WO2009059411A1 - Lignée de cellules épithéliales de poumon de porc et son utilisation dans la production et la détection du virus du syndrome reproducteur et respiratoire porcin - Google Patents

Lignée de cellules épithéliales de poumon de porc et son utilisation dans la production et la détection du virus du syndrome reproducteur et respiratoire porcin Download PDF

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WO2009059411A1
WO2009059411A1 PCT/CA2008/001953 CA2008001953W WO2009059411A1 WO 2009059411 A1 WO2009059411 A1 WO 2009059411A1 CA 2008001953 W CA2008001953 W CA 2008001953W WO 2009059411 A1 WO2009059411 A1 WO 2009059411A1
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cell line
prrs
sjpl
virus
cells
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PCT/CA2008/001953
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Carl Gagnon
Mario Jacques
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Valorisation-Recherche, Limited Partnership
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/10011—Arteriviridae
    • C12N2770/10051—Methods of production or purification of viral material
    • C12N2770/10052—Methods of production or purification of viral material relating to complementing cells and packaging systems for producing virus or viral particles

Definitions

  • the present invention relates to the use of a porcine lung epithelial cell line, such as one having the characteristics of the porcine lung epithelial cell line SJPL, deposited with the American Type Culture Collection (ATCC) on April 5, 2001 , and assigned accession number PTA-3256, in the diagnostic and production of the porcine reproductive and respiratory syndrome (PRRS) virus.
  • a porcine lung epithelial cell line such as one having the characteristics of the porcine lung epithelial cell line SJPL, deposited with the American Type Culture Collection (ATCC) on April 5, 2001 , and assigned accession number PTA-3256, in the diagnostic and production of the porcine reproductive and respiratory syndrome (PRRS) virus.
  • the porcine reproductive and respiratory syndrome is an infectious disease in swine that emerged 20 years ago in the United States and Canada, but two to three years later in the European countries.
  • PRRS porcine reproductive and respiratory syndrome
  • the disease has many clinical manifestations but the two most prevalent are severe reproductive failure in sows and gilts (characterized by late-term abortions, an increased number of stillborns, mummified and weak-born pigs), and respiratory problems in pigs of all ages associated with a non-specific lymphomononuclear interstitial pneumonitis.
  • MARC- 145 cells are still the only cell line used for vaccine production.
  • ZMAC-1 an immortalized porcine alveolar macrophages cell line (ZMAC-1) was reported to be permissive to PRRSV but seems less appropriate for vaccine production since its propagation is slow and needs growth factors (Calzada-Nova et al., 2007). This limitation concerning PRRSV permissive cell lines thus raises a significant obstacle to successful PRRS virus production in tissue culture.
  • the present invention addresses the above mentioned obstacle by providing a porcine lung epithelial cell line, such as the SJPL and derivatives thereof, for its use in a method for the production and detection of the PRRS virus.
  • PRRS porcine reproductive and respiratory syndrome
  • the present invention is also concerned with a method for the production of the porcine reproductive and respiratory syndrome (PRRS) virus, comprising the steps of:
  • the present invention is further concerned with a method for diagnosing a porcine reproductive and respiratory syndrome (PRRS) virus infection in a pig, comprising the steps of:
  • the present invention is further concerned with a method for detecting the presence or absence of anti-porcine reproductive and respiratory syndrome (PRRS) virus antibodies in a sample, comprising the steps of :
  • PRRS porcine reproductive and respiratory syndrome
  • the present invention is also concerned with a kit for determining the presence or absence of a porcine reproductive and respiratory syndrome (PRRS) virus in a sample, comprising:
  • binding means optionally a biological reference sample lacking a PRRS vims that specifically bind with said binding means; and - optionally a comparison sample comprising a PRRS virus which can specifically bind to said binding means; wherein said binding means, reagent, biological reference sample, and comparison sample are present in an amount sufficient to perform said detection.
  • FIG. 1 Cytopathic effect (CPE) observed in SJPL PRRSV-infected cells.
  • Figure 2 Detection of the viral nucleocapsid protein (N) in PRRSV-infected cells by an immunofluorescence assay (IFA).
