[Show abstract][Hide abstract] ABSTRACT: Since the emergence of highly pathogenic North American Porcine Reproductive and Respiratory Syndrome virus (H-US-PRRSV) in 2006, the classical North American PRRSV (C-US-PRRSV) and H-US-PRRSV isolates have coexisted in Chinese swine herds. A duplex real-time RT-PCR assay using minor groove binder (MGB) probes for differential detection of the two US PRRSV isolates was developed. The specificity, sensitivity, reproducibility, and interference test of this assay were validated. The sensitivity of the assay was 3.2TCID(50)/ml or 38 RNA copies/microl for C-US-PRRSV and 0.4 TCID(50)/ml or 14 RNA copies/microl for H-US-PRRSV. Both assays were 10 times more sensitive than the current methods. A total of 302 clinical samples were tested by duplex real-time RT-PCR and conventional RT-PCR assays, and the results showed over 98.7% agreement. In addition, the new assay can be completed in less than 2h. This duplex real-time RT-PCR assay is a promising tool for rapid differential detection and epidemiology of US PRRSV in China.
[Show abstract][Hide abstract] ABSTRACT: Mutants of a highly pathogenic, porcine reproductive, and respiratory syndrome virus (PRRSV), JXA1 strain, were prepared by continuous in vitro passage. Genomic sequence comparisons were made between mutants obtained at different passages and the parental strain JXA1. The mutant strain obtained at passage 80 contained a 12 nucleotide insertion and 108 nucleotide mutations that resulted in 45 amino acid changes. Most of these changes (89%) occurred between passage 10 and 45 and were genetically stable for the next 35-70 passages. A comparison of the mutants, their parental strain, and several American PRRSV strains, identified 13 characteristic amino acid changes. These sites, as well as the distinct 12 nucleotide insertion, represent possible genetic markers for the evaluation of live vaccine applications, particularly for additional studies of the safety and potency of live PRRSV vaccines.