Within the family of cholesterol oxidative derivatives known as oxysterols, several members, particularly those synthesized through enzyme-dependent reactions, have traditionally been recognized for their roles in cholesterol homeostasis and lipid metabolism. However, these molecules are now appreciated as multifunctional natural compounds, acting as critical modulators of diverse cellular and tissue functions for a review see Ref. [1,2]. Among them, in particular two oxysterols, 25-hydroxycholesterol (25OHC) and 25R,26-hydroxycholesterol, commonly referred to in the biomedical literature as 27-hydroxycholesterol (27OHC), despite the nomenclature inaccuracy, have drawn increasing attention due to their remarkable antiviral properties. These compounds exhibit broad-spectrum activity and operate through multiple mechanisms [3].

25OHC is generated by hydroxylation at the 25th carbon of the cholesterol backbone, primarily via an enzymatic process mediated by cholesterol 25-hydroxylase (CH25H), an endoplasmic reticulum-associated enzyme [4]. CH25H expression is strongly upregulated by interferons (INFs), particularly type I interferons, during immune responses [5,6]. This likely accounts for the greater scientific focus on 25OHC compared to 27OHC in studies examining the antiviral properties of these oxysterols (Fig. 1).

27OHC, a naturally occurring oxysterol as well, is generated through hydroxylation at the 27th carbon of the cholesterol structure via an enzymatic pathway mediated by the mitochondrial cytochrome P450 enzyme cholesterol 27-hydroxylase (CYP27A1) [4]. It represents the most abundant oxysterol in human peripheral blood [7] and is also detected at appreciable concentrations in other biological fluids, including cerebrospinal fluid (CSF) [8] and, unexpectedly, in maternal colostrum and mature milk [9]. In addition to its reported antiviral activity, 27OHC is primarily recognized as a key intermediate in cholesterol catabolism and bile acid biosynthesis.

A noteworthy observation is the comparison between the physiological concentration of 27OHC in peripheral blood, approximately 200–300 ng/mL [10], and the markedly lower levels of 25OHC, around 5–20 ng/mL [10]. Despite these differences, both oxysterols occur at concentrations that are higher than, or at least comparable to those of certain vitamins, such as vitamin D (20–50 ng/mL) and vitamin B12 (200–900 pg/mL). Their evolutionarily conserved presence in human biological fluids strongly suggests a physiological role in innate immunity, potentially contributing to passive antiviral protection in newborns and systemic defense in adults. Furthermore, their endogenous origin implies a favorable safety profile.

Ten years ago, we reviewed the early literature on the antiviral properties of oxysterols, particularly 25OHC and 27OHC, emphasizing their host-directed mechanism and broad-spectrum activity [3]. Since then, substantial experimental evidence has reinforced these observations and expanded the range of viral families that include at least one human pathogen significantly inhibited by these oxysterols. In this update, focused on humans viral infections, we aim to summarize recent findings, examine the diverse mechanisms underlying viral replication inhibition, and discuss why we strongly believe certain oxysterols may play a pivotal role in human defense against viral infections.