A 10-kDa structural protein of porcine reproductive and respiratory syndrome virus encoded by ORF2b

The major structural proteins of porcine reproductive and respiratory syndr ome virus (PRRSV) are derived from ORFs 5, 6, and 7. Western blots of sucro se gradient-purified virions and PRRSV-infected MARC-145 cells, probed with immune pig serum, showed the presence of an additional 10-kDa protein. Nuc leotide sequence analysis of North American PRRSV isolate SDSU-23983 reveal ed a small ORF within ORF2, named ORF2b, which, when translated, produced a 73-amino-acid nonglycosylated protein. Recombinant 2b protein expressed by a baculovirus clone, AcVR2, comigrated with the 10-kDa virus-associated pr otein. The loss of 10-kDa protein immunoreactivity after absorption of immu ne sera with lysates from AcVR2-infected insect cells demonstrated that the 2b and 10-kDa proteins are immunologically similar. Immunoblots were also used for the detection of anti-2b activity in serum samples from experiment ally infected adult pigs. Antibodies against PRRSV were apparent by 14 days postinfection, followed by anti-2b activity and serum neutralizing activit y. The putative ORF2b start codon is only 6 nucleotides downstream of the a denine of the ORF2a start codon. The expression of ORF2a and 2b as enhanced green fluorescent fusion proteins showed that both proteins were translate d; however, the ORF2b was preferentially expressed. These results suggest t hat the 2b protein is virion associated and the principal product of ORF2. (C) 2001 Academic Press.