Abstract:
A method for determining the capability of an arterivirus to replicate in a permissive cell is disclosed. The method includes determining an amino acid at a position that corresponds to an amino acid of a protein of a porcine reproductive and respiratory syndrome virus. The invention also discloses a method for determining the capability of an arterivirus to replicate in a green monkey cell line. The invention further discloses a method for producing arterivirus in a green monkey cell line wherein the virulence of the arterivirus is maintained and the virus yield is increased. Methods for determining the attenuation of an arterivirus and for attenuating the virulence of the arterivirus by changing amino acids are further disclosed.

Description:
TECHNICAL FIELD 
     The invention relates to the field of virology and more particularly, to the field of vaccine production. More specifically, the invention relates to the in vitro propagation of virus and to the adaptation of a virus to a cell line and the attenuation of a virus. 
     BACKGROUND 
     Porcine reproductive and respiratory syndrome virus (PRRSV) is a small, enveloped, positive-stranded RNA virus which belongs to the genus arterivirus. PRRSV was isolated in Europe and in the United States of America in 1991 (Collins et al., 1992, Wensvoort et al., 1991). The genus arterivirus also includes equine arteritis virus (EAV), lactate dehydrogenase-elevating virus (LDV) and simian hemorrhagic fever virus (SHFV). The genus arterivirus is the only member in the family of the Arteriviridae. The family Arteriviridae and the Coronaviridae are grouped into the order Nidovirales. 
     In vivo, PRRSV reproduces primarily in porcine alveolar lung macrophages and blood monocytes. (Wensvoort et al., 1991). Macrophages from other tissues, such as heart, tonsil, spleen, turbinates and choroid plexus are also susceptible to PRRSV infection. 
     In vitro, the porcine virus can be passaged to primary cultures of porcine alveolar lung macrophages and blood monocytes, to cultures of the African green monkey kidney cell line MA-104 and to the derivative cell lines CL2621 and MARC-145. (Collins et al., 1992, Wensvoort et al., 1991). A few cell lines exist that the porcine virus cannot penetrate, such as BHK-21 and Vero cells, but once the genome of these cell lines is brought inside the cells artificially, the porcine virus is able to replicate and produce new virus particles. (Meulenberg et al., 1998). 
     PRRSV enters porcine alveolar macrophages and MARC-145 cells via receptor-mediated endocytosis. The uptake of PRRSV by the host cell is a multi-step process in which one or two viral constituents appear to interact with at least one host proteonic factor before virus-containing clathrin-coated pits are formed. (Kreutz, 1998). The entry process of PRRSV into porcine macrophages differs from the binding and internalization of PRRSV into MARC-145 cells. A membrane protein of about 210-kDa has been identified as being a putative receptor for PRRSV on macrophages (Duan et al., 1998) while a heparin-like molecule is suggested to be important for the binding of PRRSV to MARC-145 cells. (Jusa et al., 1997). 
     PRRSV has a concise genome structure (for a review, see, Snijder, and Meulenberg, 1998) which is 15,098 nt in length without the poly-A tail and contains 9 open reading frames (ORFs) flanked by a 5′ and 3′ non-translated region (NTR) of 221 and 114 nucleotides, respectively. The two 5′-terminal ORFs designated 1A and 1B ( FIGS. 1A and 1B ) comprise about 75% of the genomic RNA. The structural region of the RNA contains 7 ORFs designated 2A, 2B, and 3 to 7 ( FIGS. 1A and 1B ) and occupies the 3′-terminal third of the genome. ORF 1A/B encodes the non-structural proteins (nsps) involved in replication of the RNA genome and the forming of a 3′ nested set of subgenomic RNAs (sgRNAs). 
     The first two N-terminal proteins named nsp 1α and nsp 1β, respectively, have been studied for PRRSV. The functions of the other nsps are based on predictions derived from sequence homologies with EAV. Nsp 1α and nsp 1β are papain-like cysteine proteases and together with nsp2 and nsp4, a cysteine protease and a serine protease, respectively, the entire polyprotein ORF1 (a/ab) is assumed to be cleaved into 12 nsps. Nsp9 and 10 are predicted to be the replicase subunits. 
     The 3′-terminal end of the genome encodes the structural proteins. The ORFs 2A to 6 encode six structural membrane-associated proteins and the most 3′-terminal ORF encodes the N protein (ORF7). The minor glycoproteins are GP2a (formerly named GP2), GP3 and GP4. The GP2a and the GP4 proteins are class I membrane proteins and have molecular masses of 27-30 and 31-35 kDa, respectively. Both N-glycosylated proteins are constituents of the virion. The other minor N-glycosylated structural protein of 42-50 kDa is GP3 and is encoded by ORF3. The nature of GP3 was unclear until the present disclosure. 
     The three major structural proteins are GP5, M and the N protein. GP5, a N-glycosylated protein of 24-26 kDa, is the most variable protein in its sequence. The most conserved protein is the non-glycosylated M protein. The M protein is a 18-19 kDa integral membrane protein and appears to have a function in viral infectivity. The M protein forms a heterodimer with GP5 which is present in the virion. The N protein is a small, highly basic, nucleocapsid protein of 14-15 kDa which interacts with the viral RNA during assembly. (Snijder &amp; Meulenberg, 1998). 
     Sequence comparisons and studies involving the antigenic nature of PRRSV strains reveal two distinct groups designated as EU (European) and US (North American) strains. (Wensvoort et al., 1992, Snijder &amp; Meulenberg, 1998). Another indication of the observed diversity between the EU strain and the US strain from the two continents is the fact that EU field isolates initially replicate easier in alveolar lung macrophages, whereas US isolates are recovered easier in the African green monkey kidney cell line MA-104 or derivatives thereof, such as CL2621 or MARC-145 cells. (Bautista et al., 1993). 
