Infectious Transcripts from Cloned Genome-Length cDNA of Porcine Reproductive and Respiratory Syndrome Virus

Abstract

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.

Attenuation of highly pathogenic porcine reproductive and respiratory syndrome virus by inserting an additional transcription unit

“…1A). BHK-21 and Marc-145 cell lines are previously used for PRRSV rescue [22]. In this study, we used 293FT cell line to rescue PRRSV.…”

Porcine DC-SIGN: Molecular cloning, gene structure, tissue distribution and binding characteristics

“…BHK-21 was shown to support enveloped PRRSV replication inside the cell but not allow cell-to-cell spread of the virus [37]. Since pDC-SIGN was expressed on PAM, the susceptible host cells for PRRSV which contains highly glycosylated envelope viral proteins, it was important to see if pDC-SIGN expressed on the cell surface was involved in PRRSV attachment and entry.…”

Infectious cDNA clones of porcine reproductive and respiratory syndrome virus and their potential as vaccine vectors

“…As with other positive-strand RNA viruses, arterivirus genomes are infectious in cells (van der Zeijst and Horzinek, 1975;Meulenberg et al, 1998), and based on these findings, full-length infectious cDNA clones have been constructed for EAV and PRRSV (Fig. 1).…”

“…1). The first infectious clone for PRRSV was developed for the European type PRRS Lelystad virus (Meulenberg et al, 1998). In this construction, a genome length cDNA copy was assembled and placed under the bacteriophage T7 RNA polymerase promoter in the low copy number plasmid pOK12 (Vierra and Messing, 1991).…”