RNA recombination plays a major role in genomic change during circulation of coxsackie B viruses

RNA recombination has been shown to occur during circulation of enteroviruses, but most studies have focused on poliovirus. To examine the role of recombination in the evolution of the coxsackie B viruses (CVB), we determined the partial sequences of four genomic intervals for multiple clinical isolates of each of the six CVB serotypes isolated from 1970 to 1996. The regions sequenced were the 5'-nontranslated region (5'-NTR) (350 nucleotides [nt]), capsid (VP4-VP2, 416 nt, and VP1, approximately 320 nt), and polymerase (3D, 491 nt). Phylogenetic trees were constructed for each genome region, using the clinical isolate sequences and those of the prototype strains of all 65 enterovirus serotypes. The partial VP1 sequences of each CVB serotype were monophyletic with respect to serotype, as were the VP4-VP2 sequences, in agreement with previously published studies. In some cases, however, incongruent tree topologies suggested that intraserotypic recombination had occurred between the sequenced portions of VP2 and VP1. Outside the capsid region, however, isolates of the same serotype were not monophyletic, indicating that recombination had occurred between the 5'-NTR and capsid, the capsid and 3D, or both. Almost all clinical isolates were recombinant relative to the prototype strain of the same serotype. All of the recombination partners appear to be members of human enterovirus species B. These results suggest that recombination is a frequent event during enterovirus evolution but that there are genetic restrictions that may influence recombinational compatibility.     

Frequency and dynamics of recombination within different species of human enteroviruses

Enteroviruses are members of the family Picornaviridae that cause widespread infections in human and other mammalian populations. Enteroviruses are genetically and antigenically highly variable, and recombination within and between serotypes contributes to their genetic diversity. To investigate the dynamics of the recombination process, sequence phylogenies between three regions of the genome (VP4, VP1, and 3Dpol) were compared among species A and B enterovirus variants detected in a human population-based survey in Scotland between 2000 and 2001, along with contemporary virus isolates collected in the same geographical region. This analysis used novel bioinformatic methods to quantify phylogenetic compatibility and correlations with serotype assignments of evolutionary trees constructed for different regions of the enterovirus genome. Species B enteroviruses showed much more frequent, time-correlated recombination events than those found for species A, despite the equivalence in population sampling, concordant with a linkage analysis of previously characterized enterovirus sequences obtained over longer collection periods. An analysis of recombination among complete genome sequences by computation of a phylogenetic compatibility matrix (PCM) demonstrated sharply defined boundaries between the VP2/VP3/VP1 block and sequences to either side in phylogenetic compatibility. The PCM also revealed equivalent or frequently greater degrees of incompatibility between different parts within the nonstructural region (2A-3D), indicating the occurrence of extensive recombination events in the past evolution of this part of the genome. Together, these findings provide new insights into the dynamics of species A and B enterovirus recombination and evolution and into the contribution of structured sampling to documenting reservoirs, emergence, and spread of novel recombinant forms in human populations.     

Evidence of recombination among enteroviruses.

Human enteroviruses consist of more than 60 serotypes, reflecting a wide range of evolutionary divergence. They have been genetically classified into four clusters on the basis of sequence homology in the coding region of the single-stranded RNA genome. To explore further the genetic relationships between human enteroviruses and to characterize the evolutionary mechanisms responsible for variation, previously sequenced genomes were subjected to detailed comparison. Bootstrap and genetic similarity analyses were used to systematically scan the alignments of complete genomic sequences. Bootstrap analysis provided evidence from an early recombination event at the junction of the 5' noncoding and coding regions of the progenitors of the current clusters. Analysis within the genetic clusters indicated that enterovirus prototype strains include intraspecies recombinants. Recombination breakpoints were detected in all genomic regions except the capsid protein coding region. Our results suggest that recombination is a significant and relatively frequent mechanism in the evolution of enterovirus genomes.     

