Genetic landscape and macro-evolution of co-circulating Coxsackieviruses A and Vaccine-derived Polioviruses in the Democratic Republic of Congo, 2008-2013

Abstract

Enteroviruses (EVs) are among the most common viruses infecting humans worldwide but only a few Non-Polio Enterovirus (NPEV) isolates have been characterized in the Democratic Republic of Congo (DR Congo). Moreover, circulating vaccine-derived polioviruses (PVs) [cVDPVs] isolated during multiple outbreaks in DR Congo from 2004 to 2018 have been characterized so far only by the sequences of their VP1 capsid coding gene. This study was carried to i) investigate the circulation and genetic diversity of NPEV and polio vaccine isolates recovered from healthy children and Acute Flaccid Paralysis (AFP) patients, ii) evaluate the occurrence of genetic recombination among EVs belonging to the Enterovirus C species (including PVs) and iii) identify the virological factors favoring multiple emergences of cVDPVs in DR Congo. The biological material considered in this study included i) a collection of 91 Sabin-like PVs, 54 cVDPVs and 150 NPEVs isolated from AFP patients between 2008 and 2012 in DR Congo and iii) a collection of 330 stool specimens collected from healthy children in 2013 in the Kasai Oriental and Maniema provinces of DR Congo. Studied virus isolates were sequenced in four distinct sub-genomic regions 5’-UTR, VP1, 2C ^ATPase and 3D ^pol. Resulting sequences were compared through comparative phylogenetic analyses. Virus isolation showed that 19.1% (63/330) healthy children were infected by EVs including 17.9% (59/330) of NPEVs and 1.2% (4/330) of type 3 Sabin-like PVs. Only one EV-C type, EV-C99 was identified among the NPEV collection from AFP patients whereas 27.5% of the 69 NPEV isolates typed in healthy children belonged to the EV-C species: CV-A13 (13/69), A20 (5/69) and A17 (1/69). Interestingly, 50 of the 54 cVDPVs featured recombinant genomes containing exogenous sequences in at least one of the targeted non-structural regions of their genomes: 5’UTR, 2C ^ATPase and 3D ^pol. Some of these non-vaccine sequences of the recombinant cVDPVs were strikingly related to homologous sequences from co-circulating CV-A17 and A20 in the 2C ^ATPase region as well as to those from co-circulating CV-A13, A17 and A20 in the 3D ^pol region. This study provided the first evidence uncovering CV-A20 strains as major recombination partners of PVs. High quality AFP surveillance, sensitive environmental surveillance and efficient vaccination activities remain essential to ensure timely detection and efficient response to recombinant cVDPVs outbreaks in DR Congo. Such needs are valid for any epidemiological setting where high frequency and genetic diversity of Coxsackieviruses A13, A17 and A20 provide a conducive viral ecosystem for the emergence of virulent recombinant cVDPVs.

Author summary

The strategy of the Global Polio Eradication Initiative is based on the surveillance of patients suffering from Acute Flaccid Paralysis (AFP) and mass vaccination with live-attenuated vaccine strains of polioviruses (PVs) in endemic areas. However, vaccine strains of PVs can circulate and replicate for a long time when the vaccine coverage of the population is low. Such prolonged circulation and replication of vaccine strains of PVs can result to the emergence of circulating vaccine-derived polioviruses [cVDPVs] that are as virulent as wild PVs. In this study, we performed the molecular characterization of a large collection of 377 virus isolates recovered from paralyzed patients between 2008 and 2012 in DR Congo and healthy children in 2013 in the Kasai Oriental and Maniema provinces of DR Congo. We found that the genetic diversity of enteroviruses of the species Enterovirus C is more important than previously reported. Interestingly, 50 of the 54 cVDPVs featured recombinant genomes containing exogenous sequences of the 2C ATPase and/or 3D polymerase coding genes acquired from co-circulating Coxsackieviruses A13, A17 and A20. Coxsackieviruses A20 strains were identified for the first time as major partners of genetic recombination with co-circulating live-attenuated polio vaccine strains.

Our findings highlight the need to reinforce and maintain high quality surveillance of PVs and efficient immunization activities in order to ensure early detection and control of emerging cVDPVs in all settings where high frequency and diversity of Coxsackieviruses A13, A17 and A20 have been documented.