AU6877401A - Vaccines inducing an immune response against viruses causing porcine respiratory and reproductive diseases - Google Patents

Description

S&FRef: 253954D2

AUSTRALIA

PATENTS ACT 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT

ORIGINAL

Name and Address of Applicants: Actual Inventor(s): Address for Service: Invention Title: Iowa State University Research Foundation, Inc.

214 O &L Ames Iowa 50011-3020 United States of America Solvay Animal Health, Inc.

1201 Northland Drive Mendota Heights Minnesota 55120-1148 United States of America Prem Sagar Paul, Patrick Gerald Halbur, Xiang-Jin Meng, Melissa Anne Lum, Young S. Lyoo Spruson Ferguson St Martins Tower,Level 31 Market Street Sydney NSW 2000 (CCN 3710000177) Vaccines Inducing an Immune Response Against Viruses Causing Porcine Respiratory and Reproductive Diseases The following statement is a full description of this invention, including the best method of performing it known to me/us:- 5845c Vaccines Inducing an Immune Response Against Viruses Causing Porcine Respiratory and Reproductive Diseases Background Of The Invention Field of the Invention The present invention concerns a vaccine which protects pigs from a disease caused by respiratory and reproductive viruses, a method of protecting a pig from a respiratory and reproductive disease, a method of producing a vaccine, and DNA obtained from a virus causing a porcine respiratory and reproductive disease.

Discussion of the Background In recent years, North American and European swine herds have been susceptible to infection by new strains of respiratory and reproductive viruses (see A.A.S.P., September/October 1991, pp. 7-11; The Veterinary Record, February 1, 1992, pp. 87-89; Ibid., November 30, 1991, pp. 495-496; Ibid., October 26, 1991, p. 370; Ibid., October 19, 1991, pp. 367-368; Ibid., August 3, 1991, pp. 102-103; Ibid., July 6, 1991; Ibid., June 22, 1991, p. 578; Ibid., June 15, 1991, p. 574; Ibid., June 8, 1991, p. 536; Ibid., June 1, 1991, p. 511; Ibid., March 2, 1991, p. 213). Among the first of the new strains to be identified was a virus associated with the so-called Mystery Swine Disease (MSD) or "blue-eared syndrome", now known as Swine InferEility afnd Respiratory Syndrome (SIRS) or Porcine Reproductive and Respiratory Syndrome (PRRS). In Europe, this disease has 20 also been called porcine epidemic abortion and respiratory syndrome (PEARS), blue abortion disease, blue ear disease abortus blau (Netherlands) and seuchenhafter spatabort der schweine (Germany), and the corresponding virus has been termed "Lelystad virus." In the this disease has also been called Wabash syndrome, mystery pig disease (MPD) and swine plague. A disease which is sometimes associated with PRRS is 00 25 proliferative interstitial pneumonia

(PIP).

Outbreaks of "blue ear disease" have been observed in swine herds in the U.K., Germany, Belgium and the Netherlands. Its outbreak in England has led to cancellation of pig shows. The symptoms of PRRS include a reluctance to eat (anorexia), a mild fever (pyrexia), cyanosis of the extremities (notably bluish ears), stillbirths, abortion, high mortality in affected litters, weak-born piglets and premature farrowing. The majority of piglets born alive to affected sows die within 48 hours. PRRS clinical signs include mild influenza-like signs, rapid respiration ("thumping"), and a diffuse interstitial pneumonitis.

PRRS virus has an incubation period of about 2 weeks from contact with an infected animal. The virus appears to be an enveloped RNA arterivirus (Ibid., February 1, 1992).

The virus has been grown successfully in pig alveolar macrophages and CL2621 cells (Benfield et al, J. Vet. Diagn. Invest., 4:127-133, 1992; Collins et al, Swine Infertility and Respiratory Syndrome/Mystery Swine Disease. Proc., Minnesota Swine Conference for Veterinarians, pp. 200-205, 1991), and in MARC-145 cells (Joo, PRRS: Diagnosis, Proc., IK:IO B168:EAR/GSA 1 68 2 Allen D. Leman Swine Conference, Veterinary Continuing Education and Extension, University of Minnesota (1993), 20:53-55). A successful culturing of a virus which causes SIRS has also been reported by Wensvoort et al (Mystery Swine Disease in the Netherlands: The Isolation of Lelystad Virus. Vet. Quart. 13:121-130, 1991).

The occurrence of PRRS in the U.S. has adversely affected the pig farming industry.

In Canada, PRRS has been characterized by anorexia and pyrexia in sows lasting up to 2 weeks, late-term abortions, increased stillbirth rates, weak-born pigs and neonatal deaths preceded by rapid abdominal breathing and diarrhea. Work on the isolation of the virus causing PRRS, on a method of diagnosing PRRS infection, and on the development of a to vaccine against the PRRS virus has been published (see Canadian Patent Publication No. 2 076 744; PCT International Patent Publication No. W093/03760; PCT International Patent Publication No. W093/06211; and PCT International Patent Publication No.

W093/07898).

A second virus strain discovered in the search for the causative agent of PRRS causes 15 a disease now known as Proliferative and Necrotizing Pneumonia (PNP). The symptoms of PNP and the etiology of the virus which causes it appear similar to PRRS and its corresponding virus, but there are identifiable differences. For example, the virus which causes PNP is believed to be a non-classical or atypical swine influenza A virus (aSIV).

The main clinical signs of PNP are fever, dyspnea and abdominal respiration. Pigs of different ages are affected, but most signs-occur in-pigs between 4 and 16 weeks of age.

Lungs of affected pigs are diffusely reddened and "meaty" in consistency (Collins, September/October 1991, pp. 7-11). By contrast, pigs affected with PRRS show no significant fever, and respiratory signs are observed mainly in neonatal pigs (less than 3 weeks old) with pulmonary lesions, characterized by a diffuse interstitial pneumonia.

25 Encephalomyocarditis virus (EMCV) is another virus which causes severe interstitial pneumonia along with severe interstitial, necrotizing and calcifying myocarditis.

Experimentally, EMCV produces reproductive failure in affected sows (Kim et al, J. Vet.

Diagn. Invest., 1:101-104 (1989); Links et al, Aust. Vet. 63 :150-152 (1986); Love et al, Aust. Vet. 63:128-129 (1986)).

Recently, a more virulent form of PRRS has been occurring with increased incidence in 3-8 week old pigs in the midwestern United States. Typically, healthy 3-5 week old pigs are weaned and become sick 5-7 days later. Routine virus identification methods on tissues from affected pigs have shown that swine influenza virus (SIV), pseudorabies virus (PRV), and Mycoplasma hyopneumoniae are not associated with this new form of PRRS.

The present invention is primarily concerned with a vaccine which protects pigs from the infectious agent causing this new, more virulent form of PRRS, with a method of producing and administering the vaccine, and with DNA encoding a portion of the genome of the infectious agent causing this new form of PRRS. However, it is believed that the information learned in the course of developing the present invention will be useful in developing vaccines and methods of protecting pigs against any and/or all porcine IK:100168:EARJGSA 2of688 3 respiratory and reproductive diseases. For example, the present Inventors have characterised the pathology of at least one PRRS virus which differs from the previously published pathology of PRRS virus(es) (see Table I below). Therefore, the present invention is not necessarily limited to vaccines and methods related to the infectious agent causing this new form of PRRS, which the present Inventors have termed the "Iowa strain" of PRRS virus (PRRSV).

Nonetheless, pessimism and scepticism has been expressed in the art concerning the development of effective vaccines against three porcine viruses (The Veterinary Record, October 26, 1991). A belief that human influenza vaccine may afford some protection against the effects of PRRS and PNP exists in the art (for example, see Ibid., July 6, 1991). However, the use of a human vaccine in a food animal is generally discouraged by regulatory and administrative agencies, and therefore, this approach is not feasible in actual practice (Ibid.).

The pig farming industry has been and will continue to be adversely affected by these porcine reproductive and respiratory diseases and new variants thereof, as they appear. Surprisingly, the market for animal vaccines in the US and worldwide is larger than the market for human vaccines. Thus, there exists an economic incentive to develop new veterinary vaccines, in addition to the substantial public health benefit which is derived from protecting farm animals from disease.

Summary of the Invention There is herein disclosed a biologically pure virus wherein said virus is the •Iowa strain of the virus which causes porcine reproductive and respiratory syndrome (PRRS), wherein inoculation of five-week-old colostrum-deprived, caesarean-derived pigs with 105 TCID 50 of said virus results in lesions in at least 51.9% of lung tissue 10 days post-infection.

There is also disclosed the virus described directly above but which is selected from the group consisting of ISU-12 (VR 2385 and VR 2386), ISU-22 (VR 2429), ISU-79 (VR 2474) and ISU-28 (VR 2549); or a virus exhibiting the identifying characteristics of a virus in said group.

Further disclosed is a biologically pure virus which causes porcine reproductive and respiratory syndrome (PRRS) and has a nested set of mRNAs, wherein said virus has either 7 or more subgenomic mRNAs or from 1 to 4 deletions in its mRNAs, relative to ISU-1894.

Still further disclosed is a vaccine which protects a pig against porcine reproductive and respiratory syndrome (PRRS), comprising an effective amount of a biologically pure virus as described above.

Further disclosed is a method of protecting a pig from a porcine reproductive and respiratory disease, comprising administering to said pig an effective amount of this vaccine.

N\LIBaa]01135:sak 4 Brief Description Of The Figures Figure 1 is a flow chart for the production of a modified live vaccine; Figure 2 is a flow chart of a process for producing an inactivated vaccine; Figure 3 is a flow chart outlining a procedure for producing a subunit vaccine; N\LIBaa]01135:sak Figure 4 is a flowchart outlining a procedure for producing a genetically engineered vaccine; Figures 5 and 6 show histological sections from the lungs of conventional pigs 10 days after infection with a sample of the infectious agent isolated from a pig infected with the Iowa strain of PRRSV; Figure 7 shows a histological section from the lung of a gnotobiotic pig 9 days after infection with a sample of infectious agent isolated from a pig infected with the Iowa strain of PRRSV; Figure 8 shows the heart lesions of a gnotobiotic pig 35 days after infection with a sample of an infectious agent isolated from a pig infected with the Iowa strain of PRRSV; Figure 9 shows bronchio-alveolar lavage cultures exhibiting extensive syncytia, prepared from a gnotobiotic pig 9 days after infection with a lung filtrate sample of an infectious agent isolated from a pig infected with the Iowa strain of PRRSV (ISU-12; see Experiment I, Section below); Figure 10 is an electron micrograph of an enveloped virus particle, about 70 nm in diameter, having short surface spicules, found in alveolar macrophage cultures of pigs infected with an infectious agent associated with the Iowa strain of PRRSV; Figure 11 is an electron micrograph of a pleomorphic, enveloped virus particle, approximately 80 X 320 nm in size, coated by antibodies, found in alveolar macrophage cultures of pigs infected with the Iowa strain of PRRSV; Figures are a series of photographs showing swine alveolar macrophage .(SAM) cultures: uninfected CPE in those infected with ISU-12 and IFA in those infected with ISU-12 (see Experiment II below); Figures are a series of photographs showing PSP-36 cell cultures: uninfected CPE in those infected with ISU-12 four DPI CPE in those infected with ISU-12 five DPI and CPE in those infected with ISU-984 at 5 DPI (a second virus isolate representing the Iowa strain of PRRSV) Figures are a series of photographs showing IFA in ISU-12 infected PSP-36 cells: uninfected infected with ISU-12 at 2.5 DPI and stained with convalescent sera infected with ISU-12 at 2.5 DPI and stained with anti-PRRSV polyclonal antibody (C) and infected with ISU-12 and stained with anti-PRRSV monoclonal antibody Figure 15 is a protein profile of ISU-12 propagated in PSP-36 cells as determined by radioimmunoprecipitation (RIP): lanes 1 and 2 are mock-infected PSP-36 cells, immunoprecipitated with anti-PRRSV polyclonal sera and convalescent sera lanes 3 and 4 are virus-infected PSP-36 cells, immunoprecipitated with anti-PRRSV polyclonal sera and convalescent sera Figure 16 is a flowchart showing a general procedure for construction of a cDNA X library of a strain of infectious agent causing PRRS; IK:)1001 :EAR/GSA 6 of 68 Figure 17 is a flowchart showing a general procedure for the identification of authentic cDNA clones of an infectious agent associated with the Iowa strain of PRRSV (ISU-12) by differential hybridization; Figures 18(A) show open reading frames (ORF's) in the nucleotide sequence of s ISU-12, subgenomic mRNA's, in which the boxed L indicates the leader sequence and (A)n indicates the poly(A) tail at the extreme 3'-end of the genome and the X cDNA clones used to obtain the 3'-terminal nucleotide sequence of ISU-12, in which the regions sequenced are shown by the solid bars and regions not sequenced are shadowed Figure 19 presents the 1938-bp 3'-terminal sequence of the genome of the infectious o1 agent associated with the Iowa strain of PRRSV; Figure 20 shows deduced amino acid sequences encoded by the DNA sequence of Figure 19, shown below the nucleotide sequence; Figure 21 compares the nucleotide sequences of the infectious agent associated with the Iowa strain of PRRSV (ISU-12) and of the Lelystad virus with regard to open reading frame-5 Figure 22 compares the nucleotide sequences of the ORF-6 of the ISU-12 virus with the ORF-6 of the Lelystad virus; Figure 23 compares the nucleotide sequences of the ORF-7 of the ISU-12 virus and the ORF-7 of the Lelystad virus; Figure 24. compares the 3'-nontranslational nucleotide sequences of the ISU-12 virus and the Lelystad virus; Figure 25 shows uninfected Trichoplusian egg cell homogenates (HI-FIVEru, Invitrogen, San Diego, California); Figure 26 shows HI-FIVE cells infected with a recombinant baculovirus containing the ISU-12 ORF-6 gene, exhibiting a cytopathic effect; Figure 27 shows HI-FIVE cells infected with a recombinant baculovirus containing the ISU-12 ORF-7 gene, also exhibiting a cytopathic effect; Figure 28 shows HI-FIVE cells infected with a recombinant baculovirus containing the **-'ISU-12 ORF-6 gene, stained with swine antisera to ISU-12, followed by staining with fluorescein-conjugated anti-swine IgG, in which the insect cells are producing a recombinant protein encoded by the ISU-12 ORF-6 gene; Figure 29 shows HI-FIVE cells infected with a recombinant baculovirus containing the ISU-12 ORF-7 gene, stained with swine antisera to ISU-12, followed by staining with fluorescein-conjugated anti-swine IgG, in which the insect cells are producing recombinant protein encoded by the ISU-12 ORF-7 gene; Figure 30 shows the results of PCR amplification of ORF-5 (lane ORF-6 (lane M) and ORF-7 (lane NP) using ISU-12 specific primers, in which lane SM contains molecular weight standards; Figure 31 shows the results of expressing recombinant baculovirus transfer vector pVL1393, containing ORF-5 (lane ORF-6 (lane M) or ORF-7 (lane NP) of the genome :)0018:EARtGSA 6 of sB of ISU-12, after cleaving plasmid DNA with BamHI and EcoRI restriction enzymes; lane SM contains molecular weight standards; Figure 32 shows a Northern blot of ISU-12 mRNA; Figures 33A and 33B show Northern blots of mRNA taken from other isolates of the s Iowa strain of PRRSV (ISU-22, ISU-55, ISU-79, ISU-1894 and ISU-3927); and Figure 34 is a bar graph of the average gross lung lesion scores (percent of lung affected) for groups of 3 week-old, PRRSV-seronegative, specific pathogen-free (SPF) pigs administered one embodiment of the present vaccine intranasally (IN) or intramuscularly and a group of control pigs (NV/CHALL).

Detailed Description Of The Preferred Embodiments In the present invention, a "porcine respiratory and reproductive disease" refers to the diseases PRRS, PNP and EMCV described above, the disease caused by the Iowa strain of PRRSV, and closely-related variants of these diseases which have appeared and which will appear in the future.

15 is A vaccine "protects a pig against a disease caused by a porcine respiratory and reproductive disease virus or infectious agent" if, after administration of the vaccine to an unaffected pig, lesions in the lung or symptoms of the disease do not appear or are not as 2 severe as in infected, unprotected pigs, and if, after administration of the vaccine to an affected pig, lesions in the lung or symptoms of the-:disease are eliminated or are not as severe as in infected, unprotected pigs. An unaffected pig is a pig which has either not been exposed to a porcine respiratory and reproductive disease infectious agent, or which has been exposed to a porcine respiratory and reproductive disease infectious agent but is not showing symptoms of the disease. An affected pig is a pig which is showing symptoms the disease. The symptoms of the porcine respiratory and reproductive disease may be 25 quantified or scored temperature/fever, lung lesions [percentage of lung tissue infected]) or semi-quantified severity of respiratory distress [explained in detail below]).

A "porcine respiratory and reproductive virus or infectious agent" causes a porcine respiratory and reproductive disease, as described above. The agent causing the new, more virulent form of PRRS has been termed the "Iowa" strain of PRRSV. The disease caused by some isolates of the "Iowa" strain of PRRS virus has symptoms similar to but more severe than other porcine respiratory and reproductive diseases. Clinical signs may include lethargy, respiratory distress, "thumping" (forced expiration), fevers, roughened haircoats, sneezing, coughing, eye edema and occasionally conjunctivitis. Lesions may include gross 3s and/or microscopic lung lesions and myocarditis. The infectious agent may be a single virus, or may be combined with one or more additional infectious agents other viruses or bacteria). In addition, less virulent and non-virulent forms of the Iowa strain have been found, which may cause a subset of the above symptoms or may cause no IK:)00 68:EAJRGSA 7 of 8 symptoms at all, but which can be used according to the present invention to provide protection against porcine reproductive and respiratory diseases nonetheless.

Histological lesions in the various porcine diseases are different. Table 1 below compares physiological observations and pathology of the lesions associated with a number s of diseases caused by porcine viruses: Table 1 Swine Viral Pneumonia Comnarativ Pathnlnov Lesion PRRS(p) PRRS(o), SIV PNP PRCV PPMV Iowa TypeII Inter. thickening Alveolar exudate Airway necrosis_ Syncytia Encephalitis Myocarditis a wherein "PRRS(p)" represents the published pathology of the PRRS virus, "PRRS(o)" represents the pathology of PRRS virus observed by the present Inventors, "SIV" o10 represents swine influenza A virus, "PRCV" represents porcine respiratory coronavirus, "PPMV" represents porcine paramyxovirus, "Iowa" refers to the new strain of PRRSV discovered by the present Inventors, "Type II" refers to Type II pneumocytes (which proliferate in infected pigs), "Inter." refers to interstitial, "Airway necrosis" refers to necrosis in terminal airways, and the symbols and through refer to a 15 comparative severity scale as follows: negative (not observed) mild (just above the threshold of observation) moderate severe' most severe The Iowa strain of PRRSV has been identified by the present Inventors in the midwestern in association with PRRS. It is not yet clear whether the disease associated with the Iowa strain of PRRSV as it is found naturally is due to a unique virus, or a combination of a virus with one (or more) additional infectious agent(s). However, plaque-purified samples of the Iowa strain of PRRSV appear to be a single, unique virus.

Therefore, "the Iowa strain of PRRSV" refers to either a unique, plaque purified virus or a tissue homogenate from an infected animal which may contain a combination of a virus IK:IOe188:EAR/GSA 8 o 68

J

with one (or more) additional infectious agent(s), and a pig infected with the Iowa strain of PRRSV shows one or more of the symptoms characteristic of the disease caused by the Iowa strain of PRRSV, as described above.

Recent evidence indicates that the Iowa strain of PRRSV differs from the infectious agent which causes conventional PRRS. For example, lesions observed in infected pigs exhibiting symptoms of the disease caused by the Iowa strain of PRRSV are more severe than lesions observed in pigs infected with a conventional, previously described PRRS virus alone, and pigs suffering from the disease caused by the Iowa strain of PRRSV are also seronegative for influenza, including viruses associated with PNP.

Referring now to Figures 1-4, flowcharts of procedures are provided for preparing various types of vaccines encompassed by the present invention. The flowcharts of Figures 1-4 are provided as exemplary methods of producing the present vaccines, and are not intended to limit the present invention in any manner.

The first step in each procedure detailed in Figures 1-4 is to identify a cell line is susceptible to infection with a porcine respiratory and reproductive virus or infectious agent. (To simplify the discussion concerning preparation of the vaccine, the term "virus" means virus and/or other infectious agent associated with a porcine respiratory and reproductive disease.) A master cell stock (MCS) of the susceptible host cell is then prepared. The susceptible host cells continue to be passaged beyond MCS. Working cell stock (WCS) is prepared from cell passages betweenrMCS and MCS+n.