Complete Genome Sequence of an Enterovirus 80 Strain Isolated in China

Enterovirus 80 (EV80) is a newly identified serotype of the species Human enterovirus B. An EV80 strain designated HZ01/SD/CHN/2004 was isolated from an acute flaccid paralysis case in Shandong, China, in 2004. Complete genome comparison revealed 79.5% similarity with the prototype strain and an insertion of 36 nucleotides in the 3′ end of the VP1 coding region. Intertypic recombination with other serotypes was observed. This is the first report of the complete genome of EV80 in China.     

Complete genome sequence of a recombinant coxsackievirus b4 from a patient with a fatal case of hand, foot, and mouth disease in guangxi, china

The coxsackievirus B4 (CVB4) belongs to human enterovirus B species within the family Picornaviridae. Here we report a novel complete genome sequence of a recombinant CVB4 strain, CVB4/GX/10, which was isolated from a patient with a fatal case of hand, foot, and mouth disease in China. The complete genome consists of 7,293 nucleotides, excluding the 3' poly(A) tail, and has an open reading frame that maps between nucleotide positions 742 and 7293 and encodes a 2,183-amino-acid polyprotein. Phylogenetic analysis based on different genome regions reveals that CVB4/GX/10 is closest to a CVB4 strain, EPIHFMD-CLOSE CONTACT-16, in the 5' half (VP4～2B) of the genome, although it is closer to a Chinese CVB5 strain, CVB5/Henan/2010, in the 3' half (2C～3D) of the genome. Furthermore, similar bootscan analysis based on the whole genomes demonstrates that recombination has possibly occurred within the 2C domain and that CVB4/GX/10 is a possible progeny of intertypic recombination of the CVB4 strain EPIHFMD-CLOSE CONTACT-16 and CVB5/Henan/2010 that occurred during their cocirculation and evolution, which is a relatively common phenomenon in enteroviruses.     

Natural recombination event within the capsid genomic region leading to a chimeric strain of human enterovirus B

Recombination between two strains is a known phenomenon for enteroviruses replicating within a single cell. We describe a recombinant strain recovered from human stools, typed as coxsackievirus B4 (CV-B4) and CV-B3 after partial sequencing of the VP1 and VP2 coding regions, respectively. The strain was neutralized by a polyclonal CV-B3-specific antiserum but not by a CV-B4-specific antiserum. The nucleotide sequence analysis of the whole structural genomic region showed the occurrence of a recombination event at position 1950 within the VP3 capsid gene, in a region coding for the 2b antigenic site previously described for CV-B3. This observation evidences for the first time the occurrence of an interserotypic recombination within the VP2-VP3-VP1 capsid region between two nonpoliovirus enterovirus strains. The neutralization pattern suggests that the major antigenic site is located within the VP2 protein.     

口蹄疫病毒Asia1/YNBS/58株全基因组序列分析

口蹄疫是由口蹄疫病毒(Foot-and-mouth dis-ease virus,FMDV)感染引起的偶蹄动物(猪、牛、羊、骆驼等)共患的一种急性、烈性、接触性传染病。FMDV是小核糖核酸病毒科(Picornaviridae)口蹄疫病毒属(Aphthovirus)的成员,有7个血清型,分别为O、A、C、Asia1、SAT1、SAT2、SAT3,完整     

Evidence for frequent recombination within species human enterovirus B based on complete genomic sequences of all thirty-seven serotypes

The species Human enterovirus B (HEV-B) in the family Picornaviridae consists of coxsackievirus A9; coxsackieviruses B1 to B6; echoviruses 1 to 7, 9, 11 to 21, 24 to 27, and 29 to 33; and enteroviruses 69 and 73. We have determined complete genome sequences for the remaining 22 HEV-B serotypes whose sequences were not represented in public databases and analyzed these in conjunction with previously available complete sequences in GenBank. Members of HEV-B were monophyletic relative to all other human enterovirus species in all regions of the genome except in the 5'-nontranslated region (NTR), where they are known to cluster with members of HEV-A. Within HEV-B, phylogenies constructed from the structural (P1) and nonstructural regions of the genome (P2 and P3) are incongruent, suggesting that recombination had occurred. Similarity plots and bootscanning analysis across the complete genome identified multiple sites at which the phylogeny of a given strain's sequence shifted, indicating potential recombination points. These points are distributed in the 5'-NTR and throughout P2 and P3, but no sites with >80% bootstrap support were identified within the capsid. Individual sequence comparisons and phylogenetic analyses suggest that members of HEV-B have recombined with one another on multiple occasions, resulting in a complex mosaic of sequences derived from multiple parental viruses in the nonstructural regions of the genome. We conclude that RNA recombination is a common mechanism for enterovirus evolution and that recombination within the nonstructural regions of the genome (P2 and P3) has been observed only among members of the same species.     

