Sequence analysis of open reading frames (ORFs) 2 to 4 of a U.S. isolate of porcine reproductive and respiratory syndrome virus

Summary The sequence of ORFs 2 to 4 of a U.S. isolate of porcine reproductive and respiratory syndrome virus (PRRSV), ATCC VR2385, was determined by analysis of a cDNA library. The cDNA clones containing PRRSV specific sequences were selected using a VR2385 ORF 4 specific PCR probe and sequenced. The ORFs 2, 3 and 4 overlapped each other and encoded polypeptides with predicted M _r of 29.5 kDa (ORF 2), 28.7 kDa (ORF 3) and 19.5 kDa (ORF 4), respectively. No overlap was found between ORFs 4 and 5, and instead there was a 10 bp sequence which separated these two ORFs. The nucleic acid homology with corresponding ORFs of the European PRRSV isolate Lelystad virus (LV) was 65% for ORF 2, 64% for ORF 3 and 66% for ORF 4. Comparison of the ORF 4 sequences of VR2385 with that of another U.S. isolate MN-1b revealed only 86% amino acid sequence homology and the presence of deletions in the ORF 4 of MN-1b. Our results further strengthen the observation that there is sequence variation between US and European PRRSV isolates.     

Construction of chimeric arteriviruses reveals that the ectodomain of the major glycoprotein is not the main determinant of equine arteritis virus tropism in cell culture

The recent development of arterivirus full-length cDNA clones makes possible the construction of chimeric arteriviruses for fundamental and applied studies. Using an equine arteritis virus (EAV) infectious cDNA clone, we have engineered chimeras in which the ectodomains of the two major envelope proteins, the glycoprotein GP(5) and the membrane protein M, were replaced by sequences from envelope proteins of related and unrelated RNA viruses. Using immunofluorescence microscopy, we monitored the transport of the hybrid GP(5) and M proteins to the Golgi complex, which depends on their heterodimerization and is a prerequisite for virus assembly. The only viable chimeras were those containing the GP(5) ectodomain from the porcine (PRRSV) or mouse (LDV) arteriviruses, which are both considerably smaller than the corresponding sequence of EAV. Although the two viable GP(5) chimeras were attenuated, they were still able to infect baby hamster kidney (BHK-21) and rabbit kidney (RK-13) cells. These cells can be infected by EAV, but not by either PRRSV or LDV. This implies that the ectodomain of the major glycoprotein GP(5), which has been postulated to be involved in receptor recognition, is not the main determinant of EAV tropism in cell culture.     

The primary neutralization epitope of porcine respiratory and reproductive syndrome virus strain VR-2332 is located in the middle of the GP5 ectodomain

Summary.  Pigs infected with porcine respiratory and reproductive syndrome virus (PRRSV) strain VR-2332 were found to generate high levels of antibodies (Abs) that bound in an indirect ELISA to synthetic peptides representing segments of the primary envelope glycoprotein (GP5) ectodomain of this virus. Use of overlapping GP5 ectodomain peptides of various length indicated that the epitope recognized by the Abs was located in the middle of the ectodomain (amino acids 36-52), in the same relative segment that contains the single linear neutralization epitope of the closely related mouse arterivirus, lactate dehydrogenase-elevating virus (LDV). The VR-2332 GP5 segment exhibits 77% amino acid homology with the corresponding GP5 ectodomain segments of both the European PRRSV strain Lelystad virus (LV) and LDV. This explains some observed crossreaction between the pig Abs and neutralizing anti-LDV monoclonal Abs with peptides representing the GP5 ectodomains of VR-2332, LV and LDV. The GP5 binding Abs of pigs seem to be the primary PRRSV neutralizing Abs, since the well timed appearance in sera of all VR-2332 infected pigs of GP5 peptide binding Abs correlated 100% with the appearance of neutralizing Abs and earlier studies indicated that GP5 of PRRSV, like that of other arteriviruses, contains the main neutralization epitope of PRRSV. In addition, one neutralizing anti-LDV monoclonal Ab that is specific for the GP5 ectodomain epitope of LDV also strongly neutralized both PRRSV strains, VR-2332 and LV. The PRRSV GP5 epitope is associated with an N-glycan that is conserved in both PRRSV genotypes and all LDV isolates. This N-glycan may impede the humoral immune control of PRRSV in infected pigs and might be responsible for the low immunogenicity of PRRSV when injected into mice. Received April 2, 2002; accepted July 9, 2002     