  • IFA immunofluorescence assay
  • MARC-145 and SJPL cells were poured into wells of an eight-well Lab-tech Chamber slide (Nalge Nunc International, USA) and incubated overnight. Next day, both cell types were infected with lAF-Klop or LV PRRSV reference strains at a multiplicity of infection (MOI) of 0.5. The infected cells were fixed with a 4% paraformaldehyde solution at 72hrs post-infection (p.i.). Subsequently, the cells were permeabilized with a 0.5% Triton X-100 solution. Then, the immunofluorescence assay was performed as previously described (Gagnon et al., 2003) and positive cells were visualized using a fluorescent microscope.
  • MOI multiplicity of infection
  • FIG. 3 PRRSV infectious viral particles production in SJPL after five passages.
  • Figure 4 PRRSV IAF-Klop strain replication kinetics in MARC-145 and SJPL cells.
  • MARC-145 and SJPL cells were infected with PRRSV IAF-Klop strain using an infectious dose of 1 MOI.
  • both supernatant (cell culture medium) and cell pellets (cells) were collected after centrifugation. Then cell pellets and supernatants were stored at -70oC until virus titration was performed in MARC-145 cells.
  • FIG. 5 PRRSV LV strain replication kinetics in MARC-145 and SJPL cells. 10 5 MARC-145 and SJPL cells were infected with PRRSV LV strain using an infectious dose of 1 MOI. At different time points post infection, both supernatant (cell culture medium) and cell pellets (cells) were collected after centrifugation. Then cell pellets and supernatants were stored at -70oC until virus titration was performed in MARC-145 cells.
  • Figure 6 PRRS virus plaque size formation in MARC-145 and SJPL infected cells.
  • SJPL cell clones Three clones (6,10 and 11) were infected with the PRRSV North American lAF-Klop strain at 1 MOI. After 3 days post-infection, an immunofluorescence assay was performed to detect PRRSV N protein expression using the ⁇ 7 specific sera as previously described (Gagnon et al., 2003). For each clone, none infected cells (Mock) was included as a negative control. In addition, the parental SJPL cells were used as a positive control.
  • porcine lung epithelial cell lines find an advantageous use in the propagation of the porcine reproductive and respiratory syndrome (PRRS) virus.
  • PRRS porcine reproductive and respiratory syndrome
  • the present inventors have discovered that a porcine lung epithelial cell line having the characteristics of the SJPL cell line is surprisingly permissive to the PRRS virus.
  • Such permissiveness to PRRS virus replication thus opens up a plurality of applications such as PRRS virus production and detection/quantification of PRRS virus.
  • the present invention contemplates of using a porcine lung epithelial cell line in the propagation or production of the porcine reproductive and respiratory syndrome (PRRS) virus or PRRSV.
  • PRRS porcine reproductive and respiratory syndrome
  • the contemplated cell line has the characteristics of the SJPL cell line (deposited with the American Type Culture Collection (ATCC) on
  • the expression "having the characteristics of the SJPL cell line” means that the use of any porcine lung epithelial cell line is within the scope of the present invention when one or more of its morphological, cell culture, virus propagation and production and response to viral infection are about the same as the SJPL cell line. It will be understood that, for the purposes of the invention, the determination of the characteristics of the cell lines contemplated herein is more qualitative than quantitative.
  • - it is a porcine lung epithelial cell line; - it is continuous, i.e., able to propagate indefinitely in tissue culture;
  • titer is used herein to refer to a quantitative amount of infectious PRRS virus particles in a cell culture or an amount of viral genome copies quantified by PCR which is transposed to an amount of infectious PRRS virus particles in a cell culture as previously described (Gagnon et al., 2008).
  • a porcine lung epithelial cell line contemplated by the present is considered to produce a "desired titers of virus” when, upon infection with a PRRS virus strain, it produces a titer suitable for vaccine production.
  • a desired titer contemplated by the present invention may be a titer of at least 10 5 TCID 50 /mL.
  • the contemplated cell line used for PRRS virus propagation or production consists of the SJPL cell line.
  • the epithelial cell line (St-Jude porcine lung (SJPL) cells) was spontaneaously generated from the normal lungs of a 4-week old female Yorkshire pig at St-Jude Children's Research Hospital.