Porcine teschoviruses comprise at least eleven distinct serotypes: molecular and evolutionary aspects

Nucleotide sequencing and phylogenetic analysis of 10 recognized prototype strains of the porcine enterovirus (PEV) cytopathic effect (CPE) group I reveals a close relationship of the viral genomes to the previously sequenced strain F65, supporting the concept of a reclassification of this virus group into a new picornavirus genus. Also, nucleotide sequences of the polyprotein-encoding genome region or the P1 region of 28 historic strains and recent field isolates were determined. The data suggest that several closely related but antigenically and molecular distinct serotypes constitute one species within the proposed genus Teschovirus. Based on sequence data and serological data, we propose a new serotype with strain Dresden as prototype. This hitherto unrecognized serotype is closely related to porcine teschovirus 1 (PTV-1, former PEV-1), but induces type-specific neutralizing antibodies. Sequencing of field isolates collected from animals presenting with neurological disorders prove that other serotypes than PTV-1 may also cause polioencephalomyelitis of swine.     

Nucleotide sequence of the 5'nontranslated and virion polypeptides regions of coxsackievirus B6.

The nucleotide sequence of coxsackievirus B6 (CVB6) has been determined, and the nucleotides encoding the 5' nontranslated region (5' NTR) and virion polypeptides (VP4, 2, 3 and 1) were compared with other serotype CVBs. An Unweighted Pair-Group Method Analysis (UPGMA) of phylogenetic trees indicated that the 5' NTR of CVB6 locates on an independent branch from the other CVBs. The tree based on the amino acid sequences showed that CVB6 has close correlation with CVB4 in the VP4 and VP2 regions, with CVB1 and CVB5 in the VP3 region, and with CVB5 in the VP1 region. Amino acid sequences of variable regions within the VP2, VP3, and VP1 of CVB6 were unique among CVBs. Thus, by comparison of the nucleotide and amino acid sequences of these variable regions, CVB6 can be easily distinguished from other serotypes. In addition, serine, instead of glycine, was found to locate at the amino-terminus of the VP1 region of CVB6, indicating that CVB6 has a unique cleavage site (i.e., glutamine/serine instead of glutamine/glycine) for proteinase 3C of Picornaviridae.     

Phylogenetic comparison of the S3 gene of United States prototype strains of bluetongue virus with that of field isolates from California.

To better define the molecular epidemiology of bluetongue virus (BTV) infection, the genetic characteristics and phylogenetic relationships of the S3 genes of the five U.S. prototype strains of BTV, the commercially available serotype 10 modified live virus vaccine, and 18 field isolates of BTV serotypes 10, 11, 13, and 17 obtained in California during 1980, 1981, 1989, and 1990 were determined. With the exception of the S3 gene of the U.S. prototype strain of BTV serotype 2 (BTV 2), these viruses had an overall sequence homology of between 95 and 100%. Phylogenetic analyses segregated the prototype U.S. BTV 2 strain to a unique branch (100% bootstrap value), whereas the rest of the viruses clustered in two main monophyletic groups that were not correlated with their serotype, year of isolation, or geographical origin. The lack of consistent association between S3 gene sequence and virus serotype likely is a consequence of reassortment of BTV gene segments during natural mixed infections of vertebrate and invertebrate hosts. The prototype strain of BTV 13, which is considered an introduction to the U.S. like BTV 2, presents an S3 gene which is highly homologous to those of some isolates of BTV 10 and especially to that of the vaccine strain. This finding strongly suggests that the U.S. prototype strain of BTV 13 is a natural reassortant. The different topologies of the phylogenetic trees of the L2 and S3 genes of the various viruses indicate that these two genome segments evolve independently. We conclude that the S3 gene segment of populations of BTV in California is formed by different consensus sequences which cocirculate and which cannot be grouped by serotype.     

