparation of the injection site, infected with 10-200 .mu.l, preferably 75-125 .mu.l, of a virus suspension. In the virus suspension, the virus is diluted in the virus replication medium to a concentration of 10.sup.1 -10.sup.7 TCID.sub.50 /ml (50% culture-infectious dose per ml of suspension=the dilution stage at which 50% of the cell cultures employed would be infected), preferably 10.sup.4 -10.sup.5 TCID.sub.50 /ml. As the virus replication medium, all cell culture media known per se, such as, in particular, the abovementioned MEM, are employed.
Infection and virus replication are carried out for several days, preferably 2-5 days, particularly preferably 3 days, under the incubation conditions indicated above.
The virus-containing allantoic fluid is obtained by aspiration after opening the calcareous shell and also the periostracum and the chorioallantoic membrane and can be further worked up e.g. by means of filtration using pore sizes of e.g. 0.1-0.45 .mu.m and/or centrifugation up to 10,000.times.g.
The preparation of attenuated, live virus is carried out in conventional manner by continuous passage and/or alternating passage on the one hand in tissue cultures of animal cells as primary cells or permanent cell lines, e.g. in porcine cells, monkey cells or bovine cells, preferably in porcine kidney cells such as e.g. the cloned, permanent porcine kidney cell PK15 (ATCC CCL33 or its derivatives) or the primary porcine kidney cell EPK or monkey kidney cells such as the permanent monkey kidney cells BGM (Flow 03-240 or its derivatives) or Vero (ATCC CCL81 or its derivatives) or bovine kidney cells such as the permanent bovine kidney cell MDBK (ATCC CCL22 or its derivatives) or canine kidney cells such as the permanent canine kidney cell MDCK (ATCC CCL34 or its derivatives) and on the other hand in embryonate hens', doves' or ducks' eggs, preferably in embryonated chicken eggs (e.g. Valo hatching eggs, Lohmann) or in experimental animals, preferably in small laboratory animals, e.g. in the guinea-pig, rat or mouse, in which the virus replicates without causing serious symptoms of disease.
Passaging in cell cultures is carried out in a manner known per se in stationary cultures in the form of monolayers. The growth media employed for the cells are all cell culture media known per se e.g. described in the product catalogue of Gibco BRL GmbH, Dieselstra.beta.e 5, 76344 Eggenstein, such as, in particular, Minimal Essential Medium (MEM), which as essential constituents contains amino acids, vitamins, salts and carbohydrates, completed with buffer substances such as e.g. sodium bicarbonate (NaHCO.sub.3) or hydroxyethylpiperazine-N-2-ethanesulphonic acid (Hepes) and optionally animal sera, such as e.g. sera from cattle, horses or their foetuses. Eagles MEM having a content of NaHCO.sub.3 of 0.1-5 g/l, preferably 0.5-3 g/l and foetal calf serum in a concentration of 1-30% by volume, preferably 2-10% by volume, is particularly preferably employed.
The cells and cell lawns used for passaging the viruses are grown in a conventional manner almost up to confluence or to the optimal cell density. Before their infection with viruses, the cell growth medium is preferably removed and the cells are preferably washed with virus replication medium. The virus replication medium employed are all cell culture media known per se, such as, in particular, the abovementioned MEM. Infection with a virus suspension is then carried out. In the virus suspension, the virus is diluted in the virus replication medium to MOI (=multiplicity of infection, corresponds to the ratio of the number of infectious virus particles to the number of cells present) of 0.01-50, preferably 0.1-10.
The replication of the viruses is carried out with or without addition of animal sera. In the case where serum is employed, it is added to the replication medium in a concentration of 1-30% by volume, preferably 2-10% by volume.
Infection and virus replication are carried out at temperatures between room temperature and 40.degree. C., preferably between 30 and 39.degree. C., for several days, preferably up to complete destruction of the infected cells.
The virus-containing medium of the infected cells is used for infection of a fresh cell culture (subsequent passage).
Passaging in embryonated poultry eggs is carried out in a manner known per se in the allantoic cavity of e.g. hens' hatching eggs which have been preincubated for 9-12 days, preferably 10 days, at a temperature of 37-39.degree. C., preferably 38.5.degree. C., and a relative humidity of 30-90%, preferably 50-60%, in a commercially available incubator, preferably a power-driven incubator.
The hatching eggs used for passaging of the viruses are placed in the incubator standing vertically on the pointed end of the egg for 1-3 hours, preferably 2 hours, before inoculation and then infected with 10-200 .mu.l, preferably 75-125 .mu.l, of a virus suspension after preparation of the injection site. In the virus suspension, the virus is present in the virus replication medium diluted to a concentration of 10.sup.1 -10.sup.7 TCID.sub.50 /ml (50% culture-infectious dose per ml of suspension=the dilution stage at which 50% of the cell cultures employed would be infected), preferably 10.sup.4 -10.sup.5 TCID.sub.50 /ml. The replication medium employed are all cell culture media known per se, such as, in particular, the abovementioned MEM.
Infection and virus replication are carried out for several days, preferably 2-5 days, particularly preferably 3 days, under the incubation conditions indicated above.
The virus-containing allantoic fluid is obtained by aspiration after opening the calcareous shell and also the periostracum and the chorioallantoic membrane. It is used for the infection of fresh preincubated, embryonate eggs (subsequent passage).
Passaging in experimental animals is carried out in a manner known per se by parenteral administration of a virus suspension and reisolation of the virus from organs and tissues of the experimental animals.
For passaging in experimental animals, juvenile, small laboratory animals are preferably employed which originate from SPF (specified pathogen-free) breeding, e.g. guinea-pigs (Hsd/Win:DH, Harlan-Winkelmann GmbH, Borchen), rats (Hsd/Win:WU, Harlan-Winkelmann GmbH, Borchen) or mouse (Hsd/Win:NMRI, Harlan-Winkelmann GmbH, Borchen; Balb/C/JICO, Iffa Credo Belgium). The experimental animals are parenterally infected with 0.1-2.0 ml of a virus suspension, e.g. by intradermal, intramuscular, intranasal, intraperitoneal, intravenous or subcutaneous administration. In the virus suspension, the virus is diluted in the virus replication medium such that the experimental animals in each case receive a virus dose of 10.sup.1 -10.sup.7 TCID.sub.50, preferably 10.sup.3 -10.sup.5 TCID.sub.50 (50% culture-infectious dose per ml of suspension=the dilution stage at which 50% of the cell cultures employed would still be infected). The virus replication medium employed are all cell culture media known per se, such as, in particular, the abovementioned MEM.
Virus replication takes place over the course of several days preferably 1-12 days.
The virus is reisolated from tissues, preferably internal organs of the experimental animals, in a manner known per ser. For this purpose, internal organs, e.g. lungs, liver or spleen are removed from the experimental animals. Fine suspensions are prepared in virus replication medium from the organs or parts of the organs by mechanical disruption, e.g. with the aid of scissors and mortars, which are further worked up, e.g. by removing the cells and cell debris by means of filtration using pore sizes of e.g. 0.1-0.45 .mu.m and/or centrifugation up to 10,000.times.g. The virus replication medium employed are all cell culture media known per se, such as, in particular, the abovementioned MEM.
The virus-containing medium obtained is used for the infection of new experimental animals (subsequent passages).
The process of subsequent passage is repeated several times, preferably 10-20 times, in the same replication system (homologous passages) or in different replication systems (heterologous passages).
Monitoring of the virus for attenuation is carried out by experimental infection of fully susceptible experimental animals, preferably pigs, with a virus suspension which originates from the last passage of a series of subsequent passages.
If typical disease symptoms still occur, e.g. abortions and stillbirths in pregnant sows or respiratory diseases, further homologous or heterologous continuous passages are carried out starting from the viruses of the last subsequent passages.
If typical disease symptoms no longer occur, the viruses of the last subsequent passage are replicated as described above and the filtrates or centrifugation supernatants of virus-containing culture supernatants or allantoic fluid are used for the production of vaccines.
Replication of the viruses for the preparation of killed virus particles is carried out in a conventional manner on the one hand in tissue cultures of animal cells as primary cells or permanent cell lines, e.g. in porcine cells, monkey cells or bovine cells, preferably in porcine kidney cells such as e.g. the cloned, permanent porcine kidney cell PK15 (ATCC CCL33 or its derivatives) or the primary porcine kidney cell EPK or monkey kidney cells such as the permanent monkey kidney cells BGM (flow 03-240 or its derivatives) or Vero (ATCC CCL81 or its derivatives) or bovine kidney cells such as the permanent bovine kidney cell MDBK (ATCC CCL22 or its derivatives) and on the other hand in embryonate hens' eggs (e.g. Valo hatching eggs, Lohmann).
Replication in cell cultures is carried out in a manner known per se in stationary roller or carrier cultures in the form of monolayers or in suspension cultures. The growing media employed for the cells are all cell culture media known per se e.g. described in the product catalogue of Gibco BRL GmbH, Dieselstra.beta.e 5, 76344 Eggenstein, such as, in particular, Minimal Essential Medium (MEM), which as essential constituents contains amino acids, vitamins, salts and carbohydrates, completed with buffer substances such as e.g. sodium bicarbonate (NaHCO.sub.3) or hydroxyethylpiperazine-N-2-ethanesulphonic acid (Hepes) and optionally animal sera, such as e.g. sera of cattle, horses or their foetuses. Eagles MEM having a content of NaHCO.sub.3 of 0.1-5 g/l, preferably 0.5-3 g/l and also foetal calf serum in a concentration of 1-30% by volume, preferably 2-10% by volume, is particularly preferably employed.
The cells and cell lawns used for replication of the viruses are grown in conventional manner almost to confluence or to the optimal cell density. Before their infection with viruses, the cell growth medium is preferably removed and the cells preferably are washed with virus replication medium. The virus replication medium employed are all cell culture media known per se, such as, in particular, the abovementioned MEM. Infection is then carried out with a virus suspension. In the virus suspension, the virus is diluted in the virus replication medium diluted to a MOI (=multiplicity of infection, corresponds to the ratio of the number of infectious virus particles to the number of cells present) of 0.01-50, preferably 0.1-10.
The replication of the viruses is carried out with or without addition of animal sera. In the case where serum is employed, it is added to the replication medium in a concentration of 1-30% by volume, preferably 2-10% by volume.
Infection and virus replication are carried out at temperatures between room temperature and 40.degree. C., preferably between 32 and 39.degree. C., particularly preferably at 37.degree. C. for several days, preferably up to complete destruction of the infected cells.
The virus-containing medium of the infected cells is worked up further, e.g. by removing the cells and cell debris by means of filtration using pore sizes of e.g. 0.1-0.45 .mu.m and/or centrifugation up to 10,000.times.g.
Replication in embryonated chicken eggs is carried out in a manner known per se in the allantoic cavity of chicken hatching eggs which have been preincubated for 9-12 days, preferably 10 days, at a temperature of 37-39.degree. C., preferably 38.5.degree. C., and a relative humidity of 30-90% preferably 50-60%, in a commercially available incubator, preferably a power-driven incubator.
The hatching eggs used for replication of the viruses are placed in the incubator standing vertically on the pointed end of the egg for 1-3 hours, preferably 2 hours, before inoculation and then infected with 10-200 .mu.l, preferably 75-125 .mu.l, of a virus suspension after preparation of the injection site. In the virus suspension, the virus is present in the virus replication medium diluted to a concentration of 10.sup.1 -10.sup.7 TCID.sub.50 /ml (50% culture-infectious dose per ml of suspension=the dilution stage at which 50% of the cell cultures employed would be infected), preferably 10.sup.4 -10.sup.5 TCID.sub.50 /ml. The virus replication medium employed are all cell culture media known per se, such as, in particular, the abovementioned MEM.
Infection and virus replication are carried out for several days, preferably 2-5 days, particularly preferably 3 days, under the incubation conditions indicated above.
The virus-containing allantoic fluid is obtained by aspiration after opening the calcareous shell and also the periostracum and the chorioallantoic membrane and can be further worked up e.g. by means of filtration using pore sizes of e.g. 0.1-0.45 .mu.m and/or centrifugation up to 10,000.times.g.
Inactivation of the viruses is carried out in a manner known per se by physical processes, e.g. by the action of heat, UV or gamma irradiation or preferably by chemical processes, e.g. by the action of ethanol, formaldehyde, .beta.-propiolactone and preferably by ethyleneamines.
Chemical inactivation is carried out in a manner known per se in suitable reaction vessels which have a device for maintaining a constant reaction temperature and also for continuous agitation of the reaction mixture (e.g. fermenters). Inactivating agents employed are preferably ethyleneamines, particularly preferably 2-bromoethylamine hydrobromide (2-BEA) in a concentration of 1-10 mmol/l, preferably 2.5-7.5 mmol/l.
A virus suspension having a concentration of 10.sup.4.0 14 10.sup.9.0 TCID.sub.50 /ml, preferably 10.sup.5.0 -10.sup.8.0 TCID.sub.50 /ml which originates from one or more virus replications is adjusted to a pH of 8.1-8.7, preferably 8.3-8.5, before addition of the 2-BEA solution.
Inactivation is carried out at 4-40.degree. C., preferably 23-37.degree. C., particularly preferably at 36-37.degree. C., for 6-48 hours, preferably 16-20 hours.
Excess 2-BEA is neutralized by addition of hydrolysing agents after conclusion of the inactivation. For this purpose, in particular, sodium thiosulphate that is added in a final concentration of 40-80 mmol/l, preferably 50 mmol/l, is suitable. Neutralization is carried out at 4-40.degree. C., preferably at 2-8.degree. C., for 2-16 hours, preferably 4-8 hours.
