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

Abstract

Background: Poliomyelitis outbreaks due to pathogenic vaccine-derived polioviruses (VDPVs) are threatening and complicating the global polio eradication initiative. Most of these VDPVs are genetic recombinants with non-polio enteroviruses (NPEVs) of species C. Little is known about factors favoring this genetic macroevolution process. Since 2001, Madagascar has experienced several outbreaks of poliomyelitis due to VDPVs, and most of VDPVs were isolated in the south of the island. The current study explored some of the viral factors that can promote and explain the emergence of recombinant VDPVs in Madagascar.

Methods: Between May to August 2011, we collected stools from healthy children living in two southern and two northern regions of Madagascar. Virus isolation was done in RD, HEp-2c, and L20B cell lines, and enteroviruses were detected using a wide-spectrum 5'-untranslated region RT-PCR assay. NPEVs were then sequenced for the VP1 gene used for viral genotyping.

Results: Overall, we collected 1309 stools, of which 351 NPEVs (26.8%) were identified. Sequencing revealed 33 types of viruses belonging to three different species: Enterovirus A (8.5%), Enterovirus B (EV-B, 40.2%), and Enterovirus C (EV-C, 51.3%). EV-C species included coxsackievirus A13, A17, and A20 previously described as putative recombination partners for poliovirus vaccine strains. Interestingly, the isolation rate was higher among stools originating from the South (30.3% vs. 23.6%, p-value = 0.009). EV-C were predominant in southern sites (65.7%) while EV-B predominated in northern sites (54.9%). The factors that explain the relative abundance of EV-C in the South are still unknown.

Conclusions: Whatever its causes, the relative abundance of EV-C in the South of Madagascar may have promoted the infections of children by EV-C, including the PV vaccine strains, and have favored the recombination events between PVs and NPEVs in co-infected children, thus leading to the recurrent emergence of recombinant VDPVs in this region of Madagascar.

Keywords: Genotype; Human enterovirus; Madagascar; Vaccine-derived poliovirus.

Fig. 1

A Map of Madagascar. Antananarivo, the capital city, and the four capitals of the districts in which the samples were collected are indicated. B Boxplot of the age of the donors in each district. C Number of donors with and without vaccination card in each district. D Proportion of donors fully vaccinated amongst the donors with vaccination card in each district

Fig. 2

Detection of viruses through screening on cell cultures and identification by molecular assays

Fig. 3

Number of EV-A, -B and -C isolates found in each sampling region

Fig. 4

Phylogenetic relationships of CVA10 strains based on the full-length VP1 sequence. The samples from this study are in red with their respective collection site. The other sequences are named by using their respective GenBank accession number, country (ISO 3166-1 alpha-3 codes) and year of sampling, when known; some lineages have been collapsed for better legibility. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches; bootstrap values < 95% were hidden

Fig. 5

Evolutionary relationships based on the full-length VP1 sequences belonging to the types E19, E2, E12 and E24. The samples from this study are in red with their respective collection site. The other sequences are named by using their respective GenBank accession number, country (ISO 3166-1 alpha-3 codes) and year of sampling, when known; some lineages have been collapsed for better legibility. The evolutionary history was inferred in MEGA X by using the Maximum Likelihood method and Tamura-Nei model. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Tamura-Nei model, and then selecting the topology with superior log likelihood value. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches; bootstrap values < 90% were hidden

Fig. 6

Phylogenetic relationships based on the VP1 sequences of CVA13s. For better legibility, some clusters were collapsed and the name of the isolate from this study are not indicated. Circles and triangles indicate the district where each isolate was sampled. For the other sequences, their respective GenBank accession number, country (ISO 3166-1 alpha-3 codes) and year of sampling are indicated, when known. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches; bootstrap values < 95% were hidden

Fig. 7

Phylogenetic relationships of EV-Cs, based on the full-length 3D-encoding region. The main tree contains all 3D-encoding sequences of EV-Cs belonging to cluster IV found in GenBank. The subtrees with Madagascan sequences are expanded. Red circles indicate isolates from this study (with the name of the corresponding district), blue circles indicate sequences sampled in Madagascar in previous studies (with their respective year of sampling). The other sequences are named by using their respective GenBank accession number, country (ISO 3166-1 alpha-3 codes) and year of sampling. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches if < 90%. The CVA1 sequence was used as outlier for rooting the tree