A quantitative assessment of the effectiveness of PRRSV vaccination in pigs under experimental conditions.

This paper presents a quantitative approach to evaluate effectiveness of vaccination under experimental conditions. We used two consecutive experimental designs to investigate whether PRRSV transmission among vaccinated pigs was reduced compared to control pigs and to estimate the reproduction parameter R. Based upon data analysis and power calculations the series of small-scale vaccination-challenge experiments ended with multiple one-to-one experiments. This new experimental design has considerable power to detect the effect of vaccination on transmission if R is close to but still above one in vaccinated pigs. The last experiment showed that transmission was not significantly reduced and the R for vaccinated pigs was estimated to be larger than 4.9. This is remarkable because duration and level of viremia were significantly reduced by vaccination.

An infectious cDNA clone of porcine reproductive and respiratory syndrome virus.

A plasmid containing a full-length cDNA copy of the Lelystad virus isolate (LV) of porcine reproductive and respiratory syndrome virus was constructed. When RNA that was transcribed in vitro from this full-length cDNA clone was transfected to BHK-21 cells, infectious LV was produced and secreted. The virus was rescued by passage to porcine alveolar lung macrophages or CL2621 cells. When infectious transcripts were transfected to porcine alveolar lung macrophages or CL2621 cells no infectious virus was produced due to the poor transfection efficiency of these cells. The growth properties of the viruses produced by BHK-21 cells transfected with infectious transcripts of LV cDNA resembled the growth properties of the parental virus from which the cDNA was derived. The infectious clone of LV enables us to mutagenize the viral genome at specific sites and thus will be useful for detailed molecular characterization of the virus, as well as for the development of a safe and effective live vaccine for use in pigs.

Infectious transcripts from cloned genome-length cDNA of porcine reproductive and respiratory syndrome virus.

The 5'-terminal end of the genomic RNA of the Lelystad virus isolate (LV) of porcine reproductive and respiratory syndrome virus was determined. To construct full-length cDNA clones, the 5'-terminal sequence was ligated to cDNA clones covering the complete genome of LV. When RNA that was transcribed in vitro from these full-length cDNA clones was transfected into BHK-21 cells, infectious LV was produced and secreted. The virus was rescued by passage to porcine alveolar lung macrophages or CL2621 cells. When infectious transcripts were transfected to porcine alveolar lung macrophages or CL2621 cells, no infectious virus was produced due to the poor transfection efficiency of these cells. The growth properties of the viruses produced by BHK-21 cells transfected with infectious transcripts of LV cDNA resembled the growth properties of the parental virus from which the cDNA was derived. Two nucleotide changes leading to a unique PacI restriction site directly downstream of the ORF7 gene were introduced in the genome-length cDNA clone. The virus recovered from this mutated cDNA clone retained the PacI site, which confirmed the de novo generation of infectious LV from cloned cDNA. These results indicate that the infectious clone of LV enables us to mutagenize the viral genome at specific sites and that it will therefore be useful for detailed molecular characterization of the virus, as well as for the development of a safe and effective live vaccine for use in pigs.

A congenital persistent infection of bovine virus diarrhoea virus in pigs: clinical, virological and immunological observations.

We report on a lifelong 'carrier' state of non-cytopathic bovine virus diarrhoea virus (BVDV) in an otherwise healthy pig. Three out of 13 pigs of a litter congenitally infected with BVDV survived for more than 3 months. One pig was BVDV seropositive at 1 month, the second seroconverted between 6 and 8 months, and the third remained viraemic and BVDV-immunotolerant until slaughter at 26 months. The latter pig, a boar, excreted virus in oropharyngeal fluid, urine and semen. Ejaculates, however, did not contain spermatozoa. The persistently infected pig had leucopenia from 3 months onwards, though differential white cell counts and thrombocyte counts remained normal. The antibody response of this pig after vaccination against foot-and-mouth disease and after infection with porcine parvovirus was not affected. The antibody response after vaccination against hog cholera, however, was delayed. In contrast, superinfection with antigenically related cytopathic BVDV strains did not alter the status of BVDV immunotolerance, nor did it induce clinical signs resembling mucosal disease as observed in cattle persistently infected with BVDV. Although the role of pigs in the epizootiology of bovine virus diarrhoea infections may be limited, the existence of healthy BVDV carrier pigs should be noted wherever control or eradication measures are applied.

