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    • 专利摘要:
    A novel attenuated strain of the virus (CNCM Institut Pasteur I-1642), which causes swine disease known as swine reproduction and respiratory distress syndrome (PRRS), is disclosed, together with a new clone (CNCM Institut) obtained from monkey kidney cell MA-104. Attenuating and replicating the strains using Pasteur I-1643) is disclosed. By being harmless to pigs and having high immunogenic activity, the new attenuated strains make it possible to obtain vaccines and diagnostic kits that enable the early diagnosis of PRRS and the effective prevention of such diseases.
    公开号:KR19980703268A
    申请号:KR1019970706672
    申请日:1996-12-04
    公开日:1998-10-15
    发明作者:부르크 레이나 알레마니；마소 엔리끄 에스뿌냐；뿌하다스 뻬레 리에라；로까 나르시스 사우비
    申请人:후안 노가레다；라보라또리오스 이쁘라 에쎄. 아.；
    IPC主号:
    专利说明:

    New attenuated strains of viruses that cause reproductive and respiratory distress syndrome (PRRS) in pigs, vaccines and diagnostic means that can be obtained, and manufacturing processes
    In 1987, the first mysterious swine disease, or MSD, was defined in North America at that time, the disease was later known as swine infertility and respiratory distress syndrome or SIRS. A very similar syndrome was first discovered in central Europe in 1990 and then spread to other European countries, including Spain. In Europe, the disease was initially named Pandemic Abortion and Respiratory Disorder Syndrome (PEARS), and eventually Pig Reproductive and Respiratory Disorder Syndrome (PRRS). This name has been accepted all over the world in regard to the above statement.
    It is already known that the etiological pathogen of the PRRS is a small virus that was first isolated in the Netherlands and wrapped in an RNA capsule named Lelystad virus. The virus has been proposed to belong to the Arterviridae group. The virus is described in PCT Patent Application Publication No. WO-92 / 21375 and European Patent EP-B-0587780 (Stichting Centraal Diegeneeskundig Instituut) derived from the invention. For the purposes of this patent application, the isolates of the above viruses have been deposited in the Institut Pasteur of Paris, number I-1102.
    The North American type was isolated at about the same time as the European virus isolation, as described in PCT Patent Application Publication No. WO-93 / 03760 (Collins et al.) And European Patent Application EP-A-0529584 (Boehringer Ing.). . For the purposes of this patent application, the isolates of the viruses have been deposited in the American Type Culture Collection (ATCC) under number VR-2332.
    European and North American viruses differ clearly in their serological reactivity as well as in the homology of the nucleotide sequences of the major RNA fragments. The first two pages of European patent application EP-A-0676467 (Akzo) describe the trajectories with reference to extensive literature on such differences. The above patent application concludes that the European and North American viruses have clearly diverged long ago. As a result, it is possible to expect that a vaccine that ultimately works against either type will have little or no effect against the other.
    Different strains were isolated from both European and American viruses. Each strain has its own characteristics, and several strains have been subject to patent applications. For example, PCT Patent Application Publication No. WO-93 / 07898 (Akzo) discloses a vaccine deposited with the number I-1140 in CNCM (Institut Pasteur) as a European strain and a vaccine derived therefrom. PCT Patent Application Publication No. WO-93 / 14196 and European Patent Application EP-A-541418 (Rhone-Merieux), both of which are derived from the same priority application, are separated from France and assigned to Institut Pasteur (CNCM) number I-1153. New strains deposited have been disclosed. European patent application EP-A-0595436 (Solvay) discloses new American strains and vaccines of the strains which are more toxic than previously described. This strain was deposited with the ATCC but the accession number was not listed in the patent application. Finally, Spanish patent application No. ES-A-2074850 (Cyanamid Iberica) discloses so-called Spanish strains that differ from European and American strains. This Spanish strain was deposited with the number V93070108 in the European Animal Cell Culture Collection (EACCC).