Replication of the viruses for the preparation of sub-units is carried out in the manner known per se on the one hand in tissue cultures of animal cells as primary cells or permanent cell lines, e.g. in porcine cells, monkey cells or bovine cells, preferably in kidney cells such as e.g. the cloned, permanent porcine kidney cell PK15 (ATCC CCL33 or its derivatives) or the primary porcine kidney cell EPK or porcine kidney cells such as the permanent porcine kidney cells BGM (Flow 03-240 or its derivatives) or Vero (ATCC CCL81 or its derivatives) or bovine kidney cells such as the permanent bovine kidney cell MDBK (ATCC CCL22 or its derivatives) and on the other hand in embryonated chicken eggs (e.g. Valo hatching eggs, Lohmann).
Replication in cell cultures is carried out in a manner known per se in stationary roller or carrier cultures in the form of monolayers or in suspension cultures. Growing media employed for the cells are all cell culture media known per se e.g. described in the product catalogue of Gibco BRL GmbH, Dieselstra.beta.e 5, 76344 Eggenstein, such as, in particular, Minimal Essential Medium (MEM), which as essential constituents contains amino acids, vitamins, salts and carbohydrates, completed with buffer substances such as e.g. sodium bicarbonate (NaHCO.sub.3) or hydroxyethyl-piperazine-N-2-ethanesulphonic acid (Hepes) and optionally animal sera, such as e.g. sera of cattle, horses or their foetuses. Eagles MEM having a content of NaHCO.sub.3 of 0.1-5 g/l, preferably 0.5-3 g/l and also foetal calf serum in a concentration of 1-30% by volume, preferably 2-10% by volume, is particularly preferably employed.
The cells and cell lawns used for replication of the viruses are grown in conventional manner almost to confluence or to the optimal cell density. Before its infection with viruses, the cell growth medium is preferably removed and the cells are preferably washed with virus replication medium. The virus replication medium employed are all cell culture media known per se, such as, in particular, the abovementioned MEM. Infection with a virus suspension is then carried out. In the virus suspension, the virus is diluted in the virus replication medium to a MOI (=multiplicity of infection, corresponds to the ratio of the number of infectious virus particles to the number of cells present) of 0.01-50, preferably 0.1-10.
Replication of the viruses is carried out with or without addition of animal sera. In the case where serum is employed, this is added to the replication medium in a concentration of 1-30% by volume, preferably 2-10% by volume.
Infection and virus replication are carried out at temperatures between room temperature and 40.degree. C., preferably between 32 and 39.degree. C., particularly preferably at 37.degree. C., for several days, preferably up to complete destruction of the infected cells.
The virus-containing medium of the infected cells is worked up further, e.g. by removal of the cells and cell debris by means of filtration using pore sizes of e.g. 0.1-0.45 .mu.m and/or centrifugation up to 10,000.times.g.
Replication in embryonated chicken eggs is carried out in a manner known per se in the allantoic cavity of chicken hatching eggs which have been preincubated for 9-12 days, preferably 10 days, at a temperature of 37-39.degree. C., preferably 38.5.degree. C. and a relative humidity of 30-90%, preferably 50-60%, in a commercially available incubator, preferably a power-driven incubator.
The hatching eggs used for replication of the viruses are placed in the incubator standing vertically on the pointed end of the egg for 1-3 hours, preferably 2 hours, before inoculation and then infected with 10-200 .mu.l, preferably 75-125 .mu.l, of a virus suspension after preparation of the injection site. In the virus suspension, the virus is diluted to in the virus replication medium diluted to a concentration of 10.sup.1 -10.sup.7 TCID.sub.50 /ml (50% culture infectious dose per ml of suspension=the dilution stage at which 50% of the cell cultures employed would still be infected), preferably 10.sup.4 -10.sup.5 TCID.sub.50 /ml. The virus replication medium employed are all cell culture media known per se, such as, in particular, the abovementioned MEM.
Infection and virus replication are carried out for several days, preferably 2-5 days, particularly preferably 3 days, under the incubation conditions indicated above.
The virus-containing allantoic fluid is obtained by aspiration after opening the calcareous shell and also the periostracum and the chorioallantoic membrane and can be further worked up e.g. by means of filtration using pore sizes of e.g. 0.1-0.45 .mu.m and/or centrifugation up to 10,000.times.g.
Virus isolation is achieved by isopycnic or zonal centrifugation in e.g. sucrose density gradients. For this purpose, the virus-containing medium or the allantoic fluid is subjected after removal of the cell debris to a zonal centrifugation at 100,000.times.g until the virus particles are pelleted. A purer preparation of the virus particles results by zonal centrifugation in an aqueous solution having a higher density than the virus-containing medium. The aqueous solution used can be e.g. a 30-60% w/w, preferably 35-50% w/w, buffered solution of sucrose. A still higher degree of purity is achieved by centrifugation in density gradients. For this purpose, the virus purified from cells and cell debris and concentrated by means of zonal centrifugation is isolated by an isopycnic or zonal density gradient centrifugation in a density gradient of e.g. 30 to 50% w/w sucrose in buffered aqueous solution at a centrifugal acceleration of e.g. 100,000 to 150,000.times.g.
The virus concentrates thus obtained are treated with detergents.
Suitable detergents are:
Anionic surfactants, such as sodium laurylsulphate, fatty alcohol ether sulphates, mono-/dialkyl polyglycol ether orthophosphate monoethanolamine salt, calcium alkylaryl-sulphonate, sodium deoxycholate, cationic surfactants, such as cetyltrimethylammonium chloride, ampholytic surfactants, such as di-sodium N-lauryl-iminodipropiont or lecithin, non-ionic surfactants, e.g. polyoxyethylated castor oil, polyoxyethylated sorbitan monooleate, sorbitan monostearate, glycerol monostearate, polyoxyethylene stearate, alkylphenol polyglycol ethers.
Non-ionic detergents may preferably be mentioned: Non-ionic, water-soluble emulsifiers having an HLB (hydrophilic-lipophilic balance) of greater than 10, e.g. emulsifier NP 40.RTM. (Bayer AG), alkylaryl polyglycol ether; Renex 678.RTM. (Atlas Chemical Industries), polyoxyethylene alkylaryl ether; Tween 20.RTM. (Atlas), polyoxyethylene sorbitan monopalmitate; Myri 53.RTM. (Atlas), polyoxyethylene stearate; Atlas G 3707.RTM., polyoxyethylene lauryl ether; Atlas G 3920.RTM., polyoxyethylene oleyl ether; Atlas G 9046 T.RTM., polyoxyethylene mannitan monolaurate; emulsifier 1371 B.RTM. (Bayer AG), alkyl polyglycol ether; emulsifier 1736.RTM. (Bayer AG), alkyl polyglycol ether (oleyl polyglycol ether); emulsifier OX.RTM. (Bayer AG), alkyl polyglycol ether (dodecyl polyglycol ether); Ninox BM-2.RTM. (Stepan Chemical Co.) ethoxyethylated nonylphenol; Triton X-100.RTM. (Rohm an Haas Co.), isooctylphenolpolyethoxy-ethanol; Cremophor EL.RTM., Nonidet P 40.RTM. (Shell).
The detergents are used in diluted form aqueous solutions. Solutions may be mentioned having a 0.1 to 10 per cent by volume, preferably having a 0.5 to 5 per cent by volume, particularly preferably about 1 per cent by volume, detergent content.
The detergent solution is added to the virus concentrate in the volume ratio of about 1:1 to about 10:1. Preferably, the ratio of detergent solution to virus concentrate is of about 3:1.
Detergent treatment is carried out continuous agitation of the mixture at temperatures between 0 and about 24.degree. C., preferably between 2 and 8.degree. C. The detergent treatment lasts 15 minutes to 2 days, preferably 6 to 18 hours. To improve the detergent treatment, the mixture can additionally be subjected to ultrasonic treatment.
The particles which are not dissolved during this treatment are removed, preferably by filtration or centrifugation at e.g. 150,000.times.g. The filtrate or the centrifugation supernatant thus obtained can be stored at low temperatures (0 to -70.degree. C.) until it is processed further.
The glycoproteins of the virus particles contained in the lysate are isolated by treatment with lectins. Lectins are proteins or glycoproteins from plants, especially their seeds, microorganisms, vertebrates and invertebrates, which specifically bind sugars and their conjugates. Lectins are used which recognize and bind glycoproteins from paramyxoviruses. Lectins are preferably used which recognize mannose and/or glucose and their conjugates. Lectins from Canavalia ensiformis, Lens culinaris, Lathygros odoratus, Pisum sativum, Vicia faba, Sambucus nigra, Glycine max, Ulex europaens, Helix promatia, Phytolacca americana, Lycopersicon esculentum, Datura stramonium, Bandeiraea simplificolia may be mentioned especially.
The lectins are used in water-soluble or water-insoluble form. In water-insoluble form they are preferably immobilized by coupling to inert matrixes such as e.g. dextrans, agaroses or celluloses as suspensions or gels. Concanavalin A-agarose, concanavalin A-Sepharose, lentil lectin-Sepharose, agarose-wheatgerm lectin and Helix pomatia lectin-Sepharose may be mentioned especially.
The lectins are employed in form of a detergent- and salt-containing solution, suspension or of a gel. For this, both the lysate and the lectin solution, lectin suspension or the lectin gel employed is previously treated with sufficient sodium chloride and the known lectin-stabilizing salts that a concentration of sodium chloride of 0.5 to 2, preferably 0.7 to 1.2, mol/l achieved. The concentration of the lysates is preferably carried out by dialysis. The concentration of the lectin-salts necessary is known from the prior art and is specific for the lectins to be employed. The lectin solution, the lectin suspension or the lectin gel is moreover treated in the same concentration with the detergent employed for the treatment of the lysates, such that lysate and lectin solution have identical concentrations of salt and detergent.
About 1 to 150 mg, preferably 1 to 50 mg, particularly preferably 5 to 20 mg, of pure lectin are used per ml of solution, suspension or gel. A sufficient amount of the lectin solution, suspension or the lectin gel is added to the lysate that 0.01 to 50 mg, preferably 0.1 to 20 mg, particularly preferably 0.5 to 5 mg, of lectin are employed per mg of total protein. The lectin treatment is carried out at 0 to about 24.degree. C., preferably at 2 to 8.degree. C. for about 10 minutes to 3 days, preferably 1 hour to 2 days.
The reaction of the lectins with the glycoproteins can also be carried out by means of column chromatography, where the lysate is being brought into contact with the lectin immobilized on a gel-like matrix in a chromatography column.
The glycoprotein-lectin complex is separated from the solution or suspension by established methods. It can be achieved by centrifugation, filtration or, in the case of chromatography, by washing.
The concentration of detergent and/or salt of the lectin-glycoprotein containing suspensions or gels obtained in these processes can be adjusted to physiologically tolerable range or eliminated by filtration, centrifugation, dialysis or other washing processes.
The suspensions or gels of the lectin-glycoprotein complexes thus obtained can be used directly as antigenic material. Depending on the content of the glycoprotein bound to lectin, they can be further concentrated or diluted.
The suspensions or gels of the lectin-glycoprotein complexes can be stored at temperatures below 8.degree. C. They can also be freeze-dried.
For the preparation of antigenic material, the glycoproteins can be isolated from the lectin-glycoprotein complex containing suspensions or gels. Therefor, the suspensions or gels are treated with a salt-containing, aqueous sugar solution.
The nature of the sugar to be employed depends on the specificity of the lectins used. The concentration of the sugar is 0.1 to 1 mol/l, preferably 0.1 to 0.5 mol/l, particularly preferably 0.3 to 0.5 mol/l. Concentration and composition of the salt content corresponds to that of the glycoprotein-lectin complex containing suspensions or gels.
The treatment of the sugar solution is carried out at 0 to about 24.degree. C., preferably at 2 to 8.degree. C. The treatment is for about 15 minutes to 4 days, preferably 1 hour to 2 days, particularly preferably 10 to 24 hours.
The glycoproteins eluted in this way are isolated from the lectins by centrifugation, filtration or by other customary separation processes (e.g. chromatography). The concentrations of detergent, salt and sugar in the resulting preparations can be adjusted as already described above.
The isolated glycoproteins thus obtained can be used as antigenic material. The glycoprotein content can be adjusted by concentration or dilution.
The preparations are stored in soluble form at temperatures below 0.degree. C. or in lyophilized form.
For the preparation of subunits of the virus particles by the recombinant route, the virus genome is obtained first.
To obtain the virus genome, replication of the viruses is carried out in a manner known per se on the one hand in tissue cultures of animal cells as primary cells or permanent cell lines, e.g. in porcine cells, simian cells or bovine cells, preferably in porcine kidney cells such as e.g. the cloned, permanent porcine kidney cell PK15 (ATCC CCL33 or its derivatives) or the primary porcine kidney cell EPK or monkey kidney cells such as the permanent monkey kidney cells BGM (Flow 03-240 or their derivatives) or Vero (ATCC CCL81 or their derivatives) or bovine kidney cells such as the permanent bovine kidney cell MDBK (ATCC CCL22 or its derivatives) and on the other hand in embryonated chicken eggs (e.g. Valo hatching eggs, Lohmann).