A master seed virus is propagated on the susceptible host cell line, between MCS and MCS+n, preferably on WCS. The raw virus is isolated by methods known in the art from appropriate, preferably homogenized, tissue samples taken from infected pigs exhibiting disease symptoms corresponding to those caused by the virus of interest. A suitable host o.0• 25 cell, preferably a sample of the WCS, is infected with the raw virus, then cultured.

S* Vaccine virus is subsequently isolated and plaque-purified from the infected, cultured host cell by methods known in the art. Preferably, the virus to be used to prepare the vaccine is plaque-purified three times.

Master seed virus (MSV) is then prepared from the plaque-purified virus by methods known in the art. The MSV(X) is then passaged in WCS at least four times through MSV(X+1), MSV(X+2), MSV(X+3) and MSV(X+4) virus passages. The MSV(X+4) is considered to be the working seed virus. Preferably, the virus passage to be used in the pig studies and vaccine product of the present invention is MSV(X+5), the product of the fifth passage.

In conjunction with the working cell stock, the working seed virus is cultured by known methods in sufficient amounts to prepare a prototype vaccine, preferably The present prototype vaccines may be of any type suitable for use in the veterinary medicine field. Suitable types include a modified live or attenuated vaccine (Figure an inactivated or killed vaccine (Figure a subunit vaccine (Figure a genetically engineered vaccine (Figure and other types of vaccines recognized in the IK:)0018:EAR/GSA 0 o1 68 O (r veterinary vaccine art. A killed vaccine may be rendered inactive through chemical treatment or heat, etc., in a manner known to the artisan of ordinary skill.

In the procedures outlined by each of Figures 1-4, following preparation of a prototype vaccine, pig challenge models and clinical assays are conducted by methods known in the art. For example, before performing actual vaccination/challenge studies, the disease to be prevented and/or treated must be defined in terms of its symptoms, clinical assay results, conditions etc. As described above, the infectious agent associated with the Iowa strain of PRRSV has been defined in terms of its symptoms and conditions. The clinical analysis of the infectious agent associated with the Iowa strain of PRRSV is io described in the Examples below.

After the disease is sufficiently defined and characterized, one can administer a prototype vaccine to a pig, then expose the pig to the virus or infectious agent which causes the disease. This is known in the art as "challenging" the pig and its immunological system. After observing the response of the challenged pig to exposure to the virus or infectious agent and analyzing the ability of the prototype vaccine to protect the pig, e efficacy studies are then performed by methods known in the art. A potency assay is then developed in a separate procedure by methods known in the art, and prelicensing serials are then produced.

In the preparation of a modified live vaccine as outlined in Figure 1, once a prototype vaccine-is prepared, cell growth conditions -and virus production are first optimized, then a production outline is prepared by methods known in the art. Once the production outline is prepared, prelicensing serials are then subsequently prepared by methods known in the art.

SPrelicensing serials refer to a large-scale production of a promising prototype vaccine, which demonstrates the ability to produce serials with consistent standards, one approach S 25 to preparing a prototype live vaccine is to subject the virus-infected cells (preferably, master seed virus-infected cells) to one or more cycles of freezing and thawing to lyse the cells. The frozen and thawed infected cell culture material may be lyophilized (freezedried) to enhance preservability for storage. After subsequent rehydration, the material is then used as a live vaccine.

The procedure for preparing prelicensing serials for an inactivated vaccine (Figure 2) is similar to that used for the preparation of a modified live vaccine, with one primary modification. After optimization of cell growth conditions and virus production protocol, a virus inactivation protocol must then be optimized prior to preparation of a suitable production outline. Virus inactivation protocols and their optimization are generally known to those in the art, and may vary in a known or predictable manner, depending on the particular virus being studied.

The preparation of a subunit vaccine (Figure 3) differs from the preparation of a modified live vaccine or inactivated vaccine. Prior to preparation of the prototype vaccine, the protective or antigenic components of the vaccine virus must be identified. Such protective or antigenic components include certain amino acid segments or fragments of the IK:)OO168:EARIGSA 10 Of 88 viral coat proteins which raise a particularly strong protective or immunological response in pigs (which are preferably at least 5 amino acids in length, particularly preferably at least amino acids in length); single or multiple viral coat proteins themselves, oligomers thereof, and higher-order associations of the viral coat proteins which form virus substructures or identifiable parts or units of such substructures; oligoglycosides, glycolipids or glycoproteins present on or near the surface of the virus or in viral substructures such as the nucleocapsid; lipoproteins or lipid groups associated with the virus, etc. These components are identified by methods known in the art. Once identified, the protective or antigenic portions of the virus (the "subunit") are subsequently purified and/or cloned by methods known in the art.

The preparation of prelicensing serials for a subunit vaccine (Figure 3) is similar to the method used for an inactivated vaccine (Figure with some modifications. For example, if the subunit is being produced through recombinant genetic techniques, expression of the cloned subunit may be optimized by methods known to those in the art (see, for example, S 15 relevant sections of Maniatis et al, "Molecular Cloning: A Laboratory Manual," Cold Spring Harbor Laboratory (1989), Cold Spring Harbor, Massachusetts). On the other hand, if the subunit being employed represents an intact structural feature of the virus, such as an entire coat protein, the procedure for its isolation from the virus must then be optimized. In either case, after optimization of the inactivation protocol, the subunit purification protocol may be optimized prior to preparation of the production outline.

Genetically engineered vaccines (Figure 4) begin with a modification of the general procedure used for preparation of the other vaccines. After plaque purification, the wildtype virus may be isolated from a suitable tissue homogenate by methods known in the art, preferably by conventional cell culture methods using PSP-36 or macrophage cells as hosts.

25 The RNA is extracted from the biologically pure virus or infectious agent by methods known in the art, preferably by the guanidine isothiocyanate method using a commercially available RNA isolation kit (for example, the kit available from Stratagene, La Jolla, California), and purified by methods known in the art, preferably by ultracentrifugation in CsCI gradient. RNA may be further purified or enriched by oligo (dT)-cellulose column chromatography.

The viral genome is then cloned into a suitable host by methods known in the art (see Maniatis et al, cited above), and the virus genome is then analyzed to determine essential regions of the genome for producing antigenic portions of the virus. Thereafter, the procedure is generally the same as for a modified live vaccine, an inactivated vaccine or a subunit vaccine. The present vaccine protects pigs against a virus or infectious agent which causes a porcine reproductive and respiratory disease. Preferably, the present vaccine protects pigs against the infectious agent associated with the Iowa strain of PRRSV.

However, the present vaccine is also expected to protect a pig against infection by exposure to closely related variants of the infectious agent associated with the Iowa strain of PRRSV.

IK:)00188:EARIGSA 11 of 88 12 Relatively few viruses are amenable to the production of live virus vaccines. The advantages of live virus vaccines is that all possible immune responses are activated in the recipient of the vaccine, including systemic, local, humoral and cell-mediated immune responses. The disadvantages of live virus vaccines lie in the potential for contamination s with live adventitious agents, such as SV40 virus and bovine viral diarrhea virus, a common contaminant of bovine fetal serum. This risk, plus the risk that the virus may revert to virulence in the field or may not be attenuated with regard to the fetus, young animals and other species, may outweigh the advantages of a live vaccine.

Inactivated virus vaccines can be prepared- by treating viruses with inactivating agents lo such as formalin or hydrophobic solvents, acid, etc., by irradiation with ultraviolet light or X-rays, by heating, etc. Inactivation is conducted in a manner understood in the art. A virus is considered inactivated if it is unable to infect a cell susceptible to infection. For example, in chemical inactivation, a suitable virus sample or serum sample containing the virus is treated for a sufficient length of time with a sufficient amount or concentration of inactivating agent at a sufficiently high (or low, depending on the inactivating agent) temperature or pH to inactivate the virus. Inactivation by heating is conducted at a temperature and. for a length of time sufficient to inactivate the'virus. Inactivation by irradiation is conducted using a wavelength of light or other energy for a length of time sufficient to inactivate the virus. Examples of inactivated vaccines for human use include influenza vaccine, poliomyelitis, 'rabies and hepatitis type B. A successful and effective example of an inactivated vaccine for use in pigs is the porcine parvovirus vaccine.

*Subunit -virus vaccines are prepared from semi-purified virus subunits b y the methods 4 described above in the discussion of Figure 3. For example, hemagglutinin isolated from influenza virus and neuraminidase surface antigens isolated from influenza virus have been prepared, and shown to be less toxic than the w hole virus. Alternatively, subunit vaccines *..can be prepared from highly purified subunits of the virus. An example in humans is the 22-nm surface antigen of human hepatitis B virus. Human herpes simplex virus subunits and many other examples of subunit vaccines for use in humans are known.

-Attenuated virus vaccines can be found in nature and may have naturally-occurring gene deletions, or alternatively, may be prepared by a variety of known methods, such as serial passage in cell cultures or tissue cultures. Viruses can also be attenuated by gene deletions or gene mutations.

Genetically engineered vaccines are produced by technigues known to those in the art.

Such techniques include those using recombinant DNA and those using live viruses. For example, certain virus genes can be identified which code for proteins responsible for inducing a stronger immune or protective response. in pigs. Such identified genes can be cloned into protein expression vectors, such as the baculovirus vector, and used to infect appropriate host cells (see,, for example, O'Reilly et al, "Baculovirus Expression Vectors: A Lab Manual," Freeman Co. (1992)). The host cells are cultured, thus expressing the (K:JOO1O8:EARIGSA 12 of88 13 desired vaccine proteins, which can be purified to a desired extent, then used to protect the pigs from a'respiratory and reproductive disease.

Genetically engineered proteins may be expressed in insect cells, yeast cells or mammalian cells. The genetically engineered proteins, which may be purified and/or isolated by conventional methods, can be directly inoculated into animals to confer protection against porcine reproductive and respiratory diseases. Envelope proteins from a porcine reproductive and respiratory disease infectious agent or virus are used in a vaccine to induce neutralizing antibodies. Nucleoproteins from a porcine reproductive and respiratory disease infectious agent or virus are used in a vaccine to induce cellular immunity.

Preferably, the present invention transforms an insect cell line (HI-FIVE) with a transfer vector containing polynucleic acids obtained from the Iowa strain of PRRSV.

Preferably, the present transfer vector comprises linearized baculovirus DNA and a plasmid containing polynucleic acids obtained from the Iowa strain of PRRSV. The host cell line may be co-transfected with the linearized baculovirus DNA and a plasmid, so that a recombinant baculovirus is made. Particularly preferably, the present polynucleic acid encodes one or more proteins of the Iowa strain of PRRSV.

Alternatively, RNA or DNA from a porcine reproductive and respiratory disease infectious agent or virus encoding one or more envelope proteins and/or nucleoproteins can 0..

be inseited into live vectors, such as a poxvirus or an adenovirus, and used as a vaccine.

Thus, the present invention further concerns a polynucleic acid isolated from a portion of the genome of a virus causing a respiratory and reproductive disease, preferably a polynucleic acid isolated from a portion of the genome of the Iowa strain of PRRSV. The phrase "polynucleic acid" refers to RNA or DNA, as well as RNA and cDNA .9 25 corresponding to or complementary to the RNA or DNA from the infectious agent. The present polynucleic acid has utility as a means for producing the present vaccine, as a means for screening or identifying infected animals, and as a means for identifying related viruses and infectious agents.

In one embodiment of the present invention, the polynucleic acid encodes one or more proteins of a virus causing a respiratory and reproductive disease, preferably one or both of the viral membrane (envelope) protein and the capsid protein (nucleoprotein). Particularly preferably, the present polynucleic acid is taken from a 2 kb fragment from the 3'-end of the genome, and encodes one or more of the envelope proteins encoded by ORF-5 and ORF-6 and/or the nucleoprotein encoded by ORF-7 of the genome of the Iowa strain of PRRSV. Most preferably, the polynucleic acid is isolated from the genome of an infectious agent associated with the Iowa strain of PRRSV; for example, the agent described in Experiments I-M below (ISU-12), and is selected from the group consisting of ORF 5 (SEQ ID NO:13), ORF 6 (SEQ ID NO:15), ORF 7 (SEQ ID NO:18) and the 1938bp 3'-terminal sequence of the ISU-12 genome (SEQ ID NO:8).

(K:)00168:EAR/GSA 13 of 14 In the context of the present application, the proteins or peptides encoded by RNA and/or DNA from a virus or infectious agent are considered "immunologically equivalent" if the polynucleic acid has 90% or greater homology with the polynucleic acid encoding the immunogenic protein or peptide. "Homology" in this application refers to the percentage s of identical nucleotide or amino acid sequences between two or more viruses of infectious agents. Accordingly, a further aspect of the present invention encompasses an isolated polynucleic,acid which is at least 90% homologous to a polynucleic acid obtained from the genome of a virus causing a respiratory and reproductive disease, preferably a polynucleic acid obtained from the genome of the infectious agent associated with the Iowa strain of to PRRSV.

Relatively short segments of polynucleic acid (about 20 bp or longer) in the genome of a virus can be used to screen or identify infected animals, and/or to identify related viruses, by methods described herein and known to those of ordinary skill in the art. Accordingly, a further aspect of the present invention encompasses an isolated (and if desired, purified) S 15 polynucleic acid consisting essentially of isolated fragments obtained from a portion of the genome of a virus causing a respiratory and reproductive disease, preferably a polynucleic acid obtained from a portion of the genome of the infectious agent associated with the Iowa o strain of PRRSV, which are at least 20 nucleotides in length, preferably from 20 to 100 nucleotides in length. Particularly preferably, the present isolated polynucleic acid 20 fragments are obtained from the 1938-bp 3Sterminafsequence of the ISU-12 genome (SEQ ID NO:8), and most preferably, are selected from the group consisting of SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7.

The present isolated polynucleic acid fragments can be obtained by digestion of the 25 cDNA corresponding to (complementary to) the viral polynucleic acids with one or more appropriate restriction enzymes, or can be synthesized using a commercially available automated polynucleotide synthesizer.

In another embodiment of the present invention, the polynucleic acid encodes one or more antigenic peptides from a virus causing a respiratory and reproductive disease, preferably the one or more antigenic peptides from the infectious agent associated with the Iowa strain of PRRSV. As described above, the present polynucleic acid encodes an antigenic portion of a protein from a virus causing a respiratory and reproductive disease, preferably from the infectious agent associated with the Iowa strain of PRRSV, at least amino acids in length, particularly preferably at least 10 amino acids in length. Methods of determining the antigenic portion of a protein are known to those of ordinary skill in the art.

The present invention also concerns a protein encoded by one or more of the ORF's of the Iowa strain of PRRSV. Preferably, the protein is encoded by a polynucleic acid sequence selected from the group consisting of SEQ ID NO: 8, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:18 and SEQ ID NO:19 (see also SEQ ID NOS:9-12). The present IK:O0168:EAR/GSA 14 o 68 proteins and antigenic peptides are useful in serological tests for screening pigs for exposure to PRRSV, particularly to the Iowa strain of PRRSV.

The present invention further concerns a biologically pure sample of a virus or infectious agent causing a porcine reproductive and respiratory disease characterised by the following symptoms and clinical signs: lethargy, respiratory distress, forced expiration, fevers, roughened haircoats, sneezing, coughing, eye edema and occasionally conjunctivitis.

The present biologically pure sample of a virus or infectious agent may be further characterised in that it causes a porcine reproductive and respiratory disease which may include the following histological lesions: gross and/or microscopic lung lesions, Type II pneumocyte, myocarditis, encephalitis, alveolar exudate formation and syncytia formation.

The phrase "biologically pure" refers to a sample of a virus or infectious agent in which all progeny are derived from a single parent. Usually, a "biologically pure" sample is achieved by 3 x plaque purification in cell culture. In particular, the present biologically pure virus or infectious agent is the Iowa strain of porcine reproductive and respiratory syndrome.

Samples of ISU-12-SAH and ISU-12 have been deposited under the terms of the Budapest Treaty at the ATCC (American Type Culture Collection), 12301 Parklawn Drive, Rockville, Maryland 20852, on 30 October, 1992 under the accession numbers VR 2385 and VR 2386, respectively. ISU-22-SAH, ISU-51-SAH, ISU-55-SAH and ISU-3927-SAH were deposited with the ATCC on 29 September 1993 under the accession numbers VR 2429, 20 VR 2428, VR 2430 and VR 2431 respectively. Samples of ISU-28, ISU-79 and ISU-1894 have been deposited under the terms of the Budapest Treaty at the ATCC (American Type Culture Collection), 10801 University Boulevard, Manassas VA 20110-2209, U.S.A. under the accession numbers VR 2549, VR 2474 and VR 2475 respectively.

°The Iowa strain of PRRSV may also be characterised by Northern blots of its mRNA.

for example, the Iowa strain of PRRSV may contain either 7 or 9 mRNA's, which may also have deletions therein. In particular, as will be described in the Experiments below, the mRNA's of the Iowa strain of PRRSV may contain up to four deletions.

The present invention further concerns a composition for protecting a pig from viral infection, comprising an amount of the present vaccine effective to raise an immunological response to a virus which causes a porcine reproductive and respiratory disease in a physiologically acceptable carrier.

An effective amount of the present vaccine is one in which a sufficient immunological response to the vaccine is raised to protect a pig exposed to a virus which causes a porcine reproductive and respiratory disease or related illness. Preferably, the pig is protected to an I:\DAYLIBibaa\08720.docsak extent in which from one to all of the adverse physiological symptoms or effects lung lesions) of the disease to be prevented are found to be significantly reduced.

The composition can be administered in a single dose, or in repeated doses. Dosages may contain, for example, from 1 to 1,000 micrograms of virus-based antigen (vaccine), but should not contain an amount of virus-based antigen sufficient to result in an adverse reaction or physiological symptoms of infection. Methods are known in the art for determining suitable dosages of active antigenic agent.

The composition containing the present vaccine may be administered in conjunction with an adjuvant. An adjuvant is a substance that increases the immunological response to I:\DAYLIB\libaa\08720.docsak 16 the present vaccine when combined therewith. The adjuvant mae adjuvant m b administered at the same time and at the same site as the vaccine or at a different time, for example, as a booster. Adjuvants also may advantageously be administered to the animal in a manner or at a site or location different from the manner, site or location in which the vaccine is administered. Adjuvants include aluminum hydroxide, aluminum potassium sulfate, heatlabile or heat-stable enterotoxin isolated from Escherichia coli, cholera toxin or the B subunit thereof, diphtheria toxin, tetanus toxin, pertussis -toxin, Freund's incomplete adjuvant, Freund's complete adjuvant, and the like. Toxin-based adjuvants, such as diphtheria toxin, tetanus toxin and pertussis toxin, may be inactivated prior to use, for example, by treatment with formaldehyde.

The present invention also concerns a method of protecting a pig from infection against a virus which causes a porcine respiratory and reproductive disease, comprising administering an effective amount of a vaccine which raises an immunological response against such a virus to a pig in need of protection against infection by such a virus. By "protecting a pig from infection" against a porcine respiratory and reproductive virus or infectious agent, it is meant that after administration of the present vaccine to a pig, the pig shows reduced (ess severe) or no clinical symptoms (such as fever) associated with the corresponding disease, relative to control (infected) pigs. The clinical symptoms may be quantified fever, antibody count, and/or lung lesions), or semi-quantified 20 severity of respiratory distress). In the present invention, a system for measuring respiratory distress in affected pigs has been developed. The present clinical respiratory scoring system evaluates the respiratory distress of affected pigs by the following scale: 0 no disease; normal breathing 1 mild dyspnea and polypnea when the pigs are stressed (forced to breathe in larger volumes and/or at an accelerated rate) 2 mild dyspnea and polypnea when the pigs are at rest 3 moderate dyspnea and polypnea when the pigs are stressed 4 moderate dyspnea and polypnea when the pigs are at rest 30 5 severe dyspnea and polypnea when the pigs are stressed 6 severe dyspnea and polypnea when the pigs are at rest In the present clinical respiratory scoring system, a score of is normal, and indicates that the pig is unaffected by a porcine respiratory and reproductive disease. A score of indicates moderate respiratory disease, and a score of indicates very severe respiratory disease. An amount of the present vaccine or composition may be considered effective if a group of challenged pigs given the vaccine or composition show a lower average clinical respiratory score than a group of identically challenged pigs not given the vaccine or composition. (A pig is considered "challenged" when exposed to a concentration of an infectious agent sufficient to cause disease in a non-vaccinated animal.) IK:100188:EAR/GSA 18 O 88 17 Preferably, the present vaccine composition is administered directly to a pig not yet exposed to a virus which causes a reproductive or respiratory disease. The present vaccine may be administered orally or parenterally. Examples of parenteral routes of administration include intradermal, intramuscular, intravenous, intraperitoneal, subcutaneous and intranasal routes of administration.

