Construction of plasmids for PRRSV rescue. A. The cDNA fragments F1, F2, F3, F4, and F5 were reversely transcribed and amplified from HP-PRRSV/SD16 genomic RNA. The CMV promoter was introduced to the 5′ end of HP-PRRSV/SD16 and the hepatitis delta virus ribozyme (HDV) and the bovine growth hormone termination and polyadenylation sequences (BGH) were included at the 3′ end of HP-PRRSV/SD16. All fragments were subcloned stepwise into the pBAC-SD16-5′-3′ vector to produce plasmid pBAC-SD16FL. B. DNA fragments Fa (from the genome position of 11,883 nt to the stop codon of M with AsiS I and Mlu I sites introduced at the 3′ end) and Fm (from start sequence of 3-UTR to the pBAC-SD16-5′-3′ vector with AsiS I and Mlu I sites introduced at the 5′ end) were PCR-amplified from pBAC-SD16FL and ligated together to form fragment Fam. The corresponding fragment of pBAC-SD16FL was replaced with Fam to construct plasmid pBAC-SD16FL-AM, which contains AsiS I and Mlu I sites between nt 15 170 and 15 171 of the HP-PRRSV/SD16 genome cDNA sequence. C. The EGFP with a copy of the transcription regulatory sequence for ORF6 (TRS6) at the 5′ end of the EGFP sequence was inserted into plasmid pBAC-SD16FL-AM to produce plasmid pBAC-SD16FL-TRS6-EGFP.