Molecular Evolution of the Human Enteroviruses: Correlation of Serotype with VP1 Sequence and Application to Picornavirus Classification

Sixty-six human enterovirus serotypes have been identified by serum neutralization, but the molecular determinants of the serotypes are unknown. Since the picornavirus VP1 protein contains a number of neutralization domains, we hypothesized that the VP1 sequence should correspond with neutralization (serotype) and, hence, with phylogenetic lineage. To test this hypothesis and to analyze the phylogenetic relationships among the human enteroviruses, we determined the complete VP1 sequences of the prototype strains of 47 human enterovirus serotypes and 10 antigenic variants. Our sequences, together with those available from GenBank, comprise a database of complete VP1 sequences for all 66 human enterovirus serotypes plus additional strains of seven serotypes. Phylogenetic trees constructed from complete VP1 sequences produced the same four major clusters as published trees based on partial VP2 sequences; in contrast to the VP2 trees, however, in the VP1 trees strains of the same serotype were always monophyletic. In pairwise comparisons of complete VP1 sequences, enteroviruses of the same serotype were clearly distinguished from those of heterologous serotypes, and the limits of intraserotypic divergence appeared to be about 25% nucleotide sequence difference or 12% amino acid sequence difference. Pairwise comparisons suggested that coxsackie A11 and A15 viruses should be classified as strains of the same serotype, as should coxsackie A13 and A18 viruses. Pairwise identity scores also distinguished between enteroviruses of different clusters and enteroviruses from picornaviruses of different genera. The data suggest that VP1 sequence comparisons may be valuable in enterovirus typing and in picornavirus taxonomy by assisting in the genus assignment of unclassified picornaviruses.Human enteroviruses (family Picornaviridae) infect millions of people worldwide each year, resulting in a wide range of clinical outcomes ranging from inapparent infection to mild respiratory illness (common cold), hand-foot-and-mouth disease, acute hemorrhagic conjunctivitis, aseptic meningitis, myocarditis, severe neonatal sepsis-like disease, and acute flaccid paralysis (reviewed in references 43 and 45). In the United States, enteroviruses are responsible for 30,000 to 50,000 meningitis hospitalizations per year as a result of 30 million to 50 million infections. Serologic studies have distinguished 66 human enterovirus serotypes on the basis of an antibody neutralization test (43), and additional antigenic variants have been defined within several of the serotypes on the basis of reduced or nonreciprocal cross-neutralization between prototype and variant strains (6, 8, 68, 71, 72). On the basis of their pathogenesis in humans and experimental animals, the enteroviruses were originally classified into four groups, polioviruses, coxsackie A viruses (CA), coxsackie B viruses (CB), and echoviruses, but it was quickly realized that there were significant overlaps in the biological properties of viruses in the different groups (8). The more recently isolated enteroviruses have been named with a system of consecutive numbers: EV68, EV69, EV70, and EV71 (42).A comparison of nucleotide and deduced amino acid sequences at the 5′ end of VP2 has identified four major phylogenetic groups within the Enterovirus genus: CA16-like viruses (cluster A), a CB-like group containing all CB and echoviruses as well as CA9 and EV69 (cluster B), poliovirus-like viruses (cluster C), and EV68 and EV70 (cluster D) (23, 24, 49, 53, 54, 73). However, pairwise alignments and phylogenetic analyses within these groups demonstrated that the VP2 sequence does not fully correlate with serotype, as viruses known to belong to the same serotype often failed to cluster together (2, 49). (E22 and E23 are genetically distinct from enteroviruses [24], and their reclassification into a separate genus has been proposed [45]).VP1 is the most external and immunodominant of the picornavirus capsid proteins (58). A number of major neutralization sites reside in the VP1 proteins of many picornaviruses (reviewed in references 40 and 44), but the specific epitopes responsible for serotype specificity and intratypic variation have not been identified. Similarly, the genetic correlates of serotype identity remain unknown. If the important serotype-specific neutralization sites reside in VP1, then the VP1 sequence or some portion thereof would be predicted to correlate with serotype. Studies on the three serotypes of poliovirus have shown that a partial VP1 sequence correlates well with serotype (32). In addition, genetic lineages based on the VP1 sequence can be used to define poliovirus reservoirs and chains of transmission (reviewed in reference 30). To test whether the VP1 sequence might be applied to the classification of nonpolio enteroviruses and to the analysis of the phylogenetic relationships among the human enteroviruses, we determined the complete VP1 nucleotide sequences for 47 human enterovirus prototypes and 10 well-characterized antigenic variants. These data, together with previously available sequences, comprise a database of complete VP1 sequences for all known human enterovirus serotypes and 12 natural antigenic variants. This database will be useful for molecular epidemiologic studies of enteroviral disease outbreaks, to obtain a better understanding of the genetic correlates of serotype, and for the development of enteroviral molecular diagnostic reagents.     