When administered as a solution, the present vaccine may be prepared in the form of an aqueous solution, a syrup, an elixir, or a tincture.

Such formulations are known in the art, and are prepared by dissolution of the antigen and other appropriate additives in the appropriate solvent systems. Such solvents include water, saline, ethanol, ethylene glycol, glycerol, Al fluid, etc. Suitable additives known in the art include certified dyes, flavors, sweeteners, and antimicrobial preservatives, such as thimerosal (sodium ethylmercurithiosalicylate). Such solutions may be stabilized, for example, by addition of partially hydrolyzed gelatin, sorbitol, or cell culture medium, and may be buffered by methods known in the art, using reagents known in the art, such as sodium hydrogen phosphate, sodium dihydrogen phosphate, potassium hydrogen phosphate and/or potassium dihydrogen phosphate.

Liquid formulations may also include suspensions and emulsions. The preparation of suspensions, for example using a colloid mill, and emulsions, for example using a homogenizer, is known in the art.

20 Parenteral dosage forms, designed for injection into body fluid systems, require proper isotonicity and pH buffering to the corresponding levels of porcine body fluids. Parenteral formulations must also be sterilized prior to use.

Isotonicity can be adjusted with sodium chloride and other salts as needed. Other solvents, such as ethanol or propylene glycol, can be used to increase solubility of ingredients of the composition and stability of the solution. Further additives which can be used in the present formulation include dextrose, conventional antioxidants and conventional chelating agents, such as ethylenediamine tetraacetic acid (EDTA).

The present invention also concerns a method of producing the present vaccine, comprising the steps of: 30 collecting a virus or infectious agent which causes a porcine respiratory and reproductive disease, and treating the virus or infectious agent in a manner selected from the group consisting of plaque-purifying the virus or infectious agent, (ii) heating the virus or infectious agent at a temperature and for a time sufficient to deactivate the virus or infectious agent, (iii) exposing or mixing the virus or infectious agent with an, amount of an inactivating chemical sufficient to inactivate the virus or infectious agent, (iv) breaking down the virus or infectious agent into its corresponding subunits and isolating at least one of the subunits, and synthesizing or isolating a polynucleic acid encoding a surface protein of the virus or infectious agent, infecting a suitable host cell with the polynucleic acid, culturing the host cell, and isolating the surface protein from the culture.

IK:)00188:EARIGSA 17 of 68 18 Preferably, the virus or infectious agent is collected from a culture medium by the steps of precipitating infected host cells, (ii) lysing the precipitated cells, and (iii) centrifuging the virus or infectious agent prior to the subsequent treatment step.

Particularly preferably, the host cells infected with the virus or infectious agent are s cultured in a suitable medium prior to collecting.

Preferably, after culturing infected host cells, the infected host cells are precipitated by adding a solution of a conventionally-used poly(ethylene glycol) (PEG) to the culture medium, in an amount sufficient to precipitate the infected cells. the precipitated infected cells may be further purified by centrifugation. The precipitated cells are then lysed by methods known to those of ordinary skill in the art.

Preferably, the cells are lysed by repeated freezing and thawing (three cycles of freezing and thawing is particularly preferred). Lysing the precipitated cells releases the virus, which may then be collected, preferably by centrifugation. The virus may be isolated and purified by centrifuging in a CsCI gradient, then recovering the appropriate is virus-containing band from the CsCI gradient.

Alternatively, the infected cell culture may be frozen and thawed to lyse the cells.

The frozen and thawed cell culture material may be used directly as a live vaccine.

Preferably, however, the frozen and thawed cell culture material is lyophilized (for storage), then rehydrated for use as a vaccine.

20 The culture media may contain buffered saline, essential nutrients and suitable sources of carbon and nitrogen recognized in the art, in concentrations sufficient to permit growth of virus-infected cells. Suitable culture media include Dulbecco's minimal essential medium (DMEM), Eagle's minimal essential medium (MEM), Ham's medium, medium 199, fetal bovine serum, fetal calf serum, and other equivalent media which support the 25 growth of virus-infected cells. The culture medium may be supplemented with fetal bovine serum (up to 10%) and/or L-glutamine (up to 2 mM), or other appropriate additives, such as conventional growth supplements and/or antibiotics. A preferred medium is DMEM.

Preferably, the present vaccine is prepared from a virus or infectious agent cultured in an appropriate cell line. The cell line is preferably PSP-36 or an equivalent cell line.

30 capable of being infected with the virus and cultured. An example of a cell line equivalent to PSP-36 is the cell line PSP-36-SAH, which was deposited under the terms of the Budapest Treaty at the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852, on October 30, 1992, under accession number CRL 11171. Another equivalent cell line is MA-104, available commercially from Whittaker Bioproducts, Inc. (Walkersville, Maryland). Preliminary results indicate that the infectious agent associated with the Iowa strain of PRRSV can be cultured in porcine turbinate cells.

After plaque purification, the infectious agent associated with the Iowa strain of PRRSV produces the lesions characterized under the heading "Iowa" in Table I above, and shown in Figs. 5-8.

[K:1001 6 8:EAR/GSA 18 of 8 Accordingly, the present invention also concerns a method of culturing a virus or infectious agent, preferably in a cell line selected from the group consisting of PSP-36 and equivalent cell lines capable of being infected with the virus and cultured. The method of culturing a virus or infectious agent according to the present invention comprises infecting cell line PSP-36 or an equivalent cell line capable of being infected with a virus or infectious agent which causes a porcine respiratory and reproductive disease and cultured, and culturing the infected cell line in a suitable medium.

Preferably, the virus or infectious agent is the Iowa strain of PRRSV, or causes a disease selected from the group consisting of PRRS, PNP, and related diseases.

Particularly preferably, the present vaccine is prepared from the Iowa strain of PRRSV, and is cultivated in PSP-36 cells.

The cell line MA-104 is obtained from monkey kidney cells, and is epithelial-like.

MA-104 cells form a confluent monolayer in culture flasks containing Dulbecco's minimal essential medium and 10% FBS (fetal bovine serum). When the monolayer is formed, the is cells are inoculated with a sample of 10% homogenized tissue, taken from an appropriate tissue (such as lung and/or heart) in an infected pig. Preferably, appropriate antibiotics are present, to permit growth of virus and host cells and to suppress growth and/or viability of cells other than the host cells bacteria or yeast).

i" Both PSP-36 and MA-104 cells grow some isolates of the PRRS virus to high titers 20 (over 107 TCIDso/ml). PSP-36 and MA-104 cells will also grow the infectious agent associated with the Iowa strain of PRRSV. MA-104 cells also are able to grow rotaviruses, polioviruses, and other viruses. CL2621 cells are believed to be of nonporcine origin and are epithelial-like, and are proprietary (Boehringer-Mannheim). By contrast to PSP-36 and MA-104, some samples of the virus which causes PRRS have been 25 unsuccessfully cultured in CL2621 cells (Bautista et al, American Association of Swine Practitioners Newsletter, 4:32, 1992).

The primary characteristics of CL2621 are that it is of non-swine origin, and is epithelial-like, growing in MEM medium. However, Benfield et al Vet. Diagn.

Invest., 1992; 4:127-133) have reported that CL2621 cells were used to propagate PRRS 30 virus, but MA-104 cells were used to control polio virus propagation, thus inferring that CL2621 is not the same as MA-104, and that the same cell may not propagate both viruses.

The infectious agent associated with the Iowa strain of PRRSV generally cannot grow in cell lines other than PSP-36, PSP-36-SAH and MA-104. As described above, however, some viruses which cause PRRS have been reported to grow in both CL2621 and primary swine alveolar macrophages, although some strains of PRRS virus do not grow in PSP-36, MA-104 or CL2621 cells.

The present vaccine can be used to prepare antibodies which may provide.

immunological resistance to a patient (in this case, a pig) exposed to a virus or infectious agent. Antibodies encompassed by the present invention immunologically bind either to a vaccine which protects a pig against a virus or infectious agent which causes a IK:100168:EARGSA 19 of 68 respiratory and reproductive disease or to the porcine respiratory and reproductive virus or infectious agent itself. The present antibodies also have utility as a diagnostic agent for determining whether a pig has been exposed to a respiratory and reproductive virus or infectious agent, and in the preparation of the present vaccine. The antibody may be used s to prepare an immunoaffinity column by known methods, and the immunoaffinity column can be used to isolate the virus or infectious agent, or a protein thereof.

To raise antibodies to such vaccines or viruses, one must immunize an appropriate host animal, such as a mouse, rabbit, or other animals used for such inoculation, with the protein used to prepare the vaccine. The host animal is then immunized (injected) with one of the types of vaccines described above, optionally administering an immune-enhancing agent (adjuvant), such as those described above. The host animal is preferably subsequently immunized from 1 to 5 times at certain intervals of time, preferably every 1 to 4 weeks, most preferably every 2 weeks. The host animals are then sacrificed, and their blood is collected. Sera is then separated by known techniques from the whole blood collected. The sera contains antibodies to the vaccines. Antibodies can also be purified by known methods to provide immunoglobulin G (IgG) antibodies.

The present invention also encompasses monoclonal antibodies to the present vaccines and/or viruses. Monoclonal antibodies may be produced by the method of Kohler et al (Nature, vol. 256 (1975), pages 495-497). Basically, the immune cells from a whole cell 20 preparation of the spleen of the immunized host animal (described above) are fused with myeloma cells by a conventional procedure to produce hybridomas. Hybridomas are cultured, and the resulting culture fluid is screened against the fluid or inoculum carrying the infectious agent (virus or vaccine). Introducing the hybridoma into the peritoneum of the host animal produces a peritoneal growth of the hybridoma. Collection of the ascites 25 fluid of the host animal provides a sample of the monoclonal antibody to the infectious agent produced by the hybridoma. Also, supernatant from the hybridoma cell culture can be used as a source of the monoclonal antibody, which is isolated by methods known to those of ordinary skill in the art. Preferably, the present antibody is of the IgG or IgM type of immunoglobulin.

o The present invention also concerns a method of treating a pig suffering from a respiratory and reproductive disease, comprising administering an effective amount of an antibody which immunologically binds to a virus which causes a porcine respiratory and reproductive disease or to a vaccine which protects a pig against infection by a porcine respiratory and reproductive virus in a physiologically acceptable carrier to a pig in need thereof.

The present method also concerns a diagnostic kit for assaying a virus which causes a porcine respiratory disease, a porcine reproductive disease, or a porcine reproductive and respiratory disease, comprising the present antibody described above and a diagnostic agent which indicates a positive immunological reaction with said antibody.

K:)00168:EAR/GSA 20 of 68 21 The present diagnostic kit is preferably based on modifications to known immunofluorescence assay (IFA), immunoperoxidase assay (IPA) and enzyme-linked immunosorbant assay (ELISA) procedures.

In IFA, infected cells are fixed with acetone and methanol solutions, and antibodies s for the convalescent sera of infected pigs are incubated with the infected cells, preferably for about 30 min. at 37 0 C. A positive immunological reaction is one in which the antibody binds to the virus-infected cells, but is not washed out by subsequent washing steps (usually 3 X with PBS buffer). A second antibody (an anti-antibody) labeled with a fluorescent reagent (FITC) is then added and incubated, preferably for another 30 min. A positive immunological reaction results in the second antibody binding to the first, being retained after washing, and resulting in a fluorescent signal, which can be detected and semiquantified. A negative immunological reaction results in little or no binding of the antibody to the infected cell. Therefore, the second, fluorescently-labeled antibody fails to bind, the fluorescent label is washed out, and little or no fluorescence is detected, compared to an appropriate positive control.

IPA and ELISA kits are similar to the IFA kit, except that the second antibody is labeled with a specific enzyme, instead of a fluorescent reagent. Thus, one adds an appropriate substrate for the enzyme bound to the second antibody which results in the production of a colored product, which is then detected and quantified by colorimetry, for 20 example.

Other features of the invention will become apparent in the course of the following descriptions of exemplary embodiments, which are given for illustration of the invention, and are not intended to be limiting thereof.

Experiment I In Example 1, a case of endemic pneumonia in 5-8 week old pigs was investigated.

Microscopic lesions of the Iowa strain of PRRSV observed in the pigs were compatible with a viral etiology. (Accordingly, hereinafter, to simplify the discussion, the terms "virus" and "viral" will refer to a virus or infectious agent in the meaning described above for the present application, or a property thereof.) The disease was experimentally S 30 transmitted to conventional and gnotobiotic pigs using lung homogenate isolated from infected pigs filtered through a 0.22 plm filter. Common swine viral respiratory pathogens were not demonstrated. Two types of virus particles were observed in cell culture by electron microscopy. One type was about 70 nm in diameter, was enveloped and had short surface spicules. The other type was enveloped, elongated, pleo-morphic, measured 80 X 320 nm and was coated by antibodies.

Materials and Methods Material from pigs infected with naturally-occurring pneumonia Tissues from three infected 6-week-old pigs from a 900-sow farrow-to-feeder-pig herd in Southwestern Iowa were collected and studied. Prior observations of the herd showed IK:)00188:EARIGSA 21 of 68

I

22 that five to seven days after weaning, 50-70% of the similarly-infected pigs became anorexic, were rough-haired, and experienced lethargy, coughing, fever, and "thumping".

Approximately 10-25% of the infected pigs had conjunctivitis. Most of the infected pigs recovered in 7-10 days but, 10-15% were severely stunted. due to secondary bacterial infections, and were not suitable for sale as feeder pigs. Swine reproductive failure, including increased stillbirths, mummified fetuses, and infertility, had occurred at the time of the original outbreak of the disease in this herd, but later diminished with time.

Respiratory disease in the nursery stage has been persistant.

Lung lesions characterized by proliferative bronchiolitis and alveolitis were observed o1 in formalin-fixed tissues from four different 6-week-old pigs. Attempts to isolate SIV, pseudorabies virus (PRV) and encephalomyocarditis virus (EMCV) were not successful.

Immunofluorescence examination of frozen sections of lung for swine influenza virus (SIV), pseudorabies virus (PRV), and Mycoplasma hyopneumoniae were negative.

Pasteurella multocida type D was isolated from the nasal cavities and Haemophilus parasuis was isolated from the lungs.

Five acutely affected 5-6 week old pigs, which had been weaned for 10 days, were subsequently obtained from the herd. All pigs had fevers of at least 40.5 0 C. The pigs were necropsied, and lung tissue samples from the pig with gross lesions most typical of a viral pneumonia were collected and prepared for immediate inoculation into conventional 20 specific pathogen-free (SPF) pigs. Lung, liver, kidney, spleen, brain, and heart tissue samples from all five acutely affected 5-6 week old pigs were cultured for common bacterial and viral pathogens. Sections of the same tissues were collected and fixed in neutral buffered formalin for histopathological examination.

Experimental transmission in conventional pigs 25 Experimental pigs Sixteen five-week old pigs were obtained from a herd free of mycoplasmas, PRV, porcine respiratory coronavirus (PRCV), and transmissible gastroenteritis virus (TGEV).

Eight pigs were placed in each of two isolated 4 X 5 meter rooms with concrete floors and automated ventilation. The pigs were fed an 18% protein corn-soybean meal ration and ao water ad libitum.

Experimental design Immediately after necropsy of the pigs with naturally occurring pneumonia, a lung homogenate was prepared in Dulbecco's modified Eagle's minimal essential medium, clarified at 1000 x g for 10 minutes, followed by centrifugation at 10,000 x g for minutes. The clarified supernatant was filtered through a 0.22 im filter. Eight pigs were inoculated intranasally with 5 ml of filtered lung homogenate. Eight control pigs .were inoculated intranasally with 5 ml of filtered lung homogenate prepared as described above from a normal uninfected gnotobiotic pig.

Clinical signs and temperatures were monitored and recorded daily. One pig from each group was euthanized and necropsied at 5, 7, 10 and 15 days post inoculation (DPI), IK:)O01B8:EAR/GSA 22 of 68 respectively. Tissues were collected at the time of necropsy for aerobic and anaerobic bacterial isolation procedures, mycoplasma isolation, detection of antigens for Mycoplasma hyopneumoniae, SIV, PRV, parainfluenza virus type 3 and bovine respiratory syncytial virus (BRSV), and for virus isolation. Tissues were fixed in 10% neutral buffered formalin for histopathological examination. Lungs were fixed by inflation with formalin at the time of necropsy.

Experimental transmission in gnotobiotic pigs Experimental pigs Eight colostrum-deprived, caesarean-derived (CDCD), crossbred, one-day-old to gnotobiotic pigs were randomly divided into two isolators (four pigs in each isolator). Pigs were fed an iron-fortified, sterilized, canned liquid milk replacer (SPF-LAC, Pet-Ag Inc, Elgin, Illinois.) Experimental design Four principal pigs were inoculated -with filtered (0.22 gim) lung homogenate intranasally (3 ml) and orally (1 ml) at 3 days of age. This filtrate was prepared from an experimentally infected conventional pig lung which had been collected 7 days postinfection (DPI). Four control pigs were inoculated with lung homogenate prepared from a normal gnotobiotic pig.

One pig from each group was killed at 5, 9, 28, and 35 DPI, respectively. Lung, 20 liver, kidney, brain, spleen, thymus, nasal-turbinates- heart, and intestines were collected and fixed in 10% neutral buffered formalin for histopathological examination. Lung, brain, spleen, and heart were collected for virus isolation. Lung, liver, and spleen were collected for bacteriologic isolation. Lung was collected immediately into Friis medium for mycoplasma isolation or was frozen at -70 0

C.

25 Microbiological assays Virus isolation Tissue suspensions (10% w/v) clarified at 1000 X g were inoculated on to cell monolayers and observed for cytopathic effect. Primary fetal swine kidney cultures, primary porcine alveolar macrophage cultures, and established cell lines of PK15, bovine 30 turbinate, baby hamster kidney (BHK), Vero, and swine testes (ST) were used for the virus isolation attempts. Direct bronchio-alveolar lavage cultures were prepared from infected and control gnotobiotic pigs. Attempts to detect virus were done by indirect immunofluorescence using reference gnotobiotic hyperimmune or convalescent swine serum to porcine parvovirus (PPV), SIV, bovine viral diarrhea virus, hemagglutinating encephalomyelitis virus (HEV), TGEV and EMCV. Filtrates were blindly passed three times by intra-allantoic inoculation of 10-day old embryonated chicken eggs and allantoic fluid tested for hemagglutinating activity after each passage.

Mycoplasma isolation Lung suspensions were inoculated into mycoplasma broth medium Friis (Frii (1975), Acta Vet. Scand., 27, 337), BHI-TS, D-TS (Ross et al (1971), Journal of Bacteriology, (K:K)0168:EARGSA 23 o 68 24 103, 707) and BHL (Yamamoto et al (1982), Proc. Int. Pig Vet. Society Congress, p. 94).

Cultures were passaged when growth was evident or on day 3, 7, 14, and 21 and identified by epiimmunofluorescence. (Del Giudice et al (1967), Journal of Bacteriology, 93, 1205).

s Bacteria isolation Nasal turbinate swabs were inoculated on two blood agar plates as well as on MacConkey, Tergitol-7 and PMD (for isolation of P. multocida.) agars. One of the blood agar plates was incubated at 37 0 C in an anaerobic environment of CO 2 and H 2 The second plate was cross-streaked with a Staphylococcus epidermidis nurse colony and to incubated with the other plates in air at 37 0

C.

Lungs were plated exactly as the nasal turbinate swabs. Liver and spleen were cultured on 2 blood agar plates (aerobic and anaerobic) and a Tergitol-7 plate. All bacterial isolates were identified by standard methods (Biberstein (1990), In: Diagnostic Procedures in Veterinary Bacteriology and Mycology, ed. Carter et al, 5th ed., pp. 129- 142, Academic Press Inc., San Diego, Cal.; and Carter (1990) In: Diagnostic Procedures in Veterinary Bacteriology and Mycology, ed. Carter G.R. and Cole 5th ed., pp.

129-142, Academic Press Inc., San Diego, Cal.).

