Porcine reproductive and respiratory syndrome vaccine virus

Abstract

The present invention relates to modified live porcine reproductive and respiratory syndrome viruses. The viruses were genetically analyzed and selected based on phylogenetic classification for modification by repeated passage in tissue culture. The modified live viruses were evaluated for their ability to confer protective immunity to heterologous viruses. The modified live viruses are useful in vaccines, particularly vaccines capable of treating infection of pigs with multiple heterologous viruses.

Description

[Background technology]

[0001] The present invention relates to modified live porcine reproductive and respiratory syndrome viruses. The modified live viruses are useful in vaccines, particularly vaccines that provide protection against heterologous viruses.

[0002] Porcine reproductive and respiratory syndrome (PRRS), originally called mystery pig disease, was first described in Europe but has now spread worldwide. PRRS causes late abortions, stillbirths, and infertility in sows of reproductive age, as well as respiratory disease, reduced growth performance, and death in growing and fattening pigs. PRRS causes significant economic losses.

[0003] Symptoms of PRRS virus infection in adult pigs include, but are not limited to, decreased appetite, lethargy, and fever. Pregnant sows may give birth early, abort the fetus, or give birth to mummified or stillborn piglets, and up to 10% of pregnant sows may die from PRRS virus infection. Infected piglets have a high preweaning mortality rate and are often weak and may have edema around the eyes. PRRS virus infection in weaned growing or fattening pigs can cause, but is not limited to, poor growth, respiratory distress, labored or rapid breathing, reddened patches of skin, and a coarse hair coat.

[0004] PRRS virus is an enveloped virus with a linear, positive-stranded RNA genome of approximately 15 kb, and the virus is classified as an Arteriviridae. At least 11 open reading frames have been identified within the genome. PRRS virus is divided into two genotypes: the European genotype, PRRS virus type 1 (PRRSV-1), is exemplified by the Lelystad strain, while the North American PRRS virus type 2 (PRRSV-2) is exemplified by the strain VR-2332.

[0005] Two genotypes can share approximately 60% sequence identity in their genomes, and even within a genotype, individual strains can vary by up to approximately 20% in their genomic identity. This variability has complicated the development of vaccines to effectively treat and / or prevent PRRS. While modified live virus (MLV) variants of PRRS virus can generate immunity against challenge with PRRS virus, vaccines are most effective when challenge is with a PRRS virus that is genetically homologous to the MLV. MLV vaccines are less effective against challenge with heterologous viruses. Furthermore, MLVs exhibit some reversion to virulence, such that the vaccine virus can cause disease in vaccinated animals. Vaccines containing inactivated (i.e., killed) PRRS virus have a better safety profile but have limited efficacy against heterologous challenge.

[0006] Current PRRS vaccines do not demonstrate sufficient safety and efficacy to reduce the economic impact of PRRS virus infection, so new and improved vaccines are needed. Preferably, these vaccines will be both safe and effective. If a vaccine contains attenuated MLVs, the attenuated MLVs should not exhibit reversion to virulence to be considered safe for use in the art. For example, by adapting a PRRS strain to growth in tissue culture cells for at least 60 passages, at least 70 passages, at least 80 passages, or preferably at least 85 passages, the MLVs should not exhibit reversion to virulence. To be effective, a vaccine virus strain must be able to induce protective immunity in swine animals against a variety of phylogenetically diverse wild-type PRRS strains. Preferably, a new PRRS vaccine virus strain will be able to induce protective immunity in swine animals against at least three phylogenetically diverse wild-type PRRS strains. Summary of the Invention

[0007] The present invention provides a modified live porcine reproductive and respiratory syndrome vaccine virus strain, wherein the consensus complementary DNA sequence of the PRRS strain is at least 90% identical to a sequence selected from the group of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4. Preferably, the modified live strain may have a consensus complementary DNA sequence that is at least 95% identical to a sequence selected from the group of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4. More preferably, the modified live strain may also have a consensus complementary DNA sequence that is at least 98% identical to a sequence selected from the group of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4. As will be appreciated by those skilled in the art, due to the high mutation rate of PRRS viruses, a modified live PRRS strain may comprise multiple subpopulations, each with a homologous, but not identical, genome.

[0008] The present invention provides a modified live porcine reproductive and respiratory syndrome (PRRS) virus strain, wherein the PRRS virus strain is a DE 14-3073, ES 13-49, IT 14-32, or PL 14-02 strain. The PRRS virus strain should preferably be passaged at least 60 times, more preferably 70 times, or even more preferably 80 times in tissue culture cells. Most preferably, the PRRS virus strain should be passaged 85 times in tissue culture cells. Such passage in tissue culture cells is useful for attenuating the modified live PRRS virus strain. Attenuated PRRS virus strains can cause subclinical but not clinical disease when administered to swine animals. Modified live PRRS virus strains that have been passaged at least 80 times are less likely to revert to wild-type virulence. Most preferably, modified live PRRS virus strains that have been passaged 85 times are less likely to revert to wild-type virulence.

[0009] The present invention provides immunogenic compositions comprising a modified live PRRS virus strain having a consensus complementary DNA sequence at least 90% identical to a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4. Preferably, the modified live strain will have a consensus complementary DNA sequence at least 95% identical to a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4. More preferably, the modified live strain will also have a consensus complementary DNA sequence at least 98% identical to a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4. As will be appreciated by those skilled in the art, due to the high mutation rate of PRRS viruses, a modified live PRRS strain may comprise multiple subpopulations, each with a homologous, but not identical, genome.

[0010] The present invention provides an immunogenic composition comprising a modified live PRRS virus strain, wherein the PRRS virus strain is DE 14-3073, ES 13-49, IT 14-32, or PL 14-02. The DE 14-3073, ES 13-49, IT 14-32, or PL 14-02 strain can be passaged at least 80 times, or preferably as many as 85 times, in tissue culture cells. Most preferably, the immunogenic composition includes at least one pharmaceutically acceptable excipient.

[0011] The immunogenic composition may also include additional antigens from different viruses, or bacterial strains, or parasites.

