Enterovirus detection in different regions of Madagascar reveals a higher abundance of enteroviruses of species C in areas where several outbreaks of vaccine-derived polioviruses occurred

Members of the species Enterovirus C (EV-C, genus Enterovirus, family Picornaviridae), the three serotypes of poliovirus (PVs) are the causal agent of poliomyelitis, an infectious disease characterized by acute flaccid paralysis. Launched in 1988 and coordinated by the World Health Organization, the Global Polio Eradication Initiative (GPEI) aims to eradicate the PVs. One GPEI pillar is mass vaccination campaigns that target children under 5 years old. Two efficient vaccines were developed in the 1950s–1960s: the injectable polio vaccine, which contains inactivated particles of each of the three serotypes and the oral polio vaccine (OPV), which contains three attenuated strains. When orally administrated, these strains replicate in the gut of the vaccine recipient and induce a strong mucosal immunity that limits the replication of PVs during subsequent infections with PVs [1, 2]. Therefore, OPV effectively block PV circulation in humans. OPV has been the main tool used by the GPEI, which succeeded in eliminating wild PV-2 and -3 while the area of circulation of wild PV-1 strains has been restricted to two countries only, Pakistan and Afghanistan.

Nonetheless, a low vaccine coverage can enable the circulation of OPV strains in non-vaccinated children. Long transmission chains allow the genetic drift of the vaccine strains, which can lose their attenuation determinants and recover a pathogenic phenotype similar to that of wild strains. Polio outbreaks due to these circulating vaccine-derived polioviruses (cVDPVs) have been regularly reported since 2000 in areas where the vaccine coverage is low [3]. These cVDPVs are threatening the benefit of the vaccination campaigns and complexifying the GPEI strategies.

Most cVDPVs detected worldwide feature recombinant genomes made of genetic sequences originating from the OPV strains and from non-polio enteroviruses (NPEVs). All cVDPVs retain the PV vaccine capsid-encoding region but large parts of the other regions of their genomes come from NPEVs [4]. In particular, CVA17 and -A20 and to some extent CVA11 and -A13 appeared to be possible recombinant partners of polioviruses.

The main factor that favors the emergence of pathogenic cVDPVs is the low vaccine coverage that allows the establishment of long transmission chains between non-immunized people. Nonetheless, it is possible that other factors promote the emergence and the circulation of cVDPVs. In particular, it is likely that recombination events between the PV vaccine strains and co-circulating EV-Cs accelerate the reversion of their attenuated phenotype.

In the 2000s and 2010s, Madagascar experienced several independent emergences of cVDPVs, of which some were responsible of poliomyelitis cases [5, 6]. All these cVDPVs featured recombinant genomes. Noteworthy, most of them were detected in three southern regions of the island. The 2001–2002 poliomyelitis outbreak took place in two Southern provinces of Madagascar, Toliara and Taolagnaro [6]. Three years later, in 2005, another outbreak occurred in Toliara province [5]. In 2013, a study in the same province highlighted the circulation of different lineages of VDPVs, although no poliomyelitis cases were linked to these viruses [7]. These regions do not have peculiar high population density that could explain the emergence of cVDPVs [8]. Huge differences regarding the vaccine coverage does not seem to explain either why the cVDPVs preferentially emerges in the southern part of the island.

Many other factors could explain discrepancy between the southern and northern regions regarding the frequency of cVDPVs emergence: climate, access to potable water, eating habits, etc. One factor could be differences in the NPEV populations circulating in children. To address this hypothesis, we compared the NPEV populations isolated from children stools collected in the meantime in four regions of Madagascar: Toliara and Taolagnaro in the South, and Mahajanga and Antseranana in the North.

Previous studies have described the frequent presence of NPEVs in Madagascar and underlined the high frequency of EVs of species C [5, 25, 35]. Nonetheless, these studies only focused on the southern part of the island, where most VDPVs were detected. Nothing was known regarding putative differences between NPEV populations circulating in different parts of Madagascar. To determine whether peculiar treats of the NPEV population circulating in the southern part of Madagascar could explain why cVDPVs preferentially emerged in this region, we conducted a comparative study based on stools collected in the same period from healthy children living in two southern and two northern regions of Madagascar. This study was carried out in 2011, when next-generation sequencing was less affordable than nowadays. For this reason, we chose to detect the NPEVs through virus isolation rather than through direct molecular screening. Because it is known that RD cells are poorly sensitive to NPEVs of the species C [36, 37], we screened each sample on RD cells and HEp-2c cells, known to be more sensitive to EV-C [38]. The parallel use of these two cell lines is expected to allow the detection of most types of EV-A, -B, C and D, including the EV-C that commonly recombine with PV vaccine strains.

