In-vitro assessment of differential cytokine gene expression in response to infections with Egyptian classic and variant strains of highly pathogenic H5N1 avian influenza virus

Peer review under responsibility of Faculty of Veterinary Medicine, Cairo University.

Figures & data

Table 1 Sequences of the primers and probes used in quantitative qRT-PCR.

Gene	Name	Sequence (5′–3′)	Refs.
IL1B	F-Primer	GCTCTACATGTCGTGTGTGATGAG	Kaiser et al. [16]
	R-Primer	TGTCGATGTCCCGCATGA
	Probe	F AM-CCACACTGCAGCTGGAGGAAGCC-(TAMRA)
IL4	F-Primer	AACATGCGTCAGCTCCTGAAT	Kumar et al. [17]
	R-Primer	TCTGCTAGGAACTTCTCCATTGAA
	Probe	FAM-AGCAGCACCTCCCTCAAGGCACC-TAMRA
IL6	F-Primer	GCTCGCCGGCTTCGA	Kaiser et al. [16]
	R-Primer	GGTAGGTCTGAAAGGCGAACAG
	Probe	FAM-AGGAGAAATGCCTGACGAAGCTCTCCA-TAMRA
IL8	F-Primer	GCCCTCCTCCTGGTTTCA G	Kogut et al. [18]
	R-Primer	TGGCACCGCAGCTCATT
	Probe	FAM-TCTTTACCAGCGTCCTACCTTGCGACA-TAMRA
IL10	F-Primer	CATGCTGCTGGGCCTGAA	Kumar et al. [17]
	R-Primer	CGTCTCCTTGATCTGCTTGATG
	Probe	FAM-CGACGATGCGGCGCTGTCA-TAMRA
IL18	F-Primer	AGGTGAAATCTGGCAGTGGAAT	Kogut et al. [18]
	R-Primer	ACCTGGACGCTGAATGCAA
	Probe	FAM-CCGCGCCTTCAGCAGGGATG-TAMRA
IFNα	F-Primer	GACAGCCAACGCCAAAGC	Eldaghayes et al. [19]
	R-Primer	GTCGCTGCTGTCCAAGCATT
	Probe	FAM-CTCAACCGGATCCACCGCTACACC-TAMRA
IFNγ	F-Primer	GTGAAGAAGGTGAAAGATATCATGGA	Kaiser et al. [16]
	R-Primer	GCTTTGCGCTGGATTCTCA
	Probe	FAM-TGGCCAAGCTCCCGATGAACGA-TAMRA
28S	F-Primer	GGCGAAGCCAGAGGAAACT	Kaiser et al. [16]
	R-Primer	GACGACCGATTTGCACGTC
	Probe	FAM-AGGACCGCTACGGACCTCCACCA-TAMRA
AIV (H5)	LH1-Primer	ACATATGACTACCCACARTATTCAG	Slomka et al. [20]
	RH1-Primer	AGACCAGCTAYCATGATTGC
	Probe	FAM-TCWACAGTGGCGAGTTCCCTAGCA-TAMRA

Table 2 Cytokines mRNA fold change at 6, 24 and 48 h after infection of HD11 cell line with 0.05 TCID50 per cell.

Cytokines	6 h		24 h		48 h
	Classical	Variant	Classical	Variant	Classical	Variant
IL1b	23.96405	28.12147	1.261552	2.211308	1.198143	5.088005
IL4	1.3297	2.39197	13.8864	21.0303	20.8171	26.4904
IL6	1.11891	1.2886	1.30279	0.99481	1.20497	1.88296
IL8	4.63992	11.1255	0.48609	0.55826	0.51939	0.82599
IL10	1.32648	2.25793	0.29968	0.26521	0.07471	0.14839
IL18	2.90291	2.46844	0.42647	0.14876	0.35792	0.18233
IFNγ	2.61389	3.52079	26.5309	8.56112	22.4773	8.5185
IFNα	1.07482	1.00292	0.95754	0.94606	1.91906	3.24204

Fig. 1 Cytokines mRNA fold change at 6, 24 and 48 h post infection (hpi) of HD11 cell line with Egyptian H5N1 variant (A/chicken/Egypt/0963S-NLQP/2009) and classical (A/chicken/Egypt/121/2012) strains. mRNAs of IL1b, IL6, IL8, IL4, IL10, IFNγ and IFNα were measured by qRT-PCR in HD11 infected cells. Data show fold change compared with uninfected control HD11 cells. Data represent the mean; error bars show SE.

Table 3 Cytokines mRNA fold change at 6, 24 and 48 h after infection of PBMC with 0.05 TCID50 per cell.

	6 h after infection		24 h after infection		48 h after infection
	Classical	Variant	Classical	Variant	Classical	Variant
IL1b	63.3819	111.4691	9.1883	20.8157	4.0424	21.6111
IL4	2.8036	1.2879	0.9764	3.1199	1.1037	3.0111
IL6	184.3879	506.8096	13.5226	24.804	12.4416	12.4554
IL8	219.4274	136.5232	34.0386	30.6009	10.1944	35.8152
IL10	4.5732	84.4368	4.1592	70.3608	3.5373	26.3768
IL18	2.6036	5.1437	1.0786	1.0449	0.3396	0.4444
IFNγ	4.5732	84.4368	0.6596	5.6859	2.0669	6.7473
IFNα	0.4411	1.1423	0.4180	1.7633	0.2595	0.8520

Fig. 2 Cytokines mRNA fold change at 6, 24 and 48 h post infection (hpi) of PBMC cell line with Egyptian H5N1 variant (A/chicken/Egypt/0963S-NLQP/2009) and classical (A/chicken/Egypt/121/2012) strains. mRNAs of IL1b, IL6, IL8, IL4, IL10, IFNγ and IFNα were measured by qRT-PCR in PBMC infected cells. Data show fold change compared with uninfected control PBMC cells. Data represent the mean; error bars show SE.