Whole Genome Sequencing of Enteroviruses Species A to D by High-Throughput Sequencing: Application for Viral Mixtures

Human enteroviruses (EV) consist of more than 100 serotypes classified within four species for enteroviruses (EV-A to -D) and three species for rhinoviruses, which have been implicated in a variety of human illnesses. Being able to simultaneously amplify the whole genome and identify enteroviruses in samples is important for studying the viral diversity in different geographical regions and populations. It also provides knowledge about the evolution of these viruses. Therefore, we developed a rapid, sensitive method to detect and genetically classify all human enteroviruses in mixtures. Strains of EV-A (15), EV-B (40), EV-C (20), and EV-D (2) viruses were used in addition to 20 supernatants from RD cells infected with stool extracts or sewage concentrates. Two overlapping fragments were produced using a newly designed degenerated primer targeting the conserved CRE region for enteroviruses A-D and one degenerated primer set designed to specifically target the conserved region for each enterovirus species (EV-A to -D). This method was capable of sequencing the full genome for all viruses except two, for which nearly 90% of the genome was sequenced. This method also demonstrated the ability to discriminate, in both spiked and unspiked mixtures, the different enterovirus types present

Recombination between Poliovirus and Coxsackie A Viruses of Species C: A Model of Viral Genetic Plasticity and Emergence

Genetic recombination in RNA viruses was discovered many years ago for poliovirus (PV), an enterovirus of the Picornaviridae family, and studied using PV or other picornaviruses as models. Recently, recombination was shown to be a general phenomenon between different types of enteroviruses of the same species. In particular, the interest for this mechanism of genetic plasticity was renewed with the emergence of pathogenic recombinant circulating vaccine-derived polioviruses (cVDPVs), which were implicated in poliomyelitis outbreaks in several regions of the world with insufficient vaccination coverage. Most of these cVDPVs had mosaic genomes constituted of mutated poliovaccine capsid sequences and part or all of the non-structural sequences from other human enteroviruses of species C (HEV-C), in particular coxsackie A viruses. A study in Madagascar showed that recombinant cVDPVs had been co-circulating in a small population of children with many different HEV-C types. This viral ecosystem showed a surprising and extensive biodiversity associated to several types and recombinant genotypes, indicating that intertypic genetic recombination was not only a mechanism of evolution for HEV-C, but an usual mode of genetic plasticity shaping viral diversity. Results suggested that recombination may be, in conjunction with mutations, implicated in the phenotypic diversity of enterovirus strains and in the emergence of new pathogenic strains. Nevertheless, little is known about the rules and mechanisms which govern genetic exchanges between HEV-C types, as well as about the importance of intertypic recombination in generating phenotypic variation. This review summarizes our current knowledge of the mechanisms of evolution of PV, in particular recombination events leading to the emergence of recombinant cVDPVs

Genetic Relationship between Cocirculating Human Enteroviruses Species C

Recombination events between human enteroviruses (HEV) are known to occur frequently and to participate in the evolution of these viruses. In a previous study, we reported the isolation of a panel of viruses belonging to the Human enterovirus species C (HEV-C) that had been cocirculating in a small geographic area of Madagascar in 2002. This panel included type 2 vaccine-derived polioviruses (PV) that had caused several cases of acute flaccid paralysis in humans. Previous partial sequencing of the genome of these HEV-C isolates revealed considerable genetic diversity, mostly due to recombination. In the work presented herein, we carried out a more detailed characterization of the genomes of viruses from this collection. First, we determined the full VP1 sequence of 41 of these isolates of different types. These sequences were compared with those of HEV-C isolates obtained from other countries or in other contexts. The sequences of the Madagascan isolates of a given type formed specific clusters clearly differentiated from those formed by other strains of the same type isolated elsewhere. Second, we sequenced the entire genome of 10 viruses representing most of the lineages present in this panel. All but one of the genomes appeared to be mosaic assemblies of different genomic fragments generated by intra- and intertypic recombination. The location of the breakpoints suggested potential preferred genomic regions for recombination. Our results also suggest that recombination between type HEV-99 and other HEV-C may be quite rare. This first exhaustive genomic analysis of a panel of non-PV HEV-C cocirculating in a small human population highlights the high frequency of inter and intra-typic genetic recombination, constituting a widespread mechanism of genetic plasticity and continually shifting the HEV-C biodiversity

Recombination between Polioviruses and Co-Circulating Coxsackie A Viruses: Role in the Emergence of Pathogenic Vaccine-Derived Polioviruses

