Attenuation of Porcine Reproductive and Respiratory Syndrome Virus by Molecular Breeding of Virus Envelope Genes from Genetically Divergent Strains

Abstract

bMolecular breeding via DNA shuffling can direct the evolution of viruses with desired traits. By using a positive-strand RNA virus, porcine reproductive and respiratory syndrome virus (PRRSV), as a model, rapid attenuation of the virus was achieved in this study by DNA shuffling of the viral envelope genes from multiple strains. The GP5 envelope genes of 7 genetically divergent PRRSV strains and the GP5-M genes of 6 different PRRSV strains were molecularly bred by DNA shuffling and iteration of the process, and the shuffled genes were cloned into the backbone of a DNA-launched PRRSV infectious clone. Two representative chimeric viruses, DS722 with shuffled GP5 genes and DS5M3 with shuffled GP5-M genes, were rescued and shown to replicate at a lower level and to form smaller plaques in vitro than their parental virus. An in vivo pathogenicity study revealed that pigs infected with the two chimeric viruses had significant reductions in viral-RNA loads in sera and lungs and in gross and microscopic lung lesions, indicating attenuation of the chimeric viruses. Furthermore, pigs vaccinated with the chimeric virus DS722, but not pigs vaccinated with DS5M3, still acquired protection against PRRSV challenge at a level similar to that of the parental virus. Therefore, this study reveals a unique approach through DNA shuffling of viral envelope genes to attenuate a positivestrand RNA virus. The results have important implications for future vaccine development and will generate broad general interest in the scientific community in rapidly attenuating other important human and veterinary viruses.

Protective humoral immune response induced by an inactivated porcine reproductive and respiratory syndrome virus expressing the hypo-glycosylated glycoprotein 5

“…The rescued mutant virus exhibited slightly delayed growth properties compared to the wt virus (data not shown). Consistent with our results, many mutant viruses have been found to exhibit reduced viral titers compared with the wt viruses in previous studies [31,46,47]. In in vivo experiments, the inactivated PRRSV vaccine based on the mutant virus induced a higher SN antibody titer and resulted in faster clearing of viremia compared to the inactivated PRRSV vaccine that was based on the wt virus.…”

The vOTU domain of highly-pathogenic porcine reproductive and respiratory syndrome virus displays a differential substrate preference

“…PRRSV virulence and attenuation have been studied extensively, with most studies implicating multiple regions of the virus in contributing to virulence, consistent with the observation that individual PRRSV vaccines appear to have become attenuated by different mechanisms (An et al, 2011; Brockmeier et al, 2012; Ellingson et al, 2010; Han et al, 2013; Kim et al, 2010; Kwon et al, 2008; Ni et al, 2013; Nielsen et al, 2001; Shi et al, 2013; Song et al, 2010; Storgaard et al, 1999; Wang et al, 2008; Yuan et al, 2001). Narrowing the focus to the PLP2/vOTU region of nsp2, previous reports have generally demonstrated deubiquitinating activity of certain strains, leading to subsequent suppression of innate immune responses in infected cells (Frias-Staheli et al, 2007; Li et al, 2010; Sun et al, 2010; van Kasteren et al, 2012), suggesting a mechanistic role in PRRSV virulence.…”

Genomic characterization and pathogenicity of a strain of type 1 porcine reproductive and respiratory syndrome virus

“…Gross lesions of lung were scored by estimating the percentage of the lung surface area affected by grossly visible pneumonia according to the method as described previously Ni et al, 2013). Meanwhile, lung and lymph node (mandibulares) samples of each piglet were collected at 21 DPI and fixed with 4% paraformaldehyde solution at room temperature for 48 h. The paraffin-embedded tissue samples were cut into 5-m-thick sections.…”