Baculovirus Expression of Proteins of Porcine Reproductive and Respiratory Syndrome Virus Strain Olot/91. Involvement of ORF3 and ORF5 Proteins in Protection

Porcine reproductive and respiratory syndrome virus (PRRSV) is a new arterivirus that has spread rapidly all around the world in the last few years. The genomic region containing open reading frames (ORFs) 2 to 7 of PRRSV Spanish isolate Olot/91 was cloned and sequenced. The genomic sequence shared 95% identity with Lelystad and Tu¨bingen isolates and between 61–64% with the ORF7 region of the American isolates. ORFs 2 to 7 were inserted into recombinant baculoviruses downstream of the polyhedrin promoter. Only ORFs 2, 3 5 and 7 were expressed in insect cells as detected by PRRS-specific pig antisera. To analyze the immunogenicity of these proteins and their ability to confer protection, Sf9 cells infected with recombinant baculoviruses expressing ORFs 3, 5 and 7 gene products were used to immunize pregnant sows, either individually or in combination. The results obtained indicate that ORFs 3 and 5 gene products could be major candidates for the development of a vaccine against PRRS since they conferred 68.4 and 50% protection, respectively, as evaluated by the number of piglets born alive and healthy at the time of weaning. In addition, piglets born to sows immunized with ORFs 3 and 5 proteins were seronegative to PRRSV after weaning, indicating absence of viral replication. ORF7 is the most immunogenic protein of PRRSV, but the antibodies induced in sows are non-protective and may even interfere with protection.     

Genetic typing of equine arteritis virus isolates from Argentina

We report the nucleotide sequence and genetic diversity of four Equine Arteritis Virus (EAV) ORF 5 and 6 from Argentina isolates, obtained from asymptomatic virus-shedding stallions. Nucleic acid recovered from the isolates were amplified by RT-PCR and sequenced. Nucleotide and deduced amino acid sequences from the Argentine isolates were compared with 17 sequences available from the GenBank. Phylogenetic analysis revealed that the Argentine isolates grouped together in a definite cluster near European strains. Despite the greater genetic variability among ORF 5 from different isolates and strains of EAV, phylogenetic trees based on ORF 5 and 6 are similar. Both trees showed that virus sequences from America and Europe segregate into distinct clades based on sequence analysis of either ORF 5 or 6. This study constitutes the first characterization of Argentine EAV isolates. M. G. Echeverría, S. Díaz, E. Nosetto Members of CONICET (Scientific Research Council)     

Sequence analysis of the NSP2, ORF5, and ORF7 genes of 11 PRRS virus isolates from China

During a tracing investigation of blue ear disease in China conducted from January to November 2008, 11 porcine reproductive and respiratory syndrome virus (PRRSV) isolates were collected from eight provinces including Liaoning, Jilin, Hebei, Shandong, Henan, Shanghai, Zhejiang, and Guangxi. The complete gene sequences of the NSP2, ORF5, and ORF7 genes from these 11 PRRSV isolates were amplified, cloned and detected by RT-PCR and then compared with the published sequences of other strains. The results showed that all of the isolates genotypically belonged to an American strain, but shared high homology with JXA1, the highly pathogenic strain endemic to China. All of the 11 PRRSV isolates demonstrated a 90-nucleotide deletion in the NSP2 gene, suggesting that the main epidemic of PRRSV in 2008 was due to this gene deletion isolate. More consistent mutations were found in specific regions of the ORF5 and ORF7 genes of the 11 PRRSV isolates, such as the signal peptide and transmembrane regions of GP5 and the Pat 7 motif of the N protein. Whether these mutations influence nuclear localization of PRRSV requires confirmation by future studies.     

