Inhibition of Coxsackievirus B3 cardiotropic strain Woodruff replication by silencing essential viral genes

Figures & data

Table 1. Reagents for extraction of total RNA with silica particles.

Buffer	Substance	Quantity
Grinding buffer (GB)	Guanadine thiocyanate	4.0 mol/L
	Na-Acetate, pH 5.2	0.2 mol/L
	EDTA	25 mmol/L
	K-Acetate	1.0 mol/L
	PVP 40	2.5 % (w/v)
	storage at 4 °C
Washing buffer (WB)	Tris-HCl, pH 7.5	10 mmol/L
	EDTA	0.5 mmol/L
	NaCl	5 mmol/L
	Ethanol	50 % (v/v)
		storage at 4 °C
NaI	Na2SO3	0.15 mol/L
	NaI	6 mol/L
	storage at 4 °C, in the dark

Table 2. Primers used in touch-down RT-PCR.

No.	Primer	Sequence (5’→ 3’)	Length (nt)	Position in the genetic region	Genetic region	Flanking fragment (bp)
1	3C For	CGG GAA TGC CCA ATC AGA AGC C	22	5297–5318	3C	663
2	3C Rev	GCG TGT TGA TGA CTG GGA ACC C	22	5959–5938
3	VP3-VP1 For	GCA ATG TTG GGT ACA CAT GT	20	2179–2199 (VP3)	VP3-VP1	677
4	VP3-VP1 Rev	TAC CGC TAC CAG TGT AGA CGC ACC T	25	2856–2832 (VP1)
5	3C For DS	TAA TAC GAC TCA CTA TAG GGC GGG AAT GCC CAA TCA GAA GCC	42	5297–5318	3C	692
6	3C Rev DS	GGA AAA AAA GCG TGT TGA TGA CTG GGA ACC C	40	5959–5938
7	VP3-VP1 For DS	TAA TAC GAC TCA CTA TAG GGG CAA TGT TGG GTA CAC ATG T	40	2179–2199 (VP3)	VP3-VP1	706
8	VP3-VP1 Rev DS	GGA AAA AAA TAC CGC TAC CAG TGT AGA CGC ACC T	34	2856–2832 (VP1)

Table 3. Program for touch-down RT-PCR.

Initial denaturation	3 min	95 °C
Denaturation	30 s	92 °C	5 cycles
Annealing	30 s	64 °C
Elongation	90 s	72 °C
Denaturation	30 s	92 °C	5 cycles
Annealing	30 s	62 °C
Elongation	90 s	72 °C
Denaturation	30 s	92 °C	10 cycles
Annealing	30 s	60 °C
Elongation	90 s	72 °C
Denaturation	30 s	92 °C	10 cycles
Annealing	30 s	55 °C
Elongation	90 s	72 °C
Final elongation	10 min	72 °C

Figure 1. Schematic illustration of in vitro dsRNA production system.

Figure 2. Cytotoxicity assay for 3C and VP1 + VP3 specific dsRNAs in HEp-2 cells.

Figure 3. Cytotoxicity assay of siRNA pools for HEp-2 cells.

Figure 4. Antiviral effect of dsRNAs against Coxsackievirus B3.

Figure 5. Antiviral effect of siRNA pools against Coxsackievirus B3.