  • SJPL cells were epithelial cell-like, and were positive for an epithelial cell marker, cytokeratin.
  • the SJPL cell line were disclosed and described in international application WO 02/089586 and in Seo et a/., 2001. It is worth noting that the term "derivative" when used in conjunction with the expression "SJPL cell line” refers to any variant or clone having about the same characteristics (e.g. permissive to PRRS virus) of the SJPL cells, such as those shown in Example 6.
  • porcine lung epithelial cell lines contemplated by the present invention may be used, for instance, in PRRS virus production methods. It is therefore an aspect of the invention to provide a method for the production of the PRRS virus.
  • the method of the invention comprises the steps of:
  • a "culture” means a propagation of cells, such as the SJPL cells, in a medium conducive to their growth.
  • the term “to culture” refers to the process by which such culture propagates.
  • the PRRS viral particles obtained in the harvesting step may be harvested by any suitable harvesting methods known to one skilled in the art, such as by concentration by column chromatography, by ultracentrifugation through, for instance, a 30% sucrose cushion or through a continuous 15 to 30% (W/V) CsCI gradient.
  • the PRRS viral particles encompass the whole PRRS virus and any of the non structural or structural components of PRRSV.
  • the PRRS viral particles may be from different PRRS genotypes, such the North American (NA) genotype or the European genotype (EU).
  • the harvested PRRS viral particles may be used in a vaccine composition in the form of a live viral vaccine, an attenuated vaccine or an inactivated vaccine.
  • the harvested PRRS viral particles are inactivated by suitable methods known to one skilled in the art, such as but not limited to, gamma irradiation, thermal treatment, chemical inactivation such as peracetic acid- ethanol treatment or binary ethylenimine.
  • suitable methods known to one skilled in the art such as but not limited to, gamma irradiation, thermal treatment, chemical inactivation such as peracetic acid- ethanol treatment or binary ethylenimine.
  • the harvested PRRS viral particles may be obtained to form part of a subunit PRRS virus vaccine. In such a case, the harvested PRRS viral particle may consist of a PRRS viral antigen.
  • the term "antigen" as used herein refers to any protein, carbohydrate, or other moiety expressed by the PRRSV that is capable of eliciting an immune response (e.g. a protective response) against the PRRSV.
  • the PRRSV antigen may or may not be a structural component of PRRSV.
  • such a PRRSV antigen may be, but not limited to, GP 5 , M and N structural proteins of PRRSV.
  • the present invention provides an efficient method for the production of vaccines for treating or preventing a PRRS virus infection.
  • infection used in conjunction with PRRS virus refers to the presence, growth or proliferation of virions of a PRRSV strain within, or on a surface of, cell cultures or an animal.
  • the contemplated porcine lung epithelial cell lines of the invention allow to produce a vaccine from a large spectrum of PRRS virus strains.
  • a vaccine may be prepared from the culture of authorized vaccinal PRRS virus strain (such as ATCC VR-2332 vaccine strain) or from the culture of the PRRS virus strain isolated from the infected animal's own tissues, secretions or blood.
  • PRRS virus observed in the contemplated porcine lung epithelial cells used in the production method of the invention as shown in Figure 2 and 6 compared to MARC- 145 PRRSV infected cells, it thus allows to efficiently produce large quantities of
  • PRRS virus suitable for vaccine production is provided.
  • porcine lung epithelial cell lines contemplated by the present invention may further be used, for instance, in PRRS virus detection and quantification methods.
  • the method of the invention comprises the steps of:
  • sample when referring to the above-mentioned detection method, refers to a variety of sample types obtained from a subject, such as a pig, and can be used in a diagnostic or detection assay.
  • the definition encompasses blood and other liquid samples of biological origin, solid tissue samples such as a biopsy specimen or tissue cultures or cells derived therefrom, and the progeny thereof.
  • Techniques for detecting the presence or absence of PRRS viral particles in the used porcine lung epithelial cell line e.g. the SJPL cells are well known to one skilled in the art.
  • the detection method of the invention may further comprise a step of determining the quantity of PRRS viral particles detected. Such a determination may be obtained by determining the PRRS viral infectious titer. Techniques for quantifying the viral titer may be the same as those defined above in relation with the detecting step of the detection method of the invention.