Genetic diversity of the ORF5 gene of porcine reproductive and respiratory syndrome virus (PRRSV) genotypes I and II in Thailand

To investigate the genetic diversity of porcine reproductive and respiratory syndrome virus (PRRSV) in Thailand, 279 ORF5 sequences of PRRSV collected during 2010–2011 from 102 swine herds in five swine-producing areas were analyzed. The co-existence of European (EU) and North American (NA) genotypes was observed in 98 % of herds investigated and was evident at the pig level. Both genotypes have evolved separately with a temporal influence on strain development. Novel introductions influence the genetic diversity of the NA genotype. Although Thai EU and NA isolates develop their own clusters that are separate from those of other countries, there was no geographic influence on strain development within Thailand.     

Comparison of nucleic and amino acid sequences and phylogenetic analysis of the GS protein of various equine arteritis virus isolates

The genetic variation in equine arteritis virus (EAV) G_S protein encoding gene was investigated. Nucleic and deduced amino acid sequences from eight different EAV isolates (one European, two American and five Canadian isolates) were compared with those of the Bucyrus reference strain. Nucleotide and amino acid sequence identities between these isolates and the Bucyrus reference strain ranged from 92.3 to 96.4%, and 93.2 to 95.5%, respectively. However, phylogenetic tree analysis and estimation of genetic distances based on the G_S protein encoding gene sequences showed that the European prototype Vienna strain, the American 87AR-A1 isolate and all other North American EAV isolates could be classified into three genetically divergent groups. Our results showed that the G_S protein-encoding gene can be subjected on the basis of phylogenetic analysis to genetic variation, as previously shown for the other three EAV structural protein (M, N and G_L)-encoding genes.     

The genetic diversity of European type PRRSV is similar to that of the North American type but is geographically skewed within Europe

Porcine reproductive and respiratory syndrome virus (PRRSV) is a recently emerged pathogen. Two PRRSV genotypes exist, North American and European, which are only 55-70% identical at the nucleotide level. Previous studies have shown high nucleotide diversity in the North American genotype and low nucleotide diversity in the European genotype. Here, we analyzed the ORF5 and ORF7 genes for a large number of new European type PRRSV isolates in conjunction with existing database sequences. This new analysis showed that contrary to previous assumptions, genetic diversity is at least as high in the European genotype as in the North American genotype. Furthermore, we showed that genetic diversity of European type PRRSV has a marked geographical pattern, with exceptionally high genetic diversity among Italian sequences. The geographical pattern of diversity in relation to the epidemiology of PRRSV in Europe is discussed. Discrepancies between ORF5- and ORF7-based genealogies were observed, and further analysis of the data set confirmed the presence of recombination. We were therefore able to report the first observation of recombination in wild-type isolates of European genotype PRRSV.     

GP5 ectodomain epitope of porcine reproductive and respiratory syndrome virus, strain Lelystad virus

Sera from pigs infected with the European porcine reproductive and respiratory syndrome virus (PRRSV), strain Lelystad virus (LV), were screened by indirect ELISA for antibodies that bound to a series of overlapping synthetic peptides covering amino acids (AA) 19-60 of the primary envelope glycoprotein (GP)5. Antibodies were detected that bound to an epitope(s) located in an ectodomain segment composed of AA 38-54. The antibodies strongly cross-reacted with peptides specific for the North American PRRSV VR2332. Antibodies with the same specificity were present in sera of pigs infected in the US with a European-like PRRSV. Competitive ELISA using overlapping 8-10-AA-long peptides confirmed that the epitope recognized by the antibodies from LV-infected pigs is located in the same segment as the neutralization epitopes previously identified for PRRSV VR2332 and the closely related arterivirus, lactate dehydrogenase-elevating virus (LDV). No antibodies were detected that bound to synthetic peptides representing further upstream GP5 segments that have been reported to carry neutralization or non-neutralization epitopes of some PRRSV isolates.     

