Date Published: August 3, 2018
Publisher: Public Library of Science
Author(s): Eirini D. Tseligka, Komla Sobo, Luc Stoppini, Valeria Cagno, Fabien Abdul, Isabelle Piuz, Pascal Meylan, Song Huang, Samuel Constant, Caroline Tapparel, Shin-Ru Shih.
Enterovirus 71 (EV71) causes hand, foot and mouth disease, a mild and self-limited illness that is sometimes associated with severe neurological complications. EV71 neurotropic determinants remain ill-defined to date. We previously identified a mutation in the VP1 capsid protein (L97R) that was acquired over the course of a disseminated infection in an immunocompromised host. The mutation was absent in the respiratory tract but was present in the gut (as a mixed population) and in blood and cerebrospinal fluid (as a dominant species). In this study, we demonstrated that this mutation does not alter the dependence of EV71 on the human scavenger receptor class B2 (SCARB2), while it enables the virus to bind to the heparan sulfate (HS) attachment receptor and modifies viral tropism in cell lines and in respiratory, intestinal and neural tissues. Variants with VP197L or VP197R were able to replicate to high levels in intestinal and neural tissues and, to a lesser extent, in respiratory tissues, but their preferred entry site (from the luminal or basal tissue side) differed in respiratory and intestinal tissues and correlated with HS expression levels. These data account for the viral populations sequenced from the patient’s respiratory and intestinal samples and suggest that improved dissemination, resulting from an acquired ability to bind HS, rather than specific neurotropism determinants, enabled the virus to reach and infect the central nervous system. Finally, we showed that iota-carrageenan, a highly sulfated polysaccharide, efficiently blocks the replication of HS-dependent variants in cells and 2D neural cultures. Overall, the results of this study emphasize the importance of HS binding in EV71 pathogenesis and open new avenues for the development of antiviral molecules that may prevent this virus’s dissemination.
Enterovirus 71 (EV71) is one of a limited number of enterovirus genotypes that have the ability to infect the central nervous system (CNS) [1–3] and has emerged as a major health-care threat across the Asia-Pacific region [4–7]. Although this virus is typically associated with mild hand, foot and mouth disease (HFMD) epidemics, EV71 has been increasingly associated with neurological disorders that range from aseptic meningitis with or without pulmonary edema to brain stem encephalitis and poliomyelitis-like acute flaccid paralysis, particularly among children less than 6 years old [8–10]. EV71 transmission depends on hygiene, water quality, and the extent of crowding . EV71 is typically transmitted through fecal-oral routes, although transmission via respiratory secretions also frequently occurs, particularly in countries with high standard sanitation [12, 13].
In 2012, we reported an EV71 genogroup C1 disseminated infection in an immunocompromised patient treated with rituximab who was hospitalized with respiratory and neurological symptoms . Although EV71 genogroup C1 infections have not been associated with severe diseases in the past, several complicated cases have been recently reported in Europe, underscoring the importance of defining EV71 virulence determinants [43, 44]. By sequencing the full genome of the virus from different samples, we identified the acquisition of a non-conservative mutation in VP1 (L97R), which was absent at the beginning of the infection in the respiratory tract but was present in a mixed population in the gastrointestinal tract and became dominant in the blood and CSF at later time points. We showed that this mutation conferred a binding advantage to the virus in certain cell lines. In this study, we extended these findings and showed that the VP1 L97R substitution, as well as an associated mutation (VP1 E167G) observed after in vitro culturing, confers an ability of the virus to use HS as an attachment receptor while it does not alter the dependence of the virus on the ubiquitously expressed SCARB2 entry receptor. Tan and colleagues recently showed that variants with VP1 E167G do not bind heparin . In addition, in our previous publication, we observed the same binding advantage of the VP197R mutant in neuroblastoma and Vero cells that was observed in this study with the VP197R167G mutant . This strongly suggests that the VP1 L97R mutation confers an HS binding ability, while the VP1 E167G mutation has a stabilizing function as proposed previously based on VP1 3D structure . Interestingly, after competition between EV71-VP197L167E and EV71-VP197R167G for binding and replication in cells pretreated with heparinase, EV71-VP197R was excluded from the viral population, while a new variant (EV71-VP198K) which is also known to promote HS binding , emerged. Thus, it is very likely that after cleavage with heparinase at the 1→4 linkages between hexosamine and glucuronic acid residues, HS is still bound by EV71-VP198K but not by EV71-VP197R, suggesting that these two variants present different HS binding specificities.