Mechanism of virulence attenuation of glycosaminoglycan-binding variants of Japanese encephalitis virus and Murray Valley encephalitis virus - PubMed (original) (raw)
Comparative Study
Mechanism of virulence attenuation of glycosaminoglycan-binding variants of Japanese encephalitis virus and Murray Valley encephalitis virus
Eva Lee et al. J Virol. 2002 May.
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.
Figures
FIG. 1.
Inhibition by heparin of infectivity of Jn and passaged viruses. Jn virus, SW13 cell-passaged viruses (Js1, Js2, and Js5), and Vero cell-passaged virus (Jv1) were incubated with heparin (200 μg/ml) for 15 min prior to their addition to Vero (open bars) and SW13 (gray bars) cell monolayers. Error bars show the standard deviation values calculated for each set of duplicates. The percent inhibition of plaque formation by heparin was calculated as follows: [(1 − plaque number in the presence of heparin)/plaque number for mock-treated control] × 100%.
FIG. 2.
Binding of parental and variant strains of JEV and MVE to heparin-Sepharose. 35S-labeled, gradient-purified virus preparations were incubated with heparin-Sepharose, and the percent counts per minute bound were determined after 2 h at 4°C. The means for two samples ± the standard deviation are presented.
FIG. 3.
Virulence of SW13 cell-passaged and parental JEV in 3-week-old Swiss outbred mice. Mortality in groups of five mice inoculated by the i.c. and i.p. routes with 10-fold serial dilutions of parental and SW13-cell passaged JEV (A) and revertants isolated from brain and liver (Jr1 and Jr3, respectively) (B) of IFN-α-R−/− mice infected with Js1.
FIG. 4.
Growth and tissue distribution of SW13 cell-passaged and parental JEV in mice after footpad inoculation. Virus titers in serum, brain, spleen and liver of 3-wk-old Swiss outbred (A and B) and IFN-α-R−/− (C and D) mice infected via the footpad with 103 PFU of Jn (open symbols) or Js1 (solid symbols).
FIG. 5.
Blood clearance of virulent and attenuated strains of JEV and MVE with distinct GAG-binding properties. Eight-week-old Swiss outbred mice were inoculated i.v. in groups of two with Jn (□; 1.7 × 107 PFU) or Js1 (○; 1.6 × 107 PFU) (A) and MVEAsp390 (▪; 2.4 × 105 PFU) or MVEGly390 (•; 2.0 × 105 PFU) (B). At 5 and 30 min p.i., blood was collected, virus titers were determined by plaque assay of Vero cell monolayers, and titers were multiplied by an estimate of the blood volume for each animal.
FIG. 6.
Inhibition by heparin of infectivity of virulent JEV SA14 and its vaccine derivative SA14-14-2. Inhibition of virus infectivity in the presence of 50 (open bars) and 200 (shaded bars) μg of heparin/ml was determined as described in the legend to Fig. 1. The error bars show the standard deviation values of each set of duplicates.
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