Qiaprep viral rna minikit

Total RNA was extracted from Marc-145 cells treated with 3000 U/mL human IFN-α (Peprotech) using QIAprep viral RNA minikit (Qiagen, Hilden, Germany) and cDNA synthesis was performed with SuperScript III Reverse Transcriptase (Invitrogen, Shanghai, China). The full-length mViperin gene was amplified with a set of primers (Table 1), and amplicons were cloned into a pEASY-Simple Blunt vector (Beijing TransGen Biotech Co. Ltd., Beijing, China) and sequenced. The determined nucleotide sequence of mViperin was compared to that found in the database (GenBank accession number: JQ437826.1). To generate the expression vector, the mViperin gene was amplified from a previously sequenced plasmid using the primers shown in Table 1. Polymerase chain reaction (PCR) products were digested with restriction enzymes and cloned into a pVAX-1 vector with the kozak sequence at the N terminus and a FLAG tag at the C terminus to produce a pVAX-mVIP plasmid. Truncations of mViperin were subcloned from the pVAX-mVIP plasmid with a FLAG tag at the N-terminus to produce pVAX-mVIP (5’Δ8, 5’Δ10, 5’Δ12, 5’Δ17, 5’Δ33 and 3’Δ143) plasmids.

The animal welfare committee of the Shanghai Veterinary Research Institute approved the animal experiments. The approve ID is SYXK-2011-0116. All animal studies were carried out in accordance with the approved guidelines and blinded to remove investigator bias. Twelve 4-week-old PRRSV-free piglets were obtained and divided randomly into three groups, i.e., four piglets in each group. Each treatment group was housed individually. We administered miR-130b or NC mimic (6 mg per dose) mixed with RNAi-Mate (GenePharma) in Opti-MEM^® I (Invitrogen) solution in a final volume of 2.5 ml intranasally to the piglets, and inoculated intranasally with 3 ml of diluted vJX143 (1 × 105 TCID₅₀) 6 h later, simulating the natural route of PRRSV infection. At 5 day post-infection (dpi), second deliveries of miR-130b or NC mimics were performed using the half dose and same route. The rectal temperature of each piglet was monitored daily until 21 dpi. Viral genomic RNA in serum samples from each piglet was isolated using a QIAprep viral RNA minikit (Qiagen) and viral load was detected at 3, 7, 10, 14, and 21 dpi using one-step RT-PCR. Specific primers for quantitative analysis of viral RNA copies and listed in Table 2.