Serology Serum neutralization test was used to test for serum antibodies to PRV, TGEV, and 20 EMCV. Hemagglutination inhibition test was used to test serum antibodies to EMCV and HEV. Indirect immunofluorescence test was used to detect serum antibodies to BRSV, PI- 3, SIV, and TGEV. Gnotobiotic sera were tested for antibodies to PRRSV. An indirect immunofluorescence assay using cell line CL2621 was used for detection of PRRSV antibodies.

25 (II) Results Naturally occurring pneumonia The lungs from acutely affected pigs did not collapse. Grossly, the lungs had moderate interlobular edema, and multifocal to coalescing linear areas of atelectasis involving all lung lobes. There was 5-15% cranioventral consolidation of the cranial and 3o middle lobes.

Histopathological examination revealed moderate, acute diffuse proliferative bronchiolitis and alveolitis. There was a mild multifocal lymphoplasmacytic myocarditis.

No lesions were seen in other organs.

Virus isolation attempts for adenovirus, PRV, SIV, HEV, porcine respiratory as coronavirus (PRCV), porcine parvovirus (PPV), EMCV, and enteroviruses were negative from the original case submission as well as from the acutely affected pigs later obtained from the herd. Immunofluorescence examination of frozen lung sections did not reveal Mycoplasma hyopneumoniae, SIV, bovine respiratory syncytial virus (BRSV), parainfluenza virus-3 PRV or TGEV antigens.

IK:)00168:EARGSA 24 of 68 Serum from one of the five conventional SPF pigs of section above gave a positive immunological reaction at a dilution of 1:20 for PRRSV by indirect immunofluorescence. Pasteurella multocida type D and Haemophilus parasuis were isolated, respectively, from the nasal turbinates and lung of this pig. No aerobic or s anaerobic bacteria were isolated from the acutely affected pig lung chosen for homogenization and inoculum (see Methods and Materials, Section above).

Conventional pig study By 7 DPI, all principal pigs had fevers of 40-41.1 0 C and were experiencing moderate respiratory distress. The pigs were anorexic and lethargic. By 15 DPI, the pigs had recovered. Macroscopic changes in the lungs were characterized by failure to collapse, mild interlobular edema, and tan-grey linear areas of atelectasis multifocally involving from 20-40% of the lung.

Microscopic examination of 7 DPI lungs revealed a patchy interstitial pneumonia characterized by type II pneumocyte proliferation, accumulation of mixed inflammatory is cells and necrotic cell debris in alveolar lumina, and infiltration of macrophages and lymphocytes in alveolar septa. Alveolar lumina contained proteinaceous fluid.

Occasionally, syncytial-like cells were seen within alveolar lumina and along septa.

Figure 5 shows a histological section from the lung of a conventional pig 10 DPI, using hematoxylin-eosin stain. There is extensive type II pneumocyte proliferation (arrow) 20 and necrotic cell debris in alveolar spaces (arrow heads). The condition and appearance of the lesions observed at day 10 were similar to those observed at day 7.

Figure 6 shows a second histological section from the lung of a conventional pig DPI, using hematoxylin-eosin stain. Syncytial-like cells (arrows) are present in alveolar spaces. Pronounced type II pneumocyte proliferation and more syncytia are observed at 25 day 10 than at day 7.

Lesions were still moderately severe at 15 DPI, yet the pigs appeared clinically normal. No bacteria or mycoplasmas were isolated from the lungs. Virus isolation .se. attempts for EMCV, PRV, PRCV, adenovirus, and SIV were negative.

Immunofluorescence examination of frozen lung sections did not demonstrate BRSV, PI-3 30 virus, PRV, SIV, TGEV, or Mycoplasma hyopneumoniae antigens.

No gross or microscopic lesions were seen in control pigs.

Gnotobiotic pig study All inoculated principal pigs were experiencing severe respiratory distress and "thumping" by 5 DPI. Temperatures were 40.5 0 C or greater, and the pigs were anorexic and lethargic. The pigs were improved clinically by 8 DPI, and appeared clinically normal by 15 DPI. No pigs died. Control pigs inoculated with normal lung homogenate filtrate remained clinically normal.

Macroscopic lesions by 5 DPI were characterized by a lung that failed to collapse, mild multifocal tan-red atelectasis and mild interlobular edema. Microscopically, there was mild diffuse interstitial pneumonia with multifocal areas of mononuclear cell IK:)00188:EAR/GSA 26 of 68 IK:)Oo 68:EARJGSA 26 of es infiltration of alveolar septae and moderate type II pneumocyte proliferation. There was accumulation of inflammatory cells, necrotic cell debris, and proteinaceous fluid in alveolar lumina. No lesions were seen in other organs.

By 9 DPI, the lung failed to collapse, had moderate interlobular edema and multifocal s 1-3 cm areas of firm tan-red atelectasis. Figure 7 shows a histological section from the lung of a gnotobiotic pig at 9 DPI, using hematoxylin-eosin stain. There is moderate type II pneumocyte proliferation (arrow heads)..and syncytial-like cell formation (arrows).

Microscopically, the lesions were similar to those observed on day 5 DPI, except that type II pneumocyte proliferation was more pronounced, and there were moderate numbers of syncytial-like cells along alveolar septa and in lumina. The kidney had dilated renal tubules, some containing a lymphoplasmacytic exudate and cell debris.

By 28 DPI, there was 20% craniovental bilateral atelectasis involving the apical and middle lobes with focal 1-2 cm areas of atelectasis in other lobes. Microscopically, the lung lesions were similar to those observed at 9 DPI, but in addition, there was mild peribronchiolar and periarteriolar lymphoplasmacytic accumulation. Mild to moderate infiltrates of lymphocytes and plasma cells were present multifocally in the choroid plexus, meninges, myocardium, and nasal turbinates.

Figure 8 shows that by 35 DPI, the lung lesions were less severe but the multifocal lymphoplasmacytic myocarditis was pronounced. Virus isolation attempts for PRV, SIV, 20 adenovirus, EMCV, HEV, PPV,. enteroviruses, -and PRCV were unsuccessful. A cytopathic effect was observed in porcine alveolar macrophages, characterized by cell rounding, lysis and cell death. Direct bronchio-alveolar lavage cultures exhibiting extensive syncytia are shown in Figure 9, which were not observed in similar cultures prepared from control pigs. Examination of these cultures by negative staining immune 25 electron microscopy revealed two types of virus-like particles. One type, shown in Figure was about 70 nm in diameter, enveloped and had short surface spicules. The other type, shown in Figure 11, was enveloped, pleomorphic, approximately 80 X 320 nm and was coated by antibodies. No bacteria were isolated from lung, liver, spleen, or brain.

Serum collected at 28 and 35 DPI had no antibody titers to SIV, EMCV, PRV,.

30 TGEV, BRSV, HEV, or PI-3 virus. These sera were positive (1:1280) for antibody to PRRS virus.

The control pigs remained normal throughout the study and had no gross or microscopic lesions in any tissue. No bacteria or viruses were isolated from the control pigs.

(MI) Discussion Lung filtrates from pigs with naturally occurring endemic pneumonia produced lung and heart lesions in experimentally inoculated conventional and gnotobiotic pigs. The lesions observed in both the natural and experimental disease were consistent with a viral etiology. No common, previously identified swine viral respiratory pathogens were isolated. A cytopathic effect was observed, characterized by cell lysis of primary porcine [K:I00168:EAGSA 20 of 88 27 alveolar macrophage cultures, consistent with the report of PRRS virus infections by Yoon et al (Journal of Veterinary Diagnostic Investigation, vol. 4 (1992), p. 139). However, the large syncytia in direct bronchio-alveolar lavage cultures seen in this study have not been previously reported with PRRS.

s Electron microscopy of infected cell culture shows two virus-like particles. A 70 nm enveloped virus-like particle with short surface spicules appears compatible with the PRRS virus as reported by Benfield et al (Journal of Veterinary Diagnostic Investigation, vol. 4 (1992), p. 117), but the other virus-like particle appears to be distinct. None of the pigs developed antibody titers to SIV, PRV, TGEV (PRCV) or EMCV. The gnotobiotic pigs did seroconvert to the PRRS virus, however.

The clinical disease reproduced in Experiment I is characterized by moderate to severe respiratory distress in all inoculated gnotobiotic and conventional pigs within 5 DPI. The disease in this Experiment is more severe than that observed in previous experiments (Collins et al and Yoon et al, supra).

is Terminal airway epithelial necrosis and proliferation, described for the recentlyidentified type A SIV variant (aSIV or a related disease thereto, supra) by Morin et al (Canadian Veterinary Journal, vol. 31 (1990), p. 837) were not observed in Experiment I.

The fibrin deposits and hyaline membranes along alveolar septa associated with aSIV (Morin et al, and Girard et al, supra) were not observed. The severe nonsuppurative 20 myocarditis observed in pigs that lived beyond 15 -DPI in Experiment I is not associated with aSIV (Morin et al, and Girard et al, supra). Pigs did not seroconvert to SIV, and no SIV was detected by passage in embryonated chicken eggs.

The predominant lung lesion seen in PRRS outbreaks and experimental inoculations is marked interstitial infiltration with mononuclear cells (Collins et al, Pol et al, supra).

Type II pneumocyte proliferation, syncytial cell formation, and myocarditis observed in the infected pigs of Experiment I have not been observed by others. The lesions consistently reproduced with the filterable infectious agent of Experiment I suggest that the disease described in this study, which we designate the Iowa strain of PRRSV, is caused by either a unique viral agent or a combination of a PRRS virus with another infectious agent.

Experiment II Materials and Methods Field Case Material and History A pig was obtained from a herd which experienced PRRS with persistent severe nursery pneumonia, and had only 20 viable pigs from the last 42 litters farrowed. The pig was necropsied, and samples of lung tissue was collected and homogenized using standard, sterile homogenization techniques. The lung homogenate (10% w/v) prepared in Eagle's minimal essential medium (MEM) and filtered through a 0.22 mp. filter was used as inoculum.

Cells ,uor. w 27 of 68 28 A continuous cell line, designated PSP-36, was derived from MA-104 cells, which were purchased from Whittaker Bioproducts, Inc. (Walkersville, Maryland). A sample of PSP-36 cells were separately propagated, and this cell line was designated PSP-36-SAH.

Swine alveolar macrophages and approximately ninety other cell lines, examples of which are described in Table 2 hereinbelow were used for virus isolation.

Table 2 Porcine Simian Canine Feline Murine Human Hamster ST-SAH Vero 76 NLDK CRFK MT U937 BHK-21 ST-ATCC BGM-70 CK65D FKCU P388D1 Hep 2 CHO-K1 ST-ISU BSC-1 MDCK FL IC-21 ST-UNE PSP 36 CT-60 NCE PU5-18 3201 L929

SL

PSP 29 PSP PSP 31 IBRS2D10 AG08114 AG08116 Bovine Invertebrate Quail Chicken Lapine Bat MDBK ASE QT-6 CU10 RK13 TblLu STAE QT-35 LMH AVE HD 11 BGE BM2L

HZM

IDE2 IDE8

RAE

Virus Isolation Lung homogenates prepared as described above were clarified either at 2,000 x g or 3,000 rpm at 4 0 C for 15 min. The supernatants were filtered through a 0.22 mp filter.

o1 The filtrates were inoculated onto each of the cell lines described in Section above.

Cultures were then maintained in appropriate media with 0-4% fetal bovine serum (FBS) and antibiotics. Cell lines were monitored daily for cytopathic effects (CPE). If CPE was not observed within eight or nine days, the cultures were blindly passed 2-3 times. If suspicious CPE was observed, cultures were examined in an indirect immunofluorescence is assay (IFA) using convalescent pig antiserum to ISU-12.

Virus Titration Serial 10-fold dilutions of ISU-12 isolate were prepared in Dulbecco's minimal essential medium (DMEM) with 2% FBS and 1 x antibiotics. Each dilution (0.2 ml) was inoculated in duplicate onto each well of PSP-36 cells and swine alveolar macrophage cultures seeded in Lab-Tek chambers. At three days post infection (DPI), the chambers were fixed with cold 80% acetone and 20% methanol solution at 4 0 C for 15 min. The IK:)00168:EARIGSA 28 o 68 chambers were then stained in an IFA using convalescent ISU-12 antiserum and anti-PRRS virus serum.

Indirect Immunofluorescence Assay (IFA) The PSP-36 cells and swine alveolar macrophage cultures were infected with ISU-12 s isolate. At 20 and 48 hours post infection, the cultures were fixed with cold 80% acetone and 20% methanol solution at 4 0 C for 15min. IFA was carried out using ISU-12 convalescent serum, anti-PRRSV serum and anti-PRRSV monoclonal antibody purchased from South Dakota State University, Brookings, South Dakota. Uninfected PSP-36 cells and macrophage cultures were used as controls.

Radioimmunoprecipitation Assay (RIP) ISU-12 isolate and mock-infected PSP-36 cells were labelled with 35 S-methionine and 35 S-cysteine. 3-day-old PSP-36 cells in 25 cm 3 flasks were infected with 0.5 ml of 104

TCID

5 0 of ISU-12 virus. At 24h post-infection, the medium was replaced with methionine-deficient and cysteine-deficient DMEM, and the cultures were incubated at 370° is C for lh. The medium was then replaced with fresh methionine-deficient and cysteinedeficient DMEM with 100 gci/ml of the 35 S-methioninre 35 Met) and 35 S-cysteine 35 Cys).

Five hours after addition of 35 Met and 35 Cys, the cells were washed three times with cold phosphate-buffered saline (PBS), pH 7.2, then scraped from the flasks and pelleted by centrifugation at 1000 x g 410min. The cell pellets containing labelled viral proteins and 20 mock-infected cell pellets were then disrupted with lysis buffer, and the cellular residues were clarified by centrifugation according to the procedure of Zhu et al (Am. J. Vet. Res., 51:232-238 (1990)). The lysates were then incubated with ISU-12 convalescent serum and *i anti-PRRS virus serum, preabsorbed with cold normal PSP-36 cell lysates at 4 0

C

overnight. Immune complexes were collected by addition of Sepharose-protein A beads (obtained from Sigma Chemical Co., St. Louis, Missouri) for 2h at room temperature.

The mixture of Sepharose-protein A beads and immune complex were then washed three times with lysis buffer and three times with distilled water. The mixture was resuspended in 50gL sample buffer, and run on an SDS-PAGE gel as described by Zhu et al, supra.

Electron Microscopy (EM) 30 The PSP-36 cells were infected with ISU-12 virus in a 25cm 2 flask. At 48h post infection, the infected cells were fixed with 3% glutaraldehyde (pH7.2) at 4 0 C for 2h. The cells were then scraped from the flask and pelleted by centrifugation. The cell pellets were processed and embedded in plastic. The plastic-embedded cell pellets were thin-sectioned, stained and then visualized under a transmission electron microscope as described by Paul et al (Am. J. Vet. Res., 38:311-315 (1976)).

(II) Experimental Reproduction of the Porcine Reproductive and Respiratory Disease Experiment 92.1 SPF IK:)01 e8:EAR/GSA 29 df 8 IK:3001 68:EAR/OSA 29.166 Lung filtrate from ISU-12 above was inoculated intranasally into six specific pathogenfree (SPF) pigs that were 5 weeks old. Pigs were killed at 3, 5, 10, 28, and 43 days post inoculation (DPI).

Experiment 92.3 SPF Six SPF crossbred pigs were inoculated intranasally at 5 weeks of age with porcine alveolar macrophage material infected with ISU-12 lung filtrate. The ISU-12 inoculated pigs were necropsied at 10 and 28 DPI.

Experiment 92.10 SPF Three 5-week old pigs were inoculated intranasally with 3mL of ISU-12 propagated on PSP-36, containing 105 TCID50/mL of virus. Two pigs served as uninoculated controls.

One principal pig was necropsied at 5, 10 and 28 DPI. One control pig was necropsied at each of 5 and 10 DPI.

Experiment 92.12 SPF Twenty-two 5-week old pigs were divided into six groups. In group I, 6 pigs (principal) were inoculated intranasally with 3mL of plaque-purified ISU-12 (plaque no. 1) virus propagated on PSP-36 containing 105 TCIDs5/mL of virus. In group II, 6 pigs were inoculated with control cell culture medium. In each of group III (plaque no. 2) and group IV (plaque no. 2 pigs were inoculated with plaque-purified ISU-12. In group V, 3 pigs were inoculated with ISU-12 which was not plaque-purified. In group VI, 3 pigs were 20 inoculated with ISU-12 tissue filtrate Two principal and two control pigs were necropsied from each of groups I and II at each of 5, 10 and 25 DPI. Two pigs inoculated with plaques no. 2 and no. 3 were each necropsied at 10 DPI. One pig from each of groups V and VI was necropsied at each of 10 and 25 DPI.

Microscopic Examination Lung, brain, heart and spleen were collected at necropsy, fixed with 10% neutral buffered formalin, embedded in paraffin, and stained with hematoxylin and eosin.

(III) Results Virus Cultivation 30 Cultivation of ISU-12 Isolate in Swine Alveolar Macrophage Cultures A cytopathic effect (CPE) was observed in swine alveolar macrophage cultures infected with ISU-12 lung filtrate beginning at 2-3 DPI. CPE was characterized by clumping of the macrophages and cell lysis. About 90% of the macrophage cultures in ISU-12 infected cultures were showing CPE by 4-5 DPI. Figure 12(A) shows that no CPE was observed in uninfected macrophage cultures. The titer of ISU-12 virus in macrophage cultures at third passage was 104-105 TCIDs/ml.

Viral antigens were detected by IFA in the cytoplasm of ISU-12 infected swine alveolar macrophage cultures using ISU-12 convalescent serum from gnotobiotic pigs, as shown in Figure 12(C). No immunofluorescence was detected in uninoculated macrophage cultures.

IK:00168:EAR/GSA 30 o 68 31 Cultivation of ISU-12 Isolate On Continuous Cell Lines Of the approximately ninety cell lines tested (see Section of "Materials and Methods" above), evidence of viral replication was noted in six cell lines, notably PSP-36, PSP-36-SAH, MA-104, synovial cells, alveolar macrophage cells and porcine turbinate cells.

Figure 13(B) shows that CPE started at 2 DPI, and was characterized by the degeneration, cell rounding and clumping of cells. At 3-4 DPI, the number of rounded cell clumps increased, and some clumps fused. Many rounded cells detached from the cell monolayer, and led to the subsequent disintegration of the monolayer. After 5 DPI, CPE became quite extensive, and involved over 95% of the monolayer typically. No CPE was observed in control PSP-36 cells, as shown in Figure 13(A). The ISU-12 isolate grew to high titers on PSP-36 cells, about 106-107 TCID50/ml at the 11th cell culture passage.

Viral antigens were detected in the cytoplasm of infected cells with convalescent sera from gnotobiotic pigs experimentally inoculated with ISU-12 lung filtrate (see Figure No fluorescence was observed in control PSP-36 cells (Figure 14(A)).

(IV) Virus Characteristics Antigenic Relatedness of ISU-12 to PRRS Virus *Monoclonal antibody to PRRS virus isolate VR-2332 (purchased from Dr. Benfield, South Dakota State University, Brookings, South Dakota) and anti-PRRCV sera (obtained 20 from the USDA National Veterinary Services Laboratory, Ames, Iowa) reacted with ISU- 12-infected PSP-36 cells, evidenced by bright cytoplasmic fluorescence during IFA (see Figure but did not react with uninfected PSP-36 cells.

Viral Proteins Anti-ISU-12 convalescent sera and anti-PRRS virus sera were used to analyze viral proteins. Both sera recognized at least 4 proteins, respectively having molecular weights of 19, 24, 32 and 61 kD (Figure 15). In Figure 15, mock infected (lanes 2 and 3) or ISU-12 .infected (lanes 4-7) were immunoprecipitated with anti-ISU-12 serum (lanes 2 and anti- PRRSV serum (lanes 3 and anti-PRRSV monoclonal antibody (lane 6) or rabbit anti- PRRSV serum (obtained from Dr. Benfield, South Dakota State University, Brookings,.

South Dakota). Lanes 1 and 8 have weight markers. These proteins were not evident in mock-infected PSP-36 cells.

Viral Structure Typical virus particles ranging from 55-85 nm were observed in ISU-12 infected PSP- 36 cells. The virus particles were enveloped, spherical and present in cytoplasmic vesicles of ISU-12 infected PSP-36 cells.

