Kate D. Ryman

Comparison of the genomes of the wild-type French viscerotropic strain of yellow fever virus with its vaccine derivative French neurotropic vaccine

The French neurotropic vaccine, or FNV, was used extensively in Africa to control yellow fever (YF). Although efficacious, the vaccine caused an unacceptable rate of post-vaccinal complications in children and was subsequently replaced by the 17D vaccine. Here we report that the genomes of the wild-type YF virus French viscerotropic virus and its attenuated vaccine derivative, FNV virus from the Institut Pasteur, Paris, (FNV-IP) differ by 77 nucleotides encoding 35 amino acid substitutions. Comparison of FNV-IP and three other isolates of FNV with other YF vaccine strains (17D-204 and 17DD derived from wild-type strain Asibi) revealed that during the two attenuation processes two common nucleotide changes arose that encode two amino acid substitutions: one is in the membrane protein at amino acid 35 (M-35), the other in non-structural (NS) protein 4B at NS4B-95. These common substitutions may be important in the process of attenuation of viscerotropic disease for humans and monkeys, and/or may be involved in loss of mosquito competence of the vaccine viruses.

YELLOW FEVER VIRUS ENVELOPE PROTEIN HAS TWO DISCRETE TYPE-SPECIFIC NEUTRALIZING EPITOPES

Two monoclonal antibody neutralization resistant (MAbR) variants of the yellow fever (YF) 17D-204 vaccine virus strain were selected using YF type-specific MAb B39. These B39R variants were compared with the variant virus selected by Lobigs et al. (Virology 161, 474-478, 1987) using a second YF-type specific MAb (2E10) which mapped to amino acid position 71/72 in the envelope (E) protein. Neutralization assays with a panel of MAbs suggested that these two YF-type-specific epitopes are located in two discrete regions of the folded E protein. Each of the B39R variants had a single nucleotide mutation which encoded an amino acid substitution at either position E-155 or E-158. Thus, YF type-specific epitopes map to both domain I (B39) and II (2E10) of the YF virus E protein. The B39 defined epitope represents the first flavivirus neutralizing epitope localized to this region of domain I of the E protein.

Genetic Relationships and Evolution of Genotypes of Yellow Fever Virus and Other Members of the Yellow Fever Virus Group within the Flavivirus Genus Based on the 3′ Noncoding Region

ABSTRACT Genetic relationships among flaviviruses within the yellow fever (YF) virus genetic group were investigated by comparing nucleotide sequences of the 3′ noncoding region (3′NCR). Size heterogeneity was observed between members and even among strains of the same viral species. Size variation between YF strains was due to duplications and/or deletions of repeated nucleotide sequence elements (RYF). West African genotypes had three copies of the RYF (RYF1, RYF2, and RYF3); the Angola and the East and Central African genotypes had two copies (RYF1 and RYF3); and South American genotypes had only a single copy (RYF3). Nucleotide sequence analyses suggest a deletion within the 3′NCR of South American genotypes, including RYF1 and RYF2. Based on studies with the French neurotropic vaccine strain, passage of a YF virus strain in cell culture can result in deletion of RYF1 and RYF2. Taken together, these observations suggest that South American genotypes of YF virus evolved from West African genotypes and that the South American genotypes lost RYF1 and RYF2, possibly in a single event. Repeated sequence elements were found within the 3′NCR of other members of the YF virus genetic group, suggesting that it is probably characteristic for members of the YF virus genetic group. A core sequence of 15 nucleotides, containing two stem-loops, was found within the 3′NCR of all members of the YF genetic group and may represent the progenitor repeat sequence. Secondary structure predictions of the 3′NCR showed very similar structures for viruses that were closely related phylogenetically.

