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Infection of equine monocyte-derived macrophages with an attenuated equine infectious anemia virus (EIAV) strain induces a strong resistance to the infection by a virulent EIAV strain.

Ma J, Wang SS, Lin YZ, Liu HF, Liu Q, Wei HM, Wang XF, Wang YH, Du C, Kong XG, Zhou JH, Wang X - Vet. Res. (2014)

Bottom Line: Further experiments indicate that the expression of the soluble EIAV receptor sELR1, Toll-like receptor 3 (TLR3) and interferon β (IFNβ) was up-regulated in eMDM infected with EIAVFDDV13 compared with eMDM infected with EIAVUK3.The knock down of TLR3 mRNA significantly impaired poly I:C-stimulated resistance to EIAV, greatly reducing the expression of sELR1 and IFNβ and lowered the level of infection resistance induced by EIAVFDDV13.These results indicate that the induction of restraining infection by EIAVFDDV13 in macrophages is partially mediated through the up-regulated expression of the soluble viral receptor and IFNβ, and that the TLR3 pathway activation plays an important role in the development of an EIAV-resistant intracellular environment.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150001, Heilongjiang, China. Jianhua_uc@126.com.

ABSTRACT
The Chinese attenuated equine infectious anemia virus (EIAV) vaccine has successfully protected millions of equine animals from EIA disease in China. Given that the induction of immune protection results from the interactions between viruses and hosts, a better understanding of the characteristics of vaccine strain infection and host responses would be useful for elucidating the mechanism of the induction of immune protection by the Chinese attenuated EIAV strain. In this study, we demonstrate in equine monocyte-derived macrophages (eMDM) that EIAVFDDV13, a Chinese attenuated EIAV strain, induced a strong resistance to subsequent infection by a pathogenic strain, EIAVUK3. Further experiments indicate that the expression of the soluble EIAV receptor sELR1, Toll-like receptor 3 (TLR3) and interferon β (IFNβ) was up-regulated in eMDM infected with EIAVFDDV13 compared with eMDM infected with EIAVUK3. Stimulating eMDM with poly I:C resulted in similar resistance to EIAV infection as induced by EIAVFDDV13 and was correlated with enhanced TLR3, sELR1 and IFNβ expression. The knock down of TLR3 mRNA significantly impaired poly I:C-stimulated resistance to EIAV, greatly reducing the expression of sELR1 and IFNβ and lowered the level of infection resistance induced by EIAVFDDV13. These results indicate that the induction of restraining infection by EIAVFDDV13 in macrophages is partially mediated through the up-regulated expression of the soluble viral receptor and IFNβ, and that the TLR3 pathway activation plays an important role in the development of an EIAV-resistant intracellular environment.

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Effects of TLR3 mRNA knockdown on sELR1 and INFβ expression in eMDM. (A) Knockdown of TLR3 expression in eMDM with siRNA. At 48 h after transfection with siRNA targeting equine TLR3 (siRNA-TLR3), the expression level of TLR3 mRNA was quantified with real-time qPCR. siRNA with a scrambled sequence (siRNA-C) was used as the control. (B) The induction of TLR3 expression by poly I:C in siRNA-TLR3-transfected eMDM. The cells were transfected with siRNA-TLR3 or siRNA-C. TLR3 mRNA was measured with real-time qPCR 12 h after stimulation with 0.5 μg/mL poly I:C. (C) Kinetics of TLR3, sELR1 and INFβ mRNA expression induced by EIAVFDDV13 in eMDM transfected with siRNA-TLR3. Cells were transfected with siRNA-TLR3 or siRNA-C. After 48 h of incubation, EIAVFDDV13 was added to the cells, and total RNA was extracted at different times after infection. The mRNA copies were specifically amplified and quantified with bDNA (for TLR3 and INFβ) and qPCR (for sELR1). The values of Y axis were treated by Log2. *P < 0.05.
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Fig5: Effects of TLR3 mRNA knockdown on sELR1 and INFβ expression in eMDM. (A) Knockdown of TLR3 expression in eMDM with siRNA. At 48 h after transfection with siRNA targeting equine TLR3 (siRNA-TLR3), the expression level of TLR3 mRNA was quantified with real-time qPCR. siRNA with a scrambled sequence (siRNA-C) was used as the control. (B) The induction of TLR3 expression by poly I:C in siRNA-TLR3-transfected eMDM. The cells were transfected with siRNA-TLR3 or siRNA-C. TLR3 mRNA was measured with real-time qPCR 12 h after stimulation with 0.5 μg/mL poly I:C. (C) Kinetics of TLR3, sELR1 and INFβ mRNA expression induced by EIAVFDDV13 in eMDM transfected with siRNA-TLR3. Cells were transfected with siRNA-TLR3 or siRNA-C. After 48 h of incubation, EIAVFDDV13 was added to the cells, and total RNA was extracted at different times after infection. The mRNA copies were specifically amplified and quantified with bDNA (for TLR3 and INFβ) and qPCR (for sELR1). The values of Y axis were treated by Log2. *P < 0.05.

