The primary GP5 neutralization epitope of North American isolates of porcine reproductive and respiratory syndrome virus

Abstract

I have used indirect ELISA with overlapping synthetic peptides representing the GP5 ectodomain to study the generation and specificity of peptide-binding Abs in pigs that were infected in utero with porcine reproductive and respiratory syndrome virus (PRRSV) strain VR2332 and in North American field sera submitted for PRRSV infection diagnosis. Peptide-binding Abs appeared in sera of the VR2332-infected pigs within about 30 days post-farrowing (dpf), reaching maximum titers 100-200 dpf and then decreasing slowly to about half of maximum titer by about 400 dpf. The formation of peptide-binding Abs and of virus neutralizing Abs correlated and their initial appearance coincided with disappearance of virus from the circulation. The Abs were specific for VR2332-specific peptides. In contrast, anti-N-protein Abs as measured by HerdCheck ELISA appeared within 7 dpf, reached maximum levels at about 100 dpf and had decreased below detectable levels by about 200 dpf. Twenty-seven field serum samples with virus neutralizing activity all possessed high levels of peptide binding Abs, but the Abs bound about equally to VR2332 and strain Lelystad virus (LV)-specific peptides. The indirect ELISA results using various large peptides and competition ELISA using small peptides (8 or 9 amino acids long) confirmed that the epitope recognized by the Abs is located in the GP5 ectodomain sequence 37SHLQLIYNL of VR2332. Use of mutated peptides in the competition ELISA showed that 42I to T and 38HL to TY substitutions blocked Ab recognition, whereas deletion of 41L had no effect. In addition, 26 serum samples submitted by two farms for diagnostic tests were found to possess low levels of Abs that bound to GP5 ectodomain peptides, even though the sera were sero-negative in the HerdChek ELISA and lacked neutralizing activity. Competition ELISA showed that the Abs recognized one or more epitopes located downstream of the PRRSV neutralization epitope. An epitope(s) located in the same area was recognized by Abs generated in mice by immunization with a GP5 ectodomain peptide conjugated to BSA. These Abs also lacked neutralizing activity.