Cytokine levels in imiquimod, poly I:C or PPRV treated PBMCs.

<p>a) <b>TNFα,</b> b) IFNα, and c) IFNγ from supernatants of PPRV infected PBMC of different goat breeds. Bars with the same superscript do not differ significantly. Significance is indicated when p<0.05. PPRV infected PBMC from Kanni and Salem black breeds show higher production of TNFα, IFNα and IFNγ than Barbari. Poly I:C and imiquimod treated PBMC, similarly, show higher production of TNFα and IFNα in Kanni and Salem black breeds than in Barbari. TNFα and IFNγ levels are expressed as the corrected mean ± SD of optical density [OD] of treatment groups from which the OD of mock infected supernatants is subtracted. IFNα concentrations in the experimental samples are expressed as pg/ml.</p

Basal expression levels of TLR3 and 7 mRNA in Barbari, Tellicherry, Kanni and Salem Black goat breeds.

<p>Significantly higher basal TLR3 (p<0.001) and TLR7 (p<0.001) mRNA expression levels observed in Kanni and Salem Black breeds, compared to Barbari. Bars with the same superscript do not differ significantly. Values represent mean ± SD of 40-corrected CT of TLR3 and 7 in nine individual animals per breed (n = 9).</p

Induction of cytokine genes of goat PBMCs with TLR3 and TLR7 agonists and PPRV.

<p>A) Fold changes in mRNA expression levels of IL1β, IL6, IL8, IL10, IL12p40, TNFα, IFNγ and IFNα in goat PBMC stimulated with a) poly I:C, b) imiquimod or c) infected with PPRV d) Goat and buffalo PBMCs infected with PPRV. Fold change was determined by the 2^−ΔΔCt formula. An upregulation in TNFα expression levels, consistent with a downregulation in IL10 levels, is observed in Kanni and Salem Black breeds of all treatment groups. In addition, upregulation of IFNα and IFNγ was observed in the PBMC of Kanni and Salem Black breeds after PPRV infection. PPRV stimulation resulted in an upregulation of IL1β and IFNα in buffalo PBMC and IL10, IL12p40 and IFNγ in goat PBMCs. Bars with the same superscript do not differ significantly. Significance is indicated when p<0.05. Values represent mean ± SD of 5 individual animals per goat breed.</p

TaqMan primer/Probe sequences for assessing basal expression levels of TLR3 and TLR7 mRNA.

<p>TaqMan primer/Probe sequences for assessing basal expression levels of TLR3 and TLR7 mRNA.</p

Antiviral activity of human IFNα against PPRV.

<p>Reduction in PPRV viral load was observed in Vero cells pretreated with different concentrations of human IFNα. All the doses tested significantly reduced PPRV viral load (mean ± SD of 40-Ct). Statistical significance was defined as follows: ***<i>P</i><0.001.</p

Role of type I IFN in limiting PPRV replication.

<p>a) Imiquimod or b) PPRV induced IFN-alpha mRNA expression in buffalo and goat PBMCs in the presence and absence of the TLR7 antagonist (IRS 661). Cytokine mRNA expression was quantified at 3, 6, 12 and 24 h post stimulation by qRT-PCR assays using SYBR Green chemistry. Fold change in mRNA expression induced by Imiquimod or PPRV stimulation was calculated using mock induced cytokine mRNA expression levels as a calibrator. c) PPRV replication in goat and buffalo PBMC (at 24 h and 48 h) in the presence of conditioned medium (CM) from PPRV infected cells. The expression levels of viral hemagglutinin (H) mRNA levels expressed as a percentage inhibition in viral replication in the presence of CM when compared with the control (PBMC+PPRV). Statistical significance at <i>P</i><0.01. Values are mean ± SD of fold change/percent inhibition. B: water buffalo, G: goat, BPBMC: buffalo PBMC, GPBMC: goat PBMC.</p

Virus replication in PBMCs stimulated with TLR3 and TLR7 agonists.

<p>a) Reduction in TCID₅₀ values of PPRV in goat PBMC on imiquimod treatment. Reduction in PPRV H gene expression levels in goat PBMC on treatment with b) poly I:C and c) imiquimod. Bars with the same superscript do not differ significantly. Significance is indicated when p<0.05. Values represent mean ± SD of 40-corrected CT in 5 individual animals per goat breed. Significantly higher PPRV viral loads observed in PBMC of Barbari and Tellicherry breeds as compared to Kanni and Salem Black. Significant reduction in PPRV levels on poly I:C and imiquimod treatment in all breeds.</p