    In conclusion, the etiological pathogens of PRRS represent many strains, and it is clear that different vaccines are needed depending on the virus strain infecting pigs in order to effectively counter the disease.
    In sows, the disease is characterized by anorexia, reproductive disorders (abortion, premature delivery, stillbirth or live births, mummified or non-mummy fetal death). Sometimes infected sows die. Less frequent symptoms appear temporarily blue in the ear, abdomen or vulva, which is why the disease was first known as Abortus blauw in Dutch, ie blue ears in English. In piglets, these symptoms depend on age. Respiratory distress and muscle spasms may be observed in newborn piglets, but acquired paralysis and ataxia are more common in older pigs. At the peak of the outbreak, death on the first day of birth is limited, but the mortality rate of piglets after 10 days can reach 80%. Temporarily infected finishing pigs eat less and show more respiratory problems.
    The disease incubation period is highly variable in the range of 5 to 37 days (I. B. Robertson Eurp. Comm. Seminar on PRRS, 11: 4-5, Brussels, 1991). Sometimes the disease spreads very slowly, but if the farm is infected, the disease can last for months (B. Thacker, Int. Symp. On SIRS, St. Paul / Minnesota, 1992).
    Antibodies against the virus were previously described by Wensvoort G. et al. (The Vet. Quart. 13: 121-130, 1990). Immunoperoxidase monolayer by day 6 post-infection. assay, hereinafter referred to as IPMA). The titer of antibodies can reach 1 / 20,000 after 5 days and generally lasts 12 months or more. However, some pigs may develop seronegative negatives after 4-5 months, and a reference report on this is reported by Meredith, M. De, Pig Dis. Info. Center Cambridge, Dec. There is 1992. The authors of the report used the virus at a titer of 10 4 TCID 50 (tissue cultured infection 50%) for six weeks post infection in lungs, serum, plasma and blood cell homogenates. The virus could be isolated after infection from different organs. This indicates that the virus and antibody can survive together for several weeks. In addition, it is well known that animals that survive outbreaks can act as infectious agents for infectious pigs. Viral infections can be detected from day 1 post-infection and are typically shorter but last up to 56 days.
    The virus in the blood is rampant so that the virus can reach the placenta of the sow in conception. It has been demonstrated that viruses can penetrate the placenta and cause death of the fetus. Fetal infectivity is most likely during the last tertiary pregnancy. In addition, the virus can replicate within the fetus without killing the fetus. However, the virus could not be isolated from the mummified or autolyzed fetus.
    In piglets, the disease occurs when the level of colostral acquired maternal antibodies is reduced. It was observed that some of the pups delivered in infected gestation in the last tertiary pregnancy had antibodies to the virus before colostrum was secreted. Typically such animals exhibit viral infection at birth (C. Terpstra et al., Vet. Q 13: 131-136, 1991).
    Although a large number of phagocytic cells were destroyed, the immunosuppressive activity of the PRRS-induced virus was not clear. However, the associated secondary infections are frequent and result in serious economic losses at pig farms.
    The PRRS virus is found today in most countries with high pig yields.
    Currently, PRRS is one of the most important diseases affecting the pig breeding sector due to direct and indirect economic losses and is caused by secondary pathogens aided by PRRS virus infection.
    Satisfactory results are obtained at the laboratory level using inactivated vaccines made from porcine alveolar macrophage (hereinafter PAM) tissues, but the effect on site is partially dependent on environmental conditions and control of vaccinated animals. Depends on.
    One of the problems that have hampered the acquisition of immunological products against the PRRS virus is the limited availability of stable substrates available for viral replication.
    Until recently, PRRS viruses could only propagate in PAM tissue (Wensvoort G. et al., In The Vet. Quart. 13: 121-130, 1991). The need to use healthy pigs of suitable age to obtain these phagocytic cells has several drawbacks. In addition, in recovered PAM, cell substrates derived from other animals are always variable and thus susceptibility to viral infections is not guaranteed. This has been addressed as a major drawback in producing antibodies of constant and uniform quality, and has to be evaluated for each batch to determine the sensitivity.
