|Chen, Z -|
|Zhou, X -|
|Lawson, S -|
|Sun, Z -|
|Brown, E -|
|Christoper-Hennings, J -|
|Knudsen, D -|
|Nelson, E -|
|Fang, Y -|
Submitted to: Journal of General Virology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: October 28, 2009
Publication Date: November 18, 2009
Repository URL: http://dx.doi.org/10.1099/vir.0.016212-0
Citation: Chen, Z., Zhou, X., Lunney, J.K., Lawson, S., Sun, Z., Brown, E., Christoper-Hennings, J., Knudsen, D., Nelson, E., Fang, Y. 2009. Immunodominant epitopes in nsp2 of porcine reproductive and respiratory syndrome virus are dispensable for replication but play an important role in viral pathogenesis. Journal of General Virology. 91:1047-57. 91:1047-1057. Interpretive Summary: Porcine reproductive and respiratory syndrome (PRRS) is the most economically important pig disease in the United States. The disease was first described in 1987 in the US and in Europe in 1990. The PRRS virus (PRRSV) was first isolated in The Netherlands in 1991, and is represented by the European prototypic strain, Lelystad or genotype (type 1) PRRSV. A US Type 1 PRRSV isolates, termed North American Type 1, was identified in U.S. swineherds in the last decade and a Type 1 PRRSV infectious clone developed in the Fang lab. In the US, the North American genotype, or type 2 PRRSV, was first isolated in 1992. In 2006 the Chinese reported outbreaks of porcine high fever disease in China, caused in part by a highly pathogenic (HP) type II PRRSV; this has posed a substantial threat to the US swine industry. The Chinese HP PRRSV was found to contain a discontinuous 30 (29 and 1) amino acid deletion in the nonstructural protein 2 (nsp2) region of the viral genome. This deletion was suspected to be a potential determining factor that causes the fatal disease. Using the infectious clone for North American Type 1 PRRSV, SD01-08, this manuscript describes the production of six different nsp2 region deletion clones that were engineered to represent each of the six previously identified immunodominant B-cell epitopes (ES2 through ES7) in the nsp2 viral gene. When tested only deletion of ES3 (dES3), dES4, or dES7 allowed the generation of viable virus and could be tested further. When compared to the parental virus, the dES3 mutant showed increased viral lytic activity and more vigorous growth in culture with MARC cells; the other 2 deleted viruses, dES4 and dES7 mutants, displayed decreased lytic activity and slower growth kinetics in MARC-145 cells. The different viruses were then compared for their ability to stimulate immunity, by testing for expression of innate (interleukin-1 IL-1b) and T helper 1 (tumor necrosis factor TNF) cytokines from mutant virus infected macrophages. The results, measured by ELISAs and real-time PCR, showed that the IL-1b and TNF expression levels were significantly down-regulated in macrophages infected with the dES3 mutant, while slightly higher with the dES7 mutant, as compared to parental virus. To confirm the in vitro results, these nsp2 epitope deletion mutant viruses were characterized in a nursery pig PRRSV disease model. As predicted, dES4 and dES7 mutant viruses exhibited attenuated phenotypes, lower virus replication, whereas the dES3 mutant produced higher peak viral loads in pigs as compared to parental virus. When blood cells from pigs infected with the parental virus were cultured in vitro, the dES3 stimulated cell expressed lower IL-1b and TNF when as compared to dES7 mutant or parental virus stimulated blood cells. Thus, these results suggest that certain regions in nsp2 are not essential for PRRSV replication but may play an important role in viral pathogenesis in vivo. The availability of these North American Type 1 infectious clone mutant viruses provides important tools for further study of the basic PRRSV biology and pathogenic mechanisms. They will be useful for designing the next generation of genetically engineered PRRSV vaccines.
Technical Abstract: The nonstructural protein 2 (nsp2) of porcine reproductive and respiratory syndrome virus (PRRSV) is the largest protein of the virus. Besides its crucial role in viral replication, recent studies indicated its involvement in modulating host immunity. In this study, each of the six identified immunodominant nsp2 B-cell epitopes (ES2 through ES7) was deleted using a type I PRRSV cDNA infectious clone. Deletion of ES3, ES4, or ES7 allowed the generation of viable virus. In comparison to the parental virus, 'ES3 mutant showed increased cytolytic activity and more vigorous growth kinetics, while 'ES4 and 'ES7 mutants displayed decreased cytolytic activity and slower growth kinetics in MARC-145 cells. The expression of innate and T helper 1 cytokines from virus infected macrophages, measured by ELISAs and real-time PCR, showed that the IL-1ß and TNF-a expression levels were significantly down-regulated in macrophages infected with the 'ES3 mutant, while slightly higher with the 'ES7 mutant, as compared to parental virus. To confirm the in vitro results, these nsp2 epitope deletion mutant viruses were characterized in a nursery pig disease model. As predicted, 'ES4 and 'ES7 mutant viruses exhibited attenuated phenotypes, whereas the 'ES3 mutant produced higher peak viral loads in pigs as compared to parental virus. Similarly, cultured blood cells from these pigs expressed lower IL-1ß and TNF-a when stimulated with 'ES3 as compared to 'ES7 mutant or parental virus. These results suggest that certain regions in nsp2 are non-essential for PRRSV replication but may play an important role in viral pathogenesis in vivo.