Experimental Reproduction of Disease Experiment 92.1 SPF Lung filtrate from ISU-12 above was inoculated intranasally into six specific pathogenfree (SPF) pigs that were 5 weeks old. Pigs were killed at 3, 5, 10, 28, and 43 days post inoculation (DPI). By 3 DPI, the ISU-12 pigs had exhibited severe respiratory distress and (K:)00168:EARIGSA 31 o 8 pyrexia. These signs persisted for 10-14 days. Gross pulmonary lesions were characterized by severe multifocal grey-tan consolidation of 60% of the lungs. There was also moderate cardiomegaly and accumulation of abdominal fluid. Microscopic changes were characterized by severe proliferative interstitial pneumonia with type II pneumocyte s proliferation, syncytial cell formation, alveolar exudation, and mild interstitial thickening with mononuclear cells. There was a mild nonsuppurative myocarditis, a severe encephalitis, and a moderate lymphoplasmacytic nephritis. The ISU-12 experimental pigs necropsied at 10 and 28 days had seroconverted to the PRRS agent as confirmed by NVSL.

Experiment 92.3 SPF All ISU-12 inoculated SPF pigs exhibited severe respiratory disease within 3 days, persisting for more than 14 days. Gross lesions were characterized by pulmonary congestion, edema and marked multifocal-diffuse hepatization. Microscopically, severe proliferative interstitial pneumonia, moderate nephritis, moderate myocarditis, and mild encephalitis were observed. The ISU-12 inoculated pigs necropsied at 10 and 28 DPI had is seroconverted to PRRS as confirmed by NVSL.

Experiment 92.10 SPF Clinical signs in inoculated pigs included severe lethargy and pyrexia, moderate anorexia, and moderate-to-severe respiratory distress, observed 5-22 DPI. Moderate *tearing was present in these pigs throughout the experiment. Microscopic lesions included mild proliferative interstitial pneumonia and severe necropurulent tonsilitis at 5 DPI.

Moderate multifocal PIP with type II proliferation, alveolar exudation, multinucleated giant cells, and syncytial cell formation was observed at 10 DPI. Moderate multifocal encephalitis with perivascular cuffs and gliosis was also observed at 10 DPI. Mild S* periportal lymphomacrophagic hepatitis, mild nonsuppurative myocarditis and rhinitis was detected at 10 DPI. At 26 DPI, there was severe interstitial pneumonia, characterized by marked multifocal interstitial thickening with mononuclear cells, moderate multifocal type S. II pneumocyte proliferation, moderate amounts of mixed alveolar exudate, and loose peribronchiolar cuffs of lymphocytes and macrophages. There was also a moderate multifocal myocarditis, a mild hepatitis, a mild nephritis and tonsilitis. The two ISU-12 s30 inoculated pigs seroconverted to PRRS at 10 DPI.

The control pigs remained clinically normal during the duration of the experiment, and exhibited neither gross nor microscopic lesions. They also remained seronegative for

PRRS.

Experiment 92.12 SPF The biologically uncloned ISU-12 was pathogenic for SPF pigs, and produced interstitial pneumonia, myocarditis and encephalitis, as described above for Experiment 92.10 SPF. Pigs inoculated with the three biological clones of ISU-12 (plaques nos. 1, 2 and 3) produced mild interstitial pneumonia, but evidence of type II pneumocyte proliferation, alveolar exudation, myocarditis and/or encephalitis were not detected in these IK:)00168:EAR/GSA 32 of 68 V' I.

33 pigs. All pigs inoculated with ISU-12, either cloned or uncloned, seroconverted to PRRS at 10 DPI. The control pigs remained free of virus infection and disease.

(VI) Summary Severe pneumonia was experimentally reproduced in five-week-old SPF pigs with lung and heart filtrates (0.22 mp) from naturally-affected pigs (ISU-12). The pneumonia produced by the Iowa strain of PRRSV (ISU-12) is characterized by interstitial pneumonia, type II pneumocyte proliferation, and syncytial cell formation. .Myocarditis and encephalitis are observed in affected pigs. ISU-12 produced cytopathic effects (CPE) in swine alveolar macrophage cultures and a continuous cell line, PSP-36. Viral antigens to were detected by indirect immunofluorescence in ISU-12-infected cultures but not in uninfected cells. ISU-12 is antigenically related to PRRS virus strain VR-2332 by indirect immunofluorescence using polyclonal and monoclonal antibodies. However, differences were observed in microscopic lesions of the pigs infected with non-plaque-purified ISU-12, thus indicating that another virus or infectious agent may be grown in PSP-36, and that the is other virus or infectious agent may be the reason that the disease and lesions caused by the Iowa strain of PRRSV is different from and more severe than that reported for PRRS virus in the literature. All pigs inoculated with ISU-12, either cloned or uncloned, seroconverted to PRRS at 10 DPI. The control pigs remained free of virus infection and disease.

Experiment III Molecular Cloning And Nucleotide Sequencing Of The 3'-Terminal Region Of The Infectious Agent Associated With The Iowa Strain Of Porcine Respiratory And Reproductive Syndrome Materials and Methods Virus Propagation and Purification 25 Hereinafter, to simplify the discussion, the terms "virus" and "viral" will refer to a virus or infectious agent in the meaning described above for the present application, or a property of the virus or infectious agent.

S.A continuous cell line, PSP-36, was used to isolate and propagate ISU-12 isolate, associated with the Iowa strain of PRRSV. The ISU-12 virus was plaque-purified 3 times on PSP-36 cells. The PSP-36 cells were then infected with the plaque-purified virus.

When more than 70% of the infected cells showed cytopathic changes, the culture was frozen and thawed three times. The culture medium was then clarified by low-speed centrifugation at 5,000 X g for 15min. at 4 0 C. The virus was then precipitated with 7% PEG-8000 and 2.3% NaCI at 4 0 C overnight with stirring, and the precipitate was pelleted by centrifugation. The virus pellets were then resuspended in 2mL of tris-EDTA buffer, and layered on top of a CsCI gradient (1.1245-1.2858g/mL). After ultracentrifugation at 28 000rpm for about 8 hours at 20 0 C, a clear band with a density of 1.15-1.18g/mL was observed and harvested. The infectivity titer of this band was determined by IFA, and the (K:100168:EAR/GSA 33 of 88 titer was found to be 10 6 TCIDso/mL. Typical virus particles were also observed by negative staining electron microscopy (EM).

Isolation of Viral RNA Total RNA was isolated from the virus-containing band in the CsC1 gradient with a s commercially available RNA isolation kit (obtained from Stratagene). Poly(A) RNA was then enriched by oligo (dT)-cellulose column chromatography according to the procedure described by the manufacturer of the column (Invitrogen).

Construction of ISU-12 cDNA library A general schematic procedure for the construction of a cDNA X library is shown in o1 Figure 16. First strand cDNA synthesis from mRNA was conducted by reverse transcription using an oligo (dT) primer having a Xho I restriction site. The nucleotide mixture contained normal dATP, dGTP, dTTP and the analog 5-methyl dCTP, which protects the cDNA from restriction enzymes used in subsequent cloning steps.

Second strand cDNA synthesis was then conducted with RNase H and DNA polymerase I. The cDNA termini were blunted (blunt-ended) with T4 DNA polymerase, ligated to EcoR I adaptors with T4 DNA ligase, and subsequently kinased phosphorylated) with T4 polynucleotide kinase. The cDNA was digested with Xho I, and the digested cDNA were size-selected on an agarose gel. Digested cDNA larger than 1 kb in size were selected and purified by a commercially available DNA purification kit 20 (GENECLEAN, available from BIO 101 Inc., LaJolla, California).

The purified cDNA was then ligated into lambda phage vector arms, engineered with Xho I and EcoR I cohesive ends. The ligated vector was packaged into infectious lambda phages with lambda extracts. The SURE strain (available from Stratagene) of E. coli cells were used for transfection, and the lambda library was then amplified and titrated in the XL-1 blue cell strain.

Screening the A Library by Differential Hybridization A general schematic procedure for identifying authentic clones of the PIP virus ISU-12 strain by differential hybridization is shown in Figure 17, and is described hereunder. The X library was plated on XL-1 blue cells, plaques were lifted onto nylon membranes in 3o duplicates, and denatured with 0.5N NaOH by conventional methodology. Messenger RNA's from both virus-infected PSP-36 cells and non-infected PSP-36 cells were isolated by oligo (dT)cellulose column chromatography as described by the manufacturer of the column (Invitrogen).

Complementary DNA probes were synthesized from mRNA's isolated from virusinfected PSP-36 cells and normal PSP-36 cells using random primers in the presence of 32P-dCTP according to the procedure described by the manufacturer (Amersham). Two probes (the first synthesized from virus-infected PSP-36 cells, the other from normal, uninfected PSP-36 cells) were then purified individually by Sephadex G-50 column chromatography. The probes were hybridized with the duplicated nylon membranes, respectively, at 42 0 C in 50% formamide. Plaques which hybridized with the probe IK:)0188:EAR/GSA 34 of 68 prepared from virus infected cells, but not with the probe prepared from normal cells, were isolated. The phagemids containing viral cDNA inserts were rescued by in vitro excision with the help of G408 helper phage. The rescued phagemids were then amplified on XL-1 blue cells. The plasmids containing viral cDNA inserts were isolated by Qiagen column s chromatography, and were subsequently sequenced.

Nucleotide Sequencing and Sequence Analysis Plasmids containing viral cDNA inserts were purified by Qiagen column chromatography, and sequenced by Sanger's dideoxy method with universal and reverse primers, as well as a variety of internal .oligonucleotide primers. Sequences were obtained from at least three separate clones. Additional clones or regions were sequenced when ambiguous sequence data were obtained. The nucleotide sequence data were assembled and analyzed independently using two computer software programs, GENEWORRS (IntelliGenetics, Inc., Mountain View, California) and MACVECTOR (International Biotechnologies, Inc., New Haven, Connecticut).

1s Oligonucleotide Primers Oligonucleotides were synthesized as single-stranded DNA using an automated DNA synthesizer (Applied Biosystems) and purified by HPLC. Oligonucleotides PP284 CGGCCGTGTG GTTCTCGCCA AT-3'; SEQ ID NO: 1) and PP285 CTCTAGCGAC TG-3'; SEQ ID NO: 2) were synthesized for PCR amplification. A DNA 20 probe was generated with these two primers from-the extreme 3' end of the viral genome for Northern blot analysis (see discussion below). Oligonucleotides PP286 GCCGCGGAAC CATCAAGCAC-3'; SEQ ID NO: 3) and PP287 CTATGTGAGC-3'; SEQ ID NO: 4) were synthesized for PCR amplification. A DNA S probe generated by these two primers was used to further screen the X library.

Oligonucleotides PP288 (5'-GCGGTCTGGA TTGACGACAG-3'; SEQ ID NO: PP289 (5'-GACTGCTAGG GCTTCTGCAC-3'; SEQ ID NO: PP386 .TCACATAGCG-3'; SEQ ID NO: PP286 and PP287 were used as sequencing primers to obtain internal sequences.

Northern Blot Analysis S 30 A specific DNA fragment from the extreme 3' end of the ISU-12 cDNA clone was amplified by PCR with primers PP284 and PP285. The DNA fragment was excised from an agarose gel with a commercially available DNA purification kit (GENECLEAN, obtained from Bio 101), and labeled with 32P-dCTP by random primer extension (using a kit available from Amersham). Total RNA was isolated from ISU-12-infected PSP-36 cells at 36 hours post-infection, using a commercially available kit for isolation of total RNA according to the procedure described by the manufacturer (Stratagene). ISU-12 subgenomic mRNA species were denatured with 6M glyoxal and DMSO, and separated on a 1% agarose gel. (Results from a similar procedure substituting a 1.5% agarose gel are described in Experiment VIII below and shown in Figure 32.) The separated subgenomic mRNA's were then transferred onto nylon membranes using a POSIBLOT' pressure IK:)00168:EARIGSA 36 of 68 blotter (Stratagene). Hybridization was carried out in a hybridization oven with roller bottles at 42 0 C and 50% formamide.

(II) Results Cloning, Identification and Sequencing of ISU-12 3' Terminal Genome An oligo (dT)-primed cDNA X library was constructed from a partially purified virus, obtained from ISU-12-infected PSP-36 cells. Problems were encountered in screening the cDNA A library with probes based on the Lelystad virus sequence. Three sets of primers were prepared. The first set (PP105 and PP106; SEQ ID NOS: 21-22) correspond to positions 14577 to 14596 and 14977 to 14995 of the Lelystad genomic sequence, located in the nucleocapsid gene region. The second set (PP106 and PP107, SEQ ID NOS: 22-23) correspond to positions 14977 to 14995 and 14054 to 14072 of the Lelystad genomic sequence, flanking ORF's 6 and 7. The third set (PM541 and PM542; SEQ ID NOS: 24correspond to positions 11718 to 11737 and 11394 to 11413 of the Lelystad genomic sequence, located in the ORF-lb region.

PP105: 5'-CTCGTCAAGT ATGGCCGGT-3' (SEQ ID NO: 21) PP106: 5'-GCCAITCGCC TGACTGTCA-3' (SEQ ID NO: 22) PP107: 5'-TTGACGAGGA CTTCGGCTG-3' (SEQ ID NO: 23) PM541: 5'-GCTCTACCTG CAATTCTGTG-3' (SEQ ID NO: 24) PM542: 5'-GTGTATAGGA CCGGCAACCG-3' (SEQ ID NO: 20 All attempts to generate probes by PCR from the ISU-12 infectious agent using these three sets of primers were unsuccessful. After several attempts using the differential hybridization technique, however, the authentic plaques representing ISU-12-specific cDNA were isolated using probes prepared from ISU-12-infected PSP-36 cells and normal PSP-36 cells. The procedures involved in differential hybridization are described and set forth in Figure 17.

Three positive plaques X-75 and X-91) were initially identified. Phagemids containing viral cDNA inserts within the X phage were rescued by in vitro excision with the help of G408 helper phages. The inserts of the positive clones were analyzed by restriction enzyme digestion and terminal sequencing. The specificity of the cDNA clones was further confirmed by hybridization with RNA from PSP-36 cells infected with the Iowa strain of PRRSV, but not with RNA from normal PSP-36 cells. A DNA probe was then generated from the 5'-end of clone X-75 by PCR with primers PP286 and PP287. Further positive plaques (X-229, X-268, X-275, X-281, X-323 and X-345) were identified using this probe. All X cDNA clones used to obtain the 3'-terminal nucleotide sequences are presented in Fig. 18. At least three separate clones were sequenced to eliminate any mistakes. In the case of any ambiguous sequence data, additional clones and internal primers (PP288, PP289, PP286, PP287 and PP386) were used to determine the sequence.

The 1938-bp 3'-terminal sequence (SEQ ID NO:8) is presented in Figure 19, and the deduced amino acid sequences (SEQ ID NOS: 9-12) are presented in Fig. IK:IO1168:EARfGSA 36 of 88 A Nested Set of Subgenomic mRNA Total RNA from virus-infected PSP-36 cells was separated on 1% glyoxal/DMSO agarose gel, and blotted onto nylon membranes. A cDNA probe was generated by PCR with a set of primers (PP284 and PP285) flanking the extreme 3'-terminal region of the viral genome. The probe contains a 3'-nontranslational sequence and most of the ORF-7 sequence. Northern blot hybridization results show that the pattern of mRNA species from PSP-36 cells infected with the Iowa strain of PRRSV is very similar to that of Lelystad virus equine arteritis virus (EAV), lactate dehydrogenase-elevating virus (LDV) and coronavirus, in that virus replication required the formation of subgenomic mRNA's.

o1 The results also indicate that ISU-12-specific subgenomic mRNA's represent a 3'nested set of mRNA's, because the Northern blot probe represents only the extreme 3' terminal sequence. The size of ISU-12 viral genomic RNA (14 kb) and 6 subgenomic mRNA's (RNA 2 (3.0 kb), RNA 3 (2.5 kb), RNA 4 (2.2 kb), RNA 5 (1.8 kb), RNA 6 (1.3 kb) and RNA 7 (0.98 kb)) resemble those of LV (Fig. 18), although there are is differences in both the genome and in subgenomic RNA species. Differences were also observed in the relative amounts of the subgenomic mRNA's, RNA 7 being the most predominant subgenomic mRNA.

Analysis of Open Reading Frames Encoded by Subgenomic RNA Three large ORF's have been found in SEQ ID NO:8: ORF-5 (nt 239-901; SEQ ID 20 NO: 13), ORF 6 (nt 889-1403; SEQ ID NO: 15) and ORF 7 (nt 1403-1771; SEQ ID NO: 18). ORF 4, located at the 5' end of the resulting sequence, is incomplete in the 1938-bp 3'-terminal sequence of SEQ ID NO: 8. ORF'S 5, 6 AND 7 each have a coding capacity of more than 100 amino acids. ORF 5 and ORF 6 overlap each other by 10 bp, and ORF 6 and ORF 7 overlap each other by 5 bp. Two smaller ORF's located entirely within ORF 7 have also been found, coding for only 37 aa and 43 aa, respectively. Another two short ORF's overlap fully with ORF 5. The coding capacity of these two ORF's is only 29 aa and 44 aa, respectively. No specific subgenomic mRNA's were correlated to these smaller ORF's by Northern Blot analysis. ORF 6 and ORF 7 are believed to encode the viral membrane protein and capsid protein, respectively.

Consensus Sequence for Leader Junction Sequence analysis shows that a short sequence motif, AACC, may serve as the site in the subgenomic mRNA's where the leader is added during transcription (the junction site).

The junction site of ORF 6 is found 21 bp upstream from the ATG start codon, and the junction site of ORF 7 is found 13 bp upstream from the ATG start codon, respectively.

No AACC consensus sequence has been identified in ORF 5, although it has been found in ORF 5 of LV. Similar junction sequences have been found in LDV and EAV.

3'-Nontranslational Sequence and Poly Tail A 150 nucleotide-long (150 nt) nontranslational sequence following the stop codon of ORF 7 has been identified in the genome of the ISU-12 virus, compared to 114 nt in LV, 80 nt in LDV and 59 nt in EAV. The length of the poly tail is at least 13 nucleotides.

K:100168:EAR/GSA 37 of 88 There is a consensus sequence, CCGG/AAATT-poly among PIP virus ISU-12, LV and LDV in the region adjacent to the poly tail.

Sequence Comparison of ISU-12 and LV Genomes Among ORF's 5, 6 and 7, and Among the Nontranslational Sequences s A comparison of the ORF-5 regions of the genomes of ISU-12 and of the Lelystad viruses is shown in Figure 21. The corresponding comparisons of the ORF-6 region, the ORF-7 region, and the nontranslational sequences are respectively shown in Figures 22, 23 and 24. The results of the comparison are presented in Table 3 below. Consistent with the description above, a virus is considered immunologically equivalent if it has 90% or greater homology with an immunogenic virus. The nucleotide sequence homologies between LV and ISU-12 of the ORF 5, ORF 6, ORF 7 and the nontranslational sequences are 60%, 68%, 60% and 58%, respectively. Accordingly, LV and ISU-12 are not immunogenic equivalents.

The size of ORF's 5 and 6 in LV is 61 nt and 3 nt smaller than ORF's 5 and 6 in ISU- 12, respectively. In contrast, the size of ORF 7 in LV.is 15 nt larger than that in ISU-12.

Also, the 3'-terminal nontranslational sequence is different in length (150 nt in ISU-12, but only 114 nt in LV). Like LV, the junction sequence, AACC, has also been identified in the genome of the loaw strain of PRRS virus isolate ISU-12, except for ORF 5. The junction sequence of ORF 6 in ISU-12 is 21 nt upstream from the ATG start codon, 20 whereas the junction sequence of ORF.6 is 28 nt upstream from ATG in LV.

Table 3 Characteristics of the ORFs and Nontranslational Sequence of Lelystad Virus and ISU-12 Lelystad Virus __PRRSV ISU-12 Size (bp) Junction Seq. nt Sequence Size (bp) Junction Seq. (nt from ATG) Homology from ATG) 605 AACC (ATG-36) 60 666 No ORF-6 521 AACC (ATG-28) 68 525 AACC (ATG-21) (Env) ORF-7 386 AACC (ATG-13) 60 371 AACC (ATG-13) NT 113 58 150 NT 113 58 Experiment IV Expression of Iowa Strain Infectious Agent Genes In Insect Cells Production of Recombinant Baculovirus The ORF-5, ORF-6 and ORF-7 sequences were individually amplified by PCR using primers based on the ISU-12 genomic nucleotide sequence. ORF-5 was amplified using the following primers: TATTTGGCAA TGTGTC-3' (SEQ ID NO:26) (K:]O0168:EARIGSA 38 of 68 39 3'-GGGAATTCGC CAAGAGCACC TTTTGTGG-5' (SEQ ID NO:27) ORF-6 was amplified using the following primers: AGTYTCAGCG G-3' (SEQ ID NO:28) 3'-GGGAATTCTG GCACAGCTGA TTGAC-5' (SEQ ID NO:29) s ORF-7 was amplified using the following primers: GTTAAATATG CC-3' (SEQ ID 3'-GGGAATTCAC CACGCATTC-5' (SEQ ID NO:31) The amplified DNA fragments were cloned into baculovirus transfer vector pVL1393 (available from Invitrogen). One gig of linearized baculovirus AcMNPV DNA (commercially available from Pharmingen, San Diego, California) and 2 ig of PCRamplified cloned cDNA-containing vector constructs were mixed with 50 gl of lipofectin (Gibco), and incubated at 22 0 C for 15 min. to prepare a transfection mixture.

