First evidence of enterovirus A71 and echovirus 30 in Uruguay and genetic relationship with strains circulating in the South American region.

Human enteroviruses (EVs) comprise more than 100 types of coxsackievirus, echovirus, poliovirus and numbered enteroviruses, which are mainly transmitted by the faecal-oral route leading to diverse diseases such as aseptic meningitis, encephalitis, and acute flaccid paralysis, among others. Since enteroviruses are excreted in faeces, wastewater-based epidemiology approaches are useful to describe EV diversity in a community. In Uruguay, knowledge about enteroviruses is extremely limited. This study assessed the diversity of enteroviruses through Illumina next-generation sequencing of VP1-amplicons obtained by RT-PCR directly applied to viral concentrates of 84 wastewater samples collected in Uruguay during 2011-2012 and 2017-2018. Fifty out of the 84 samples were positive for enteroviruses. There were detected 27 different types belonging to Enterovirus A species (CVA2-A6, A10, A16, EV-A71, A90), Enterovirus B species (CVA9, B1-B5, E1, E6, E11, E14, E21, E30) and Enterovirus C species (CVA1, A13, A19, A22, A24, EV-C99). Enterovirus A71 (EV-A71) and echovirus 30 (E30) strains were studied more in depth through phylogenetic analysis, together with some strains previously detected by us in Argentina. Results unveiled that EV-A71 sub-genogroup C2 circulates in both countries at least since 2011-2012, and that the C1-like emerging variant recently entered in Argentina. We also confirmed the circulation of echovirus 30 genotypes E and F in Argentina, and reported the detection of genotype E in Uruguay. To the best of our knowledge this is the first report of the EV-A71 C1-like emerging variant in South-America, and the first report of EV-A71 and E30 in Uruguay.

Molecular epidemiology of enteroviruses associated with severe hand, foot and mouth disease in Shenzhen, China, 2014-2018.

In this study, we investigated the epidemiology and molecular characteristics of enteroviruses associated with severe hand, foot and mouth disease (HFMD) in Shenzhen, China, during 2014-2018. A total of 137 fecal specimens from patients with severe HFMD were collected. Enterovirus (EV) types were determined using real-time reverse transcription polymerase chain reaction (RT-PCR), RT nested PCR, and sequencing. Sequences were analyzed using bioinformatics programs. Of 137 specimens tested, 97 (70.8%), 12 (8.8%), and 10 (7.3%) were positive for EV-A71, coxsackievirus A6 (CVA6), and CVA16, respectively. Other pathogens detected included CVA2 (2.9%, 4/137), CVA10 (2.9%, 4/137), CVA5 (0.7%, 1/137), echovirus 6 (E6) (0.7%, 1/137) and E18 (0.7%, 1/137). The most frequent complication in patients with proven EV infections was myoclonic jerk, followed by aseptic encephalitis, tachypnea, and vomiting. The frequencies of vomiting and abnormal eye movements were higher in EV-A71-infected patients than that in CVA6-infected or CVA16-infected patients. Molecular phylogeny based on the complete VP1 gene revealed no association between the subgenotype of the virus and disease severity. Nevertheless, 12 significant mutations that were likely to be associated with virulence or the clinical phenotype were observed in the 5'UTR, 2Apro, 2C, 3A, 3Dpol and 3'UTR of CVA6. Eight significant mutations were observed in the 5'UTR, 2B, 3A, 3Dpol and 3'UTR of CVA16, and 10 significant mutations were observed in the 5'UTR, VP1, 3A and 3Cpro of CVA10. In conclusion, EV-A71 is still the main pathogen causing severe HFMD, although other EV types can also cause severe complications. Potential virulence or phenotype-associated sites were identified in the genomes of CVA6, CVA16, and CVA10.

Complete genome sequence of a coxsackievirus type A24 variant causing an outbreak of acute haemorrhagic conjunctivitis in southeastern Mexico in 2017.

Cases of acute haemorrhagic conjunctivitis (AHC) caused by a coxsackie virus A24 variant (CV-A24v) in Mexico have been reported since 1987; however, no molecular data on the causative strains have been available. Here, we report the identification of the etiological agent responsible for the most recent AHC outbreak in southeastern Mexico (at the end of 2017) as well as the complete genome sequences of seven isolates, using next-generation sequencing (NGS). Phylogenomic analysis of the CV-A24v sequences reported here showed similarity to contemporary strains causing AHC outbreaks in French Guiana and Uganda, forming a novel clade related to genotype IV. Moreover, a specific mutational pattern in the non-structural proteins was identified in the 2017 isolates. This is the first report of genetic characterization of CV-A24v isolates obtained in Mexico.

