GB2282811A

GB2282811A - Viral vaccine for the prevention of porcine reproductive and respiratory syndrome

Info

Publication number: GB2282811A
Application number: GB9418775A
Authority: GB
Inventors: Duran D Juan Plana
Original assignee: Cyanamid Iberica SA
Current assignee: Wyeth Farma SA
Priority date: 1993-09-17
Filing date: 1994-09-16
Publication date: 1995-04-19
Anticipated expiration: 2014-09-16
Also published as: DE4432338C2; NL9401414A; ITTO940713A1; ITTO940713A0; NL194940C; FR2709966B1; GB9418775D0; NL194940B; DE4432338A1; DK173128B1; IT1267448B1; ES2074950A1; GB2282811B; ES2074950B1; FR2709966A1; DK102894A

Description

2282811 1 VACCINE FOR THE PREVENTION OF PORCINE REPRODUCTIVE AND

RESPIRATORY SYNDROME SCOPE OF THE INVENTION This irivention relates to a vaccine capable of preventing porcine reproductive and respiratory syndrome, in particular, to an inactivated vaccine containing the causative virus of the said disease in inactivated form.

BACKGROUND OF THE INVENTION is The disease known as porcine reproductive and respiratory syndrome (PRRS) affects pregnant sows in which it can provoke anorexia, abortions, stillbirths, mummified fetuses, weak piglets that die in a few hours or days of life, respiratory post farrowing problems and breeding problems (Loula, T., "Clinical Presentation of Mystery Pig Disease in the breeding herd and suckling piglets", Proceedings of the Mystery Swine Disease Committee Meeting, October 6, 1990, Denver, Colorado, Livestock Conservation Institute, Madison, WI, (USA). Some cases have been described in which infected sows present blue spots on the ears for which reason the disease has also been known as "Blue abortion", or "Blue-eared pig disease" (Veterinary Record, Vol. 130, no. 3, January 18, 1992). Other names given to the disease are "Mystery Pig Disease" (MPD), "Mystery Swine Disease" (MSD), "Mysterious Reproductive Syndrome" (MRS), "Swine Infertility and Respiratory Syndrome (SIARS) or "Porcine Epidemic Abortion and Respiratory Syndrome" (PEARS).

The first epizootic outbreaks of this disease appeared in the United States and Canada in 1987. In Europe, the first outbreak was detected in Germany in 1990, from where it spread to the Netherlands and Belgium late in 1990 and early in 1991. In Spain, the first cases of the disease were detected in mid- 2 January 1991, when important respiratory alterations were observed in a 300-piglet batch imported from Germany (Plana et al., Med. Vet., Vol. 8, no. 11, 1991). Shortly afterwards, in two breeding herds that were situated 500 meters from the herd where the initial problem had appeared, a disease was detected characterized by an abnormally high number of abortions during the last phase of gestation, as well as 70% mortality in piglets. Analysing thle observed clinical signs, and bearing in mind that (i) these herds were subjected to an intensive vaccination program against porcine parvovirosis, Aujeszky, s disease and swine influenza, and that (ii) laboratory trials had discarded the presence of other abortive diseases, the clinical presence of PRRS was suspected. In samples from these farms the causative agent of the disease has been isolated, as described in further detail below.

A number of agents have been correlated with this infectious process, among which are: encephalomyocarditis virus, swine influenza, classic swine fever, African swine fever, mucosal disease, Aujeszkyls disease, brucellosis, leptospirosis, Q fever, parvovirosis and chlamydia disease, although some authors have also related it to mycotoxins (Loula, T., "Clinical Presentation of Mystery Pig Disease in the breeding herd and suckling piglets", Proceedings of the Mystery Swine Disease Committee Meeting, supra, Mengeling, W.L. and Lager, K.M., "Mystery Pig Disease: Evidence and considerations for its etiology", in Proceedings of the Mystery Swine Disease Committee Meeting, supra; Dea et al., "Virus isolation from farms in Quebec experiencing severe outbreaks of respiratory and reproductive problems", Proceedings of the Mystery Swine Disease Committee Meeting, supra; Van Alstine, W., "Past diagnostic approaches and findings and potential useful diagnostic strategies", in Proceedings of the Mystery Swine Disease Committee Meeting, supra; Loula, T. Agri-Practice, 12(l): 23-33, 1991; Woolen, N.

et al., J. Am. Vet. Assoc. 197:600-601, 1990).

The PCT patent request published under publication number 11 3 WO 92/21375, in the name of Stitching Centraal Diergeneeskundig Instituut (CDI), describes the isolation of a virus denominated I'Lelystad Agent" (LA) or I'Lelystad Virus (LV) which is identified by them as the causative agent of the disease denominated at that time as MSD. The LV isolated when inoculated intranasally in pregnant sows produces loss of appetite and even refusal to ingest during days 4 to 10-12 post inoculation, reproductive disorders, ind a blue colouring on the ears of some of the infected sows of days 9-10 post inoculation. However, it is also observed that in 2 of the 8 experimentally infected sows the disease does not reproduce (see Table 6 of the said PCT request), since in one of the sows the number of piglets born alive and surviving the first week is large (6 out 9, sow 1305) and another sow had two stillborn piglets whereas the other 9 survived the is first week (sow 1065). The LV virus isolated by CDI belongs to the Arteriviridae genus, has a genome made up of one polyadenylated RNA molecule 14. 5 to 15. 5 kb in length (determined in neutral agarose gel), which replicates by means of a set of subgenomic RNAs at the 31 end. The above-indicated PCT request shows the LV genome nucleotide sequence, the 8 possible open reading frames (ORF), the amino acid sequence deduced from the identified ORFs and the putative sites of N-glycosilation. Posteriorly, other viruses causative of PRRS have been isolated in German, French and Spanish farms. The virus isolated in Tubingen, Germany (TV) [ (Virology, 193, 329-339 (1993)l presents 99.3-0. homology to LV at the nucleotide level contained in ORFs 2 to 7 (there are 24 different base pairs (bp) out of the total of 3316 bp included in that region). The deduced TV amino acid sequence presents 99.20-. homology to LV (there are only 10 different amino acids in the deduced amino acid sequences coded by ORFs 2 to 5, since there are no differences in the sequences deduced from ORFs 6 and 7).

on the other hand, the homology found between LV and TV 35 against the virus isolated at our laboratory (Spanish strain or SV) is smaller. Up till now, only the nucleotide and amino acid sequences corresponding to ORFs 3 to 7 have been compared, with 4 the following results:

1) There is 95.5% homology at nucleotide level of SV in front of LV or TV (out of a total of 2599bp there are 144 that 5 are different); and 2) There is 94.9% homology at amino acid level of SV in front of LV or TV (out of a total of 955 amino acids, a total of 47 amino acids are observed to be different).

These amino acid variations may be related to the higher pathogenicity of a strain in comparison with another, for reason that the virus isolated at our laboratories (Spanish strain) is more pathogenic than other known PRRS viruses. For example, the virus isolated in France (French strain), as shown in Example 8 of this description, where it can be seen that the percentage of piglets born alive to a sow infected with the French strain is 75-. whereas the percentage is 9.511 when sows are infected with the Spanish strain (Tables 12 and 14). The percentage is 611 when sows are infected with LV (Table 6 of the PCT request no. WO 92/21375) since 58 piglets were born alive out of a total of 95 piglets.

The disease causes severe losses to the porcine industry as it can provoke, in acute outbreaks, mortality of 7096 of the piglets in a litter. A means to solve the problem created would be to conduct a suitable vaccination program that would allow the prevention of the appearance of the disease. To that end, vaccines, capable of bringing about effective prevention of PRRS would be required.

The PCT patent request published under no. WO 93/06211 in the name of Collins, J.E., and Benfield, D.A., refers to a vaccine against MSD containing an infectious agent isolated from lungs of pigs infected with MSD. However, the said PCT request does not describe the characterization or identification of the infectious agent, nor has it been deposited to any Authorized Deposit Institution. For all this, the realization of the knowledge derived from that PCT request presents serious reproducibility problems because the product described in the PCT request can not be verified nor the obtained results compared with those obtained by the petitioners of the PCT. Likewise, in this PCT request are not clearly expressed the antigen nor the adjuvant used in the formulation of the vaccine which, as will be verified, posteriorly, play a very important part, nor are examples described that demonstrate the potency and efficacy of the said vaccines.

The PCT request published under no. WO 93/07898 refers to, among other things, the identification of the agent causative of PRRS and to vaccines derived from it. The isolated virus has characteristics similar to LV but, differently from the virus isolated at our laboratory, it is not capable of growing on ST cells (swine testis cells) at detectable levels. Apparently, the vaccine has been efficacious, but the level of protection has only been effective in approximately 520- . of vaccinated animals only, which can be considered as a relatively low level of protection, especially if we bear in mind the high viral titre employed in the vaccinal dose. Additionally, this PCT request does not afford any information on the organization of the virus genome nor on the proteins coded for which reason the comparison between this virus and the virus obtained at our laboratories cannot be carried out or may be carried out but by exerting an undue research effort. Therefore, the need for vaccines capable of preventing PRRS continues to exist. In order to solve this problem, this invention provides a vaccine capable of efficaciously protecting sows against the infectious disease. The antigenic phase of this vaccine is composed of an inactivated Spanish isolate. The invention also provides combinations of the isolated PRRS viral antigen (Spanish strain) together with different porcine pathogens with the purpose of providing bi- or multivalent vaccines.

6 BRIEF DESCRIPTION OF THE FIGURES

Figures 1 to 5 show the cDNA sequences corresponding to ORFs 3 to 7, respectively, of the PRRS virus (Spanish strain) as well as the sequence deduced from amino acids coded by each ORF.