Replication in cell cultures is carried out in a manner known per se in stationary roller or carrier cultures in the form of monolayers or in suspension cultures. Growing media employed for the cells are all cell culture media known per se e.g. described in the product catalogue of Gibco BRL GmbH, Dieselstra.beta.e 5, 76344 Eggenstein, such as, in particular, Minimal Essential Medium (MEM), which as essential constituents contains amino acids, vitamins, salts and carbohydrates, completed with buffer substances such as e.g. sodium bicarbonate (NaHCO.sub.3) or hydroxyethyl-piperazine-N-2-ethanesulphonic acid (Hepes) and optionally animal sera, such as e.g. sera of cattle, horses or their foetuses. Eagles MEM having a content of NaHCO.sub.3 of 0.1-5 g/l, preferably 0.5-3 g/l and also foetal calf serum in a concentration of 1-30% by volume, preferably 2-10% by volume, is particularly preferably employed.
The cells and cell lawns used for replication of the viruses are grown in a conventional manner almost to confluence or to the optimal cell density. Before their infection with viruses, the cell growth medium is preferably removed and the cells are preferably washed with virus replication medium. The virus replication medium employed are all cell culture media known per se, such as, in particular, the abovementioned MEM. Infection is then carried out using a virus suspension. In the virus suspension, the virus is diluted in the virus replication medium to a MOI (=multiplicity of infection, corresponds to the ratio of the number of infectious virus particles to the number of cells present) of 0.01-50, preferably 0.1-10.
Replication of the viruses is carried out with or without addition of animal sera. In the case where serum is employed, it is added to the replication medium in a concentration of 1-30% by volume, preferably 2-10% by volume.
Infection and virus replication are carried out at temperatures between room temperature and 40.degree. C., preferably between 32 and 39.degree. C., particularly preferably at 37.degree. C. for several days, preferably up to complete destruction of the infected cells.
The virus-containing medium of the infected cells is further worked up, e.g. by removal of the cells and cell debris by means of filtration using pore sizes of e.g. 0.1-0.45 .mu.m and/or centrifugation at up to 10,000.times.g.
Replication in embryonated chicken eggs is carried out in a manner known per se in the allantoic cavity of chicken hatching eggs which have been preincubated for 9-12 days, preferably 10 days, at a temperature of 37-39.degree. C., preferably 38.5.degree. C., and a relative humidity of 30-90%, preferably 50-60%, in a commercially available incubator, preferably a power-driven incubator.
The hatching eggs used for replication of the viruses are stored in the incubator standing vertically on the pointed end of the egg for 1-3 hours, preferably 2 hours, before inoculation and then infected with 10-200 .mu.l, preferably 75-125 .mu.l, of a virus suspension after preparation of the injection site. In the virus suspension, the virus is in the virus replication medium diluted to a concentration of 10.sup.1 -10.sup.7 TCID.sub.50 /ml (50% culture-infectious dose per ml of suspension=the dilution stage at which 50% of the cell cultures employed would be infected), preferably 10.sup.4 -10.sup.5 TCID.sub.50 /ml. The virus replication medium employed are all cell culture media known per se, such as, in particular, the abovementioned MEM.
Infection and virus replication are carried out for several days, preferably 2-5 days, particularly preferably 3 days, under the incubation conditions indicated above.
The virus-containing allantoic fluid is obtained by aspiration after opening the calcareous shell and also the periostracum and the chorioallantoic membrane and can be further worked up e.g. by means of filtration using pore sizes of e.g. 0.1-0.45 .mu.m and/or centrifugation at 10,000.times.g.
Virus purification or isolation is achieved by isopycnic or zonal centrifugation in e.g. sucrose density gradients. For this purpose, the virus-containing medium or the allantoic fluid is subjected after removal of the cell debris to a zonal centrifugation at 100,000.times.g until the virus particles are pelleted. A purer preparation of the virus particles results by zonal centrifugation in an aqueous solution having a higher density than the virus-containing medium. The aqueous solution used can be e.g. a 30-60% w/w, preferably 35-50% w/w, buffered solution of sucrose. A still higher degree of purity is achieved by centrifugation in density gradients. For this purpose, the virus purified from cells and cell debris and concentrated by means of zonal centrifugation is isolated by an isopycnic or zonal density gradient centrifugation in a density gradient of e.g. 30 to 50% w/w sucrose in buffered aqueous solution at a centrifugal acceleration of e.g. 100,000 to 150,000.times.g.
To obtain suitable genes which code for immunogenic proteins, the virus genome is first isolated from the purified virus particles. The native virus RNA is preferably obtained by treatment of the purified virus particles with detergent- and protease-containing aqueous solutions.
Anionic, cationic, amphoteric and non-ionic detergents are employed. Ionic detergents, preferably sodium dodecylsulphate, are preferably employed in a concentration 0.1-10% by volume, preferably 0.5-3% by volume.
Proteases employed are those which act in the presence of detergents, such as e.g. pronase and, preferably, proteinase K. The proteases are employed in a concentration 0.01-10 mg/ml, preferably 0.05-0.5 mg/ml.
Preferably, aqueous, buffered solutions with supplemented RNase inhibitors are used.
Buffer substances used are salts of weak acids with strong bases such as e.g. tris(hydroxymethyl)-aminomethane, and salts of strong acids with weak bases such as e.g. primary phosphates or mixtures thereof. Tris(hydroxymethyl)-aminomethane is preferably used. The buffer substances are employed in concentrations which ensure a pH at which the RNA is not denatured. pHs of 6-8.5 are preferred, particularly preferably of 7-8.
RNase inhibitors used are e.g. ribonucleoside-vanadyl complexes, protein inhibitors (e.g. RNAguard.RTM./Pharmacia) or preferably diethyl pyrocarbonate (DEPC) in concentrations of 0.01-2% by volume, preferably 0.1-0.5% by volume.
The lipophilic substances of the virus lysate are then extracted using solvents such as e.g. phenol, chloroform or mixtures thereof. Extraction is carried out in one or more stages.
The RNA is precipitated out of the remaining aqueous phase by means of aqueous solutions containing alcohols such as e.g. ethanol or isopropanol and monovalent chloride or acetate salts such as e.g. sodium chloride, sodium acetate or potassium acetate.
The concentration of the alcohols is between 40 and 100% by volume, preferably 60 and 80% by volume and that of the chloride or acetate salts is between 0.01 and 1 mol/l, preferably 0.1 to 0.8 mol/l.
The precipitated RNA is recovered from the aqueous solution e.g. by centrifugation and dissolved again in an aqueous solution e.g. DEPC-water. This aqueous solution preferably contains buffer substances such as e.g. tris(hydroxymethyl)-aminomethane in concentrations of 1-100 mmol/l, preferably 10-50 mmol/l, possibly supplemented with ethylenediamine tetraacetate (EDTA) in concentrations of 0.1-10 mmol/l, preferably 1-10 mmol/l or dithiothreitol (DTT) in concentrations of 0.1-10 mmol/l, preferably 1-10 mmol/l.
The isolated RNA is stored at temperatures below -65.degree. C.
Another method for RNA isolation is e.g. RNA extraction using guanidinium thiocyanate and subsequent caesium chloride density gradient centrifugation of the virus lysate.
Methods for RNA isolation are described in: J. Sambrook, E. F. Fritsch and T. Maniatis (Editor), Molecular Cloning, A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, 1989.
The identification of suitable genes is carried out using the isolated virus genome e.g. by:
a) RNA/DNA hybridization of the genome using known gene probes. Suitable gene probes used are DNA probes having nucleotide sequences of known genes for immunogens of related virus strains, such as e.g. simian virus 5 or canine parainfluenza virus 2.
b) Preparation of a complementary DNA (cDNA), cloning of the CDNA in e.g. bacterial plasmids such as e.g. pBR322 to concentrate viral DNA and hybridization of the cloned DNA by means of known gene probes. Suitable gene probes used are DNA probes having nucleotide sequences of known genes for immunogens of related virus strains, such as e.g. simian virus 5 or canine parainfluenza virus 2.
c) Preparation of a complementary DNA (cDNA) and cloning of the cDNA in plasmid expression vectors such as e.g. pUC18/19 or puC 118/119 or in .lambda.-bacteriophage expression vectors such as e.g. .lambda.gt11 and its derivatives or .lambda.ZAP or .lambda.ORF8. The identification of the genes is carried out by detection of their expressed immunogens with the aid of antibodies which are detected directly or indirectly e.g. by means of immunofluorescence or immunoprecipitation. Suitable antibodies are those which react with immunogens of related virus strains, such as e.g. simian virus 5 or canine parainfluenza virus 2.
d) Preparation of a complementary DNA (CDNA) and cloning of the cDNA in e.g. bacterial plasmids to concentrate viral DNA. The viral DNA of the clones is sequenced and investigated for sequence homologies using known genes of related virus strains, such as e.g. simian virus 5 or canine parainfluenza virus 2.
e) Sequencing of the cDNA during its preparation and investigation for sequence homologies with known genes of related virus strains, such as e.g. simian virus 5 or canine parainfluenza virus 2.
f) Combinations of the methods a) to e).
Methods for RNA/DNA and DNA/DNA hybridization, preparation of cDNA, cloning of DNA in plasmid and bacteriophage vectors, sequencing of DNA and methods for the immunological detection of expressed immunogens are described in:
J. Sambrook, E. F. Fritsch and T. Maniatis (Editor), Molecular Cloning, A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, 1989
F. M. Ausubel, Current protocols in molecular biology 1987-1988, John Wiley & Sons, New York, 1987
A. Mayr, Virologische Arbeitsmethoden (Virological Working Methods), Volume III, Gustav Fischer Verlag, Stuttgart, 1989
Those genes are selected in which, using the above-mentioned methods, a nucleotide sequence can be detected which codes for one or more immunogens.
As an example, in the sequence listing (below) are given the nucleotide sequence and the corresponding amino acid sequence of the neuraminidase-hemagglutinin and of the fusion-protein-gene of the Parainfluenza virus type 2 geposed at CNCM under the number I-1331.
The expression of the genes for the production of the immunogens is carried out e.g. by:
a) Stable integration of the genes in the form of complementary DNA into cellular DNA of cells. The genes are cloned beforehand into suitable shuttle vectors. A suitable virus for this is, for example, simian virus 40 (SV40) as well as plasmid expression vectors, which are suitable to be selected and replicated in procaryotes (e.g. E. coli) and possess regulatory elements for the expression of foreign DNA in higher cells.
Suitable plasmid expression vectors are e.g. plasmid vectors based on SV40, such as pMSG, pSVT7 or pMT2, or plasmid vectors based on Ebbstein-Barr virus such as pHEBo or p205.
The cloned DNA is isolated and purified by means of the methods described above and inserted in eucaryotic cells by transfection.
Suitable cells are animal cells, in particular permanent cell lines, such as e.g. the porcine kidney cell PK15 (ATCC CCL33 or its derivatives), the monkey kidney cell BGM (Flow 03-240 or its derivatives) or Vero (ATCC CCL81 or its derivatives), the bovine kidney cell MDBK (ATCC CCL22 or its derivatives), the canine kidney cell MDCK (ATCC CCL34 or its derivatives) or the rabbit kidney cell RK-13 (ATCC CCL37).
Transfection is carried out e.g. in the form of calcium phosphate-DNA coprecipitates or by the DEAE/dextran method, the liposome method or by electroporation.
Methods for cloning of the selected genes in suitable vectors and for transfection of the cloned genes in higher cells are described in detail in J. Sambrook, E. F. Fritsch and T. Maniatis (Editor), Molecular Cloning, A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, 1989 and F. M. Ausubel, Current protocols in molecular biology 1987-1988, John Wiley & Sons, New York, 1987.
Cell culture supernatants or cell lysates of cells treated in such a way are tested for the presence of expressed immunogens with the aid of antibodies which are detected directly or indirectly e.g. by means of immunofluorescence or immunoprecipitation. Suitable antibodies are those which react with immunogens of related virus strains, such as e.g. simian virus 5 or canine parainfluenza virus 2.
b) Cloning of the genes in the form of complementary DNA in suitable expression vectors for procaryotic or eucaryotic cells.
Suitable vectors are e.g. (i) bacterial plasmid expression vectors, (ii) viral expression vectors for bacteria or (iii) viral expression vectors for eucaryotic cells in which the cloned gene is expressed.
re (i)
Suitable bacterial plasmid expression vectors are e.g. pUC18/19 or pUC 118/119. After cloning of the DNA into the plasmid, it is inserted in procaryotic cells, preferably bacteria, and replicated. A suitable bacterium is e.g. Escherichia coli K12 and its derivatives.
The plasmid is incorporated in the procaryotic cell by e.g. calcium phosphate-DNA coprecipitation or electroporation.
re (ii)
Suitable viral expression vectors for bacteria are .lambda.-bacteriophage vectors such as e.g. .lambda.gt11 and derivatives, .lambda.ZAP or .lambda.ORF8. The replication of the .lambda.-bacteriophage vectors is in particular carried out in Escherichia coli e.g. E. coli K12 and its derivatives.
re (iii)
Suitable viral expression vectors for eucaryotic cells are e.g. simian virus 40, adenoviruses, herpes simplex virus or baculoviruses. The replication of the viral vectors is carried out in appropriate cell systems. Methods for cloning of the selected genes in suitable expression vectors and their use in appropriate expression systems are described in detail in J. Sambrook, E. F. Fritsch and T. Maniatis (Editor), Molecular Cloning, A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, 1989 and F. M. Ausubel, Current protocols in molecular biology 1987-1988, John Wiley & Sons, New York, 1987.
The expressed immunogens are used as antigens either directly in the form of the expression systems (culture substrate and/or cells) or after preparation and purification by means of biochemical and/or immunological methods and optionally after concentration or dilution.
Suitable processes for purification are e.g. affinity or gel chromatography processes in which the immunogens are separated or isolated from the expression system, optionally after detergent treatment.