This study revealed an extensive circulation of NPEVs among healthy children, with an overall isolation rate of 26.8% (351/1309). This value is in the range of NPEV isolation rates already reported in healthy people, but the isolation range notably varies from study to study: 8.9% in children under 15 years old in the border area of China and Myanmar in 2009 [39], 24.7% in children under 6 years in the Philippines [40], 24.2% in children under 8 years old in Ghana [41], and 51% in people under 21 years old in Mongolia [42].

The molecular characterization of NPEVs showed that the EV-C species were the most prevalent followed by EV-B species and in the last the EV-A species. No EV-D species were detected during our investigation. Since members of this species generally produce clear CPE when grown in RD and L20B cells [11], it can be assumed that EV-D were not actively circulating when the study was conducted.

Interestingly, some VP1-encoding sequences fell in phylogenetic clusters that exclusively contain sequences of viruses isolated in Madagascar. These findings could be due to Madagascar geographic isolation, which could favor the appearance of topotypes in absence of the recent introduction of strains from other countries. Nonetheless, it is also possible that the genetic data available in public databases provides a biased overview of the actual area of circulation of some EV types and lineages. Indeed, only a few sequences are available for some EV types, such as for instance the types E2 or E12. It is to noted that CVA7 was the most prevalent EV-A type detected in this study although this type is rarely reported nowadays [43], although it was widely detected in the 1950s and 1960s during outbreaks of flaccid paralysis [44, 45].

The fact that most of the non-structural sequences from EV-C sampled in Madagascar in the 2000s and in 2011 cluster together in a branch that does not contain any sequences from viruses sampled in other countries suggests that EV-C mainly evolved during this period without massive importation of strains from other geographic regions. Whether it means that EV-C importations are rare in Madagascar or whether foreign strains are outcompeted by the indigenous ones remains to be determined.

No differences regarding the types that circulate in the northern and the southern districts was noted. Importantly, the usual partners of the PV vaccine strains (CVA11, CVA13, CVA17 and CVA20) were found both in the North and in the South. Inside each type, no obvious clustering based on the origin (North vs South of the island) was observed, which indicates the probable co-circulation of different lineages through the island during the same year. By contrast, the northern and southern districts differed by two important features: first, the isolation rate was higher in the South; second, the relative abundance of EV-B and EV-C greatly differs between the North and the South. The donors were randomly selected in all sites, which is expected to limit sampling bias. As all the samples were processed using the same protocols in the same lab, technical biases can be ruled out. Because this study was conducted on a short period of time, it is impossible to determine whether this huge discrepancy is constant over the time. Nonetheless, a previous study conducted 10 years before in the district of Taolagnaro also showed a predominance of EV-C among circulating NPEVs since they accounted for two-thirds of the NPEVs detected by using both RD and HEp-2c cell lines [35]. The climate could be one of the factors that explain the difference observed between the northern districts and the southern one. Madagascar has a subtropical climate with a warm rainy season (summer, from November to March) and a cooler dry season (winter, from April through October). There are huge differences between the North and the South in the island: the south is an arid region and the district of Toliara is the driest zone of the island.

The reasons that explain this discrepancy remain to be deciphered but the fact that EV-C were majority in regions where cVDPVs repeatedly emerged is striking. The unknown factors responsible for the high frequency of EV-Cs in this region could also favor the circulation of vaccine PV strains. Furthermore, the high frequency of EV-C could facilitate the recombination of vaccine PV strains with NPEV partners, particularly CVA13, 17 or 20. Indeed, the production of recombinant genomes requires co-infections, which are probably more likely with majority types. It is known that most VDPVs feature recombinant genomes and it has been shown that NPEV sequences play an important role in the VDPV neurovirulence level [46‐48]. Therefore, environmental surveillance constitutes a precious tool in the GPEI strategy as it enables the early detection of the circulation of Sabin strains but also provides clues for the presence in the same area of non-polio EV-Cs that frequently recombine with them.

This study demonstrated that the exposure to NPEVs is not uniform in Madagascar. Coupled with the low immunity against poliovirus, the particular enteroviral ecosystem that exists in the South of Madagascar could be a favorable factor for increasing the emergence risk of recombinant VDPVs in this region.