Characterization of Enteroviruses circulating among Farm Animals and Children in Central African Republic.

AbstractTo characterize enteroviruses (EVs) circulating in farm animals in Central African Republic (CAR), we screened 192 stools of animals under 12 months belonging to family farms located in or near Bangui. To assess whether EV exchanges exist between these animals and humans, we also screened 197 stools of children who lived in contact with farm animals, as well as control stools of 256 children with no contact with farm animals. EVs were typed based on their capsid sequences. In children, all EVs belonged to species A, B and C, with EV-Cs accounting for 60%. Some EV-Cs shared recent common ancestors with lineages of vaccine-derived poliovirus that emerged in the country in 2019-2020. In animals, we identified EV-Gs that belonged to 10 different types, including a previously unknown one that we named EV-G28, while no EV-E or EV-F were observed. The CAR EV-Gs were genetically closely related to specimens sampled in other continents and some of them harboured the torovirus-derived insertion already reported in some EV-Gs. The worldwide circulation of EV-Gs is likely due the massive international trade of live animals. Besides, two human EV-Cs (coxsackievirus A17 and coxsackievirus A24) were detected in pigs, suggesting that these viruses could cross the species barrier. Our work provides original data on the epidemiology and ecology of EVs circulating among herd animals in Africa.

Neutralization of African enterovirus A71 genogroups by antibodies to canonical genogroups.

Enterovirus 71 (EV-A71) is a major public health problem, causing a range of illnesses from hand-foot-and-mouth disease to severe neurological manifestations. EV-A71 strains have been phylogenetically classified into eight genogroups (A to H), based on their capsid-coding genomic region. Genogroups B and C have caused large outbreaks worldwide and represent the two canonical circulating EV-A71 subtypes. Little is known about the antigenic diversity of new genogroups as compared to the canonical ones. Here, we compared the antigenic features of EV-A71 strains that belong to the canonical B and C genogroups and to genogroups E and F, which circulate in Africa. Analysis of the peptide sequences of EV-A71 strains belonging to different genogroups revealed a high level of conservation of the capsid residues involved in known linear and conformational neutralization antigenic sites. Using a published crystal structure of the EV-A71 capsid as a model, we found that most of the residues that are seemingly specific to some genogroups were mapped outside known antigenic sites or external loops. These observations suggest a cross-neutralization activity of anti-genogroup B or C antibodies against strains of genogroups E and F. Neutralization assays were performed with diverse rabbit and mouse anti-EV-A71 sera, anti-EV-A71 human standards and a monoclonal neutralizing antibody. All the batches of antibodies that were tested successfully neutralized all available isolates, indicating an overall broad cross-neutralization between the canonical genogroups B and C and genogroups E and F. A panel constituted of more than 80 individual human serum samples from Cambodia with neutralizing antibodies against EV-A71 subgenogroup C4 showed quite similar cross-neutralization activities between isolates of genogroups C4, E and F. Our results thus indicate that the genetic drift underlying the separation of EV-A71 strains into genogroups A, B, C, E and F does not correlate with the emergence of antigenically distinct variants.

Estimating prevalence of Enterovirus D111 in human and non-human primate populations using cross-sectional serology.

Enteroviruses primarily affect young children with a varying severity of disease. Recent outbreaks of severe respiratory and neurological disease due to EV-D68 and EV-A71, as well as atypical hand-foot-and-mouth-disease due to CVA6, have brought to light the potency of enteroviruses to emerge as severe human pathogens. Enterovirus D111 (EV-D111) is an enteric pathogen initially detected in Central Africa in human and wildlife samples and was recently detected in environmental samples. The natural history and epidemiology of EV-D111 are poorly studied. Here, the presence of serum neutralizing antibodies to EV-D111 was estimated in human and wildlife samples from five countries. We report high prevalence of neutralizing antibodies measured against EV-D111 in human populations (range, 55-83 %), a proxy for previous infection, which indicates active virus circulation in absence of detection in clinical cases and a high number of undiagnosed infections. Notably, seroprevalence in samples from the UK varied by age and was higher in children and older adults (1-5 and >60 years old), but lower in ages 11-60. EV-D111 seroprevalence in apes and Old World monkeys was 50 % (33-66 %), which also suggests prior exposure and supports existing knowledge of enterovirus circulation in wild and captive apes and Old World monkeys. Generally, reported cases of infection likely underestimate the prevalence of infection particularly when the knowledge of community transmission is limited. Continued serologic surveillance and detection of EV-D111 in clinical and environmental samples will allow for a more robust assessment of EV-D111 epidemiology.