两株PRRSV分离株ORF5与Nsp2基因部分序列分析

目的分析沈阳和甘肃发病猪场的2株猪繁殖与呼吸综合征病毒(PRRSV)的ORF5基因和Nsp2基因变异情况。方法采用RT-PCR方法,对ORF5基因序列和Nsp2基因部分序列进行扩增、克隆并测序。并用DNAStar软件将测序结果与国内外发表的10株参考毒株进行比对分析。结果 2株分离株ORF5基因与国内外其它美洲型分离株核苷酸的同源性为88.6%～98.7%,推导的氨基酸的同源性为86.6%～98.0%;Nsp2基因的核苷酸同源性为73.4%～99.8%,推导的氨基酸的同源性为68.6%～99.5%,且该基因与国内其他变异株有完全一致的缺失特征。结论这两株分离株均属于PRRSV美洲型变异株,为该病的防治及疫苗的设计奠定理论基础。     

Genetic characterization of the Korean porcine reproductive and respiratory syndrome viruses based on the nucleocapsid protein gene (ORF7) sequences

To investigate the genetic characteristics of the Korean porcine reproductive and respiratory syndrome virus (PRRSV), we determined the complete sequence of the nucleocapsid protein gene (ORF7) from 105 PRRSV isolates from all nine Korean prefectures during the years 2003 through 2006. These sequences were then analyzed along with the published ORF7 sequences for two Korean PRRS viruses (PL97-1/1997 and LMY/2002) and 36 non-Korean viruses. The ORF7 nucleotide sequence identities among the 107 Korean PRRS viruses ranged from 86.2 to 100%, corresponding to 85.4 to 100% identity at the amino acid level. All of the Korean isolates examined belonged to the North American genotype. The ORF7 gene sequence from the North American prototype virus (VR-2332) and its derived vaccine virus (Ingelvac PRRS MLV) was 90.0–100% identical to the various ORF7 sequences of the Korean isolates, with corresponding amino acid identities from 91.0 to 100%. In the phylogenetic tree obtained by neighbor-joining analysis, all of the Korean PRRSVs were divided into four groups. Our ORF7 sequence data also revealed no correlations between the date or place of collection and the distribution of PRRSV in Korea. North American genotype PRRSVs may have been introduced into Korean swine herds some time ago; these viruses apparently radiated nationwide within a relatively short period of time. Within the North American genotype PRRSVs from around the world, the Korean PRRSVs did not emerge as a single independent clade overall, and their immediate relationships with the PRRSVs from other countries could not be determined.     

Large Envelope Glycoprotein and Nucleocapsid Protein of Equine Arteritis Virus (EAV) Induce an Immune Response in Balb/c Mice by DNA Vaccination; Strategy for Developing a DNA-Vaccine Against EAV-Infection

Equine arteritis virus (EAV) is a member of the Arteriviridae family, that includes lactate dehydrogenase-elevating virus (LDV), porcine reproductive and respiratory syndrome virus (PRRSV), and simian haemorrhagic fever virus (SHFV). Equine arteritis is a contagious disease of horses and is spread via respiratory or reproductive tract. The objective of the present study is to evaluate the possibility for developing a model system for prevention horses against an EAV infection by DNA vaccination. A cDNA bank from the RNA of EAV was established. This gene library contains the translation unit of the EAV open reading frames (ORF) 1 to 7. The identity of the cDNA was confirmed by nucleotide sequence analysis. Using this defined EAV cDNA gene library the cDNA sequence of the viral ORFs were molecularly cloned into the corresponding sites of well characterized and powerful expression vectors (pCR3.1, pDisplay, and/or pcDNA3.1/HisC).The capability of these recombinant plasmids expressing the gene products of the individual viral ORFs 3 to 5, and 7 in induction of an immune response in mouse system was investigated. The Balb/c mice (ten mice per assay) were inoculated with the DNA of the constructed expression vectors harboring and expressing the EAV cDNA of the viral ORFs. The Balb/c mice were injected with about 100 g DNA diluted in 100 l PBS. The DNA was injected subcutaneously and into the tibialis cranialis muscle (Musculus gastrocnemius). The mice were boosted 3 to 5 times with the same quantities of DNA and under the same conditions at about two week intervals. Control mice received the same amount of parental expression vectors via an identical route and frequency.The pre- and post-vaccinated sera of the individual animals were screened by neutralization tests (NT). Neutralizing antibodies against EAV were detected when the animals were inoculated with the DNA of the expression vectors harboring cDNA of the EAV ORFs 5 and 7. Highest NT-titers were observed when the animals were administered with the cDNA of ORF 5 and/or with the cDNA of the neutralization determinants of EAV that is located on the N-terminal ectodomain of the gene product of ORF 5 between the amino acid positions 1–121. These results obtained from these studies justified proofing the capability of the EAV cDNA sequences of the viral genes including ORFs 5 and 7 in the autologous animal system horse.     