Circulation of enteroviruses in Cyprus assessed by molecular analysis of clinical specimens and sewage isolates

Aims: To study the circulation of non‐polio enteroviruses in the Cypriot population and assess the clinical relevance of different serotypes by the analysis of clinical specimens and environmental samples. Methods and Results: Sewage samples were collected on a monthly basis for 2 years from all five districts of Cyprus. Enteroviruses were isolated using the VIRADEN method and typed by partial VP1 region sequencing. In addition, all enterovirus‐positive clinical samples received during this 2‐year period were typed, and a phylogenetic comparison of clinical and sewage samples based on the partial VP1 sequences was made. A significant difference between the most common serotypes found in sewage and clinical samples was observed. While Coxsackieviruses B constituted the most frequent serotypes in sewages, Echoviruses 30 and 18 prevailed in clinical samples. Conclusions: The phylogenetic analysis revealed that certain enterovirus strains circulate in the population over long period of time, while others are observed only sporadically and disappear quickly. For some serotypes, it was observed that several strains were cocirculating in the population but only some of them being detected also in clinical specimens. Significance and Impact of the Study: This study, for the first time, compares enteroviruses isolated from environmental samples and clinical specimens on a molecular level, which allowed for strain identification and discrimination. A more comprehensive molecular analysis of these strains will help identify factors, which determine different degrees of pathogenicity.     

Exchange of the VP5 of infectious bursal disease virus in a serotype I strain with that of a serotype II strain reduced the viral replication and cytotoxicity

Infectious bursal disease virus (IBDV), belonging to Avibirnavirus genus in the Birnaviridae family, consists of two segments of double-strand RNA. There are two distinct serotypes of IBDV, the pathogenic serotype I and the non-pathogenic serotype II. Comparison of the deduced amino acid sequences of a panel of VP5 genes retrieved from GenBank revealed a high identity among strains within the serotype I or serotype II group but a low identity between strains across two serotypes. In this study, we rescued two mosaic viruses, rGtGxVP5 and rGt2382VP5 by exchanging the VP5 gene of a cell culture-adapted serotype I Gt strain with its counterpart of the very virulent IBDV Gx strain, or a non-pathogenic 23/82 strain of the serotype II. In comparison to the parental strain rGt virus, the rGtGxVP5 showed the similar viral replication, cytotoxicity and the ability of inducing apoptosis; however, the other mosaic virus rGt2382VP5 had a lower titer and a reduced cytotoxicity. Although exchange of VP5 within serotype I group did not alter the viral replication and cytotoxicity of Gt strain, exchange of VP5 in the serotype I with that of a serotype II reduced the viral replication and cytotoxicity on chicken embryo fibroblast (CEF) cells. Therefore, the VP5 of serotype II may be one of the factors responsible for the distinct pathogenic features of two serotypes.     