Figures 6 to 10 show the homology and existing differences between LV and the virus isolated at our laboratories, at the level of amino acid sequences deduced from ORFs 3 to 7. The amino acids are expressed in accordance with a one letter code. The upper line of each two lines corresponds to the LV amino acid sequence, whereas the lower line corresponds to the virus isolated at our laboratories (Spanish strain). The homologous amino acids are represented by two vertical lines, while when there are substitutions of some amino acids by others they are represented by two vertical dots in cases of conservative substitutions, that is the substitution of an amino acid by another one functionally equivalent. The absence of vertical lines and dots between two amino acids represents the existence of non- conservative substitutions.

DETAILED DESCRIPTION OF THE INVENTION

1. Samples 1.1 Animals chosen for the isolation of the virus mummified fetuses, stillborn piglets and living but weak piglets of 1 to 10 days of age, progeny of f ield sows with clinical problems due to PRRS, were chosen.

1.2 Preparation of the samples Lung, spleen, liver, kidney, brain and heart samples from the piglets were obtained by means of necropsy. In one 7 particular case, samples were prepared from the lung of a stillborn piglet born to a sow suspected of being affected with PRRS. With that lung, a homogenate was prepared with DMEM (GIBCO), (10 g of lung in 90 ml DMEM) supplemented with a solution of antibiotics (PEG) composed of 1000 IU/ml of penicillin, 1 mg/ml of streptomycin and 0.5 mg/ml of gentamicin. The resulting suspension was allowed to stand for 1 hour at 41C, was frozen and thawed twice, centrifuged and the supernatant obtained stored at -700C to be used in the infection of pig's lung alveolar macrophages. In another case lung samples were prepared from a piglet born alive but which died within a few hours after birth, by means of a similar process.

Additionally, blood was extracted from the animals by 15 puncturing the vena cava to obtain (i) blood plasma, which was stored at -700C and used for virus isolation, and (ii) serum, which was used to carry out antibody titration.

2. Pig's lung alveolar macrophages 2.1 Obtainment Pigs seronegative to Aujeszky's disease, porcine parvovirosis, foot-and- mouth disease, classic swine fever, swine influenza (types H1N1 and H3N2) and transmissible gastroenteritis were used. The age of the pigs used ranged between 7 and 8 weeks. The animals were anaesthetized with phenobarbital sodium before the extraction of the lungs, which was done by first ligating the trachea below the epiglottis and by sectioning above the ligature. Once the lung had been extracted, it was washed externally with physiological saline solution, and then PBS and PEG solution of antibiotics were introduced by means of successive washings. The cells obtained from these washings were subjected to centrifugation for 10 minutes at 300g and resuspended in DMEMs medium [(DMEM medium supplemented with unessential amino acids (GIBCO), 1-'. of sodium pyruvate 1 mM and 1% of glutamine 2mM) 1, 10% fetal calf serum (FCS) and PEG 8 solution of antibiotics at 1-%. Newbauer chambers.

2.2 Sterility Control is The cell count was done in It is verified that the pig's lung alveolar macrophages are free of contamination by bacteria, fungi and other viruses, by means of the suitable tests. Likewise the good general state of the cells is verified by optical microscopy.

The absence of contamination by mycoplasmas is also verified by cytochemical detection with DAPI (4.6-diamidine-2-phenylindole) which binds selectively to DNA and forms DNA-DAPI complexes of high fluorescence and high specificity.

3.

Isolation of the virus 3.1 Isolation of the virus and viral growth on pig's lung alveolar macrophaqes A culture of pig's lung alveolar macrophages, previously prepared in DMEMs medium and FCS at 10%, was infected with a homogenate of a sample suspected of containing the causative agent of PRRS. The sample was composed, in one case, of a lung isolate from a stillborn piglet born to a sow that presented clinical signs of PRRS, whereas in other cases isolate was used from a piglet born alive but which had died after a few hours, progeny of a sow with PRRS symptoms, as well as an isolate from blood plasma. The homogenate was kept in contact with the macrophage culture at 370C for 1 hour. Afterwards, the inoculum was removed and fresh DMEMs with 20-h FCS and 10-h PEG solution were added, buffering the culture withC02 and allowing to incubate at 370C for several days during which the cytopathic effect (CPE) produced by the virus on the macrophages was observed under microscope: at 3-4 days post infection (dpi) CPE was 70-800-. (giant and deformed cells appeared). The cultures were frozen at 800C.

iR 9 Simultaneously, a pig' s lung alveolar macrophage culture free of PRRS was prepared to be used as negative control.

Subcultures were prepared with the isolated virus and it was observed that there was 1OM; CPE from the second dpi. The virus was frozen at -800C for its posterior identification and characterization.

Similarly, the virus was isolated from blood plasma and other samples from stillborn piglets or living but weak piglets born to experimentally infected sows.

one of the isolated viruses, in particular the virus denominated PRRS-CY218-JPD-PS-6-91, was isolated from a stillborn piglet's lung. It is capable of experimentally reproducing the disease, has the characteristics mentioned in Section 4 and was deposited at the European Collection of Animal Cell Cultures (ECACC), Salisbury (United kingdom), on June 29, 1993 with accession no. V93070108.

3.2 viral growth in other cell systems Infections with the isolated virus (Spanish strain) have been carried out in pig's lung alveolar macrophage and ST cell [Swine Testis continuous. cell line) ATCC CRL 1745 ST] cocultures, as a first step for the adapting of the virus to ST cells. After several serial passages (5-6) on the ST cell and macrophage cocultures and cultures of ST alone, the virus infectious titres were of the order of 106 TCID.Jml when macrophage-ST cell co-cultures were infected, and of the order of 104---5 TCID.Jml for the virus obtained in ST cells alone. [TCID., tissue culture infectious dose 50%].

Additionally, pig's lung alveolar macrophages can also be 35 immortalized by fusing them with ST cells by means of hybridization.

Alternatively, pig's lung alveolar macrophages can be immortalized by fusing them with L-14 cell line (porcine peripheral blood B cells) ECACC no. 91012317, or with cell line Jag-1 (porcine trophoblast cell line) supplied by Dr Jag 5 Ramsoondar The fusion procedures are done following conventional techniques of use in this field.

Alternatively, viruses can be grown on ST cells or on any other porcine cell line in which have been introduced the genes coding for pig's lung alveolar macrophage membrane receptors for the PRRS virus.

is The viruses produced with these cell systems are susceptible of being used in the formulation of both living as well as inactivated vaccines.

4 Identification and characterisation of the virus 4.1 Denomination and depositinq of the -.,irus The virus isolated from a stillborn piglet's lung, denominated PRRS-CY-218-L7PD-PS-6-91, was deposited at the European Collection of Animal Cell Cultures (ECACC), Salisbury (United Kingdom), on June 29, 1993 with accession no. V93070108.

In the present description, occasionally, the virus is described without distinction as Spanish virus (SV) or Spanish strain.

4.2 Characteristics of the isolated virus This virus (PRRS-CY-218-JPD-PS-6-91) presents the following characteristics:

the production of slight CPE on a continuous ST cell line 11 d) e) f) g) h) i) j) m) k) 1) n) (ATCC CRL 1746 ST) (fetal swine testis) with average titre of 104---5 TCID.50/m1 and on pig's lung alveolar macrophages with average titres of 10-5--5 TCID.Jml.; b) whenit infects pig's lung alveolar macrophage and ST cell co-cultures, average titres of 106.3 TCID.Jml are obtained [which titres represent a logarithm unit (1 log,,)) higher than when only pig's lung alveolar macrophages are infected]; cytoplasmic replication; production of cytoplasmic vacuolation; lipid envelope; 40-50 nm size; no hemadsorption or hemagglutination observed with chicken, guinea pig, pig or human group 0 red blood cells; loss of infectivity at acid pH (pH <5); production of microscopic (interstitial pneumonia) and macroscopic lesions in 2-month old piglets; production of adverse reproductive effects in pregnant sows with stillbirths, mummified fetuses and live but weak piglets; cross-reaction with Lelystad reference serum (IPMA = Immuno peroxidase monolayer assay); cross-reaction with sera from animals with clinical field infections (IPMA); serum from sows infected with this virus cross-reacts with LV; RNA polyadenylated genome of approximately 15000 bp in length (neutral agarose gel); replication by means of a subgenomic RNA set present at end 3'; nucleotide sequence having 8 ORFs; in a purified suspension subjected to electrophoresis in polyacrylamide gel and posterior transference by immunoblot were detected, by means of a specific serum, prepared in sows from our own laboratory and which cross-reacts with the Lelystad PRRS virus, 4 majority bands corresponding to protein of apparent molecular weights of 15000, 23000, 12 54000 and 66000 Daltons, which were not detected in negative controls (uninfected macrophages); r) when ORFs 3 to 7 of this virus are compared with those of LV and/or TV, 95.5-,, homology is observed at nucleotide level (there are 114 different bp out of a total of 2599 bp) and 94.950k homology at amino acid level (51 different amino acids out of a total of 955); and S) the virus belongs to the Arterivirus genus.