[0012] The present invention provides a vaccine comprising a modified live PRRS virus strain, wherein the consensus complementary DNA sequence of the PRRS strain is at least 90% identical to a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4. Preferably, the modified live strain has a consensus complementary DNA sequence that is at least 95% identical to a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4. Most preferably, the modified live strain also has a consensus complementary DNA sequence that is at least 98% identical to a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4. The vaccine may further comprise an adjuvant. The vaccine may further comprise a pharmaceutically acceptable excipient, stabilizer, solubilizer, or diluent. The vaccine may include additional antigens from different viruses, bacterial strains, or parasites.

[0013] The present invention provides a vaccine for use in preventing porcine reproductive and respiratory syndrome (PRRS) in swine animals. Because PRRS is caused by the PRRS virus, the present invention provides a vaccine for use in preventing PRRS virus infection. The present invention also provides a vaccine for use in swine animals to reduce symptoms caused by PRRS virus infection. The infection may be caused by a wild-type virulent strain of the PRRS virus. Symptoms may include, but are not limited to, decreased appetite, lethargy, fever, premature birth, abortion, stillbirth, edema, poor growth, coughing, difficulty breathing, labored or rapid breathing, patchy redness of the skin, a coarse hair coat, lung lesions, viral shedding, and death. The present invention provides a vaccine for use in preventing PRRS in swine animals. Preferably, the vaccine comprises a modified live PRRS strain having a consensus complementary DNA sequence that is at least 90%, at least 95%, or at least 98% identical to a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4. Most preferably, the vaccine comprises a modified live PRRS strain that is strain DE 14-3073, strain ES 13-49, strain IT 14-32, or strain PL 14-02. The vaccine may further comprise a pharmaceutically acceptable excipient.

[0014] The vaccine may further comprise an adjuvant. The vaccine may comprise additional antigens from different viruses, or bacterial strains, or parasites.

[0015] The present invention provides a method for preventing symptoms of porcine reproductive and respiratory syndrome in porcine animals, comprising administering to the porcine animal an immunogenic composition comprising a modified live PRRS virus strain. The present invention also provides a method for preventing porcine reproductive and respiratory syndrome in porcine animals, comprising administering to the porcine animal an immunogenic composition comprising a modified live PRRS virus strain. Preferably, the modified live PRRS virus strain for use in the method will have a consensus complementary DNA sequence at least 90% identical to a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4. More preferably, the modified live PRRS virus strain for use in the method will have a consensus complementary DNA sequence at least 95% identical to a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4. Most preferably, the modified live PRRS virus strain for use in the method will have a consensus complementary DNA sequence at least 98% identical to a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4. The immunogenic composition may further comprise a pharmaceutically acceptable excipient. The immunogenic composition may include additional antigens from different viruses, or bacteria, or parasites.

[0016] The present invention provides a method for preventing porcine reproductive and respiratory syndrome (PRRS) in porcine animals, comprising administering to the porcine animal an immunogenic composition containing a modified live porcine reproductive and respiratory syndrome (PRRS) virus strain, wherein the PRRS virus strain for use in the method is the DE 14-3073, ES 13-49, IT 14-32, or PL 14-02 strain, which can be passaged at least 80 times, or preferably as many as 85 times, in tissue culture cells. The immunogenic composition can further comprise a pharmaceutically acceptable excipient. The immunogenic composition can also comprise additional antigens from different viruses, bacterial strains, or parasites.

[0017] The present invention provides a method for preventing symptoms caused by PRRS virus infection in porcine animals, comprising administering to the porcine animal an immunogenic composition comprising a modified live porcine reproductive and respiratory syndrome (PRRS) virus strain, wherein the consensus complementary DNA sequence of the PRRS strain is preferably at least 90% identical to a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4. More preferably, the modified live strain for use in the method may also have a consensus complementary DNA sequence that is at least 95% identical to a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4. Most preferably, the modified live strain for use in the method may also have a consensus complementary DNA sequence that is at least 98% identical to a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4. The immunogenic composition may further comprise a pharmaceutically acceptable excipient. The immunogenic composition may also comprise additional antigens from different viruses, bacterial strains, or parasites.

[0018] The present invention provides a method for reducing symptoms caused by PRRS virus infection in porcine animals, comprising administering to the porcine animal an immunogenic composition containing a modified live porcine reproductive and respiratory syndrome (PRRS) virus strain, wherein the PRRS virus strain for use in the method is the DE 14-3073, ES 13-49, IT 14-32, or PL 14-02 strain, which can be passaged at least 80 times, or preferably as many as 85 times, in tissue culture cells. The immunogenic composition can further comprise a pharmaceutically acceptable excipient. The immunogenic composition can also contain additional antigens from different viruses, bacterial strains, or parasites. The PRRS virus infection can be with a virulent PRRS virus heterologous to the modified live PRRS virus strain in the immunogenic composition. Two PRRS virus strains are considered heterologous if the genome consensus sequences of each virus strain map to different phylogenetic groups. Two PRRS virus strains are considered heterologous if the complementary DNA consensus sequences of each virus strain map to different phylogenetic groups.

[0019] The present invention provides the use of a modified live PRRS virus strain in the manufacture of a medicament for preventing or reducing symptoms of PRRS, wherein the modified live PRRS virus comprises a consensus complementary DNA sequence that is at least 90% identical to a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4. Preferably, the modified live strain may also have a consensus complementary DNA sequence that is at least 95% identical to a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4. More preferably, the modified live strain may also have a consensus complementary DNA sequence that is at least 98% identical to a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4.

[0020] The present invention provides the use of a modified live PRRS virus strain, including strain DE 14-3073, strain ES 13-49, strain IT 14-32, or strain PL 14-02, in the manufacture of a medicament for preventing or reducing PRRS symptoms. The modified live PRRS virus strain should be passaged at least 80 times, or preferably as many as 85 times, in tissue culture cells. Such passage in tissue culture cells is useful for properly attenuating the modified live PRRS virus strain. Attenuated PRRS virus strains can cause subclinical, but not clinical, disease when administered to swine animals. Modified live PRRS virus strains that have been passaged at least 80 times are less likely to revert to wild-type virulence.

[0021] The present invention provides the use of an immunogenic composition comprising a modified live PRRS virus strain in the manufacture of a medicament for preventing PRRS virus infection, wherein the modified live PRRS virus strain comprises a consensus complementary DNA sequence that is at least 90% identical to a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4. The modified live strain for such use may also have a consensus complementary DNA sequence that is at least 95% identical to a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4. The modified live strain for such use may also have a consensus complementary DNA sequence that is at least 98% identical to a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4.