    For all of the above reasons, the fact that the availability of stable and continuous host cells has been reduced has been a serious obstacle in studying strategies for obtaining strains based on PRRS virus replication and attenuated modifications in cell substrates.
    One of the major problems to be solved is the need to neutralize highly infectious viruses while avoiding replication in PAM because viral replication destroys PAM.
    As a result, by adapting the virus to stable substrates derived from transformed cell lines, an appropriate tool is provided for the acquisition of attenuated strains and for the preparation of inactive vaccines, thus eliminating the dependence and variability on PAM. will be.
    PCT Patent Application Publication No. WO-94 / 18311 (Miles), derived from a commercial cell line known as MA-104, derived from an African green monkey kidney cell line, which the patent applicant named line MA-104 (M). Proliferation of PRRS virus strains in special clones named clone 900B has been proposed. In the patent application, the deposit of the special clone is not mentioned, so that the execution and reproduction of the patent is difficult.
    The present invention relates to a novel attenuated strain of a virus that causes a disease in pigs known as pig reproductive and respiratory distress syndrome (PRRS). The process of attenuating and replicating the virus strain using a novel cell clone obtained from monkey kidneys has led to the preparation of vaccines and diagnostic kits that enable early diagnosis of PRRS and effective precautions against the above diseases. Make it possible.
    Four figures are shown on two pages herein. The following are illustrated in the drawings and the present invention is not limited thereby.
    1 is a diagram showing rectal temperature change in two dimensions in pregnant sows receiving the attenuated strains according to the present invention by intranasal route.
    Figure 2 is a view showing the two-dimensional weight change of the piglets born to sows inoculated with attenuated strains according to the present invention.
    Fig. 3 is a two-dimensional diagram showing antibody dynamics of colostrum determined by IPMA in the offspring of sows vaccinated with the attenuated strains according to the present invention. The offspring of sows 1 and 10 show low titers at birth because some of their offspring have not already sucked colostrum at the moment of draining the blood.
    Figure 4 is a diagram showing the three-dimensional change in body fluid response in piglets inoculated with attenuated strains according to the invention 200 TCID 50 , 2,000 TCID 50 and 20,000 TCID 50 in the intramuscular route.
    The object of the present invention is a novel attenuated strain of PRRS virus which enables to obtain in a stable and reproducible manner a non-toxic vaccine for pigs with high potency in the prevention of PRRS.
    Another object of the present invention is a novel cell clone derived from stable monkey kidney cells, which supports the PRRS virus to grow at a high titer and to harvest a stable virus of selected attenuated strains. Acquisition process.
    Still another object of the present invention is an efficient vaccine against PRRS name and a process for acquiring the new strain, which can be obtained from the new attenuated strain and its variants.
    Still another object of the present invention is a diagnostic kit for a PRRS disease and an acquisition process of the new strain, which can be obtained from the new attenuated strain and its variants.
    The attenuated strain of PRRS virus according to the present invention is derived by attenuating a highly infectious strain isolated from infected pigs on a Spanish farm. In order to meet the relevance of the description, the attenuated strain was deposited with Accession No. I-1642 in Collection Nationale de Cultures de Microorganismes (CNCM) from Institut Pasteur. The date of deposit is November 23, 1995.
    Attenuation of highly infectious strains and replication of the attenuated strains was also an object of the present invention and proceeded by successive passages within cell clones named clone-8 by the author. This clone is derived from a commercial monkey kidney cell line known as MA-104. It was also deposited with CNCM from Institut Pasteur under accession number I-1643 on the same day as clone-8 above, in order to satisfy the relevance of the description.
    Previously, the commercial cell line MA-104 was cloned for the purpose of selection and isolation of clone-8. This cloning suspends the cells in a suitable growth medium (e.g. Earle's MEM) with fetal bovine serum (FBS) and suspensions at various concentrations. By plating, selecting and trypsinizing the clones, and spreading the result into a culture flask The clones obtained in this way were described above until a well-mutated clone was obtained. It was cloned continuously using.
