Complete genome comparison of porcine reproductive and respiratory syndrome virus parental and attenuated strains

Abstract

Two full-length porcine reproductive and respiratory syndrome virus (PRRSV) genomes, strain VR-2332 and its cell culture passaged descendent RespPRRS vaccine strain, were compared and analyzed in order to identify possible sites of attenuation. Of the 44 nucleotide changes, 13 resulted in conservative changes and 18 produced non-conservative changes. The results suggest that key amino acids in ORF1 may contribute to the phenotype of RespPRRS, which includes increased growth rate on MA-104 cells and decreased virulence in swine. The results provide a genetic basis for future manipulation of a PRRSV reverse genetics system.

Fig. 1

5′-terminal nucleotides of strains VR-2332 and RespPRRS. VR-2332 (VR) or RespPRRS (R) from different passages were subjected to rapid amplification of cDNA ends (RACE) and products were cloned and sequenced as described in Section 2. VR-2332 represents sequence submitted to GenBank prior to elucidation of the 5′ terminal nucleotides. Strain VR-2332-infected brain homogenate was passaged two times on two separate occasions (VR-2a, VR-2b), or plaque purified and passaged 24 (VR-24) or 31 (VR-31) times. RespPRRS was passaged four (R-4) or 12 (R-12) times. Bold letters indicate previously unresolved bases and letters in italics refer to the non-viral nucleotides (T or C) arising from the RACE procedure.

Fig. 2

Nucleotide differences between strain VR-2332 and RespPRRS. (A) Full-genome schematic of the nucleotide differences between VR-2332 and RespPRRS reveals several changes occurred in ORF1a, a cluster of changes were seen in the 3′-terminus of ORF1b, and discrete changes were seen in all envelope glycoproteins (ORFs 2–5) and in the membrane protein (ORF6). When RespPRRS is similarly compared to strain 16244B, many nucleotide changes are seen throughout the genome. (B) Each region of the PRRSV genome was analyzed for the number of nucleotide changes and the corresponding ORF percent identity between VR-2332 and RespPRRS.

Fig. 3

ORF1 protein amino acid changes between VR-2332 and RespPRRS. ORF1 protein schematic shows identified domains: papain-like cysteine proteases α and β (PCPα and PCPβ), Cysteine protease (CP), serine protease/3c-like protease (SP/3CP), polymerase (POL), cysteine/histidine-rich domain (C/H), helicase domain (HEL) and coronavirus-like domain (CORONA). The schematic also shows possible nonstructural proteins (1–12) produced during cleavage by ORF1-encoded proteases at proposed cleavage sites (grey arrows). Seventeen amino acid changes occurred during in vitro passaging of strain VR-2332 to produce attenuated strain RespPRRS. Interesting mutations mentioned in the text are indicated by an asterisk.

Fig. 4

Viral growth curves of PRRSV strains VR-2332 (○) and RespPRRS (▵) reveal that RespPRRS has enhanced growth kinetics in vitro. Results are representative of three separate experiments. The number of plaques were determined in triplicate for two separate viral dilutions and were found to deviate by less than 5% in each individual experiment.