4.3 Techniques used for virus identification 4.3.1 Experimental reproduction of the disease in precrnant sows German Landrace x Large White cross sows were used, originating from farms with systematic serological control against the viruses of Aujeszkyls disease, footand-mouth disease, porcine parvovirosis, classic swine fever, swine influenza (types HiNi and H3N2) and transmissible gastroenteritis. Additionally, the antibody titration test against the causative virus of PRRS was carried out. Between days 77 and 90 of gestation the animals were infected, with an isolate obtained on pig's lung alveolar macrophages in one case, intravenously (M and intranasally (IN), whereas in another case, only via intranaasal route (Example 2.1). Throughout the entire experiment feed intake, rectal temperature and clinical state of the animals were monitored. With the purpose of excluding the above mentioned agents, blood samples were taken from all the sows before infection. They proved to be seronegative to all the agents. Likewise, after the experimental infection, all the sows were still seronegative to all the said viruses and seropositive against PRRS (verified with the reference LV). The results obtained are shown in Table 1.

41 TABLE 1

TESTS CO)UCTED t) is AD FMD pp CSF 25 SF TG PRRS ELISA (4) ND INFE. AGENT AD FMD pp CSF SF TG PPRS HAI 30 NPLA ELISA SN IPMA (1) (2) (3) HAI (1) ND ND ND ND HAI (2) ND ND ND ND NPLA (3) ND ND ND j ND KEY SN (5) ND ND ND ND SN (6) IPMA 1(7) ND ND - ND ND ND ND ND ND ND ND ND ND ND Aujeszkyls disease.

Foot-and-mouth disease.

Porcine parvovirosis.

Classic swine fever.

Swine influenza (H1M, H2N3).

Transmissible Gastroenteritis.

Porcine reproductive and respiratory syndrome.

Hemagglutination Peroxidase-linked Assay.

Neutralizing Peroxidase-linked Assay.

Enzyme-linked Immunosorbent Assay.

Seroneutralization.

Immuno peroxidase monolayer Assay.

Vannier et al., Rec. Med. Vet. 155 (2), 151 158 (1979).

Charley B., Doctoral Thesis, Alfort (1976).

Trepsta et al., Vet. Microb. 9, 113-120 14 (4) (5) (6) (7) (-) ND (1984). Elliot M., J. Rech. Porc., 20, 141-146. Lucam F., "Diagnostic sero-immunologique des viroses humains et animals", F. Bricout; L. Joubert; J.M. Huraux (1977). Jim6nex et al., J. Virol., 60, 131-139 (1986). Wensvoort et al., Vet. QUARTERLY, Vol. 13, nO 3 (July 1991). Negative. Positive. Not done.

4.3.2 Experimental reproduction of the disease in piglets The objective of this experiment was to verify if the causative virus of reproductive alterations in sows was capable of producing in 2-month old piglets respiratory symptoms and macroscopic and microscopic lesions at lung level. To that end, a number of piglets were infected with virus (Spanish strain), IN route, which were then sacrificed on different post-infection days (Example 2.2).

The most relevant resulting data demonstrate that this virus produces at macroscopic level multiple foci of consolidation as well as interstitial pneumonia at microscopic level (Table 4). From the health point of view, no relevant clinical signs were observed.

4.3.3 Sensitivity to chloroform test This test was conducted to determine if the isolated virus had lipid envelope. To that end the Feldman, H. and Wang, S., method was used, described in "A manual of basic virological 3S techniques", Prentice-Hall Inc., New Jersey, 14G-148 (1978). The results obtained demonstrate that the untreated virus was a titre of 10S. 6 TCIDsO/ml, whereas after treatment with chloroform the c z titre is lower than 101.3 TCID50/ml, based on which it can be stated that the isolated virus has lipid envelope.

4.3.4 Secruencin-: of the viral genome i) ii) iii) iv) 10 v) i) Purification of the virus Purification of the viral RNA cDNA synthesis Cloning and characterization of the cDNA clones Sequencing and comparing of the sequences with those of LV Purification of the virus is Virus replicated on pig's lung alveolar macrophages was clarified, and concentrated by filtration using MILLIPORE filters. Afterwards, the virus was subjected to centrifugation in 10i to 50% metrizamide gradient (SIGMA). Once centrifugation was completed, a band has obtained which was concentrated by 20 centrifugation. With the purified virus, an electrophoresis in polyacrylamide gel was done and an immunoblot was developed with a specific serum, showing proteins whose apparent molecu'lar weights were 15, 23, 54 and 66 K Daltons.

ii) Purification of the viral RNA A technique was used for selection and purification of the RNA based on the fact that the RNA contains poly (A) sequence at the 31 end. For that purpose, a commercial kit (Pharmacia) was 30 used which exclusively allowed the binding of the RNA poly (A) chain to a cellulose-oligo (dT) matrix and its posterior elution.

iii) cDNA synthesis A commercial kit was used (Boehringer Mannheim), following the manufacturer's instructions. Briefly, the viral genomic RNA 16 was incubated in the presence of an oligo (dT), dATP, dCTP, dGTP, dTTP and reverse transcriptase.

iv) Cloninq and characterization of the cDRA clones The cDNA was cloned in a vector derived from pUC18 and a series of clones was obtained containing the complete sequence of nucleotidbs corresponding to ORFs 3 to 7.

v) Secruencinq and comparincr of the secruences with those of W v. a.) Sequencing The region corresponding to ORFs 3-7 of the virus isolated 15 at our laboratories has been sequenced completely. Figures 1 to 5 show the sequences of cDNA obtained as well as the sequences deduced from amino acids coded by each ORF. The total sequenced region length is of approximately 2599 nucleotides (nt).

As can be seen, ORF 3 has a length of about 798 nt and codes for a protein of 266 amino acid residues.

-ely 552 nt and codes f ORF 4 has a length of approximall For a protein of 183 amino acid residues. The beginning of this ORF 4 is located in the ATG codon located at about 540 bp from the initial ORF 3 ATG codon. ORF3 and ORF 4 share a sequence of about 246 nt.

ORF S has a length of about 606 nt and codes for a protein of 200 amino acid residues. The initial codon of this ORF S practically overlaps the ORF 4 end codon (they share the TG nucleotides, the ATG codon at the beginning of ORF S and the ORF 4 TGA end codon). The ORF 5 ATG initial codon is located about 1092 nt from the ORF 3 initial ATG codon.

ORF 6 has a length of about 522 nt and codes for a protein of 173 amino acid residues. This ORF G initial ATG codon is 17 located 8 nt upstream from the beginning of the ORF 5 termination TAG codon (at about 1682 nt approximately from the initial ORF 3 ATG codon).

ORF 7 has a length of some 387 nt and codes for a protein of 129 amino acid residues. This ORF 7 initial ATG codon is located 5 nt upstream from the beginning of the ORF 6 termination TAA codon (at about 2193 nt approximately from the ORF 3 initial ATG codon).

The proteins coded by ORFs 3 to 6 are membrane proteins, whereas the protein coded by ORF 7 is a nucleocapsid protein v.b.) Comparison with LV Comparing the cDNA sequences of ORFs 3-7 of LV with those of the virus isolated at our laboratories it is observed that:

i) ii) at nucleotide level, out of the 2599 nucleotides compared 114 are different, which represents approximately 95.5% homology, at amino acid level, out of the 955 amino acids compared, there are 47 different ones, representing 94.9% homology approximately, of the 47 different amino acids there are 35 considered as non- conservative substitutions, of which there are: 12 in the product of the ORF 3 gene (Figure 6); 9 in the product of the ORF 4 gene (Figure 7); 10 in the product of the ORF 5 gene (Figure 8), although it is convenient to point out that the product of the LV ORF 5 gene contains one amino acid more than the product of the Spanish virus ORF 5, specifically amino acid 35 (Asn) of the LV ORP 5 product is not present in the product 18 expressed by the Spanish virus; 4 in the product of the ORF 6 gene (Figure 9) whereas, the product of the ORF 7 gene does not contain any non-conservative substitution (Figure 10), iv) partial homology of each one of the products expressed by the different ORFs of the Spanish virus and LV is the 93.6% for the ORP 3 and ORF 5 products,94.00-. for the ORF 4 product, 96.60-h for the ORF 6 product and 99.2% for the ORF 7 product.

As mentioned above, the changes in the amino acids may be connected to the higher pathogenicity of one strain in comparison 15 with another, since the virus isolated at our laboratories (Spanish strain) is more pathogenic than other known PRRS viruses like, for example, the virus isolated in France (Example 8) and LV (Table 6 of PCT request no. WO 92/21375).

5 Vaccines The invention provides a vaccine capable of preventing porcine reproductive and respiratory syndrome (PRRS). The vaccine has proved to be efficacious in preventing reproductive alterations in sows, such as stillborn piglets, mummified piglets or live but weak piglets, return to service and similar problems, produced by the virus causative of PRRS. Likewise, it has been verified that the vaccine induces cellular immunity in vaccinated animals.

The vaccine contains a suitable amount of PRRS viral antigen, Spanish strain, inactivated, as well as an adjuvant and a preservative.

Tests conducted with these vaccines have demonstrated the efficacy of the vaccine, as manifested by Examples 7 and 8. Additionally, the vaccine has demonstrated to be effective in 19 avoiding return to service, which appears in infected sows. Actually, the sows vaccinated with the vaccines resultant from this invention and infected with the causative virus of PRRS mated and became pregnant at the first post-partum ovulation and 5 weaning of the piglets.

5.1 Components 5.1.a Antiqenic phase An active component, the vaccine contains inactivated PRRS viral antigen, Spanish strain, at a concentration higher or equal to 105.5 TCID_,,, per vaccinal dose. The inactivation can be done by chemical means that include treatment with 9-propiolactone or with other conventional inactivating agents such as ethylenimine or formaldehyde, or by physical means.

5.1.b Adjuvants Although it is possible to use adjuvants of aluminum hydroxide type, Quil A or their mixtures, as well as oily adjuvants, for the formulation of the vaccine, it has been possible to verify that the best results are obtained when an oily adjuvant is used (Example 4). In particular, it has been verified that an oily adjuvant constituted by a mixture of Marcol 52, Simulsol S100 and Montanide 888 affords very good results.

