No abstract

No abstract

The in vivo mechanism for virulence attenuation of laboratory-derived variants of two flaviviruses in the Japanese encephalitis virus (JEV) serocomplex is described. Host cell adaptation of JEV and Murray Valley encephalitis virus (MVE) by serial passage in adenocarcinoma cells selected for variants characterized by (i) a small plaque phenotype, (ii) increased affinity to heparin-Sepharose, (iii) enhanced susceptibility to inhibition of infectivity by heparin, and (iv) loss of neuroinvasiveness in a mouse model for flaviviral encephalitis. We previously suggested that virulence attenuation of the host cell-adapted variants of MVE is a consequence of their increased dependence on cell surface glycosaminoglycans (GAGs) for attachment and entry (E. Lee and M. Lobigs, J. Virol. 74:8867-8875, 2000). In support of this proposition, we find that GAG-binding variants of JEV and MVE were rapidly removed from the bloodstream and failed to spread from extraneural sites of replication into the brain. Thus, the enhanced affinity of the attenuated variants for GAGs ubiquitously present on cells and extracellular matrices most likely prevented viremia of sufficient magnitude and/or duration required for virus entry into the brain parenchyma. This mechanism may also account, in part, for the attenuation of the JEV SA14-14-2 vaccine, given the sensitivity of the virus to heparin inhibition. A pronounced loss of the capacity of the GAG-binding variants to produce disease was also noted in mice defective in the alpha/beta interferon response, a mouse strain shown here to be highly susceptible to infection with JEV serocomplex flaviviruses. Despite the close genetic relatedness of JEV and MVE, the variants selected for the two viruses were altered at different residues in the envelope (E) protein, viz., Glu 306 and Asp 390 for JEV and MVE, respectively. In both cases the substitutions gave the protein an increased net positive charge. The close spatial proximity of amino acids 306 and 390 in the predicted E protein structure strongly suggests that the two residues define a receptor-binding domain involved in virus attachment to sulfated proteoglycans.Altered virulence properties of viral variants are the basis for their application as live attenuated vaccines. Therefore, it is of particular interest to elucidate first the mechanisms for loss of the capacity of the variants to produce disease in a host and then the viral molecular determinants for the altered virulence phenotypes. Potential mechanisms for the attenuation of viral virulence include (i) changes in viral binding and penetration properties on host membranes resulting in altered tissue tropisms, (ii) a reduction of viral replication rate in vivo, (iii) a decreased efficiency of virus spread in the host, and (iv) an increased susceptibility of variant viruses to host antiviral responses (reviewed in reference 35). Virulence attenuation has traditionally been achieved by serial passage of virus in cultured cells or laboratory animals, a selection process that can give rise...

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...

No abstract