Human Enterovirus C105, China, 2017.

We report a case of enterovirus C105 infection in an 11-year-old girl with lower respiratory tract symptoms that was identified through the Respiratory Virus Surveillance System, which covers 30 sentinel hospitals in all 16 districts of Beijing, China. The presence of this virus strain in China confirmed its geographically wide distribution.

Identification and complete genome characterization of human enterovirus 117 from a child with pneumonia in China.

In this study, human enterovirus C117 (EV-C117) was detected in a 3-month-old boy diagnosed with pneumonia in China. A phylogenetic analysis showed that this strain was genetically closer to the Lithuanian strain than to the USA strain.

Re-emergence of a coxsackievirus A24 variant causing acute hemorrhagic conjunctivitis in Brazil from 2017 to 2018.

A large outbreak (over 200,000 cases) of acute hemorrhagic conjunctivitis (AHC) took place in Brazil during the summer of 2017/2018, seven years after a nationwide epidemic, which occurred in 2011. To identify the etiological agent, 80 conjunctival swabs from patients with a clinical presentation suggestive of AHC were analyzed at the national enterovirus laboratory. Real-time RT-PCR for human enteroviruses was performed, and enterovirus RNA was detected in 91.25% (73/80) of the specimens. Twenty-nine swab fluids were used to inoculate cell cultures (RD and Hep2C), and 72.4% (21/29) yielded a cytopathic effect. Genotype IV coxsackievirus A24v (CV-A24v) was identified as the causative agent of the outbreak. Phylogenetic analysis based on the VP1 gene revealed that Brazilian isolates were genetically related to strains that caused an outbreak in French Guiana in 2017. Our results show the re-emergence of CV-A24v causing AHC outbreaks in Brazil between the end of 2017 and the beginning of 2018.

Abundance of enterovirus C in RD-L20B cell culture-negative stool samples from acute flaccid paralysis cases in Nigeria is geographically defined.

PURPOSE: We recently showed that enteroviruses (EVs) andenterovirus species C (EV-C) in particular were abundant in faecal samples from children who had been diagnosed with acute flaccid paralysis (AFP) in Nigeria but declared to be EV-free by the RD-L20B cell culture-based algorithm. In this study, we investigated whether this observed preponderance of EVs (and EV-Cs) in such samples varies by geographical region. METHODOLOGY: One hundred and eight samples (i.e. 54 paired stool suspensions from 54 AFP cases) that had previously been confirmed to be negative for EVs by the WHO-recommended RD-L20B cell culture-based algorithm were analysed. The 108 samples were made into 54 pools (27 each from North-West and South-South Nigeria). All were subjected to RNA extraction, cDNA synthesis and the WHO-recommended semi-nested PCR assay and its modifications. All of the amplicons were sequenced, and the enteroviruses identified, using the enterovirus genotyping tool and phylogenetic analysis. RESULTS: EVs were detected in 16 (29.63 %) of the 54 samples that were screened and successfully identified in 14 (25.93 %). Of these, 10 were from North-West and 4 were from South-South Nigeria. One (7.14 %), 2 (14.29 %) and 11 (78.57 %) of the strains detected were EV-A, EV-B and EV-C, respectively. The 10 strains from North-West Nigeria included 7 EV types, namely CV-A10, E29, CV-A13, CV-A17, CV-A19, CV-A24 and EV-C99. The four EV types recovered from South-South Nigeria were E31, CV-A1, EV-C99 and EV-C116. CONCLUSION: The results of this study showed that the presence of EVs and consequently EV-Cs in AFP samples declared to be EV-free by the RD-L20B cell culture-based algorithm varies by geographical region in Nigeria.

High frequency of Polio-like Enterovirus C strains with differential clustering of CVA-13 and EV-C99 subgenotypes in a cohort of Malawian children.