Ten outbreaks of poliomyelitis caused by pathogenic circulating vaccine-derived polioviruses (cVDPVs) have recently been reported in different regions of the world. Two of these outbreaks occurred in Madagascar. Most cVDPVs were recombinants of mutated poliovaccine strains and other unidentified enteroviruses of species C. We previously reported that a type 2 cVDPV isolated during an outbreak in Madagascar was co-circulating with coxsackieviruses A17 (CA17) and that sequences in the 3′ half of the cVDPV and CA17 genomes were related. The goal of this study was to investigate whether these CA17 isolates can act as recombination partners of poliovirus and subsequently to evaluate the major effects of recombination events on the phenotype of the recombinants. We first cloned the infectious cDNA of a Madagascar CA17 isolate. We then generated recombinant constructs combining the genetic material of this CA17 isolate with that of the type 2 vaccine strain and that of the type 2 cVDPV. Our results showed that poliovirus/CA17 recombinants are viable. The recombinant in which the 3′ half of the vaccine strain genome had been replaced by that of the CA17 genome yielded larger plaques and was less temperature sensitive than its parental strains. The virus in which the 3′ portion of the cVDPV genome was replaced by the 3′ half of the CA17 genome was almost as neurovirulent as the cVDPV in transgenic mice expressing the poliovirus cellular receptor gene. The co-circulation in children and genetic recombination of viruses, differing in their pathogenicity for humans and in certain other biological properties such as receptor usage, can lead to the generation of pathogenic recombinants, thus constituting an interesting model of viral evolution and emergence

Poliovirus, la menace fantôme

International audienceLa poliomyélite est une maladie caractérisée par des paralysies irréversibles dues à la destruction de motoneurones par un virus appartenant à un des trois sérotypes de poliovirus (PV). Après l’infection par voie orale, les PV gagnent l’intestin qui constitue leur site principal de réplication. Les poliovirus peuvent se disséminer dans l’organisme par voie sanguine jusqu’à atteindre le système nerveux central et notamment les motoneurones des cornes antérieures de la moelle épinière

Intérêt de la surveillance environnementale pour le programme d'éradication des poliovirus

International audienc

Exchanges of genomic domains between poliovirus and other cocirculating species C enteroviruses reveal a high degree of plasticity

International audienceThe attenuated Sabin strains contained in the oral poliomyelitis vaccine are genetically unstable, and their circulation in poorly immunized populations can lead to the emergence of pathogenic circulating vaccine-derived polioviruses (cVDPVs). The recombinant nature of most cVDPV genomes and the preferential presence of genomic sequences from certain cocirculating non-polio enteroviruses of species C (EV-Cs) raise questions about the permissiveness of genetic exchanges between EV-Cs and the phenotypic impact of such exchanges. We investigated whether functional constraints limited genetic exchanges between Sabin strains and other EV-Cs. We bypassed the natural recombination events by constructing 29 genomes containing a Sabin 2 capsid-encoding sequence and other sequences from Sabin 2 or from non-polio EV-Cs. Most genomes were functional. All recombinant viruses replicated similarly in vitro, but recombination modulated plaque size and temperature sensitivity. All viruses with a 5′UTR from Sabin 2 were attenuated in mice, whereas almost all viruses with a non-polio 5′UTR caused disease. These data highlight the striking conservation of functional compatibility between different genetic domains of cocirculating EV-Cs. This aspect is only one of the requirements for the generation of recombinant cVDPVs in natural conditions, but it may facilitate the generation of viable intertypic recombinants with diverse phenotypic features, including pathogenicity. Recombination between viral RNA genomes is a well-known phenomenon in poliovirus (PV) and other entero-viruses (EVs). Intra-and intertypic recombination shapes the genetic diversity of these viruses, which often have mosaic genomes. Nevertheless, little is known about the permissiveness of intertypic genetic exchanges and their possible impact on phenotype. PV is the etiological agent of paralytic poliomyelitis, a disease characterized by acute flaccid paralysis due to the destruction of motor neurons following PV replication 1. The three serotypes of PV belong to Enterovirus species C (EV-C) (genus Enterovirus, family Picornaviridae). In addition to PV, the EV-C species includes more than 20 other serotypes defined according to the genomic sequence encoding their capsid proteins. EVs are small non-enveloped viruses containing a single-stranded RNA genome of positive polarity of approximately 7.5 kb in length. This genome consists of two untranslated regions (5′ UTR and 3′ UTR) flanking a single large open reading frame. The 5′ UTR and 3′ UTR contain secondary structures involved in initiating the translation and replication of the genome 2–4. The open reading frame is translated into a single polyprotein that is proteolytically processed to yield the four capsid proteins (VP1–4), which are encoded by the P1 region of the genome, and the non-structural proteins, encoded by the P2 and P3 regions. The non-structural proteins are involved in viral replication; they include proteases and the RNA-dependent RNA polymerase 3D. The World Health Organization program for the global eradication of poliomyelitis, which was launched in 1988, has been largely successful. This program has mostly been based on vaccination with the oral polio vaccine (OPV), which is composed of live attenuated strains of the three PV serotypes, the Sabin 1, 2 and 3 strains. Most of the determinants of vaccine strain attenuation, impairing replication in the nervous system, are located in the 5′ UTR and the region encoding the capsid proteins 5. Many of these determinants also confer a temperature-sensitive (ts) phenotype on the strains 6. The Sabin strains can replicate to high titers only in the digestive tract, conferring strong systemic and intestinal immunity, limiting subsequent PV replication and vira

First Full Genome Sequence of a Human Enterovirus A120, Isolated in Madagascar

International audienceWe report the first complete genome sequence of an enterovirus isolate belonging to the human enterovirus A species of the Pi-cornaviridae family and to type A120 (EV-A120). The EV-A120 isolate MAD-2741-11 was obtained from the stool of a healthy child living on Madagascar Island. The isolate genome was amplified by a reverse transcription-PCR method, and the consensus sequence was determined