Concurrent porcine circovirus type 2a (PCV2a) or PCV2b infection increases the rate of amino acid mutations of porcine reproductive and respiratory syndrome virus (PRRSV) during serial passages in pigs

Porcine reproductive and respiratory syndrome virus (PRRSV) has a high degree of genetic and antigenic variability. The purpose of this study was to determine if porcine circovirus type 2 (PCV2) infection increases genetic variability of PRRSV during serial passages in pigs and to determine if there is a difference in the PRRSV mutation rate between pigs concurrently infected with PCV2a or PCV2b. After 8 consecutive passages of PRRSV alone (group 1), PRRSV with PCV2a (group 2), or PCV2b (group 3) in pigs, the sequences of PRRSV structural genes for open reading frame (ORF) 5, ORF6, ORF7 and the partial non-structural protein gene (Nsp) 2 were determined. The total number of identified amino acid mutations in ORF5, ORF6, ORF7 and Nsp2 sequences was 30 for PRRSV infection only, 63 for PRRSV/PCV2a concurrent infection, and 77 for PRRSV/PCV2b concurrent infection when compared with the original VR2385 virus used to infect the passage 1 pigs. Compared to what occurred in pigs infected with PRRSV only, the mutation rates in ORF5 and ORF6 were significantly higher for concurrent PRRSV/PCV2b infected pigs. The PRRSV/PCV2a pigs had a significantly higher mutation rate in ORF7. The results from this study indicated that, besides ORF5 and Nsp2, the PRRSV structural genes ORF6 and ORF7 were shown to mutate at various degrees when the PRRSV was passaged over time in vivo. Furthermore, a significantly higher mutation rate of PRRSV was observed when pigs were co-infected with PCV2 highlighting the importance of concurrent infections on PRRSV evolution and control.     

Genetic variation and phylogenetic relationships of 22 porcine reproductive and respiratory syndrome virus (PRRSV) field strains based on sequence analysis of open reading frame 5

Summary.  Porcine reproductive and respiratory syndrome virus (PRRSV) strains from 13 states in the United States, Guatemala and Canada were used to compare the envelope glycoprotein gene (ORF 5) nucleotide and deduced amino acid sequences. The gene was the same size, 603 nt, for all the 22 field strains. These strains had 89–94% amino acid identity compared to reference strain VR 2332. A putative signal sequence and cleavage site between residues 31 and 32 was identified and three potential glycosylation sites were present on all but two strains. Hydrophobicity/hydrophilicity and surface probability analyses reveal a primary structure for the envelope glycoprotein (E protein) with six potential surface regions that could be antigenic sites. Similar E protein structural features are conserved for the prototype European PRRSV – Lelystad virus. Accepted December 18, 1996 Received August 26, 1996     

Genetic diversity of the ORF5 gene of porcine reproductive and respiratory syndrome virus isolates in China from 2006 to 2008

Since April 2006, swine herds have experienced the outbreaks of a highly pathogenic porcine reproductive and respiratory syndrome (PRRS) in China. To explore the possible mechanism of the emergence of the highly pathogenic PRRS and more fully understand the extent of genetic diversity of PRRSV in China, we analyzed the ORF5 gene sequences of 159 representative PRRSV isolates in 16 provinces from 2006 to 2008. Sequence and phylogenetic analyses showed that all these 159 isolates belonged to the North American genotype and were further divided into six subgenotypes; 140 of 159 isolates were closely related to the highly pathogenic PRRSV with 98.5–100% nucleotide and 98.3–100% amino acid sequence identities and belonged to Subgenotype I; and 3, 8, 4, 3, 1 of 159 isolates were part of Subgenotypes II–VI, respectively. Amino acid analysis of the GP5 protein revealed that all the isolates in Subgenotypes I–III were found to be highly variable in the primary neutralizing epitope; most of the isolates in Subgenotypes I and IV had more glycosylation sites than those in Subgenotypes II, III, V and VI; and 1, 5, and 9 unique amino acid mutations were observed in Subgenotypes I, IV and VI, respectively. In conclusion, our study provides the evidence of coexistence of six different subgenotype isolates in pigs in China from 2006 to 2008, and emphasizes the importance of reinforcing PRRSV surveillance, especially after the emergence of highly pathogenic PRRS in China.     

