Astrocytic growth through the autocrine/paracrine production of IL-1β in the early infectious phase of fowl glioma-inducing virus

Figures & data

Table 1. Sequence of the oligonucleotide primers used in quantitative RT-PCR.

Target gene	Primer (5′ to 3′)		Accession number	Reference
β-actin	Forward	TAT CCG TTA GGA TCT GTA TG	L08165	Nakamura et al. (2011)
	Reverse	ATC TCG TCT TGT TTT ATG CG
ALV-A	Forward	TGG AAA GGT GAG CAA GAA GGA	AB112960	Nakamura et al. (2011)
	Reverse	CAC ACA AGA CCA GGA CAC CAA
IL-1β	Forward	GCT CTA CAT GTC GTG TGT GAT GAG	AJ245728	Shini et al. (2010)
	Reverse	TGT CGA TGT CCC GCA TGA
TGF-β	Forward	AGG ATC TGC AGT GGA AGT GGA T	M31160	Shini et al. (2010)
	Reverse	CCC CGG GTT GTG GTT GGT
IL-4	Forward	AAC ATG CGT CAG CTC CTG AAT	AJ624249	Shini et al. (2010)
	Reverse	TCT GCT AGG AAC TTC TCC ATT GAA
IL-6	Forward	GCT CGC CGG CTT CGA	AJ250838	Kaiser et al. (2000)
	Reverse	GGT AGG TCT GAA AGG CGA ACA G
Interferon-α	Forward	ATG CCA CCT TCT CTC ACG AC	EU367971	Adams et al. (2009)
	Reverse	AGG CGC TGT AAT CGT TGT CT
Interferon-β	Forward	CCT CAA CCA GAT CCA GCA TT	AY831397	Adams et al. (2009)
	Reverse	GGA TGA GGC TGT GAG AGG AG
Interferon-γ	Forward	GTG AAG AAG GTG AAA GAT ATC ATG GA	Y07922	Shini et al. (2010)
	Reverse	GCT TTG CGC TGG ATT CTC A

Figure 1. Nodular astrocytic growth in the brain of a chicken inoculated in ovo with 5 × 10⁴ TCIU Sp-53. 1a: Protoplasmic astrocytes proliferate around vessels. Haematoxylin and eosin stain. Bar = 20 µm. 1b: Astrocytes of nodules show positivity for glial fibrillary acidic protein. Immunohistochemistry. Bar = 20 µm.

Table 2. Histological score, viral RNA and cytokine expression (relative units) in brain of chickens inoculated with different titres of Sp-53.

	Mock	5 × 10^2 TCIU	5 × 10^4 TCIU	5 × 10^6 TCIU
n	3	6	3	5
Histological score	0.00 ± 0.00	1.33 ± 0.49	4.67 ± 0.33	4.00 ± 0.45
Viral RNA	0.00 ± 0.00	1.26 ± 0.17	1.95 ± 0.39	2.13 ± 0.24
IL-1β	0.76 ± 0.12	1.40 ± 0.19	2.77 ± 0.84	2.84 ± 0.78*
TGF-β	0.94 ± 0.04	0.92 ± 0.08	1.25 ± 0.03*	0.91 ± 0.10**
Interferon-α	0.96 ± 0.08	1.79 ± 0.41	2.11 ± 0.33	1.71 ± 0.37
Interferon-β	0.89 ± 0.08	1.44 ± 0.36	1.47 ± 0.16	1.26 ± 0.18
Interferon-γ	5.36 ± 3.23	30.1 ± 11.7	11.9 ± 4.98	26.3 ± 14.2
IL-6	1.53 ± 0.29	1.82 ± 0.31	2.62 ± 0.68	2.00 ± 0.06
IL-4	0.82 ± 0.13	0.88 ± 0.16	2.03 ± 0.47	1.10 ± 0.31

Histological scores were defined as described in Materials and Methods. The level of viral RNA and each cytokine mRNA was calculated by the –ΔΔCt method using β-actin as an internal control. All results were obtained in duplicate.

* P < 0.05 compared with 5 × 10² TCIU as determined by analysis of variance and the Kruskal–Wallis test.

** P < 0.05 compared with 5 × 10⁴ TCIU as determined by analysis of variance and the Kruskal–Wallis test.

Table 3. Histological score, viral RNA and cytokine expression (relative units) in brain of chickens infected with Sp-53 and RCAS(A).

	Sp-53	RCAS(A)
n	5	5
Histological score	4.00 ± 0.45*	1.00 ± 0.45
Viral RNA	2.13 ± 0.24*	0.86 × 10^−2 ± 0.37 × 10^−2
IL-1β	2.84 ± 0.78	1.99 ± 0.41
TGF-β	0.91 ± 0.10*	0.46 ± 0.03
Interferon-α	1.71 ± 0.37	1.11 ± 0.18
Interferon-β	1.26 ± 0.18	1.48 ± 0.18
Interferon-γ	26.3 ± 14.2	29.7 ± 7.31
IL-6	2.00 ± 0.06	2.85 ± 0.20
IL-4	1.10 ± 0.31*	2.26 ± 0.14

Histological scores were defined as described in Materials and Methods. The level of viral RNA and each cytokine mRNA was calculated by the –ΔΔCt method using β-actin as an internal control.

All results were obtained in duplicate.

* P < 0.05 compared with RCAS(A) as determined by Student's t test.

Figure 2. Viral replication in cultured cells. 2a: Viral RNA levels of DF-1 infected with Sp-53 and RCAS(A). 2b: Antigen p27 in cell supernatant of DF-1 infected with Sp-53 and RCAS(A). 2c: Viral RNA levels of primary astrocytes infected with Sp-53 and RCAS(A). 2d: Antigen p27 in cell supernatant of primary cultured astrocytes infected with Sp-53 and RCAS(A). Error bars represent the means ± standard error of the means (n = 2).

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