    After eliminating clones that showed irregular morphology or difficult to propagate, the selected clones were tested for susceptibility to early virulent strains of the PRRS virus. Clone-8 was chosen because of its high sensitivity to viral strain replication (high TCID 50 ) and the reproducibility and consistency of the viral harvest obtained.
    The virulent strains were attenuated by sequential replication in Clone-8 culture, preferably at 34 ° C. The contents of infectious viral particles were analyzed to assess the viability of the replicas, and the cytopathic effect (CPE) was tested to predict adaptability. The results show that the virus can replicate for at least 20 passages in clone-8 without losing viability, and with the attenuation achieved, a truly harmless virus with the activity of the antigen can be obtained.
    As a result, the PRRS virus attenuated strains according to the present invention are obtained in a stable and industrially reproducible manner, thus facilitating the use of these strains for the production of both PRRS vaccines and PRRS diagnostic kits.
    Comparative tests between swine populations infected with toxic strains or any of the attenuated strains clearly showed that the attenuated strains are harmless to the animals infected with them. On the other hand, the attenuated strain according to the present invention shows high replication efficiency in seronegative pigs, and can induce seroconversion in animals inoculated with a small amount of 200 TCID50 via the intramuscular route. Antibodies derived in this manner persist for at least 80 days.
    As a result, the attenuated strains according to the present invention are an excellent basis for preparing vaccines for the preventive measures of swine against PRRS. Vaccines as described above may be prepared by any method well known to those skilled in the art, and may also be prepared in different known forms, such as in water soluble suspension forms, oil emulsion forms, liposome mixtures, lyophilized forms, and the like. The composition of the vaccine can be completed with different adjuvants such as immunostimulants, emulsifiers, stabilizers and the like. The vaccine can be administered by intramuscular or subcutaneous, intranasal, intratracheal, volumetric, transdermal or intradermal routes.
    Effective vaccine dosages are highly variable within the preferred form between 10 2 and 10 6 TCID 50 of the attenuated strains according to the invention.
    The obtained vaccine, which is one of the objects of the present invention, may be formulated as a polyvalent vaccine with another live or inactivated porcine virus, or with a live or inactivated bacterium.
    As will be apparent to those skilled in the art, vaccines may also be prepared containing viral antigens derived from viral strains according to the invention. For example, vaccines, capsules or RNA fragments of such strains containing the strains in a form that is thoroughly inactivated using known methods such as thermal or chemical methods.
    The attenuated strains according to the present invention can also be used to prepare appropriate diagnostic kits containing antigenic elements capable of detecting antibodies in seronegative animals by applying conventional techniques. For example, an IPMA process for the detection of a PRRS antibody may comprise the following steps.
    a) The attenuated virus object according to the invention is adapted in such a way that each well is infected with about 20-40 infectious particles in a culture microplate against a stable cell culture, preferably clone-8. Steps.
    b) fixing the infected cells in a solid phase with a known fixative.
    c) detecting porcine serum antibodies by staining of the antibodies in the microplates with IPMA technology after culturing in the microplates.
    Several process examples are disclosed to illustrate a more accurate description of the invention. Such examples should not be considered as limiting the scope of the invention.
    Example 1 Acquisition of Cell Clones from Stable Monkey Kidney Cell Line MA-104
    Six passages of selected PRRS viral strains were obtained as static culture cell monolayers grown in plastic culture flasks in Earle's MEM medium supplemented with 10% fetal bovine serum (FBS) without CO 2 supplementation at 37 ° C. Stable monkey kidney cell line MA-104, supplied by the European Animal Cell Culture Collection (EACCC), deposited accession number 85102918. Viral harvests were collected 6-7 days after inoculation at each passage.
    To study the yield of the viral harvest the cell lines were inoculated with different MOI (hereinafter referred to as MOI) viral strains. The yield of viral harvest obtained was low (between 10 3 and 10 4 TCID 50 / ml) and insufficient for use in vaccine production even after four adaptive passages.