One hour after seeding HI-FIVE cells, the medium was replaced with fresh Excell 400 insect cell culture medium (available from JR Scientific and the transfection mixture was added drop by drop. The resulting mixture was incubated at 28 0 C for six hours.

Afterwards, the transfection medium was removed, and fresh Excell 400 insect cell culture medium was added. The resulting mixture was then incubated at 28 0

C.

Five days after transfection, the culture medium was collected and clarified. Ten-fold *dilutions of supernatants were inoculated onto HI-FIVE cells, and incubated for 60 min. at 20 room temperature. After the inoculum was discarded, an overlay of 1.25% of agarose was applied onto the cells. Incubation at 28 0 C was conducted for four days. Thereafter, clear plaques were selected and picked using a sterile Pasteur pipette. Each plaque was mixed with 1 ml of Grace's insect medium into a 5 ml snap cap tube, and placed in a refrigerator overnight to release the virus from the agarose. Tubes were centrifuged for 30 minutes at 2000 x g to remove agarose, and the supernatants were transferred into new sterile tubes.

Plaque purification steps were repeated three times to avoid possible wild-type virus contamination. Pure recombinant clones were stored at -80 0 C for further investigation.

Expression of Recombinant Iowa Strain Infectious Agent Proteins Indirect immunofluorescence assay and radioimmunoprecipitation tests were used to 30 evaluate expression.

Indirect immunofluorescence assay: Hi-five insect cells, shown in Figure 25, in a 24well cell culture cluster plate were infected with wild-type baculovirus or recombinant baculovirus, or were mock-infected. After 72 hours, cells were fixed and stained with appropriate dilutions of swine anti-ISU-12 polyclonal antibodies, followed by fluorescein isothiocyanate-labelled (FITC-labelled) anti-swine IgG. As shown in Figures 26-29, immunofluorescence was detected in cells infected with the recombinant viruses, but not in mock-infected cells or cells inoculated with wild-type baculovirus. For example, Figure 26 shows HI-FIVE cells infected with the recombinant baculovirus containing the ISU-12 ORF-6 gene (Baculo.PRRSV.6), which exhibit a cytopathic effect. Figure 27 shows HI- FIVE cells infected with another recombinant baculovirus containing the ISU-12 ORF-7 IK:)00168:EAR/GSA 39 of 68 IK:)OO 1 e8:EARIGsA 39 of 68 gene (Baculo.PRRSV.7), which also exhibit a cytopathic effect. Similar results were obtained with recombinant baculovirus containing ORF-5 (Baculo.PRRSV.5, data not shown). Figures 28 and 29 show HI-FIVE cells infected with a recombinant baculovirus containing the ISU-12 ORF-6 gene and ISU-12 ORF-7 gene, respectively, stained with swine antisera to ISU-12, followed by fluorescein-conjugated anti-swine IgG, in which the insect cells are producing recombinant Iowa strain infectious agent protein. Similar results were obtained with recombinant baculovirus containing Radioimmunoprecipitation: Radioimmunoprecipitation was carried out with each recombinant virus (Baculo.PRRSV.5, Baculo.PRRSV.6 and Baculo.PRRSV.7) to further determine the antigenicity and authenticity of the recombinant proteins. HI-FIVE insect cells were mock-infected, or alternatively, infected with each of the recombinant baculoviruses. Two days after infection, methionine-free medium was added. Each mixture was incubated for two hours, and then proteins labeled with 35 S-methionine (Amersham) were added, and the mixture was incubated for four additional hours at 28 0

C.

Radiolabeled cell lysates were prepared by three cycles of freezing and thawing, and the cell lysates were incubated with preimmune or immune anti-ISU-12 antisera. The immune complexes were precipitated with Protein A agarose and analyzed on SDS-PAGE after boiling. X-ray film was exposed to the gels at -80 0 C, and developed. Bands of expected size were detected with ORF-6 (Figure 30) and ORF-7 (Figure 31) products.

20 ExperimentV Other samples of PRRSV, described in Table 4 below, were plaque-purified three times. Plaque purification was performed by culturing a clarified tissue homogenate on PSP-36-SAH cells and selecting a single plaque, assuming one plaque is produced by a single virus. The selected plaque was then cultured, and a single plaque was again selected, then cultured a third time. IFA was carried out using anti-PRRSV monoclonal antibody purchased from South Dakota State University, Brookings, South Dakota.

Table 4 PRRSV 3X Plaque-Purified Isolates PRRSV Isolate Date frozen stock PRRS Monoclonal IFA Titer prepared result ISU-22 9/15/92 105-57 0.15 ISU-28 9/15/92 105-14 0.28 ISU-12 9/17/92 104-33 0.21 ISU-3927 9/21/92 103-56 0.17 ISU-984 9/21/92 103-89 0.24 ISU-7229 9/22/92 103-45 0.20 ISU-92-11581 9/22/92 102-39 0.17 ISU-695 10/01/92 104-49 0.20 ISU-79 10/01/92 105-69 0.25 ISU-412 10/01/92 105-31 0.50 10/01/92 105-54 ±0O i IK:100168:EAR/GSA 40 of 88 ISU-33 10/05/92 105-36 0.21 ISU-1894 10/27/92 105-18 0.33 ISU-04 10/27/92 105-78 0.24 ISU-51 2/07/93 104-59 0.15 ISU-30262 4/01/93 105-99 0.24 NOTE: All virus isolates were plaque-purified and propagated on PSP-36-SAH cells.

Some isolated samples selected for further study are identified in Table 5 below, and are characterized by their pathogenicity and number of mRNA's.

Table Isolate Pathogenicity No. of mRNA's ISU-12 Very pathogenic 7 ISU-984 Very pathogenic 7 ISU-3927 Mildly pathogenic 7* ISU-51 Mildly pathogenic 7 ISU-22 very pathogenic 9 Mildly pathogenic 9 ISU-79 Very pathogenic 9 s Some mRNA's exhibited deletions.

Samples of each of unplaque-purified ISU-12, plaque-purified ISU-12, ISU-22, ISU- 51, ISU-55 and ISU-3927 have been deposited under the terms of the Budapest Treaty at the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852, on 30 October, 1992 under the accession numbers VR 2386 and VR 2385, and deposited on 29 September, 1993 under the accession numbers VR 2429, VR 2428, VR 2430 and VR 2431.

The mRNA's of ISU-3927 exhibited deletions in four of the seven mRNA's. mRNA's 4, 5, 6 and 7 of ISU-3927 migrated faster than those of ISU-12, and hence, are smaller than those of ISU-12. This feature may possibly be related to the lower virulence of ISU- 15 3927.

The pathogenicity of six isolates was compared in five-week-old CDCD pigs. Fifteen pigs were inoculated with 105 TCID 50 of virus. Ten pigs were necropsied at 10 DPI, and five pigs were necropsied at 28 DPI. Virus isolates ISU-12, ISU-22 and ISU-28 were the most pathogenic, whereas ISU-51 and ISU-55 were of low pathogenicity. In a previous study, ISU-3927 was only mildly pathogenic for 5-week old pigs.

Lesions caused by ISU-22 and unplaque-purified isolated infectious agent which was not plaque-purified) ISU-12 persist for longer periods than those caused by plaquepurified viruses. The plaque-purified isolates produce mild myocarditis and encephalitis.

Unplaque-purified isolates produced slightly more severe disease than the corresponding plaque-purified isolates.

CDCD piglets provide an excellent model for evaluation of the pathogenicity and efficacy of candidate vaccines. The isolates ISU-12, ISU-22 and ISU-984 produce similar IK:100188:EAR/GSA 41 of 68

CO

lesions, and can be used to evaluate vaccine efficacy, based on examinations of gross and microscopic lesions. ISU-3927 is less virulent, but is adequate for evaluating a vaccine against pathogenic strains of PRRSV.

Pigs infected with plaque-purified ISU-12 gained an average of 9.9 pounds less than s control pigs (challenged with uninfected PSP-36 cells) over a time period of 28 days.

Preliminary results indicate that a lymphopenia and neutrophilia appear from 2-10 DPI.

Only those pigs infected with unplaque-purified ISU-12 developed significant encephalitis. No rhinitis was observed in any pig challenged with biologically cloned (plaque-purified) Iowa strain isolates. By contrast, rhinitis was severe when tissue filtrates (unplaque-purified isolates) were used as inocula.

The pathology and histology of CDCD pigs infected with ISU-12 unplaque-purified, ISU-12 plaque-purified, ISU-22, ISU-984, ISU-3927 and uninfected PSP-36 cells are summarized in Tables 6-12 below. In these Tables, gross lung lesion scores represent the percentage of lung consolidation the percentage of lung tissue diseased with pneumonia, showing lesions). A score is based on a scale of from 0 to 100% consolidation. "ND" means the gross lung lesion score was not determined.

Table 6 Isolate average average average average average average score, score, 7 score, 10 score, 21 score, 28 score, 36 3DPI DPI DPI DPI DPI DPI ISU-12 unpl. 29 56.3 77.3 37.25 6.0 ND ISU-12 20.5 35.5 77.5 25.0 0.0 0 ISU-22 26.5 35.0 64.75 36.5 11.0 0 ISU-984 7.25 21.75 76.0 21.0 0.5 0 ISU-3927 13.S 20.0 10.5 0 0.0 0 PSP-36 0 0 0 0 0 0 Uninoc. 0 0 0 0 0 0 In Table 6 above, "unpl." means unplaque-purified, and "uninoc." means uninoculated.

The results in Table 6 above show that ISU-12 and ISU-22 produce lesions which persist longer than other isolates. The lesions produced by ISU-12, ISU-22 and ISU-984 are of similar severity. The lesions produced by ISU-3927 are much less severe, and are resolved earlier than lesions produced by other isolates. All gross lesions were resolved by 36 DPI.

The pathology results presented in Tables 7-12 below are based on the same scale of severity presented for Table 1 above. In Tables 7-12 below, "Int. thick." means interstitial thickening, "alv. exud." means alveolar exudate, and "encephal." means encephalitis.

Table 7 IK: 1168:EARIGSA 42 of 68 9**9 9@ 9**9 Microscopic lesions at 3 DPI Lesion ISU-12 ISU- 12 ISU-22 1ISU-984 ISU-3927 PSP-36 unpI. Control TypeIf Syncytia Int. thick. alv. exud. myocarditis encephal.

Table 8 Microscopic lesions at 7 DPI Lesion ISU-12 ISU-12 ISU-22 ISU-984 ISU-3927 PSP-36 I unpl. control Type Syncytia Int. thick. alv. exud. myocarditis encephal.

Table 9 Microscopic lesions at 10 DPI Lesion ISU-12 ISU-12 ISU-22 ISU-984 ISU-3927 PSP-3 F un-pl contro Type II Snctia Int. thick. alv. exud. myocarditis encephal. Table Microscopic lesions at 21 DPI Lesion ISU-12 ISU-12 ISU-22 ISU-984 ISU-3927 PSP-36 unpl. 11 control Type II Syncytia Int. thick. alv. exud. myocarditis encephal. E E] IK:100168:EARJGSA 43 of188 44 Table 11 Microscopic lesions at 28 DPI Lesion I SU-12 I SU-12 flISUJ-22 Il SU-984 flISU-3927 PSP-36 1unpil. fl 1J V IiIIrcontro Typel+l+ synctia Int. thick. alv. exud. myocarditis encephal. Table 12 Microscopic lesions at 36 DPI Lesion ISU-12 ISU- 12 ISU-22 I SU-9 84 flISU-3927 PSP-36 I jjunpi. .I 1 V control Type Il ND+1+1+1 syncytia ND--- Int. thick. 1 alv. exud. ND myocarditis encephal.

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OS *500 0@*O 5 By- 7 DPI, lung lesions produced by -ISU-12, -ISU-22 and ISU-984 -are severe, and similar to each other. Lung lesions produced by ISU-3927 are only mild or moderately severe by 7 DPI.

By 10 DPI, the lung lesions produced by ISU-12, ISU-22 and ISUJ-984 are similar to those at 7 DPI, but a little more severe. Only pigs infected by unpiaque-purified ISU-12 lo exhibit mild encephalitis and myocarditis. By 10 DPI, lesions produced by ISU-3927 are nearly resolved.

By 21 DPI, myocarditis produc ed by unplaque-purified ISU-12 is severe, whereas myocarditis produced by ISU-12, ISU-22 and ISU-984 is moderate. Only pigs infected by unpiaque-purified ISU-12 exhibit moderate encephalitis at 21 DPI.

15 At 28 DPI, lung lesions are still moderate in pigs infected by unplaque-purified ISU- 12 and ISU-22. These isolates also produce severe myocarditis at 28 DPI. However, lung lesions produced by ISU-12, ISU-984 and ISU-3927 are nearly resolved at 28 DPI.' By 36 DPI, all lesions are essentially resolved. Only 1 pig per group was examined at 3 6 DPI.

Experiment VI An in vivo cross-neutralization study was performed. CDCD pigs were inoculated intranasally first with an isolate selected from ISU-12, ISU-22, ISU-984 and ISU-3927, then four weeks later, the pigs were challenged with ISU-12. Lung lesions and other IK:IS6:EAM/SA 406 44 of a8 1 disease symptoms were examined 8 DPI after challenging with ISU-12. Control pigs were only challenged with ISU-12. The results are presented in Table 13 below.

The pathology results presented in Table 13 below are based on the same scale of severity presented for Table 1 above. In Table 13 below, "Int. thick." means interstitial thickening, "alv. exud." means alveolar exudate, and "encephal." means encephalitis.

Table 13 In vivo cross neutralization Lsion 1-1 2 then -12 Cont. then 1-12 1-22 then 1-12 1-984 then I- 3927 then 1-12 12 TypeII syncytia Int. thick. alv. exud. myocarditis encephal. The data in Table 13 above demonstrate that ISU-12 provides protection for pigs against most symptoms of the disease caused by ISU-12. ISU-984 provides protection 10 against some symptoms and clinical signs of PRRS caused by ISU-12, which is among the most virulent strains of PRRSV virus known.

However, ISU-3927, a mildly pathogenic variant of the Iowa strain of PRRS virus, provides the greatest protection of the isolates studid as a live vaccine against a subsequent challenge with ISU-12. Thus, ISU-3927 may show commercial promise for use as a live vaccine.

Experiment VII Groups of 10 CDCD pigs were inoculated with isolates of the Iowa strain of PRRSV listed in Table 14 below, or with uninfected PSP-36 cells as a control. The pigs were weeks old when challenged intranasally with 105 TCID 50 of each virus isolate listed in 20 Table 14 below. The pigs were necropsied at 10 DPI.

The mean gross lung lesion score 10 DPI is provided in Table 13 below as an indication of the pathogenicity of the isolate. The standard deviation (SD) is provided as an indication of the statistical significance of the mean gross lung lesion score.

Table 14 Inocula N Mean gross lung score 10 DPI SD PSP-36 10 0.0 0.0 ISU-28 10 62.4 20.9 ISU-12 10 54.3 9.8 ISU-79 10 51.9 13.5 ISU-1894 10 27.4 11.7 10 20.8 15.1 ISU-51 10 16.7 IK:)00168:EAR/GSA 45 of 68 46 A statistical comparison of the gross lung lesion scores is provided in Table 15 below.

Table Statistical comparison of eross lung lesion scnres Comparison Value of t p I t I Control vs 12 9.43 p .001 Control vs 28 10.83 p .001 Control vs 51 2.89 .01 Control vs 55 3.61 .001 Control vs 1894 4.76p .001 Conerol vs 79 9.oo00p .001 12 vs 28 1.41 p .2 12 vs 51 6.54 p .001 12 vs 55 5.82 p .001 12 vs 79 0.43 p 12 vs 1894 4.76 p .001 28 vs 51 7.94 p .001 28 vs 55 7.22 p .001 28 vs 79 1.83 p .1 28 vs 1894 6.06 p .001 51 vs 55 0.72 p 51 vs 79 6.11 p .001 51 vs 1894 1.87 p .1 vs 79 5.39 p .001 55 vs 1894 1.15 p .3 79 vs 1894 4.24 p .001 a In addition, each group of pigs was examined for respiratory distress according to the clinical respiratory scoring system described above (see "Clinical score mean" in Table 16 below). "Gross Score" refers to the gross lung lesion score described above. "Enceph.", "myocard." and "rhinitis" refer to the number of pigs in each group exhibiting lesions of encephalitis, myocarditis and rhinitis, respectively. "Micro Score" refers to a score based on the following scale, used to evaluate and compare microscopic lesions of interstitial o1 pneumonia in lung tissue: 0 no disease; normal lung tissue 1 mild multifocal microscopic lesions 2 mild diffuse microscopic lesions 3 moderate multifocal microscopic lesions 4 moderate diffuse microscopic lesions severe multifocal microscopic lesions 6 severe diffuse microscopic lesions Microscopic lesions may be observed in tissues which do not exhibit gross lesions.

Thus, the "micro score" provides an additional means for evaluating and comparing the IK:)00168:EARJGSA 48 of 88 47 pathogenicity of these isolates, in addition to gross lung lesions, respiratory distress, fever, etc.

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IK:)Oo i 6:EARtGsA 4 18 47 of 68 48 Table 16 Isolate ll 5DPI 10 DPI Jj10 DPI 10 DPI 28 DPI 28 DPI [IEnceph. Myocard. 0Rhinitis I Clnc~Za sCreinia Gross score IIMicro score JjGross score Micro scoreV_ PSP-36 0. 0 0 0 0 0.2 1/15 4/15 1/15 ISU-51 0.1 0.2 19.4 2.5 10.0 1.0 2/12 2/12 1/12 1.1 1.5 20.9 2.5 14.4 1.6 8/15 6/15 6/15 ISU-1894 2.5 1.1 26.1 2.3 46.6 2.4 7/15 4/15- 9/15 ISU-79 3.5 2.9 51.9 3.2. 32.0 3.0 6/15 11/15 4/15 ISU-12 1.5 1.4 54.3 4.0 43. 3.0 .9/15 3/15 4/15 ISU-28 1.0 13.1 64.5 3.8 8.6 1.9 10/15 10/15 18/15 IK:300108:EAR/GSA 4 f0 40 of 00 Experiment VIII The mRNA from PSP-36 cells infected with each of ISU-12, ISU-22, ISU-55, ISU- 79, ISU-1894 and ISU-3927 was isolated and separated on a 1.5% agarose gel, to achieve better separation of subgenomic mRNA's. Two groups of migration patterns were observed.

Group I includes isolates ISU-12, ISU-1894, ISU-3927 and possibly, ISU-51. The Northern blot of ISU-12 is shown in Figure 32, and the Northern blots of ISU-1894, ISU- 3927 and ISU-51 are shown in Figure 33. Like the Lelystad virus, seven subgenomic mRNA's (labelled 1-7 in Figures 32 and 33) were found in each of these isolates. The sizes of the subgenomic mRNA's (SgRNA's) are similar to those of the Lelystad virus.

Group II includes isolates ISU-22, ISU-55 and ISU-79. Each of these isolates have nine SgRNA' S, instead of seven. SgRNA's 1, 2, 3, 6 and 7 of Group II are the same as those in Group I, but two additional SgRNA's were found between SgRNA's 3 and 6 of Group I, indicated by the arrows in Figure 33.

Preliminary results indicate that the virus of Group II may replicate better than the isolates of Group I, with the possible exception of ISU-12 in PSP-36 cells. However, in some cases, even ISU-12 may replicate poorly, compared to the isolates of Group II.