Mentions: To confirm that up-regulated TLR3 expression plays a crucial role in the induction of infection resistance in EIAVFDDV13-infected eMDM, TLR3 expression in eMDM was knocked down with siRNA. As shown in Figure 5A, TLR3 transcription was reduced by approximately 65% in eMDM transfected with horse TLR3 siRNA (siRNA-TLR3) compared with cells transfected with scrambled RNA control siRNA-C. When the siRNA-TLR3-transfected cells were treated with 0.5 μg/mL poly I:C for 12 h, only a slight up-regulation of TLR3 mRNA expression (0.5-fold) was observed, whereas an approximately 8.0-fold increase in TLR3 mRNA expression was detected in siRNA-C-transfected cells (Figure 5B). These results indicate that TLR3 expression was substantially suppressed by its specific siRNA. The effect of EIAVFDDV13 on TLR3, sELR1 and IFNβ expression was evaluated after TLR3 knockdown. As shown in Figure 5C, compared with the effect of EIAVFDDV13 on eMDM that were not treated with siRNA, the up-regulation of these three factors was greatly diminished in cells transfected with specific siRNA-TLR3.Figure 5


Infection of equine monocyte-derived macrophages with an attenuated equine infectious anemia virus (EIAV) strain induces a strong resistance to the infection by a virulent EIAV strain.

Ma J, Wang SS, Lin YZ, Liu HF, Liu Q, Wei HM, Wang XF, Wang YH, Du C, Kong XG, Zhou JH, Wang X - Vet. Res. (2014)