Dynamics and evolution of porcine reproductive and respiratory syndrome virus (PRRSV) ORF5 following modified live PRRSV vaccination in a PRRSV-infected herd

The objective of this study was to investigate the dynamics and evolution of porcine reproductive and respiratory syndrome virus (PRRSV) ORF5 following the use of a modified live PRRSV (MLV) vaccine. A PRRSV-positive farm with coexistence of types 1 and 2 and no history of MLV vaccination was investigated. Vaccination with a type 2 MLV (Ingelvac PRRS MLV, Boehringer Ingelheim, USA) was implemented. All sows were vaccinated at monthly intervals for two consecutive months and then every third month. Piglets were vaccinated once at 7-10 days of age and weaned to nursery facilities at 21-23 days of age. Serum samples were collected monthly before and after vaccination from four population groups, including replacement gilts and suckling, nursery and finishing pigs, and assayed by PCR. After a year of blood collection, amplified products were sequenced, resulting in 277 complete ORF5 gene sequences from 145 type 1 and 132 type 2 isolates. Prior to and following vaccination, both type 1 and type 2 PRRSV were isolated and found to coexist in an individual pig. Each genotype evolved separately without influencing the strain development of the other. Although the substitution rates of both genotypes were relatively similar, MLV vaccination appears to increase the heterogenicity of type 2 PRRSV, resulting in the emergence of three novel type 2 PRRSV clusters in the herd, including an MLV-like cluster, which disappeared within the month following whole-herd vaccination. Two additional clusters included one related to the MLV vaccine and one related to the endemic cluster of the herd.     

Heterogeneity in Nsp2 of European-like porcine reproductive and respiratory syndrome viruses isolated in the United States

Recently, isolates of porcine reproductive and respiratory syndrome virus (PRRSV) that possess nucleotide sequences similar to European isolates have been reported in United States herds. The origin, diversity and prevalence of European-like North American PRRSV isolates in the U.S. remain unknown. Nucleotide sequence analysis of the 12 kb ORF1 of a North American isolate, SDPRRS 01-08 (01-08), showed 93.7% identity with Lelystad virus (LV), the prototypic European isolate, but only 58% identity with VR-2332, the prototypic North American isolate. Comparisons between LV and 01-08 at the peptide sequence level of the predicted non-structural proteins (Nsp) showed that Nsp9 (98.9% amino acid identity) was the most conserved and the least conserved was Nsp2 at 90.6% identity. For the purpose of comparison, GP5, the principal envelope structural protein, showed a 93.5% identity between 01-08 and LV. The most dramatic differences between the Nsp2 proteins of LV and 01-08 were a single 17 amino acid deletion between residues 734 and 750, as well as several amino acid differences. The same deletion was identified in the Nsp2 in five of seven other EuroPRRSV isolates submitted to the South Dakota Animal Disease Research and Diagnostic Laboratory. The remaining two isolates contained small deletions, but in other regions of Nsp2. Peptide sequence diversity in the form of hypervariability and deletions in Nsp2 demonstrate that European-like PRRSV isolates in the USA represent a heterogeneous group. Furthermore, areas in Nsp2 with deletions and amino acid hypervariability localize to regions that are predicted to be immunologically important.     