Seroprevalence of porcine reproductive and respiratory syndrome virus in Dutch weaning pigs.

To determine whether under Dutch field conditions PRRSV infection occurs in weaning pigs before the finishing period, a cross-sectional study was performed on 32 breeding farms to estimate the seroprevalence of antibodies directed against PRRSV in 4- to 5-week-old and 8- to 9-week-old pigs. Farms were visited twice within 5 months, and during each sampling an average of 20 sera were randomly collected from a unit of 4- to 5-week-old and a unit of 8- to 9-week-old pigs. The sera (n = 2568) were tested in the IDEXX-ELISA for the presence of antibodies directed against PRRSV. The seroprevalence of PRRSV in 4- to 5-week-old pigs and 8- to 9-week-old pigs varied between both samplings for each farm. The seroprevalence in the younger pigs was significantly higher than in the older pigs for both samplings (p < 0.05), suggesting the presence of maternal antibodies. In addition, a longitudinal study was performed to evaluate the IDEXX-ELISA in detecting maternal antibodies directed against PRRSV and to determine the rate of decline of these antibodies in field sera. From serological results of eight litters, an average decay function was computed to quantify the maternal immunity to PRRSV. A seroprevalence in 8- to 9-weeks-old pigs of > or = 0.20 was calculated to indicate an active immune response to PRRSV. In the cross-sectional study in the pigs twenty-three percent of the units with 8- to 9-week-old pigs were considered to have an active serological response against PRRSV. We conclude that most Dutch pigs are seronegative for PRRSV at the start of the finishing period, since the results of this study showed that 77% of the units with 8- to 9-week-old pigs had a seroprevalence < 0.20.

Molecular characterization of Lelystad virus.

Lelystad virus (LV), the prototype of porcine reproductive respiratory syndrome virus, is a small enveloped virus, containing a positive strand RNA genome of 15 kb. LV is tentatively classified in the family Arteriviridae, which consists of lactate dehydrogenase-elevating virus (LDV), equine arteritis virus (EAV) and simian hemorrhagic fever virus (SHFV). These viruses have a similar genome organization and replication strategy as coronaviruses, but the size of the genome is much smaller (12-15 kb) and they have different morphological and physicochemical properties. The genome of LV contains eight open reading frames (ORFs) that encode the replicase genes (ORFs 1a and 1b), envelope proteins (ORFs 2 to 6) and the nucleocapsid protein (ORF7). Genomic comparison of European and North American isolates has shown that the structural proteins encoded by ORFs 2 to 7 vary widely. The amino acid sequences of ORFs 2 to 7 of North American strains share only 55 to 79% identical amino acids with those of European strains. Using polyvalent porcine anti-LV serum, gene-specific anti-peptide sera and monoclonal antibodies, we have identified six structural proteins of LV and their corresponding genes. These are: the 15 kDa unglycosylated nucleocapsid protein (N) encoded by ORF7, an 18 kDa unglycosylated integral membrane protein M encoded by ORF6, a 25 kDa N-glycosylated protein encoded by ORF5, a 31-35 kDa N-glycosylated protein encoded by ORF4, a 45-50 kDa N-glycosylated protein encoded by ORF3 and a 29-30 kDa N-glycosylated protein encoded by ORF2. A nomenclature for these structural proteins is proposed.

Dual infections of PRRSV/influenza or PRRSV/Actinobacillus pleuropneumoniae in the respiratory tract.

To study the effect of a previous porcine respiratory and reproductive syndrome-infection (PRRS) of the respiratory tract on influenza virus and Actinobacillus pleuropneumoniae (App) infections, 3-week-old specific-pathogen-free (spf) piglets were intranasally infected with PRRS virus. One week later, when the lung alveolar macrophages of PRRSV infected pigs were lowest in number, a second infection was applied by intranasal aerosol of influenza virus H3N2 or by endobronchial instillation of a mildly virulent App. The first experiment consisted of two groups (only influenza infection or dual PRRSV/influenza infection). A second experiment consisted of 4 groups (only influenza infection, only PRRSV infection, dual PRRSV/influenza infection and uninfected controls). At day 2, 4, 14 and 21 after influenza infection, two pigs were killed and sampled for virological and histopathological examination. Influenza H3N2 virus caused only a mild inflammation of the smaller bronchioli. Previous PRRSV infection did not influence clinical signs during influenza infection. Next, we studied in two experiments the effect of dual PRRSV/App infection during the acute stage at two days after App infection. In a third experiment, the influence of PRRSV on more chronic stages of App infection was studied at two weeks after the App infection. At the end of the experiments, the pigs were killed. Lungs were ranked according to size and kind of the lesions. Lesions were cut and measured, samples were taken for virological and histopathological examination. Statistical analysis of the ranked lung-lesions in the first experiment showed a distinct but small effect of previous PRRSV infection on the development of App-lesions. In PRRSV infected pigs. App produced a more severe disease. The second and third experiment however failed to show any influence of the previous PRRSV infection on the App infection. We conclude that previous PRRSV infection of the respiratory tract of spf pigs does not necessarily enhance the severity of secondary infections of the respiratory tract.

