Abstract Porcine reproductive and respiratory syndrome (PRRS), one of the leading outbreaks on a world scale in swine farms, is caused by Porcine reproductive and respiratory syndrome virus (PRRSV). This study aimed to construct reverse genetic system of highly pathogenic PRRSV (HP-PRRSV) strain HN07-1. The whole genome of HN07-1 was first obtained by a method in which the genome was segmently cloned and sequenced. Based on the reverse genetics technique, fragments of the whole genome were cloned into pcDNA3.1(+) vector and full-length infectious clone of HN07-1 strain, designated as pcDNA3.2-rHN07-1, were constructed. Besides, the recombinant plasmid pcDNA3.2-rHN07-1-EGFP was constructed by inserting EGFP between PRRSV ORF1b and ORF2a. The recombinant viruses were rescued by transfecting the constructed plasmids into Marc-145 cells and identified by immunofluorescence assay (IFA) and Western blot. The results showed that the full-length of HN07-1 genome was 15 345 nt. The typical cytopathic effect (CPE) was observed at 48 h of culture after one blind passage on Marc-145 cells. The results of IFA and Western blot indicated that PRRSV N protein expression of the rescued viruses and parent strain was confirmed. Moreover, the rescued viruses exhibit similar growth dynamic characteristics with its parent strain. Taken together, these results indicate that the infectious clones can be used for reverse genetic operation, which lays a foundation for the research of pathogenesis and vaccine development of PRRSV.
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