     The candidate proteins which will adsorb to the host cell surface of permissive cells are GP2a, GP2b, GP3, GP4, GP5, the M protein or a heterodimer of GP5 and the M protein. 
     Typically, when viruses are replicated in vitro in cell lines derived from a different species, the viruses tend to attenuate in the process of adapting to the cell line. This adaptation characteristic is widely used in vaccine development and is also true for PRRSV replicated in monkey cell lines (Collins et al. 1992). 
     SUMMARY OF THE INVENTION 
     In one embodiment, changes in the PRRSV genome that occur during the adaptation process are related to adaptation to a cell. This aspect is useful because the PRRSV can be adapted to replicate in a permissive cell without further loss of virulence. 
     In another embodiment, changes in other sites of the genome are more related to attenuation. This aspect is useful because it enables a person skilled in the art to control the level of attenuation of a PRRSV. 
     A method to determine the capability of an arterivirus to replicate in a permissive cell, such as a green monkey cell, comprising determining the amino acid positions that correspond to the amino acid positions 75-107 of GP2a of PRRSV isolate I-1102 is disclosed. In a further aspect of the present invention, a method for determining the amino acid positions that correspond to the amino acid position 88 and/or amino acid position 95 of GP2a of PRRSV isolate I-1102 is disclosed. The knowledge and methods disclosed herein will enable a person of ordinary skill in the art to identify the capability of a PRRS virus to replicate in a permissive cell, such as a green monkey cell. 
     Further, a method for increasing the capability of an arterivirus to replicate in a permissive cell, such as a green monkey cell, comprising changing the amino acids at amino acid positions 75-107 is disclosed. In another aspect, the method comprises changing the amino acid at amino acid position 88 from a valine to any other amino acid, such as a phenylalanine, and/or changing the amino acid at amino acid position 95 from a phenylalanine to any other amino acid, such as a leucine is disclosed. 
     In a further embodiment, the concomitant amino acid changes in the two sites of GP2a enhance the adaptation of a PRRSV for a permissive cell, such as a green monkey cell. The method is suited for the production of an arterivirus and/or a nucleic acid in a permissive cell and/or cell line, wherein the virulence of the arterivirus is maintained while the virus and/or nucleic acid yield from the permissive cell and/or cell line is increased. 
     In another embodiment, a method for determining the attenuation of an Arterivirus comprising determining the amino acids positions that correspond to the amino acid positions 121-148 of GP5 of PRRSV isolate I-1102 is disclosed. The method further comprises determining the amino acid position that corresponds to the amino acid position 136 of GP5 of PRRSV isolate I-1102. A method to control and/or increase the attenuation of an arterivirus comprising changing amino acids at amino acid positions 121-148 of GP5 of PRRSV isolate I-1102 by changing the amino acid at amino acid position 136 from a cysteine to any other amino acid, such as a tyrosine, is disclosed. 
     In a further embodiment, a method for determining the attenuation of an Arterivirus comprising determining the amino acids at positions that correspond to the amino acid positions 651-675 and/or amino acid positions 2331-2355 of ORFlab of PRRSV isolate I-1102 is disclosed. The method further comprises determining the amino acid at a position that corresponds to the amino acid position 663 and/or amino acid position 2343 of ORFlab of PRRSV isolate I-1102. A method to control and/or increase the attenuation of an arterivirus comprising changing amino acids at amino acid positions 121-148 of GP5 of PRRSV isolate I-1102 by changing the amino acid at amino acid position 136 from a cysteine to any other amino acid, such as a tyrosine, is disclosed. 
     In another embodiment, a method for determining the attenuation of an Arterivirus comprising determining the amino acids positions that correspond to the amino acid positions 651-675 and/or amino acid positions 2331-2355 of ORFlab of PRRSV isolate I-1102 is disclosed. In a further aspect, the method comprises determining the amino acid at positions that corresponds to the amino acid position 663 and/or amino acid position 2343 of ORFlab of PRRSV isolate I-1102. 
     A method to control and/or increase the attenuation of an arterivirus comprising changing the amino acids at positions that correspond to the amino acid positions 651-675 and/or amino acid positions 2331-2355 of ORFlab of PRRSV isolate I-1102 is disclosed. In a further aspect, the method comprises changing the amino acid at amino acid position 663 from a glutamic acid to any other amino acid, such a lysine, and/or changing the amino acid at amino acid at position 2343 from a valine to any other amino acid, such as an alanine. The methods also allow for the production of an attenuated arterivirus. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIGS. 1A and 1B : Schematic representation of amino acid differences between the LV4.2.1 strain, which is adapted to MA-104 cells, and strain pABV437, which is the infectious cDNA clone of the field strain Ter Huurne is shown in (A). An overview of generated full length constructs based on pABV437 to observe the responsible mutations for the adaptation phenotype is shown in (B). The numbers in the bar correspond to the 9 open reading frames. 
         FIG. 2A : Multi-step growth curves on MARC-145 cells. Virus titer is in TDID50/ml. 
         FIG. 2B : Multi-step growth curves on PAMs. Virus titer is in TCID50/ml. 
         FIG. 2C : Immunostaining of infected cells using a PRRSV-specific Mab (122.17) against the nucleocapsid protein. The total amount of virus positive cells was counted per 2 cm 2 . 
         FIG. 3 : Mean virus titer per group of pigs after inoculation with vABV688, vABV707, vABV746 and vABV437 as determined by end point dilution. Significance of the differences between the-virus groups (p≦0.05) was determined using the Chi test. 
     
