Construction of an infectious bacterial artificial chromosome clone of a pseudorabies virus variant: Reconstituted virus exhibited wild-type properties in vitro and in vivo

Since late 2011, a pseudorabies virus (PRV) variant with increased virulence in old pigs had caused major disease outbreaks and great economic losses to the pig industry in China. The gene mutations that contributed to the increased virulence were unclear. To study the basis of the enhanced pathogenicity, an infectious bacterial artificial chromosome (BAC) clone consisting of the complete genome of the PRV variant was developed. Using homologous recombination and Cre/LoxP recombination, the recombinant virus rJS-2012-BAC carrying a BAC insertion downstream of the open reading frame (ORF) of gG was constructed. The circular genome of rJS-2012-BAC was extracted from infected Vero cells and transformed into Escherichia coli DH10B, generating the BAC clone pBAC-JS2012. The loxP sites flanking the BAC vector were used to excise the BAC sequences using Cre recombinase. The reconstituted BAC-excision virus, vJS2012 L, from pBAC-JS2012 exhibited similar biological properties to the wild-type virulent strain JS-2012. To manipulate the BAC clone pBAC-JS2012, the galK selection system was adopted to delete the gI/gE gene from pBAC-JS2012 in E. coli and to generate the gI/gE-deleted virus vJS2012-ΔgE/gI. The BAC clone, pBAC-JS2012, retained the same level of virulence as its parent strain and was easily manipulated using a galK system which would facilitate the study of the enhanced pathogenicity of the PRV variant as well as other studies on PRV.