Marcol 52 is a low-density mineral oil manufactured by ESSO ESPAEOLA, S.A. Simulsol S100 is a polyethoxy oleate ether commercialized by SEPIC; and Montanide 888 is an anhydrous mannitol ether octadecenoate of high purity, commercialized by SEPIC.

It has been possible to verify that the adjuvant plays an essential part in the efficacy of the vaccine. Thus, a challenge test (Example 6) was conducted using sows vaccinated with two different vaccines, one sow with the oily adjuvant indicated above (Ref. 1) and the other sow using the adjuvant Munokynin R (Ref. 2). Although both vaccines produce seroconversion, it was verified in an experimental infection test that the sows vaccinated with vaccine Ref. 1 were protected against experimental infection with PRRS virus, whereas the other sows, those vaccinated with vaccine Ref. 2, were not protected in spite of the fact that at the time of challenge they had antibodies against the -rirus. This establishes that a suitable adjuvant can play a very important part in connection with the modulation and stimulation of the immune response, principally at cellular immunity level. Additionally, this aspect has been confirmed by means of the challenge test carried out using the Spanish strain of the PRRS virus, since the sows vaccinated and revaccinated with Ref. 1 vaccine did not present serological response at the time of infection but, nevertheless, they were protected (Example 7. Table 8).

However, it could also be possible that Ref. 2 vaccine (with Munokynin') might provoke cellular immunity. To that end, it would be necessary to add substances that potentiate cell response (CRP) that is substances that potentiate helper T-cell subpopulations (Th, and Th2). such as IL-1 (interleukin-1), IL-2, IL-4, IL-5, IL-6, IL-12, g-IFN (gamma interferon), cellular necrosis factor and similar substances. Evidently, it would also be possible to add these CRP substances to vaccines with oily adjuvant, in which case their cell immune effect would be potentiated.

Other types of adjuvant may also be used that modulate and immunostimulate cell response, such as MDP (muramyl dipeptide), ISCOM (Immuno Stimulant Complex) or liposomes.

5.1.c Preservatives Any of the preservatives habitually used in the formulation of vaccines may be used. One of these is Thimerosal [sodium salt of (2-carboxy- phenylthio) ethyl-mercury] (ALDRICH).

2 21 5.2 Method for the preparation of the vaccine The vaccines resultant from this invention can be obtained by the mixing of an antigenic phase containing inactivated viral antigen and another phase, as adjuvant, which may or may not be oily, depending on the adjuvant chosen. Optionally, CRP substances could be added to any of the two phases. When the adjuvant is 'Oily, an emulsion is formed which, in a particular and preferable case (when the adjuvant is a mixture of Marcol 52, Simulsol 5100 and Montanide 888), is a double W/O/w (water/oil/water) emulsion.

5.3 Vaccine control tests In addition to the conventional tests the vaccine must pass before its administration, for (i) purity (against bacteria, fungi, mycoplasmas and foreign viruses) (ii) identification, (iii) safety, (iv) potency and M physico-chemical controls, a number of field trials (Example 5.b) have been carried out in relation to the safety and efficacy of the vaccine in a total of 5 farms, of which 508 sows were vaccinated and revaccinated with one of the vaccines resultant of this invention, whereas the remaining 472 sows were not vaccinated and were kept as control sows, making it possible to observe that the vaccine is safe and 2S at the same time effective, for in some of the farms the natural disease PRRS was detected in unvaccinated animals after the process of vaccination.

5.4 Posology and instructions for the administration of the 30 vaccine It has been possible to verify that one dose of 2 ml of oily vaccine with a concentration of inactivated viral antigen equal to or higher than 105. 5 TCID.., administered via deep intramuscular route, is capable of protecting a very high percentage of vaccinated animals against PRRS.

22 The following vaccination programm is advisable:

First vaccination: vaccinate all breeding animals (sows and boars) and revaccinate 21 days later. Afterwards, administer one dose during every lactation (sows), and every 6 months (boars).

Posterior vaccinations is Animals intended for reproduction: The first vaccination should be at least 6 months of age, revaccinating 21 days later.

Sows: It is advisable to vaccinate during the period of lactation, if possible 15 days before mating.

Boars: Vaccinate twice a year (every 6 months).

Alternatively, if no CRP substance has been included in the vaccine, these substances may be injected at a site different to the site of inoculation but simultaneously.

6. Polyvalent vaccines -h;s By means of an additional ob)ective attaine:3 from t invention, combinations are provided of the different porcine pathogens containing in addition to inactivated viral PRRS antigen (Spanish strain) one or more of the pathogens listed below, in order to enable the preparation of bi- or multivalent vaccines.

This way bi- or multivalent vaccines may be prepared containing inactivated viral PRRS antigen, and one or more of the following pathogens: Actinobacillus Dleuropneumoniae, Haemophilus parasuis, Porcine Parvovirus, Lei:)tospira, Escherichia coli, Erysipelothrix rhusiopathiae, Pasteurella multocida, Bordetella bronchiseptica, Porcine respiratory coronavirus. Rotavirus or - against the pathogens causative of Aujeszky' s disease, swine influenza or transmissible gastroenteritis.

2 23 EXAMPLES

Example 1 Isolation of the virus 1.A Preparation of the samples From the lung of a stillborn piglet, progeny of a sow with the classic PRRS symptoms [the sow was f ree of antibodies against Auj eszky, s disease, porcine parvovirosis, foot-and-mouth disease, classic swine fever, swine influenza (types H1N1 and H3N2) and transmissible gastroenteritis], a 10'k. suspension was made in DMEM culture medium, supplemented with a solution of antibiotics (PEG) composed of 1000 IU/ml of penicillin, 1 mg/ml of streptomycin and 0.5 mg/ml of gentamicin, at a ratio lung:DMEM solution of 1:10 (W/V). The suspension produced was homogenized and left to stand for 1 hour at room temperature (20-22OC). The homogenate was frozen and thawed twice, centrifuged and the supernatant obtained stored at -700C, to be used in the infection of pig's lung alveolar macrophages. Similarly, samples were prepared f rom the lung of a piglet born alive but which died within a few hours. Additionally, blood was extracted and mixed with and without anticoagulant and used for virus isolation (from blood plasma) and to obtain serum, respectively.

1.B Obtainment of piqls lung alveolar macrophages Alveolar macrophages were obtained from the lungs of pig,s seronegative to Aujeszky's disease, porcine parvovirosis, foot- and-mouth disease, classic swine fever, swine influenza (types H1N1 and H3N2) and transmissible gastroenteritis. The age of the pigs used ranged between 7 and 8 weeks. Prior to the extraction of the lungs, the animals were anaesthetized with phenobarbital sodium and then sacrificed. Immediately, the lungs together with the trachea were extracted after ligating it below the epiglottis. The extracted lung was washed externally with physiological saline solution, and in successive washings 50 ml 24 of PBS supplemented with 20-. of PEG solution of antibiotics were introduced until a total of 500 ml of PBS had been introduced. The cells obtained from these washings were centrifuged for 10 minutes at 300g. This step of washing centrifugation was repeated two more times. The cells obtained were washed with PBS and PEG solution of antibiotics and resuspended in DMEMs medium [DMEM supplemented with non-essential amino acids (GIBCO), 10-. sodium pyruvate 1mM and l-. of glutamine 2mMI, 10-. fetal calf serum (FCS) and PEG solution of antibiotics at 1-0.. The cell count was done in Newbauer chambers and to that end 1/10 dilution of the macrophage suspension was prepared by adding 0.4 ml of DMEMs and 0. 5 ml of trypan blue solution to 0 - 1 ml of macrophage suspension. A number of cells ranging between 1 and 1.2 x 109 was obtained.

1.C Isolation of the virus A culture flask of 25 cm' surface containing a culture of pig's lung alveolar macrophages previously prepared (:3 X 106 cells/ml) in DMEMs medium and 10% FCS was infected with 1 ml of the homogenate of a sample originating from the lung of a stillborn piglet (Example 1.M. The homogenate was left in contact with the macrophage culture for 1 hour, at 370C, buffered with C02 at pH7.0-7.4, and incubated at 370C for several days during which the CPE produced by the virus on the macrophages was observed. At 3-4 dpi, CPE was observed to be 70-80%, for which reason the cultures were frozen at -800C.

Simultaneously, a culture was prepared of uninfected pig's lung alveolar macrophages, used as negative control.

Subcultures with the isolated virus were done in which it was observed that CPE starting from the second dpi was 1000.-. The virus was frozen at -800C for its posterior identification and characterisation. After the fourth passage in macrophages the corresponding titrations in 96-well microplaques were done, obtaining an average titre of 105.6 TCID5./M1 in accordance with T 0.. c the Reed & Muench method [Am. J. Hyg., 27: 493-497 (1938)].

A sample of isolated virus (Spanish strain) denominated PRRS-CY-218-JPDPS-6-91, isolated from a stillborn piglet's lung, is capable of experimentally reproducing the disease and was deposited at the ECACC on July 1, 1993, under corresponding accession no. V93070108. under the terms of the Budapest Treaty.

In a similar way, the virus was isolated in live and stillborn piglets, progeny of sows infected experimentally.

Example 2 Identification and characterisation of the virus Example 2. 1 Experimental reproduction of the disease in iDreqnanr sows Twelve sows, German Landrace X Large White cross were used, originating from farms with systematic serological control against the viruses of Aujeszky's disease, disease, porcine pa"rvovirosis, classic swine influenza (types H1N1 and H3N2) and foot-and-mouth fever, swine rransmissible gastroenteritis. Additionally, the antibody evaluation test against the causative virus of PRRS was conducted.