    
    
     DETAILED DESCRIPTION 
     Materials and Methods. 
     Cells and Viruses. 
     Porcine alveolar lung macrophages (PAMs) were maintained in MCA-RPMI-1640 medium containing 5% FBS, 100 U/ml penicillin and 100 U/ml streptomycin. CL2621-cells were propagated in Eagle&#39;s minimal essential medium supplemented with Hanks salts (Gibco BRL), 10% FBS, 100 U/ml penicillin, 100 U/ml streptomycin, 1.5% sodium bicarbonate and 1% L-glutamine. Serial passage of the recombinant PRRSVs vABV688 and vABV437 was performed as described. (Meulenberg et al, 1998). Challenge virus LV-Ter Huurne, a virulent European wild type isolate of PRRSV, was isolated during the 1991 epizootic from a clinical case of PRRSV in the Netherlands and propagated on PAMs. (Wensvoort et al, 1991). SDSU# 73, a virulent American wild type isolate of PRRSV (passage 3 on CL2621-cells), was kindly provided by Dr. E. Vaughn (Boehringer Ingelheim, Animal Health, Ames, Iowa) and propagated on MA-104 cells. Virus titers (expressed as 50% tissue culture infective doses (TCID 50 ) per ml) were determined on PAMs by end point dilution (Wensvoort et al, 1986) and calculated according to Reed and Muench. (Reed et al, 1938). 
     Inoculation of pigs with PRRSV recombinants and test on their in vivo stability. 
     Three groups of three 8-week-old Dutch Landrace/Yorkshire (LY) SPF pigs, tested free of antibodies against PRRSV, were inoculated on the same day intranasally with 2 ml of a recombinant virus stock of passage 5 each containing a titer of 10 5  TCID 50 /ml. The three groups were housed in isolated pens. Experimental procedures and animal management procedures were undertaken according to the Dutch legislation animal experiments. Serum samples were collected at day 0, 2, 4, 7, 9, 11, 14, 16, 18 and 21. At day 21, the pigs were sacrificed. 
     Immunization of pigs with vABV688 and challenge with LV-Ter Huurne and SDSU#73. 
     For each recombinant virus, two groups of five 8-week-old Dutch LY SPF pigs, lacking antibodies against PRRSV, were immunized intramuscularly (half-way between the pinna of the right ear and the cranial ridge of the right shoulder blade) with 2 ml of a virus stock of 10 5  TCID 50 /ml. All groups were housed in isolated pens. Experimental procedures and animal management procedures were undertaken according to the Dutch legislation animal experiments. In order to determine the transmission of recombinant virus from the inoculated pigs, one naive sentinel pig was introduced into each group of inoculated pigs 24 hours post-vaccination and sacrificed 28 days thereafter. At day 28 post-vaccination, two animals were separated from one group of each mutant and challenged intranasally with 2 ml 10 5  TCID 50 /ml LV-Ter Huurne. Similarly, two animals were separated from the other group and challenged with 2 ml 10 5  TCID 50 /ml SDSU#73. The two challenged animals joined the other three vaccinates after 24 hours. At 28 days after challenge, all pigs were sacrificed. 
     A schematic representation of the amino acid changes is depicted in  FIGS. 1A and 1B . To confirm the efficacy of challenge, two non-inoculated animals were either inoculated intranasally with 2 ml 10 5  TCID 50 /ml LV-Ter Huurne or SDSU#73. These challenged animals joined three sentinel animals after 24 hours and were monitored for two weeks starting at the moment of challenge. Serum samples were collected for all animals three times a week starting on day 0. During the experiment, the animals were observed daily for signs of disease, i.e., fever (a rectal body temperature higher than 39.7° C.), diarrhea and respiratory distress. 
     Analysis of the genetic stability of the recombinant viruses. 
     The genetic stability of the recombinant viruses in pigs was tested by inoculation of PAMs with serum from these pigs taken at the last-virus-positive day, followed by sequence analysis of the viral RNA. In short, as soon as cytopathogenic effect (cpe) was detected, the culture supernatant was harvested and the viral RNA was isolated as described (16). The RNA was reverse transcribed with primer LV76 (5′-TCTAGGAATTCTAGACGATCG(T) 40 -3′ (SEQ ID NO: 1); antisense; nucleotide (nt) 15088). The region flanking the introduced mutations was amplified by PCR using primers LV9 (5′-CTGCCGCCCGGGCAAGTGCC-3′ (SEQ ID NO: 2); sense; nt 11746) and LV22 (5′-CATAATAACCCTCAAGTTG-3′ (SEQ ID NO: 3); antisense; nt 12715) for vABV688. Nt numbers are based on the sequence of the LV isolate as deposited in GenBank, Accession number M96262 (SEQ ID NO: 4). 
     The amplified fragments were analyzed in 2% agarose gels and the PCR fragments were excised from the gel and purified with SpinX columns (Costar). Sequence analysis of the fragments was performed using the antisense primer of the PCR, except for vABV688, for which primer LV24 (5′-AATCGGATCCTCAGGAAGCGTGCACACTGATGA-3′ (SEQ ID NO: 5); antisense; nt 12419) was used. The PRISM Ready Dye Deoxy Terminator cycle sequencing kit and the ABI PRISM 310 Genetic Analyzer (Perkin Elmer) were used to determine the sequences. 
     Virus Isolation. 
     10 5  PAMs/96 mm 2  were seeded in 96-wells plates and after one day, 50 μl of a 10-fold and 100-fold serial dilution of each serum sample was used to infect PAMs. After 48 hours, 25 μl of the culture supernatant was transferred to new PAMs seeded 16-24 hours prior to incubation. After 24 hours, the medium was discarded, the cells were washed with 0.05 M NaCl, dried and frozen for IPMA. (Wensvoort, 1986). 
     Virus Titration. 
     Virus titers were determined by end-point dilution on PAMs. (Wensvoort et al., 1986). Samples containing virus titers below the detection level (1,8) were considered negative. From the area under the curve of titer against time, the integrated virus titers after vaccination were determined. Statistical analysis was performed by the one-sided paired student&#39;s t-test. Results were considered statistically significant when the P-value was ≦0.05. 
     Immunoperoxidase Monolayer Assay (IPMA). 
     Immunostaining of PAMs was performed according to the method described. (Wensvoort et al, 1986). The expression of the EU- and US-PRRSV N protein was detected with monoclonal antibody (MAb) 122. 
     Detection of Antibodies. 
     The presence of antibodies against PRRSV in pig sera was determined by ELISA (IDEXX, Westbrook, Minn., US). 
     EXAMPLE 1 
     Identification of Adaptation Sites. 
     To identify important genomic regions for adaptation, the field isolate Lelystad virus was passaged 6 times on CL2621 cells followed by plaque purification for 3 times on the CL2621 cells and the virus was designated LV4.2.1. The entire sequence of the genome of the cell line-adapted strain LV4.2.1 was determined. RNA isolation, RT-PCR and PCR was performed yielding overlapping cDNA fragments of about 1.5 kb in length. All fragments were cloned into pGEM-T vector of PROMEGA using the TA-cloning strategy. When sequencing the whole sequence, every nucleotide was determined at least twice. To exclude mutations introduced by PCR-mismatches, a third or even a fourth cDNA-construct was made. When compared to the field strain Lelystad virus, 27 nucleotide differences in the coding region were identified and no nucleotide differences were identified in the 5′ and 3′ non-translated region. After translation, 8 amino acid differences resulted (Table 1). 
     