PASSAGE OF JAPANESE ENCEPHALITIS VIRUS IN HELA CELLS RESULTS IN ATTENUATION OF VIRULENCE IN MICE

Of four wild-type strains (Nakayama-original, SA14, 826309 and Beijing-1) of Japanese encephalitis (JE) virus that were passaged six times in HeLa cells (HeLa p6), two (Nakayama-original and 826309) became attenuated for mice. In the case of strain Nakayama-original, the virulence for mice was markedly reduced and attenuation was retained on passage in primary chicken embryo fibroblast, LLC-MK2 and C6/36 cells. The binding of non-HeLa-passaged Nakayama virus to mouse brain membrane receptor preparations could be differentiated from binding by Nakayama HeLa p6 virus, suggesting that the envelope (E) protein is involved in the attenuated phenotype. Both of the attenuated viruses can be distinguished from the virulent non-HeLa-passaged parental viruses by examination with E protein reactive vaccine and wild-type-specific monoclonal antibodies (MAbs). The vaccine-specific MAb V23, which is only reactive with the SA14 series of live vaccine viruses, recognized the HeLa cell-attenuated Nakayama-original and 826309 viruses, whereas two wild-type-specific MAbs (MAbs K13 and K39) lost reactivity. Comparison of the nucleotide sequences of the structural protein genes of the 826309 and Nakayama-original virulent parent and attenuated HeLa p6 viruses revealed that the viruses differed by 37 and 46 nucleotides coding for eight and nine amino acid mutations, respectively. However, other than one amino acid in the E protein, the membrane and E protein amino acid sequences of the two attenuated HeLa p6 viruses were identical.

Interaction of Yellow Fever Virus French Neurotropic Vaccine Strain with Monkey Brain: Characterization of Monkey Brain Membrane Receptor Escape Variants

ABSTRACT Binding of yellow fever virus wild-type strains Asibi and French viscerotropic virus and vaccine strains 17D and FNV to monkey brain and monkey liver cell membrane receptor preparations (MRPs) was investigated. Only FNV bound to monkey brain MRPs, while French viscerotropic virus, Asibi, and FNV all bound to monkey liver MRPs. Four monkey brain and two mouse brain MRP escape (MRPR) variants of FNV were selected at pH 7.6 and 6.0. Three monkey brain MRPR variants selected at pH 7.6 each had only one amino acid substitution in the envelope (E) protein in domain II (E-237, E-260, or E274) and were significantly attenuated in mice following intracerebral inoculation. Two of the variants were tested in monkeys and retained parental neurotropism following intracerebral inoculation at the dose tested. We speculate that this region of domain II is involved in binding of FNV E protein to monkey brain and is, in part, responsible for the enhanced neurotropism of FNV for monkeys. A monkey brain MRPR variant selected at pH 6.0 and two mouse brain MRPR variants selected at pH 7.6 were less attenuated in mice, and each had an amino acid substitution in the transmembrane region of the E protein (E-457 or E-458).

Mutation in NS5 protein attenuates mouse neurovirulence of yellow fever 17D vaccine virus

The 17D-204 vaccine manufactured in South Africa (17D-204-SA) and a large plaque variant (17D-LP) derived from it were highly virulent in adult mice. The LD50 of 17D-LP virus was 0-2 p.f.u. for mice following intracerebral inoculation. In comparison, a medium plaque variant derived from 17D-LP, termed 17D-MP virus, was found to be attenuated in adult mice following the same route of inoculation (> 10(4) p.f.u./LD50). Replication of 17D-MP virus was decreased in infected mouse brains compared to 17D-LP virus. Also, 17D-MP virus was slightly temperature sensitive at 39.5 degrees C. Compared to its parent viruses, 17D-204-SA and 17D-LP, 17D-MP virus had one unique mutation at nt 8045 in the genome which resulted in a single amino acid substitution (Pro --> Ser) at residue 137 of the NS5 protein and appeared to be the mutation responsible for the attenuation of 17D-MP virus. This is the first time that altered virulence of a flavivirus caused by mutation in a non-structural protein gene, other than NS1, has been reported.