Sequence analysis of VP7 gene of Indian bluetongue virus serotype-23 shows its close phylogenetic relationship to Australian and Chinese serotypes.

Bluetongue, an arthropod borne viral disease of wild and domestic ruminants, causes heavy economic losses throughout the world. In the present study, full-length VP7 gene of Indian bluetongue virus (BTV) serotype 23 was sequenced and compared with prototype strains of BTV reported from different countries. Nucleotide sequence analysis of VP7 gene revealed Indian BTV serotype 23 to have 1154 nucleotides with the deletion of two nucleotides at 3' non-coding region and a unique amino acid change 211S-N. The Indian virus also demonstrated a maximum similarity of 94.2% with Australian serotype 1 and a minimum similarity of 67.4% with Australian serotype 15. However, at deduced amino acid level, it had maximum similarity of 99.7% and a minimum of 82.5% with Chinese serotypes 1, 2 and 4 and Australian serotype 15, respectively. Deduced amino acid sequence analysis of putative receptor binding domain (121-249) revealed all the nine hydrophilic domains to be conserved across the serotypes. Functional motifs present in VP7 protein were also conserved in almost all the BTV serotypes including Indian serotype 23. Phylogenetic analysis based on VP7 gene sequence revealed Indian BTV serotype 23 segregating into a monophyletic group along with Australian serotype 1 and Chinese serotypes 1, 2 and 4, indicating its close evolutionary relationship with these Australian and Chinese serotypes.     

Complete nucleotide sequences of all three poliovirus serotype genomes. Implication for genetic relationship, gene function and antigenic determinants

The complete nucleotide sequences of the genomes of the type 2 ( P712 , Ch, 2ab ) and type 3 (Leon 12a1b ) poliovirus vaccine strains were determined. Comparison of the sequences with the previously established genome sequence of type 1 (LS-c, 2ab ) poliovirus vaccine strain revealed that 71% of the nucleotides in the genome RNAs were common, that the 5' and 3' termini of the genomes were highly homologous, and that more than 80% of the nucleotide differences in the coding region occurred in the third letter position of in-phase codons, resulting in a low frequency of amino acid difference. These results strongly suggested that the serotypes of poliovirus derived from a common prototype. A comparison of the amino acid sequences predicted from the genome sequences showed highest variation in the capsid protein region, whereas non-structural proteins are highly conserved. Initiation of polyprotein synthesis occurs in all three strains more than 740 nucleotides downstream from the 5' end. An analysis of the non-coding region suggests that small peptides that could potentially originate from this region are conserved. The amino acid sequences immediately surrounding the cleavage signals, however, show a higher than average degree of variation. The analysis of the amino acid sequences of the capsid protein VP1 of all serotypes has led to the prediction of potential antigenic sites on the virion involved in neutralization.     

Identification of enteroviruses by hemagglutination-inhibition

Kern, Jerome (Pacific Research Section, Honolulu, Hawaii), and Leon Rosen. Identification of enteroviruses by hemagglutination-inhibition. J. Bacteriol. 91:1936-1942. 1966.-Approximately 40% of a group of 906 enterovirus isolates cytopathic for monkey cell cultures were found to possess hemagglutinins for human erythrocytes when tested at temperatures of 4 and 37 C and at pH 5.8 and 7.3. The hemagglutinating isolates could be classified by relatively simple techniques into 18 serotypes. Four of these serotypes, echovirus type 24 and coxsackievirus B types 1, 5, and 6, had not previously been known to include hemagglutinating strains. One serotype, Toluca-3, represented a previously unrecognized enterovirus, and two other serotypes may also represent previously unrecognized enteroviruses.     