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Amino acid differences between LV4.2.1, the field virus Lelystad virus 
               
               
                 strain Ter Huurne, the infectious cDNA clone pABV437 and the 
               
               
                 US prototype field virus strain ATCC VR2332. 
               
             
          
           
               
                   
                 ORFIA polyprotein 
                 GP2A 
                 GP2B 
                 GP5 
               
             
          
           
               
                   
                 aa 663 
                 aa 1084 
                 aa 2343 
                 aa 29 
                 aa 88 
                 aa 95 
                 aa 27 
                 aa 136 
               
               
                   
                   
               
             
          
           
               
                 LV4.2.1 
                 Lys 
                 Leu 
                 Ala 
                 Ser 
                 Phe 
                 Leu 
                 Val 
                 Tyr 
               
               
                 Ter Huurne 
                 Glu 
                 Pro 
                 Val 
                 Pro 
                 Val 
                 Phe 
                 Ala 
                 Cys 
               
               
                 pABV437 
                 Glu 
                 Pro 
                 Val 
                 Ser 
                 Val 
                 Phe 
                 Val 
                 Cys 
               
               
                 ATCC-VR2332 
                 Gln 
                 Thr 
                 Val 
                 Leu 
                 Thr 
                 Leu 
                 Ile 
                 Trp 
               
               
                   
               
             
          
         
       
     