To prepare virus antigens expressed by vector systems the virus genome is obtained first.
To obtain the virus genome, replication of the viruses is carried out in a manner known per se on the one hand in tissue cultures of animal cells as primary cells or permanent cell lines, e.g. in porcine cells, simian cells or bovine cells, preferably in porcine kidney cells such as e.g. the cloned, permanent porcine kidney cell PK15 PK15 (ATCC CCL33 or its derivatives) or the primary porcine kidney cell EPK or monkey kidney cells such as the permanent monkey kidney cells BGM (Flow 03-240 or its derivatives) or Vero (ATCC CCL81 or its derivatives) or bovine kidney cells such as the permanent bovine kidney cell MDBK (ATCC CCL22 or its derivatives) and on the other hand in embryonated chicken eggs (e.g. Valo hatching eggs, Lohmann). Replication in cell cultures is carried out in a manner known per se in stationary roller or carrier cultures in the form of monolayers or in suspension cultures. The growing media employed for the cells are all cell culture media known per se e.g. described in the product catalogue of Gibco BRL GmbH, Dieselstra.beta.e 5, 76344 Eggenstein, such as, in particular, Minimal Essential Medium (MEM), which as essential constituents contains amino acids, vitamins, salts and carbohydrates, completed with buffer substances such as e.g. sodium bicarbonate (NaHCO.sub.3) or hydroxyethyl-piperazine-N-2-ethanesulphonic acid (Hepes) and optionally animal sera, such as e.g. sera of cattle, horses or their foetuses. Eagles MEM having a content of NaHCO.sub.3 of 0.1-5 g/l, preferably 0.5-3 g/l and also foetal calf serum in a concentration of 1-30% by volume, preferably 2-10% by volume, is particularly preferably employed.
The cells and cell lawns used for replication of the viruses are grown in a conventional manner almost to confluence or to the optimal cell density. Before their infection with viruses, the cell growth medium is preferably removed and the cells are preferably washed with virus replication medium. The virus replication medium employed are all cell culture media known per se, such as, in particular, the abovementioned MEM. Infection is then carried out using a virus suspension. In the virus suspension, the virus is diluted in the virus replication medium to a MOI (=multiplicity of infection, corresponds to the ratio of the number of infectious virus particles to the number of cells present) of 0.01-50, preferably 0.1-10.
Replication of the viruses is carried out with or without addition of animal sera. In the case where serum is employed, it is added to the replication medium in a concentration of 1-30% by volume, preferably 2-10% by volume.
Infection and virus replication are carried out at temperatures between room temperature and 40.degree. C., preferably between 32 and 39.degree. C., particularly preferably at 37.degree. C. for several days, preferably up to complete destruction of the infected cells.
The virus-containing medium of the infected cells is further worked up, e.g. by removal of the cells and cell debris by means of filtration using pore sizes of e.g. 0.1-0.45 .mu.m and/or centrifugation at up to 10,000.times.g.
Replication in embryonated chicken eggs is carried out in a manner known per se in the allantoic cavity of chicken hatching eggs which have been preincubated for 9-12 days, preferably 10 days, at a temperature of 37-39.degree. C., preferably 38.5.degree. C., and a relative humidity of 30-90%, preferably 50-60%, in a commercially available incubator, preferably a power-driven incubator.
The hatching eggs used for replication of the viruses are stored in the incubator standing vertically on the pointed end of the egg for 1-3 hours, preferably 2 hours, before inoculation and then infected with 10-200 .mu.l, preferably 75-125 .mu.l, of a virus suspension after preparation of the injection site. In the virus suspension, the virus is diluted to in the virus replication medium in a concentration of 10.sup.1 -10.sup.7 TCID.sub.50 /ml (50% culture-infectious dose per ml of suspension=the dilution stage at which 50% of the cell cultures employed would be infected), preferably 10.sup.4 -10.sup.5 TCID.sub.50 /ml. The virus replication medium employed are all cell culture media known per se, such as, in particular, the abovementioned MEM.
Infection and virus replication are carried out for several days, preferably 2-5 days, particularly preferably 3 days, under the incubation conditions indicated above.
The virus-containing allantoic fluid is obtained by aspiration after opening the calcareous shell and also the periostracum and the chorioallantoic membrane and can be further worked up e.g. by means of filtration using pore sizes of e.g. 0.1-0.45 .mu.m and/or centrifugation at 10,000.times.g.
Virus purification or isolation is achieved by isopycnic or zonal centrifugation in e.g. sucrose density gradients. For this purpose, the virus-containing medium or the allantoic fluid is subjected after removal of the cell debris to a zonal centrifugation at 100,000.times.g until the virus particles are pelleted. A purer preparation of the virus particles results by zonal centrifugation in an aqueous solution having a higher density than the virus-containing medium. The aqueous solution used can be e.g. a 30-60% w/w, preferably 35-50% w/w, buffered solution of sucrose. A still higher degree of purity is achieved by centrifugation in density gradients. For this purpose, the virus purified from cells and cell debris and concentrated by means of zonal centrifugation is isolated by an isopycnic or zonal density gradient centrifugation in a density gradient of e.g. 30 to 50% w/w sucrose in buffered aqueous solution at a centrifugal acceleration of e.g. 100,000 to 150,000.times.g.
To obtain suitable genes which code for immunogenic proteins, the virus genome is first isolated from the purified virus particles. The native virus RNA is preferably obtained by treatment of the purified virus particles with detergent- and protease-containing aqueous solutions.
Anionic, cationic, amphoteric and non-ionic detergents are employed. Ionic detergents, preferably sodium dodecylsulphate, are preferably employed in a concentration of 0.1-10% by volume, preferably 0.5-3% by volume.
Proteases employed are those which act in the presence of detergents, such as e.g. pronase and, preferably, proteinase K. The proteases are employed in a concentration 0.01-10 mg/ml, preferably 0.05-0.5 mg/ml.
Preferably, aqueous, buffered solutions supplemented with RNase inhibitors are used.
Buffer substances used are salts of weak acids with strong bases such as e.g. tris(hydroxymethyl)-aminomethane, and salts of strong acids with weak bases such as e.g. primary phosphates or mixtures thereof. Tris(hydroxymethyl)-aminomethane is preferably used. The buffer substances are employed in concentrations which ensure a pH at which the RNA is not denatured. pHs of 6-8.5 are preferred, particularly preferably of 7-8.
RNase inhibitors used are e.g. ribonucleoside-vanadyl complexes, protein inhibitors (e.g. RNAguard.RTM./Pharmacia) or preferably diethyl pyrocarbonate (DEPC) in concentrations of 0.01-2% by volume, preferably 0.1-0.5% by volume.
The lipophilic substances of the virus lysate are then extracted using solvents such as e.g. phenol, chloroform or mixtures thereof. Extraction is carried out in one or more stages.
The RNA is precipitated out of the remaining aqueous phase, by means of aqueous solutions containing alcohols such as e.g. ethanol or isopropanol and monovalent chloride or acetate salts such as e.g. sodium chloride, sodium acetate or potassium acetate.
The concentration of the alcohols is between 40 and 100% by volume, preferably 60 and 80% by volume and that of the chloride or acetate salts is between 0.01 and 1 mol/l, preferably 0.1 to 0.8 mol/l.
The precipitated RNA is recovered from the aqueous solution e.g. by centrifugation and dissolved again in an aqueous solution e.g. DEPC-water. This aqueous solution preferably contains buffer substances such as e.g. tris(hydroxymethyl)-aminomethane in concentrations of 1-100 mmol/l, preferably 10-50 mmol/l, possibly supplemented with ethylenediamine tetraacetate (EDTA) in concentrations of 0.1-10 mmol/l, preferably 1-10 mmol/l or dithiothreitol (DTT) in concentrations of 0.1-10 mmol/l, preferably 1-10 mmol/l.
The isolated RNA is stored at temperatures below -65.degree. C.
Another method for RNA isolation is e.g. RNA extraction using guanidinium thiocyanate and subsequent caesium chloride density gradient centrifugation of the virus lysate.
Methods for RNA isolation are described in: J. Sambrook, E. F. Fritsch and T. Maniatis (Editor), Molecular Cloning, A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, 1989.
The identification of suitable genes is carried out using the isolated virus genome e.g. by:
a) RNA/DNA hybridization of the genome using known gene probes. Suitable gene probes used are DNA probes having nucleotide sequences of known genes for immunogens of related virus strains, such as e.g. simian virus 5 or canine parainfluenza virus 2.
b) Preparation of a complementary DNA (cDNA), cloning of the cDNA in e.g. bacterial plasmids such as e.g. pBR322 to concentrate viral DNA and hybridization of the cloned DNA by means of known gene probes. Suitable gene probes used are DNA probes having nucleotide sequences of known genes for immunogens of related virus strains, such as e.g. simian virus 5 or canine parainfluenza virus 2.
c) Preparation of a complementary DNA (cDNA) and cloning of the cDNA in plasmid expression vectors such as e.g. pUC18/19 or pUC 118/119 or in .lambda.-bacteriophage expression vectors such as e.g. .lambda.gt11 and its derivatives or .lambda.ZAP or .lambda.ORF8. The identification of the genes is carried out by detection of their expressed immunogens with the aid of antibodies which are detected directly or indirectly e.g. by means of immunofluorescence or immunoprecipitation. Suitable antibodies are those which react with immunogens of related virus strains, such as e.g. simian virus 5 or canine parainfluenza virus 2.
d) Preparation of a complementary DNA (CDNA) and cloning of the CDNA in e.g. bacterial plasmids to concentrate viral DNA. The viral DNA of the clones is sequenced and investigated for sequence homologies using known genes of related virus strains, such as e.g. simian virus 5 or canine parainfluenza virus 2.
e) Sequencing of the cDNA during its preparation and investigation for sequence homologies with known genes of related virus strains, such as e.g. simian virus 5 or canine parainfluenza virus 2.
f) Combinations of the methods a) to e).
Methods for RNA/DNA and DNA/DNA hybridization, preparation of cDNA, cloning of DNA in plasmid and bacteriophage vectors, sequencing of DNA and methods for the immunological detection of expressed immunogens are described in:
J. Sambrook, E. F. Fritsch and T. Maniatis (Editor), Molecular Cloning, A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, 1989
F. M. Ausubel, Current protocols in molecular biology 1987-1988, John Wiley & Sons, New York, 1987
A. Mayr, Virologische Arbeitsmethoden (Virological Working Methods), Volume III, Gustav Fischer Verlag, Stuttgart, 1989
Those genes are selected in which, using the above-mentioned methods, a nucleotide sequence can be detected which codes for one or more immunogens.
As example, in the sequence listing (below) are given the nucleotide sequence and the corresponding amino acid sequence of the neuraminidase-hemagglutinin- and of the fusion-protein-gene of the Parainfluenza virus type 2 deposited at CNCM under the number I-1331.
These genes, which code for one or more immunogens (foreign DNA), are inserted in a genome vector which expresses the foreign gene during the infection of a cell or of an organism. Vector viruses and vector bacteria are suitable for this purpose. For example, DNA viruses are used which have a stable genome with known insertion sites for the acceptance of 0.1 up to 20 kB (1,000 base pairs) foreign DNA, such as e.g. vaccinia viruses, herpes viruses or adenoviruses.
For this purpose it is necessary (a) first to insert the gene(s) in a shuttle vector which contains the foreign DNA flanked by DNA sequences of the vector virus. Subsequently, (.beta.) the gene(s) is inserted in the vector virus genome e.g. by means of cotransfection of the shuttle vector and of the vector virus.
Suitable shuttle vectors are plasmid or bacteriophage vectors.
Examples of standard plasmid vectors are pBR322, pUC18/19, pAT153, pACYC184 or pSP64/65 and for bacteriophage vectors .lambda.gt10/11, .lambda.ZAP or M13mp18/19.
(.alpha.) Insertion of the gene(s) into a shuttle vector
The insertion site carrying DNA fragment which carries the of the vector virus is first inserted in the shuttle vector DNA. For this purpose, both the DNA sequences of known insertion sites of the genome of the vector virus and the DNA of the shuttle vector are treated with restriction endonucleases (restriction enzymes) in order to produce ends suitable for insertion. The shuttle vector DNA prepared in such a way is mixed with an excess of the DNA fragment to be inserted, e.g. approximately in a ratio of 1:5. The DNA mixture is treated with DNA ligases in order to bind the DNA fragment covalently in the vector.
When using a shuttle plasmid, it is incorporated in pro- or eucaryotic cells, preferably bacteria, and replicated. Suitable is e.g. Escherichia coli K12 and its derivatives.
Bacteria which carry fragment-containing plasmids are selected.
The cloning of the insertion site in the shuttle plasmid genome, its insertion in pro- or eucaryotic cells, replication and selection of the transformed bacteria are described in detail in J. Sambrook, E. F. Fritsch and T. Maniatis (Editor), Molecular Cloning, A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, 1989 and F. M. Ausubel, Current protocols in molecular biology 1987-1988, John Wiley & Sons, New York, 1987.
If necessary, so-called "polylinkers" are inserted in the insertion sites of the vector virus. Polylinkers are DNA sequences having at least two defined restriction enzyme cleavage sites in sequence.
To do this, the insertion site carrying DNA fragment is treated with such a restriction enzyme that the fragment is only opened at one site. The fragment thus prepared is incubated together with the polylinker and DNA ligase for the specific insertion of defined restriction enzyme cleavage sites.
The polylinker can be inserted in the isolated DNA fragment or the insertion site carrying DNA fragment cloned in shuttle vectors.
