Common E Protein Determinants for Attenuation of Glycosaminoglycan-Binding Variants of Japanese Encephalitis and West Nile Viruses

Abstract

Natural isolates and laboratory strains of West Nile virus (WNV) and Japanese encephalitis virus (JEV)were attenuated for neuroinvasiveness in mouse models for flavivirus encephalitis by serial passage in human adenocarcinoma (SW13) cells. The passage variants displayed a small-plaque phenotype, augmented affinity for heparin-Sepharose, and a marked increase in specific infectivity for SW13 cells relative to the respective parental viruses, while the specific infectivity for Vero cells was not altered. Therefore, host cell adaptation of passage variants was most likely a consequence of altered receptor usage for virus attachment-entry with the involvement of cell surface glycosaminoglycans (GAG) in this process. In vivo blood clearance kinetics of the passage variants was markedly faster and viremia was reduced relative to the parental viruses, suggesting that affinity for GAG (ubiquitously present on cell surfaces and extracellular matrices) is a key determinant for the neuroinvasiveness of encephalitic flaviviruses. A difference in pathogenesis between WNV and JEV, which was reflected in more efficient growth in the spleen and liver of the WNV parent and passage variants, accounted for a less pronounced loss of neuroinvasiveness of GAG binding variants of WNV than JEV. Single gain-ofnet-positive-charge amino acid changes at E protein residue 49, 138, 306, or 389/390, putatively positioned in two clusters on the virion surface, define molecular determinants for GAG binding and concomitant virulence attenuation that are shared by the JEV serotype flaviviruses.Japanese encephalitis virus (JEV) and West Nile virus (WNV) are mosquito-borne flaviviruses grouped in the JEV serocomplex, which also includes Murray Valley encephalitis virus (MVE) and St. Louis encephalitis virus (14,29). Most members of this serocomplex can cause disease in humans, ranging from mild febrile illness to fatal encephalitis. Entry of JEV serotype flaviviruses into mammalian cells is by receptormediated endocytosis (reviewed in reference [20]), however, the specific receptor(s) used for virus adsorption and uptake remains elusive.We and others have shown that flaviviruses have a variable affinity for glycosaminoglycans (GAG) and speculated that this property contributes to the attachment of flaviviruses to host cells (4,9,10,12,13,16,17,19,24,35). GAG are unbranched polysaccharides ubiquitously found on the cell surface and in the extracellular matrix and are usually bound to core proteins (heparan sulfate proteoglycans) (reviewed in reference 2). They are negatively charged due to various degrees of sulfation; accordingly, GAG binding domains typically contain positively charged amino acids. It is most likely that, in addition to attachment of flaviviruses to cell surface GAG, subsequent interaction of attached virus with other host cell receptors is critical for productive virus infection.Adaptation of flaviviruses to growth in tissue culture cells has been shown to elicit amino acid substitutions that increase the net positive charge of t...

In vitro and in vivo characterization of chimeric duck Tembusu virus based on Japanese encephalitis live vaccine strain SA14-14-2

“…This finding suggests chimerization itself failed to attenuate DTMUV, which is different from previous findings from other flavivirus members (Li et al, 2013b, c;Wang et al, 2014). Compared with the JEV vaccine strain, ChinDTMUV exhibited obvious mouse neurovirulence, indicating the prM-E proteins of DTMUV may be the major determinants of neurovirulence, which has been documented for other flaviviruses (Gualano et al, 1998;Holzmann et al, 1990;Lee & Lobigs, 2002;Lee et al, 2004;Li et al, 2006). Sequence alignment of flavivirus E proteins revealed a total of 141 amino acid substitutions in the DTMUV sequence relative to that of JEV vaccine strain (Fig.…”

Encephalitic Arboviruses: Emergence, Clinical Presentation, and Neuropathogenesis

“…Indeed, mice lacking caspase-3 displayed tissue, placenta, and amniotic fluid of newborns with microcephaly and in the stillborn infants of women infected by Zika during pregnancy [28]. The envelope protein of encephalitic arboviruses is the major determinant of neurotropism and neurovirulence, owing to its role in receptor binding and virus entry [177][178][179]. However, sequence analysis of virulent and attenuated strains of these viruses revealed that other regions of the viral genome, including the 3´ untranslated region and nonstructural proteins, also contribute to neurovirulence and viral tropism [180].…”

Prevention and Control of Japanese Encephalitis Viruses: A Challenged Issue

“…Thus, it is quite interesting to find a small plaque size variant to use for the development of the vaccine. It has been shown that there is a good correlation between small plaque size and attenuation of viruses including several members of flavivirus, i.e., Dengue virus type 2, West Nile virus, Tick-borne encephalitis virus, and Murray Valley encephalitis virus (Tsai, 2000;Blaney et al, 2002;Hanley et al, 2002;Lee et al, 2004;Lee & Lobigs, 2002;Mandl et al, 2001;Huang et al, 2003). The vaccine strain, SA 14-14-2 of JEV, also has a small plaque size and grows quite well in PHK cells.…”

“…It has been shown that temperature sensitive (ts) mutants of dengue type 4 virus and dengue 2 virus which have small plaque phenotype appeared to be attenuated in mice that may be useful for the development of a recombinant live attenuated vaccine of dengue virus , Hanley et al, 2002, Huang et al, 2003. The small plaque mutants of other flaviviruses including West Nile virus (WNV) and tick-borne encephalitis virus (TBE), Murray Valley encephalitis virus (MVE), and JEV that are increase in cells infectivity and increase in affinity of glycosaminoglycan binding, have shown their virulence were attenuated in mouse model (Lee et al, 2004, Lee & Lobigs, 2002, Mandl et al, 2001). In addition, almost all strains of JEV, which are small plaque size variants on BHK-21 cells, except for JaGAr 01, are found to be correlated with the virulence attenuation of the viruses in mice (Kimura et al, 1973).…”