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.
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.
Infection of cells with Classical swine fever virus (CSFV) is mediated by the interaction of envelope glycoprotein Erns and E2 with the cell surface. In this report we studied the role of the cell surface glycoaminoglycans (GAGs), chondroitin sulfates A, B, and C (CS-A, -B, and -C), and heparan sulfate (HS) in the initial binding of CSFV strain Brescia to cells. Removal of HS from the surface of swine kidney cells (SK6) by heparinase I treatment almost completely abolished infection of these cells with virus that was extensively passaged in swine kidney cells before it was cloned (clone C1.1.1). Infection with C1.1.1 was inhibited completely by heparin (a GAG chemically related to HS but sulfated to a higher extent) and by dextran sulfate (an artificial highly sulfated polysaccharide), whereas HS and CS-A, -B, and -C were unable to inhibit infection. Bound C1.1.1 virus particles were released from the cell surface by treatment with heparin. Furthermore, C1.1.1 virus particles and CSFV E rns purified from insect cells bound to immobilized heparin, whereas purified CSFV E2 did not. These results indicate that initial binding of this virus clone is accomplished by the interaction of E rns with cell surface HS. In contrast, infection of SK6 cells with virus clones isolated from the blood of an infected pig and minimally passaged in SK6 cells was not affected by heparinase I treatment of cells and the addition of heparin to the medium. However, after one additional round of amplification in SK6 cells, infection with these virus clones was affected by heparinase I treatment and heparin. Sequence analysis of the E rns genes of these virus clones before and after amplification in SK6 cells showed that passage in SK6 cells resulted in a change of an Ser residue to an Arg residue in the C terminus of E rns (amino acid 476 in the polyprotein of CSFV). Replacement of the E rns gene of an infectious DNA copy of C1.1.1 with the E rns genes of these virus variants proved that acquisition of this Arg was sufficient to alter an HSindependent virus to a virus that uses HS as an E rns receptor.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.