Genetic characterization of G12P[6] and G12P[8] rotavirus strains collected in six African countries between 2010 and 2014

Diarrhoeal disease is a major cause of death in infants and young children below the age of 5 years and rotavirus is the most significant pathogen associated with that mortality [1]. Rotavirus is estimated to cause 122,232–215,757 Under-5 deaths annually [2, 3]. Furthermore, it has been estimated that diarrhoeal diseases are significantly more severe in immunocompromised children, especially those infected with HIV which is relevant to sub-Saharan Africa [4]. Recent estimates showed that the introduction of rotavirus vaccines globally has resulted in a relative reduction of 59% of rotavirus hospitalizations and 36% of all cause acute gastroenteritis hospitalizations, respectively [5]. Before the introduction of rotavirus vaccines in many African countries, it was estimated that almost 40% of all diarrhoeal cases on the continent were due to rotavirus infection [4]. The introduction of rotavirus vaccines into 29 sub-Saharan African countries before 2016, resulted in a reduction of approximately 21,000 deaths and 135,000 hospitalizations in 2016 alone [6], highlighting the major impact that rotavirus vaccines have had on rotavirus diarrhoea.

Rotaviruses are double-stranded RNA (dsRNA) viruses, which belong to the family Reoviridae [7]. The viral genome comprises of eleven segments which code for six structural viral proteins (VP; labelled VP1-VP4, VP6-VP7) and six non-structural proteins (NSP; NSP1-NSP6). Two of the structural proteins (VP7 and VP4) form the outer capsid of the virus, which are used in the binomial classification of rotavirus strains into G (for the VP7 glycoprotein) and P (for the VP4 protease-sensitive) types, respectively. According to the Rotavirus Classification Working Group of the International Committee on Taxonomy of Viruses (ICTV), there are 36 G and 51 P rotavirus types causing diarrhoea in humans, animals and avian species [7‐9]. Of these, only six genotypes - G1P[8], G2P[4], G3P[8], G4P[8], G9P[8] and G12P[8] - are responsible for over 90% of rotavirus-related diarrhoea in humans globally [10‐12].

On the African continent the dominant VP7 genotypes of rotavirus are G1, G2, G3, G4, G8, G9 and G12; G4 strains predominated in the 1980s and 1990s but have subsequently diminished dramatically [13]. The common VP4 genotypes of human rotaviruses circulating are P[8], P[6] and P[4] [14‐16], with an unusually high prevalence of P[6] in Africa [16]. The emergence and rapid spread of G12 rotavirus strains has been widely observed globally [17]. A decade after the first report of G12 strains in the Philippines in 1987 [18], which was believed to be a zoonotic transmission to a child, the widespread circulation of this genotype was reported in South and North America, Asia and Europe [11, 18‐21]. In sub-Saharan Africa, G12 strains initially emerged in combination with the VP4 P[6] genotype and were first reported in South Africa in 2004 during a hospital-based rotavirus surveillance study [22]. Subsequently, G12 rotavirus strains were reported in other African countries such as Malawi, Nigeria, Ghana, Cameroon, Kenya, Tanzania, Ethiopia, Zambia, Togo, and Zimbabwe [23‐29].

Interestingly, genotype G12 strains were prevalent in Africa during the evaluation of the available rotavirus vaccines (i.e. Rotarix and RotaTeq), in large safety and efficacy studies [30, 31]. Neither of these vaccines contain the G12 VP7 genotype although both have a VP4 P[8] genotype. RotaTeq (Merck & Co., White River, Pennsylvania, USA) is a pentavalent bovine-human mono-reassortant vaccine containing 4 VP7 reassortants carrying the human G1 - G4 encoding genes and a VP4 reassortant carrying the human P[8] encoding gene, all on the genetic background of bovine rotavirus WI79 (G6 P[5]) strain [32]. Rotarix (GSK Biologicals, Rixensart, Belgium) is a human rotavirus strain bearing a G1P[8] genotype. The strain was isolated in 1989 [33].