[0022] The present invention provides the use of an immunogenic composition containing a modified live PRRS virus strain, including strain DE 14-3073, strain ES 13-49, strain IT 14-32, or strain PL 14-02, in the manufacture of a medicament for preventing PRRS virus infection. The PRRS virus strain should be passaged at least 80 times, or preferably as many as 85 times, in tissue culture cells. Such passage in tissue culture cells is useful for properly attenuating the modified live PRRS virus strain. Attenuated PRRS virus strains can cause subclinical but not clinical disease when administered to swine animals. Modified live PRRS virus strains that have been passaged at least 80 times are less likely to revert to wild-type virulence. Modified live PRRS virus strains that have been passaged 85 times are less likely to revert to wild-type virulence.

[0023] The present invention provides the use of an immunogenic composition comprising a modified live PRRS virus strain in the manufacture of a medicament for protecting porcine animals from PRRS virus infection, wherein the modified live PRRS virus strain comprises a consensus complementary DNA sequence that is at least 90% identical to a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4. The modified live strain for such use may also have a consensus complementary DNA sequence that is at least 95% identical to a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4. The modified live strain for such use may also have a consensus complementary DNA sequence that is at least 98% identical to a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4.

[0024] The present invention provides the use of an immunogenic composition containing a modified live PRRS virus strain, including strain DE 14-3073, strain ES 13-49, strain IT 14-32, or strain PL 14-02, in the manufacture of a medicament for protecting swine animals from PRRS virus infection. The PRRS virus strain should be passaged at least 80 times, or preferably as many as 85 times, in tissue culture cells. Such passage in tissue culture cells is useful for properly attenuating the modified live PRRS virus strain. Attenuated PRRS virus strains can cause subclinical but not clinical disease when administered to swine animals. Modified live PRRS virus strains that have been passaged at least 80 times are less likely to revert to wild-type virulence. Modified live PRRS virus strains that have been passaged 85 times are less likely to revert to wild-type virulence. [Brief explanation of the drawings]

[0025] [Figure 1] Phylogenetic analysis of PRRSV-1 strains DE 14-3073, ES 13-49, IT 14-32, or PL 14-02, as well as commercial vaccines and isolates selected based on ORF5 nucleotide sequences constructed by the neighbor-joining method. DETAILED DESCRIPTION OF THE INVENTION

[0026] As used in the following discussion, the terms "a" or "an" should be understood to include one or more, unless otherwise specified.

[0027] As used herein, the term "virus" can refer to either a species of virus or, interchangeably, an individual infectious unit that can contain nucleic acid and proteins. Individual infectious units are also called "virus particles" or "virions," the latter terms being synonyms.

[0028] As used herein, a viral "strain" or "isolate" refers to a collection of genetically homologous virions. If two viruses map to the same phylogenetic group, they would be considered "homologous." If two viruses map to different phylogenetic groups, they would be considered "heterologous." Because PRRS viruses have a high mutation rate, it is understood that a single PRRS strain contains individual virions with related but variable gene sequences. Thus, subpopulations of strains exist within each PRRS strain, and the gene sequence of a PRRS strain is a consensus sequence such that the gene sequence of an individual member of the PRRS strain cannot be identical to the consensus sequence for that strain. A "consensus" sequence is a nucleic acid sequence in which each nucleic acid residue at a given position is present in >51% of polynucleotides within a PRRS virus strain or isolate.

[0029] "Percent identity" can be determined by calculating the number of identical nucleotides or amino acids at the same position in a nucleic acid or protein. Calculating percent identity involves determining the optimal alignment between two or more sequences. Alignment can take into account insertions and deletions (i.e., "gaps") in each of the tested sequences, including, but not limited to, non-coding regions of the nucleic acid and truncations or extensions of the polypeptide sequence. Percent identity can be determined using computer programs and algorithms such as the Basic Local Alignment Search Tool (BLAST). BLAST is one of many resources provided by the US National Center for Biotechnology Information. Because the genetic code is degenerate and more than one codon can encode a given amino acid, the coding regions of nucleic acids are considered identical if they encode identical polypeptides. Therefore, percent identity can also be calculated based on the polypeptides encoded by the nucleic acids. Percent identity can be calculated based on the full-length consensus genome sequence or a portion of the genome sequence, for example, but not limited to, individual open reading frames (ORFs).

[0030] As used herein, the term "modified live virus" applies to any individual virus particle (i.e., "virion") or a number of virus particles whose genetic sequence has been altered from that of a native wild-type virus that still induces protective immunity against the wild-type virus. Alterations include, but are not limited to, genetic mutations such as nucleotide insertions and deletions, and transitions and transversions that change one nucleotide to another. Alterations can be achieved by adapting a wild-type virus to replicate in a tissue culture system and continuing to passage the virus in the tissue culture system, which allows the virus to accumulate genetic mutations. Alterations can also be achieved using molecular techniques. Attenuated viruses form a subset of modified live viruses.

[0031] As used herein, the term "attenuated" or "attenuation" means that the ability of a virus to cause or exacerbate clinical disease is reduced or eliminated. An attenuated virus can still infect host cells either in vitro or in vivo, and the infection may result in subclinical effects in the host organism, but the infection does not result in one or more clinical disease symptoms.

[0032] In contrast, as used herein, an "inactivated" virus refers to a virus that is no longer capable of replicating within a host. An inactivated virus is considered to be a killed or dead virus. Inactivation can be achieved by a variety of methods, including, but not limited to, chemical modification of viral proteins, chemical or physical modification of the structure of the virion, or chemical or physical modification of viral nucleic acid.

[0033] An "antigen" is any molecule that can be specifically detected by an organism's immune system. Typically, viral antigens are viral proteins encoded by or derived from the viral genome. The presence of viral antigens can be specifically detected by surface antigen receptors on both host T and B lymphocytes, as well as antibody molecules synthesized by host cells.