If the polylinker is inserted in isolated DNA fragments, they have to be inserted in a shuttle vector. When using a shuttle plasmid, it is incorporated and replicated in pro- or eucaryotic cells, preferably bacteria. Suitable is e.g. Escherichia coli K12 and its derivatives. Bacteria which contain the plasmids containing DNA fragments are selected.
If the polylinker is inserted into the DNA fragment cloned in shuttle vectors, these are replicated and selected.
Genes which code for one or more immunogens (foreign DNA) are inserted in the insertion sites.
If necessary, partial sequences of the insertion site carrying the DNA fragment are previously removed. To do this, the DNA fragment is treated with restriction enzymes and the resulting DNA fragments are separated.
To insert the foreign DNA, the isolated DNA fragment or the insertion site carrying the DNA fragment cloned in shuttle vectors, is treated with one or more restriction enzymes and the fragment is opened at the insertion site or at the inserted polylinker. The foreign DNA is inserted in the insertion site prepared in this way, for example, with the aid of DNA ligases.
If the foreign DNA is inserted in isolated DNA fragments, these then have to be inserted in a shuttle vector. When using a shuttle plasmid this is incorporated in pro- or eucaryotic cells, preferably bacteria, and replicated. Suitable is e.g. Escherichia coli K12 and its derivatives. Bacteria which contain plasmids containing foreign DNA are selected.
If the foreign DNA is inserted in the DNA fragment cloned in shuttle vectors, these are replicated and selected.
Methods for the preparation of shuttle vectors are described in detail in J. Sambrook, E. F. Fritsch and T. Maniatis (Editor), Molecular Cloning, A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, 1989 and F. M. Ausubel, Current protocols in molecular biology 1987-1988, John Wiley & Sons, New York, 1987.
(.beta.) Insertion of the foreign DNA in the vector virus genome
The following methods can be used for insertion of the foreign DNA in the vector virus genome
(i) Cotransfection of suitable cells with the shuttle vector DNA and the isolated, native vector virus DNA,
(ii) Transfection of suitable cells with the shuttle vector DNA and infection with the vector virus,
(iii) Infection of suitable cells with the vector virus and transfection with the shuttle vector DNA.
Methods suitable for this purpose are described in detail in J. Sambrook, E. F. Fritsch and T. Maniatis (Editor), Molecular Cloning, A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, 1989 and F. M. Ausubel, Current protocols in molecular biology 1987-1988, John Wiley & Sons, New York, 1987.
The method (i), which is carried out in the form of the calcium phosphate DNA precipitation technique, is preferably employed. To this end, the following steps are necessary:
(1) The shuttle vector is replicated, isolated and further purified. Purification of the shuttle vector DNA is carried out e.g. by means of isopycnic density gradient centrifugation, e.g. in a caesium chloride density gradient.
The vector virus is replicated and purified. The viral genome is isolated and further purified. Purification of the vector virus DNA is carried out e.g. by means of isopycnic density gradient centrifugation, e.g. in a caesium chloride density gradient.
(2) For cotransfection, circular or preferably linearized shuttle vector DNA is used.
The linearized shuttle vector DNA is obtained e.g. by treatment of the purified DNA with restriction enzymes. Restriction enzymes are preferred which have no recognition site in the inserted foreign DNA, i.e. the foreign DNA sequence is not divided.
(3) The vector virus DNA and the shuttle vector DNA are mixed e.g. in a ratio of 0.01 to 0.1.times.10.sup.-12 mol/l of vector virus DNA to 1 to 3.times.10.sup.-12 mol/l of shuttle vector DNA.
(4) The DNA mixture is coprecipitated with e.g. calcium phosphate and transferred to suitable cells.
Suitable cells are animal cells, in particular permanent cell lines, such as e.g. the porcine kidney cell PK15 (ATCC CCL33 or its derivatives), the monkey kidney cell BGM (Flow 03-240 or its derivatives) or Vero (ATCC CCL81 or its derivatives), the bovine kidney cell MDBK (ATCC CCL22 or its derivatives), the canine kidney cell MDCK (ATCC CCL34 or its derivatives) or the rabbit kidney cell RK-13 (ATCC CCL37).
Cotransfection can also be carried out by means of other methods. Those which may be mentioned are e.g. the DEAE/dextran method, the liposome method or electroporation.
(5) The cells are cultured, e.g. according to the methods described further above. If a cythopatho-genetic effect occurs, clones of the vector virus are isolated by means of the individual plaque purification methods and further replicated.
Methods for individual plaque purification are described in A. Mayr, Virologische Arbeitsmethoden �Virological Working Methods!, Volume I, Gustav Fischer Verlag, Stuttgart, 1974.
(6) The selection of recombinant vector viruses is carried out (i) by detection of the expression of the foreign gene or (ii) by detection of the inserted foreign DNA in the vector virus genome e.g. by DNA/DNA hybridization.
(i)
Detection of the expression of the foreign DNA is carried out, for example, with the aid of antibodies. Suitable antibodies are those which react with immunogens of related virus strains, such as e.g. simian virus 5 or canine parainfluenza virus 2. The gene product of the foreign DNA can be detected e.g. by means of immunofluorescence or immunoprecipitation.
(ii)
Detection of the inserted foreign DNA is carried out by hybridization with gene probes of the corresponding foreign gene.
Stable recombinant vector viruses are replicated in known, conventional processes, such as described further above, isolated and further worked up used as antigenic material.
In the vaccines according to the invention, the antigenic material is present on its own or in formulations. Ingredients of those formulations which may be mentioned are pharmacologically tolerable solvents or diluents, adjuvants, preservatives, and suspension promoters or solubilizers such as emulsifiers.
The antigenic material is employed as a biologically active substance in the formulation of vaccines.
To prepare a live vaccine, the antigenic material is used in the form of live virus particles, to which additives and optionally also antifoams and preservatives are added for stabilization. To improve storage ability, the live vaccine is freeze-dried. Before use of this vaccine the lyophilized product is reconstituted with a solvent, such as e.g. aqua dest., aqua purificata or 0.9% saline solution.
The virus particles purified from the cell substrate are mixed in a concentration of at least 10.sup.6 CID.sub.50 /ml together with protective colloids or stabilizers, such as e.g. celluloses, dextrans, gelatins, collidones or stearates and optionally supplemented with antifoams, such as e.g. tributyl phosphate, isopropanol or silicone oil and also preservatives, such as e.g. merthiolate or thimerosal in an aqueous pH-buffered solution, filled into appropriate containers and freeze-dried.
To prepare inactivated vaccines, the antigenic material used is complete, killed virus particles in a concentration of 10.sup.4.0 -10.sup.9.0 CID.sub.50 /ml, preferably 10.sup.5.0 -10.sup.8.0 CID.sub.50 /ml before inactivation or fragments (subunits) of the virus particles in a concentration that 10-250 mg of protein, preferably 10-100 mg of protein, are contained per vaccine dose. The antigenic material is present in the vaccine in a formulation using substances such as solvents and diluents, adjuvants, preservatives, suspension promoters or solubilizers, pH-regulating agents and optionally antifoams.
Solvents and diluents which may be mentioned are aqua dest., aqua purificata, physiologically tolerable saline solutions and cell culture media. In particular, the abovementioned E-MEM and phosphate-buffered saline solution (PBS) are used.
Adjuvants which may be mentioned are:
1.) Mineral salts such as aluminium hydroxide, aluminium phosphate, calcium phosphate, kaolin or silicon. 10-50% by volume, preferably 25-35% by volume, of an aluminium hydroxide gel having a content of 1-5% (w/v), preferably 2-3% (w/v), of aluminium hydroxide are preferably employed.
2.) Oily adjuvants such as non-toxic mineral oils (e.g. Draceol.RTM., liquid paraffin), vegetable oils (e.g. lecithins, groundnut oils) or animal oils (squalanes, squalenes), which are employed in a concentration 1-40% by volume, preferably 1-15% by volume.
3.) Hydrophilic and hydrophobic polymers such as polyoxyethylene and polyoxypropylene. Synthetically prepared block copolymers (e.g. Pluronic.RTM. L101, Pluronic.RTM. L121, Pluronic.RTM. L122, Tetronic.RTM. 1501) in a concentration of 1-10% by volume are preferably employed.
4.) Adjuvants of bacterial origin such as pertussis toxin (Bordatella pertussis), Salmonella typhimurium mitogen or bacterial endotoxins such as lipopolysaccharides (LPS, e.g. from mycobacteria or salmonellae) and also LPS analogues or derivates such as e.g. lipid A, monophosphoryl lipid A (MPL), diphosphoryl lipid A, (DPL), trehalose dimycolate (TDM), muramyl dipeptide (MDP) or adamantyl dipeptide (AdDP) and their derivatives. MDP derivatives or AdDP in a concentration of 0.0001-10% (w/v) are preferably employed.
5.) Organic water-dispersible adjuvants such as cholesterol, gelatin, phosphatidylcholine, polysaccharides (e.g. zymosan, agar), aliphatic amines (e.g. dimethyldioctadecylamine/DDA, N,N.diotadecyl-N',N'-bis(2-hydroxyethyl)propanediamine/Avridin.RTM.), DEAE-dextrans or saponin (from the bark of Quillaja saponaria Molina) and saponin derivates (Quil A).
6.) Monokines and lymphokines, such as e.g. interleukin-1, interleukin-2 or .gamma.-interferon.
7.) Possible combinations of 1.) to 6.)
Preservatives which may be mentioned are formalin in concentrations up to 1%, phenol and benzyl alcohol in concentrations up to 0.5%, sorbic acid, benzoic acid, sodium benzoate, and their derivatives such as e.g. the sodium salt of 2-(ethylmercurio-thio)-benzoic acid (merthiolate, thimerosal, thiomersal) or the sodium salt of 4-(ethylmercurio-thio)-benzenesulphonic acid (thiomerfonate). Merthiolate is preferably employed in concentrations of 0.01% to 0.5%.
Suspension promoters and solubilizers which may be mentioned are non-toxic surface-active substances such as vegetable proteins, alginates, celluloses, phospholipids and in particular substances based on glycol ethers such as polyethylene glycols and their derivatives. Polyethylene glycol (PEG) 200, 300, 400, 600 and 900 and PEG derivatives (Span.RTM., Arlacel.RTM., Tween.RTM., Myri.RTM., Brij.RTM.) are preferably employed, particularly preferably Tween.RTM. 80 in a concentration of 0.05-5% by volume, preferably 0.2-1% by volume.
pH-regulating substances which may be mentioned are e.g. sodium hydroxide and potassium hydroxide, sodium carbonate and potassium carbonate, acetic, tartaric and citric acid or hydroxyethylpiperazine-N-2-ethanesulphonic acid (HEPES).
Antifoams which may be mentioned are tributyl phosphate, isopropanol, silicone oil, Antifoam.RTM. or Baysilon.RTM. antifoamer EBZ.
Parainfluenza viruses according to the invention which can cause diseases of the respiratory and reproductive tract of pigs are obtained e.g. as follows:
Organs are removed from diseased pigs showing PRRS-like symptomatology and subjected to a virus isolation. Weak or ill piglets from infected stocks are particularly suitable. The internal organs, in particular the lungs, liver, kidneys and spleen are removed from a suitable animal. Parts of these organs or organ mixtures are homogenized in physiologically tolerable aqueous solutions to give suspensions, the proportion of organs amounting to about 10% (w/v). A particularly suitable solution is the Eagles Minimum Essential Medium (E-MEM) described above. The suspensions are purified from cells and cell debris by centrifugation at about 1500.times.g. A further purification of the centrifugation supernatant can be carried out by filtering. Suitable filters having a pore size of 0.2-5 .mu.m, preferably 0.2-0.45 .mu.m.
From the organs, preferably the lungs, a primary cell culture can also be made which is investigated for the occurrence of cythopathogenic effects (CPE). To do this, the chopped tissue is subjected to an enzymatic by proteases. Trypsin in a concentration of 0.1-0.5% (w/v), preferably 0.125-0.25% (w/v) in a physiological, aqueous solution is particularly suitable for this purpose. The trypsin digestion is carried out at 20-37.degree. C., preferably at room temperature, in the course of 2-8 hours. Undigested tissue are separated by pieces of coarse filtration. The trypsinized cells are recovered by centrifugation at 500-1500.times.g. The cell sediment is resuspended in a suitable growth medium, such as e.g. the E-MEM described, and inoculated into culture vessels in a concentration of 10.sup.5 -10.sup.6 cells/ml of medium. Depending on the growth rate, the growth medium is exchanged every 3-7 days. The growth of the cells and the occurrence of a CPE is observed daily. The cell culture supernatant may additionally be checked for haemagglutinating properties at fixed time intervals of 2-7 days.
The centrifugation supernatants or filtrates of the organ homogenates and the cell culture supernatants of the primary organ cultures are applied in a dilution of 1:1 to 1:1000, preferably 1:10 to 1:100, to primary or permanent mammalian cell cultures and incubated at 32-39.degree. C., preferably 37.degree. C., for several days. Cell lawns are used for this purpose which are grown to 20-100%, preferably 80-100%, confluence. The cell cultures are checked daily for the occurrence of a CPE. The cell culture supernatant may additionally be checked for haemagglutinating properties at fixed intervals of 2-7 days. If no signs of virus replication occur, the cell culture supernatants are passaged further in the dilutions mentioned on fresh cell cultures. This process can be repeated two or more times.
If signs of virus replication occur, the virus is adapted to the cell culture used by further passages.
