PORCINE LUNG EPITHELIAL CELL LINE AND ITS USE IN PRODUCTION AND DETECTION OF PORCINE REPRODUCTIVE AND RESPIRATORY SYNDROME

VIRUS

FIELD OF THE INVENTION

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.

BRIEF DESCRIPTION OF THE PRIOR ART

The porcine reproductive and respiratory syndrome (PRRS) 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. Today, PRRS is endemic in many, if not all swine-producing countries. 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.

It is widely recognized that very few cell lines are permissive to PRRSV. Indeed, the first cell line discovered to be permissive to PRRSV was a primary cell line, the porcine alveolar macrophages (PAM) (Wensvoort et al., 1991). Later on, other primary cell lines were found to be permissive to PRRSV (Rossow, 1998).

Because of their nature (primary cell culture), it could not efficiently be used in large scale virus production for vaccine formulation. Few years later after PAM discovery, an immortalized monkey kidney cell line (MA-104 cells and its derivatives like MARC- 145 cells) was discovered to be permissive to PRRSV (Kim et al., 1993) and was subsequently used in modified live vaccine production process. Until now, MARC- 145 cells are still the only cell line used for vaccine production. Recently, 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.

SUMMARY

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.

More particularly, it is an aspect of the present invention to provide an isolated porcine lung epithelial cell line for use in the production of the porcine reproductive and respiratory syndrome (PRRS) virus.

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:

- culturing a cell from a porcine lung epithelial cell line in the presence of a sample containing a PRRS virus under conditions suitable for efficient PRRS viral replication to obtain PRRS viral particles; and

- harvesting the PRRS viral particles.

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:

- culturing a cell from a porcine lung epithelial cell line in the presence of a sample suspected of containing a PRRS virus under conditions suitable for efficient

PRRS viral replication; and

- detecting the presence or absence of PRRS viral particles in said cells.

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 :

- contacting a sample to be tested with a porcine reproductive and respiratory syndrome (PRRS) virus produced by the PRRSV production method as defined above, under conditions sufficient to form an immune complex; and

- detecting the presence or absence of the immune complex.

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:

- cells from a porcine lung epithelial cell line as defined above; - a binding means capable of specifically bind to a PRRS virus;

- a reagent to detect binding means-PRRS virus complex;

- 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.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 : Cytopathic effect (CPE) observed in SJPL PRRSV-infected cells.

Compared to mock and NPTr none permissive PRRSV-infected cells, only SJPL lAF-Klop infected cells develop CPE and it is higher when the cells are infected with a higher MOI.

Figure 2: Detection of the viral nucleocapsid protein (N) in PRRSV-infected cells by an immunofluorescence assay (IFA).

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.

Figure 3: PRRSV infectious viral particles production in SJPL after five passages.

Initially, cells were infected with 0.005 MOI of lAF-Klop PRRSV strain and the amount of virus detected in the inoculum (Inocul.) is indicated in the graph. Infectious viral particles production in SJPL and quantified by PRRSV real-time PCR assay

(SJPL/by PCR); infectious viral particles production in SJPL and quantified by TCID ₅₀ /mL in MARC-145 infected cells (SJPL/by MARC-145); infectious viral particles production in MARC-145 cells and quantified by PRRSV real-time PCR assay (MARC-145/by PCR); and infectious viral particles production in MARC-145 cells and quantified by TCID ₅₀ /mL in MARC-145 infected cells (MARC-145/by MARC- 145).

Figure 4: PRRSV IAF-Klop strain replication kinetics in MARC-145 and SJPL cells.

10 ⁵ MARC-145 and SJPL cells were infected with PRRSV IAF-Klop 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 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.

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. At 5 days post-infection, the infected cells were fixed and colored to visualize the plaque formation.

Figure 7: PRRSV cell permissivity of different SJPL cell clones obtained following end point dilution cloning method.

An end point dilution method was used to obtain 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 α7 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.

DETAILED DESCRIPTION OF THE INVENTION

The present inventors have surprisingly discovered that porcine lung epithelial cell lines find an advantageous use in the propagation of the porcine reproductive and respiratory syndrome (PRRS) virus. Indeed, 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.