Effects of TLR3 mRNA knockdown on sELR1 and INFβ expression in eMDM. (A) Knockdown of TLR3 expression in eMDM with siRNA. At 48 h after transfection with siRNA targeting equine TLR3 (siRNA-TLR3), the expression level of TLR3 mRNA was quantified with real-time qPCR. siRNA with a scrambled sequence (siRNA-C) was used as the control. (B) The induction of TLR3 expression by poly I:C in siRNA-TLR3-transfected eMDM. The cells were transfected with siRNA-TLR3 or siRNA-C. TLR3 mRNA was measured with real-time qPCR 12 h after stimulation with 0.5 μg/mL poly I:C. (C) Kinetics of TLR3, sELR1 and INFβ mRNA expression induced by EIAVFDDV13 in eMDM transfected with siRNA-TLR3. Cells were transfected with siRNA-TLR3 or siRNA-C. After 48 h of incubation, EIAVFDDV13 was added to the cells, and total RNA was extracted at different times after infection. The mRNA copies were specifically amplified and quantified with bDNA (for TLR3 and INFβ) and qPCR (for sELR1). The values of Y axis were treated by Log2. *P < 0.05.
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Fig5: Effects of TLR3 mRNA knockdown on sELR1 and INFβ expression in eMDM. (A) Knockdown of TLR3 expression in eMDM with siRNA. At 48 h after transfection with siRNA targeting equine TLR3 (siRNA-TLR3), the expression level of TLR3 mRNA was quantified with real-time qPCR. siRNA with a scrambled sequence (siRNA-C) was used as the control. (B) The induction of TLR3 expression by poly I:C in siRNA-TLR3-transfected eMDM. The cells were transfected with siRNA-TLR3 or siRNA-C. TLR3 mRNA was measured with real-time qPCR 12 h after stimulation with 0.5 μg/mL poly I:C. (C) Kinetics of TLR3, sELR1 and INFβ mRNA expression induced by EIAVFDDV13 in eMDM transfected with siRNA-TLR3. Cells were transfected with siRNA-TLR3 or siRNA-C. After 48 h of incubation, EIAVFDDV13 was added to the cells, and total RNA was extracted at different times after infection. The mRNA copies were specifically amplified and quantified with bDNA (for TLR3 and INFβ) and qPCR (for sELR1). The values of Y axis were treated by Log2. *P < 0.05.
Mentions: To confirm that up-regulated TLR3 expression plays a crucial role in the induction of infection resistance in EIAVFDDV13-infected eMDM, TLR3 expression in eMDM was knocked down with siRNA. As shown in Figure 5A, TLR3 transcription was reduced by approximately 65% in eMDM transfected with horse TLR3 siRNA (siRNA-TLR3) compared with cells transfected with scrambled RNA control siRNA-C. When the siRNA-TLR3-transfected cells were treated with 0.5 μg/mL poly I:C for 12 h, only a slight up-regulation of TLR3 mRNA expression (0.5-fold) was observed, whereas an approximately 8.0-fold increase in TLR3 mRNA expression was detected in siRNA-C-transfected cells (Figure 5B). These results indicate that TLR3 expression was substantially suppressed by its specific siRNA. The effect of EIAVFDDV13 on TLR3, sELR1 and IFNβ expression was evaluated after TLR3 knockdown. As shown in Figure 5C, compared with the effect of EIAVFDDV13 on eMDM that were not treated with siRNA, the up-regulation of these three factors was greatly diminished in cells transfected with specific siRNA-TLR3.Figure 5

Bottom Line: Further experiments indicate that the expression of the soluble EIAV receptor sELR1, Toll-like receptor 3 (TLR3) and interferon β (IFNβ) was up-regulated in eMDM infected with EIAVFDDV13 compared with eMDM infected with EIAVUK3.The knock down of TLR3 mRNA significantly impaired poly I:C-stimulated resistance to EIAV, greatly reducing the expression of sELR1 and IFNβ and lowered the level of infection resistance induced by EIAVFDDV13.These results indicate that the induction of restraining infection by EIAVFDDV13 in macrophages is partially mediated through the up-regulated expression of the soluble viral receptor and IFNβ, and that the TLR3 pathway activation plays an important role in the development of an EIAV-resistant intracellular environment.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150001, Heilongjiang, China. Jianhua_uc@126.com.

ABSTRACT
The Chinese attenuated equine infectious anemia virus (EIAV) vaccine has successfully protected millions of equine animals from EIA disease in China. Given that the induction of immune protection results from the interactions between viruses and hosts, a better understanding of the characteristics of vaccine strain infection and host responses would be useful for elucidating the mechanism of the induction of immune protection by the Chinese attenuated EIAV strain. In this study, we demonstrate in equine monocyte-derived macrophages (eMDM) that EIAVFDDV13, a Chinese attenuated EIAV strain, induced a strong resistance to subsequent infection by a pathogenic strain, EIAVUK3. Further experiments indicate that the expression of the soluble EIAV receptor sELR1, Toll-like receptor 3 (TLR3) and interferon β (IFNβ) was up-regulated in eMDM infected with EIAVFDDV13 compared with eMDM infected with EIAVUK3. Stimulating eMDM with poly I:C resulted in similar resistance to EIAV infection as induced by EIAVFDDV13 and was correlated with enhanced TLR3, sELR1 and IFNβ expression. The knock down of TLR3 mRNA significantly impaired poly I:C-stimulated resistance to EIAV, greatly reducing the expression of sELR1 and IFNβ and lowered the level of infection resistance induced by EIAVFDDV13. These results indicate that the induction of restraining infection by EIAVFDDV13 in macrophages is partially mediated through the up-regulated expression of the soluble viral receptor and IFNβ, and that the TLR3 pathway activation plays an important role in the development of an EIAV-resistant intracellular environment.

Show MeSH
Related in: MedlinePlus