Current knowledge on the structural proteins of porcine reproductive and respiratory syndrome (PRRS) virus: comparison of the North American and European isolates

Summary.  Porcine reproductive and respiratory syndrome virus (PRRSV) belongs to the recently recognized Arteriviridae family within the genus Arterivirus, order Nidovirales, which also includes equine arteritis virus (EAV), lactate dehydrogenase-elevating virus (LDV), and simian hemorrhagic fever virus (SHFV). Mature viral particles are composed of an envelope 50–72 nm in diameter, with an isometric core about 20–30 nm enclosing a linear positive-stranded RNA genome of approximately 15 kb. The virions are assembled by the budding of preformed nucleocapsids into the lumen of the smooth endoplasmic reticulum and/or Golgi apparatus. The mature virions are then released by exocytosis. The viral genome contains eight open reading frames (ORFs) which are transcribed in cells as a nested set of subgenomic mRNAs. The ORF1a and ORF1b situated at the 5′end of the genome represent nearly 75% of the viral genome and code for proteins with apparent replicase and polymerase activities. The major structural proteins consist of a 25 kDa envelope glycoprotein (GP₅), an 18–19 kDa unglycosylated membrane protein (M), and a 15 kDa nucleocapsid (N) protein, encoded by ORFs 5, 6 and 7, respectively. The N protein is the more abundant protein of the virion and is highly antigenic, which therefore makes it a suitable candidate for the detection of virus-specific antibodies and diagnosis of the disease. Four to five domains of antigenic importance have been identified for the N protein, a common conformational antigenic site for European and North American strains being localized in the central region of the protein. In cells and virions, both M and GP₅ occur in heterodimeric complexes linked by disulfide bonds. The expression products of ORFs 2 and 4 are also incorporated into virus particles as additional minor membrane-associated glycoproteins designated as GP₂ and GP₄, with M_r of 29 and 31 kDa, respectively. The structural nature of the ORF3 product, a highly glycosylated protein with an apparent M_r of 42 kDa, is still being debated, in view of the apparently conflicting data on its presence in virus particles. Nonetheless, the GP₃ of North American and European strains has been shown to be antigenic, providing protection for piglets against PRRSV infection in the absence of a noticeable neutralizing humoral response. Pigs exposed to the native form of GP₅ by means of DNA immunization develop specific neutralizing and protecting antibodies. The GP₅ is involved in antigenic variability, apoptosis, and possibly antibody-dependent enhancement phenomena. The GP₄ also possesses antigenic determinants that trigger the immune system to produce neutralizing antibodies. Each of the PRRSV structural proteins carries common and type-specific antigenic determinants that permit the ability to differentiate between European and North American strains. The potential use of the PRRSV structural proteins in subunit recombinant-type vaccines is also discussed. Received August 30, 1999/Accepted September 29, 1999     

Antigenic variability among North American and European strains of porcine reproductive and respiratory syndrome virus as defined by monoclonal antibodies to the matrix protein.

Two hybridoma cell lines producing monoclonal antibodies (Mabs) to the 19-kDa matrix (M) protein of porcine reproductive and respiratory syndrome virus (PRRSV) were obtained from BALB/c mice that were immunized with a reference Quebec tissue culture-adapted strain (strain IAF-Klop). The polypeptide specificities of the MAbs were determined by immunoblotting and immunoprecipitation tests with concentrated and purified preparations of the virus and by determining their reactivities with the Escherichia coli-expressed gene products of open reading frames 5 to 7. The two anti-M protein MAbs (MAbs IAFK3 and IAFK6) and another MAb (MAb IAFK8) directed to the 15-kDa nucleocapsid (N) protein were devoid of virus-neutralizing activity. A library of four anti-N protein MAbs (MAbs IAFK8, SDOW17, VO17, and EP147) and two anti-M protein MAbs (MAbs IAFK6 and IAFK3) was used to investigate, by an indirect fluorescent-antibody assay, the antigenic diversity of 15 Canadian PRRSV isolates, in comparison with those of the U.S. ATCC VR2332 attenuated vaccine strain and two reference European (Lelystad and Weybridge) PRRSV strains. The North American and European PRRSV isolates tested shared the epitopes recognized by anti-N protein MAbs IAFK8 and SDOW17, but three distinct patterns could be identified on the basis of their reactivities with the other anti-PRRSV MAbs. No reactivity to the anti-M protein MAbs was observed by either European PRRSV isolate or the attenuated U.S. vaccine strain.