[0034] "Immunogenicity" refers to the ability of an antigen to elicit an immune response, including both antigen-specific and non-antigen-specific responses, or innate immune responses. "Protective immunity" is an immune response that can reduce or prevent clinical symptoms when an immunized animal is challenged or exposed to a pathogenic virus strain. As those skilled in the art will understand, protective immunity may decline over time or with the increasing age of the immunized animal. As used herein, protective immunity should be effective for at least four months, but preferably at least six months, from the last day of immunization. Protective immunity can be induced with a single dose of vaccine. A second or further dose can be used to increase or prolong the protective immune response. For example, increasing the protective immune response of breeding sows can result in increased levels of maternal antibodies in piglets.

[0035] In contrast to antigens, "adjuvants" are nonspecific stimulators of the immune response. Adjuvants can stimulate the innate immune response by binding and activating pattern recognition receptors (PRRs). Such PRR stimulators can be, for example, viral or bacterial nucleic acids, lipids from bacteria or parasites, bacterial proteins or toxins, or any artificially constructed mimics of such molecules. Adjuvants also include, but are not limited to, inorganic compounds that aggregate antigens to facilitate recognition by B lymphocytes or uptake by phagocytes, such as alum, aluminum hydroxide, aluminum phosphate, calcium hydroxide phosphate, or ammonium sulfate; oils; and detergents. Adjuvants can also be host mediators of immune signaling, such as, but not limited to, cytokines, lymphokines, chemokines, interferons, anaphylatoxins, growth factors, differentiation factors, and adhesion molecules.

[0036] As used herein, an "immunogenic composition" is a composition that induces an immune response when administered to an animal. The immunogenic composition comprises at least one antigen and at least one pharmaceutically acceptable excipient. The antigen can be a live or inactivated whole virus, bacterium, or other pathogen. The antigen can also be an antigen molecule isolated, purified, or partially purified from a virus, bacterium, or other pathogen. The antigen can be a polypeptide, polysaccharide, nucleic acid, or lipid.

[0037] As used herein, a "vaccine" is an immunogenic composition that, when administered to an animal, confers protection against, resistance to, prevention against, or reduction of disease symptoms caused by a pathogenic organism, such as a virus. A PRRS vaccine may include, but is not limited to, live or inactivated viral antigens or intact virions in a composition together with at least one pharmaceutically acceptable excipient.

[0038] As used herein, the terms "treating," "to treat," or "treatment" include, but are not limited to, inhibiting, slowing, arresting, reducing, alleviating, or reversing the progression or severity of an existing symptom, disorder, condition, or disease. Treatment may be applied or administered therapeutically.

[0039] As used herein, the terms "preventing," "to prevent," or "prevention" include, but are not limited to, lowering, reducing, or alleviating the risk of a symptom, disorder, condition, or disease, and protecting an animal from a symptom, disorder, condition, or disease. Prevention may be applied or administered prophylactically.

[0040] As used herein, "administering to an animal" includes, but is not limited to, cutaneous, subcutaneous, intramuscular, mucosal, submucosal, transdermal, oral, or intranasal administration. Administration may include injection or topical administration.

[0041] The term "pharmaceutically acceptable excipient" refers to those typically used in the preparation of veterinary and pharmaceutical compositions, and should be pure and non-toxic in the amounts used. In certain embodiments, pharmaceutical compositions may contain excipients to modify, maintain, or preserve, for example, the pH, osmolality, viscosity, clarity, isotonicity, sterility, stability, adsorption, or permeability of the composition. Some examples of acceptable excipients can be found, for example, in Remington's Pharmaceutical Sciences and the Handbook of Pharmaceutical Excipients, 18 th Edition, (AR Genrmo, ed.), 1990, Mack Publishing Company and later editions and Remington: The Science and Practice of Pharmacy, Lloyd V. Allen, ed., Pharmaceutical Press, London, UK, 22 nd The following list may be found in the 2012 edition, which includes diluents, vehicles, carriers, stabilizers, preservatives, solvents, suspending agents, emulsifiers, antibacterial agents, antioxidants, buffers, chelating agents, complexing agents, carbohydrates, proteins, diluents, and / or pharmaceutical adjuvants. In certain embodiments, the primary vehicle or carrier in a pharmaceutical composition may be either aqueous or non-aqueous in nature. For example, a suitable vehicle or carrier may be water for injection, a saline solution with other ingredients commonly used in compositions for parenteral administration.

[0042] As used herein, the term "porcine animal" refers to any pig, i.e., animal of the genus Sus within the family Suidae, an artiodactyla.

[0043] The following examples are illustrative of modified live PRRS viruses. The following examples are also illustrative of immunogenic compositions comprising modified live PRRS viruses. The following examples are also illustrative of using modified live PRRS viruses to prevent or reduce symptoms of PRRS in swine animals. Other embodiments and uses will be apparent to those skilled in the art, and it will be understood that the present invention is not limited to these specific illustrative examples or preferred embodiments.

[0044] Example 1 The objective of this study was to identify potential strains for vaccine development. A total of 36 PRRS type 1 field isolates were evaluated for their vaccine potential. Following are pre-master seed virus (pre-MSV) preparations for four type 1 (European) PRRS virus (PRRSV-1) strains: DE 14-3073, ES 13-49, IT 14-32, and PL 14-02.

[0045] Initial isolation of PRRSV-1 strains was performed using serum and lung tissue from pig herds diagnosed with PRRSV-positive test results in Europe between 2013 and 2014. The herds had not been vaccinated against PRRSV but experienced clinical signs typical of PRRSV infection, including reproductive failure in gilts and sows (i.e., late abortions, premature births, birth of vulnerable piglets and stillbirths, and / or increased preweaning mortality) and / or growth retardation and respiratory disease problems in gilts.

[0046] Virus isolation was performed on primary cultures of porcine alveolar macrophages (PAM). Three-week-old piglets were used as donors to obtain PAM cultures. Briefly, piglets were humanely euthanized, and then lungs were obtained under sterile conditions after flushing with phosphate-buffered saline to recover PAM cells. The resulting cell suspension was centrifuged at 800 × g for 15 minutes at 4°C, and the supernatant was discarded. After the above centrifugation conditions, the pelleted cells were resuspended in Dulbecco's modified Eagle's medium (DMEM) and washed twice using DMEM as a diluent.

[0047] Culture 3 × 10 cells in DMEM supplemented with 10% fetal bovine serum (FBS) and antibiotic-antimycotic solution (100 units / mL penicillin, 100 μg / mL streptomycin, and 0.25 μg / mL amphotericin B). 6 Cells were counted and seeded onto different supports (i.e., cell culture flasks or plates of different sizes) at a final concentration of 100 cells / mL. Cells were cultured at 37°C in an atmosphere with 5% CO2.