Experiment IX A porcine reproductive and respiratory syndrome virus (PRRSV) modified live 20 vaccine efficacy study was conducted in 3-week-old, PRRSV-seronegative, SPF pigs. The .*'.*vaccine consisted of 105.8 TCID 50 of plaque-purified PRRSV ISU-12 (Iowa strain) per 2 ml dose. Nine pigs were given a single vaccine dose by intranasal route 7 pigs were given a single vaccine dose by intramuscular route and 9 pigs served as nonvaccinated challenge controls (NV/CHALL). Vaccinates and controls were challenged on post-vaccination day 35, then scored for gross lung lesions (percent of lung affected) on post-challenge day The average gross lung lesion scores for each group of pigs are shown by the number above each bar in Figure 3 4 Vaccine efficacy was evaluated by reduction in lung lesion score. Both vaccinate groups demonstrated significantly lower (p 0.01) gross lung o30 lesion scores than non-vaccinated controls. Significant differences in scores were not found between vaccinate groups. The ISU-12 PRRSV vaccine was proven efficacious in three-week-old pigs, at the 105.8 TCID 50 dose.

Other Observations ISU-12 virus is enveloped, as it is sensitive to chloroform treatment. Replication of ISU-12 is resistant to 5-bromodeoxyuridine treatment. Therefore, ISU-12 is not a DNA virus. ISU-12 lacks hemagglutinating activity.

Obviously, numerous modifications and variations of the present invention are possible in light of the above teachings. It is therefore to be understood that, within the scope of the appended claims, the invention may be practiced otherwise than as specifically described herein.

IK:001688:EAR/GSA 40 o 88 49a The present claim invention represents a significant advantage in the development of effective PRRSV protection. The virus isolates disclosed in the present specification (both high and low virulence) are effective in protecting pigs against highly virulent forms of PRRSV. To date no one has demonstrated efficiency of any other virus or vaccine protecting against highly virulent forms of PRRSV. Indeed, strains designated ISU-12, ISU-51, and ISU-55, and ISU-3927 have been shown to provide protection for pigs against disease caused by ISU-12, one of the most virulent strains of PRRSV virus known, as shown in Table 17 below: TABLE 17 Vaccine Group Gross Lung Lesion Score@ Control 47.2 ISU-12 p 2 6 7.2 ISU-51 p 15 8.1 p 15 16.6 ISU-3927 p 16 20.9 @Gross lung lesion scores represents the percent of lung affected the percent of lesions in the lung. The score is based on a scale of from 0 to 100% of lung affected..

V Indeed, the variations between the presently claimed viruses and prior viruses could not have been predicted based on the knowledge in the field prior to the present invention.

Tables 18 and 19 (which follow) compare genes of PRRSV isolates of the present invention to those of the prior art. Table 18 compares genes of isolate ATCC VR2385 with those of European isolate Lelystad virus. Table 19 shows a pairwise comparison of the amino acid sequence encoded by ORFs 6 and 7 among members of the arteri virus group.

Thus, the viruses previously known, VR-2332 and Lelystad (European) strain of PRRSV are different from the viruses discovered by the present inventors. VR-2332 is a low virulence isolate and thus does not meet the requirements of claim 1 of this specification nor the requirements of claim 3, which was directed to specific low-virulence strains of PRRSV, which differ in sequence from those of VR-2332. Lelystad virus is a low virulence isolate which is only about 60% homologous to the presently disclosed PRRS isolates.

[R.M18AAaOe99.daSAX 49b TABLE 18 Comparison of genes of U.S. PRRSV isolate ATCC with those of European ipolate Ielystad virus' VR 2385 Gene 1NA. Bsismcd ORP YiR 2315 Lymad Homology RNA -im bemtwee Cb) Siz N-gSyc Pid. She Ngo- Prod VR 2315 Amkuo $yAaion protn ImkIo Iylaio pirvee Lecytzd scidi es Sim %cids ies sbz 1.9 5 200 2 2 201 22.4 53 6 I.4 6 174 1 3191 173 2 11.9 78 7 7 0.9 7 123 2- 13.6 I- 1 13.1 58 NTR 5 NA 114 0 NA )o- Based on data presented by Conzelmann et al, Virology, 193, 329-339 (1993), Meuleflercret al, Vixx'laogy, -192, 62-72 (1993), and the results prepented herein.

S.

V. TABLE 19 Paizwise comparison of the amino acid sequences among membranp proteins of the proposed arterivirus group the putative nucleocapsid and VMUs ViuS VR2385 IS-2 ISU-55 ISU-79 TSU-1894 ISU-3927 VR2332 LV PRRSV-I0 LDV-P LDV-C BAY VR2385 98 96 98 98 96 96 57 57 49 49 22 SU-22 99 98 100 100 98 98 57 57 49 49 23 99 to -98 *98 97 96 59 49 49 23 ISU-79 98 99 99 100 98 98 57 57 49 49 23 ISU-1894 99 100 100 99 98 98 57 57 49 49 23 .ISV-3927 96 97 97 97 97 96 59 59 49 49 23 VR2332 N(A N!A NIA NA NIA NIA .57 57 50 49 22 LV 78 79 79 79 79 81 NIA 99 41 40 23 78 79 79 79 79 81 N(A 100 41 40 23 Lt)V-P 50 .51 51 .51 51 51 NIA 53 53 98 23 LDlY-C 49 50 50 50 so so NA 52 t2 96 24 BAY 16 16 16. 16 16 15 NIA 17 17 16 17 te. Ze s I te (ae are the per etago identit of amixip ackct sequences. WAnot mvaiuabie.

IT$udeocapeld protela conparlsons are presened In Vhe upper right half and membrxne protein cornparsois are presvited In the lower Ileft half.

Page(s)6 -gj are claims pages they appear after the sequence listing

I

Sequence Listing INFORMATION FOR SEQ ID NO: 1: SEQUENCE CHARACTERISTICS: LENGTH: 22 base pairs TYPE: nucleic acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: ORGANISM: Porcine reproductive and respiratory syndrome virus STRAIN: Iowa INDIVIDUAL ISOLATE: ISU-12 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l: CGGCCGTGTG GTTCTCGCCA AT 22 INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 22 base pairs TYPE: nucleic acid STRANDEDNESS: unknown 20 TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: ORGANISM: Porcine reproductive and respiratory syndrome virus STRAIN: Iowa 25 INDIVIDUAL ISOLATE: ISU-12 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: CCCCATTTCC CTCTAGCGAC TG 22 INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: 30 LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: ORGANISM: Porcine reproductive and respiratory syndrome virus STRAIN: Iowa INDIVIDUAL ISOLATE: ISU-12 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: GCCGCGGAAC CATCAAGCAC IK:)00168:EAR/GSA 50 of 88 4I) 51 INFORMATION FOR SEQ ID NO:4: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid s STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: ORGANISM: Porcine reproductive and respiratory syndrome virus STRAIN: Iowa INDIVIDUAL ISOLATE: ISU-12 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: CAACTTGACG CTATGTGAGC INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: unknown TOPOLOGY: unknown 20 <ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: ORGANISM: Porcine reproductive and respiratory syndrome virus STRAIN: Iowa INDIVIDUAL ISOLATE: ISU-12 25 (xi) SEQUENCE DESCRIPTION: SEQ ID GCGGTCTGGA TTGACGACAG INFORMATION FOR SEQ ID NO:6: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs 30 TYPE: nucleic acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: ORGANISM: Porcine reproductive and respiratory syndrome virus STRAIN: Iowa INDIVIDUAL ISOLATE: ISU-12 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: GACTGCTAGG GCTTCTGCAC INFORMATION FOR SEQ ID NO:7: IK:100168:EAR/GSA 61 of 68 SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: unknown s TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: ORGANISM: Porcine reproductive and respiratory syndrome virus STRAIN: Iowa INDIVIDUAL ISOLATE: ISU-12 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: GCCATTCAGC TCACATAGCG INFORMATION FOR SEQ ID NO:8: SEQUENCE CHARACTERISTICS: LENGTH: 1938 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA 20 (vi) ORIGINAL SOURCE: ORGANISM: Porcine reproductive and respiratory syndrome virus STRAIN: Iowa INDIVIDUAL ISOLATE: ISU-12 (ix) FEATURE: 25 NAME/KEY: CDS LOCATION: 1..255 (ix) FEATURE: NAME/KEY: CDS LOCATION: 239..901 30 (ix) FEATURE: NAME/KEY: CDS LOCATION: 1403..1771 (ix) FEATURE: NAME/KEY: CDS LOCATION: 889..1410 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: GGC ACG AGC TTT GCT GTC CTC CAA GAC ATC AGT TGC CTT AGG CAT CGC 48 Gly Thr Ser Phe Ala Val Leu Gin Asp lie Ser Cys Leu Arg His Arg 1 5 10 IK:100168:EAR/GSA 52 o 68 E2 of88 53 MOC TOG GCC TOT GAG GOG AUT OGO AMA GTO: OOT OAG TGC OGO AOG GOG Asn Ser Ala Ser Glu Ala Ilie krg Lys V/al Pro Gin Cys krg Thr Ala 25 ATA GGG ACA 000 GTG TAT ATO ACT GTO: ACA GOC MAT GHT ACC GAT. GAG Ile Gly Thr Pro Val Tyr Ile Thr Val Thr Ala Asn Val Thr Asp Glu .40 MAT TAT HTG OAT TOO TOT GAT OHT OTO ATG OHT TOT TOT TGO OTT TTO Asn Tyr Leu His Ser Ser Asp Leu Leu Met Leu Ser Ser Oys Leu Phe 55 TAT GOT TOT GAG ATG AGT GMA MG GGA Mi MAG GTG GTA M~ GGO MAT Tyr Ala Ser Glu Met Ser Glu Lys Gly Phe Lys Val Val Phe Gly Asn 70 75 GTG TOA GGO ATO lTr TAGOOTGTOT TFTGOGATT OTGTTGGCAM TTTGMATGHT Val Ser Gly Ilie Phe 96 144 192 240 295

S

.555

HMAGTATGT

TGTATOGTGO

TTAOAGOTGA

AAATGACT

TATGGTGOOO

GOCTGGGTrG

GOGTTGAMT

ACOAGATATA

GTOATOATAG

GTTGTGOHTG

TGGGGAAATG, C1TGAOCGCG, OGTOTTGTTT TGTTGOGCTC 1TAAOTT GAOGOTATGT GGGOAGTGGA GTG1TTGTO TCAOTACTAG CCATHOOCTT.

HTCACGGGCG GTATGTTCTG GCHOCGTCAT TAGGCTTGCG OOMOCTTTCT TCTGGACACT AGAAAAGGGG OAAAGTTGAG ATGGHOCCGO GGCTACCCCT

GGOTGTTGOT

GTOAGOGOCA

GAGOTGAATG

ATTMCTG

GACACAdfOG

AGTAGCATGT

MAGMHTGCA

MAGGGOAGAC

GTCGMAGGTC

GTMACCAGAG

CGCAATGOT

ACGGGMACAG

GOACAGAU-G

TGTTGAOTCA

GTCTGGTCAO

ACGOGGTOTG

TGTCOTGGOG

TCTATOGTTG

ACOTGATOGA

MCrAGOGGA 1TTTGTGG 355 OGGCTOMAAT 415 GCTAGOTMAT 475 OAT-TGTOTOT 535 TGTGTOTAOO 595 TGCOTGGOT 655 OTAOTOATGT 715 GOGGTOGCOT 775 CCTOMAAAGA 835 ACA ATG 891 GAG TOG TOO HTA GAT GAO HOC TGT Glu Ser Ser Leu Asp Asp Phe Cys 5 GTG OTO HTG GOG M~ TOT AHT ACC Val Leu Leu Ala Phe Ser Ilie Thr 25 OAT GAT AGO AOG GOT OOA CAM MG His Asp Ser Thr Ala Pro GIn Lys 10 TAO AOG OCA GTG ATG ATA TAT GC Tyr Thr Pro Val Met Ilie Tyr Ala OTA MAG GTG AGI OGO GO OGA OTG OTA GGG OHT OTG CAC OHT HG GTO 1035 Lou Lys Vai Ser krg Gly krg Leu Leu Gly Leu Leu His Leu Lou Val 40 HOC OTG MAT TGT GOT HOC ACC HOC GGG TAO ATG ACA HOC GTG CAC iTT 1083 Phe Lou Asn Cys Ala Phe Thr Phe Gly Tyr Met Thr Phe Val His Phe 55 60 IK:IOO1 88:EARJGSA 83 of 88 CAG AGT ACA MAT MAG GTC GOG CTC ACT ATG GGA GCA GTA GiT GCA CTC 1131 Gin Ser Thr Asn Lys Vai Ala Leu Thr Met Gly Ala Vai Vai Ala Leu 75 CTT TGG GGG GTG TAC TCA GCC ATA GMA AGO TGG AAA HOC ATO ACO TOO 1179 Leu Trp Gly Val Tyr Ser Ala Ilie Giu Thr Trp Lys Phe Ile Thr Ser 90 AGA TGC OGT HTG TGC H TG OTA GGC CGC MAG TAO ATT CTG GOC COT GCC 1227 Akg Cys krg Leu Cys Leu Leu Gly Arg Lys Tyr Ilie Leu Ala Pro Ala *5 S 555555 a.> S 100. 105 110 CAO GAG GTT GMA AGT GOC GCA GGO Mi CAT COG AUT GOG GOA MAT GA His His Val Giu Ser Ala Ala Gly Phe His Pro Ilie Ala Ala Asn As 115 120 125 MAC CAC GCA M~ GTC GTC OGG CGT CCC GGO TOO ACT ACG GTC MAC GG Asn His Ala Phe Val Vai krg krg Pro Gly Ser Thr Thr Val Asn Gi 130 135 140 .14~ ACA HTG GTG COO GGG HTA AAA AGO OTO GTG HTG GGT GGO AGA AAA GC~ Thr Leu Val Pro Gly Leu Lys Ser Leu Val Leu Gly Gly krg Lys Al~ 150 155 160 GH AAA CAG GGA GTG GIA MOC OH GTT MAA TAT GOC AAA TMACGCGGC Val Lys Gin Gly Val *Vai Asn Ld-u Vai Lys Tyr Ala Lys 165 .170 MAGCAGOAGA AGAGAAAGMA GGGGGATGGO CAGCGAGTOA ATCAGCTGTG COAGATGGTG GGTMAGATOA TOGOTOACCA MAACCAGTOC AGAGGOMAGG GACCGGGAAA GAMAAATMAG MAGAAAAAOC CGGAGMAGOC COATTTOCCT OTAGOGAOTG MAGATGATGT CAGACATOAG 1TTACCCCTA GTGAGOGTCA ATTGTGTCTG TCGTCMATCC AGACOGCHT TMATOMGGC GOTGGGACHT GOAOOOTGTO AGAHOCAGGG AGGATMOGTI AOAOTGTGGA GTAG1TrG COTACGOATC ATAOTGTGOG OOTGATOOGC GTOAOAGCAT GACOOTOAGO ATGATGGGCT GGCAHOHTG AGGOATOOCA GTG1TGMAT TGGAAGMATG OGTGGTGMAT GGOACTGAHT GAOAHTGTGC OTOTMAGTCA CCTAHTCMT TAGGGCGACC GTGTGGGGGT MAGA1TTA TGGCGAGMAC CACACGGOGG AAA1TAAAAA MAAAAA INFORiMATION FOR SEQ ID NO:9: SEQUENCE CHARACTERISTICS: LENGTH: 85 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: Gly Thr Ser Phe Ala Val Leu Gin Asp Ilie Ser Cys Leu krg His Arg 1 5 10 y 3 1323 1371 1420 1480 1540 1600 1660 1720 1780 1840 1900 1938 T1275 p t6:EAR/GsA 54 of 18 J Asn Ser Ada Ser Giu Ada Ilie krg Lys Val Pro Gin Cys krg Thr Ala .25 Ilie Gly Thr Pro Vai Tyr Ilie Thr Val Thr Ala Asn Val Thr Asp Glu 40 Asn Tyr Leu His Set Ser Asp Leu Lou Met Lou Ser Set Cys Leu Phe 55 Tyr, Ala Ser Giu Met Ser Giu Lys Gly Phe Lys Val Val Phe Gly Asn 70 75 Val Set Gly Ilie Phe INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 22'1 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: Met Cys Gin Ala Set Phe Set Leu Set Phe Cys Asp Set Vai Gly Asn Leu Asn Val Lou Set Met Leu Gly Lys Cys Leu Thr Ala Gly Cys Cys *20 25 Set Gin Lou Leu Phe Lou Trp Cys Ilie Val Pro Ser Cys Phe Vai Ada 40 Leu Val Set Ala Asn Giy Asn Set Giy Ser Asn Leu Gin Lou Ilie Tyr 55 *Asn Leu Thr. Leu Cys Giu Leu Asn Giy Thr Asp Tip Leu Ada Asn Lys 70 75 Phe Asp Tip Ala Vai Giu Cys Phe Val Ilie Phe Pro Val Leu Thr His 90 Ilie Val Set Tyr- Giy Ala Leu Thr Tht Set His Phe Leu Asp Tht Val 100 105 110 Gly Leu Val Tht Val Set 'Tht Ala Gly Phe Vai His Gly krg Tyr Val 115 120 125 Lou Set Set Met Tyr Ada Val Cys Ada Leu Ala Ada Lou Ilie Cys Phe 130 135 140 Val Ilie krg Leu Ala Lys Asn Cys Met Set Tip krg Tyr Set Cys Thr 145 150 155 160 krg Tyt, Thr Asn Phe Leu Leu Asp Thr Lys Gly krg Leu Tyr krg Tip 165 170 175 IK:100188:EARIGSA 66 of as 56 Arg Ser Pro Val lie lie Glu Lys Arg Gly Lys Val Glu Val Glu Gly 180 185 190 His Leu lie Asp Leu Lys Arg Val Val Leu Asp Gly Ser Ala Ala Thr 195 200 205 Pro Val Thr Arg Val Ser Ala Glu Gin Trp Ser Arg Pro 210 215 220 INFORMATION FOR SEQ ID NO: 11: SEQUENCE CHARACTERISTICS: LENGTH: 174 amino acids TYPE: amino acid s TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: Met Glu Ser Ser Leu Asp Asp Phe Cys His Asp Ser Thr Ala Pro Gin 1 5 10 Lys Val Leu Leu Ala Phe Ser lie Thr Tyr Thr Pro Val Met lie Tyr 25 Ala Leu Lys Val Ser Arg Gly Arg Leu Leu Gly Leu Leu His Leu Leu 35 40 Val Phe Leu Asn Cys Ala Phe Thr Phe Gly Tyr Met Thr Phe Val His 55 Phe Gin Ser Thr Asn Lys Val Ala Leu Thr Met Gly Ala Val Val Ala 70 75 Leu Leu Trp Gly Val Tyr Ser Ala lie Glu Thr Trp Lys Phe lie Thr 90 Ser Arg Cys Arg Leu Cys Leu Leu Gly Arg Lys Tyr lie Leu Ala Pro 100 105 110 Ala His His Val Glu Ser Ala Ala Gly Phe His Pro lie Ala Ala Asn 115 120 125 Asp Asn His Ala Phe Val Val Arg Arg Pro Gly Ser Thr Thr Val Asn 130 135 140 Gly Thr Leu Val Pro Gly Leu Lys Ser Leu Val Leu Gly Gly Arg Lys 145 150 155 160 Ala Val Lys Gin Gly Val Val Asn Leu Val Lys Tyr Ala Lys 165 170 INFORMATION FOR SEQ ID NO: 12: SEQUENCE CHARACTERISTICS: 1 o LENGTH: 123 amino acids TYPE: amino acid [K:IO0168:EARtGSA 68 68

C,

57 TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: Met Pro Msn Msn Thr Gly Lys Gin Gin Lys Arg Lys- Lys Gly 1 .5 10 Gin Pro Vai Msn Gin Leu Cys Gin Met Leu Giy Lys Ilie Ilie 25 Gin Msn Gin Ser Arg Giy Lys Giy Pro Gly Lys Lys Msn Lys 40 Asn Pro Giu Lys Pro His Phe Pro Leu Ala Thr Giu Asp Asp 55 His His Phe Thr Pro Ser Giu krg Gin Leu Cys Leu Ser Ser 70 75 Thr Ala Phe Msn Gin Giy Ala Giy Thr Cys Thr Leu Ser Asp 90 Arg Ilie Ser Tyr Thr Val Giu Phe Ser Leu Pro Thr His His Asp Gly Ala His Lys Lys Vai krg Ilie Gin Ser Giy Thr Val krg Leu Ilie krg Vai Thr Ala Ser Pro Ser Ala 0* *5

S

S.

*5 S S

*SSSS*

S

*5eS 5550 INFORMATION FOR SEQ ID NO: 13: 5 SEQUENCE CHARACTERISTICS: LENGTH: 667 base pairs TYPE: nucleic acid STRANDEDNESS: unknown TOPOLOGY: unknown 10 (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: ORGANISM: Porcine reproductive and respiratory syndrome virus STRAIN: Iowa.