The sows were moved to the safety stables of the research center one week before infection and placed in separate stables. Between days 77 and 90 of gestation, the sows identified with numbers 53, 76, 8, 62, 91, 93 and 19 were infected with 5 ml intravenous route (IV) and with 5 ml intranasal route (IN) of PRRS virus, Spanish strain, isolated on pig's lung alveolar macrophages identified as PRRS-CY-218-JPD-P5-6-91, from a fourth passage on macrophages filtered through a 200 nm filter and with a titre of 105.6 TCID,,/ml. The S remaining sows (nos. 14, 40, 13 30 and 85) were inoculated between days 65 and 85 of gestation with S ml via IN (only) of the virus.

26 During the experiment feed intake, rectal temperature and the clinical state of the animals were monitored daily, as well as reproductive alterations (premature parturitions, delayed parturitions, -ive but weak piglets, stillborn piglets, mummified, and healthy piglets.).

In order to proceed with the exclusion of the above mentioned pathogens, blood samples were taken from the sows pre and post infection, with the result that the animals were seronegative to the pathogens prior to and after infection and seropositive against PRRS after infection (see Table 1, section 4.3.1).

The reproductive results appear in Tables 2 and 3. As shown in Table 2, of the 93 piglets farrowed, 15 were mummified, 35 stillborn, 22 were born alive but died on the third day and 21 survived 7 days of life.

Some sows manifested inappetence for 2-4 days, on days 6 and 8 post infection, whereas other sows manifested inappetence on the second day post infection. Hyperthermia was not observed in any case. In 4 sows (nos. 8, 62, 92 and 93) farrowing was 1 to 6 days premature, whereas in the other 3 sows farrowing was delayed l or 2 days.

of the piglets born alive, 1 or 2 per litter manifested oedema in the eyes. The weak piglets manifested incoordination, paresis of the hind quarter, bristling hair and myoclonia. In the necropsy effected on some of the stillborn and weak piglets the presence of abundant clear liquid was observed in the thoracic cavity. Healthy piglets born to infected mothers sacrificed at 8-12 days of life manifested gray foci of consolidation. Under microscopic observation the most significant change was a slight multifoci interstitial pneumonia with enlargement of alveolar septi because of the infiltration of mononuclear cells. These lesions appeared in all the animals analyzed in the experiment.

27 As seen in Table 3, out of 65 piglets farrowed, 36 were stillborn, 26 were born alive but weak, dying on the second day of life, and 3 survived the first week of life. one sow farrowed 12 days prematurely (no. 40) whereas the others farrowed 1 or 2 days prematurely. The clinical signs of the piglets born weak are similar to that obtained via IN+IV. Interstitial pneumonia was observed also. The infected sows did not manifest inappetenceOr hyperthermia.

The most relevant difference between the two systems of infection is that via IN + IV mummified piglets were observed and that 1 to 2 of the piglets born alive in each litter manifested edema around the eyes.

In view of the results obtained, it can be concluded by stating that the model for experimental infection in sows, at about 80 days of gestation via both routes of infection (IN and IV) with the virus isolated (Spanish strain) from animals infected naturally, reproduces the disease PRRS in pregnant sows and provokes a high proportion of mummified fetuses, stillborn piglets and live but weak piglets very similarly to the proportion observed in acute natural infection outbreaks. It is advisable to infect artificially via IN route for reason that this is the natural route of infection in the field and therefore the most appropriate way to evaluate the efficacy of the vaccine.

By means of this experiment it has also been possible to set up a model for experimental infection in pregnant sows that allows for the verification of the efficacy of the vaccine.

28 TAB _LE 2 IN + IV is 0 1 1 H 11 A B c D E F G hi h2 I 53 77 115 9 3 2 0 4 4/4 76 77 116 11 2 4 1 4 NT 8 77 110 12 2 4 1 5 5/5 62 80 113 16 - 3 6 4 3 3/3 91 90 109 14 - 1 8 1 4 NT 93 88 110 17 4 2 11 - - NT 88 116 14 4 1 8 1 NT is 17 EE] 1 21 1 35 TABLE 3

IN A B c D E F G hl h2 1-4 65 ill is 12 - 3 NT 82 102 12 - 12 - - _NT 13 80 113 12 10 2 - NT 80 1 3 is 12 3 - NT 112 11 L- L 4 7 - - 29 A:

B:

C:

D:

is Time of infection (days of gestation) E:

F:

G:

H:

I:

Farrowing (days of gestation) Total pigl,::s Mummified piglets Weak piglets dead by 48 h.

Stillborn piglets Piglets'apparently in good health. hl: Dead between days 2 and 7.

h2: Living after one week.

Interstitial pneumonia.

NT: Not tested.

Example 2.2 Experimental reproduction of the disease in piqlets This experiment was designed with the purpose of verifying (Spanish strain), which produces that the isolated virus reproductive alterations in sows, is capable of producing clinical signs as well as macroscopic and microscopic lesions in the lungs of 2-month- old piglets. To that end, 10 piglets were infected via IN route with 5 ml of virus, Spanish strain, with a titre of 105 6 TCID50/ml and 6 other piglets were left as controls (all of them originated from 2 litters). The animals were sacrificed on days 3, 7, 8, 9 and 11 post infection. The 25 results obtained are shown in Table 4.

TABLE 4

NQ_ 11-C Antibodies I. P. V. I 2 3 2+ NT 12 3 2+ NT 11 c 3 NL NT 1 1 7 1: 8 0 4+ + 14 1 7 1 8 0 2+ + 13 c 7 NL 1 8 1 160 4+ + is 1 8 1 16 0 2+ + 8 c 8 NL 17 c 8 NL 4 1 9 1 160 4+ + c 9 NL 1 11 1: 16 0 4+ + 18 1 11 1 3 2 0 3+ + 9 1 11 1 3 2 0 3+ 6 c 11 NL No.: Pig number I/C: Infected (I) or Control (C) DPI Days post infection I.P. Interstitial pneumonia V.I. Virus isolation 2+ Slight interstitial pneumonia 3+ Iritermediate interstitial pneumonia 4+ Serious interstitial pneumonia NT Not tested NL No lesions During the 11 days of the experiment, no clinical respiratory signs or hyperthermia were observed, although there was loss of weight in the infected animals in comparison with uninfected animals. Under microscopic observations, the most relevant aspect was the presence of multiple foci of consolidation in the lungs, congested lymph nodes in the mandible and some intestinal haemorrhages. At microscopic level interstitial pneumonia was observed.

Virus was isolated from the solutions obtained from the washings of infected animals, lungs on a fresh macrophage culture, but virus was not isolated from the control animals, in which seroconversion was not observed nor macroscopic or microscopic lesions at lung level.

When cell counts were done from the washings of infected animals' lungs, 30-. dead cells (macrophages) was observed, which may constitute a factor of importance in secondary infections because of the destruction of a key immunologic defense element (macrophages).

The absence of respiratory signs may be due to the fact that the experimental infections were carried out in stables with continuous disinfection treatments and, therefore bacterial concentration was much smaller when compared with that existing p 31 in herds under field conditions.

Example 2.3

Sensitivity of chloroform test The Feldman, H. and Wang, S. method (Section 4.3.3) was used resulting in the knowledge that the isolated virus has lipid envelope since there is a drop in titre of 4 loglo from the control cultures of those treated with chloroform.

Example 2.4

Secruencinq of the viral qenome i) Purification of the virus The virus, replicated on pig, s lung alveolar macrophages was purified by filtration and centrifugation in 10% to 50% metrizamide gradient (SIGMA), resulting in a band which was centrifuged again as mentioned in section 4. 3.4. With the purified virus was conducted an electrophoresis in polyacrylamide gel, and an immunoblot developed with a specific serum, showing proteins with apparent molecular weights of 15, 23, 54 and 66 K Daltons.

ii) Purification of the viral RNA The viral RNA was. purified using a commercial kit (PHARMACIA) that enables the binding of the RNA poly (A) tail to cellulose-oligo (dT) matrix and its posterior elution.

iii) cDNA synthesis A commercial kit was used (BOEHRINGER MANNHEIM) (section 4.3.4 iii).

iv) Cloning and characterization of the cDNA clones The cDNA was cloned in a vector derived from pUC18 and a 32 series of clones was obtained containing the complete nucleotide sequence corresponding to ORFs 3 to 7.

v) of LV is Sequencing and comparing of the sequences with those The results of the sequencing of the cDNA of the virus isolated at our laboratories (ORFs 3-7) as well as the comparison between that sequence and the LV sequence are mentioned in section 4.3.4.v, where it can be seen that, at amino acid level, there is approximately 94.9% degree of homology and a total of 47 different amino acids of which 35 correspond to nonconservative substitutions. These differences at amino acid level may be responsible for the different pathogenicities that exist between the various PRRS virus strains isolated.

Example 3 Formulation of a vaccine A vaccine, capable of protecting against PRRS, is prepared in emulsion form following the procedure described below.

A pig's lung alveolar macrophage culture is infected with MOI (multiplicity order infection) of 0.001 and incubated at 370C for 24 hours, at the end of which the culture medium is substituted by infection medium (DMEM supplemented with 2% FCS). The culture is incubated for 4 days at 370C until 70-80-'6 CPE is observed. Once this period is completed an IPMA test is conducted in order to confirm identification. The virus is collected by vacuum aspiration and frozen at -800C.

