Genetic and biologic characterization of a porcine reproductive and respiratory syndrome virus isolate attentuated by cell culture passage

A highly virulent porcine reproductive and respiratory syndrome virus (PRRSV) isolate was serially passaged 84 times in a continuous cell line in order to produce an attenuated strain. Naive pigs were inoculated with VR-2385 passage 8 (p8) or passage 85 (p85) in order to determine if cell culture passage resulted in decreased virulence as compared to the parent isolate. The pigs inoculated with p8 VR-2385 showed moderate clinical respiratory disease and had an elevated temperature 5--11 days post-infection (DPI), whereas the pigs inoculated with p85 VR-2385 had only mild respiratory disease and no fever. Long term cell culture passage resulted in significantly (P ≤ 0.05) reduced gross lung lesions, microscopic lung lesions, and microscopic lymph node lesions at 10 and 31 DPI, as compared to the p8-infected group. At 42 DPI, however, interstitial pneumonia lesions were moderate in both the p8 and p85-infected groups. This suggests that there are temporal differences in the course of infection for the two passages of the virus. Serum virus titers were determined using selected pigs at 10, 28, and 35 DPI. The results of virus isolation and titration from sera indicated that p8 VR-2385 may replicate better in vivo than the p85 virus. Growth curves were conducted in both a continuous cell line and primary cells. VR-2385 p85 replicated to a higher titer in both cell types, indicating that replication in cell culture did not correlate with in vivo virulence. The complete genome of VR-2385 p85 was sequenced and compared to the sequence of the parental strain. The two strains of VR-2385 had greater than 99% nucleotide sequence identity. The structural proteins have amino acid identities of 95--98.3%, while the replicase proteins have identities of 99.0% for ORF1a and 99.5% for ORF1b. No clear genetic determinant of virulence was identified, indicating that the differences in virulence are due to small genetic changes. A model of high and low virulence strains from the same lineage was established to further investigate potential virulence markers.