INDIVIDUAL ISOLATE: ISU-12 15 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: 0@ 5

S

MATGTGTCAG GCATC1TMA GCCTGTC1T, AAGTATGTTG GGGAAATGCT TGACCGCGGG TATCGTGCCG TCTTGTlTG TTGCGCTCGT ACAGCTGATT TACMACTTGA CGCTATGTGA ATGACTGG GCAGTGGAGT G1TJGTCAT TGGTGCCCTC ACTACTAGOC ATCCTTGA TGGG11TG1T CAOGGGCGGT ATGTTCTGAG G1TGATGC TTCGTCA1TA GGCTTGCGAA CAGATATACC MC1TFCTTC TGGACACTMA CATCATAGAG AAAAGGGGCA MAG1TGAGGT TGTGCTTGAT GGTTCCGCGG CTACCCCTGT

TCCTTAG

1TGCGATTCT GTTGGCAATT TGAATGTF CTGTTGCTCG CMATTGCMT 1TTGTGGTG CAGCGCCMAC GGGMACAGCG GCTCMA1TM GCTGAATGGC ACAGATTGGC TAGCTMATM T1TTTCCTGTG TTGACTCACA TTGTCTCTfA CACAGTCGGT CTGGTCACTG TGTCTACCGC TAGCATGTAC GCGGTCTGTG CCCTGGCTGC GM1TGCATG TCCTGGCGCT ACTCATGTAC GGGCAGACTC TATCGTTGGC GGTCGCCTGT CGMAGGTCAC CTGATCGACC TCAAMAGAGT MACCAGAGTT TCAGCGGMAC MATGGAGTCG 120 180 240 300 360 420 480- 540 600 660 667 (K:IOO1 68:EAR1GSA 6 f8 67of68 INFORMATION FOR SEQ ID NO: 14: SEQUENCE CHARACTERISTICS: LENGTH: 605 base pairs TYPE: nucleic acid STRANDEDNESS: unknown TOPOLOGY: unknown GOi MOLECULE TYPE:- cDNA (vi) ORIGINALS OURCE: ORGANISM: Porcine reproductive and respiratory syndrome virus STRAIN: Lelystad (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:

ATGAGATGTT

TTGCTGTG

ACCMATACAT

ATTGG1TrG TGGG1TFCT CAGGA1TGT

CGTTCGTATG

CCCGGMTAC

TAGTGGTAGA

TCGTCCTCGA

CCTAG

CTCACAAATT

TACCGGCTFrG

ATATMACTTG

GGCAGTCGAG

CACMACAAGC

TGGCGGGCGG

1TrGTCATC CAACT'rCATT

AMAATTGGGC

AGGGGTTAAA

GGGGCGMTC

TCCTGGTCCT

ACGATATGCG

ACC1TGTGC

CATTTTTG

TACGTACTCT

CGTGCTGCTA

GTGGACGACC

AAAGCCGAAG

GCTCAACCCT

TTGACTCCGC

T-TGCCGATGG

AGCTGMATGG

1TACCCGGT

ACGCGCTCGG

GCAGCGTCTA

AAAATTGCAT

GGGGGAGAGT

TCGATGGCMA

TGACGAGGAC

ACTCTTGC1T

OMOCGGOGAC

GACCGACTGG

TGCCACTCAT

TOTOGGOGOT

CGGCGCTTGT

GGCCTGCCGC

TCATCGATGG

CCTCGTCACC

TTCGGCTGAG

CTGGTGGCTT

AGCTCGACAT

TTGTCCAGCC

ATCCTCTCAC

GTATCCACTG

GC1TCGCAG

TATGCCCGTA,

MAGTCTCCMA

ATCAAACATG

CAATGGGAGG

0 .00.

00..

0 0 0@ 000 9090 00 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 526 base pairs TYPE: nucleic acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: ORGANISM: Porcine reproductive and respiratory syndrome virus STRAIN: Iowa INDIVIDUAL ISOLATE: ISU-12- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:

MATGGAGTCG

GGCG1TMCT

ACTGCTAGGG

GACA1TCGTG ACTCC11GG

TTGTGCTTG

AGGCTTCAT

CACTACGGTC

AGCTGTrAAA

TCCTTAGATG

ATTACOTACA

CTTCTGCACC

CACTUCAGA

GGGGTGTACT

CTAGGCCGCA

CCGA1TGCGG

MACGGCACAT

CAGGGAGTGG

ACTTCTGTCAA

CGCCAGTGAT

1TIGGTCTT

GTACAAATAA

CAGCCATAGA

AGTACATTCT

CMAATGATMA

TGGTGCCCGG

TAAACCTTGT

TGATAGCACG

GATATATGCC

CCTGMATTGT

GGTCGCGCTC

MACCTGGAAA

GGCCCCTGCC

CCACGCA1TT

GTMMAAAGC

TAAATATGCC

GCTCCACAMA

CTAAAGGTGA

GCMTCACCT

ACTATGGGAG

TTCATCACCT

CACCACG1TG

GTCGTCCGGC

CTCGTGTTGG

AAATAA

AGGTGCTCTT

GTCGCGGCCG

TCGGGTACAT

CAGTAGTTGC

CCAGATGCCG

AAAGTGCCGC

GTCCCGGCTC

GTGGCAGA

120 180 240 300 360 420 480- 526 INFORMATION FOR SEQ ID NO: 16: SEQUENCE CHARACTERISTICS: IK:)OO1 88:EARJGSA 68s of 88 LENGTH: 522 base pair TYPE: nucleic acid STRANDEDNESS: unknown TOPOLOGY: unknown.

(ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: ORGANISM: Porcine reproductive and respiratory syndrome virus STRAIN: Lelystad (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:

ATGGGAGGCC

MTAGCATCA

CTGGGGOTGT

TATGTGCA1T

CTGTGGGGTG

TGTTGCC1TG

CTCCATTCAA

TCAGTGMACG

GTTAAACGAG

TAGACGA1T

CATACACACC

TGCACATCCT

TCATCCAC

M1ACAGCTT

GCCGGCGATA

TCTCAGCGTC

GCACTCTAGT

GAGTGGTTMA

TTGCMACGAT

TATAATGATA

MTAMTCTG

CMACCGTGTC

OACAGAGTCA

CATTCTGGCC

TGGTMACCGA

ACCAGGACiT

CCTCGTCAAG

CCTATCGCCG

TACGCCCTTA

MACTGTTCCT

GCACTTACCC

TGGMG1TMA

CCTGCCCATC

GCATACGCTG

CGGAGCCTCG

TATGGCCGGT

CACAAAAGCT

AGGTGTCACG

TTACATTCGG

TGGGGGCTGT

TCACTTCCAG

ACGTAGAAAG

TGAGAAAGCC

TGCTGGGCGG

CGTGCTAGCC

CGGCCGACTC

ATACATGACA

TGTCGCCCTT

ATGCAGA1-TG

TGCTGCAGGT

CGGACTMACA

CAMACGAGCT

INFORMATION FOR SEQ ID NO: 17: SEQUENCE CHARACTERISTICS: LENGTH: 372 base pairs TYPE: nucleic acid STRANDEDNESS: unknown 15 TOPOLOGY: unknown- (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: ORGANISM: Porcine reproductive and respiratory syndrome virus STRAIN: Lelystad (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:

ATGCCAAATA

CAGCTGTGCC

CCGGGAAACA

GATGATGTCA

ACCGCC1TA

ACTGTGGAGT

CCCTCAGCAT

ACACCGGCAA

AGATGCTGGG

AMAATMAGAA

GACATCACTT

ATCAAGGCGC

TTAGMTGCC

GA

GCAGCAGMAG,

TAAGATCATC

GMAAAACCCG

TACCCCTAGT

TGGGAC1TGC

TACGCATCAT

AGMAAGMGG

GCTCACCA

GAGAAGCCCC

GAGCGTCMAT

ACCCTGTCAG

ACTGTGCGCC

GGGATGGCCA

ACCAGTCCAG

A1TCCCTCT

TGTGTCTGTC

A1TCAGGGAG

TGATCCGCGT

GCCAGTCMAT

AGGCMAGGGA

AGCGACTGMA

GTCMATCCAG

GATMAG1TAC

CACAGCATCA

INFORMATION FOR SEQ ID NO: 18: SEQUENCE CHARACTERISTICS: LENGTH: 387 base pairs TYPE: nucleic acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: tK:0oi 88.EARlGSA 6 f6 69 of 68 ORGANISM: Porcine reproductive and respiratory syndrome virus STRAIN: Iowa INDIVIDUAL ISOLATE: ISU-12 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18: ATGGCCGGTA MAAACCAGAG CCAGMAGAAA MAGAAAAGTA CAGCTCCGAT GGGGMAT CAGCCAGTCA ATCMACTGTG CCAGTTGCTG GGTGCMATGA TMAAGTCCCA GCGCCAG CCTAGGGGAG GACAGGCCMA MAGAAAMAG CCTGAGAAGC CACATMCC CCTGGCT( GAAGATGACA TCCGGCACCA CCTCACCCAG ACTGMACGCT CCCTCTGC1T GCMTCG) CAGACGGC1T TCMATCMAGG CGCAGGMACT GCGTCGC1T CATCCAGCGG GAAGGTC) 1TCAGGTTG AG1TATGCT. GCCGG1TGCT CATACAGTGC GCCTGATTCG CGTGACT1 ACATCCGCCA GTCAGGGTGC AAGTPA FOR SEQ ID. NO: 19: SEQUENCE CHARACTERISTICS: LENGTH: 164 base pairs TYPE: nucleic acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: ORGANISM: Porcine reproductive and respiratory syndrome virus STRAIN: Iowa 15 INDIVIDUAL ISOLATE: ISU-12 (xi) SEQUENCE DESCRIPTION;"SEQ ID NO: 19: TGGGCTGGCA TTCTTGAGGC ATCCCAGTGT TTGAA1TGGA AGAATGCGTG GTGMATGG CTGATTGACA TTGTGCCTCT MAGTCACCTA TTCM1TTAGG GCGACCGTGT GGGGGTMA 1TFMTTGGC GAGMACCACA CGGCCGMAT TAAM AAA INFORMATION FOR SEQ ID, SEQUENCE CHARACTERISTICS: LENGTH: 127 base pairs 20 TYPE: nucleic acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: eDNA (vi) ORIGINAL SOURCE: ORGANISM: Porcine reproductive and respiratory syndrome virus STRAIN: Lelystad (xi) SEQUENCE DESCRIPTION: SEQ ID

GGC

CAA

3CT

TC

~GT

CT

120 180 240 300 360 387 120 164

CA

~GA

1TGACAG 'TC AGGTGMATGG 'CCGCGATTGG CGTGTGGCCT CTGAGTCACC GGGCGATCAC ATGGGGGTCA TACTrAMTCA GGCAGGMACC ATGTGACCGA

AAAA

INFORMATION FOR SEQ I1D NO:21: SEQUENCE CHARACTERISTICS:

TATOCAATA

AATTAAAAA 120 127 lK:)01 8EARIGSA 60 .168 LENGTH: 19 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Other nucleic acid; DESCRIPTION: DNA (synthetic) (vi) ORIGINAL SOURCE: ORGANISM: Porcine reproductive and respiratory syndrome virus STRAIN: Lelystad 1o (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: CTCGTCAAGT ATGGCCGGT 19 INFORMATION FOR SEQ ID NO:22: SEQUENCE CHARACTERISTICS: LENGTH: 19 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Other nucleic acid; DESCRIPTION: DNA (synthetic) (vi) ORIGINAL SOURCE: ORGANISM: Porcine reproductive and respiratory syndrome virus (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: GCCATTCGCC TGACTGTCA 19 INFORMATION FOR SEQ ID NO:23: 25 SEQUENCE CHARACTERISTICS: LENGTH: 19 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear 30 (ii) MOLECULE TYPE: Other nucleic acid; DESCRIPTION: DNA (synthetic) (vi) ORIGINAL SOURCE: ORGANISM: Porcine reproductive and respiratory syndrome virus (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: TTGACGAGGA CTTCGGCTG 19 INFORMATION FOR SEQ ID NO:24: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single [K:)00168:EAR/GSA of 62 TOPOLOGY: linear (ii) MOLECULE TYPE: Other nucleic acid; DESCRIPTION: DNA (synthetic) (vi) ORIGINAL SOURCE: s ORGANISM: Porcine reproductive and respiratory syndrome virus (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: GCTCTACCTG CAATTCTGTG INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: 1o LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Other nucleic acid; DESCRIPTION: DNA (synthetic) (vi) ORIGINAL SOURCE: ORGANISM: Porcine reproductive and respiratory syndrome virus (xi) SEQUENCE DESCRIPTION: SEQ ID SGTGTATAGGA CCGGCAACAG INFORMATION FOR SEQ ID NO:26: S. SEQUENCE CHARACTERISTICS: LENGTH: 26 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Other nucleic acid; DESCRIPTION: DNA (synthetic) (vi) ORIGINAL SOURCE: ORGANISM: Porcine reproductive and respiratory syndrome virus .30 STRAIN: Iowa INDIVIDUAL ISOLATE: ISU-12 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: GGGGATCCGG TATTTGGCAA TGTGTC 26 INFORMATION FOR SEQ ID NO:27: SEQUENCE CHARACTERISTICS: LENGTH: 28 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Other nucleic acid; IK:100168:EAR/GSA 02 o 88 63 DESCRIPTION: DNA (synthetic) (vi) ORIGINAL SOURCE: ORGANISM: Porcine reproductive and respiratory syndrome virus STRAIN: Iowa INDIVIDUAL ISOLATE: ISU-12 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: GGTGTITCC ACGAGAACCG CTTAAGGG 28 INFORMATION FOR SEQ ID NO:28: SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Other nucleic acid; is DESCRIPTION: DNA (synthetic) (vi) ORIGINAL SOURCE: ORGANISM: Porcine reproductive and respiratory syndrome virus STRAIN: Iowa INDIVIDUAL ISOLATE: ISU-12 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28: SGGGGATCCAG AGTTTCAGCG G 21 INFORMATION FOR SEQ ID NO:29: SEQUENCE CHARACTERISTICS: LENGTH: 25 base pairs 25 TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Other nucleic acid; DESCRIPTION: DNA (synthetic) 30 (vi) ORIGINAL SOURCE: ORGANISM: Porcine reproductive and respiratory syndrome virus STRAIN: Iowa INDIVIDUAL ISOLATE: ISU-12 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29: CAGTTAGTCG ACACGGTCTT MAAGGG INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 22 base pairs TYPE: nucleic acid STRANDEDNESS: single IK:)00168:£AR/GSA 63 of 8 TOPOLOGY: linear (ii) MOLECULE TYPE: Other nucleic acid; DESCRIPTION: DNA (synthetic) (vi) ORIGINAL SOURCE: ORGANISM: Porcine reproductive and respiratory syndrome virus STRAIN: Iowa INDIVIDUAL ISOLATE: ISU-12 (xi) SEQUENCE DESCRIPTION: SEQ ID GGGGATCCTT GTTAAATATG CC 22 INFORMATION FOR SEQ ID NO:31: SEQUENCE CHARACTERISTICS: LENGTH: 19 base pairs TYPE: nucleic acid STRANDEDNESS: single is TOPOLOGY: linear (ii) MOLECULE TYPE: Other nucleic acid; DESCRIPTION: DNA (synthetic) (vi) ORIGINAL SOURCE: ORGANISM: Porcine reproductive and respiratory syndrome virus STRAIN: Iowa INDIVIDUAL ISOLATE: ISU-12 SEQUENCE DESCRIPTION: SEQ ID NO:31: CTTACGCACC ACTTAAGGG 19 (K:100188:EARIGSA 84 of 68

Claims (21)

1. A naturally occurring isolated virus selected from the group consisting of ISU-12 (VR 2385 and VR 2386) isolates and isolates exhibiting the identifying characteristics of ISU-12 (VR 2385 or VR 2386) isolate.

2. A naturally occurring isolated virus selected ISU-22 (VR 2429) isolate and isolates exhibiting the ISU-22 (VR 2429) isolate.

3. A naturally occurring isolated virus selected ISU-51 (VR 2428) isolate and isolates exhibiting the ISU-51 (VR 2428) isolate.

4. A naturally occurring isolated virus selected (VR 2430) isolate and isolates exhibiting the (VR 2430) isolate. A naturally occurring isolated virus selected from the group consisting of an identifying characteristics of an from the group consisting identifying characteristics from the group consisting identifying characteristics of an of an of an of an from the group consisting of an *0 ISU-3927 (VR 2431) isolate and isolates exhibiting the identifying characteristics of an ISU-3927 (VR 2431) isolate.

6. The virus of any one of the preceding claims wherein said virus has less than 90% of polynucleotide identity to Lelystad virus in any one of open reading frames (SEQ ID NO:13), 6 (SEQ ID NO:15) or 7 (SEQ ID NO:18).

7. The virus of claim 1 or claim 2 which causes porcine reproductive and respiratory syndrome (PRRS), wherein inoculation of five-week-old colostrum-deprived, caesarean-derived pigs with 105 TCID 50 of said virus results in lesions in at least 51.9% of lung tissue 10 days post-infection.

8. A composition comprising an isolated virus of any one of claims 1 to 7 and a 25 physiologically acceptable carrier.

9. A vaccine which protects a pig against porcine reproductive and respiratory syndrome (PRRS), the vaccine comprising an effective amount of an isolated virus of any one of claims 1 to 7 or of a composition of claim 8. The vaccine of claim 9, wherein said virus is live, inactivated or attenuated.

11. A vaccine which protects a pig against porcine reproductive and respiratory syndrome (PRRS), the vaccine comprising an inactivated or attenuated virus and a physiologically acceptable carrier, wherein prior to inactivation or attenuation, said virus is of any one of claims 1 to 7.

12. The vaccine of claim 10, wherein said virus is attenuated and is prepared by serial passage in cell culture. I:\DAYLIB\libaa\08720.docsak

13. The vaccine of any one of claims 9 to 12, comprising an effective amount of said virus which lowers the average clinical respiratory score of a group of five-week-old colostrum-deprived, caesarean-derived pigs inoculated with said vaccine, then subsequently challenged with live PRRS virus, relative to a group of identically challenged colostrum- deprived, caesarean-derived pigs not inoculated with the vaccine.

14. The vaccine of claim 13, wherein lung lesions in said five-week-old colostrum- deprived, caesarean-derived pigs are reduced by a statistically significant amount, wherein said amount is significant at a p value of less than 0.01, relative to lung lesions in uninoculated five-week-old colostrum-deprived, caesarean-derived pigs.

15. The vaccine of any one of claims 9 to 14 further comprising an adjuvant.

16. A method of protecting a pig from a porcine reproductive and respiratory disease, the method comprising administering to said pig an effective amount of the vaccine of any one of claims 9 to

17. The method of claim 16, wherein said vaccine is administered orally or is parenterally.

18. The method of claim 16, wherein said vaccine is administered intramuscularly, intradermally, intravenously, intraperitoneally, subcutaneously or intranasally.

19. The method of any one of claims 16 to 18, wherein said pig is a sow.

20. The vaccine of any one of claims 9 to 15 when used for protecting a pig from a 20 porcine reproductive and respiratory disease.

21. A method of producing the vaccine of any one of claims 12 to 15, the method comprising the steps of: collecting a sufficiently large sample of the virus, and treating said virus in a manner selected from the group consisting of plaque- 25 purifying the virus, (ii) heating said virus at a temperature and for a length of time sufficient to inactivate said virus, (iii) exposing or missing said virus with an amount of an inactivating chemical sufficient to inactivate said virus, (iv) breaking down said virus into its corresponding subunits and isolating at least one of said subunits, and synthesizing or isolating a polynucleic acid encoding a surface protein of said virus, infecting a suitable host cell with said polynucleic acid, culturing said host cell, and isolating said surface protein from said culture.

22. The method of claim 21 wherein said virus or infectious agent is collected from a source selected from the group consisting of culture medium, cells infected with said virus or infectious agent, and both a culture medium and cells infected with said virus or infectious agent. I:\DAYLIB\libaa\08720.docsak 67

23. The method of claim 22 further comprising the step of culturing said virus or infectious agent in a suitable medium prior to said collecting step. Dated 5 September 2001 Iowa State University Research Foundation, Inc. s Solvay Animal Health, Inc. Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON ot**f oo o ftf f f I:\DAYLIB\libaa\08720.docsak