Development of a heteroduplex mobility assay to identify field isolates of porcine reproductive and respiratory syndrome virus with nucleotide sequences closely related to those of modified live-attenuated vaccines

Abstract

Porcine reproductive and respiratory syndrome has been devastating the swine industry since the late 1980s. The disease has been controlled, to some extent, through the use of modified live-attenuated (MLV) vaccines once available. However, such a practice periodically resulted in isolation or detection of vaccine-like viruses from pigs as determined by a partial genomic sequencing. In this study, we developed a heteroduplex mobility assay (HMA) for quickly identifying porcine reproductive and respiratory syndrome virus (PRRSV) isolates with significant nucleotide sequence identities (>/=98%) with the modified live-attenuated vaccines. The major envelope gene (ORF5) of 51 PRRSV field isolates recovered before and after the introduction of the vaccines was amplified, denatured, and reannealed with the HMA reference vaccine strains Ingelvac PRRS MLV and Ingelvac PRRS ATP, respectively. Nine of the 51 field isolates and the VR2332 parent virus of Ingelvac PRRS MLV, which were all highly related to Ingelvac PRRS MLV with </=2% nucleotide sequence divergence as determined by sequence analysis, were all identified by the HMA to form homoduplexes with the reference Ingelvac PRRS MLV. No homoduplex-forming field isolate was identified when Ingelvac PRRS ATP was used as the HMA reference except for its parent virus JA142. Other field isolates with more than 2% nucleotide sequence divergence with the respective reference vaccine strain resulted in the formation of heteroduplexes with reduced mobility in polyacrylamide gel electrophoresis. The HMA results also correlated well with the results of phylogenetic analyses. The data indicated that the HMA developed in the study may be a rapid and efficient method for large-scale screening of potential vaccine-like PRRSV field isolates for further genetic characterization.

FIG. 1.

(A) HMA results for selected PRRSV isolates when Ingelvac PRRS MLV vaccine was used as the reference. Lanes: L, 1-kb DNA ladder; lane 1, reference plus reference; lane 2, reference plus VR2332; lane 3, reference plus 16727-01; lane 4, reference plus 44010A-01; lane 5, reference plus 47450-01; lane 6, reference plus 27011-01; lane 7, reference plus 18087B-01; lane 8, reference plus ISU3927; lane 9, reference plus ISU55; lane 10, reference plus JA142; lane 11, reference plus Ingelvac PRRS ATP; lane 12, reference plus 4519-01. Percent identity of nucleotide sequence between the reference and the PRRSV isolates is listed below each lane. (B) HMA results for selected PRRSV isolates when Ingelvac PRRS ATP vaccine was used as the reference. Lanes: L, 1-kb DNA ladder; lane 1, reference plus reference; lane 2, reference plus JA142; lane 3, reference plus 97-27796-4; lane 4, reference plus ISU3927; lane 5, reference plus ISU28; lane 6, reference plus ISU30262; lane 7, reference plus 98-4236; lane 8, reference plus 98-13795-3; lane 9, reference plus 93-36048; lane 10, reference plus 93-4506; lane 11, reference plus Ingelvac PRRS MLV; lane 12, reference plus VR2332. Percent identity of nucleotide sequence between the reference and the PRRSV isolates is listed below each lane.

FIG. 2.

Phylogenetic tree based on the ORF5 region of PRRSV isolates analyzed in the study: two MLV vaccines (Ingelvac PRRS MLV and Ingelvac PRRS ATP, designated with a *) used as the HMA references, 51 PRRSV field isolates analyzed in the study, including the parent strains of the two vaccines, VR2332 and JA142, and the European Lelystad virus (LV) as the outgroup. Homoduplex-forming isolates are shown in boldface. The tree was constructed with the PAUP program with a heuristic search option with midpoint rooting and 1,000 replicates.