The viral suspension destined to the formulation of vaccine should have 105's TCID.Jm1 minimum titre (prior to its inactivation) and should not be contaminated by bacteria, fungi, mycoplasmas or other viruses. In the case that the titre may be lower, it should be adjusted by concentrating the antigen.

1 33 For the inactivation of the viral suspension, 2-0. 9 propiolactone solution is added and stirred at 40C for one night, maintaining the pH at 7.4 by adding 0.5M NaOH. Once the inactivation period is completed, the viral suspension is 5 maintained at 370C for 1 hour.

The following is then prepared: a) an antigenic phase of the viral antigen inactivated with minimum concentration of 105's TCID50/dose and the preserver; and b) an oily phase composed of Marcol 52, Simulsol 1500 and Montanide 888.

The aqueous phase, maintained in stirring, is added slowly to the reactor containing the oily phase maintained also in stirring. once the aqueous phase has been added completely stirring is continued for 10 minutes.

In a particular, preferred case vaccines have been prepared capable of preventing PRRS, comprising per dose of 2 ml:

a) 53% of an antigenic phase, containing:

i) PRRS viral antigen in DMEM culturemedium, Spanish strain inactivated with 9-propiolactone, at minimum concentration of 105.5 TCID5, and ii) thimerosal 0.01%; and 47% of an oily phase, containing:

i) Marcol 52 ii) Simulsol 5100 iii) Montanide 888 b) .... 790.0 mg 70. 0 mg 80.0 mg The oily Phase/Aqueous Phase ration is a weight/volume (W/V) ratio. This vaccine has been denominated MSD Ref. 1. The obtained vaccine is subjected to the corresponding control tests prior to use.

Another vaccine was similarly prepared using Munokynin' 34 (aluminium hydroxide and Quil A, supplied by AMERICAN CYANAMID) as adjuvant, maintaining the same amount of inactivated virus. This vaccine has been denominated MSD Ref. 2.

Example 4 Evaluation of adjuvant A field trial was conducted with a total of 128 sows out of which 49 wer6 vaccinated with one dose of the vaccine denominated MSD Ref. 1, 50 sows were vaccinated with one dose of the vaccine denominated MSD Ref. 2 (Munokynin R) and the remaining 29 were not vaccinated and were kept as controls. After 22 days, the sows were revaccinated with one dose of the corresponding vaccine.

The following parameters were evaluated.

is 1) Serological response by means of IPMA determination at the following times:

T, vaccination and bleeding T22 revaccination and bleeding Tr,1 bleeding at So days post vaccination General type reactions (appetence for feed, hyperthermia, etc.).

2) The results obtained are shown in Tables 5 and 6 which reflect the percentage of sows with positive serological reaction (Table 5) and the arithmetic mean of the serological titres reached (Table 6).

TABLE 5

1 OF ANIMALS WITH SEROLOGICAL REACTION TO T22 T51 MSD Ref- 1 59-15 100% SD Ref. 2 40%- 87% 1 Controls 1 1- - - 1 - N TABLE 6

ARITHMETIC MEAN OF THE SEROLOGICAT, TITRES TO T22 T51 MSD Ref. 1 - 58 200 ef. 2 37 133 Controls - - No significant local or general type reactions were observed. It can be affirmed, based on the results obtained, that positive seroconversion is produced with both vaccines, although somewhat higher when the vaccine MSD REF. I (oily adjuvant) is used. At revaccination, a higher seroconversion percentage is observed and the arithmetic mean of the titres obtained is higher in animals vaccinated with MSD Ref. 1.

Example 5 Safety in sows Example S.A At laboratory level Example S.A.1 Primiparous sows Eighteen primiparous sows were chosen (German Landrace x Large White cross) from the porcine production farm and were distributed in two stables at the rate of 9 sows per stable, so that sows vaccinated with the same vaccine (MSD Ref. 1 or MSD Ref. 2) obtained in Example 3 above were housed in the same stable.

Nine sows were vaccinated via deep IM route with one dose of 2 ml vaccine MSD Ref. 1 containing 105.5 TCID.50/dose inactivated virus titre, and were revaccinated with another dose of the same 36 titre 20 days later. The other 9 sows were vaccinated and revaccinated on the same days with the vaccine MSD Ref. 2 (2 ml doses, 105-5 TCID.Jdose inactivated virus titre).

During the first 5 days post inoculation the following observations were done:

a) Local reaction ConsistIng of macroscopic observation of the site of inoculation and palpation, noting down the degree of inflammation 10 in comparison with objects of known size.

b) General reaction Consisting of macroscopic observation of the animals and verification of their appetence for feed. In negative case, rectal temperature is checked every 12 hours until hyperthermia 15 or any other unfavourable signs have disappeared.

The obtained results reflect that there was a slight inflammatory reaction in some sows, prominent at the site of inoculation, disappearing in every case within a few days; no purulent formations were observed. Only one of the sows refused to ingest the totality of the feed in the first postinoculation feeding, but feed ingestion was normal at the following feeding so that it was not necessary to take rectal temperature. No substantial differences in the response to the different tested vaccines were detected, based on which it can be affirmed that both vaccines are safe.

Example S.A.2 Pregnant sows Z Seven pregnant sows (German Landrace x Large White cross) from a porcine production farm were chosen at random: 6 primiparous sows of about 9 months of age and 1 multiparous sow, 3 years and 7 months old. The vaccine denominated as MSD Ref. 1 was used exclusively.

The sows were vaccinated via deep IM route with a dose of 2 ml of vaccine that contained inactivated virus titre of 10 c 37 TCID.Jml, and 15 days later they were revaccinated with one dose of the same titre.

During the first 5 days post inoculation the following observations were done:

a) Local reactions Consisting of macroscopic observations of the site of inoculation, and palpation taking note of the degree of inflammation in comparison with objects of well-known size.

b) Inappetence Consisting of macroscopic observation of the animals and checking for loss of appetence for feed.

c) Rectal temperature Measuring of the rectal temperature at 24 hours post vaccination and 24 hours post revaccination.

The results obtained reflect that there was a slight local reaction in two of the animals which was not serious because of its small size, disappearing in a few days. Inappetence or hyperthermia were not observed in any case. Based on this, it can be affirmed that the vaccine is safe.

Example S.B

Safety and efficacy field trial

This experiment was carried out in the 5 farms listed below. A variable number of sows from each farm was vaccinated via deep IM with one dose of 2 ml of the vaccine MSD Ref. 1 containing a titre of 105's TCID,,/dose of inactivated virus, and revaccinated 30 21 days later with another dose of same titre, whereas the other sows were not vaccinated and were used as controls:

FARM VACCINATED CONTROL TOTAT, 1 19 11 30 11 46 34 80 111 153 147 300 IV 127 123 250 V 163 157 320 TL 508 472 980 38 TL I II III IV V Total Farm "RAMON DEL QUINTAII (Banyoles) Farm "CAL SABATER11 (Orriols) Farm "E. CANELAII (Preixana) Farm "R. CUNILLERA" (L'Albi) Farm "INVERSORS PICBER11 (Bellpuig) It has, been possible to verify that the vaccine is safe af ter the observation of general and local reactions. Local reactions were only observed in 1-. of the animals. In connection with the productive parameters observed in the above-mentioned f arms, no variations were observed when compared with their clinical histories.

is Regarding serological response. some farms have seroconversion to the vaccine while, in others, the response is negative. This is not indicative of low level of protection since in the experimental infection tests in the laboratory, seronegative animals resist experimental infection (Examples 7 and 8).

In connection with the transmission of maternal immunity from vaccinated animals to their progeny, there is a big drop in antibody titres at one month of age.

In vaccinated and revaccinated sows that are serologically positive there is a big drop in antibody titres at 2 months from revaccination.

Example 6 Verification of cell immunity Five pregnant sows (German Landrace x Large White cross) from a porcine production farm were used. The animals were moved to the research center safety stables.

Two sows were chosen at random and were vaccinated with the vaccine denominated MSD Ref. 1. Another sow was vaccinated with 1 39 the -accine denominated MSD Ref. 2. The 2 remaining sows were not - accinated.

The sows were vaccinated deep IM route with one dose of 2 ml of vaccine MSD Ref. 1 or vaccine MSD Ref. 2 containing inactivated virus titre 105.5 TCID50, and 20 days later the sows were vaccinated with another dose of the same titre.

Afterwards, between days 77 and 90 of pregnancy all the sows 10 were infected via IN route with Sml of the virus PRRS-CY-218-JPDPS-6-91 with titre of 105,8 TCID,Jml. At the time of infection, it was verified that all the vaccinated sows presented antibodies against the causative virus of PRRS (positive serology). Table 7 shows the reproductive results obtained as a whole:

is (A) (B) (C) 25 (D) (E) (F) (G) IBI 2 1 2 MSD Ref. 1 MSD Ref. 2 6 -- 6 7 -- 17 No. of sows Vaccine used Total number of piglets No. of piglets born alive in good health No. of piglets born alive but weak No. of piglets alive after the 1st week No. of stillborn piglets TABLE 7 (C) -WI IúL (F) (G) 23 20 -- 20 3 12 24 The results obtained demonstrate that the sows vaccinated with vaccine MSD Ref. 1 (oily adjuvant) resist infection better than the sows vaccinated with vaccine MSD Ref. 2 (aqueous adjuvant), which could mean that the adjuvant plays an important part in the establishment of cell immunity.

Example 7 Efficacy in pregnant sows Eleven breeding sows were used (German Landrace x Large White cross) from a porcine production farm. The animals were 5 moved to the research center safety stables.

