Determinants of attenuation in the envelope protein of the flavivirus Alfuy

Abstract

Murray Valley encephalitis virus (MVEV) is a mosquito-borne flavivirus endemic to Australia and Papua New Guinea. Most strains of MVEV cause potentially fatal cases of encephalitis in humans and horses, and have been shown to be highly neuroinvasive in weanling mice. In contrast, the naturally occurring subtype Alfuy virus (ALFV) has never been associated with human disease, nor is it neuroinvasive in weanling mice, even at high doses. To identify viral factors associated with ALFV attenuation, a chimeric infectious clone was constructed containing the structural genes premembrane (prM) and envelope (E) of ALFV swapped into the MVEV genome. The resulting virus (vMVEV/ALFVstr) was no longer neuroinvasive in mice, suggesting that motifs within prM-E of ALFV confer attenuation. To define these motifs further, mutants were constructed by targeting divergent sequences between the MVEV and ALFV E proteins that are known markers of virulence in other encephalitic flaviviruses. MVEV mutants containing a unique ALFV sequence in the flexible hinge region (residues 273-277) or lacking the conserved glycosylation site at position 154 were significantly less neuroinvasive in mice than wild-type MVEV, as determined by delayed time to death or increased LD 50 . Conversely, when the corresponding MVEV sequences were inserted into the vMVEV/ALFVstr chimera, the mutant containing the MVEV hinge sequence was more neuroinvasive than the parental chimera, though not to the same level as wild-type MVEV. These results identify the hinge region and E protein glycosylation as motifs that contribute to the attenuation of ALFV.

Substitution of the premembrane and envelope protein genes of Modoc virus with the homologous sequences of West Nile virus generates a chimeric virus that replicates in vertebrate but not mosquito cells

“…These findings differ from most other studies which compare the plaque sizes of chimeric flaviviruses generated by prM-E gene substitutions to those of both parental viruses. Usually prM-E gene substitutions generate chimeric viruses that produce plaques that are indistinguishable from one of the parental viruses [51, 62–64] or are smaller than both parental viruses [46, 65, 66]. However, replacement of the prM-E genes of JEV with those of DENV-4 produced a chimeric virus which, like our chimeric virus, exhibited an intermediate plaque phenotype; the chimeric virus produced plaques that were smaller than JEV but larger than DENV-4 in mammalian cells [67].…”

Characterization of Virulent West Nile Virus Kunjin Strain, Australia, 2011

“…To examine glycosylation of the E protein, viral proteins from cultures of infected C6/36 cells were digested as described ( 18 ). Proteins were separated and analyzed by Western blot.…”

The effect of riboflavin and ultraviolet light on the infectivity of arboviruses

“…MVEV is transmitted through mosquito vectors, with C. annulirostris being the most common mosquito species and, like WNV, birds are the most common reservoir hosts . The incubation and presymptomatic periods in humans are believed to be similar to those observed for RRV infection . MVEV can cause encephalitis, with permanent neurologic sequelae or death, and has a high fatality rate in clinical cases .…”