1. Cell lines contemplated by the present invention

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.

In an aspect of the invention, the contemplated cell line has the characteristics of the SJPL cell line (deposited with the American Type Culture Collection (ATCC) on

April 5, 2001 , and assigned accession number PTA-3256). It will be understood that

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.

In this connection and as one skilled in the art may appreciate, the specific characteristics of a cell line contemplated by the present invention are that:

- it is a porcine lung epithelial cell line; - it is continuous, i.e., able to propagate indefinitely in tissue culture;

- it is morphologically similar to SJPL; and

- it produces desired titers of PRRS virus.

The term "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. For instance, a desired titer contemplated by the present invention may be a titer of at least 10 ⁵ TCID ₅₀ /mL.

In a specific aspect of the invention, the contemplated cell line used for PRRS virus propagation or production consists of the SJPL cell line.

Briefly, 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.

2. Production method of the invention

The 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:

- culturing a cell from a porcine lung epithelial cell line as defined above in the presence of a sample containing a PRRS virus under conditions suitable for efficient PRRS viral replication to obtain PRRS viral particles; and - harvesting the PRRS viral particles.

As used herein, 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.

It will be understood that the "conditions suitable for efficient PRRS viral replication" are known to one skilled in the art. Such conditions may be for instance those described in the Example section.

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. In the case of 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. It will be further understood that 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. For instance, such a PRRSV antigen may be, but not limited to, GP ₅ , M and N structural proteins of PRRSV.

In view of the above, it may be appreciated that the present invention provides an efficient method for the production of vaccines for treating or preventing a PRRS virus infection. As used herein, the term "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.

Due to the adequate permissiveness to PRRS virus, the contemplated porcine lung epithelial cell lines of the invention allow to produce a vaccine from a large spectrum of PRRS virus strains. Indeed, such 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.

Furthermore, due to the fact that there is substantially less cytopathic effect of

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.

3. Detection and quantification methods of the invention The porcine lung epithelial cell lines contemplated by the present invention may further be used, for instance, in PRRS virus detection and quantification methods.

In this connection, it is another aspect of the invention to provide a method for diagnosing a PRRS virus infection in an animal, such as a pig. The method of the invention comprises the steps of:

- culturing a cell from a porcine lung epithelial cell line as defined above in the presence of a sample suspected of containing a PRRS virus under conditions suitable for efficient PRRS viral replication to obtain PRRS viral particles; and

- detecting the presence or absence of PRRS viral particles in said cells. The term "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. These techniques include, but are not limited to, cell cytopathic effect assay, immunofluorescence assay (IFA) and PCR.

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.

More particularly, 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. Such a method comprises the steps of :

- contacting a sample to be tested with a porcine reproductive and respiratory syndrome (PRRS) virus produced by the PRRSV production method of the invention, under conditions sufficient to form an immune complex; and

- detecting the presence or absence of the immune complex.

Techniques for detecting the presence or absence of anti-PRRSV antibodies are well within the knowledge to one skilled in the art. These techniques include, but are not limited to, immunofluorescence assay (IFA) and ELISA.

The term "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.

In this connection, it is another aspect of the invention to provide a kit for determining the presence or absence of a porcine reproductive and respiratory syndrome (PRRS) virus in a sample. The kit comprises:

- cells from a porcine lung epithelial cell line as defined above;

- a binding means capable of specifically bind to a PRRS virus;

- a reagent to detect binding means-PRRS virus complex; - optionally a biological reference sample lacking a PRRS virus 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.

With respect to the binding means contemplated by the present invention, 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.

As mentioned above, such kits typically comprise two or more components necessary for performing a diagnostic or quantification assays. Components may be compounds, reagents, containers and/or equipment. For example, 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. Alternatively, a kit may be designed to detect the PRRS virus genetic material in a biological sample. Such 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. 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.