An improved ELISA for the detection of serum antibodies directed against classical swine fever virus.

The complex-trapping-blocking (CTB) ELISA for detection of antibodies against classical swine fever virus (CSFV) using two monoclonal antibodies (mAbs) directed against envelope glycoprotein E2, has been improved using recombinant CSFV E2-antigen. The newly developed Ceditest ELISA for CSFV-Ab is a modification of the CTB-ELISA as described by Wensvoort et al. (1988) and Bloemraad et al. (1993). The old CTB-ELISA format comprised of a two-step, single-dilution test which had to be performed by hand at 37 degrees C. The Ceditest ELISA is a one-step, single-dilution test which can be performed by hand, or automated at room temperature. A set of 505 pig sera was tested for CSFV antibodies in the CTB-ELISA, in the Ceditest ELISA and in the neutralizing peroxidase-linked assay (NPLA). Concordance between the two ELISAs was 96.7%. Sensitivity and specificity of the Ceditest ELISA were 99% and 99% compared to the CTB-ELISA. The Ceditest ELISA discriminates between antibodies directed against bovine viral diarrhea virus (BVDV) and CSFV. The discrimination between antibodies directed against some Border disease virus (BDV) strains and CSFV is as inconsistent as with the NPLA. We conclude that the Ceditest ELISA for CSFV-Ab is a promising tool for the fast and simple screening of large numbers of pig sera for detection of antibodies directed against CSFV.

Classical swine fever virus (CSFV) envelope glycoprotein E2 containing one structural antigenic unit protects pigs from lethal CSFV challenge.

Envelope glycoprotein E2, formerly called E1 or gp51-54, of classical swine fever virus (CSFV) expressed in insect cells protects swine from classical swine fever. Monoclonal antibodies directed against epitopes of domains B and C and subdomain A1 are neutralizing. The domains are located on two structural antigenic units in a proposed model of the antigenic structure of E2. One unit consists of nonconserved antigenic domains B and C and the other contains highly conserved antigenic domain A. We produced several mutant E2 proteins by use of the baculovirus expression system. Two selected mutants were E2 proteins in which one of the two structural antigenic units, unit B/C or unit A, was deleted. The protective capacity of the mutant E2 proteins was investigated in an immunization experiment in pigs. Titres of the neutralizing responses in pigs immunized with mutant E2 proteins were all comparable with that of intact E2. These vaccinated pigs were protected against an intranasal lethal CSFV challenge, indicating that the immune response induced by one structural antigenic unit of E2 can protect pigs against classical swine fever.

Proteins encoded by open reading frames 3 and 4 of the genome of Lelystad virus (Arteriviridae) are structural proteins of the virion.

Four structural proteins of Lelystad virus (Arteriviridae) were recognized by monoclonal antibodies in a Western immunoblotting experiment with purified virus. In addition to the 18-kDa integral membrane protein M and the 15-kDa nucleocapsid protein N, two new structural proteins with molecular masses of 45 to 50 kDa and 31 to 35 kDa, respectively, were detected. Monoclonal antibodies that recognized proteins of 45 to 50 kDa and 31 to 35 kDa immunoprecipitated similar proteins expressed from open reading frames (ORFs) 3 and 4 in baculovirus recombinants, respectively. Therefore, the 45- to 50-kDa protein is encoded by ORF3 and the 31- to 35-kDa protein is encoded by ORF4. Peptide-N-glycosidase F digestion of purified virus reduced the 45- to 50-kDa and 31- to 35-kDa proteins to core proteins of 29 and 16 kDa, respectively, which indicates N glycosylation of these proteins in the virion. Monoclonal antibodies specific for the 31- to 35-kDa protein neutralized Lelystad virus, which indicates that at least part of this protein is exposed at the virion surface. We propose that the 45- to 50-kDa and 31- to 35-kDa structural proteins of Lelystad virus be named GP3 and GP4, to reflect their glycosylation and the ORFs from which they are expressed. Antibodies specific for GP3 and GP4 were detected by a Western immunoblotting assay in swine serum after an infection with Lelystad virus.

