Treatment with hypertonic saline is recommended in patients with upper respiratory tract infections bronchiolitis and cystic fibrosis1. It is commonly believed that this effect is due to the mucolytic activity of hypertonic saline.
We recently published evidence that sodium chloride (NaCl) and other chloride salts have a direct, dose-dependent inhibitory effect on viruses2. NaCl was broadly inhibitory against human/animal viruses, including DNA/RNA viruses, enveloped/non-enveloped viruses (e.g. adenovirus, herpes simplex virus type 1 (HSV-1), murine herpes virus, influenza A virus, respiratory syncytial virus, coronavirus and enterovirus ). In a pilot randomised controlled trial (RCT) done in parallel we showed that hypertonic saline nasal irrigation and gargling was effective in reducing the duration of the common cold in adults by two days.
Viral inhibition by NaCl was chloride ion (Cl-) dependent (since inhibition is reversed by chloride channels inhibitors). NaCl neither had a direct effect on virus particles nor on viral attachment and uptake. The inhibition required the conversion of Cl- to hypochlorous acid (HOCl) which was observed within 2-6 hours after infection. In myeloid cells myeloperoxidase (MPO) converts Cl- to HOCl, but the enzyme which converts Cl- to HOCl in non-myeloid cells is currently unknown.
HSV-1 which causes oral, genital infections as well as infections of the braincan be effectively treated by acyclovir/its derivatives. But resistant viruses can develop. In our experiments, Cl- mediated inhibition of HSV-1 was seen in various cell types and cell lines with few exceptions. Interestingly, we identified two neuroblastoma cells lines, SH-N-SH (parental cell line) and SH-SY5Y (mutant subclone derived from SH-N-SH), in which HSV-1 showed a differential sensitivity to salt inhibition, indicating that the genetic background of the host cell plays an important role in NaCl susceptibility. Interestingly, acyclovir had no effect on replication in all neuroblastoma cell lines tested even at high concentrations, whereas chloride was able to inhibit HSV-1 in most cell lines.
The aim of this PhD project is to further investigate the mechanism by which Cl- blocks viral replication, to test whether it can be a therapeutic agent against animal/human viruses. In particular, we will test at which stage the block of replication occurs, which cellular proteins and enzymes (e.g. ion channels, myeloperoxidase and superoxide dismutase) are involved. We will also investigate the significance of the viral/host genetic background for chloride-mediated viral inhibition. By comparing the whole genome sequence of the SH-N-SH and SH-SY5Y cell line pairs we will try to identify the gene(s) responsible for chloride sensitivity. We will also perform a genome-wide RNA interference screen to identify the gene(s) responsible. Furthermore, we will test whether chloride and acyclovir are effective in different types of differentiated glioma cells derived from induced pluripotent stem cells (iPSC). We will also generate cell lines by CRISPR/Cas9 gene editing in which the gene(s) responsible for chloride sensitivity will be knocked-out or overexpressed. In summary, a broad range of different molecular, biochemical and cellular methods will be applied, including cutting edge technologies such as genome-wide RNAi screens and CRISPR/Cas9 gene editing.
1 Ramalingam S, Graham C, Dove J, Morrice L, Sheikh A. A pilot, open labelled, randomised controlled trial of hypertonic saline nasal irrigation and gargling for the common cold. Sci Rep. 2019;9(1):1015. 2 Ramalingam S, Cai B, Wong J, et al. Antiviral innate immune response in non-myeloid cells is augmented by chloride ions via an increase in intracellular hypochlorous acid levels. Sci Rep. 2018;11;8(1):13630. 3 Griffiths SJ, Koegl M, Boutell C, et al. A systematic analysis of host factors reveals a Med23-interferon-lambda regulatory axis against Herpes simplex virus Type 1 replication. PLoS pathogens 2013; 9(8): e1003514.