    In these experiments, it was observed that only a portion of infected cells were susceptible to toxic viruses and the remaining cells remained resistant to infection. Because of this, the cell lines have been cloned for the purpose of selecting cell populations that can be fully infected with the viral strain.
    The selection of clones was done as follows.
    Suspension of the cell line was diluted in Earle's MEM with 20% FBS, and various dilutions were carried out in 96 well microplates (NUNC). Plated). Plates were incubated for 8 days at 37 ° C., 5% CO 2 . Wells containing only one cell on day 3 were selected by microscopy and trypsinized on day 8. In this way 44 clones were obtained. The clones thus obtained were then augmented until a suspension of 5 × 10 7 cells / ml was obtained in the culture flask. These cells were then cloned second and third in the same process.
    After the third cloning, 44 obtained clones were developed until 25 ml of cell suspension of clones containing 6 × 10 6 cells / ml each were obtained. The medium used throughout the process contained 25% FBS.
    These clones were subsequently selected according to growth and viability characteristics.
    Example 2. Selection of Cell Clone Clon-8
    The clones obtained in the previous examples were evaluated according to their growth efficiency, except those exhibiting proliferation problems or irregular morphology, and those difficult to maintain at 37 ° C. After this preselection 35 clones were excluded and the remaining 9 clones were selected.
    The selected cell clones were infected with 8 viral passages (P-8) in PAM culture as a suspension of PRRS virus virulent strains (Bloemberg, M. et at., Vet. Microb. 42, 361-371, See description in 1994). The adaptation process for the cell clones was done in advance as follows: The virus was kept in contact with the viral suspension for 80 days at 80-90% confluent monolayer, then frozen at -80 ° C and 24 h It was replicated at 37 ° C. for 3 passages later by the melting process.
    Earle's MEM supplemented with 10% FBS and gentamicin (0.4 mg / ml) was used as infection medium. No antifungal compound or anti yeast compound was used. Each viral harvest obtained in this manner was titrated in the corresponding cell clone. After analyzing the titration results, it was concluded that nine clones were susceptible to viral infection by showing titers of 10 4.2 TCID 50 / ml.
    The results are shown in Table I.
    TABLE I
    Sensitivity of Cell Clones Acquired for Selected Viral Strains
    As can be seen in Table I, the non-cloned cell line shows very low virus-sensitivity, so its use as an antigen for obtaining an effective vaccine does not seem to be suitable.
    Table I also shows that some clones, especially Clon-8, are more sensitive to virus than uncloned cell lines. Clon-8 is particularly noticeable. Titration values up to 10 6 TCID 50 / ml were obtained in a viral harvest from Clon-8.
    Clon-8 was chosen according to the results described above. Several analyzes were performed to examine the reliability of this clone. In various analyzes, the titer of viral harvest was highly reproducible and was in the range of 10 5 and 10 7 TCID 50 / ml.
    Example 3. Attenuated Viral Strains
    Attenuated viral strains according to the present invention were obtained by replicating the selected viral strains in a Clon-8 cell culture at 34 ° C.
    Viral Clon-8 cell monolayers were maintained at 34 ° C. until CPE expanded. The CPE was always detected 24 to 48 hours later compared to when in a culture maintained at 37 ° C. However, no significant difference was found in the extended CPE properties at these two different temperatures.
    The viral harvest obtained was titrated using Clon-8 cell monolayer. In addition, the identity of the virus was also investigated by IPMA.
    The virus was inoculated near the confluence of the Clon-8 70 cm 2 cell monolayer and left to absorb at 34 ° C. for 2 hours. Next, the infection medium was added to the monolayer. Infection medium was previously set at 34 ° C. as modified Earle's Mini Essential Medium supplemented with 10% FBS.