Animal enteroviruses: a glimpse of a wide evolutionary landscape.

The genus Enterovirus (family Picornaviridae) contains numerous viruses, most of which have been identified in humans. Among them, the three serotypes of poliovirus, coxsackieviruses A and B, echoviruses, rhinoviruses and other enteroviruses (EVs) responsible in humans for a wide spectrum of clinical manifestations. There are also 60 identified EVs in different mammals. Some have been found in both humans and animals, demonstrating the possibility of zoonotic transmission of certain EVs. Compared to human EVs, genetic and epidemiological data about animal EVs are scarce. However, the detection of EVs in various species of mammals and their presence on all continents suggest that the number of EVs still to be discovered is very important. Some EVs found in animals have characteristics never seen in human EVs. Furthermore, the unique phylogenetic relationships observed between some animal EVs raise interesting questions about the rules that govern the evolution of these viruses. The aim of this review is to present the salient data on animal EVs and to highlight the questions they raise.

[Animal enteroviruses: a glimpse of a wide evolutionary landscape]. / Entérovirus des animaux : lumière sur l'immensité de l'espace évolutif des entérovirus.

The genus Enterovirus (family Picornaviridae) contains numerous viruses, most of which have been identified in humans. Among them, the three serotypes of poliovirus, coxsackieviruses A and B, echoviruses, rhinoviruses and other enteroviruses (EVs) responsible in humans for a wide spectrum of clinical manifestations. There are also 60 identified EVs in different mammals. Some have been found in both humans and animals, demonstrating the possibility of zoonotic transmission of certain EVs. Compared to human EVs, genetic and epidemiological data for animal EVs are scarce. However, the detection of EV in various species of mammals and their presence on all continents suggest that the number of EV still to be discovered is very important. Some EVs found in animals have characteristics never seen in human EVs. Furthermore, the unique phylogenetic relationships observed between animal EVs raise interesting questions about the rules that govern the evolution of these viruses. The aim of this review is to present the salient data on animal EVs and to highlight the questions they raise.

Chronic Aichi Virus Infection As a Cause of Long-Lasting Multiorgan Involvement in Patients With Primary Immune Deficiencies.

BACKGROUND: Metagenomic next-generation sequencing (mNGS) was used to assess patients with primary or secondary immune deficiencies (PIDs and SIDs) who presented with immunopathological conditions related to immunodysregulation. METHODS: Thirty patients with PIDs or SIDs who presented with symptoms related to immunodysregulation and 59 asymptomatic patients with similar PIDs or SIDs were enrolled. mNGS was performed on organ biopsy. Specific Aichi virus (AiV) reverse-transcription polymerase chain reaction (RT-PCR) was used to confirm AiV infection and screen the other patients. In situ hybridization (ISH) assay was done on AiV-infected organs to identify infected cells. Virus genotype was determined by phylogenetic analysis. RESULTS: AiV sequences were detected using mNGS in tissue samples of 5 patients and by RT-PCR in peripheral samples of another patient, all of whom presented with PID and long-lasting multiorgan involvement, including hepatitis, splenomegaly, and nephritis in 4 patients. CD8+ T-cell infiltration was a hallmark of the disease. RT-PCR detected intermittent low viral loads in urine and plasma from infected patients but not from uninfected patients. Viral detection stopped after immune reconstitution obtained by hematopoietic stem cell transplantation. ISH demonstrated the presence of AiV RNA in hepatocytes (n = 1) and spleen tissue (n = 2). AiV belonged to genotype A (n = 2) or B (n = 3). CONCLUSIONS: The similarity of the clinical presentation, the detection of AiV in a subgroup of patients suffering from immunodysregulation, the absence of AiV in asymptomatic patients, the detection of viral genome in infected organs by ISH, and the reversibility of symptoms after treatment argue for AiV causality.

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.

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.

Strengthened surveillance revealed a rapid disappearance of the poliovirus serotype 2 vaccine strain in Madagascar after its removal from the oral polio vaccine.

To assess circulation of the Sabin 2 poliovirus vaccine strain in Madagascar after its withdrawal from the oral polio vaccine in April 2016, a reinforced poliovirus surveillance was implemented in three regions of Madagascar from January 2016 to December 2017. Environmental samples and stool specimens from healthy children were screened using the Global Polio Laboratory Network algorithm to detect the presence of polioviruses. Detected polioviruses were molecularly typed and their genomes fully sequenced. Polioviruses were detected during all but 4 months of the study period. All isolates were related to the vaccine strains and no wild poliovirus was detected. The majority of isolates belong to the serotype 3. The last detection of Sabin 2 occurred in July 2016, 3 months after its withdrawal. No vaccine-derived poliovirus of any serotype was observed during the study. Only few poliovirus isolates contained sequences from non-polio origin. The genetic characterization of all the poliovirus isolates did not identify isolates that were highly divergent compared to the vaccine strains. This observation is in favor of a good vaccine coverage that efficiently prevented long-lasting transmission chains between unvaccinated persons. This study underlines that high commitment in the fight against polioviruses can succeed in stopping their circulation even in countries where poor sanitation remains a hurdle.