Comparison of a commercial ELISA and an immunoperoxidase monolayer assay to detect antibodies directed against porcine respiratory and reproductive syndrome virus.

A commercially available enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies against porcine respiratory and reproductive syndrome virus (PRRSV) was compared to an immunoperoxidase monolayer assay (IPMA). Serum samples used were collected from pigs experimentally infected with either the American or European antigenic type of PRRSV, and also from piglets born to sows that had been experimentally infected with the European antigenic type of PRRSV. In addition, three sets of European field sera (n = 275, n = 68, n = 349) were tested and evaluated using the IPMA as the gold standard. Results showed that both the IPMA and the ELISA were able to detect antibodies against the two antigenic types of PRRSV. When sera of experimentally infected pigs were tested, the IPMA with homologous antigen detected antibodies 2 to 3 days earlier than the ELISA, and was more sensitive in detecting maternal antibodies. The ELISA was slightly more sensitive for detecting antibodies against the American type than for the European type. When sets of field sera were tested, the relative sensitivity of the ELISA ranged between 0.68 and 0.91, and the relative specificity ranged between 0.75 and 0.97. However, in two of these sets (n = 275, n = 349) we determined that a decrease of the threshold value of ELISA (from 0.4 to 0.3) increased sensitivity without loss of specificity. We concluded that the ELISA is an easy, quick and reliable test to diagnose PRRSV infection in swine herds.

Comparison of the protective efficacy of recombinant pseudorabies viruses against pseudorabies and classical swine fever in pigs; influence of different promoters on gene expression and on protection.

The glycoprotein E (gE) locus in the genome of pseudorabies virus (PRV) was used as an insertion site for the expression of glycoprotein E1 of classical swine fever virus (CSFV). Transcription of E1 in the recombinants M401, M402 or M403 was regulated by the gD promoter of PRV, the immediate early gene promoter of human cytomegalovirus, or the gE promoter of PRV, respectively. Groups of four pigs were vaccinated once intramuscularly with 10(6) plaque forming units (p.f.u.) of the recombinant viruses and challenged intranasally with 100 50% lethal doses of virulent CSFV and with 10(5) p.f.u. of virulent PRV. All pigs vaccinated with M402 were fully protected against both classical swine fever and pseudorabies.

Six antigenic groups within the genus pestivirus as identified by cross neutralization assays.

Antigenic differences between pestivirus isolates of ruminant and porcine origin were characterized by neutralization assays. First, six different clusters of pestiviruses were identified by clustering cross-neutralization results of 13 pestivirus strains tested against 23 sera. Cluster I consisted of four strains of bovine viral diarrhoea virus (BVDV) of bovine origin and two BVDV isolates of porcine origin. Cluster II consisted of one sheep isolate and two porcine BDV isolates. Cluster III consisted of one classical swine fever virus strain and cluster IV, V, and VI each consisted of one strain isolated from a giraffe, a deer, and a pig. After the clusters were identified, one-way neutralization tests were used to test a total of 45 pestivirus isolates. Although the same six groups were found, results of some individual strains differed from previous cross-neutralization results and the results obtained by typing with monoclonal antibodies. The discrepancy between one way neutralization tests and cross-neutralisation tests is demonstrated clearly by recalculation of the cross-neutralization results without the classical swine fever sera. We conclude that neutralization tests are only suitable to characterize antigenic differences when virus strains are tested in a cross-neutralization test.

Nucleocapsid protein N of Lelystad virus: expression by recombinant baculovirus, immunological properties, and suitability for detection of serum antibodies.

