Variation in porcine reproductive and respiratory syndrome virus open reading frame 5 diagnostic sequencing

Objective: To assess porcine reproductive and respiratory syndrome virus (PRRSV) open reading frame 5 (ORF5) sequencing variation, within and among state diagnostic laboratories, that may contribute to observed differences in sequence homology among isolates. Materials and methods: PRRS viruspositive blood samples were collected from individual pigs on three different farms and submitted on three independent occasions to three diagnostic laboratories for PRRSV ORF5 nucleotide sequencing. The PRRSV isolates on each farm were genetically disparate. Vaccine viruses (Ingelvac PRRS MLV and Ingelvac PRRS ATP; Boehringer Ingelheim Vetmedica, Inc, St Joseph, Missouri) were submitted as positive controls. Results: Full-length ORF5 sequences were obtained from all samples. Positive-control vaccine virus sequencing was precise and highly accurate, with all laboratories on all occasions obtaining nearly identical sequences. The analytical specificity of field PRRSV sequencing was robust, with a median variation among laboratories for the same farm sample, across all pigs and submission dates, of one base difference per 603-base sequence (0.2%). Seventy-five percent of sequences had fewer than six base differences, and the greatest difference was 2.2%. However, 16% of samples in one submission from one farm appeared to be misidentified in the reports of one laboratory. Implications: Inter- and intra-laboratory ORF5 sequencing results are reproducible, reliable, and do not contribute significantly to estimated PRRSV diversity. Tracking errors may occur which can lead to confusion or inappropriate reaction by key decision makers. Submitters should retain aliquots of all samples to enable further investigation of a diagnostic error not related to the sequencing procedure.