  • the porcine lung epithelial cell lines contemplated by the present invention may further be used, for instance, in anti-PRRS virus antibodies detection, methods.
  • the present invention provides for a method for detecting the presence or absence of anti-porcine reproductive and respiratory syndrome (PRRS) virus antibodies in a sample.
  • PRRS anti-porcine reproductive and respiratory syndrome
  • PRRS porcine reproductive and respiratory syndrome
  • sample when referring to the above-mentioned anti-PRRSV antibodies detection method, refers to a variety of sample types obtained from a subject, such as a pig, and can be used in a diagnostic or detection assay.
  • the definition encompasses blood and any other liquid samples of biological origin. 4. Kits of the invention
  • the present invention further provides kits for use within any of the above diagnostic and quantification methods.
  • kits for determining the presence or absence of a porcine reproductive and respiratory syndrome (PRRS) virus in a sample comprises:
  • a binding means capable of specifically bind to a PRRS virus
  • binding means-PRRS virus complex - optionally a biological reference sample lacking a PRRS virus that specifically bind with said binding means;
  • a comparison sample comprising a PRRS virus which can specifically bind to said binding means; wherein said binding means, reagent, biological reference sample, and comparison sample are present in an amount sufficient to perform said detection.
  • the expression “specifically binds to” refers to a binding means (e.g. antibody) that binds with a relatively high affinity to one or more surface proteins or polypeptides of the tested PRRS virus, but which does not substantially recognize and bind to other viruses or bacteria.
  • a binding means e.g. antibody
  • kits typically comprise two or more components necessary for performing a diagnostic or quantification assays.
  • Components may be compounds, reagents, containers and/or equipment.
  • one container within a kit may contain a porcine lung epithelial cell line such as SJPL cells.
  • One or more additional containers may enclose elements, such as PRRS virus antigen harvested from PRRS virus SJPL infected cells, binding reagents such as monoclonal antibodies or fragment thereof that specifically binds to PRRS virus, reagents or buffers, to be used in the assays.
  • Such kits may also, or alternatively, contain a detection reagent that contains a reporter group suitable for direct or indirect detection of antibody binding as exemplified in the Example Section.
  • kits may be designed to detect the PRRS virus genetic material in a biological sample.
  • kits generally comprise at least one oligonucleotide probe or primer, as shown in the example Section, that hybridizes to a polynucleotide such as one encoding a PRRS virus protein.
  • a polynucleotide such as one encoding a PRRS virus protein.
  • Such an oligonucleotide may be used, for example, within a PCR or hybridization assay. Additional components that may be present within such kits include a second oligonucleotide and/or a diagnostic reagent or container to facilitate the detection of a PRRS virus polynucleotide.
  • the North American (NA) genotype IAF-Klop cytopathogenic reference strain of porcine reproductive and respiratory syndrome virus (PRRSV) (Gagnon et al., 2003) was propagated in MARC-145 cells, a clone of MA-104 cells highly permissive to PRRSV (Kim et al., 1993), as previously described (Mardassi et al., 1994).
  • PRRSV porcine reproductive and respiratory syndrome virus
  • the European genotype (EU) PRRSV reference strain, the Lelystad virus (LV) was propagated following the same method as for the IAF-Klop strain.
  • the infectious dose of the virus stocks were calculated from a 96 wells microplate of MARC-145 infected cells following the determination of the cytopathic effect (CPE) by the method of Karber (Payment and Trudel, 1989).
  • Virus titers were expressed in tissue culture infectious dose 50 per mL (TCID 50 /mL).
  • the epithelial SJPL (St. Jude porcine lung) cell line was cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 1% sodium pyruvate, 1% L-glutamine, 1.4% MEM nonessential amino acids, and 1% antibiotic-antimycotic solution as previously described (Seo et al., 2001).
  • DMEM Dulbecco's modified Eagle's medium
  • the NPTr (newborn pig trachea) cell line was used as negative control since it can replicate most of the porcine viruses except the PRRSV (Ferrari et al., 2003).
  • the NPTr cell line was cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 1% sodium pyruvate, 1 % L-glutamine, 1.4% MEM nonessential amino acids, and 1 % antibiotic-antimycotic solution.