[A cold case: non-replicative recombination in positive-strand RNA viruses]. / Une affaire non résolue : la recombinaison non réplicative chez les virus à ARN de polarité positive.

Genetic recombination is a major force driving the evolution of some species of positive sense RNA viruses. Recombination events occur when at least two viruses simultaneously infect the same cell, thereby giving rise to new genomes comprised of genetic sequences originating from the parental genomes. The main mechanism by which recombination occurs involves the viral polymerase that generates a chimera as it switches templates during viral replication. Various experimental systems have alluded to the existence of recombination events that are independent of viral polymerase activity. The origins and frequency of such events remain to be elucidated to this day. Furthermore, it is not known whether non-replicative recombination yields products that are different from recombinants generated by the viral polymerase. If this is the case, then non-replicative recombination may play a unique role in the evolution of positive sense RNA viruses. Finally, the sparse data available suggest that non-replicative recombination does not necessarily involve only virus-specific sequences. It is thus possible that the non-replicative recombination observed in virus-focused studies may in fact reveal a more generalized mechanism that is non-specific to virus RNAs.

A cold case: non-replicative recombination in positive-strand RNA viruses.

Genetic recombination is a major force driving the evolution of some species of positive sense RNA viruses. Recombination events occur when at least two viruses simultaneously infect the same cell, thereby giving rise to new genomes comprised of genetic sequences originating from the parental genomes. The main mechanism by which recombination occurs involves the viral polymerase that generates a chimera as it switches templates during viral replication. Various experimental systems have alluded to the existence of recombination events that are independent of viral polymerase activity. The origins and the frequency of such events remain to be elucidated to this day. Furthermore, it is not known whether non-replicative recombination yields products that are different from recombinants generated by the viral polymerase. If this is the case, then non-replicative recombination may play a unique role in the evolution of positive sense RNA viruses. Finally, the sparse data available suggest that non-replicative recombination does not necessarily involve only virus-specific sequences. It is thus possible that the non-replicative recombination observed in virus-focused studies may in fact reveal a more generalized mechanism that is non-specific to virus RNAs.

Vaccine-Derived Polioviruses, Central African Republic, 2019.

Since May 2019, the Central African Republic has experienced a poliomyelitis outbreak caused by type 2 vaccine-derived polioviruses (VDPV-2s). The outbreak affected Bangui, the capital city, and 10 districts across the country. The outbreak resulted from several independent emergence events of VDPV-2s featuring recombinant genomes with complex mosaic genomes. The low number of mutations (<20) in the viral capsid protein 1-encoding region compared with the vaccine strain suggests that VDPV-2 had been circulating for a relatively short time (probably <3 years) before being isolated. Environmental surveillance, which relies on a limited number of sampling sites in the Central African Republic and does not cover the whole country, failed to detect the circulation of VDPV-2s before some had induced poliomyelitis in children.

[New oral polio vaccine: A turning point for the global polio eradication initiative?] / Le nouveau vaccin antipoliomyélitique oral : Un tournant décisif pour le programme d'éradication ?

Launched in 1988, the Global Polio Eradication Initiative (GPEI) aims to eradicate polioviruses, which are the etiologic agents of poliomyelitis. Coordinated by the World Health Organization, this program relies on two pillars: mass vaccination campaigns that target children and active surveillance of the virus circulation. The GPEI has led to the eradication of two out of three serotypes of wild polioviruses and to the containment of the last serotype in two countries.Two polio vaccines exist: the injectable vaccine and the oral one. Both induce an efficient protection against poliomyelitis, but only the oral vaccine is able to stop poliovirus transmission chains. Therefore, the oral vaccine is essential to contain polioviruses and, finally, to eradicate them. In some contexts where the vaccine coverage is not sufficient, the attenuated strains contained in the oral vaccine can circulate for months and recover a pathogenic phenotype through genetic drift. In order to prevent this phenomenon, a new vaccine strain has been developed through genetic engineering: it has been designed to be as immunogenic as the historical vaccine strain, but more genetically stable to prevent the loss of its attenuation determinants. After being evaluated in vitro and through clinical trials, the novel strain has been rolled out in several African countries and in Tajikistan in 2021.

