Complete genome sequence analysis of candidate human rotavirus vaccine strains RV3 and 116E

Abstract

Rotaviruses (RVs) cause severe gastroenteritis in infants and young children; yet, several strains have been isolated from newborns showing no signs of clinical illness. Two of these neonatal strains, RV3 (G3P[6]) and 116E (G9P[11]), are currently being developed as live-attenuated vaccines. In this study, we sequenced the eleven-segmented double-stranded RNA genomes of cell culture-adapted RV3 and 116E and compared their genes and protein products to those of other RVs. Using amino acid alignments and structural predictions, we identified residues of RV3 or 116E that may contribute to attenuation or influence vaccine efficacy. We also discovered residues of the VP4 attachment protein that correlate with the capacity of some P[6] strains, including RV3, to infect newborns versus older infants. The results of this study enhance our understanding of the molecular determinants of RV3 and 116E attenuation and are expected to aid in the ongoing development of these vaccine candidates.

Fig. 1

Phylogenetic relationships of RV3 and 116E genome segments to those of other RVs. The neighbor-joining trees were constructed using the individual ORF nucleotide sequences for each isolate and are out-group rooted to DS-1 for purposes of clarity. Horizontal branch lengths are drawn to scale (nucleotide substitutions per base), and bootstrap values are shown as percentages for key nodes. Strain names are colored accordingly: animal strains (green), human neonatal strains (red), and human strains isolated from older infants/children (black). Strains RV3 and 116E are highlighted in yellow.

Fig. 1

Phylogenetic relationships of RV3 and 116E genome segments to those of other RVs. The neighbor-joining trees were constructed using the individual ORF nucleotide sequences for each isolate and are out-group rooted to DS-1 for purposes of clarity. Horizontal branch lengths are drawn to scale (nucleotide substitutions per base), and bootstrap values are shown as percentages for key nodes. Strain names are colored accordingly: animal strains (green), human neonatal strains (red), and human strains isolated from older infants/children (black). Strains RV3 and 116E are highlighted in yellow.

Fig. 1

Phylogenetic relationships of RV3 and 116E genome segments to those of other RVs. The neighbor-joining trees were constructed using the individual ORF nucleotide sequences for each isolate and are out-group rooted to DS-1 for purposes of clarity. Horizontal branch lengths are drawn to scale (nucleotide substitutions per base), and bootstrap values are shown as percentages for key nodes. Strain names are colored accordingly: animal strains (green), human neonatal strains (red), and human strains isolated from older infants/children (black). Strains RV3 and 116E are highlighted in yellow.

Fig. 2

Genome constellations of several human and animal RVs. The schematic illustrates the genotype of each genome segment for several RV strains. The strain name is listed to the left of the corresponding genome constellation, and the protein encoded by each gene is listed at the top. RV strain names are colored accordingly: animal strains (green), human neonatal strains (red), and human strains isolated from older infants/children (black). Strains RV3 and 116E are highlighted in yellow. All genotype 1 genes are shown as white boxes, non-genotype 1 genes are shown as grey boxes, and dotted boxes indicated genome segments in which no or only partial ORF sequences are available. Asterisks (*) indicate sequences that were omitted from the phylogenetic trees for purposes of clarity.

Fig. 3

Surface-exposed VP7 amino acids unique to RV3 or 116E. (A) Architecture of a RV virion (modified with permission from B.V.V. Prasad), showing the positions of VP7 and VP4. (B and C) Three-dimensional locations of amino acid changes in RV3 or 116E. In both images, a surface representation of the VP7 trimer crystal structure (PDB 3FMG) is shown. Residues comprising the putative neutralization domains have been colored as follows: 7-1A (red), 7-1B (salmon), and 7-2 (purple). Amino acids unique to RV3 or 116E, not represented in any other G-type-matched strains, are shown in cyan and are labeled for a single monomer of the trimer.

Fig. 4

Alignment of P[6] VP4 showing residues that correlate with neonatal infection. The VP4 amino acid sequences of several representative P[6] strains are shown. The strain names are to the left of each sequence and are colored according to Fig. 1. Grey shading indicates conservation of amino acid identity. Residues associated with most P[6] strains isolated from neonates are labeled. Blue arrows show the region of VP4 comprising the VP8* core.

Fig. 5

Location of surface-exposed P[6] VP8* residues that correlate with neonatal infection. The left image shows a surface representation of the VP4 crystal structure (PDB 1KQR). A white box defines the position of VP8*. The right images show the VP8* core from two different viewpoints (front or back). The front viewpoint is rotated 90° to the right compared with the image in the white box. The back viewpoint is rotated 180° to the left compared with the front. Residues comprising the putative neutralization domains of VP8* have been colored as follows: pink (8-1), salmon (8-2), purple (8-3) and green (8-4). Residues that correlate with the capacity of P[6] strains to infect neonates are shown in cyan.