[0048] Clinical samples were processed according to different procedures depending on their nature. Serum samples were filtered through a 0.22 μm sterile syringe filter and kept at -80°C until used for virus isolation. Meanwhile, tissue samples, including tonsils and lungs, were homogenized at a 1:10 ratio using DMEM as the diluent. The homogenates were clarified by centrifugation at 2500 × g for 15 minutes, and the supernatants were filtered through a 0.22 μm sterile syringe filter and kept at -80°C until used for virus isolation.

[0049] To infect PAM cultures, the medium was removed and clinical samples (i.e., processed serum samples or processed tissue samples) were added in variable amounts depending on the support used. After 1.5 hours of adsorption at 37°C, the cultures were washed and fresh DMEM supplemented for cell maintenance was added. Cultures were observed daily for cytopathic effect (CPE). Once CPE was observed, the cultures were harvested. After three cycles of freezing and thawing, cellular debris was removed by centrifugation at 2500 × g for 15 minutes at 4°C, and the supernatant was frozen and stored at -80°C. The presence of porcine reproductive and respiratory syndrome virus (PRRSV) in the cultures was confirmed by reverse transcription and polymerase chain reaction (RT-PCR).

[0050] If no CPE was observed, the culture was considered negative, and the original clinical sample (i.e., serum or tissue sample) was used in a bioassay to inoculate 3-week-old piglets housed in isolation. For this purpose, the clinical sample was filtered and injected intramuscularly into the pigs. One week later, blood samples were taken from the exposed pigs to confirm viremia by RT-PCR. Pigs confirmed to be virologically infected were euthanized, and blood, tonsil, and lung samples were collected at necropsy and used as inoculum for a second attempt at virus isolation in PAM cultures according to previously described methods.

[0051] Growth of PRRSV isolates in the MARC-145 cell line was attempted only for isolates that grew well in PAM and were capable of producing seed stocks. For this purpose, MARC-145 cells were cultured at 5 × 10 5 25 cm at a concentration of cells / flask 2 Cell culture flasks were seeded and maintained at 37°C in an atmosphere with 5% CO2 in DMEM supplemented with 10% FBS and an antibiotic-antimycotic solution (100 units / mL penicillin, 100 μg / mL streptomycin, and 0.25 μg / mL amphotericin B). Pre-injection cultures were infected with the supernatant of positive PAM cultures according to the same protocol described for infection of PAM cultures. After adsorption, cells were washed and cultured in DMEM supplemented with 5% FBS and the aforementioned antibiotic-antimycotic solution.

[0052] After the initial isolation, the virus was amplified to produce a primary virus stock (with a volume of at least 100 mL) for each isolate in PAM culture. To this end, PAM cells were cultured at 3 × 10 in DMEM supplemented with 10% FBS and the antibiotic-antimycotic solution described above. 6 cells / mL concentration, 75 cm 2The cells were cultured in a bottle in an atmosphere containing 5% CO2 at 37°C for 24 hours. The medium was then discarded, the cells were washed with fresh DMEM, and the virus inoculum was added. After 1.5 hours of adsorption at 37°C, the cultures were washed and fresh DMEM supplemented for cell maintenance was added. The cultures were observed daily for CPE. When most of the cells in the culture were killed, the cultures were harvested. After three cycles of freezing and thawing, cell debris was removed by centrifugation at 2500 × g for 15 minutes at 4°C, and the virus-containing supernatant was frozen and stored at -80°C. Virus titers were calculated according to the Reed-Münch method (1938) and expressed as log TCID 50 The virus stocks were kept at -80°C and used in different studies.

[0053] The same procedure was used for MARC-145 cell cultures. MARC-145 cell cultures were inoculated for initial isolation as described above and maintained at 37°C in a 5% CO2 atmosphere. After 5 days of incubation, if CPE was evident (i.e., approximately 70-80% of the monolayer was affected), or if no CPE was observed, the cultures were subjected to three cycles of freezing and thawing, clarified by centrifugation at 2500 × g for 15 min, and the supernatant was used to inoculate fresh MARC-145 cell cultures. The remaining supernatant from each passage was stored at -80°C. A total of 60 passages in the MARC-145 cell line were performed for each PRRSV isolate.

[0054] At passages 30 and 50, virus stocks were cloned by plaque purification according to standard methodologies. 1 ~10 6Six-well plates previously seeded with MARC-145 were inoculated with serial dilutions of each virus stock. After 1.5 hours of adsorption, the inoculum was removed from each well, and the cells were overlaid with fresh DMEM medium supplemented with 5% FBS, the antibiotic-antimycotic solution described above, and 1% low-melting agarose. After 2–4 days of culture, individual plaques were selected and picked using a phase-contrast microscope, depending on the isolate. Based on complete isolation within the monolayer, at least five plaques were selected for each virus in each purification round to ensure the clonality of the selected viruses. Selected plaques were used as inoculum for the next purification round. This procedure was repeated three times to confirm that the resulting viral progeny were derived from a single virus.

[0055] Example 2 The goal of this study was to further characterize the PRRS-1 isolate. The portion of ORF1 encoding nsp2 and ORFs 2–7 were amplified by RT-PCR using a previously designed set of primers. To this end, total RNA was obtained from all viral stocks using the QIAMP® Viral RNA Mini Kit (Qiagen, USA) according to the manufacturer's instructions. 15 μL of total RNA was used as a template for reverse transcription and polymerase chain reaction (RT-PCR). Reactions were performed using a commercially available one-step RT-PCR kit (SuperScript III OneStep RT-PCR PLATINUM TAQHIFI®, Invitrogen, USA) according to the manufacturer's instructions. RT-PCR products were purified using a commercially available kit (QIAQUICK® Purification Gel Kit, Qiagen, USA) according to the manufacturer's instructions. The individual sequences of both strands of DNA for each PCR product were determined using the same primer pairs used for RT-PCR. Samples were amplified by asymmetric PCR with fluorescent terminators, and the products were analyzed by electrophoresis on an ABI prism 310 Genetic Analyzer (Applied Biosystems, USA). At least two different RT-PCR products were sequenced to verify that no errors had occurred during DNA amplification and that the resulting sequences were correct. Sequences were manually corrected to remove errors and aligned using Clustal Omega software. The resulting sequences were compared to the genotype 1 PRRSV prototype Lelystad virus and the vaccine strains that form the basis of the vaccines PORCILIS® PRRS (MSD Animal Health) (DV strain), UNISTRAIN® PRRS (Laboratorios Hipra) (VP-046 BIS strain), and INGELVAC PRRSFLEX® EU (Boehringer Ingelheim) (strain 94881).In addition, they are compared to the genotype 2 prototype strain VR-2332, the strain in the INGELVAC PRRS® MLV vaccine (Boehringer Ingelheim), which is also commercially available in Europe.

