Single Mutation in the Flavivirus Envelope Protein Hinge Region Increases Neurovirulence for Mice and Monkeys but Decreases Viscerotropism for Monkeys: Relevance to Development and Safety Testing of Live, Attenuated Vaccines

Abstract

A chimeric yellow fever (YF) virus/Japanese encephalitis (JE) virus vaccine (ChimeriVax-JE) was constructed by insertion of the prM-E genes from the attenuated JE virus SA14-14-2 vaccine strain into a full-length cDNA clone of YF 17D virus. Passage in fetal rhesus lung (FRhL) cells led to the emergence of a small-plaque virus containing a single Met3Lys amino acid mutation at E279, reverting this residue from the SA14-14-2 to the wild-type amino acid. A similar virus was also constructed by site-directed mutagenesis (J. Arroyo, F. Guirakhoo, S. Fenner, Z.-X. Zhang, T. P. Monath, and T. J. Chambers, J. Virol. 75:934-942, 2001). The E279 mutation is located in a beta-sheet in the hinge region of the E protein that is responsible for a pH-dependent conformational change during virus penetration from the endosome into the cytoplasm of the infected cell. In independent transfection-passage studies with FRhL or Vero cells, mutations appeared most frequently in hinge 4 (bounded by amino acids E266 to E284), reflecting genomic instability in this functionally important region. The E279 reversion caused a significant increase in neurovirulence as determined by the 50% lethal dose and survival distribution in suckling mice and by histopathology in rhesus monkeys. Based on sensitivity and comparability of results with those for monkeys, the suckling mouse is an appropriate host for safety testing of flavivirus vaccine candidates for neurotropism. After intracerebral inoculation, the E279 Lys virus was restricted with respect to extraneural replication in monkeys, as viremia and antibody levels (markers of viscerotropism) were significantly reduced compared to those for the E279 Met virus. These results are consistent with the observation that empirically derived vaccines developed by mouse brain passage of dengue and YF viruses have increased neurovirulence for mice but reduced viscerotropism for humans.The study of chimeric viruses has afforded new insights into the molecular basis of virulence and new prospects for vaccine development. For example, molecular clones of positive-strand alphaviruses (29, 39) and flaviviruses (4, 7, 13, 15) have been modified by insertion of structural genes encoding the viral envelope and determinants involved in neutralization, cell attachment, fusion, and internalization. The replication of these chimeric viruses is controlled in part by nonstructural proteins and the noncoding termini expressed by the parental strain, while the structural proteins from the donor genes afford specific immunity. The biological characteristics of chimeric viruses are determined by both the donor and recipient virus genes. By comparing constructs with nucleotide sequence differences across the donor genes, it is possible to dissect out the functional roles of individual amino acid residues in virulence and attenuation.Using a chimeric yellow fever (YF) virus that incorporated the prM-E genes from an attenuated strain (SA14-14-2) of Japanese encephalitis (JE) virus, a detailed examination of the roles of 10 a...

In vitro growth, pathogenicity and serological characteristics of the Japanese encephalitis virus genotype V Muar strain

“…The growth rate of Muar was less than that of the (Tajima et al, 2010). Other reports have also highlighted the importance of the E protein as a determinant of pathogenicity (Monath et al, 2002;Sumiyoshi et al, 1995;Zhao et al, 2005). In contrast, our group determined that an amino acid substitution in the JEV NS4A protein alters virulence in mice (Yamaguchi et al, 2011).…”

Molecular Basis for Adaptation of a Chimeric Dengue Type‐4/Japanese Encephalitis Virus to Vero Cells

“…Residues E136 and E138 lie in close proximity and are located within the so-called "hinge" region at the domain I-II interface of the E protein, which is responsible for a pH-dependent conformational change during virus penetration from the endosome into the cytoplasm of an infected cell (24). Other studies have provided data indicating that mutations within this region modulate virulence phenotypes in mice, and this modulation is believed to occur through effects of such mutations on the low-pH-induced dimer-to-trimer structural transition of the E protein associated with virus entry (3,9,12,19,20,28).…”

“…The study of chimeras has afforded new insights into the molecular basis of virulence and new prospects for vaccine development (19). Detailed investigation of virus adaptation to different hosts and tissues remains to be conducted.…”

Secretion of dengue virus envelope protein ectodomain from mammalian cells is dependent on domain II serotype and affects the immune response upon DNA vaccination

“…DIII is also believed to contain the receptor-binding sites to the host cell (Erb et al, 2010;Mukhopadhyay et al, 2005) and has been implicated in determining host range, tropism and virulence (Lindenbach et al, 2007). A hinge region formed by the four strands that span between the different DI and DII coding segments provides the flexibility for E conformational changes during virus maturation (Butrapet et al, 2011;Monath et al, 2002), while a linker of 11 aa connects DI to DIII and is fundamental for proper E folding (de Wispelaere & Yang, 2012). …”