Enteroviruses (EVs) are among the most commonly detected viruses infecting humans worldwide. Although the prevalence of EVs is widely studied, the status of EV prevalence in sub-Saharan Africa remains largely unknown. The objective of our present study was therefore to increase our knowledge on EV circulation in sub-Saharan Africa. We obtained 749 fecal samples from a cross-sectional study conducted on Malawian children aged 6 to 60 months. We tested the samples for the presence of EVs using real time PCR, and typed the positive samples based on partial viral protein 1 (VP1) sequences. A large proportion of the samples was EV positive (89.9%). 12.9% of the typed samples belonged to EV species A (EV-A), 48.6% to species B (EV-B) and 38.5% to species C (EV-C). More than half of the EV-C strains (53%) belonged to subgroup C containing, among others, Poliovirus (PV) 1-3. The serotype most frequently isolated in our study was CVA-13, followed by EV-C99. The strains of CVA-13 showed a vast genetic diversity, possibly representing a new cluster, 'F'. The majority of the EV-C99 strains grouped together as cluster B. In conclusion, this study showed a vast circulation of EVs among Malawian children, with an EV prevalence of 89.9%. Identification of prevalences for species EV-C comparable to our study (38.5%) have only previously been reported in sub-Saharan Africa, and EV-C is rarely found outside of this region. The data found in this study are an important contribution to our current knowledge of EV epidemiology within sub-Saharan Africa.

A novel Enterovirus 96 circulating in China causes hand, foot, and mouth disease.

Enterovirus 96 (EV-96) is a recently described member of the species Enterovirus C and is associated with paralysis and myelitis. In this study, using metagenomic sequencing, we identified a new enterovirus 96 strain (EV-96-SZ/GD/CHN/2014) as the sole pathogen causing hand, foot, and mouth disease (HFMD). A genomic comparison showed that EV-96-SZ/GD/CHN/2014 is most similar to the EV-96-05517 strain (85% identity), which has also been detected in Guangdong Province. This is the first time that metagenomic sequencing has been used to identify an EV-96 strain shown to be associated with HFMD.

A cluster of coxsackievirus A21 associated acute respiratory illness: the evidence of efficient transmission of CVA21.

In March 2016, a cluster of unexplained respiratory illnesses was reported by the acute respiratory infections (ARI) surveillance system of Guangdong Province, China. Twenty-three high school students and one teacher from the four neighboring classes were admitted to a hospital. CVA21 was found in eight of fourteen patients. Phylogenetic analysis suggested that the CVA21 outbreak was most likely caused by transmission of the virus from person to person. This is the first report of an ARI outbreak caused by CVA21, which suggests that CVA21 has the potential to be transmitted efficiently from person to person and should be closely monitored by clinicians and public health agencies.

Circulation of multiple serotypes of highly divergent enterovirus C in the Xinjiang Uighur Autonomous Region of China.

Poliomyelitis associated with circulating vaccine-derived polioviruses (cVDPVs) is a serious public health issue in the post-eradication era, and the occurrence of recombinant cVDPVs emphasizes the need to elucidate enterovirus C (EV-C) epidemiology. Stool samples were collected from 826 healthy children in Southern Xinjiang in 2011 to investigate EV-C circulation and epidemiology. Thirty-six EV-Cs were isolated and assigned to eight EV-C serotypes by molecular serotyping, suggesting the circulation of diverse EV-Cs in Xinjiang. Phylogenetic analysis showed that the Xinjiang EV-C strains had larger variation compared to the prototype and other modern strains. Additionally, the results showed unique characteristics of Xinjiang EV-Cs, such as the cytopathicity of CV-A1 strains to RD cells; the high divergence in CV-A11, CV-A13, CV-A17, and CV-A20 strains; the divergence of Xinjiang CV-A24 from AHC-related CV-A24 variant stains distributed worldwide; and the circulation of two novel EV-C serotypes (EV-C96 and EV-C99). Evaluations of this dense and diverse EV-C ecosystem will help elucidate the processes shaping enteroviral biodiversity. This study will improve our understanding of the evolution of enteroviruses and the recombination potential between polioviruses and other EV-Cs.

A major outbreak of conjunctivitis caused by coxsackievirus A24, Réunion, January to April 2015.

From January to April 2015, Réunion experienced a major outbreak of acute haemorrhagic conjunctivitis (AHC) caused by coxsackievirus A24, which heavily impacted the healthcare system. According to the general practitioners' (GP) sentinel network, the number of medical consultations due to conjunctivitis during this period was estimated at ca 100,000. This report describes the characteristics of the outbreak, which were obtained through several different yet complementary surveillance systems on the island. These included the network of hospital emergency departments (OSCOUR network), the GPs' sentinel network, an Internet-based population cohort ('Koman i lé') participating in a survey on distinct symptoms including 'red eyes' and the monitoring of eye drop sales. Overall the results of the different surveillance approaches were in good agreement regarding the outbreak dynamic. A peak of patients with conjunctivitis was detected in the first 15 days of March (week 10 and 11), coinciding with increased eye drop sales on the island. Strains recovered from outbreak cases belonged to genotype IV and were most closely related to strains identified in AHC outbreaks in China, Egypt and Japan since 2010. Continued surveillance of AHC in Réunion remains important not only locally, but also because frequent exchanges between the island and mainland France may lead to introduction of this virus in Europe.