  • DMEM Dulbecco's modified Eagle's medium
  • the PK15A (porcine kidney) cells were used to propagate porcine circovirus type 2 (PCV-2).
  • the PK15A cells a subclone of PCV noninfected PK15 cells (Racine et al., 2004), were maintained in Earle's minimal essential medium (MEM; Invitrogen Corporation, GibcoBRL, Grand Island, NY, USA), supplemented with 10% fetal bovine serum (FBS), 300 U/mL of penicillin, 300 mg/mL of streptomycin, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 2.5 ⁇ g/mL of amphotericin B, and 10 mM HEPES buffer.
  • MEM Earle's minimal essential medium
  • FBS fetal bovine serum
  • the virus was purified following ultracentrifugation on a 30% sucrose cushion using the SW28 Beckman Coulter rotor at 25 000 RPM during 4 hours.
  • the virus pellets were resuspended in 2 mL of a phosphate buffer solution containing 2% fetal bovine serum and aliquots of the virus stocks were than conserved at -70 0 C for future use.
  • the infectious dose of the PCV-2 virus stock (strain FMV-06-1717 classified within the genotype PCV-2b) (Gagnon et al., 2007; Gagnon et al., 2008) was calculated from a 96 wells microplate of PK15A infected cells following immunofluorescence assay (Racine et al., 2004) by the method of Karber (Payment and Trudel, 1989). Virus titers were expressed in tissue culture infectious dose 50 per mL (TCID 50 /mL).
  • Immunofluorescence assay The presence of PRRSV antigens in infected cells was determined by an immunofluorescence assay (IFA). Briefly, PRRSV infected cells were fixed with an 80% acetone cold solution. After an incubation period of 30 minutes at room temperature, the acetone solution was removed and the cells were dried and kept at -20oC for future use. Alternatively, infected cells were fixed with a 4% paraformaldehyde (PFA) solution prepared as previously described (Ausubel et al., 2002). After an incubation period of 30 minutes at room temperature, the PFA solution was removed and cells were washed three times with a phosphate buffer saline solution (PBS).
  • IFA immunofluorescence assay
  • the ⁇ 7 rabbit monospecific antisera (specific anti-nucleocapsid (N) protein of PRRSV) (Gagnon et al., 2003; Mardassi, Massie, and Dea, 1996) was diluted 1/200 in the washing buffer and added to the cells and incubated at room temperature for a 30 minutes period. Cells were then washed three times with the washing buffer. Afterward, cells were incubated for 30 minutes with the washing buffer containing a 1/100 dilution of anti- rabbit specific antisera FITC conjugated (Sigma Aldrich). Then, cells were washed three times with the washing buffer followed by two washing steps with PBS.
  • N nucleocapsid
  • PRRSV cell infections 10 4 NPTr, MARC-145 and SJPL cells per well were seeded in an 8 wells cell culture slide (Labtech, Nunc) and were infected with an amount of 0.5 and 0.05 MOI (multiplicity of infection - equal the amount of TCID 50 /cells) of IAF- Klop strain.
  • MOI multiplicity of infection - equal the amount of TCID 50 /cells
  • the CPE was observed by light microscopy every day until the end of the experiment at 5 days post-infection. Also, at different time post-infection, cells were fixed and prepared for the IFA to detect the expression of the nucleocapsid protein (N) of PRRSV.
  • PRRSV replication in SJPL 25 cm 2 Flasks were seeded with 10 6 MARC- 145 and SJPL cells and those cells were infected with 0.005 MOI of IAF-Klop PRRSV strain.
  • the cytopathic effect (CPE) was observed by light microscopy every day until the end of the experiment at 5 days post-infection. Then, cells with their supernatants were submitted to three cycles of freeze and thaw at -70oC and the virus stock solutions were kept at -70oC for future use.
  • Four subsequent viral passages in MARC-145 and SJPL were done as previously described except that a dilution of 1/20 of the previous viral stock solutions was used for cell infection.