Development of a New Internally Controlled One-Step Real-Time RT-PCR for the Molecular Detection of Enterovirus A71 in Africa and Madagascar.

Enterovirus A71 (EV-A71) is a leading cause of hand-foot-and-mouth disease (HFMD) and can be associated with severe neurological complications. EV-A71 strains can be classified into seven genogroups, A-H, on the basis of the VP1 capsid protein gene sequence. Genogroup A includes the prototype strain; genogroups B and C are responsible of major outbreaks worldwide, but little is known about the others, particularly genogroups E and F, which have been recently identified in Africa and Madagascar, respectively. The circulation of EV-A71 in the African region is poorly known and probably underestimated. A rapid and specific assay for detecting all genogroups of EV-A71 is required. In this study, we developed a real-time RT-PCR assay with a competitive internal control (IC). The primers and TaqMan probe specifically target the genomic region encoding the VP1 capsid protein. Diverse EV-A71 RNAs were successfully amplified from the genogroups A, B, C, D, E, and F, with similar sensitivity and robust reproducibility. Neither cross reaction with other EVs nor major interference with the competitive IC was detected. Experimentally spiked stool and plasma specimens provided consistent and reproducible results, and validated the usefulness of the IC for demonstrating the presence of PCR inhibitors in samples. The analysis in an African laboratories network of 1889 untyped enterovirus isolates detected 15 EV-A71 of different genogroups. This specific real-time RT-PCR assay provides a robust and sensitive method for the detection of EV-A71 in biological specimens and for the epidemiological monitoring of EV-A71 including its recently discovered genogroups.

Reinforced poliovirus and enterovirus surveillance in Romania, 2015-2016.

Due to the risk of poliovirus importation from Ukraine in 2015, a combined surveillance program monitoring the circulation of enteroviruses (EVs) in healthy children from at-risk areas and in the environment was conducted in Romania. Virological testing of stool samples collected from 155 healthy children aged from two months to six years and of 186 sewage water samples collected from different areas was performed. A total of 58 (37.42%) stool samples and 50 (26.88%) sewage water samples were positive for non-polio EVs, but no poliovirus was detected. A high level of circulation of echovirus (E) types 6 and 7 and coxsackievirus (CV) type B5 was observed.

Whole genome sequencing and phylogenetic characterization of a novel bat-associated picornavirus-like virus with an unusual genome organization.

The order Picornavirales is one of the most important viral orders in terms of virus diversity and genome organizations, ranging from a mono- or bi-cistronic expression strategies to the recently described poly-cistronic Polycipiviridae viruses. We report here the description and characterization of a novel picorna-like virus identified in rectal swabs of frugivorous bats in Cambodia that presents an unusual genome organization. Kandabadicivirus presents a unique genome architecture and distant phylogenetic relationship to the proposed Badiciviridae family. These findings highlight a high mosaicism of genome organizations among the Picornavirales.

Genetic and phenotypic characterization of recently discovered enterovirus D type 111.

Members of the species Enterovirus D (EV-D) remain poorly studied. The two first EV-D types (EV-D68 and EV-D70) have regularly caused outbreaks in humans since their discovery five decades ago but have been neglected until the recent occurrence of severe respiratory diseases due to EV-D68. The three other known EV-D types (EV-D94, EV-D111 and EV-D120) were discovered in the 2000s-2010s in Africa and have never been observed elsewhere. One strain of EV-D111 and all known EV-D120s were detected in stool samples of wild non-human primates, suggesting that these viruses could be zoonotic viruses. To date, EV-D111s are only known through partial genetic sequences of the few strains that have been identified so far. In an attempt to bring new pieces to the puzzle, we genetically characterized four EV-D111 strains (among the seven that have been reported until now). We observed that the EV-D111 strains from human samples and the unique simian EV-D111 strain were not phylogenetically distinct, thus suggesting a recent zoonotic transmission. We also discovered evidences of probable intertypic genetic recombination events between EV-D111s and EV-D94s. As recombination can only happen in co-infected cells, this suggests that EV-D94s and EV-D111s share common replication sites in the infected hosts. These sites could be located in the gut since the phenotypic analysis we performed showed that, contrary to EV-D68s and like EV-D94s, EV-D111s are resistant to acid pHs. We also found that EV-D111s induce strong cytopathic effects on L20B cells, a cell line routinely used to specifically detect polioviruses. An active circulation of EV-D111s among humans could then induce a high number of false-positive detection of polioviruses, which could be particularly problematic in Central Africa, where EV-D111 circulates and which is a key region for poliovirus eradication.