Complete genome analysis of coxsackievirus A24 isolated in Yunnan, China, in 2013.

Human coxsackievirus A24 (CVA24) belongs to the species Enterovirus C, and variants of this virus frequently cause acute hemorrhagic conjunctivitis (AHC). The complete genome of the K282/YN/CHN/2013 strain, isolated from a healthy child in Yunnan, China, in 2013, is reported here for the first time. The strain showed 80.0 % and 79.9 % nucleotide sequence identity to CVA24 prototype strain Joseph and CVA24 variant prototype EH24, respectively. The K282/YN/CHN/2013 strain belongs to the CVA24 serotype. Twelve amino acid differences, most of which are in structural regions, were found between the CVA24 and CVA24v strains. In the whole-length genome sequence, only the structural region of K282/YN/CHN/2013 was similar to that of the CVA24 strains; the other genome regions were more similar to those of other members of the species Enterovirus C. Recombination analysis showed evidence of recombination with other viruses of the same species.

[Molecular Identification and Phylogenetic Analyses of Coxsackievirus A24v Causing an Outbreak of Acute Hemorrhagic Conjunctivitis in Jiangxi, China, in 2010].

To identify the cause of an outbreak of acute hemorrhagic conjunctivitis (AHC) in Jiangxi (China) in 2010, 20 eye conjunctival swabs were first collected from AHC patients. Then, viruses were isola- ted and tested for human enterovirus 70, coxsackievirus A24 variant (CV-A24v) and adenovirus using the polymerase chain reaction. All CV-A24v isolates underwent sequencing of 3C and VP1 coding regions. Then, a phylogenetic tree was constructed for Jiangxi CV-A24v and worldwide CV-A24v based on,3C and VP1 regions, respectively. Ten out of 20 specimens were positive for CV-A24v, implying that the outbreak was caused by CV-A24v. The phylogenetic tree based on the 3C region showed that Jiangxi CV- A24v belonged to cluster 5 in genotype IV (GIV-C5) with strains isolated throughout the world after 2010, and were divided further into A and B lineages. Phylogenetic analyses of the VP1 region showed that all of the worldwide CV-A24v strains isolated after 2000 could be divided into five groups (1-5). Jiangxi CV-A24v was classified into group 5 and also divided further into A and B lineages upon analyses of the 3C region. These data suggested that CV-A24v causing AHC outbreaks in China in 2010 belonged to GIV-C3 and GIV-C5. At least two transmission lineages were circulated in Jiangxi in 2010. The classification of CV-A24v isolated after 2010 worldwide using the phylogenetic tree based on the VP1 region was almost consistent with that based on the 3C region and also had significant chronological clustering.

Acute Flaccid Paralysis Associated with Novel Enterovirus C105.

An outbreak of acute flaccid paralysis among children in the United States during summer 2014 was tentatively associated with enterovirus D68 infection. This syndrome in a child in fall 2014 was associated with enterovirus C105 infection. The presence of this virus strain in North America may pose a diagnostic challenge.

Epidemic outbreak of acute haemorrhagic conjunctivitis caused by coxsackievirus A24 in Thailand, 2014.

Acute haemorrhagic conjunctivitis outbreaks are often attributed to viral infection. In 2014, an unprecedented nationwide outbreak of infectious conjunctivitis occurred in Thailand, which affected >300 000 individuals over 3 months. To identify and characterize the virus responsible for the epidemic, eye swab specimens from 119 patients were randomly collected from five different provinces. Conserved regions in the enteroviral 5'-UTR and adenovirus hexon gene were analysed. Enterovirus was identified in 71·43% (85/119) of the samples, while no adenovirus was detected. From enterovirus-positive samples, the coxsackievirus A24 variant (70·59%, 84/119) and echovirus (0·84%, 1/119) were identified. Additional sequencing of full-length VP1 and 3C genes and subsequent phylogenetic analysis revealed that these clinical isolates form a new lineage cluster related to genotype IV-C5. In summary, the coxsackievirus A24 variant was identified as an aetiological agent for the recent acute haemorrhagic conjunctivitis outbreak in Thailand.

Circulation of two Enterovirus C105 (EV-C105) lineages in Europe and Africa.