  • the amount of virus production at each passage was determined by TCID 50 /mL in MARC- 145 and by a quantitative PRRSV real-time PCR assay as previously described (Gagnon et al., 2008). Briefly, the PCR quantification of PRRSV was determined with the Tetracore PRRSV real-time PCR diagnostic kit (Tetracore Inc.) by comparing the sample results with a standard curve based on the amount of serially diluted IAF-Klop PRRSV reference strain (Gagnon et al., 2003) titrate following infection of MARC-145 cells and expressed as TCID 50 /mL. Thermocycling were performed in a SmartCycler system (Cepheid, Sunnyvale, California, USA).
  • PRRSV replication inhibition by PCV-2 10 5 MARC-145, SJPL and PK15A cells were infected in suspension with a MOI of 1 by IAF-Klop PRRSV strain and FMV-06- 1717 PCV-2 strain individually and simultaneously to determine if PCV-2 could inhibit the replication of PRRSV in SJPL as previously reported in MARC-145 cells (Chang et al., 2005).
  • the infected cells were seeded in 24 wells plates.
  • the CPE was evaluated at different times post-infection and the cells were fixed with PFA at 96 hrs post-infection and IFA was realized to detect the expression of the N protein of both viruses.
  • SJPL cells efficacy to detect PRRSV viremia.
  • Five weeks old piglets were inoculated intramuscularly with 10 5 TCID 50 of a PRRSV field isolate obtained following virus isolation from tissues of PRRS sick animals.
  • blood samples were collected and the obtained sera were submitted to several analyses such as PRRSV PCR detection (Tetracore inc.) and virus isolation in MARC-145 and SJPL cells.
  • 25 cm 2 Flasks (Corning) were seeded with MARC-145 and SJPL cells and those cells were incubated with 0.1 mL of two infected pigs sera.
  • cells and supernatant were collected and submitted to one freeze and thaw cycle at -70oC.
  • PRRSV strains replication kinetics assay 10 5 MARC-145 and SJPL cells were infected with PRRSV IAF-Klop or LV strain using an infectious dose of 1 MOI. The inoculum were removed after 4hrs of incubation. Cells were washed 3 times with culture medium and fresh cell culture medium was added. At different time point post infection (0, 4, 9, 12, 18, 24, 48, 72, 96 and 120hrs p.L), both supernatant (cell culture medium) and cell pellets (cells) were collected after centrifugation. Then cell pellets and supernatants were stored at -70oC until virus titration was performed in MARC- 145 cells (see above).
  • Plaque assays Plaque assay was performed in six-well tissue culture plates (Corning, USA) to compare the PRRSV plaque size formation in MARC-145 and SJPL infected cells. Confluent monolayer of MARC-145 and SJPL cells were infected with 10 4 TCID 50 (none diluted) or serially diluted PRRSV European LV reference strain or North American IAF-Klop reference strain and incubated overnight with constant agitation. After the absorption period, the virus inoculums were removed and cell monolayers were covered with medium containing 1.5% agarose.
  • the infected cells were fixed with a solution of 4% paraformaldehyde (PFA) in PBS (pH7.4) at room temperature for 40 min and stained with 0.1% of crystal violet solution at room temperature for 5 min.
  • PFA paraformaldehyde
  • SJPL cells cloning by end point dilution assay were end point dilution assay.
  • An end point dilution method was used to obtain SJPL cell clones. Briefly, fresh trypsinized SJPL cells were 10 fold diluted (from 1 to 10 "8 ) and seeded in 96-well plate with a volume of 100uL/well. After one day of incubation at 37oC and 5% CO 2 atmosphere, wells containing only one cell were selected. Then, culture medium was removed and new cell culture medium containing 15% FBS was poured onto each clone cells. After cells were confluent, they were trypsinized and transferred to a 24-well plate with 1 mL/well fresh culture medium.
  • EXAMPLE 1 Replication of PRRSV IAF-Klop North American and LV European reference strains in SJPL. As illustrated in Figure 1 , a very light cytopathic effect (CPE) was observed in SJPL cells and not in NPTr cells following PRRSV IAF-Klop infection at 96 hrs pi. These latest results indicate that the PRRSV may be able to replicate in SJPL like in MARC-145 cells since it as been previously reported that PRRSV replicates in MARC-145 cells and induces CPE by necrosis and apoptosis (Gagnon et al., 2003; Miller and Fox, 2004).
