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.

Identification and characterization of a sixth structural protein of Lelystad virus: the glycoprotein GP2 encoded by ORF2 is incorporated in virus particles.

Previously we have shown that Lelystad virus (LV) contains five structural proteins, a nucleocapsid protein N, an integral membrane protein M, and three glycoproteins GP3, GP4, and GP5. In this study we identified a sixth structural protein of Lelystad virus. The protein has an apparent molecular weight of 29 to 30 kDa, was recognized by an ORF2-specific antipeptide serum in Western immunoblotting using sucrose gradient purified LV virions, and was shown to be N-glycosylated. It was therefore designated GP2. The GP2 protein was also immunoprecipitated by ORF2-specific antipeptide serum from lysates and extracellular virus of CL2621 cells infected with LV. A fraction of the GP2 protein present in these lysates contained an intrachain disulfide bond. Endoglycosidase H treatment of immunoprecipitates indicated that the endoglycosidase H-sensitive N-glycans of the GP2 protein become endoglycosidase H-resistant during passage through the Golgi compartment in cells infected with LV. In contrast, the N-glycans of the GP2 protein expressed individually in a recombinant Semliki Forest virus remained endoglycosidase H-sensitive, indicating that the GP2 protein was retained in the endoplasmic reticulum. LV is the first arterivirus which has been demonstrated to contain six virion-associated proteins, one nucleocapsid protein, one integral membrane protein, and four glycoproteins.

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.

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.

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.

Lelystad virus, the causative agent of porcine epidemic abortion and respiratory syndrome (PEARS), is related to LDV and EAV.

The genome of Lelystad virus (LV), the causative agent of porcine epidemic abortion and respiratory syndrome (previously known as mystery swine disease), was shown to be a polyadenylated RNA molecule. The nucleotide sequence of the LV genome was determined from a set of overlapping cDNA clones. A consecutive sequence of 15,088 nucleotides was obtained. Eight open reading frames (ORFs) that might encode virus-specific proteins were identified. ORF1a and ORF1b are predicted to encode the viral RNA polymerase because the amino acid sequence contains sequence elements that are conserved in RNA polymerases of the torovirus Berne virus (BEV), equine arteritis virus (EAV), lactate dehydrogenase-elevating virus (LDV), the coronaviruses, and other positive-strand RNA viruses. A heptanucleotide slippery sequence (UUUAAAC) and a putative pseudoknot structure, which are both required for efficient ribosomal frameshifting during translation of the RNA polymerase ORF1b of BEV, EAV, and the coronaviruses, were identified in the overlapping region of ORF1a and ORF1b of LV. ORFs 2 to 6 probably encode viral membrane-associated proteins, whereas ORF7 is predicted to encode the nucleocapsid protein. Comparison of the amino acid sequences of the ORFs identified in the genome of LV, LDV, and EAV indicated that LV and LDV are more closely related than LV and EAV. A 3' nested set of six subgenomic RNAs was detected in LV-infected cells. These subgenomic RNAs contain a common leader sequence that is derived from the 5' end of the genomic RNA and that is joined to the 3' terminal body sequence. Our results indicate that LV is closely related evolutionarily to LDV and EAV, both members of a recently proposed family of positive-strand RNA viruses, the Arteriviridae.

Lelystad virus, the cause of porcine epidemic abortion and respiratory syndrome: a review of mystery swine disease research at Lelystad.

This paper reviews the laboratory investigations that led us to isolate the Lelystad virus and demonstrate that this virus causes mystery swine disease. We describe: 1) isolating the virus from the disease; 2) characterizing the virus as a new enveloped RNA virus; 3) reproducing the disease experimentally with the isolated Lelystad virus; 4) isolating the virus from the experimentally induced disease.

Antigenic comparison of Lelystad virus and swine infertility and respiratory syndrome (SIRS) virus.

This study reports the antigenic relatedness of isolates of Lelystad virus collected in the Netherlands, Germany, and the United States. The binding of antibodies directed against these isolates was tested in a set of field sera collected during outbreaks of porcine epidemic abortion and respiratory syndrome in Europe and outbreaks of swine infertility and respiratory syndrome (SIRS) in North America. Two sets of sera from pigs experimentally infected with Lelystad virus or SIRS virus were also tested. Although all 7 isolates reacted with anti-Lelystad virus sera, antigenic variation was considerable. The 4 European isolates resembled each other closely, but differed from the American isolates, and the 3 American isolates differed antigenically from each other. To reliably diagnose Lelystad virus infection, a common antigen must first be identified.

Mystery swine disease in The Netherlands: the isolation of Lelystad virus.

In early 1991, the Dutch pig-industry was struck by the so-called mystery swine disease. Large-scale laboratory investigations were undertaken to search for the etiological agent. We focused on isolating viruses and mycoplasmas, and we tested paired sera of affected sows for antibodies against ten known pig viruses. The mycoplasmas M. hyosynoviae, M. hyopneumoniae, and Acholeplasma laidlawii, and the viruses encephalomyocarditis virus and porcine enterovirus types 2 and 7 were isolated from individual pigs. An unknown agent, however, was isolated from 16 of 20 piglets and from 41 of 63 sows. This agent was characterised as a virus and designated Lelystad virus. No relationship between this virus and other viruses has yet been established. Of 165 sows reportedly afflicted by the disease, 123 (75 per cent) seroconverted to Lelystad virus, whereas less than 10 per cent seroconverted to any of the other virus isolates or to the known viral pathogens. Antibodies directed against Lelystad virus were also found in pigs with mystery swine disease in England, Germany, and in the United States. We conclude that infection with Lelystad virus is the likely cause of mystery swine disease.