[0056] Nucleotide similarity between the resulting PRRSV isolates was calculated, as well as between each field isolate and the vaccine strain available at the time of isolation. The purpose was to confirm that the isolates were unrelated to each other and were not derivatives of the commercially available vaccine strain at the time of clinical specimen collection. Furthermore, a phylogenetic tree was constructed using the neighbor-joining method, including VR-2332, the prototype American genotype, as an outgroup to determine the subtype to which the Utopia isolate belonged. To assess the statistical reliability of the dendrogram, a bootstrapping value was calculated (random number seed: 123; 1,000 replicates). All phylogenetic analyses were performed using MEGA 5.0 software.

[0057] PRRSV-1 was further attenuated by passage 10 times (to P70) on MARC-145 cells in growth medium OPTI-MEM® I (catalog no. 31985, Life Technologies) supplemented with 2% fetal bovine serum (FBS; Sigma catalog nos. 12003C and 12007C, and Gibco catalog no. 04-4000DJ) and 50 μg gentamicin / mL (catalog no. 15750, Life Technologies), followed by an additional 15 times (to P85) in the same growth medium supplemented with 2% FBS without gentamicin. The identity of the 85th passage (P85) PRRSV-1 was confirmed by indirect immunofluorescence assay (IFA) using a PRRSV-specific monoclonal antibody, and P85 PRRSV-1 was considered the pre-master seed virus (pre-MSV).

[0058] To determine the titer of PRRSV-1, the following procedure was used: MARC-145 cells were cultured at 0.75-1.5 x 10 in 100 μL of growth medium (OPTI-MEM® I medium supplemented with 5% FBS and 50 μg / mL gentamicin). 4 Cells were seeded into 96-well plates at a density of 1000 x g. Cells were incubated for 48-72 hours in a 37 ± 2°C and 5 ± 1% CO2 incubator until cells were >95% saturated. On the day of titration, all medium was removed from the 96-well plates and replaced with 100 µL of fresh growth medium.

[0059] Ten-fold serial dilutions of PRRSV-1 were prepared in diluent (OPTI-MEM® I medium, 50 μg / mL gentamicin) and transferred to corresponding wells on the plates prepared above, along with a negative control consisting of diluent alone and a positive control with a known titer. The titration plates were incubated at 37 ± 2°C in a 5 ± 1% CO2 incubator for 4 days. At the end of the incubation period, each plate was observed using an inverted microscope for the presence of virus-induced cytopathic effect (CPE) in each sample well. The 50% tissue culture infectious dose (TCID 50 ) and calculate the titer using the Reed-Muench method. 10 TCID 50 The titer range of P85 PRRSV-1 was 8.1 to 8.5 log 10 TCID 50 / mL.

[0060] Example 3 The goal of this study was to sequence pre-MSV. To determine the genome sequence of each viral isolate, viruses grown in MARC-145 cells were concentrated and purified by ultracentrifugation on a sucrose cushion, and RNA was extracted using the MINELUTE Virus Spin Kit (Qiagen) and TRIZOL LS (Invitrogen). The complete genome sequence was determined by multiple runs of next-generation sequencing (NGS) using the ILLUMINA® MISEQ® platform and / or NEXTSEQ500 system at Bioreliance (Rockville, MD) and ACGT (Wheeling, IL). Variable and uncertain sequences and gaps were fixed and confirmed by Sanger dideoxy sequencing to generate a consensus full genome sequence. Nucleotide sequences were aligned and compared to selected known PRRSV-1 and commercial vaccine viruses using the neighbor-joining tree nucleotide alignment tool in Geneious 10.1.3 software (Figure 1).

[0061] The PRRSV-1 MLV strain designated "pre-MSV" is being deposited under conditions that will ensure access to the culture during the pendency of this patent application as determined by the Commissioner of Patents and Trademarks under 37 C.F.R. § 1.14 and 35 U.S.C. § 122. The deposit will be made available as required by foreign patent laws in countries in which counterparts of the subject application, or progeny thereof, are being filed. However, it is understood that the availability of the deposit does not constitute a license to practice the subject invention in infringement of patent rights granted by governmental action. The subject culture deposit will be kept in accordance with the provisions of the Budapest Treaty for the Deposit of Microorganisms and will be made available to the public, i.e., it will be preserved with all care necessary to keep it viable and uncontaminated for at least five years from the most recent request to furnish a sample of the deposit, and, in any event, for at least 30 years from the date of deposit, or for the term of any patent that may be issued disclosing the deposited culture. The depositor acknowledges its obligation to replace the deposit if, due to the condition of the deposit, the depository is unable to provide samples upon request. All restrictions on the public availability of the subject culture deposit will be irrevocably lifted upon the issuance of a patent disclosing it. The PRRSV-1 pre-MSV deposit was placed in the permanent collection of the American Type Culture Laboratory Patent Depository, 10801 University Blvd., Manassas, Va., 20110-2209, USA, under the terms of the Budapest Treaty, on March 7, 2019, and the strains were subsequently assigned repository accession numbers PTA-125490 (DE 14-3073), PTA-125489 (ES 13-49), PTA-125488 (IT 14-32), and PTA-125487 (PL 14-02).

[0062] The safety and efficacy of modified live porcine reproductive and respiratory syndrome virus immunogenic compositions and vaccines can be determined by methods well known in the art, including dose response, onset of immunity, duration of immunity, and shedding and transmission of porcine reproductive and respiratory syndrome virus. The lack of reversion to virulence of any pre-MSV can also be readily determined.