     It was concluded that the observed difference in growth properties between LV4.2.1 and the field virus strain Lelystad virus was due to a different amino acid sequence in a non-structural protein, a structural protein or in a combination of two or more amino acid changes. 
     Construction of full length cDNA with non-structural or structural gene mutations. 
     A set of full length constructs (pABV647, 688, 689, and 690) was constructed based on pABV437 in which mutations were introduced. In vitro transcribed full length RNAs were transfected into the non-permissive BHK-21 cells using lipofectin as a cationic reagent. After 24 h, the supernatants (p0) were transferred to MARC-145 cells and after 24 h to 48 h, immunostaining was performed using MAb 122.17 directed against the N protein to detect the effect of the introduced mutations. Recombinant viruses containing substitutions responsible for adaptation showed cell line adapted phenotypes on MARC-145 cells similar to that of the positive control LV4.2.1. For the positive control, PAMs were infected with the same amount of p0 to show infection on the primary target cell of PRRSV. The supernatant p0 of pABV437 and LV4.2.1 virus served as positive controls in infection experiments. 
     Two full length cDNA clones were constructed in which either mutations in the non-structural or in the structural region were introduced and designated as pABV647 and pABV690, respectively ( FIG. 1B ). In vitro analysis of these two constructs revealed that the RNA-transcript of pABV690 resulted in a phenotype similar to LV4.2.1. It was concluded that the mutations responsible for adaptation are located in the structural part of the genome. 
     The next step was to determine whether the mutations in GP2a or in GP5 had any effect on the observed cell line-adaptation. pABV688 contains two amino acid differences in GP2a, while pABV689 contains only the amino acid change in GP5 ( FIG. 1B ). After performing the screening assay, pABV688 with the two mutations in GP2a resulted in a cell line-adapted phenotype similar to LV4.2.1 and the GP5 recombinant pABV689 did not. It was concluded that adaptation to the cell line is in majority caused by the two mutations in GP2a. 
     Full Length Genome Constructs with Mutations in GP2a. 
     Two amino acid differences in GP2a were studied. Two new constructs were generated. pABV772 contains a phenylalanine at amino acid 88 and pABV773 contains a leucine at position 95 in GP2a ( FIG. 1B ). In vitro, both substitutions were better adapted for growth in MARC-145 cells than the positive control pABV437, but the substitutions caused less cell pathologic effects (CPE) than vABV688. 
     A multi-step and a one-step growth curve were performed to investigate the effect of the introduced amino acid residues on the infectivity process. The p 0 -medium of the recombinant viruses derived from pABV437/688/772/773 were harvested, transferred to PAMs and passaged three times (p3) in parallel with LV4.2.1 to increase the amount of virus. For further growth characterization, the presence of the introduced mutations was confirmed by sequence analysis and the TCID50/ml was determined (data not shown). Multi-step growth curves on both PAMs and MARC-145 were performed using a multiplicity of infection (m.o.i) of 0.05 with vABV437/688/772/773 and LV4.2.1. 10 6  cells were used in growth curves per 2 cm 2 . At different time points, virus was harvested and stored until the virus titer in TCID50/ml was determined. 
     Introduction of a phenylalanine and a leucine in GP2a at amino acid 88 and 95, respectively, had a positive effect on the growth characteristics of a European strain of PRRSV on MARC-145 cells ( FIG. 2A ). No differences were observed when performing multi-step growth curves on PAMs ( FIG. 2B ). To secure the observed results, a total amount of 10 6  MARC-145 cells per 2 cm 2  were infected with a m.o.i. of 0.1 with supernatant p3 of vABV437/688/772/773 and LV4.2.1. After 12 h, an immunostaining was performed using a PRRSV-specific MAb (122.17) against the nucleocapsid protein and the total amount of positive cells were counted per 2 cm 2  ( FIG. 2C ). Based on the results, it was concluded that both amino acid residues at position 88 and 95 in the minor glycoprotein GP2a of PRRSV are important for adaptation to MARC-145 cells. 
