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Use of reverse genetics to study porcine reproductive and respiratory syndrome virus virulence
The efforts to identify important virulence determinants of the porcine reproductive and respiratory syndrome virus (PRRSV) began by comparing sequences between virulent and their avirulent derivative strains. This approach could provide an a priori clue to the candidate gene(s) that contribute to PRRSV attenuation by mutation. However, these changes may not necessarily represent the relevant acquisition of attenuation in vivo since many of these mutations may just reflect the necessary adaptation to grow in susceptible cell lines. The recent advances in reverse genetics system in PRRSV research can appropriately be used to understand the molecular basis of PRRSV virulence. The goal of this dissertation was to use a reverse genetics system to study the molecular basis of virulence and attenuation, and to identify candidate virulence determinant(s). Using Prime Pac (PP) vaccine strain as a donor and a highly virulent infectious clone (FL12) derived virus as a recipient, a series of chimeric viruses representing multiple or a single gene(s) of PP vaccine, were generated and characterized to map the virulence gene(s). These chimeric constructs were also used to generate a full-length cDNA clone (PP18) of the PP vaccine strain by serially replacing the genomic fragments of the FL12 with the corresponding regions from the PP strain. These 2 phenotypically different clone-derived viruses mimicked the in vitro (in growth kinetics in MARC-145 and PAM cells) and in vivo (in sows reproductive failure model) biological properties of their parental viruses. The results showed that the chimeric viruses comprising NSP3-8 and structural genes ORFs 2-7 were most attenuated. A fine mapping of structural gene regions by generating additional chimeric viruses with the individual structural genes and their reciprocal constructs, showed that ORF5 gene encoding major glycoprotein (GP) 5 is the most important structural gene for PRRSV virulence. In addition, ORF2 encoding GP2 and protein E, also contributed to virulence.^
Biology, Molecular|Agriculture, Animal Pathology|Biology, Virology|Biology, Veterinary Science
Kwon, Byungjoon, "Use of reverse genetics to study porcine reproductive and respiratory syndrome virus virulence" (2006). ETD collection for University of Nebraska - Lincoln. AAI3237555.