[0063] The cDNA consensus sequences of four PRRS virus isolates at passage 85 (P85) were deposited in the GenBank gene sequence database, an annotated collection of all publicly available nucleic acid sequences. The GenBank database is maintained by the National Center for Biotechnology Information (NCBI), part of the United States National Institutes of Health (NIH). GenBank is part of the International Nucleotide Sequence Database Collaboration.

[0064] The cDNA consensus sequence of PRRS strain DE14-3073 in P85 has been assigned GenBank accession number MK024324 (SEQ ID NO: 1). The cDNA consensus sequence designated SEQ ID NO: 1 is as follows:

[0065] TIFF0007747527000001.tif19142Sequence List 1-2

[0066] TIFF0007747527000002.tif222143Sequence List 1-3

[0067] TIFF0007747527000003.tif222143Sequence List 1-4

[0068] TIFF0007747527000004.tif222143Sequence Tables 1-5

[0069] TIFF0007747527000005.tif192143

[0070] The cDNA consensus sequence of PRRS strain ES13-49 at P85 has been assigned GenBank accession number MK024325 (SEQ ID NO: 2). The cDNA consensus sequence designated SEQ ID NO: 2 is as follows:

[0071] TIFF0007747527000006.tif10146Sequence List 2-2

[0072] TIFF0007747527000007.tif223144Sequence List 2-3

[0073] TIFF0007747527000008.tif223144Sequence Table 2-4

[0074] TIFF0007747527000009.tif222144Sequence Table 2-5

[0075] TIFF0007747527000010.tif199144

[0076] The cDNA consensus sequence of PRRS strain IT 14-32 in P85 has been assigned GenBank accession number MK024326 (SEQ ID NO: 3). The cDNA consensus sequence designated SEQ ID NO: 3 is as follows:

[0077] TIFF0007747527000011.tif223144Sequence List 3-2

[0078] TIFF0007747527000012.tif223144Sequence List 3-3

[0079] TIFF0007747527000013.tif223144Sequence Table 3-4

[0080] TIFF0007747527000014.tif195144

[0081] The cDNA consensus sequence of PRRS strain PL 14-02 at P85 has been assigned GenBank accession number MK024327 (SEQ ID NO: 4). The cDNA consensus sequence designated SEQ ID NO: 4 is as follows:

[0082] TIFF0007747527000015.tif223145Sequence List 4-2

[0083] TIFF0007747527000016.tif222145Sequence Table 4-3

[0084] TIFF0007747527000017.tif223145Sequence Table 4-4

[0085] TIFF0007747527000018.tif206144

[0086] Those skilled in the art will recognize that the polyadenosine tail of each of the genomic consensus sequences, if present, may vary in length from the sequence reported above.

Claims

1. 1. A modified live porcine reproductive and respiratory syndrome (PRRS) virus strain, wherein the consensus complementary DNA sequence of said PRRS strain is at least 95% identical to a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4, wherein: SEQ ID NO: 1 is the consensus complementary DNA sequence of strain DE 14-3073, designated PTA-125490; SEQ ID NO:2 is the consensus complementary DNA sequence of strain ES 13-49, designated PTA-125489; SEQ ID NO:3 is the consensus complementary DNA sequence of strain IT 14-32, designated PTA-125488, and SEQ ID NO:4 is the consensus complementary DNA sequence of strain PL 14-02, designated PTA-125487, wherein: The modified live PRRS virus strain maintains the same attenuated phenotype and titer as the original strain, the pre-master seed virus, and wherein: The premaster seed virus is strain DE 14-3073, strain ES 13-49, strain IT 14-32 or strain PL 14-02; The modified live PRRS virus strain.

2. 2. The modified live PRRS virus strain of claim 1, wherein the consensus complementary DNA sequence of said PRRS strain is at least 98% identical to a sequence selected from the group consisting of: SEQ ID NO: 1: Consensus complementary DNA sequence of the DE 14-3073 strain designated PTA-125490; SEQ ID NO: 2: Consensus complementary DNA sequence of strain ES 13-49, designated PTA-125489; SEQ ID NO: 3: Consensus complementary DNA sequence of strain IT 14-32, designated PTA-125488; and SEQ ID NO: 4: Consensus complementary DNA sequence of strain PL 14-02, designated PTA-125487.

3. 3. The PRRS virus strain of claim 1 or 2, wherein the PRRS virus strain is passaged at least 85 times in tissue culture cells.

4. 4. Use of the modified live PRRS virus strain of any one of claims 1 to 3 in the manufacture of a medicament for treating porcine reproductive and respiratory syndrome (PRRS).

5. 1. An immunogenic composition comprising a modified live PRRS virus strain having a consensus complementary DNA sequence at least 90% identical to a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4, and a pharmaceutically acceptable excipient, stabilizer, solubilizer, or diluent, wherein SEQ ID NO: 1 is the consensus complementary DNA sequence of strain DE 14-3073, designated PTA-125490; SEQ ID NO:2 is the consensus complementary DNA sequence of strain ES 13-49, designated PTA-125489; SEQ ID NO:3 is the consensus complementary DNA sequence of strain IT 14-32, designated PTA-125488, and SEQ ID NO:4 is the consensus complementary DNA sequence of strain PL 14-02, designated PTA-125487, wherein: The modified live PRRS virus strain maintains the same attenuated phenotype and titer as the original strain, the pre-master seed virus, and wherein: The premaster seed virus is strain DE 14-3073, strain ES 13-49, strain IT 14-32 or strain PL 14-02; The immunogenic composition.

6. 1. An immunogenic composition comprising a modified live PRRS virus strain having a consensus complementary DNA sequence at least 95% identical to a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4, and a pharmaceutically acceptable excipient, stabilizer, solubilizer, or diluent, wherein: SEQ ID NO: 1 is the consensus complementary DNA sequence of strain DE 14-3073, designated PTA-125490; SEQ ID NO:2 is the consensus complementary DNA sequence of strain ES 13-49, designated PTA-125489; SEQ ID NO:3 is the consensus complementary DNA sequence of strain IT 14-32, designated PTA-125488, and SEQ ID NO:4 is the consensus complementary DNA sequence of strain PL 14-02, designated PTA-125487, wherein: The modified live PRRS virus strain maintains the same attenuated phenotype and titer as the original strain, the pre-master seed virus, and wherein: The premaster seed virus is strain DE 14-3073, strain ES 13-49, strain IT 14-32 or strain PL 14-02; The immunogenic composition.