     The arteriviral reproduction process in permissive cells can be defined in seven distinct steps. The first two processes are called the viral entry and include (A) attachment of the virus to the cell surface of PAMs or MA-104 (or derivatives thereof) and (B) penetration of the virus through the cell membrane. Next, (C) the arterivirus needs to be uncoated in the cytoplasm before (D) replication, transcription and translation can occur. The next step is (E) RNA encapsidation and assembly, followed by (F) release of mature virions into the extracellular space which occurs before (G) spread of the virus can take place. Using a 12 h-time point in the one-step growth curves and knowing that the viral reproduction cycle of PRRSV takes about 10 hours, the observed difference in the amount of positive stained cells is caused by an increased number of cells being successfully infected (A-D). The observed difference is not due to a difference in encapsidation of the RNA, release of the newly produced virions or spread of the virus through a monolayer of MARC-145 cells (E-G). Since a role of glycoprotein GP2a in release of-viral RNA into the cytoplasm during uncoating (C) or in transcription and translation (D) is most unlikely, it was concluded that both residues, or a domain in which both residues are present, are important for the entry process, i.e., attachment and/or penetration of the virus (A and B). 
     EXAMPLE 2 
     Animal Experiments with Cell Line-Adapted Recombinant PRRSV vABV688. 
     In recombinant vABV688, amino acids 88 and 95 of the minor envelope glycoprotein GP2a were mutated and resulted in improved growth on MARC-145 cells. This characteristic facilitates the production of virus. In cell culture, these recombinant viruses were shown to be genetically stable and able to grow to virus titers sufficient to perform animal experiments. The properties of these PRRSV recombinants were studied with regard to safety and protective efficacy in animal experiments. The properties of the recombinant viruses were compared with those of virus derived from an infectious cDNA copy, vABV437. This virus is identical to wild type virus except for a PacI-restriction site and is, therefore, assumed to have similar properties as wild type virus. First, the in vivo genetic stability of vABV688 was determined in 8-week-old pigs. Subsequently, the immunogenicity, attenuation and efficacy of these viruses were tested in a homologous and heterologous immunization-challenge experiment in young pigs. 
     Genetic Stability of the PRRSV Recombinant vABV688 in 8-Week-Old Pigs. 
     In the first experiment, the genetic stability of the PRRSV recombinants was determined in vivo. Sequence analysis of viral RNA isolated from serum of all pigs inoculated from 14 days post inoculation (DPI) with the recombinant viruses at the last virus-positive day was performed. Sequence analysis of the fragments obtained by RT-PCR showed that the introduced mutations were present and that no additional changes were introduced in the domain of GP2a indicating that the recombinant viruses were genetically stable in vivo. 
     Seroconversion of Virus inoculated Pigs and Sentinel Pigs. 
     In the second experiment, it was determined whether pigs inoculated with the PRRSV recombinants and, subsequently, the sentinel pigs introduced into the inoculated groups had seroconverted. The presence of PRRSV antibodies was measured in IDEXX ELISA. Antibodies were detected in pigs inoculated with recombinant virus (i.e., ELISA sample-to-positive (S/P) ratio&gt;0.4) which indicated proper exposure of the viruses to the animals. Virus transmission is one of the characteristics of virulence. The sentinels had seroconverted at day 14 (vABV688) indicating that transmission of virus from the inoculated animals to these sentinels and, thus, conservation of the virulence. 
     Duration and Height of Viremia after Inoculation. 
     To determine the presence of virus in the serum of inoculated animals, virus isolation was performed for all sera collected. From the virus positive sera, virus titers were determined ( FIG. 3 ). All viruses induced viremia in the pigs and ranged from 2 dpi to 25 dpi. 
     Viremia after Transmission of Virus to Sentinel Pigs. 
     To determine spread of the recombinant virus to non-inoculated pigs, one sentinel animal was introduced into each inoculated group at 24 hours after inoculation. The virus titration of all virus positive sera indicated that the maximum virus titers did not differ between inoculated pigs and sentinels (data not shown). 
     Challenge of virus-inoculated animals with homologous and heterologous PRRSV and transmission of challenge virus. 
     To test whether inoculation with the recombinant virus protects pigs against wild type PRRSV, the inoculated pigs were challenged with EU- and US-PRRSV. Pigs inoculated with vABV437 remained virus negative after challenge with LV-Ter Huurne. Only one animal of the vABV688-inoculated pigs became virus positive for one day. After challenge with SDSU#73, all inoculated animals became viremic. All inoculated, unchallenged animals remained virus negative after they were united with their group members that were challenged with LV-Ter Huurne. In pigs inoculated with vABV688, viremia was induced after the inoculated pigs were united with SDSU#73-challenged pigs. In pigs used as challenge control, viremia was induced in all LV-Ter Huurne and SDSU#78-challenged pigs as well as in all sentinel animals (data not shown). 
     Clinical Observations. 