    A 75 cm 2 flask was placed in a 34 ° C. incubator and checked daily until clear CPE was observed. Typically, the viral harvest was collected when 80-95% of the cell monolayers showed CPE between days 5 and 7 after infection. The viral harvest was then centrifuged at 2,000 rpm and the supernatant titrated to determine virus titer (TCID 50 / ml).
    20 passes were executed using the method described above. Virus contents were evaluated in passages P.1, P.5, P.10 and P.20. The results demonstrated that the virus could replicate at least up to passage P.20 in the Clon-8 cell monolayer at 34 ° C. without any loss of viability (see Table II).
    TABLE II
    Evaluation of Virus Proliferated in Clon-8 at 34 ° C from Passage P.1 to Passage P.20
    Further results confirm that Clon-8 cell monolayers can be degraded at 0.001 MOI.
    Experiments performed as described in the Examples herein showed that the P.20 induced viral strain was harmless to swine.
    Example 4 Biological Properties of Attenuated Viral Strains and Effects of Vaccination with the Same
    The starting viral strain used to obtain the attenuated strains of the invention is a toxic strain. The main effects on gestational sows are early delivery, weak childbirth, stillbirth and / or mummified newborn piglets. Infected sows also showed mild anorexia 3-5 days after depression and 4-5 days after deafness.
    Four pregnant sows (ref. 01, 02, 03 and 06) were infected by the intranasal route with a starting toxic strain of 10 6.6 TCID 50 . Two uninfected sows (ref. 73 and 74) were used for control. The results are shown in Table III.
    TABLE III
    Effects on Pregnant Sows Offspring Inoculated with Toxic Virus
    Only one infected sow was delivered on expected date. Infected sows gave birth to 2, 0, 5 and 6 stillborn piglets, respectively, and 2, 4, 2 and 1 weak piglets. The weak piglets died within days of birth. An average of 11 piglets survived in each control sow during weaning. Only an average of 6.5 piglets of each infected sow survived weaning. The mean weights of the piglets at weaning were 4.621 g (chicken of infected sows) and 5.365 g (chicken of control sows). Toxic PRRS virus was isolated from homogenized lungs of piglets given medication. After challenge infection both infected sows and their progeny were seroconverted.
    Biological characterization of the attenuated viral strains according to the invention was performed using both male and female pigs according to specific tests. A harmless test was performed on pregnant sows with three PRRS seronegative negatives from small farms, as long as no occurrence of PRRS was detected. The sows were inoculated at the last third trimester, i.e. at the lowest sensitivity to the virus. An amount of 10 6 TCID 50 per sow was administered via the intranasal route between 78 and 93 days after pregnancy. After inoculation of the virus, the physiological constants of the three sows remained unchanged and the rectal temperature was within the normal count range (see FIG. 1). Three sows were also delivered on expected date. The results obtained are summarized in Table IV.
    TABLE IV
    Effects on Offspring of Pregnant Sows inoculated with Attenuated Virus
    The three sows gave birth to 13, 14 and 16 piglets, respectively. The viability of newborn piglets was judged normal. However, several mummified piglets were found in the offspring of two sows. This can be considered normal given the many litters from sows. The change in weight of piglets was observed to be normal in all cases (see FIG. 2) and remained within the normal range until the end of the observation period (45 days). Weak constitution and disease symptoms that could be associated with PRRS virus infection were not found in any piglets. In addition, PRRS virus was not detected at all in blood and serum collected from newborn embryos.
    No PRRS virus was found in blood and serum collected from pregnant sows 21-36 days after the virus inoculation. All of these facts, strictly speaking, demonstrate the harmlessness of the attenuated PRRS strain in pregnant sows, the category most susceptible to infection with wild-type viruses. All serum samples taken from newborn piglets given to pregnant sows were found to be negative when screened for the presence of PRRS virus in the PAM culture using known techniques. Three sows inoculated with the attenuated virus had humoral antibodies with stable values of IPMA titers of 1/480 at 45 days postpartum and little change thereafter. The sows were found to be seropositive as soon as 21 days after inoculation. As can be seen in Figure 3, the inoculated sows were delivered in colostrum (初乳) and showed a positive serum response and remained positive until at least 75 days after delivery.
