2009年云南省2株肠道病毒71型分离株全基因组序列遗传特性分析

对2009年云南省肠道病毒71型分离株KMM09和KM186-09进行全基因组序列测序,并与我国及其它国家流行的EV71基因型进行比较和进化分析。KMM09和KM186-09基因组长为7 409bp,编码2 193个氨基酸,VP1系统进化分析显示2009年云南分离株属于C4基因型的C4a亚型。在结构区,与其它基因型相比较,C基因型之间的核苷酸和氨基酸的同源性高于其它基因型;而在非结构区,C4与B基因型和CA16原型株G10同源性高于其它C基因亚型。通过RDP3重组软件和blast比对分析,发现EV71C4基因型与B3基因型,与CA16原型株G10的基因组在非结构区存在重组。EV71全基因组序列的比较和分析,对了解引起我国手足口病暴发或流行C4基因亚型EV71毒株的遗传特性具有重要意义。     

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.     

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.     

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.     

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.     

Microsatellites: genomic distribution,putative functions and mutational mechanisms: a review

Microsatellites, or tandem simple sequence repeats (SSR), are abundant across genomes and show high levels of polymorphism. SSR genetic and evolutionary mechanisms remain controversial. Here we attempt to summarize the available data related to SSR distribution in coding and noncoding regions of genomes and SSR functional importance. Numerous lines of evidence demonstrate that SSR genomic distribution is nonrandom. Random expansions or contractions appear to be selected against for at least part of SSR loci, presumably because of their effect on chromatin organization, regulation of gene activity, recombination, DNA replication, cell cycle, mismatch repair system, etc. This review also discusses the role of two putative mutational mechanisms, replication slippage and recombination, and their interaction in SSR variation.     

Recombination in circulating enteroviruses

Recombination is a well-known phenomenon for enteroviruses. However, the actual extent of recombination in circulating nonpoliovirus enteroviruses is not known. We have analyzed the phylogenetic relationships in four genome regions, VP1, 2A, 3D, and the 5' nontranslated region (NTR), of 40 enterovirus B strains (coxsackie B viruses and echoviruses) representing 11 serotypes and isolated in 1981 to 2002 in the former Soviet Union states. In the VP1 region, strains of the same serotype expectedly grouped with their prototype strain. However, as early as the 2A region, phylogenetic grouping differed significantly from that in the VP1 region and indicated recombination within the 2A region. Moreover, in the 5' NTR and 3D region, only 1 strain of 40 grouped with its prototype strain. Instead, we observed a major group in both the 5' NTR and the 3D region that united most (in the 5' NTR) or all (in the 3D region) of the strains studied, regardless of the serotype. Subdivision within that major group in the 3D region correlated with the time of virus isolation but not with the serotype. Therefore, we conclude that a majority, if not all, circulating enterovirus B strains are recombinants relative to the prototype strains, isolated mostly in the 1950s. Moreover, the ubiquitous recombination has allowed different regions of the enterovirus genome to evolve independently. Thus, a novel model of enterovirus genetics is proposed: the enterovirus genome is a stable symbiosis of genes, and enterovirus species consist of a finite set of capsid genes responsible for different serotypes and a continuum of nonstructural protein genes that seem to evolve in a relatively independent manner.     

Co-circulation and evolution of polioviruses and species C enteroviruses in a district of Madagascar

Between October 2001 and April 2002, five cases of acute flaccid paralysis (AFP) associated with type 2 vaccine-derived polioviruses (VDPVs) were reported in the southern province of the Republic of Madagascar. To determine viral factors that favor the emergence of these pathogenic VDPVs, we analyzed in detail their genomic and phenotypic characteristics and compared them with co-circulating enteroviruses. These VDPVs appeared to belong to two independent recombinant lineages with sequences from the type 2 strain of the oral poliovaccine (OPV) in the 5'-half of the genome and sequences derived from unidentified species C enteroviruses (HEV-C) in the 3'-half. VDPV strains showed characteristics similar to those of wild neurovirulent viruses including neurovirulence in poliovirus-receptor transgenic mice. We looked for other VDPVs and for circulating enteroviruses in 316 stools collected from healthy children living in the small area where most of the AFP cases occurred. We found vaccine PVs, two VDPVs similar to those found in AFP cases, some echoviruses, and above all, many serotypes of coxsackie A viruses belonging to HEV-C, with substantial genetic diversity. Several coxsackie viruses A17 and A13 carried nucleotide sequences closely related to the 2C and the 3D(pol) coding regions of the VDPVs, respectively. There was also evidence of multiple genetic recombination events among the HEV-C resulting in numerous recombinant genotypes. This indicates that co-circulation of HEV-C and OPV strains is associated with evolution by recombination, resulting in unexpectedly extensive viral diversity in small human populations in some tropical regions. This probably contributed to the emergence of recombinant VDPVs. These findings give further insight into viral ecosystems and the evolutionary processes that shape viral biodiversity.     