     After inoculation with the virus, no severe clinical signs of the disease were monitored. Only moderate rectal temperature increases were noted in all inoculated groups (data not shown). After challenge with LV-Ter Huurne in vABV688-inoculated animals and all SDSU#73-challenged animals, no temperature increases were measured. 
     EXAMPLE 3 
     Attenuation of PRRS mutant virus strains with mutations in GP5 and/or ORFlab in addition to the mutations in GP2a (LV4.2.1). Comparison of virulence of 3 PRRS virus strains in an animal model. 
     The clinical signs of PRRSV may differ among strains after exposure of pigs to PRRSV. The US PRRSV strains often cause respiratory and reproductive problems, whereas EU PRRSV strains predominantly cause reproductive problems. (Steverink et.al. 1999). Respiratory signs and the extent of lung involvement may vary per strain and are not consistently reproduced under experimental conditions. An infection model should include parameters to study and quantify the virulence of the virus based on frequency and severity of clinical signs, viral parameters (viremia, virus excretion and virus transmission) and seroconversion. Furthermore, duration, height and frequency of viremia and viral excretion after vaccination could also be important parameters to study vaccine safety. The same parameters after PRRSV challenge of vaccinated animals are important for vaccination efficacy since reducing virus shedding and virus transmission among pigs is an important target aiming at the control of PRRSV. Therefore pigs of 6 to 8 weeks old and 6 months old were infected with 3 different PRRSV strains, i.e., LV-Ter Huurne, SDSU#73 and LV4.2.1. 
     The animals were conventional Landrace pigs obtained from a PRRSV-free farm in Denmark and born from PRRSV-unvaccinated sows. The pigs were free from antibodies against PRRSV as measured by an ELISA (IDEXX). Viremia was first detected in all pigs at 3 dpi except for the 6 month-old group infected with LV4.2.1 where viremia was first detected at 7 dpi and in one animal of this group, no viremia was detected. In 6-8 week-old pigs, viremia was detected until 42 dpi with a maximum frequency of positive animals of 88% and a virus titer ranging from 3, 4, 7, 10, 11 or 14 dpi and from 2.1-2.6. 
     In 6 month-old pigs, viremia was detected until 28 dpi with a maximum frequency of 65% positive animals per time-point and virus titer ranging from 0.7-1. After 24 dpi, both LV-Ter Huurne and SDSU#73 were intermittently detected in the serum of pigs. LV4.2.1-infected pigs showed a shorter duration of viremia which was last detected at 21 dpi. The frequency of positive pigs at 7 and 10 dpi was significantly lower, had a maximum of 41% (p&gt;0.05) and a lower  10 log virus titer as compared to LV-Ter Huurne and SDSU#73. Kinetics of viremia was significantly different at 14 dpi for LV-Ter Huurne-infected pigs and SDSU#73-infected pigs. 
     More pigs of 6-8 weeks excrete virus (maximum of positive animals per time-point was 50%) than pigs of 6 months (maximum of 37%). Furthermore, 6-8 week-old pigs showed a higher virus  10 log titer (between 0.2 and 1.9) in tonsillar swabs than 6 month-old pigs (ranging from 0.0 to 0.3) at day 3, 4, 7, 10 or 11 and 14. Per time-point, more pigs infected with LV-Ter Huurne excrete virus (maximum 100%) as compared to LV4.2.1 (maximum 17%) and SDSU#73 (maximum 50%). In addition, all pigs infected with LV-Ter Huurne excreted virus, but not all pigs infected with LV4.2.1 or SDSU#73 excreted virus. The  10 log virus titer in tonsillar swabs was higher for LV-Ter Huurne-infected pigs ranging from 0.0 to 3.0, while the  10 log titer of the LV4.2.1 infected group was 0.0 and the  10 log titer of the SDSU#73 infected pigs ranged from 0.0 to 0.5. At 14 dpi, pigs were still excreting virus. 
     LV-Ter Huurne and SDSU#73 appear to be the most virulent due to the occurrence of a high level of virus-positive pigs, a long duration of viremia of both virus strains, the instigation of most severe clinical signs of SDSU#73 and a high level of virus excretion for LV-Ter Huurne. LV4.2.1 was less virulent since it showed less clinical signs and a low viremia and virus excretion. The reduced virulence of LV 4.2.1 was confirmed by impairment in its ability to cause reproductive problems for gestation sows as compared to LV-Ter Huurne. (Steverink et al. 1999). Sows infected with LV-Ter Huurne and LV4.2.1 resulted in the same number of piglets, but the piglets of the sows infected with the higher virulent strain were weaker. In addition, the group infected with the higher virulent virus LV-Ter Huurne showed a higher frequency of virus positive piglets until 28 days post farrowing. (Steverink et al 1999). Apparently, the lower virulence of LV4.2.1 is not related to macrophage infection since the growth curve of LV4.2.1 in vitro on macrophages is comparable to the growth curve of LV-Ter Huurne. 
     REFERENCES 
     