  • CPE cytopathic effect
  • EXAMPLE 2 PRRSV replication in SJPL cells is not inhibited by PCV-2 coinfection compared to the observations made in coinfected MARC-145 cells.
  • interferon alpha IFN ⁇
  • porcine circovirus type 2 PCV-2
  • PCV-2 porcine circovirus type 2
  • EXAMPLE 3 SJPL efficacy to isolate PRRSV from pig sera.
  • Results presented in Table 2 indicate that SJPL cells are more efficient for the detection of viremia for at least one PRRSV NA strain since CPE and positive IFA results were detected in SJPL incubated with PRRSV PCR positive pig sera compared to MARC-145 cells where no CPE and negative IFA results were obtained.
  • Example 4 PRRS virus replication kinetics in MARC-145 and SJPL cells.
  • Example 5 Plaque assays comparison results of PRRS virus plaque size formation in MARC-145 and SJPL infected cells.
  • the amount of virus plaque formation in SJPL cells is lower or none existing compared to MARC-145 cells for both PRRSV IAF- Klop and LV strains, respectively, even when the same amount of virus has been used to infect both cell lines.
  • this latest result confirms that SJPL cells develop less CPE when infected with PRRSV compared to MARC-145 cells.
  • this phenomenon does not seem to affect the amount of infectious viral particles production as illustrated in Figures 4 and 5.
  • This interesting characteristic of the SJPL cells advantageously opens up the possibility of establishing a continuous cell line expressing constitutively PRRSV particles which will eliminate the need of a PRRSV viral stock for the production of PRRSV vaccine antigens.
  • the present inventors have surprisingly found that the use of the SJPL cell line and derivatives thereof for the production of PRRSV has at least the following advantages over the prior art cell lines, such as the MARC-145 cell line:
  • Adenoviral-expressed GP5 of porcine respiratory and reproductive syndrome virus differs in its cellular maturation from the authentic viral protein but maintains known biological functions.

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Abstract

La présente invention porte sur l'utilisation d'une lignée de cellules épithéliales de poumon de porc qui a les caractéristiques de la lignée de cellules épithéliales de poumon de porc SJPL dans le diagnostic et la production du virus du syndrome reproducteur et respiratoire porcin (PRRS).
PCT/CA2008/001953 2007-11-06 2008-11-05 Lignée de cellules épithéliales de poumon de porc et son utilisation dans la production et la détection du virus du syndrome reproducteur et respiratoire porcin WO2009059411A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011098615A1 (fr) 2010-02-15 2011-08-18 Universite Claude Bernard Lyon 1 Cellules modifiees pour la production virale par l'inhibition du gene hipk2
WO2018086686A1 (fr) * 2016-11-09 2018-05-17 Probiogen Ag Nouvelle lignée cellulaire porcine pour la production de virus

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998035023A1 (fr) * 1997-02-07 1998-08-13 Origen, Inc. Procede servant a cultiver le virus du syndrome respiratoire et reproductif porcin afin de l'utiliser en tant que vaccin et dans des essais diagnostiques
WO2002089586A1 (fr) * 2001-05-10 2002-11-14 St. Jude Children's Research Hospital Lignee cellulaire epitheliale pulmonaire pour propagation de virus

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998035023A1 (fr) * 1997-02-07 1998-08-13 Origen, Inc. Procede servant a cultiver le virus du syndrome respiratoire et reproductif porcin afin de l'utiliser en tant que vaccin et dans des essais diagnostiques
WO2002089586A1 (fr) * 2001-05-10 2002-11-14 St. Jude Children's Research Hospital Lignee cellulaire epitheliale pulmonaire pour propagation de virus

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011098615A1 (fr) 2010-02-15 2011-08-18 Universite Claude Bernard Lyon 1 Cellules modifiees pour la production virale par l'inhibition du gene hipk2
WO2018086686A1 (fr) * 2016-11-09 2018-05-17 Probiogen Ag Nouvelle lignée cellulaire porcine pour la production de virus
US11060069B2 (en) 2016-11-09 2021-07-13 Probiogen Ab Porcine cell line for virus production

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