2008～2010年河南省人肠道病毒71型基因特征和重组分析

对河南省2008～2010年河南省人肠道病毒71型进行基因特征及重组特点研究。对河南省2008～2010年分离的5株肠道病毒EV71型构建VP1序列系统进化树并分析其全基因组序列的重组特点。VP1序列系统进化分析显示2008～2010年河南株均属于C4基因型的C4a亚群,Bootscan分析和5’NCR、P1、P2、P3区的进化树证实C4基因型在2A～2B处存在EV71的B基因型和C基因型的型内重组及在3B～3C处存在EV71的B基因型和CA16/G-10间的型间重组。2008～2010年河南EV71分离株为C4基因型的C4a亚群,与2004年以来的中国大陆优势株流行趋势完全一致,EV71C4基因型存在基因型内和型间双重组现象。     

Identification and High-Density Mapping of Gene-Rich Regions in Chromosome Group 5 of Wheat

The distribution of genes and recombination in the wheat genome was studied by comparing physical maps with the genetic linkage maps. The physical maps were generated by mapping 80 DNA and two phenotypic markers on an array of 65 deletion lines for homoeologous group 5 chromosomes. The genetic maps were constructed for chromosome 5B in wheat and 5D in Triticum tauschii. No marker mapped in the proximal 20% chromosome region surrounding the centromere. More than 60% of the long arm markers were present in three major clusters that physically encompassed <18% of the arm. Because 48% of the markers were cDNA clones and the distributions of the cDNA and genomic clones were similar, the marker distribution may represent the distribution of genes. The gene clusters were identified and allocated to very small chromosome regions because of a higher number of deletions in their surrounding regions. The recombination was suppressed in the centromeric regions and mainly occurred in the gene-rich regions. The bp/cM estimates varied from 118 kb for gene-rich regions to 22 Mb for gene-poor regions. The wheat genes present in these clusters are, therefore, amenable to molecular manipulations parallel to the plants with smaller genomes like rice.     

Haplotype block structure is conserved across mammals

Genetic variation in genomes is organized in haplotype blocks, and species-specific block structure is defined by differential contribution of population history effects in combination with mutation and recombination events. Haplotype maps characterize the common patterns of linkage disequilibrium in populations and have important applications in the design and interpretation of genetic experiments. Although evolutionary processes are known to drive the selection of individual polymorphisms, their effect on haplotype block structure dynamics has not been shown. Here, we present a high-resolution haplotype map for a 5-megabase genomic region in the rat and compare it with the orthologous human and mouse segments. Although the size and fine structure of haplotype blocks are species dependent, there is a significant interspecies overlap in structure and a tendency for blocks to encompass complete genes. Extending these findings to the complete human genome using haplotype map phase I data reveals that linkage disequilibrium values are significantly higher for equally spaced positions in genic regions, including promoters, as compared to intergenic regions, indicating that a selective mechanism exists to maintain combinations of alleles within potentially interacting coding and regulatory regions. Although this characteristic may complicate the identification of causal polymorphisms underlying phenotypic traits, conservation of haplotype structure may be employed for the identification and characterization of functionally important genomic regions.     

Transcription-related mutations and GC content drive variation in nucleotide substitution rates across the genomes of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> and <Emphasis Type="Italic">Arabidopsis lyrata</Emphasis>

Background  

There has been remarkably little study of nucleotide substitution rate variation among plant nuclear genes, in part because orthology is difficult to establish. Orthology is even more problematic for intergenic regions of plant nuclear genomes, because plant genomes generally harbor a wealth of repetitive DNA. In theory orthologous intergenic data is valuable for studying rate variation because nucleotide substitutions in these regions should be under little selective constraint compared to coding regions. As a result, evolutionary rates in intergenic regions may more accurately reflect genomic features, like recombination and GC content, that contribute to nucleotide substitution.     