Three sows were chosen at random (sows no. 57, 63 and 74) and were vaccinated with he vaccine denominated MSD Ref. 1. Three sows (no. 15, 18 and 23) were vaccinated with the vaccine 10 denominated MSD Ref. 2, and the remaining 5 sows (no. 14, 40, 13, and 85) were not vaccinated.

The sows were vaccinated via deep IM route with one dose of 2 ml of the vaccine denominated MSD Ref. 1 or the vaccine MSD 15 Ref. 2, containing inactivated virus titre of 105-5 TCID../dose, and revaccinated with another dose of the same titre 20 days later. Local and general reactions were observed.

Serological response in the animals were verified by means of the IPMA test in accordance with the following program:

TO T20 T42 25 T78 T8 T25 T50 Bleeding and vaccination Bleeding and revaccination Bleeding Bleeding and experimental infection Post experimental infection bleeding Post experimental infection bleeding Post experimental infection bleeding Experimental infection was carried out in the research center safety stables. All the animals were infected at the rate of 5 ml of PRRS-CY-218- JPD-PS-6-91 virulent virus with titre of 105-8 TCID.50/ml via IN route. Serological response was noted down as well as the number of piglets born alive and stillborn to each sow. Pre and post colostrum bleedings were done in the piglets, and lung and brain samples were taken from the stillborn piglets and from the piglets sacrificed on different days for isolation of virus and histologic cLLts.

is The results obtained are shown on Tables 8-10 below:

TABLE 8

Serological results No. TO T20 T42 T78 T8 T25 T50 is ' - - 1/160 11160 1/640 1/1280 NT 18 -- 1/80 1/80 1/320 1/640 1/320 23 1/160 1/160 NP NP NP NP 57 1/160 1/160 1/320 1/320 1/1280 11160 63 1/80 1/80 1/160 1/640 1/1280 11160 74 1/80 NT 1/160 1/640 1/2560 NT c14 -- -- 1/1280 1/2560 NT c40 NT 1/1280 NT c13 NT 1/320 NT c30 NT 1/320 NT c85 NT 1/320 NT NT NP Not tested Not pregnant TABLE 9 Results of farrowing NO. (A) (B) (C) (D) (E) (F) (G) (H) (I) (i) 83 112 13 -- -- -- -- 18 78 ill -- 11 8 3 2 23' 57 63 108 13 7 6 - 63 79 114 13 8 1 2 2 2 74 83 112 6 -- -- -- -- - c14 65 ill -- is 3 12 c40 82 102 12 -- 12 c13 80 113 12 10 2 3 0 80 113 is 12 3 1 CC85 113 11 4 7 4 42 (D) (E) (F) (G) (H) (I) (i) Not pregnant These sows died because of environmental temperature.

112 days of gestation.

excessively high Cesarean was performed at (A): Date of infection (days of gestation) (B): F4rrowing (days of gestation) (C): Total number of piglets (cesarean) Total number of piglets (born) Piglets born in normal health Piglets born weak Live piglets born splay-legged Stillborn piglets (dead) Stillborn piglets (mummified) Piglets dead by the first week TABLE 10

Serology of the piglets A. Vaccinated sows 1 2. 31 A.5 NT NT NT - 3 Day-a 10 Dayi 18 1 NT NT - 2 NT NT - 7 NT 1/640 1/160 - 8 NT 1/2560 1/320 - 9 NT 1/1280 1/640 NT 1/1280 1/320 11 NT 1/1280 - 1/2560 1/640 - 12 NT 1/2560 1/640 - 13 NT ->1/2560 1/160 - 14 NT 1/1280 1/160 - is NT 1/2560 1/160 23 NP NP NP NP 4 Days 8 Days 57 1 - 1/320 - 1/640 1/320 - 2 - 1/320 - 1/640 1/160 1/320 - 3 - 1/640 NT - 4 - 1/320 - 1/640 NT - - 1/640 NT - S c 1 1 43 1 63 1 2 3 4 6 7 NT 8 NT 7 4 ') 1 Dav NT NT NT NT 1 ti/2560:1/2560 1/2560 1/2560 1/1280 1/2560 1/640 - 1/1280 1/1280 NT B. Control sows (nnt va_-,inated) 8 Day.9 1/640 1/320 1/320 - 1/640 1/640 - 1/1280 1/320 1/640 1/320 NT is 1. 21 12 H. 38 H. 4 5 days 9 Davs c14 1 NT ?t1/2560 >_1/2560 2 >_1/2560 >_1/2560 3 zl/2560 >_1/2560 1/1280 5_ 1/1280 1/640 1/1280 1/1280 1/1280 1/1280 c40 NT 9 D a v,I c13 1 - 1/160 + 2 - 1/160 3 - 1/320 4 1/320 C30 - N T N T c85 NT N T N T Key:

These sows died because of excessively high environmental temperature.

112 days of gestation.

1 Number assigned to each animal.

Piglets (the number assigned corresponds to the order of birth) 3 Pre-colostrum serology Post-colostrum serology Isolation of the virus Cesarean was performed at to each piglet 44 H 5 NP Not tested Negative Positive Hours Not pregnant It is evident from the results obtained that there is positive setoconversion against the causative virus of PRRS. Additionally, there is a satisfactory behaviour against experimental infection, in comparison with the control animals in which death was produced in the majority of the fetuses. Consequently, it can be affirmed that this vaccination is an efficacious measure for the prevention of PRRS.

Example 8 Efficacy of the vaccine against experimental infections with another agent causative of PRRS (cross-protection) This experiment was designed for the verification of the efficacy of the vaccine identified as MSD Ref. 1 in an experimental infection test using 2 pathogenic strains of the causative virus of PRRS.

T úhe pathogenic PRRS strains used were:

j) Spanish strain, PRRS-CY-218-JPD-PS-6-91; and ii) French strain, SDRP II 8B, provided by Dr. E. Albina of I'Laboratoire Central de Recherches Avicole et Porcine", Ploufragan, France The vaccine used was the vaccine denominated MSD Ref. 1, of 30 which the formula is given in Example 5.

Thirty sows seronegative to the causative viruses of PRRS were used, at reproductive cycle not comprised between 10 days prior to nor 10 days posterior to mating, nor 10 days prior to 35 nor 10 days posterior to farrowing.

It F-our groups of animals were formed:

A: 10 sows vaccinated and revaccinated via IM route and infected via IN route with Spanish strain PRRS-CY-218JPD-P5-691 of the virus.

B: 5 sows not subjected to any vaccination and infected via IN route with Spanish strain PRRS-CY-218-JPD-PS-691.

sows vaccinated and revaccinated via IM route and infected via IN route with French strain SDRP II 8B; sows not subjected to any vaccination and infected via IN route with French strain SDRP II 8B.

C:

D:

Experiment carried out is Twenty sows of the Groups A and C mentioned above were vaccinated with one dose of 2 ml of the vaccine denominated MSD Ref. 1 via deep IM route and revaccinated 21 days later with the same dose of vaccine.

Afterwards, between days 70 and 80 of gestation, all the 20 sows were infected experimentally with:

Groups A and B:

Groups C and D:

Parameters evaluated ml of virus PRRS-CY-218-JPD-PS-6-91 with titre of 105.8 TCID../ml via IN route; and 5 ml of virus SDRP II 8B with titre of 105' TCID.Jml via IN route.

1. Serological response by IPMA assay, at:

T, bleeding and vaccination T21 bleeding and revaccination T41 bleeding T, bleeding and infection TI-7 bleeding at 7 days post infection 2. Evaluation of rectal temperature, local reaction and 46 general reaction, during the 4 days posterior to vaccination and revaccination, or until the temperature or clinical signs, if any, disappear.

3.

is Evaluation of rectal temperature, feed intake and clinical signs during the 6 days following experimental infection.

Detection of antibodies in serum and isolation of virus in serum and in monocytes extracted from whole blood.

5.

Reproduction parameters at the time of farrowing, such as the number of piglets born alive, number of stillborn or mummified piglets and number of piglets born alive but weak that died within the first week.

Determination of the amount of virus present in serum by means of titrations on macrophages based on CPE.

Determination of virus in pleural liquid and lungs of the piglets.

The reproductive parameter results are shown in Tables 11 - 12 14 (chai.'LF-n:.-.e (challenae with PRRS-CY-218-JPD-PS-6-91) and 13- with SDRP II 8B).

TABLE 11 (Vaccinated sows) Challenge with PRRS-CY-218-JPD-PS-6-91 (1) (2) (3) (4) (5) (6) (7) 37') 14 39 6 68 12 45 10 19 10 38 9 41 7 71 11 36 10 26 b) TL 55 6 8 7 9 8 5 6 1 6 1 1 2 1 1 1 1 2 1 2 4 2 6 2 12 d 7 6 8 8 5 6 6 51 47 TL Total % of live piglets: 68% (75 born / 51 survive) 68'-'. protection is observed when comparing the piglets born alive with those surviving 7 days of life. The fact that experimental infection i's much more potent than natural infection in the field, added to the above results, make it possible to foresee that the prospects for protection are even better.

TABLE 12 (Unvaccinated sows) Challenqe with PRRS-CY-218-JPD-P5-6-91 is (1) (2) (3) (4) (5) (6) (7) 63 9 1 8 88 6 3 3 79 8 3 5 22 12 2 10 1 28 7 3 4 3 TL 42 5 6 1 30 4 TL Total % of live piglets: 9.5% (42 born / 4 survive) The Spanish strain used for infection has an extremely high pathogenic potency (90.5".-) since only 4 piglets survived the first week out of the 42 piglets born.