The ORF7 gene, encoding the nucleocapsid protein N of Lelystad virus (LV), was inserted downstream of the P10 promoter into Autographa californica nuclear polyhedrosis virus (baculovirus). The resulting recombinant baculovirus, designated bac-ORF7, expressed a 15-kDa protein in insect cells. This protein was similar in size to the N protein expressed by LV in CL2621 cells when it was analyzed on sodium dodecyl sulfate-polyacrylamide gels. The N protein expressed by bac-ORF7 was immunoprecipitated with anti-ORF7 was immunoprecipitated with anti-ORF7 peptide serum, porcine convalescent-phase anti-LV serum, and N protein-specific monoclonal antibodies, indicating that this N protein had retained its native antigenic structure. The recombinant N protein was immunogenic in pigs, and the porcine antibodies raised against this protein recognized LV in an immunoperoxidase monolayer assay. However, pigs vaccinated twice with approximately 20 micrograms of N protein were not protected against a challenge with 10(5) 50% tissue culture infective doses of LV. Experimental and field sera directed against various European and North American isolates reacted with the N protein expressed by bac-ORF7 in a blocking enzyme-linked immunosorbent assay. Therefore, the recombinant N protein may be useful for developing diagnostic assays for the detection of serum antibodies directed against different isolates of LV.

Characterization of proteins encoded by ORFs 2 to 7 of Lelystad virus.

The genome of Lelystad virus (LV), a positive-strand RNA virus, is 15 kb in length and contains 8 open reading frames (ORFs) that encode putative viral proteins. ORFs 2 to 7 were cloned in plasmids downstream of the Sp6 RNA polymerase promoter, and the translation of transcripts generated in vitro yielded proteins that could be immunoprecipitated with porcine anti-LV serum. Synthetic polypeptides of 15 to 17 amino acids were selected from the amino acid sequences of ORFs 2 to 7 and antipeptide sera were raised in rabbits. Antisera that immunoprecipitated the in vitro translation products of ORFs 2 to 5 and 7 were obtained. Sera containing antibodies directed against peptides from ORFs 3 to 7 reacted positively with LV-infected alveolar lung macrophages in the immunoperoxidase monolayer assay. Using these antipeptide sera and porcine anti-LV serum, we identified three structural proteins and assigned their corresponding genes. Virions were found to contain a nucleocapsid protein of 15 kDa (N), an unglycosylated membrane protein of 18 kDa (M), and a glycosylated membrane protein of 25 kDa (E). The N protein is encoded by ORF7, the M protein is encoded by ORF6, and the E protein is encoded by ORF5. The E protein in virus particles contains one or two N-glycans that are resistant to endo-beta-N-acetyl-D-glucosaminidase H. This finding indicates that the high-mannose glycans are processed into complex glycans in the Golgi compartment. The protein composition of the LV virions further confirms that LV is evolutionarily related to equine arteritis virus, simian hemorrhagic fever virus, and lactate dehydrogenase-elevating virus.

Characterization of structural proteins of Lelystad virus.

The genome of Lelystad virus (LV), a positive-strand RNA virus, is 15 kb in length and contains 8 open reading frames that encode putative viral proteins. Synthetic polypeptides of 15 to 17 amino acids were selected from the amino acid sequences of ORFs 2 to 7 and anti-peptide sera were raised in rabbits. Using these anti-peptide sera and porcine anti-LV serum, we identified three structural proteins and assigned their corresponding genes. Virions were found to contain a nucleocapsid protein of 15 kDa (N), an unglycosylated membrane protein of 18 kDa (M), and a glycosylated membrane protein of 25 kDa (E). The N protein is encoded by ORF7, the M protein is encoded by ORF6, and the E protein is encoded by ORF5.

Porcine reproductive and respiratory syndrome: temperature and pH stability of Lelystad virus and its survival in tissue specimens from viraemic pigs.

We investigated the growth of Lelystad virus (LV) in porcine alveolar macrophages, the thermal and pH stability of the virus in cell culture medium, and its survival in tissue specimens from viraemic pigs. Lelystad virus grew to titres of 10(6) TCID50/ml, which were found at 40 h after virus inoculation when the macrophage cultures showed a cytopathic effect of approximately 40%. In culture medium at pH 7.5, LV was stable for prolonged periods of storage at -70 degrees C and -20 degrees C. At higher temperatures the half-life of LV was 140 h at 4 degrees C, 20 h at 21 degrees C, 3 h at 37 degrees C and 6 min at 56 degrees C. The half-life of LV, both at 4 degrees C and 37 degrees C, changed considerably when the pH of the medium was varied. At 4 degrees C and pH 6.25 a maximum half-life of 50 h and at 37 degrees C and at pH 6.0 a maximum half-life of 6.5 h was observed. However, increasing or decreasing the pH of the medium rapidly decreased the half-life of LV at both temperatures. Although, LV proved to be more stable at pH 6.00 than at pH 7.5, it did not replicate at pH 6.0. We also tested various tissue specimens from viraemic pigs for the presence of LV. The virus was detected in tonsils, lymph nodes, lungs, serum, and sporadically, albeit at low titres, in muscle tissue. The titre of virus in muscle tissue and organs was only minimally affected by storage for up to 48 h at 4 degrees C.