The coding sequences of five human enterovirus (HEV)-C genotype 105 strains recovered in Italy, Romania and Burundi from patients with upper and lower respiratory tract infections were analysed and phylogenetically compared with other circulating HEV-C strains. The EV-C105 was closely related to EV-C109 and EV-C118 strains. The European strains were similar to other circulating EV-C105 strains, while the two African EV-C105 clustered in separate bootstrap-supported (>0.90) branches of the P2 and P3 region trees. Minor inconsistencies in the clustering pattern of EV-C105 in the capsid region (P1) and non-capsid region (P3) suggest that recombination may have occurred in EV-C105 group B viruses. In conclusion, phylogenetic analysis revealed the circulation of two distinct EV-C105 lineages in Europe and Africa. A different pattern of evolution could be hypothesized for the two EV-C105 lineages.

Complete coding regions of the prototypes enterovirus B93 and C95: phylogenetic analyses of the P1 and P3 regions of EV-B and EV-C strains.

Complete coding regions were sequenced for two new enterovirus genomes: EV-B93 previously identified by VP1 sequencing, derived from a child with acute flaccid paralysis in the Democratic Republic of Congo; and EV-C95 from a French soldier with acute gastroenteritis in Djibouti. The EV-B93 P1 had more than 30% nucleotide divergence from other EV-B types, with highest similarity to E-15 and EV-B80. The P1 nucleotide sequence of EV-C95 was most similar, 71%, to CV-A21. Complete coding regions for the new enteroviruses were compared with those of 135 EV-B and 176 EV-C strains representing all types available in GenBank. When strains from the same outbreak or strains isolated during the same year in the same geographical region were excluded, 27 of the 58 EV-B, and 16 of the 23 EV-C types were represented by more than one sequence. However, for EV-B the P3 sequences formed three clades mainly according to origin or time of isolation, irrespective of type, while for EV-C the P3 sequences segregated mainly according to disease manifestation, with most strains causing paralysis, including polioviruses, forming one clade, and strains causing respiratory illness forming another. There was no intermixing of types between these two clades, apart from two EV-C96 strains. The EV-B P3 sequences had lower inter-clade and higher intra-clade variability as compared to the EV-C sequences, which may explain why inter-clade recombinations are more frequent in EV-B. Further analysis of more isolates may shed light on the role of recombinations in the evolution of EV-B in geographical context.

Recombination among human non-polio enteroviruses: implications for epidemiology and evolution.

Human enteroviruses (EV) belong to the Picornaviridae family and are among the most common viruses infecting humans. They consist of up to 100 immunologically and genetically distinct types: polioviruses, coxsackieviruses A and B, echoviruses, and the more recently characterized 43 EV types. Frequent recombinations and mutations in enteroviruses have been recognized as the main mechanisms for the observed high rate of evolution, thus enabling them to rapidly respond and adapt to new environmental challenges. The first signs of genetic exchanges between enteroviruses came from polioviruses many years ago, and since then recombination has been recognized, along with mutations, as the main cause for reversion of vaccine strains to neurovirulence. More recently, non-polio enteroviruses became the focus of many studies, where recombination was recognized as a frequent event and was correlated with the appearance of new enterovirus lineages and types. The accumulation of multiple inter- and intra-typic recombination events could also explain the series of successive emergences and disappearances of specific enterovirus types that could in turn explain the epidemic profile of circulation of several types. This review focuses on recombination among human non-polio enteroviruses from all four species (EV-A, EV-B, EV-C, and EV-D) and discusses the recombination effects on enterovirus epidemiology and evolution.

Genomic characterization of coxsackievirus type A24 strains associated with acute flaccid paralysis and rarely identified Hopkins syndrome.

The full-length genome sequence analysis of four coxsackievirus A24 (CV-A24) strains, detected in three paralytic and one post-asthmatic paralytic (Hopkins syndrome) cases, is reported here for the first time. A phylogenetic tree constructed on the basis of entire genomes displayed topology similar to that of the full-VP1 tree, classifying the study strains in genogroup CV-A24vGIV along with their temporal counterparts in strains from non-paralytic cases. The strains of the study formed a single genetic cluster C4 within CV-A24vGIV and showed 3.5-19.4 % nucleotide sequence divergence, with 2-4 novel nucleotide mutations in the 5'NCR and 3-8 unique amino acid substitutions in the polyprotein, with respect to the CV-A24 strains associated with non-paralytic cases. Among the nucleotide mutations, A299U was identified in the 5'NCRs of all of the study strains. CV-A24v strains of the same genogroup with few genomic variations but different disease manifestations need to be explored to investigate the molecular basis of evolution of neurovirulence.