Discovery of novel antiviral treatments and development of new antiviral platforms for enteroviruses

Abstract

Enterovirus A71 (EV-A71) has emerged as a public health threat in the Asia-Pacific region. Over the past decades, EV-A71 associated hand, foot and mouth disease (HFMD) epidemics recurred with high frequency and disease severity. Infection with EV-A71 has been of particular concern as it can cause severe cardiopulmonary and neurological complications, which have been commonly reported as causes of mortality in complicated HFMD cases. However, no effective antiviral drug is available currently. To foster the antiviral development, we focused on developing a new antiviral platform using small intestinal organoids, and identifying a novel host factor that can be targeted for antiviral treatment for enteroviral infections.

In our study, we first investigated the susceptibility of small intestinoids to infections by different enterovirus species. We characterized the viral replication kinetics and expression profiles of different enterovirus receptors. We found that small intestinoids supported replications of EV-A71, coxsackievirus B2 (CVB2) and poliovirus type 3 (PV-3) but not EV-D68, which is a respiratory virus. In addition, we showed that the system exhibited satisfactory consistency and reproducibility as demonstrated by different antiviral assays. Hence, we also provided a new piece of antiviral data of itraconazole against EV-A71 replication in the physiological relevant small intestinoid model.

Next, we identified GADD34 as a novel host dependency gene that facilitated EV-A71 replication using the transcriptomic approach. To investigate the effect of GADD34 expression on EV-A71 replication, we assessed the viral load change after promoting and suppressing GADD34 expression by siRNA knockdown and overexpression of GADD34. Expression of GADD34 was found to be directly correlated with the viral replication. Furthermore, it was shown that EV-A71 infection induced up-regulation of GADD34 expression, which reduced eIF-2α phosphorylation and promoted the viral replication. We found that suppressed expression of GADD34 protein led to an elevated level of eIF-2α phosphorylation and reduction of viral protein synthesis. These observations suggested that GADD34 promoted EV-A71 replication via the reduction of eIF-2α phosphorylation. Sephin1 is a selective inhibitor of GADD34. It has currently completed Phase I clinical trial with a promising pharmacokinetic profile for targeting the central and peripheral nervous system. To explore the potential of Sephin1 being re-purposed into an antiviral drug against enteroviral infections, we further assessed the antiviral activity of Sephin1 in RD cell line, small intestinoid and iPSC-derived human neural progenitor cell models. We demonstrated that Sephin1 treatment significantly inhibited EV-A71 replication in these models. Importantly, we also showed that Sephin1 elicited broad-spectrum antiviral activity against a variety of enterovirus species including CVB2, PV-3 and EV-D68. Interestingly, it was also reported Sephin1 inhibited replications of a variety of RNA and DNA viruses. Collectively, the broad antiviral spectrum of Sephin1 indicates it has a high potential to be developed into a broad-spectrum antiviral drug against a wide range of viruses. The significance of the control by eIF-2α phosphorylation over the viral replications is also denoted, warranting further in-depth investigation.

Taken together, our findings advance our understanding of EV-A71 pathogenesis and the antiviral development against enteroviral infections.