Comparative Analysis of G1P[8] Rotaviruses Identified Prior to Vaccine Implementation in Pakistan With Rotarix™ and RotaTeq™ Vaccine Strains

Abstract

Group A rotavirus (RVA) is the leading cause of severe childhood diarrhea globally, even with all effective interventions, particularly in developing countries. Among the diverse genotypes of RVA, G1P[8] is a common genotype that has continued to pervade around the world, including Pakistan. Two universally accepted rotavirus vaccines-Rotarix™ and RotaTeq™ contain the genotype G1P[8]. The current work was aimed at identifying differences between antigenic epitopes of Pakistan's G1P[8] strains and those of the two licensed vaccines. We sequenced 6 G1P[8] rotavirus strains previously reported in Rawalpindi, Islamabad, Pakistan in 2015 and 2016 for their outer capsid genes (VP7 and VP4). Phylogenetic analysis was then conducted in order to classify their specific lineages and to detect their association with strains isolated throughout world. Compared with the Rotarix™ and RotaTeq™ vaccine strains (G1-lineage II, P[8]-lineage III), our study G1-lineage I, P[8]-lineage IV strains showed 3 and 5 variations in the VP7 epitopes, respectively, and 13 and 11 variations in the VP4 epitopes, respectively. The G1 lineage II strains showed no single amino acid change compared to Rotarix™ (lineage II), but exhibited changes at 2 positions compared to RotaTeq™ (lineage III). So, this has been proposed that these G1 strains exist in our natural setting, or that they may have been introduced in Pakistan from other countries of the world. The distinct P[8]-lineage IV (OP354-like) strains showed twelve and thirteen amino acid variations, with Rotarix™ and RotaTeq™ (lineages II and III) strains, respectively. Such findings have shown that the VP4-P[8] component of the G1P[8] strains circulating in Pakistan differs considerably from that of the vaccine viruses compared to that of the VP7-G1. To monitor the long-term effects of vaccines on the emergence of G1P[8] strains with different lineages, routine and successful monitoring of these strains will be crucial.

Keywords: G1P[8]; antigenic epitopes; emergence; genotype; rotavirus.

Figure 1

The nucleotides and amino acids differences of VP7 and VP4 proteins of Pakistani G1P[8] strains with rotavirus vaccine strains. The similarities between G1 lineage I to Rotarix™ and RotaTeq™ vaccine strains is shown in green and purple colours, respectively. While, the similarities between G1 lineage II to Rotarix™ and RotaTeq™ vaccine strains is shown in blue and orange colours, respectively.

Figure 2

Maximum Likelihood tree of the VP7 protein of G1P[8] group A rotavirus (RVA) strains reported in Pakistan and the vaccine strains Rotarix™ and RotaTeq ™. Maximum Likelihood trees were generated using Kimura-2-parameter model in MEGA 6. Coloured branches represent different lineages. The lineage I is represented by purple colour branch including strains isolated from all over the world and our study strains represented by green triangles in the same branch. The lineage II is represented by red colour branch including strains isolated from all over the world and our study strains represented by green triangle in the same branch. Yellow colour branch includes strains belong to lineage III. Aqua colour branch includes strains belong to lineage IV. Blue colour branch includes strains belong to lineage V. G2P[4] RVA strain from Bangladesh is used as an out-group represented by green colour branch. Bootstrap replicates (1000) below than 60% are not shown in the tree.

Figure 3

Maximum Likelihood Phylogenetic tree of the VP4 protein of G1P[8] Group A rotavirus (RVA) strains reported in Pakistan and the vaccine strains Rotarix™ and RotaTeq ™. Maximum Likelihood trees were generated using Kimura-2-parameter model in MEGA 6. Coloured branches represent different lineages. Yellow colour branch includes strains belong to lineage I and orange colour branch represent lineage II strains. The lineage III is represented by Purple colour branch including strains isolated from all over the world and our study strains represented by green triangles in the same branch. The lineage IV is represented by Red colour branch including strains isolated from all over the world and our study strains represented by green triangle in the same branch. RVA strain (G12P[6]) from Bangladesh is used as an as out-group is represented by green colour branch in the tree. Bootstrap replicates (1000) below than 60% are not shown in the tree.

Figure 4

Alignment of the antigenic epitopes of VP7-G1 of the Pakistani group A rotavirus (RVAs) with those of Rotarix™ and RotaTeq™ vaccines. (A) The whole antigenic region is divided into three epitopes 7-1a, 7-1b, and 7-2. Amino acid residues highlighted in green/orange colours are those different from both Rotarix™ and RotaTeq ™, amino acid residues different from Rotarix™ and RotaTeq™ are marked with green and orange colours, respectively and amino acid residues believed to induce escape from the mAbs neutralization are marked with red triangle (40). (B) Surface representation of VP7 trimer (PDB 3FMG). Antigenic epitopes 7-1a, 7-1b, and 7-2 are marked in blue, yellow and purple colours, respectively. Cyan colour shows surface exposed residues which differ between our G1 strains and vaccine strains.

Figure 5

Alignment of the antigenic epitopes of VP4-P[8] of the Pakistani group A rotavirus (RVAs) with those of Rotarix™ and RotaTeq™ vaccines. (A) The whole antigenic region is divided into four epitopes 8-1, 8-2, 8-3, and 8-4 for VP8* and five epitopes for VP5*. Amino acid residues shading in green and orange are those different from Rotarix™ and RotaTeq™, respectively, amino acid residues highlighting in green/orange colour are those different from both Rotarix™ and RotaTeq™ and amino acid residues believed to induce escape from the mAbs neutralization are marked with red triangle (41). (B) Representation of surface exposed amino acid residues of VP4 core VP8* core (PDB 1KQR). Antigenic epitopes 8-1, 8-2, 8-3, and 8-4 are represented by blue, pink, yellow, and green colours, respectively. Red colour shows surface exposed residues which differ between our P[8] strains and vaccine strains.