    Another vaccination test was performed on a group of 12 sows in the final third trimester. Eight sows were vaccinated intramuscularly with 39 vaccine doses (10 5 TCID 50 ) and the remaining four were used as controls to assess the effect of attenuated strains on reproductive parameters. No changes were observed in the physiological parameters of vaccination sows delivered at the expected delivery date. The results are shown in Table V. As shown in Table V, both inoculated 8 sows and 4 control sows gave birth to the normal number of piglets. The piglets also had normal viability.
    TABLE V
    Effect of Attenuated Virus Inoculation on Birth Parameters in the Last Third Pregnancy of Pregnancy Sows
    The attenuated viral body according to the invention replicates efficiently in PRRS-serum-reactive negative pigs. This is evidenced by the fact that as little as 200 TCID 50 administered intramuscularly can be replicated in pigs and induce seroconversion. As shown in FIG. 4, the induced antibodies survived for 52 days in the inoculated animals.
    The attenuated virus body according to the present invention does not spread to unwatched pigs in the role of four watchmen housed with a group of eight piglets inoculated intramuscularly. The fact that the virus does not spread to unvaccinated animals illustrates that the attenuated strain is suitable as a vaccine. Furthermore, no leukemia or any other clinical symptoms were found in vaccinated pigs, indicating that the attenuated virus is harmless when administered intramuscularly. An average of 86% of the inoculated piglets became seropositive as soon as 11 days after inoculation.
    The attenuated viral body according to the present invention induces an immune protective response that prevents the clinical impact of challenge infection with viral PRRS strains in inoculated pigs. Therefore, no clinical symptoms were observed when the vaccinated piglets, 4 weeks old, were infected with the toxic virus. On the other hand, as shown in Table VI, 80% of the unvaccinated control offspring after experimental infection showed a significant rectal temperature rise. In addition, autopsies performed 19 days after the experimental infection showed that the vaccinated piglets showed significantly less lung damage than the unvaccinated control piglets. In the same way, only 25% of vaccinated animals after experimental infection were found to be toxic, whereas in 80% of unvaccinated control animals, toxic viruses could be detected at least 12 days after infection.
    Table VI
    Vaccination and challenge hyperthermia on both sides of control piglets after challenge infection
    Data on deposited microorganisms
    According to the Budapest Convention, viral strains and cell Clon-8 objects according to the present invention were deposited with the international organization Collection Nationale de Cultures de Microorganismes (CNCM) of the Institut Pasteur.
    The deposit shall be entrusted to public disposal under the conditions laid down in the Budapest Convention. This should not be construed as a license for infringement of the right attributable to the applicant of the present invention by carrying out the object of the present invention.
    权利要求:
    Claims (20)
    [1" claim-type="Currently amended] Attenuated strain of virus that causes swine vaginal known as swine reproduction and respiratory distress syndrome (PRRS), essentially corresponding to the November 23, 1995 deposit, access number I-1642 of the CNCM of Institut Pasteur.
    [2" claim-type="Currently amended] A vaccine for protecting swine against a disease known as swine reproduction and respiratory distress syndrome (PRRS),
    A vaccine comprising the viral strain according to claim 1 and / or at least one viral antigen obtainable from the strain.
    [3" claim-type="Currently amended] The method of claim 2,
    The viral antigen is an inactivated virus.
    [4" claim-type="Currently amended] The method of claim 2,
    A vaccine comprising attenuated viral strains between 10 2 and 10 6 TCID 50 .
    [5" claim-type="Currently amended] The method of claim 2 or 3,
    Wherein the vaccine dose contains an amount of viral antigen equal to the amount produced by an attenuated viral strain between 10 2 and 10 6 TCID 50 .
    [6" claim-type="Currently amended] The method according to any one of claims 2-5,
    A vaccine comprising additional immunostimulatory adjuvant and / or emulsifiers and / or stabilizers.