Both rotavirus vaccines demonstrated homotypic and heterotypic protection against the common circulating strains in multiple studies in diverse geographies, including the circulating genotype G12 strain [34‐38]. Furthermore, various post-marketing surveillance studies have reported that the vaccine confers heterotypic protection against novel strains carrying neither VP4 nor VP7 antigens found in the vaccines [39]. Nevertheless, there is concern in the scientific community about the issue of “vaccine-induced immune pressure” driving the emergence of novel strains that may evade vaccine protection [40]. As G12 rotavirus strains are the most recent to emerge and spread globally and are circulating in several African countries that have introduced the vaccine, and as information about the temporal genetic diversity of the circulating G12 strains in Africa is still limited, we sought to investigate the genetic variability of the two recognized neutralization antigens, VP7 and VP4, of G12 strains from across the continent. Thus, this study investigated the genetic variability of the gene segments 4 (encoding VP4) and 9 (encoding VP7) of G12 strains identified in several African countries and analysed the putative neutralization epitopes in an effort to provide insights on the evolutionary mechanisms and possible origins of the G12 strains in Africa.

This study analysed circulating G12P[6] and G12P[8] rotaviruses from several African countries collected 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 [52‐54]. 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 with the exception of Rwanda which initially introduced RotaTeq vaccine and switched to Rotarix in 2018.

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% [37]. Similar results were observed with the RotaTeq vaccine study in three African countries [38]. Importantly, rotavirus vaccines have been shown to exercise protection via the immune responses to the VP4 neutralization antigens also [55], 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]) [17, 56, 57]. Lineage III strains, on the other hand, are the mostly contemporary G12 strains detected since the early-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 [58‐60]. The evolutionary rate of G12 genotype (1.016 × 10–3 nucleotide substitutions/site/year) observed in this study is well within the ranges that have been reported by several investigators [17, 61, 62]. The estimated time to the most recent ancestor of lineage III strains is 1992 which is similar to a previous estimate of 1995 [47] and the African strains - although scattered within the three sub-clusters - show their most recent ancestor to be from the late 1990s. This reflects the epidemiologic data, which reported the first isolation of G12 strains in the African continent in 2004. The observed diversification of our African strains in three sub-clusters is not due to their country of isolation, but more likely due to three different ancestral strains emerging at approximately the same time. This trend can be applied to globally circulating G12 strains belonging to lineage III [62].

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 between the prototype lineage I detected in 1987 and the porcine lineage IV. The lineages were further characterised by the amino acid substitutions A125S in VR6 and V142I in antigenic region B detected only in the current circulating lineage III strains. This 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 [63]. 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 [47]. 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 [64]. Zeller and colleagues proposed that differences in the neutralizing epitopes in VP4 could undermine the vaccines effectiveness [47]. 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.

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 and 2014 and the G12 strains analysed in this study were collected between 2010 and 2014. To substantiate further, various studies from Ethiopia have reported G12 rotaviruses as a dominant strain both pre- and post-vaccine introduction [65].

It is therefore not possible to conclude that the prevalence of G12 strains was affected by vaccine introduction. 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.

The study findings suggest that the novel G12 strains circulating in African countries are highly similar at the nucleotide and amino acid level, irrespective of geographical distribution and year of detection. The African G12P[8] and G12P[6] rotavirus strains belonging to lineage III circulating in these countries are not unique and are the same as the globally circulating rotavirus G12 strains and there is no evidence of molecular evolutionary pressure from widespread vaccine use. The antigenic epitopes display limited diversity to each other and other global strains, including to the two rotavirus vaccines (RotaTeq and Rotarix), indicating that this is unlikely to be associated with sustained circulation over time in Africa. The G12 strains diversified into three sub-clusters with ancestries estimated to the late 1990s and are evolving at 1.016 × 10^− 3 nucleotide substitutions/site/year sustaining this strain overtime. However, as new rotavirus vaccines which do not carry the common human rotavirus VP4 genotype (P[8]), such as RotaVac and RotaSIIL from India [66, 67] have been pre-qualified by WHO and are introduced in selected African countries, it will be imperative to continue genotypic surveillance to identify and monitor emerging strains.