This study analysed circulating G12P[6] and G12P[8] rotaviruses from several African countries during the period 2010–2014 and prior to widespread use of rotavirus vaccines on the continent. Genotype G12 strains, which emerged approximately two decades ago, have been reported to be the cause of severe dehydrating diarrhoea in vaccinated children in several countries, particularly in Latin America which started vaccination about six years prior to sub-Saharan Africa [41–43]. However, if one looks at a temporal association of the emergence of the G12 strains, it is associated with the global spread of these strains, rather than causally associated with wide-spread vaccine use. Nevertheless, with the introduction of rotavirus vaccine in 2012–2014 in many of the African countries included in this study, the opportunity existed to conduct an analysis of circulating G12P[6] and G12P[8] strains in several countries, just prior to and as vaccines were introduced and to evaluate whether these strains might become predominant due to evading the vaccine. Five of the six studied countries had introduced the Rotarix vaccine. The exception is Rwanda which uses RotaTeq vaccine.
Clearly, G12 strains do not share the VP7 G-specificity with vaccine strains; however, both licenced rotavirus vaccines (RotaTeq and Rotarix) have demonstrated clinical protection against heterotypic strains, including G12 strains. For instance, the phase III Rotarix® clinical trial conducted in Malawi and South Africa showed cross protection against diverse rotavirus strains, including G12 with vaccine efficacy of 51.5% [33]. Similar results were observed with the RotaTeq vaccine study in three African countries [34]. However, rotavirus vaccines have also been shown to exercise protection via the immune responses to the VP4 neutralization antigens [44], and the VP4 P[8] is shared between both vaccines and a proportion of the G12 strains evaluated, those with G12P[8]. Thus, understanding the genetic variability of both the VP4 and VP7 genes of the circulating G12 rotavirus strains should provide insights into the evolutionary relationships and potential biological advantages of these strains in Africa.
Phylogenetic analysis of G12 rotavirus strains globally, shows segregation of the strains into four lineages (I –IV). Lineage I is the prototype strain L26 identified in 1987 and which was not apparently biologically competitive in humans and did not spread; lineage II is the G12P[9] strains from Asia which appear to be a unique class of natural reassortants with a VP4 P[9]; and lineage IV includes the only porcine strain (G12P[7]) [19, 45, 46]. Lineage III strains, on the other hand, are the mostly contemporary G12 strains detected since the mid-2000’s and which are now globally prevalent in most continents. This analysis confirms that the genotype G12 strains circulating in these six sub-Saharan African countries (Ethiopia, Kenya, Rwanda, Tanzania, Togo and Zambia) clustered in lineage III with strains circulating all over the world, showing the dominance and biological competitiveness of these strains, which have persisted over the last two decades, in most continents [47–49].
Evidence of genetic variation was observed amongst the four G12 lineages in this study. Amino acid substitution S25N (VR2), N87S (antigenic region A) and A213T (antigenic region C) in lineages II & III segregate the prototype lineage I detected in 1987 and the porcine lineage IV. The lineages were further characterised by the amino acid substitutions A125S in VR 6 and V142I in antigenic region B detected only in the current circulating lineage III strains. The latter change from Valine to Isoleucine, where the amino acids share similar chemical properties, might not impose a conformational change to the VP7 protein. However, the A125S substitution, in which Alanine acquired a hydroxyl group to change to Serine over the period of early 2000s to late 2000s could influence the capsid structure. The mechanism of rotaviruses mutating to advance epidemiological spread was observed with recent G2 rotavirus strains belonging to lineage IVa that spread globally. All these strains exhibited an amino acid substitution D96N which seemed to confer survival advantage to these lineage IVa G2 rotavirus strains [50]. It needs to be investigated further whether the A125S amino acid substitution observed in lineage III G12 strains has contributed to its competitiveness and spread. The amino acid substitutions and phylogenetic clustering of the study strains away from the porcine lineage IV, indicates that they are not genetically related although animal-human rotavirus transmission is often reported in the African continent.
Amino acids changes within the antigenic regions of VP7 can result in alteration to the antigenicity of the virus and potentially enhance immunity [51]. It has been shown that the antibodies targeting neutralization epitopes stabilize the capsid and prevent uncoating of the virus which is required for viral replication [52]. The observed amino acid substitutions in the antigenic region A-C within VP7 of different G12 lineages might not substantially explain the increased detection of G12 strains which have evolved naturally but might support the continual detection of more competitive G12 strains belonging to lineage III globally.
Zeller and colleagues proposed that differences in the neutralizing epitopes in VP4 could undermine the vaccines effectiveness [51]. If the vaccine efficacy is mediated through the VP4 antigen, then considering these mutations may provide further insight. The study strains had similar amino acids in most of the antigenic epitopes to the VP4 P[8] gene of RotaTeq, with some differences to Rotarix, which is the preferred vaccine in most African countries. The major amino acid substitution is in position 131, in which Rotarix had a Serine and RotaTeq and study strains had an Arginine. Arginine is a positively charged amino acid able to interact with negatively charged particles, and such change from a non-essential to essential amino acid in the VP4 rotavirus protein involved in virus entry might cause conformational changes leading to neutralizing antibody escape.
The G12 rotaviruses appear to have emerged irrespective of the use of rotavirus vaccines and continue circulating in countries that have not introduced the vaccines, indicating the natural circulation and competitiveness of these human viral strains. For example, rotavirus vaccines were introduced in the six countries included in this study between 2012–2014 and the G12 strains analysed in this study were isolated between 2010–2014. To substantiate further, various studies from Ethiopia have reported G12 rotaviruses as a dominant strain both pre- and post-vaccine introduction [53].
It is therefore not possible to draw conclusions that the prevalence of G12 strains was affected by vaccine introduction. The amino acid changes in the neutralization epitopes of VP7 and VP4 may cause conformational changes that provide selective advantages of the new strain and establish continued infection within the population, particularly with high vaccine induced antibody levels and should be monitored prospectively. Possibly, assessing the G12 strains that have emerged in Latin America and Africa at different stages after rotavirus vaccine introduction might shed light on the evolutionary pressure exerted by the vaccines.