         
         Bautista, E. M., Goyal, S. M., Yoon, I. J., Joo, H. S. &amp; Collins, J. E. (1993). Comparison of porcine alveolar macrophages and CL 2621 for the detection of porcine reproductive and respiratory syndrome (PRRS) virus and anti-PRRS antibody.  J Vet Diagn Invest  5, 163-5. 
         Collins, J. E., Benfield, D. A., Christianson, W. T., Harris, L., Hennings, J. C., Shaw, D. P., Goyal, S. M., McCullough, S., Morrison, R. B., Joo, H. S. &amp; et al. (1992). Isolation of swine infertility and respiratory syndrome virus (isolate ATCC VR-2332) in North America and experimental reproduction of the disease in gnotobiotic pigs.  J Vet Diagn Invest  4, 117-26. 
         Duan, X., Nauwynck, H. J., Favoreel, H. W. &amp; Pensaert, M. B. (1998). Identification of a putative receptor for porcine reproductive and respiratory syndrome virus on porcine alveolar macrophages.  J Virol  72, 4520-3. 
         Jusa, E. R., Inaba, Y., Kouno, M. &amp; Hirose, O. (1997). Effect of heparin on infection of cells by porcine reproductive and respiratory syndrome virus.  Am J Vet Res  58, 488-91. 
         Kreutz, L. C. (1998). Cellular membrane factors are the major determinants of porcine reproductive and respiratory syndrome virus tropism. Virus Res 53, 121-8. 
         Meulenberg, J. J., Bos de Ruijter, J. N., van de Graaf, R., Wensvoort, G. &amp; Moormann, R. J. (1998). Infectious transcripts from cloned genome-length cDNA of porcine reproductive and respiratory syndrome virus. J Virol 72, 380-7. 
         Reed L J, Muench H. A simple method of estimating fifty percent end points. Am J Hyg 1938;27: 709-16. 
         Snijder, E. J. &amp; Meulenberg, J. J. (1998). The molecular biology of arteriviruses.  J Gen Virol  79, 961-79. 
         Wensvoort G, Terpstra C, Boonstra J, Bloemraad M, Van Zaane D. Production of monoclonal antibodies against swine fever virus and their use in laboratory diagnosis. Vet Microbiol 1986;12: 101-8. 
         Wensvoort, G., de Kluyver, E. P., Luijtze, E. A., den Besten, A., Harris, L., Collins, J. E., Christianson, W. T. &amp; Chladek, D. (1992). Antigenic comparison of Lelystad virus and swine infertility and respiratory syndrome (SIRS) virus.  J Vet Diagn Invest  4, 134-8. 
         Wensvoort, G., Terpstra, C., Pol, J. M., ter Laak, E. A., Bloemraad, M., de Kluyver, E. P., Kragten, C., van Buiten, L., den Besten, A., Wagenaar, F. &amp; et al. (1991). Mystery swine disease in The Netherlands:the isolation of Lelystad virus.  Vet Q  13, 121-30. 
         Steverink, P. J. G. M., Pol, J. M. A., Bos-De Ruijter, J. N. A. &amp; Meulenberg, J. J. M. Virulence of vABV414, the virus derived from the infectious cDNA clone of Lelystad virus, for third trimester pregnant gilts.  Proc. PRRS  &amp;  Aujeszky&#39;s disease  1999, 119.