Biological and phylogenetic characteristics of yellow fever virus lineages from West Africa

Abstract

The yellow fever virus (YFV), the first proven human-pathogenic virus, although isolated in 1927, is still a major public health problem, especially in West Africa where it causes outbreaks every year. Nevertheless, little is known about its genetic diversity and evolutionary dynamics, mainly due to a limited number of genomic sequences from wild virus isolates. In this study, we analyzed the phylogenetic relationships of 24 full-length genomes from YFV strains isolated between 1973 and 2005 in a sylvatic context of West Africa, including 14 isolates that had previously not been sequenced. By this, we confirmed genetic variability within one genotype by the identification of various YF lineages circulating in West Africa. Further analyses of the biological properties of these lineages revealed differential growth behavior in human liver and insect cells, correlating with the source of isolation and suggesting host adaptation. For one lineage, repeatedly isolated in a context of vertical transmission, specific characteristics in the growth behavior and unique mutations of the viral genome were observed and deserve further investigation to gain insight into mechanisms involved in YFV emergence and maintenance in nature.

Fig 1

Differences in the amino acid sequence throughout the viral polyprotein among the YFV strains that were completely sequenced in this study in comparison to the reference strain Asibi. Differences from the reference strain Asibi are highlighted in gray.

Fig 2

Majority rule consensus tree of the Bayesian analysis, with 10 million generations based on YFV full genome sequences. Posterior probability values are displayed beside the related node. Bootstrap values of the ML analysis are given in parentheses. Note that unsupported values of 50/0.5 or less are indicated by a minus sign. The appropriate lineages of the already classified YF strains are specified at the end of the labels as L1 to L6.

Fig 3

RNA sequence alignment of the 3′-nontranslated region (3′-NTR) of 18 West African YFV strains. Dots indicate concordance with the reference strain Asibi.

Fig 3

RNA sequence alignment of the 3′-nontranslated region (3′-NTR) of 18 West African YFV strains. Dots indicate concordance with the reference strain Asibi.

Fig 4

Growth kinetics of the strains Asibi (a), 357 (lineage 4) (b), and 333 (lineage 3) (c). The diagrams show the amount of viral RNA equivalents isolated from supernatants (•) and cells (■) (genome equivalents [GE]/ml), as well as the number of infectious viral particles (▲) (PFU/ml) over a time period of 146 h postinfection. The experiments were performed with Ap61 cells (dotted lines) and HepG2 cells (continuous lines). Range values of two independent experiments are indicated by the error bars in the lines reflecting RNA equivalents isolated from supernatants.

Fig 5

Immunofluorescence assay of Ap61 and HepG2 cells infected with YFV Asibi or YFV 357 within a time period of 50 to 146 h postinfection. Staining occurred against the viral E protein (MAK6330) and the nucleus (DAPI) as described above.

Fig 6

Growth curves of the different YF lineages in HepG2 cells. Analyzed parameters are the quantity of viral RNA copies isolated from cell culture supernatants (a) and from cells directly (b) (GE/ml) and the quantity of infectious viral particles (c) (PFU/ml). Range values of two independent experiments are indicated by the error bars in panel a.

Fig 7

Growth curves of all tested strains determined with Ap61 cells. Analyzed parameters are the quantity of viral RNA copies isolated from cell culture supernatants (a) and from cells (b) (GE/ml) and the quantity of infectious viral particles (c) (PFU/ml). Range values of two independent experiments are indicated by the error bars in panel a.