M mlv system

The RNA were extracted from the three viral stocks (AnD133719, SHM172805 and ArD141967) and were used as templates for RT-PCR. Specific primers (Table 2), the M-MLV system (Invitrogen, Carlsbad, CA, USA), and the Go-Taq PCR Kit (Promega, Madison, WI, USA) were used for cDNA synthesis and amplification, according to the manufacturer’s instructions. All primers used to amplify the S and M segments were designed according to RVFV sequences available in GenBank. Accession numbers of all RVFV sequences used to design the primers are presented in Additional file 1: Table S1. The PCR products of the expected sizes were purified directly from the agarose gel using a QIAgen Gel extraction kit and sequenced by Cogenics (Beckman Coulter Genomics, Essex, United Kingdom). Sequencing was performed in both directions, using the original reverse and forward primers as for the amplification.Table 2

List of primers used

Nom	Segment	Position	Sequence	Tm
NSng	S	31–48	TATCATGGATTACTTTCC	48
NSca	S	841–824	CCTTAACCTCTAATCAAC	50
M1F	M	3–22	ACAAAGACCGGTGCAACTTC	53.9
M1R	M	1120–1140	CCAYGCAAAGGGTATGCAAT	53.2
M2F	M	1035–1054	TGAGGACTCTGAATTRCACCT	48.7
M2R	M	2395–2415	TCCAGAGAGTTGAGCCTTGC	53.3
MRV1a	M	3050–3068	CAAATGACTACCAGTCAGC	44.6
MRV2g	M	2262–2292	GGTGGAAGGACTCTGCGA	52.5
M3F	M	2979–2998	CAGTCCTCAGTGAGCYCATA	46.1
M3R	M	3763–3782	TCTCGGTTCTGGRGTGTGAA	52.5

RNA of cells was extracted using Trizol reagent (Invitrogen, Carlsbad, CA, USA). Concentration and purity were measured by spectrophotometer (Thermo Fisher Scientific Inc., MA, USA). Totally, 500 ng RNA was reverse transcribed into cDNA by M-MLV system (Cat no. C28025-011; Invitrogen, China). PCR reactions were performed by the 7900HT Fast Real-Time PCR System (Applied Biosystems, Waltham, MA, USA) with SYBR-green (TAKARA, Japan) in a 10 μl reaction mixture. The primers are shown in additional information (Supplementary Table 2).

Total RNA was purified using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Reverse transcription was performed using the M‐MLV system (Cat no. C28025‐011; Invitrogen, China). qRT‐PCR quantified RNA expression using the SYBR Green Master Mix (Takara, Japan). ACTB and U6 were used as internal controls for mRNAs and miRNAs, respectively. The relative RNA abundance was calculated using the standard 2^‐ΔΔCt method. The primers used in the present study are listed in Table S2. Plasmid extraction was performed according to the manufacturer’s protocols (Omega Bio‐tek, Norcross, GA, USA).