Characterization of Rotavirus Strains Responsible for Breakthrough Diarrheal Diseases among Zambian Children Using Whole Genome Sequencing

Abstract

The occurrence of rotavirus (RV) infection among vaccinated children in high-burden settings poses a threat to further disease burden reduction. Genetically altered viruses have the potential to evade both natural infection and vaccine-induced immune responses, leading to diarrheal diseases among vaccinated children. Studies characterizing RV strains responsible for breakthrough infections in resource-limited countries where RV-associated diarrheal diseases are endemic are limited. We aimed to characterize RV strains detected in fully vaccinated children residing in Zambia using next-generation sequencing. We conducted whole genome sequencing on Illumina MiSeq. Whole genome assembly was performed using Geneious Prime 2023.1.2. A total of 76 diarrheal stool specimens were screened for RV, and 4/76 (5.2%) were RV-positive. Whole genome analysis revealed RVA/Human-wt/ZMB/CIDRZ-RV2088/2020/G1P[4]-I2-R2-C2-M2-A2-N2-T2-E2-H2 and RVA/Human-wt/ZMB/CIDRZ-RV2106/2020/G12P[4]-I1-R2-C2-M2-A2-N1-T2-E1-H2 strains were mono and multiple reassortant (exchanged genes in bold) respectively, whilst RVA/Human-wt/ZMB/CIDRZ-RV2150/2020/G12P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1 was a typical Wa-like strain. Comparison of VP7 and VP4 antigenic epitope of breakthrough strains and Rotarix strain revealed several amino acid differences. Variations in amino acids in antigenic epitope suggested they played a role in immune evasion of neutralizing antibodies elicited by vaccination. Findings from this study have the potential to inform national RV vaccination strategies and the design of highly efficacious universal RV vaccines.

Keywords: Zambia; breakthrough; children; reassortment; rotavirus; vaccine.

Figure 1

Study flow Chart.

Figure 2

Maximum likelihood phylogenetic tree between the VP7 gene of the breakthrough G1 and G12 strains as well as global strains. Green-filled triangles represented vaccine sequences, whilst breakthrough strains were represented by blue-filled triangles. The scale at the bottom indicates nucleotide substitutions per site, whereas bootstrap values ≥ 70 were shown on the branch nodes.

Figure 3

Amino acid alignment of the antigenic sites of the G1 and G12 strains are highlighted in black, reference strains green and red for the Rotarix vaccine. Blue-filled triangles represented amino residue positions that have been shown to escape neutralization with monoclonal antibodies. Dots represented conserved amino acids.

Figure 4

Maximum likelihood phylogenetic tree between the VP4 gene of the breakthrough P[8] and P[4] strains as well as global strains. Green-filled triangles represented vaccine sequences, whereas blue-filled triangles represented breakthrough strains. The scale at the bottom indicates nucleotide substitutions per site, whilst bootstrap values ≥ 70 were shown on the branch nodes.

Figure 5

Amino acid alignment of the antigenic sites of the Rotarix vaccine highlighted in red, P[4] and P[8] breakthrough strains highlighted in blue and reference strains. Blue-filled triangles represented amino residues that have been shown to escape neutralization with monoclonal antibodies. Dots represented conserved amino acids.