Antigenic structure of envelope glycoprotein E1 of hog cholera virus.

Envelope glycoprotein E1 (gp51 to gp54) is the most antigenic protein of hog cholera virus or classical swine fever virus (CSFV). Four antigenic domains, A to D, have been mapped on E1 with a panel of monoclonal antibodies (MAbs) raised against CSFV strain Brescia. The boundaries of these domains have been established by extensive studies on binding of MAbs to transiently expressed deletion mutants of E1 (P. A. van Rijn, E. J. de Meijer, H. G. P. van Gennip, and R. J. M. Moormann, J. Gen. Virol. 74:2053-2060, 1993). In this study, we used neutralizing MAbs of domains A, B, and C to isolate MAb-resistant mutants (MAR mutants) of CSFV strain Brescia and Chinese vaccine strain ("C"). The E1 genes of MAR mutants were cloned in a eukaryotic expression vector, and the effects of MAR mutations on epitopes were studied with a panel of 19 MAbs by immunostaining of COS1 cells transiently expressing these mutant E1s. Except for the MAR mutation Cys-->Arg at position 792, which abolished binding of all MAbs of domains A and D, amino acid substitutions affected only MAbs belonging to the same domain as the MAb used to select the MAR mutant. However, a MAR mutation in a particular domain did not per se abolish binding of all MAbs recognizing that domain. Furthermore, MAR mutants possessed conservative as well as nonconservative amino acid substitutions. To investigate the significance of a secondary structure for the binding of MAbs, all cysteine residues in the N-terminal antigenic part of E1 were mutated to serine. We found that the cysteines at positions 693 and 737 were essential for binding by MAbs of domains B and C, whereas those at positions 792, 818, 828, and 856 appeared to be essential for the binding of most MAbs of domains A and D. These results fully comply with the previously proposed two-unit structure of the N-terminal half of E1. One unit consists of antigenic domains B and C, whereas the other unit consists of the highly conserved domain A and domain D. We conclude that the first six cysteines are critical for the correct folding of E1. A model of the antigenic structure of E1 is presented and discussed.

Lelystad virus belongs to a new virus family, comprising lactate dehydrogenase-elevating virus, equine arteritis virus, and simian hemorrhagic fever virus.

Lelystad virus (LV) is an enveloped positive-stranded RNA virus, which causes abortions and respiratory disease in pigs. The complete nucleotide sequence of the genome of LV has been determined. This sequence is 15.1 kb in length and contains a poly(A) tail at the 3' end. Open reading frames that might encode the viral replicases (ORFs 1a and 1b), membrane-associated proteins (ORFs 2 to 6) and the nucleocapsid protein (ORF7) have been identified. Sequence comparisons have indicated that LV is distantly related to the coronaviruses and toroviruses and closely related to lactate dehydrogenase-elevating virus (LDV) and equine arteritis virus (EAV). A 3' nested set of six subgenomic RNAs is produced in LV-infected alveolar lung macrophages. These subgenomic RNAs contain a leader sequence, which is derived from the 5' end of the viral genome. Altogether, these data show that LV is closely related evolutionarily to LDV and EAV, both members of a recently proposed family of positive-stranded RNA viruses, the Arteriviridae.

Differentiation of U.S. and European isolates of porcine reproductive and respiratory syndrome virus by monoclonal antibodies.

Monoclonal antibodies (MAbs) to two U.S. isolates of porcine reproductive and respiratory syndrome (PRRS) virus were prepared. Two MAbs specifically recognized a conserved epitope on the putative 15-kDa nucleocapsid protein of U.S. and European isolates of PRRS virus. Four other MAbs recognized epitopes on the 15-kDa protein of U.S. but not European isolates of PRRS virus. Collectively, this indicates that PRRS viruses contain both conserved and divergent epitopes on the 15-kDa viral protein.