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Journal of Virology, March 2003, p. 3702-3711, Vol. 77, No. 6
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.6.3702-3711.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Generation of an Infectious Clone of VR-2332, a Highly Virulent North American-Type Isolate of Porcine Reproductive and Respiratory Syndrome Virus

H. S. Nielsen,1,2,{dagger} G. Liu,3 J. Nielsen,1,{dagger} M. B. Oleksiewicz,1,{ddagger} A. Bøtner,1 T. Storgaard,4* and K. S. Faaberg3

Danish Veterinary Institute, Lindholm,1 Laboratory for Virology and Immunology, Royal Veterinary and Agricultural University, Frederiksberg,2 Applied Trinomics, Novo Nordisk A/S, Måløv, Denmark,4 Department of Veterinary Pathobiology, University of Minnesota, St. Paul, Minnesota3

Received 15 October 2002/ Accepted 9 December 2002

A full-length cDNA clone of the prototypical North American porcine reproductive and respiratory syndrome virus (PRRSV) isolate VR-2332 was assembled in the plasmid vector pOK12. To rescue infectious virus, capped RNA was transcribed in vitro from the pOK12 clone and transfected into BHK-21C cells. The supernatant from transfected monolayers were serially passaged on Marc-145 cells and porcine pulmonary alveolar macrophages. Infectious PRRSV was recovered on Marc-145 cells as well as porcine pulmonary macrophages; thus, the cloned virus exhibited the same cell tropism as the parental VR-2332 strain. However, the cloned virus was clearly distinguishable from the parental VR-2332 strain by an engineered marker, a BstZ17I restriction site. The full-length cDNA clone had 11 nucleotide changes, 2 of which affected coding, compared to the parental VR-2332 strain. Additionally, the transcribed RNA had an extra G at the 5' end. To examine whether these changes influenced viral replication, we examined the growth kinetics of the cloned virus in vitro. In Marc-145 cells, the growth kinetics of the cloned virus reflected those of the parental isolate, even though the titers of the cloned virus were consistently slightly lower. In experimentally infected 5.5-week-old pigs, the cloned virus produced blue discoloration of the ears, a classical clinical symptom of PRRSV. Also, the seroconversion kinetics of pigs infected with the cloned virus and VR-2332 were very similar. Hence, virus derived from the full-length cDNA clone appeared to recapitulate the biological properties of the highly virulent parental VR-2332 strain. This is the first report of an infectious cDNA clone based on American-type PRRSV. The availability of this cDNA clone will allow examination of the molecular mechanisms behind PRRSV virulence and attenuation, which might in turn allow the production of second-generation, genetically engineered PRRSV vaccines.

* Corresponding author. Mailing address: Applied Trinomics, Preclinical Development, Novo Nordisk A/S, Novo Nordisk Park, DK-2760 Måløv, Denmark. Phone: 45 44 43 61 24. Fax: 45 44 43 45 58. E-mail: TSTS{at}novonordisk.com.

{dagger} Present address: Preclinical Development; Novo Nordisk A/S, Måløv, Denmark.

{ddagger} Present address: Symphogen A/S, Lyngby, Denmark.

Journal of Virology, March 2003, p. 3702-3711, Vol. 77, No. 6
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.6.3702-3711.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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