EXAMPLES

General Materials and Methods Cells and viruses. 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). The European genotype (EU) PRRSV reference strain, the Lelystad virus (LV) (Wensvoort, 1993; Wensvoort et al., 1992; Wensvoort et al., 1991) 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). In some experiments, 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. 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 μg/mL of amphotericin B, and 10 mM HEPES buffer. Following propagation of the PCV-2 in PK15A cells, 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). Then, cells were incubated during 10 minutes at room temperature with a PBS solution containing 1% Triton X-100. After removing the Triton X-100 solution, the cells were washed three times with a PBS-Tween 20 solution (PBS containing 0.02% Tween 20) and cells were kept at 4ºC for future use. Acetone and PFA fixed cells were washed three times with a washing buffer (PBS containing 0.02% Tween 20 and 1 % bovine serum). Then, the α7 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. Finally, cells were visualized using a DMI 4000B reverse fluorescence microscope, image of the cells were taking with a DFC 490 digital camera and the image were analyzed using the Leica Application Suite Software, version 2.4.0 (Leica Microsystems). The same techniques was used to detect the nucleocapsid protein (N) of PCV-2, except that a specific polyclonal pig serum was used as the primary antibodies (diluted 1/200) and an anti-swine PE conjugated antibodies (Abeam, USA) was used as the secondary antibodies (diluted as suggested by the manufacturer).

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. 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 (Coming) 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. At different time postinfection, 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. At 6 days post-infection, cells and supernatant were collected and submitted to one freeze and thaw cycle at -70ºC. Then, 0.5 mL of cells lysates were collected to infect one well/sample of a newly seeded MARC-145 and SJPL 6 wells cell culture plate. Two rounds of subsequent cell infections with 0.5 mL of supernatant of the previous infected cell cultures were used to infect one well/sample of newly seeded 6 wells cell culture plate using the same protocol. The CPE was observed by light microscopy every day until the end of the cell infection experiment at 5 days post-infection. At the final fourth passage, the cells and supernatants were submitted to one freeze and thaw cycle at -70ºC. Then, 8 wells/samples of a newly cells seeded 96 wells plate were infected with 10 μL/well of cell lysates to realize IFA to detect the expression of the N protein.

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). Thereafter, three cycles of frozen-thaw were performed to release infectious viral particles from the supernatants and cell pellets. Afterwards, supernatants and cell pellets were centrifuged at 4000 rpm at 4ºC during 10 min to remove cellular debris. Mock-infected controls were included in each experiment. All experiments were repeated two times in triplicate.

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. At 5 days post-infection, 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.

SJPL cells cloning by 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. After 3 consecutive cell passages, three clone cells were tested for their ability to permit PRRSV replication. Three clones (6,10 and 11) were seeded in a 24- well plate (1X10 ⁵ cells/well), and were infected with the PRRSV North American IAF-

Klop strain at 1 MOI. After 3 days post-infection, the infected cells were fixed with 4% PFA solution and permeabilized with a 0.5% Triton X-100 solution. Then, the PRRSV N protein expression was detected by a specific immunofluorescence assay using the α7 anti-N protein of PRRSV antibody. For each clone, none infected cells (Mock) was included as a negative control. In addition, the parental SJPL cells were use as a positive control.

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). To further establish if the IAF-Klop NA strain could replicate in SJPL, IFA was realized to detect the expression of viral protein such as the nucleocapsid protein (N) of the virion. Following the IFA, the N protein was detected only in SJPL infected cells (Figure 2) and not in mock infected cells. Moreover, N protein expression was not detected in PRRSV NPTr infected cells (data not shown). Furthermore, results presented in Figure 2 indicated that N protein was also expressed in LV SJPL infected cells. This latest result shows that PRRSV LV European strain is able to infect SJPL cells indicating that both genotypes of PRRSV are able to infect SJPL cells and subsequently expressed viral proteins in infected cells. Even if some viral proteins could be detected in infected cells, this finding does not guaranty the production of PRRSV infectious viral particles in SJPL infected cells. Consequently, another experiment was conducted to demonstrate without any doubt the production of PRRSV infectious particles in SJPL infected cells. As illustrated in Figure 3, a very small amount of IAF-Klop PRRSV strain was used to infect SJPL cells. Interestingly, no infectious virus could be detected following titration in MARC-145 cells in the initial inoculum. Then, after one passage in SJPL cells, the amount of virus increased dramatically and significantly from 0 to 1 X 10 ⁸ TCID ₅₀ AnL (Figure 3). This latest result indicates that SJPL cells could efficiently amplify and produce PRRSV infectious viral particles. Moreover, since the same inoculum was used to infect SJPL and MARC-145 cells, the amount of virus recovered after the first passage in SJPL was 4.37 X 10 ² times higher compared to the amount of virus recovered from MARC-145 infected cells (Figure

3). This latest result suggests that the SJPL cells is believed to be more suitable for PRRSV replication and infectious virions production than MARC-145 cells.