7. 1. An immunogenic composition comprising a modified live PRRS virus strain having a consensus complementary DNA sequence at least 98% identical to a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4, and a pharmaceutically acceptable excipient, stabilizer, solubilizer, or diluent, wherein: SEQ ID NO: 1 is the consensus complementary DNA sequence of strain DE 14-3073, designated PTA-125490; SEQ ID NO:2 is the consensus complementary DNA sequence of strain ES 13-49, designated PTA-125489; SEQ ID NO:3 is the consensus complementary DNA sequence of strain IT 14-32, designated PTA-125488, and SEQ ID NO:4 is the consensus complementary DNA sequence of strain PL 14-02, designated PTA-125487, wherein: The modified live PRRS virus strain maintains the same attenuated phenotype and titer as the original strain, the pre-master seed virus, and wherein: The premaster seed virus is strain DE 14-3073, strain ES 13-49, strain IT 14-32 or strain PL 14-02; The immunogenic composition.

8. 8. The immunogenic composition of any one of claims 5 to 7, wherein the modified live PRRS virus strain is DE 14-3073 strain, designated PTA-125490, ES 13-49 strain, designated PTA-125489, IT 14-32 strain, designated PTA-125488, or PL 14-02 strain, designated PTA-125487.

9. The immunogenic composition of any one of claims 5 to 8, further comprising an adjuvant.

10. 1. A vaccine comprising a modified live PRRS virus strain, wherein the PRRS virus strain is DE 14-3073 strain designated PTA-125490, ES 13-49 strain designated PTA-125489, IT 14-32 strain designated PTA-125488, or PL 14-02 strain designated PTA-125487.

11. A vaccine comprising the modified live PRRS virus strain of any one of claims 1 to 3 for use in treating or preventing symptoms of porcine reproductive and respiratory syndrome in porcine animals.

12. 1. A method of treating or preventing symptoms of porcine reproductive and respiratory syndrome (PRRS) in a porcine animal, comprising administering to said porcine animal an immunogenic composition comprising a modified live PRRS virus strain having a consensus complementary DNA sequence that is at least 98% identical to a sequence selected from the group consisting of: SEQ ID NO: 1: Consensus complementary DNA sequence of the DE 14-3073 strain designated PTA-125490; SEQ ID NO: 2: Consensus complementary DNA sequence of strain ES 13-49, designated PTA-125489; SEQ ID NO: 3: Consensus complementary DNA sequence of strain IT 14-32, designated PTA-125488; and SEQ ID NO: 4: Consensus complementary DNA sequence of strain PL 14-02, designated PTA-125487; wherein the modified live PRRS virus strain maintains an attenuated phenotype and titer equivalent to the original strain, the pre-master seed virus, and wherein: The premaster seed virus is strain DE 14-3073, strain ES 13-49, strain IT 14-32 or strain PL 14-02; The method.

13. 13. The method of claim 12, wherein the PRRS symptoms are caused by PRRS virus type 1 infection in porcine animals.

14. 1. A method of treating or preventing symptoms of porcine reproductive and respiratory syndrome (PRRS) in a porcine animal, comprising administering to the porcine animal a vaccine comprising a modified live PRRS virus strain, wherein the PRRS virus strain is DE 14-3073 strain designated PTA-125490, ES 13-49 strain designated PTA-125489, IT 14-32 strain designated PTA-125488, or PL 14-02 strain designated PTA-125487. method.

15. 15. The method of claim 14, wherein the PRRS symptoms are caused by PRRS virus type 1 infection in porcine animals.

16. 1. Use of an immunogenic composition comprising a modified live PRRS virus strain having a consensus complementary DNA sequence that is at least 98% identical to a sequence selected from the group consisting of: SEQ ID NO: 1: Consensus complementary DNA sequence of the DE 14-3073 strain designated PTA-125490; SEQ ID NO: 2: Consensus complementary DNA sequence of strain ES 13-49, designated PTA-125489; SEQ ID NO: 3: Consensus complementary DNA sequence of strain IT 14-32, designated PTA-125488; and SEQ ID NO: 4: Consensus complementary DNA sequence of strain PL 14-02, designated PTA-125487; wherein the modified live PRRS virus strain maintains an attenuated phenotype and titer equivalent to the original strain, the pre-master seed virus, and wherein: The premaster seed virus is strain DE 14-3073, strain ES 13-49, strain IT 14-32 or strain PL 14-02; The above use.

17. 17. The use according to claim 16, wherein the PRRS symptoms are caused by PRRS virus type 1 infection in porcine animals.

18. 1. Use of a vaccine comprising a modified live porcine reproductive and respiratory syndrome (PRRS) virus strain, wherein the PRRS virus strain is DE 14-3073 strain designated PTA-125490, ES 13-49 strain designated PTA-125489, IT 14-32 strain designated PTA-125488, or PL 14-02 strain designated PTA-125487, for treating or preventing symptoms of PRRS in porcine animals.

19. 19. The use of claim 18, wherein the PRRS symptoms are caused by PRRS virus type 1 infection in porcine animals.

20. 2. The modified live PRRS virus strain of claim 1, wherein the consensus complementary DNA sequence of the PRRS strain comprises SEQ ID NO:

1.

21. 2. The modified live PRRS virus strain of claim 1, wherein the consensus complementary DNA sequence of the PRRS strain comprises SEQ ID NO:

2.

22. 2. The modified live PRRS virus strain of claim 1, wherein the consensus complementary DNA sequence of the PRRS strain comprises SEQ ID NO:

3.

23. 2. The modified live PRRS virus strain of claim 1, wherein the consensus complementary DNA sequence of the PRRS strain comprises SEQ ID NO:

4.

24. 12. The vaccine of claim 11, wherein the consensus complementary DNA sequence of the PRRS strain comprises SEQ ID NO:

1.

25. 12. The vaccine of claim 11, wherein the consensus complementary DNA sequence of the PRRS strain comprises SEQ ID NO:

2.

26. 12. The vaccine of claim 11, wherein the consensus complementary DNA sequence of the PRRS strain comprises SEQ ID NO:

3.

27. 12. The vaccine of claim 11, wherein the consensus complementary DNA sequence of the PRRS strain comprises SEQ ID NO:4.

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