    [7" claim-type="Currently amended] The method according to any one of claims 2-6,
    Vaccine, characterized in that it is in one form or in combination, and further contains live or inactivated porcine viruses.
    [8" claim-type="Currently amended] The method according to any one of claims 2-6,
    A vaccine comprising live or inactivated bacteria.
    [9" claim-type="Currently amended] Cell clone derived from stable monkey kidney cell line MA-104, essentially corresponding to the November 23, 1995 deposit of Access No. I-1642 of the CNCM of Institut Pasteur.
    [10" claim-type="Currently amended] In the process of obtaining an attenuated strain of PRRS virus,
    A process comprising essentially the modification of a toxic strain by successive passages in the cell clone according to claim 9.
    [11" claim-type="Currently amended] The method of claim 10,
    Maintaining a temperature of 34 ° C. in a continuous passage.
    [12" claim-type="Currently amended] In the process for preparing an active vaccine against PRRS,
    Fixation essentially comprising propagation of the viral strain of claim 1 in a cell clone of claim 9.
    [13" claim-type="Currently amended] The method of claim 12,
    Process for growing the viral strain at 34 ℃.
    [14" claim-type="Currently amended] The method according to claim 12 or 13,
    The composition of the obtained attenuated strain is a water-soluble suspension or oil emulsion, liposome composition, or lyophilized form, with or without the adjuvant according to claim 6.
    [15" claim-type="Currently amended] The method according to any one of claims 12-14,
    Inactivating said attenuated strain by thermal or chemical methods until the attenuated strain obtained is completely deactivated.
    [16" claim-type="Currently amended] PRRS diagnostic process comprising the attenuated strain according to claim 1 and / or at least one viral antigen obtained from the strain.
    [17" claim-type="Currently amended] As a process for detecting a PRRS antibody,
    a) adapting the attenuated viral strain to grow in a stable cell culture in the culture microplate in such a way that each well is infected with about 20-40 infectious particles;
    b) fixing the infected cells to a solid phase using known fixatives,
    c) detecting the antibody from pig serum by culturing in a microplate and staining using IPMA techniques.
    [18" claim-type="Currently amended] The method of claim 17,
    The stable cell culture is a cell clone according to claim 9.
    [19" claim-type="Currently amended] A method of using the attenuated viral strain according to claim 1 for producing a vaccine for preventing a pig disease known as PRRS.
    [20" claim-type="Currently amended] A method of using the attenuated viral strain according to claim 1 for producing a diagnostic kit for detecting the name of a pig known as PRRS.
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    同族专利:
    公开号 | 公开日
    ES2102971B1|1998-03-01|
    WO1997027288A1|1997-07-31|
    EP0835929A1|1998-04-15|
    JPH10507372A|1998-07-21|
    UA68326C2|2004-08-16|
    JP3068204B2|2000-07-24|
    HU9801124A2|1998-08-28|
    ES2102971A1|1997-08-01|
    PL185841B1|2003-08-29|
    BR9604888A|1998-12-15|
    PL322455A1|1998-02-02|
    US6001370A|1999-12-14|
    CN1185806A|1998-06-24|
    CZ286766B6|2000-06-14|
    CA2216436A1|1997-07-31|
    HU9801124A3|1999-09-28|
    CZ337797A3|1998-03-18|
    MX9707302A|1998-06-30|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    法律状态:
    1996-01-25|Priority to ESP9600168
    1996-01-25|Priority to ES09600168A
    1996-12-04|Application filed by 후안 노가레다, 라보라또리오스 이쁘라 에쎄. 아.
    1998-10-15|Publication of KR19980703268A
    优先权:
    申请号 | 申请日 | 专利标题
    ESP9600168|1996-01-25|
    ES09600168A|ES2102971B1|1996-01-25|1996-01-25|New attenuated strain of the virus causing the respiratory and reproductive syndrome , the vaccines and diagnostic media obtainable with the same and the procedures for its obtaining.|
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