Mmessage high yield capped rna transcription kit

To rescue the chimeric viruses, each full-length cDNA clone plasmid was separately linearized using the restriction enzyme PacI (for cDNA clones with RvJXwn as the backbone) or RsrII (for cDNA clones with RvHB-1/3.9 as the backbone). Then, the purified full-length cDNA clone was transcribed and capped using a mMessage high-yield capped RNA transcription kit (Ambion) according to the manufacturer's protocol. The purified RNAs were transfected into BHK-21 cells as previously described [40] (link). At 24 h post-transfection, the supernatants were harvested and serially passaged for three times in MARC-145 cells. The MARC-145 cells infected with the rescued viruses were examined by indirect immunofluorescence assay (IFA) using the monoclonal antibody (McAb) SDOW17 (Rural Technologies), which is specific for the PRRSV N protein [59] (link), [60] (link). To further verify the correctness of the exchanged regions, the RNAs of third-passage chimeric viruses were extracted and subjected to RT-PCR, followed by sequencing.

In vitro transcription and virus recovery were made as described before [26 ]. Briefly, chimeric full-length cDNA clones were linearized by cleavage with the restriction enzyme SwaI downstream from the poly (A) tail, then linearized plasmid DNA were transcribed in vitro according to the procedure of mMessage High Yield Capped RNA Transcription kit (Ambion, Austin, TX, USA). To rescue the viruses, the synthetic RNA was transfected into BHK-21 cells then passaged on MARC-145 cells. The rescued viruses were confirmed using an indirect immunofluorescence assay (IFA) with anti-PRRSV serum and sequencing, and named rHuN4-F5-ORF1a, rHuN4-F5-ORF1b, rHuN4-F5-ORF2-7 (genetic backbone of HuN4-F5 with 5′UTR + ORF1a, ORF1b or ORF2-7 + 3′UTR from HuN4-F112), and rHun4-F112-ORF1a, rHun4-F112-ORF1b, rHun4-F112-ORF2-7 (genetic backbone of HuN4-F112 with 5′UTR + ORF1a, ORF1b, or ORF2-7 + 3′UTR from HuN4-F5), respectively. The 4th passage viruses (P5) on MARC-145 cells were used on the following studies.

The protocol for the rescue of the chimeric and parental viruses was performed as previously described [32 (link)]. First, each full-length cDNA clone plasmid was separately linearized using the restriction enzyme NotI (New England Biolabs). The linearized plasmid DNA was transcribed and capped using the mMessage High Yield Capped RNA Transcription kit (Ambion) according to the manufacturer’s instructions. The synthetic RNA was transfected into BHK-21 cells using the DMRIE-C reagent (Invitrogen) according to the manufacturer’s protocol. Finally, the supernatants were harvested 24 h post-transfection and serially passaged on MARC-145 cells.