Effect of interferon alpha and cyclosporine treatment separately and in combination on Middle East Respiratory Syndrome Coronavirus (MERS-CoV) replication in a human in-vitro and ex-vivo culture model

Abstract

Middle East Respiratory Syndrome Coronavirus (MERS-CoV) has emerged as a coronavirus infection of humans in the past 5 years. Though confined to certain geographical regions of the world, infection has been associated with a case fatality rate of 35%, and this mortality may be higher in ventilated patients. As there are few readily available animal models that accurately mimic human disease, it has been a challenge to ethically determine what optimum treatment strategies can be used for this disease. We used in-vitro and human ex-vivo explant cultures to investigate the effect of two immunomodulatory agents, interferon alpha and cyclosporine, singly and in combination, on MERS-CoV replication. In both culture systems the combined treatment was more effective than either agent used alone in reducing MERS-CoV replication. PCR SuperArray analysis showed that the reduction of virus replication was associated with a greater induction of interferon stimulated genes. As these therapeutic agents are already licensed for clinical use, it may be relevant to investigate their use for therapy of human MERS-CoV infection.

Keywords: Cyclosporine; Ex vivo explants; Middle East Respiratory Syndrome Coronavirus (MERS-CoV); Type I interferon.

Copyright © 2018 Elsevier B.V. All rights reserved.

Figures

Fig. 1

Evaluation of the changes in MERS-CoV replication after the addition of IFN-α1, CsA and a combination of the two agents in Vero cells. 9 μM CsA and/or 2.4 × 104 U/ml of IFN-α1 were used. The data shown are the mean ± standard error of the mean in three representative experiments, which are analyzed by two-way ANOVA followed by Bonferroni's post hoc test (*/#P < 0.05, **/##P < 0.01, ***/###P < 0.001).

Fig. 2

Evaluation of the changes in MERS-CoV replication after the addition of IFN-α1, CsA and a combination of the two agents in (A) human bronchus and (B) human lung explant cultures. 9 μM CsA and/or 2.4 × 104 U/ml of IFN-α1 were used. The data shown are the mean ± standard error of the mean in at least three independent experiments, which are analyzed by two-way ANOVA followed by Bonferroni's post hoc test (*/#P < 0.05, **/##P < 0.01, ***/###P < 0.001).

Fig. 3

IFN-α1 and/or CsA inhibited MERS-CoV infection in bronchus & lung explant culture. 9 μM CsA and/or 2.4 × 104 U/ml of IFN-α1 were used. Sections of infected human bronchus and lung were stained with a polyclonal antibody against the MERS-CoV nucleocapsid protein. Positive cells were identified with a reddish brown colour. Magnification: x200. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4

IFN-α1 and/or CsA reduced apoptosis caused by MERS-CoV infection in bronchus & lung explant culture. 9 μM CsA and/or 2.4 × 104 U/ml of IFN-α1 were used. Sections of infected human bronchus and lung were stained with a monoclonal anti-cleaved caspase 3 antibody. Positive cells were identified with a Vector Red staining (pink colour). Magnification: x200. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 5

Combined treatment of IFN-α1 and CsA induced highest level of interferon stimulated genes (ISGs). 9 μM CsA and 2.4 × 104 U/ml of IFN-α1 were used. Distribution of gene expression was shown in the clustergram (A), the X axis indicates the normalized expression level of EMC infected lung or bronchus tissue without treatment, and the Y axis indicates the normalized expression level of EMC infected lung or bronchus tissue with different treatments; each dot on the plot represents the corresponding expression of a gene, the central line indicates no differences in gene level between two groups, while the boundary lines indicates the fold-change threshold (>=2 folds). The red dots lie above the upper boundary line are the up-regulated genes in EMC infected tissues with different treatments as compared to EMC infected tissues without treatment; and the green dots in lower section of the plot are the down-regulated genes. Gene expression of the combined treatment group in human bronchus (B) and lung (C) explant culture relative to the no treatment group (n = 3). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 6

Replication of MERS-CoV with different treatments in human microvascular endothelial cells (A), 9 μM CsA and/or 2.4 × 104 U/ml of IFN-α1 were used. Effect of IFN-α1 and/or CsA on protein expression level of MERS-CoV nucleocapsid and phosphorylation/activation of STAT1 protein (B); phosphorylation/activation level of protein involve in PI3K/Akt/mTOR and p38 MAPK pathways (C) were shown. Bands from three independent experiments were quantified by densitometry using ImageJ software, normalized expression/activation levels were indicated on the histogram. 9 μM CsA and/or 2.4 × 104 U/ml of IFN-α1 were used.

figs1

The trend of cell survival by MTT assay. Regimens of 9 μM CsA and/or 2.4 x 104 U/ml of IFN-α was applied on Vero cells (A); and human lung microvascular endothelial cells (B).

figs2

Verification of ISG expression using qPCR in human bronchus tissue (A). The corresponding MERS-CoV UpE level is also shown (B). The data shown are the mean ± standard error of the mean in three representative experiments, which are analyzed by two-way ANOVA followed by Bonferroni’s post hoc test (*P<0.05, **P<0.01, ***P<0.001 ).

figs3

Verification of ISG expression using qPCR in human lung tissue (A). The corresponding MERS-CoV UpE level is also shown (B).The data shown are the mean ± standard error of the mean in three representative experiments, which are analyzed by two-way ANOVA followed by Bonferroni’s post hoc test (*P<0.05, **P<0.01, ***P<0.001 ).

figs4

Schematic diagram of the proposed pathways that may be involved in the IFN-α and/or CsA control of ISGs transcription and translation.