An unborn piglet was removed from the uterus of an aborting sow which originated from a herd with PRRS-like symptomatology. From a primary lung cell culture and by subsequent passaging of the culture supernatant cell lines it was possible to isolate a cythopathogenic agent from the lungs of this piglet. It was characterized as an enveloped haemagglutinating single-stranded RNA virus of about 200 nm in diameter, which showed by electron microscopic examinations the morphology of a paramyxovirus. Proteins of this virus were detected in its Western Blots an antiserum against a parainfluenza virus type 2 (Pl-2). In the same test system, an antiserum prepared against the isolated virus detected Pl-2 strain "SV5", by which the serological relationship of the isolated virus to parainfluenza virus type 2 is confirmed.
This parainfluenza isolate with the notation "SER" was deposited on Dec. 6, 1993 in the "Collection Nationale de Cultures et de Microorganismes" of the "Institut Pasteur", Paris, France under the number I-1331.
The isolated virus can be replicated on a large scale using animal cell cultures. Purified antigen preparations can be prepared from virus suspensions produced in this way by means of suitable technical processes (centrifugation, tangential filtration). These can be used as starting material for the diagnosis and for the prevention of respiratory and reproductive diseases of the pig, in particular PRRS.

EXAMPLE 1
Isolation of the parainfluenza isolate "SER"
It was possible to isolate the parainfluenza isolate "SER" from the lungs of a piglet which had been removed from the uterus by section of an aborting sow originating from a herd showing PRRS-like symptomatology, which had been euthanized.
______________________________________PK15 cells (cloned porcine kidney cell, ATCC No.CCL 33)Eagles Minimum Essential Medium containing Earl'ssalts (E-MEM):E-MEM - powder containing Phenol Red for 100 l(e.g. Gibco BRL 072-0110)Non-essential amino acids, stock solution 1000 ml100.times.Neomycin sulphate 3 gPolymyxin B sulphate 3 MUAqua purificata (EP 8) to 100 lNon-essential amino acids, stocksolution 100.times.:Alanine (EP 752) 8.9 gAsparagine monohydrate (GP 10) 15.0 gL-Aspartic acid 13.2 gGlycine (EP 614) 7.5 gGlutamic acid (EP 750) 14.7 gProline (EP 785) 11.5 gSerine (EP 788) 10.5 gAqua purificata (EP 8) to 10 lFoetal calf serum (FCS, e.g. Gibco BRL 012-06290)Growth medium: E-MEM containing 2.0 g/l of sodiumbicarbonate and 2% FCSMaintenance medium: E-MEM containing 0.85 g/l ofsodium bicarbonate and 5% FCS0.25% trypsin solution (e.g. Gibco BRL 043-05050)PBS buffer (Phosphate Buffered Saline):NaCl 8.0 gKCl 0.2 gKH.sub.2 PO.sub.4 0.2 gNa.sub.2 HPO.sub.4 .times. 12 H.sub.2 O 2.9 gAqua purificata (EP 8) to 1000 mlTissue culture flask, 80 cm.sup.2 (Roux flask,e.g. Greiner 658 170)______________________________________
A part of the piglet lung removed under sterile conditions was chopped using scissors and subjected to an enzymatic digestion in 0.25% trypsin solution. The trypsinization was carried out with stirring at room temperature for 4 hours. Undigested large pieces of tissue were separated in a sterile gauze filter. The filtrate was washed three times in PBS by low-speed centrifugation (1000.times.g, 10 minutes). The cells were inoculated in 80 cm.sup.2 culture flasks in E-MEM and addition of 5% FCS in a concentration of 10.sup.5 cells/ml and incubated at 37.degree. C. The growth medium was changed every 4-5 days. The growth of the primary culture was observed daily by microscopic examination. Attention was paid in particular to the occurrence of cytopathogenic effects (CPE). After about 2 weeks, a CPE in the form of rounding, shrinking cells having a slow tendency to spread could be observed. The culture supernatant of the primary cells was diluted 1:10 with maintenance medium and inoculated onto confluent monolayers of PK-15 cell cultures in 80 cm.sup.2 culture flasks (incubation volume: 40 ml). A non-infected culture of each cell was additionally carried out as a control. After incubation for 7 days, a CPE develops with beginning destruction of the monolayer. The culture was subjected to a freeze-thaw process and the supernatant was diluted 1:10 inoculated onto a fresh culture whose supernatant was tested after 6-7 days in the haemagglutination test using chicken erythrocytes. In the 4th passage, the cultures after incubation for 6 days exhibited a cytopathogenic effect to approximately 100%. Culture supernatants which were positive in the haemagglutination test were stored at -70.degree.C.
EXAMPLE 2
Replication of the parainfluenza isolate "SER"
Material
______________________________________Parainfluenza isolate "SER", masterseedPK-15 cells (cloned porcine kidney cell,ATCC No. CCL 33)Eagles Minimum Essential Medium containing Earle'ssalts (E-MEM):E-MEM - powder containing Phenol Red (e.g. for 100 lGibco BRL 072-0110)Non-essential amino acids, stock solution 100.times. 1000 mlNeomycin sulphate 3 gPolymyxin B sulphate 3 MUAqua purificata (EP 8) to 100 lFoetal calf serum (FCS, e.g. Gibco BRL 012-06290)Growth medium: E-MEM containing 2.0 g/l of sodiumbicarbonate and 2% FCSMaintenance medium: E-MEM containing 0.85 g/l ofsodium bicarbonate and 5% FCSTissue culture flask, 80 cm.sup.2 (Roux flask,e.g. Greiner 658 170)multi tray-disk, 6,000 cm.sup.2 (e.g. Nunc 164 327)______________________________________
Methodology
The growth medium of a tissue culture flask grown to confluence with PK-15 cells is discarded and the latter covered with 40 ml of the virus masterseed diluted 1:50 in maintenance medium. After incubation for 7 days at 37.degree. C., the contents of the tissue culture flask, subjected to a freeze-thaw process and suspended by ultrasound, are made up to a volume of 3,000 ml with maintenance medium and herewith a multi tray-disk grown to confluence with PK-15 cells is inoculated. After incubation at 37.degree. C. for 7 days, the culture supernatant is harvested and stored at -70.degree. C. until further processing.
EXAMPLE 3
Production of inactivated vaccine (parainfluenza isolate "SER")
Material
______________________________________Parainfluenza isolate "SER", cell culture supernatantfrom virus replication(s)2-Bromoethylamine hydrobromide 0.5 M stock(2-BEA) solution:2-Bromoethylamine hydrobromide 102.5 g(2-BEA, BrCH.sub.2 CH.sub.2 NH.sub.2 HBr, Merck 820176)Aqua purificata (EP 8) to 1000 mlSodium thiosulphate 2.5 M stock solution:Na.sub.2 S.sub.2 O.sub.3 .times. 5H.sub.2 O (EP 414) 620.5 gAqua purificata (EP 8) to 1000 mlAluminium hydroxide suspension 3% (e.g. Superfos)Quil A 1% stock solutionQuil A (e.g. Superfos) 10.0 gAqua purificata (EP 8) to 1000 mlThimerosal 2% stock solutionThimerosal (C.sub.9 H.sub.9 HgNaO.sub.2 S) 50.0 gAqua purificata (EP 8) to 1000 mlPBS buffer (Phosphate Buffered Saline):NaCl 8.0 gKCl 0.2 gKH.sub.2 PO.sub.4 0.2 gNa.sub.2 HPO.sub.4 .times. 12 H.sub.2 O 2.9 gAqua purificata (EP 8) to 1000 ml______________________________________
The supernatant of the virus replicated in cell cultures is purified from cells and cell debris by centrifugation at 10,000.times.g. A virus suspension purified in such a way with a concentration of virus particles of 10.sup.6.0 CID.sub.50 /ml, which originates from one or more virus harvests, is transferred to a sterile vessel. The pH is adjusted to 8.4 using sodium hydroxide solution (2N NaOH). Such an amount of 0.5M 2-bromoethylamine hydrobromide solution (2-BEA) is added with continuous stirring until a final concentration of 5 mmol/l of 2-BEA is achieved. Inactivation of the virus is carried out in the course of 18 hours at 37.degree. C. The inactivating agent is then neutralized at 4.degree. C. by addition of a 2.5M sodium thiosulphate solution up to a final concentration of 50 mmol/l.
62 ml of the inactivated virus suspension are added to 31 ml of a sterile aluminium hydroxide suspension (3% Al(OH).sub.3, pH 7.3) and the mixture is stirred at 4.degree. C. for 2 hours. After addition of 1.25 ml of Quil A (2% solution) and 0.1 ml of thimerosal (2% solution), the mixture is filled up to 100 ml with PBS buffer and stirred at 4.degree. C. for a further 20 hours. The finished vaccine is filled into multiple-dose containers and stored at 4.degree. C.
Inoculation of pigs of all age groups is carried out by subcutaneous administration of 2 ml of this vaccine.
Publications containing published nucleotide sequences which code for immunogens of simian virus 5
Hiebert, S. W., Paterson, R. G. & Lamb, R. A. (1985). Hemagglutinin-neuraminidase protein of the paramyxovirus simian virus 5: nucleotide sequence of the mRNA predicts a N-terminal membrane anchor. Journal of Virology, 54, 1-6.
Hiebert, S. W., Paterson, R. G. & Lamb, R. A. (1985). Identification and predicted sequence of a previously unrecognized small hydrophobic protein, SH, of the paramyxovirus simian virus 5. Journal of Virology, 55, 744-751.
Paterson, R. G., Harris, T. J. R. & Lamb, R. A. (1984). Analysis and gene assignment of mRNAs of a paramyxovirus, simian virus 5. Virology, 138, 310-323.
Paterson, R. G., Harris, T. J. R. & Lamb, R. A. (1984). Fusion protein of the paramyxovirus simian virus 5: nucleotide sequence of mRNA predicts a highly hydrophobic glycoprotein. Proc. Natl. Acad. Sci. USA, 81, 6706-6710.
Paterson, R. G., Hiebert, S. W. & Lamb, R. A. (1985). Expression at the cell surface of biologically active fusion and hemagglutinin/neuraminidase proteins of the paramyxovirus simian virus 5 from cloned cDNA. Proc. Natl. Acad. Sci. USA, 82, 7520-7524.
Thomas, S., Lamb, R. A. & Paterson, R. G. (1988). Two mRNAs that differ by two nontemplated nucleotides encode the amino coterminal proteins P and V of the paramyxovirus SV5. Cell, 54, 891-902.