A Predicted Secondary Structural Domain within the Internal Ribosome Entry Site of Echovirus 12 Mediates a Cell-Type-Specific Block to Viral Replication

The enterovirus 5' nontranslated region (NTR) contains an internal ribosome entry site (IRES), which facilitates translation initiation of the viral open reading frame in a 5' (m(7)GpppN) cap-independent manner, and cis-acting signals for positive-strand RNA replication. For several enteroviruses, the 5' NTR has been shown to determine the virulence phenotype. We have constructed a chimera consisting of the putative IRES element from the Travis strain of echovirus 12 (ECV12), a wild-type, relatively nonvirulent human enterovirus, exchanged with the homologous region of a full-length infectious clone of coxsackievirus B3 (CBV3). The resulting chimera, known as ECV12(5'NTR)CBV3, replicates similarly to CBV3 in human and simian cell lines yet, unlike CBV3, is completely restricted for growth on two primary murine cell lines at 37 degrees C. By utilizing a reverse-genetics approach, the growth restriction phenotype was localized to the predicted stem-loop II within the IRES of ECV12. In addition, a revertant of ECV12(5'NTR)CBV3 was isolated which possessed three transition mutations and had restored capability for replication in the utilized murine cell lines. Assays for cardiovirulence indicated that the ECV12 IRES is responsible for a noncardiovirulent phenotype in a murine model for acute myocarditis. The results indicate that the 5' NTRs of ECV12 and CBV3 exhibit variable intracellular requirements for function and serve as secondary determinants of tissue or species tropism.     

A retrospective overview of enterovirus infection diagnosis and molecular epidemiology in the public hospitals of Marseille, France (1985-2005)

Human enteroviruses (HEV) are frequent human pathogens and, associated in particular with large outbreaks of aseptic meningitis. Here, we have compiled a database of clinical HEV isolates from the Public Hospitals of Marseille, from 1985 to 2005. Amongst 654 isolates that could be characterized by complete sequencing of the VP1 gene, 98% belonged to species HEV-B; the most frequently isolated serotypes were Echovirus E30, E11, E7, E6 and E4. The high incidence of E30 and the recent emergence of E13 are consistent with reports worldwide and peak HEV isolation occurred mostly in the late spring and summer months. The proportion of echoviruses has decreased across the years, while that of coxsackieviruses has increased. Stool (the most frequent sample type) allowed detection of all identified serotypes. MRC5 (Human lung fibroblasts) cell line was the most conducive cell line for HEV isolation (84.9% of 10 most common serotype isolates, 96.3% in association with BGM (Buffalo green monkey kidney cells)). Previous seroneutralization-based serotype identification demonstrated 55.4% accuracy when compared with molecular VP1 analysis. Our analysis of a large number of clinical strains over 20 years reinforced the validity of VP1 serotyping and showed that comparative p-distance scores can be coupled with phylogenetic analysis to provide non-ambiguous serotype identification. Phylogenetic analysis in the VP1, 2C and 3D regions also provided evidence for recombination events amongst clinical isolates. In particular, it identified isolates with dissimilar VP1 but almost identical nonstructural regions.     

Recombination in the Evolution of Enterovirus C Species Sub-Group that Contains Types CVA-21, CVA-24, EV-C95, EV-C96 and EV-C99

Genetic recombination is considered to be a very frequent phenomenon among enteroviruses (Family Picornaviridae, Genus Enterovirus). However, the recombination patterns may differ between enterovirus species and between types within species. Enterovirus C (EV-C) species contains 21 types. In the capsid coding P1 region, the types of EV-C species cluster further into three sub-groups (designated here as A–C). In this study, the recombination pattern of EV-C species sub-group B that contains types CVA-21, CVA-24, EV-C95, EV-C96 and EV-C99 was determined using partial 5′UTR and VP1 sequences of enterovirus strains isolated during poliovirus surveillance and previously published complete genome sequences. Several inter-typic recombination events were detected. Furthermore, the analyses suggested that inter-typic recombination events have occurred mainly within the distinct sub-groups of EV-C species. Only sporadic recombination events between EV-C species sub-group B and other EV-C sub-groups were detected. In addition, strict recombination barriers were inferred for CVA-21 genotype C and CVA-24 variant strains. These results suggest that the frequency of inter-typic recombinations, even within species, may depend on the phylogenetic position of the given viruses.