EXAMPLE 2: PRRSV replication in SJPL cells is not inhibited by PCV-2 coinfection compared to the observations made in coinfected MARC-145 cells. As previously reported by others, interferon alpha (IFNα) could inhibit the PRRSV replication in MARC-145 cells (Chang et al., 2005). Also, it was found that porcine circovirus type 2 (PCV-2) viral stock could be contaminated with IFNα and consequently following PRRSV and PCV-2 coinfection, the PRRSV replication is inhibited in MARC-145 infected cells (Chang et al., 2005). Thus, it was interesting to establish if PCV-2 viral stock could inhibit the replication of PRRSV lAF-Klop strain in SJPL cells. Consequently an experiment was set up to determine the impact of PRRSV and PCV-2 coinfection in SJPL cells. As previously reported, PCV-2 inhibit the replication of PRRSV in MARC-145 cells since no PRRSV N protein expression was detected in coinfected MARC-145 cells (Table 1). Interestingly, PCV-2 did not affect the PRRSV N expression in SJPL suggesting that IFNα does not inhibit the replication of PRRSV in SJPL cells (Table 1). This finding is believed to have an important impact since even if PRRSV viral stock are contaminated with PCV-2 and IFNα, such will not have an affect on the viral production in SJPL cells.

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.

To establish if their was any differences in the efficiency of virus particles production of both PRRSV genotypes in SJPL cells compared to MARC-145 cells, the PRRSV lAF-Klop and LV reference strains replication kinetics were evaluated in both MARC-145 and SJPL cell lines. The statistical analyses were performed using the GraphPad Prism version 4 software to determine if there was statistically significant differences between MARC-145 and SJPL cell lines with regards to their efficiency to allow the replication of PRRSV. Using the regular two-way ANOVA combined with the Bonferroni post-tests models, statistically significant differences between MARC-145 and SJPL cells were found at some time points with regards to viruses replication kinetics (such as for LV at 96 and 120 hrs post-infection where there was a higher amount of LV infectious viral particles production in the supernatant of SJPL infected cells compared to MARC- 145 infected cells supernatant: P<0.01). Nonetheless, and as illustrated in Figures 4 and 5, no significant difference was observed between MARC-145 and SJPL cells with regards to viruses kinetic replication for both PRRSV lAF-Klop and LV reference strains. These latest results indicate that SJPL cells are as efficient as the MARC-145 cells for infectious viral particles production.

Example 5: Plaque assays comparison results of PRRS virus plaque size formation in MARC-145 and SJPL infected cells.

As illustrated in Figure 6, 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. Interestingly, this latest result confirms that SJPL cells develop less CPE when infected with PRRSV compared to MARC-145 cells. Furthermore, 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.

Example 6: End point dilution SJPL cells cloning result

When different SJPL cell clones were tested for their permissivity to PRRSV IAF-klop infection, it was found that each cell clone possesses its own capacity level to permit the expression of PRRSV viral proteins as illustrated in

Figure 7. Interestingly, one clone was not permissive to PRRSV (clone 10), another clone (clone 6) has a lower capacity for PRRSV permissivity compared to the parental SJPL cells, and another clone (clone 11) expressed higher amount of viral proteins then the parental SJPL cells. Consequently, this result indicates that by selecting appropriate SJPL cell clones, it is possible to increase PRRSV infectious viral particles production.

BRIEF CONCLUSION

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:

- more efficient for the production of PRRSV; and

- more efficient detection of viremia; and

- PRRSV replication is not affected by the presence of PCV-2 and subsequently IFNα.

Moreover, the use of the SJPL cell line is efficient for the production of both genotypes of PRRSV (NA and EU).

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