__________________________________________________________________________# SEQUENCE LISTING- (1) GENERAL INFORMATION:- (iii) NUMBER OF SEQUENCES: 4- (2) INFORMATION FOR SEQ ID NO: 1:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 1698 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: DNA (genomic)- (iii) HYPOTHETICAL: NO- (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION:1..1695#1: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- ATG GTT GCA GAA GAT GCC CCT GTT AGG GGC AC - #T TGC CGA GTA TTA TTT 48Met Val Ala Glu Asp Ala Pro Val Arg Gly Th - #r Cys Arg Val Leu Phe# 15- CGA ACA ACA ACT TTA ATT TTT CTA TGC ACA CT - #A CTA GCA TTA AGC ATC 96Arg Thr Thr Thr Leu Ile Phe Leu Cys Thr Le - #u Leu Ala Leu Ser Ile# 30- TCT ATC CTT TAT GAG AGT TTA ATA ACC CAA AA - #G CAA ATC ATG AGC CAC 144Ser Ile Leu Tyr Glu Ser Leu Ile Thr Gln Ly - #s Gln Ile Met Ser His# 45- GCA GGA TAC ACT CGA TCT AAT TCT AGA TTA GG - #A AGT ATC ACT GAT CTT 192Ala Gly Tyr Thr Arg Ser Asn Ser Arg Leu Gl - #y Ser Ile Thr Asp Leu# 60- CTT AAT AAT ATT CTC TCT GTC GCA AAT CAG AT - #T ATA TAT AAC TCT GCA 240Leu Asn Asn Ile Leu Ser Val Ala Asn Gln Il - #e Ile Tyr Asn Ser Ala# 80- GTC GCT CTA CCT CTA CAA TTG GAC ACT CTT GA - #A TCA ACA CTC CTT ACA 288Val Ala Leu Pro Leu Gln Leu Asp Thr Leu Gl - #u Ser Thr Leu Leu Thr# 95- GCC ATT AAG TCT CTT CAA ACC AGT GAC AAG CT - #A GAA CAG AAC TGC TCG 336Ala Ile Lys Ser Leu Gln Thr Ser Asp Lys Le - #u Glu Gln Asn Cys Ser# 110- TGG GGT GCT GCA CTG ATT AAT AAT AAT AGA TA - #C ATT AAT GGC ATC AAT 384Trp Gly Ala Ala Leu Ile Asn Asn Asn Arg Ty - #r Ile Asn Gly Ile Asn# 125- CAG TTC TAT TTT TCA ATT GCT GAG GGT CGC AA - #T CTG ACA CTT GGC CCA 432Gln Phe Tyr Phe Ser Ile Ala Glu Gly Arg As - #n Leu Thr Leu Gly Pro# 140- CTT CTT AAT ATA CCT AGT TTC ATT CCA ACT GC - #C ACG ACA CCA GAG GGC 480Leu Leu Asn Ile Pro Ser Phe Ile Pro Thr Al - #a Thr Thr Pro Glu Gly145 1 - #50 1 - #55 1 -#60- TGC ACC AGG ATC CCA TCA TTC TCG CTC ACC AA - #G ACA CAC TGG TGT TAT 528Cys Thr Arg Ile Pro Ser Phe Ser Leu Thr Ly - #s Thr His Trp Cys Tyr# 175- ACA CAC AAT GTT ATC CTG AAT GGA TGC CAG GA - #T CAT GTA TCC TCA AAT 576Thr His Asn Val Ile Leu Asn Gly Cys Gln As - #p His Val Ser Ser Asn# 190- CAA TTT GTT TCC ATG GGA ATC ATT GAA CCC AC - #T TCT GCC GGG TTT CCA 624Gln Phe Val Ser Met Gly Ile Ile Glu Pro Th - #r Ser Ala Gly Phe Pro# 205- TCC TTT CGA ACC CTA AAG ACT CTA TAT CTC AG - #C GAT GGG GTC AAT CGT 672Ser Phe Arg Thr Leu Lys Thr Leu Tyr Leu Se - #r Asp Gly Val Asn Arg# 220- AAG AGC TGC TCT ATC AGT ACA GTT CCG GGG GG - #T TGT ATG ATG TAC TGT 720Lys Ser Cys Ser Ile Ser Thr Val Pro Gly Gl - #y Cys Met Met Tyr Cys225 2 - #30 2 - #35 2 -#40- TTT GTC TCT ACT CAA CCA GAG AGG GAT GAC TA - #C TTT TCT ACC GCT CCT 768Phe Val Ser Thr Gln Pro Glu Arg Asp Asp Ty - #r Phe Ser Thr Ala Pro# 255- CCA GAA CAA CGA ATT ATT ATA ATG TAC TAT AA - #T GAT ACA ATC GTG GAG 816Pro Glu Gln Arg Ile Ile Ile Met Tyr Tyr As - #n Asp Thr Ile Val Glu# 270- CGC ATA ATT AAT CCA CCC GGG GTA CTA GAT GT - #A TGG GCA ACA TTG ACC 864Arg Ile Ile Asn Pro Pro Gly Val Leu Asp Va - #l Trp Ala Thr Leu Thr# 285- CCA GGA ACA GGA AGC GGG GTA TAT TAT TTA GG - #T TGG GTG CTC TTT CCA 912Pro Gly Thr Gly Ser Gly Val Tyr Tyr Leu Gl - #y Trp Val Leu Phe Pro# 300- ATA TAT GGC GGC GTG ATT AAA GAT ACG AGT TT - #A TGG AAT AAT CAA GCA 960Ile Tyr Gly Gly Val Ile Lys Asp Thr Ser Le - #u Trp Asn Asn Gln Ala305 3 - #10 3 - #15 3 -#20- AAT AAA TAC TTT ATC CCC CAG ATG GTT GCT GC - #T CTC TGC TCA CAA AAC1008Asn Lys Tyr Phe Ile Pro Gln Met Val Ala Al - #a Leu Cys Ser Gln Asn# 335- CAG GCA ACT CAA GTC CAA AAT GCT AAG TCA TC - #A TAC TAT AGC AGC TGG1056Gln Ala Thr Gln Val Gln Asn Ala Lys Ser Se - #r Tyr Tyr Ser Ser Trp# 350- TTT GGC AAT CGA ATG ATT CAG TCT GGG ATC CT - #G GCA TGT CCT CTT CAA1104Phe Gly Asn Arg Met Ile Gln Ser Gly Ile Le - #u Ala Cys Pro Leu Gln# 365- CAG GAT CTA ACC AAT GAG TGT TTA GTT CTG CC - #C TTT TCT AAT GAT CAG1152Gln Asp Leu Thr Asn Glu Cys Leu Val Leu Pr - #o Phe Ser Asn Asp Gln# 380- GTG CTT ATG GGT GCT GAA GGG AGA TTA TAC AT - #G TAT GGT GAC TCG GTG1200Val Leu Met Gly Ala Glu Gly Arg Leu Tyr Me - #t Tyr Gly Asp Ser Val385 3 - #90 3 - #95 4 -#00- TAT TAC TAC CAA AGA AGC AAT AGT TGG TGG CC - #T ATG ACC ATG CTG TAT1248Tyr Tyr Tyr Gln Arg Ser Asn Ser Trp Trp Pr - #o Met Thr Met Leu Tyr# 415- AAG GTA ACC ATA ACA TTC ACT AAT GGT CAG CC - #A TCT GCT ATA TCA GCT1296Lys Val Thr Ile Thr Phe Thr Asn Gly Gln Pr - #o Ser Ala Ile Ser Ala# 430- CAG AAT GTG CCC ACA CAG CAG GTC CCT AGA CC - #T GGG ACA GGA GCC TGC1344Gln Asn Val Pro Thr Gln Gln Val Pro Arg Pr - #o Gly Thr Gly Ala Cys# 445- TCT GCA ACA AAT AGA TGT CCC GGT TTT TGC TT - #G AAA GGA GTG TAT GCT1392Ser Ala Thr Asn Arg Cys Pro Gly Phe Cys Le - #u Lys Gly Val Tyr Ala# 460- GAT GCC TGG TTA CTG ACC AAC CCT TCG TCT AC - #C AGT ACA TTT GGA TCA1440Asp Ala Trp Leu Leu Thr Asn Pro Ser Ser Th - #r Ser Thr Phe Gly Ser465 4 - #70 4 - #75 4 -#80- GAA GCA ACC TTC ACT GGT TCT TAT CTC AAC GC - #A GCA ACT CAG CGT ATC1488Glu Ala Thr Phe Thr Gly Ser Tyr Leu Asn Al - #a Ala Thr Gln Arg Ile# 495- AAT CCG ACG ATG TAT ATC GCG AAC AAC ACA CA - #G ATC ATA AGC TCA CAG1536Asn Pro Thr Met Tyr Ile Ala Asn Asn Thr Gl - #n Ile Ile Ser Ser Gln# 510- CAA TTT GGA TCA AGC GGT CAA GAA GCA GCA TA - #T AGC CAC ACA ACT TGT1584Gln Phe Gly Ser Ser Gly Gln Glu Ala Ala Ty - #r Ser His Thr Thr Cys# 525- TTT AGG GAC ACA GGC TCT GTT ATG GTA TAC TG - #T CTC TAT ATT ATT GAA1632Phe Arg Asp Thr Gly Ser Val Met Val Tyr Cy - #s Leu Tyr Ile Ile Glu# 540- TTG TCC TCA TCT CTC TTA GGA CAA TTT CAG AT - #T GTC CCA TTT ATC CGT1680Leu Ser Ser Ser Leu Leu Gly Gln Phe Gln Il - #e Val Pro Phe Ile Arg545 5 - #50 5 - #55 5 -#60#1698 CC TAAGln Val Thr Leu Ser 565- (2) INFORMATION FOR SEQ ID NO: 2:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 565 amino (B) TYPE: amino acid (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein#2: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- Met Val Ala Glu Asp Ala Pro Val Arg Gly Th - #r Cys Arg Val Leu Phe# 15- Arg Thr Thr Thr Leu Ile Phe Leu Cys Thr Le - #u Leu Ala Leu Ser Ile# 30- Ser Ile Leu Tyr Glu Ser Leu Ile Thr Gln Ly - #s Gln Ile Met Ser His# 45- Ala Gly Tyr Thr Arg Ser Asn Ser Arg Leu Gl - #y Ser Ile Thr Asp Leu# 60- Leu Asn Asn Ile Leu Ser Val Ala Asn Gln Il - #e Ile Tyr Asn Ser Ala# 80- Val Ala Leu Pro Leu Gln Leu Asp Thr Leu Gl - #u Ser Thr Leu Leu Thr# 95- Ala Ile Lys Ser Leu Gln Thr Ser Asp Lys Le - #u Glu Gln Asn Cys Ser# 110- Trp Gly Ala Ala Leu Ile Asn Asn Asn Arg Ty - #r Ile Asn Gly Ile Asn# 125- Gln Phe Tyr Phe Ser Ile Ala Glu Gly Arg As - #n Leu Thr Leu Gly Pro# 140- Leu Leu Asn Ile Pro Ser Phe Ile Pro Thr Al - #a Thr Thr Pro Glu Gly145 1 - #50 1 - #55 1 -#60- Cys Thr Arg Ile Pro Ser Phe Ser Leu Thr Ly - #s Thr His Trp Cys Tyr# 175- Thr His Asn Val Ile Leu Asn Gly Cys Gln As - #p His Val Ser Ser Asn# 190- Gln Phe Val Ser Met Gly Ile Ile Glu Pro Th - #r Ser Ala Gly Phe Pro# 205- Ser Phe Arg Thr Leu Lys Thr Leu Tyr Leu Se - #r Asp Gly Val Asn Arg# 220- Lys Ser Cys Ser Ile Ser Thr Val Pro Gly Gl - #y Cys Met Met Tyr Cys225 2 - #30 2 - #35 2 -#40- Phe Val Ser Thr Gln Pro Glu Arg Asp Asp Ty - #r Phe Ser Thr Ala Pro# 255- Pro Glu Gln Arg Ile Ile Ile Met Tyr Tyr As - #n Asp Thr Ile Val Glu# 270- Arg Ile Ile Asn Pro Pro Gly Val Leu Asp Va - #l Trp Ala Thr Leu Thr# 285- Pro Gly Thr Gly Ser Gly Val Tyr Tyr Leu Gl - #y Trp Val Leu Phe Pro# 300- Ile Tyr Gly Gly Val Ile Lys Asp Thr Ser Le - #u Trp Asn Asn Gln Ala305 3 - #10 3 - #15 3 -#20- Asn Lys Tyr Phe Ile Pro Gln Met Val Ala Al - #a Leu Cys Ser Gln Asn# 335- Gln Ala Thr Gln Val Gln Asn Ala Lys Ser Se - #r Tyr Tyr Ser Ser Trp# 350- Phe Gly Asn Arg Met Ile Gln Ser Gly Ile Le - #u Ala Cys Pro Leu Gln# 365- Gln Asp Leu Thr Asn Glu Cys Leu Val Leu Pr - #o Phe Ser Asn Asp Gln# 380- Val Leu Met Gly Ala Glu Gly Arg Leu Tyr Me - #t Tyr Gly Asp Ser Val385 3 - #90 3 - #95 4 -#00- Tyr Tyr Tyr Gln Arg Ser Asn Ser Trp Trp Pr - #o Met Thr Met Leu Tyr# 415- Lys Val Thr Ile Thr Phe Thr Asn Gly Gln Pr - #o Ser Ala Ile Ser Ala# 430- Gln Asn Val Pro Thr Gln Gln Val Pro Arg Pr - #o Gly Thr Gly Ala Cys# 445- Ser Ala Thr Asn Arg Cys Pro Gly Phe Cys Le - #u Lys Gly Val Tyr Ala# 460- Asp Ala Trp Leu Leu Thr Asn Pro Ser Ser Th - #r Ser Thr Phe Gly Ser465 4 - #70 4 - #75 4 -#80- Glu Ala Thr Phe Thr Gly Ser Tyr Leu Asn Al - #a Ala Thr Gln Arg Ile# 495- Asn Pro Thr Met Tyr Ile Ala Asn Asn Thr Gl - #n Ile Ile Ser Ser Gln# 510- Gln Phe Gly Ser Ser Gly Gln Glu Ala Ala Ty - #r Ser His Thr Thr Cys# 525- Phe Arg Asp Thr Gly Ser Val Met Val Tyr Cy - #s Leu Tyr Ile Ile Glu# 540- Leu Ser Ser Ser Leu Leu Gly Gln Phe Gln Il - #e Val Pro Phe Ile Arg545 5 - #50 5 - #55 5 -#60- Gln Val Thr Leu Ser 565- (2) INFORMATION FOR SEQ ID NO: 3:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 1656 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: DNA (genomic)- (iii) HYPOTHETICAL: NO- (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION:1..1653#3: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- ATG GGT ACT ATA ATT CAA TTT CTG GTG GTC TC - #C TGT CTA TTG GCA GGA 48Met Gly Thr Ile Ile Gln Phe Leu Val Val Se - #r Cys Leu Leu Ala Gly# 580- GCA GGC AGC CCT GAT CCA GCA GCC CTC ATG CA - #A ATC GGT GTC ATT CCA 96Ala Gly Ser Pro Asp Pro Ala Ala Leu Met Gl - #n Ile Gly Val Ile Pro# 