TABLE 13 (Vaccinated sows) Challenge with SDRP II 8B (1) (2) (3) (4) (5) (6) (7) 51 13 10 2 1 10 27 14 12 1 1 11 16 9 8 -- 1 7 17 is 13 2 12 1 7 7 8 is 13 2 13 57 10 9 1 8 61 12 8 1 3 8 78 10 10 -- -- 10 52c) TL 105 83 4 11 86 48 TL Total % of live piglets: 82% (105 born / 86 survive) 82-'6 approximate protection is observed TABLE 14 (Unvaccinated sows) Challenge with SDRP II 8B k (1) (2) (3) (4) (5) (6) (7) 62 12 7 1 4 9 81 15 13 2 13 4 12 12 11 94 14 9 1 4 9 1 13 8 1 4 8 TL 66 49 3 8 6 50 is TL Total % of live piglets: 75.7% (66 born / 50 survive) 24.396 approximate mortality is observed. The pathogenicity of the French strain is very weak (24.3%) when comparing with the results obtained from the challenge with Spanish strain (mortality 90.51s) The results used for the evaluation of efficacy comparing vaccinated and unvaccinated sows are not very significant in the case of the French strain. However and in any case, pathogenicity in the vaccinated animals is reduced to 17.8-0.- Key to Table 11 - 14 ' b (1) 35 (2) (3) (4) A different disease (not PRRS) (the piglets are not included) Sick, died before infection Sick, died by reason of another cause Sow reference Total number of piglets Number of healthy piglets born Number of weak piglets born 1 49 (5) (6) (7) Number of live splay-legged piglets born Number of stillborn piglets Number of piglets alive after the 1st week

Claims

PATENT CLAIMS

1) A vaccine capable of preventing porcine reproductive and 5 respiratory syndrome (PRRS), characterized on account of the fact that it comprises a suitable quantity of PRRS viral antigen or virus, Spanish strain, inactivated, as well as a suitable adjuvant, and optionally, a preserver.

2) Vaccine as per patent Claim 1, characterized on account of the fact that the said PRRS virus, Spanish strain, is the strain denominated PRRSCY-218-JPD-PS-6-91, deposited at ECACC, with accession number V93070108.

is 3) Vaccine as per patent Claim 1, characterized on account of the fact that it contains a quantity of inactivated virus of, at least, 105.5 TCID5./dose.

4) Vaccine as per patent Claim 1, characterized on account of 20 the fact that the said virus has been grown on a pig's lung alveolar macrophage culture.

7act 5) Vaccine as per Claim 1, characterized on account of the 'L that the said virus has been grown on a pig's lung alveolar 25 macrophage and ST cell (ATCC CRL 1746 ST) co-culture.

6) Vaccine as per Claim 1, characterized on account of the fact that the said virus has been grown on ST cell (ATCC CRL 1746 ST) culture.

7) vaccine as per patent Claim 1, characterized on account of the fact that the said virus has been grown on pigs lung alveolar macrophage hybrid cells fused with ST cells by means of hybridization.

8) vaccine as per patent Claim 1, characterized on account of the fact that the said virus has been grown on pigs lung 51 alveolar macrophage hybrid cells fused with L-14 cell line (ECACC no. 91012317) or with cell line Jag-1.

9) Vaccine as per patent Claim 1, characterized on account of the fact that the said virus has been grown on ST cells or any other porcine cell line into which have been introduced the genes coding for pig's lung alveolar macrophage membrane receptors for the PRRS virus.

10) Vaccine as per patent Claim 1, characterized on account of the fact that the said adjuvant is an oily adjuvant.

11) Vaccine as per patent Claim 10, characterized on account of the fact that the said oil adjuvant is gonstituted by a mixture ( P14- -,/Vk) of Marcoi/52, Simulsol 5100 and Montanide/ 6.

12) Vaccine as per patent Claim 1, characterized on account of the fact that it is an emulsion of (i) an aqueous antigenic phase containing the inactivated virus, and (ii) an oily phase containing the adjuvant.

13) Vaccine as per patent Claim 12, characterized on account of the fact that the said emulsion is composed of 53% by volume of an aqueous phase containing the inactivated virus and 47% by weight of an oily phase containing the adjuvant.

14) Vaccine as per any of the previous patent claims, characterized on account of the fact that it is capable of inducing cellular immunity in the vaccinated animal.

15) Vaccine as per patent Claim 14, characterized on account of the fact that it contains additionally cell response potentiation substances (CRP) that potentiate cell immune effect, such as IL1, IL-2, IL-4, IL-5, IL-6, IL-12, g-IFN, cell necrosis factor and similar substances.

16) Vaccine as per patent Claim 1, characterized on account of 5- the fact that the adjuvant is an aqueous adjuvant.

17) Vaccine as per patent Claim 16, characterized on account of the fact that it con%-ains additionally cell response potentiation 5 substances (CRP) that induce cell immune effect, such as IL-1, IL-2, IL-4, IL-5, IL6, IL-12, g-IFN, cell necrosis factor and similar substances.

18) Vaccine as per patent Claim 1, characterized on account of 10 the fact that the adjuvant is an adjuvant that can modulate and immunostimulate cell response, such as MDP, ISCOM or liposomes.

19) Vaccine as per any of the previous patent claims, characterized on account of the fact that it is capable of avoiding return to service in the vaccinated animal.

20) A vaccine capable of preventing porcine reproductive and respiratory syndrome (PRRS) characterized on account of the fact that it is an emulsion comprising:

a) 532k of an aqueous phase containing PRRS viral antigen in DMEM culture medium, Spanish strain, inactivated, at minimum concentration of 1055 TCID.Jdose, and b) 47% of an oily phase containing a mixture oft: Marcol(Z-1") CRrt^) 52, Simulsol 5100 and Montanide/88B.

21) Vaccine as per patent Claim 20, characterized on account of the fact that the said viral antigen is the PRRS virus, Spanish strain, denominated PRRS-CY-218-JPD-P5-6-91, deposited at ECACC, with accession number V93070108.

22) Vaccine as per any of patent Claims 20 or 21, characterized on account of the fact that it is capable of inducing cell immunity in the vaccinated animal.

23) Vaccine as per patent Claim 22, characterized on account of the fact that it contains additionally cell response potentiation substances (CRP) that potentiate cell immune effect, such as IL- D ' 1, IL-2, IL-4, IL-S, IL-6, IL-12, g-IFN, cell necrosis factor and similar substances.

24) Vaccine as per any of patent Claims 20 to 23, characterized 5 on account of the fact that it is capable of avoiding return to service in the vaccinated animal.

25) A bior multivalent vaccine capable of preventing porcine reproductive and respiratory syndrome and another or other porcine infections, characterized on account of the fact that it contains a suitable quantity of PRRS viral antigen or virus, Spanish strain, inactivated, plus one or more porcine pathogens.

26) Vaccine as per patent Claim 25, characterized on account of 15 the fact that the said viral antigen is the PRRS virus, Spanish strain, denominated PRRS-CY-218-JPD-P5-6-91, deposited at ECACC, with accession number V93070108.

27) Vaccine as per patent Claim 25, characterized on account of 20 the fact that it includes, at least, one porcine pathogen selected from the group made up of Actinobacillus pleurolDneumoniae, Haemophilus parasuis, Porcine -1Darvovirus, Lentospira, Escherichia coli, Erysipelothrix rhusiopathiae, Pasteurella multocida, Bordetella bronchiseptica, Porcine respiratory coronavirus, Rotavirus, or against the pathogens causative of Aujeszky's disease, swine influenza or transmissible gastroenteritis.

28) A procedure for the preparation of a vaccine capable of 30 preventing porcine reproductive and respiratory syndrome (PRRS), containing a suitable quantity of PRRS virus, Spanish strain, inactivated, plus a suitable adjuvant and, optionally, a preservative, characterized on account of the fact that it comprises:

1) the growing of the causative virus of PRRS, Spanish strain, in a suitable cell system, 51 % 3) 4) 2) the collection of the virus from the said cell system when minimum titre of 105.5 TCID,,,/ml has been attained, the inactivation of virus by means of physical or chemical methods, and the mixing of the inactivated virus with the adjuvant and the preservative.

29) Procedure as per patent Claim 28, characterized on account of the fact that the said PRRS virus, Spanish strain, is the virus denominated PRRS-CY-218-JPD-PS-6-91, deposited at ECACC, with accession number V93070108.

is 30) Procedure as per patent Claim 28, characterized on account of the fact that the said virus has been grown on:

i) pigrs lung alveolar macrophage culture, or on ii) a co-culture of pig's lung alveolar macrophages and ST cells (ATCC CRL 1746 ST), or on iii) ST cell culture (ATCC CRL 1746 ST), or on iv) pig's lung alveolar macrophage hybrid cells fused with ST cells, or on v) pig's lung alveolar macrophage hybrid cells fused with L-14 cell line (ECACC no. 91012317) or with cell line Jag-1, or on vi) ST cells or on any other porcine cell line into which have been introduced genes coding for pig's lung alveolar macrophage membrane receptors for the PRRS virus.

31) DNA sequence of the virus causative of PRRS, Spanish strain, comprising essentially the DNA sequences shown in Figures 1 to 5.

32) Virus causative of PRRS, Spanish strain, whose characteristics essentially correspond to those of the virus denominated PRRS-CY-218-JPD-PS-6-91, deposited at ECACC, with accession no. V93070108.

1 1% 1 33) Virus as per patent Claim 32, inactivated, capable of being put to use in the formulation of vaccines capable of preventing porcine reproductive and respiratory syndrome.

34) Virus as per patent Claim 32, inactivated, capable of being put to use in the formulation of bi-or multivalent vaccines capable of preventing porcine reproductive and respiratory syndrome and'other porcine infections.