595- ACA AAT GTC CGG CAA CTT ATG TAT TAT ACT GA - #G GCC TCA TCA GCA TTC 144Thr Asn Val Arg Gln Leu Met Tyr Tyr Thr Gl - #u Ala Ser Ser Ala Phe# 610- ATT GTT GTG AAG TTA ATG CCT ACA ATT GAC TC - #G CCG ATT AGT GGA TGT 192Ile Val Val Lys Leu Met Pro Thr Ile Asp Se - #r Pro Ile Ser Gly Cys# 625- AAT ATA ACA TCA ATT TCA AGC TAT AAT GCA AC - #A CTG ACA AAA CTC CTA 240Asn Ile Thr Ser Ile Ser Ser Tyr Asn Ala Th - #r Leu Thr Lys Leu Leu630 6 - #35 6 - #40 6 -#45- CAG CCG ATC GGT GAG AAT TTG GAA ACG ATT AG - #G AAC CAG TTG ATT CCA 288Gln Pro Ile Gly Glu Asn Leu Glu Thr Ile Ar - #g Asn Gln Leu Ile Pro# 660- ACT CGG AGG AGA CGC CGG TTT GCA GGG GTG GT - #G ATT GGA TTA GCT GCA 336Thr Arg Arg Arg Arg Arg Phe Ala Gly Val Va - #l Ile Gly Leu Ala Ala# 675- TTA GGA GTA GCT ACT GCC GCA CAG GTC ACT GC - #C GCA GTA GCA CTA GTA 384Leu Gly Val Ala Thr Ala Ala Gln Val Thr Al - #a Ala Val Ala Leu Val# 690- AAG GCA AAT AAA AAT GCT GCG GCT ATA CTC AA - #T CTC AAA AAT GCA ATC 432Lys Ala Asn Lys Asn Ala Ala Ala Ile Leu As - #n Leu Lys Asn Ala Ile# 705- CAA AAA ACA AAT ACA GCA GTT GCA GAT GTG GT - #C CAG GCC ACA CAA TCA 480Gln Lys Thr Asn Thr Ala Val Ala Asp Val Va - #l Gln Ala Thr Gln Ser710 7 - #15 7 - #20 7 -#25- CTA GGA ACG GCA GTT CAA GCA GTT CAA GAT CA - #C ATA AAC AGT GTG GTA 528Leu Gly Thr Ala Val Gln Ala Val Gln Asp Hi - #s Ile Asn Ser Val Val# 740- AGT CCA GCA ATT ACA GCA GCC AAT TGT AAG GC - #C CAA GAT GCT ATC ATT 576Ser Pro Ala Ile Thr Ala Ala Asn Cys Lys Al - #a Gln Asp Ala Ile Ile# 755- GGC TCA ATC CTC AAT CTC TAT TTG ACC GAG TT - #G ACA ACT ATC TTC CAC 624Gly Ser Ile Leu Asn Leu Tyr Leu Thr Glu Le - #u Thr Thr Ile Phe His# 770- AAT CAA ATT ACA AAC CCT GCA TTG AGT CCT AT - #T ACA ATT CAA GCT TTA 672Asn Gln Ile Thr Asn Pro Ala Leu Ser Pro Il - #e Thr Ile Gln Ala Leu# 785- AGG ATC CTA CTG GGG AGT ACC TTG CCG ACT GT - #G GTC GAA AAA TCT TTC 720Arg Ile Leu Leu Gly Ser Thr Leu Pro Thr Va - #l Val Glu Lys Ser Phe790 7 - #95 8 - #00 8 -#05- AAT ACC CAG ATA AGT GCA GCT GAG CTT CTC TC - #A TCA GGG TTA TTG ACA 768Asn Thr Gln Ile Ser Ala Ala Glu Leu Leu Se - #r Ser Gly Leu Leu Thr# 820- GGC CAG ATT GTG GGA TTA GAT TTG ACC TAT AT - #G CAG ATG GTC ATA AAA 816Gly Gln Ile Val Gly Leu Asp Leu Thr Tyr Me - #t Gln Met Val Ile Lys# 835- ATT GAG CTG CCA ACT TTA ACT GTA CAA CCT GC - #A ACC CAG ATC ATA GAT 864Ile Glu Leu Pro Thr Leu Thr Val Gln Pro Al - #a Thr Gln Ile Ile Asp# 850- CTG GCC ACC ATT TCT GCA TTC ATT AAC AAT CA - #A GAA GTC ATG GCC CAA 912Leu Ala Thr Ile Ser Ala Phe Ile Asn Asn Gl - #n Glu Val Met Ala Gln# 865- TTA CCA ACA CGT GTT ATG GTG ACT GGC AGC TT - #G ATC CAA GCC TAT CCC 960Leu Pro Thr Arg Val Met Val Thr Gly Ser Le - #u Ile Gln Ala Tyr Pro870 8 - #75 8 - #80 8 -#85- GCA TCG CAA TGC ACT ATT ACA CCC AAC ACT GT - #G TAC TGT AGG TAT AAT1008Ala Ser Gln Cys Thr Ile Thr Pro Asn Thr Va - #l Tyr Cys Arg Tyr Asn# 900- GAT GCC CAA GTA CTC TCA GAT GAT ACG ATG GC - #T TGC CTC CAA GGT AAC1056Asp Ala Gln Val Leu Ser Asp Asp Thr Met Al - #a Cys Leu Gln Gly Asn# 915- TTG ACA AGA TGC ACC TTC TCT CCG GTG GTT GG - #G AGC TTT CTC ACT CGA1104Leu Thr Arg Cys Thr Phe Ser Pro Val Val Gl - #y Ser Phe Leu Thr Arg# 930- TTC ATG CTG TTC GAT GGA ATA GTT TAT GCA AA - #T TGC AGG TCG ATG TTA1152Phe Met Leu Phe Asp Gly Ile Val Tyr Ala As - #n Cys Arg Ser Met Leu# 945- TGC AAG TGC ATG CAG CCT GCT GCT GTG ATC CT - #A CAG CCG AGT TCA TCC1200Cys Lys Cys Met Gln Pro Ala Ala Val Ile Le - #u Gln Pro Ser Ser Ser950 9 - #55 9 - #60 9 -#65- CCT GTA ACT GTC ATT GAC ATG TAC AAA TGT GT - #G AGT CTG CAG CTT GAC1248Pro Val Thr Val Ile Asp Met Tyr Lys Cys Va - #l Ser Leu Gln Leu Asp# 980- AAT CTC AGA TTC ACC ATC ACT CAA TTG GCC AA - #T GTA ACC TAC AAT AGC1296Asn Leu Arg Phe Thr Ile Thr Gln Leu Ala As - #n Val Thr Tyr Asn Ser# 995- ACC ATC AAG CTT GAA ACA TCC CAG ATC TTG CC - #T ATT GAT CCG TTG GAT1344Thr Ile Lys Leu Glu Thr Ser Gln Ile Leu Pr - #o Ile Asp Pro Leu Asp# 10105- ATA TCC CAG AAT CTA GCT GCG GTG AAT AAG AG - #T CTA AGT GAT GCA CTA1392Ile Ser Gln Asn Leu Ala Ala Val Asn Lys Se - #r Leu Ser Asp Ala Leu# 10250- CAA CAC TTA GCA CAA AGT GAC ACA TAC CTT TC - #T GCA ATC ACA TCA GCT1440Gln His Leu Ala Gln Ser Asp Thr Tyr Leu Se - #r Ala Ile Thr Ser Ala# 10451035 - # 1040- ACG ACT ACA AGT GTA TTA TCC ATA ATG GCA AT - #C TGT CTT GGA TCG TTA1488Thr Thr Thr Ser Val Leu Ser Ile Met Ala Il - #e Cys Leu Gly Ser Leu# 10605- GGT TTA ATA TTA ATA ATC TTG CTC AGT GTA GT - #T GTG TGG AAG TTA TTG1536Gly Leu Ile Leu Ile Ile Leu Leu Ser Val Va - #l Val Trp Lys Leu Leu# 10750- ACC ATT GTC ACT GCT AAT CGA AAT AGA ATG GA - #G AAT TTT GTT TAT CAT1584Thr Ile Val Thr Ala Asn Arg Asn Arg Met Gl - #u Asn Phe Val Tyr His# 10905- AAT TCA GCA TTC CAC CAC TCA CGA TCT GAT CT - #C AGT GAG AAA AAT CAA1632Asn Ser Ala Phe His His Ser Arg Ser Asp Le - #u Ser Glu Lys Asn Gln# 11050# 1656GA ACA AGA TAAPro Ala Thr Leu Gly Thr Arg1110 1115- (2) INFORMATION FOR SEQ ID NO: 4:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 551 amino (B) TYPE: amino acid (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein#4: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- Met Gly Thr Ile Ile Gln Phe Leu Val Val Se - #r Cys Leu Leu Ala Gly# 15- Ala Gly Ser Pro Asp Pro Ala Ala Leu Met Gl - #n Ile Gly Val Ile Pro# 30- Thr Asn Val Arg Gln Leu Met Tyr Tyr Thr Gl - #u Ala Ser Ser Ala Phe# 45- Ile Val Val Lys Leu Met Pro Thr Ile Asp Se - #r Pro Ile Ser Gly Cys# 60- Asn Ile Thr Ser Ile Ser Ser Tyr Asn Ala Th - #r Leu Thr Lys Leu Leu# 80- Gln Pro Ile Gly Glu Asn Leu Glu Thr Ile Ar - #g Asn Gln Leu Ile Pro# 95- Thr Arg Arg Arg Arg Arg Phe Ala Gly Val Va - #l Ile Gly Leu Ala Ala# 110- Leu Gly Val Ala Thr Ala Ala Gln Val Thr Al - #a Ala Val Ala Leu Val# 125- Lys Ala Asn Lys Asn Ala Ala Ala Ile Leu As - #n Leu Lys Asn Ala Ile# 140- Gln Lys Thr Asn Thr Ala Val Ala Asp Val Va - #l Gln Ala Thr Gln Ser145 1 - #50 1 - #55 1 -#60- Leu Gly Thr Ala Val Gln Ala Val Gln Asp Hi - #s Ile Asn Ser Val Val# 175- Ser Pro Ala Ile Thr Ala Ala Asn Cys Lys Al - #a Gln Asp Ala Ile Ile# 190- Gly Ser Ile Leu Asn Leu Tyr Leu Thr Glu Le - #u Thr Thr Ile Phe His# 205- Asn Gln Ile Thr Asn Pro Ala Leu Ser Pro Il - #e Thr Ile Gln Ala Leu# 220- Arg Ile Leu Leu Gly Ser Thr Leu Pro Thr Va - #l Val Glu Lys Ser Phe225 2 - #30 2 - #35 2 -#40- Asn Thr Gln Ile Ser Ala Ala Glu Leu Leu Se - #r Ser Gly Leu Leu Thr# 255- Gly Gln Ile Val Gly Leu Asp Leu Thr Tyr Me - #t Gln Met Val Ile Lys# 270- Ile Glu Leu Pro Thr Leu Thr Val Gln Pro Al - #a Thr Gln Ile Ile Asp# 285- Leu Ala Thr Ile Ser Ala Phe Ile Asn Asn Gl - #n Glu Val Met Ala Gln# 300- Leu Pro Thr Arg Val Met Val Thr Gly Ser Le - #u Ile Gln Ala Tyr Pro305 3 - #10 3 - #15 3 -#20- Ala Ser Gln Cys Thr Ile Thr Pro Asn Thr Va - #l Tyr Cys Arg Tyr Asn# 335- Asp Ala Gln Val Leu Ser Asp Asp Thr Met Al - #a Cys Leu Gln Gly Asn# 350- Leu Thr Arg Cys Thr Phe Ser Pro Val Val Gl - #y Ser Phe Leu Thr Arg# 365- Phe Met Leu Phe Asp Gly Ile Val Tyr Ala As - #n Cys Arg Ser Met Leu# 380- Cys Lys Cys Met Gln Pro Ala Ala Val Ile Le - #u Gln Pro Ser Ser Ser385 3 - #90 3 - #95 4 -#00- Pro Val Thr Val Ile Asp Met Tyr Lys Cys Va - #l Ser Leu Gln Leu Asp# 415- Asn Leu Arg Phe Thr Ile Thr Gln Leu Ala As - #n Val Thr Tyr Asn Ser# 430- Thr Ile Lys Leu Glu Thr Ser Gln Ile Leu Pr - #o Ile Asp Pro Leu Asp# 445- Ile Ser Gln Asn Leu Ala Ala Val Asn Lys Se - #r Leu Ser Asp Ala Leu# 460- Gln His Leu Ala Gln Ser Asp Thr Tyr Leu Se - #r Ala Ile Thr Ser Ala465 4 - #70 4 - #75 4 -#80- Thr Thr Thr Ser Val Leu Ser Ile Met Ala Il - #e Cys Leu Gly Ser Leu# 495- Gly Leu Ile Leu Ile Ile Leu Leu Ser Val Va - #l Val Trp Lys Leu Leu# 510- Thr Ile Val Thr Ala Asn Arg Asn Arg Met Gl - #u Asn Phe Val Tyr His# 525- Asn Ser Ala Phe His His Ser Arg Ser Asp Le - #u Ser Glu Lys Asn Gln# 540- Pro Ala Thr Leu Gly Thr Arg545 5 - #50__________________________________________________________________________

Number	Date	Country
0 013 188 A2	Jan 1979	EPX
0013188	Sep 1980	EPX
8808718	Nov 1988	WOX
WO 8808718	Nov 1988	WOX
9002566	Mar 1990	WOX
WO 9002566	Mar 1990	WOX
9307898	Apr 1993	WOX
WO 9307898	Apr 1993	WOX

Porcine parainfluenza virus type 2

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Date Issued

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Abstract

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Priority Claims (1)

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Foreign Referenced Citations (8)

Non-Patent Literature Citations (9)

Entry
Chanock et al Parainfluenza virus in Virology Ed Fields Raven Press New York p. 1250, 1985.
Itoh et al. Single amino substitution of sendai virus at the cleavage site of the fusion confers trypsin resistance. J. Gen. Virology 68:2939-2944, 1987.
Christianson, W.T. et al. Swine Health and Production, vol. 2, No. 2, p. 10-28, Apr. 1994.
Crandell, R.A. et al. Journal of Clinical Microbiology, vol. 7, No. 2, pp. 214-218, Feb. 1978.
Fenner, F.J. et al. Veterinary Virology, second edition, pp. 476-478. Academic Press, Inc., San Diego CA, 1993.
Goyal, S.M. et al. The Veterinary Record, vol. 119, No. 14, p. 363, Oct. 1986.
Guenov, I. et al. Zentralblatt fur Veterinarymedizin, vol. 19B (Heft 6), pp. 437-444, abstract only, 1972.
Hernandez Jauregui, P. et al. Veterinaria Mexico, vol. 23, pp. 217-222, abstract only, 1992.
The Veterinary Record, vol. 129, No. 1, p. 19, London Jul. 6, 1991.