Human Enterovirus 109: a Novel Interspecies Recombinant Enterovirus Isolated from a Case of Acute Pediatric Respiratory Illness in Nicaragua

Enteroviruses (Picornaviridae family) are a common cause of human illness worldwide and are associated with diverse clinical syndromes, including asymptomatic infection, respiratory illness, gastroenteritis, and meningitis. In this study, we report the identification and complete genome sequence of a novel enterovirus isolated from a case of acute respiratory illness in a Nicaraguan child. Unbiased deep sequencing of nucleic acids from a nose and throat swab sample enabled rapid recovery of the full-genome sequence. Phylogenetic analysis revealed that human enterovirus 109 (EV109) is most closely related to serotypes of human enterovirus species C (HEV-C) in all genomic regions except the 5′ untranslated region (5′ UTR). Bootstrap analysis indicates that the 5′ UTR of EV109 is likely the product of an interspecies recombination event between ancestral members of the HEV-A and HEV-C groups. Overall, the EV109 coding region shares 67 to 72% nucleotide sequence identity with its nearest relatives. EV109 isolates were detected in 5/310 (1.6%) of nose and throat swab samples collected from children in a pediatric cohort study of influenza-like illness in Managua, Nicaragua, between June 2007 and June 2008. Further experimentation is required to more fully characterize the pathogenic role, disease associations, and global distribution of EV109.The genus Enterovirus (EV) in the family Picornaviridae is a group of related viruses that are associated with a spectrum of disease, ranging from subclinical infections to acute respiratory and gastrointestinal illness to more severe manifestations, such as aseptic meningitis, encephalitis, and acute flaccid paralysis (16, 32). Enteroviruses are small, nonenveloped viruses that share a genomic organization. The RNA genome is a ∼7.5 kb single-stranded, positive-sense, polyadenylated molecule, with a single, long open reading frame flanked by 5′ and 3′ untranslated regions (UTRs). The 5′ UTR is ∼700 nucleotides in length and contains highly structured secondary elements with internal ribosomal entry site (IRES) function. The ∼2,200-amino-acid (aa) polyprotein is cotranslationally processed by viral proteases to yield structural (VP4, VP2, VP3, and VP1) and nonstructural (2A, 2B, 2C, 3A, 3B, 3C, and 3D) proteins (32). Current enterovirus classification is based on the high sequence divergence within the VP1 capsid region, which has been shown to correspond with serotype neutralization (27, 28). Human enterovirus (HEV) types are currently classified into four species, human enterovirus A (HEV-A), HEV-B, HEV-C (including poliovirus), and HEV-D, based on the four phylogenetic clusters observed in comparisons of the coding region sequences. An enterovirus is considered a new type within a species if it possesses <75% nucleotide identity and <85% amino acid identity with known members across the VP1 sequence (27, 30). Molecular identification methods play a crucial role in rapid, sensitive enterovirus diagnostics and have led to the recent discovery of several novel enteroviruses (29, 31, 40, 42, 44). Most approaches target a limited number of conserved regions in the 5′ UTR and VP4-VP2 junction or seek to ascertain serotype information by probing antigenic regions, such as VP1 (5).Picornavirus RNA-dependent RNA polymerases are highly error prone and lack proofreading ability, resulting in a misincorporation frequency of 1 per 10³ to 10⁴ nucleotides (48). The relative infidelity of these polymerases is believed to enable rapid adaptability under selective pressure. Large-impact evolutionary events, such as recombination within and between enterovirus serotypes, also contribute to their evolution and genetic diversity (3, 8, 26, 39) and may lead to changes in disease associations with human enterovirus infections. Human enteroviruses are classified into four species based on coding region sequence phylogeny, and intraspecies recombination events between enteroviruses that are closely related in the coding region are well documented (26, 38, 39). All known enterovirus 5′ UTR sequences, however, cluster into two groups containing either HEV-A and -B sequences or HEV-C and -D sequences. Recent findings have described enterovirus genomes with a coding region that clusters with one species and a 5′ UTR that clusters with a different species, suggesting possible interspecies recombination events (41, 44). Understanding the recombination-driven evolution of HEV-C viruses is of particular public health concern due to the viruses'' ability to recombine with vaccine poliovirus, resulting in circulating, highly neurovirulent vaccine-derived polioviruses (17, 21, 34). It is unclear whether recombination events between poliovirus and HEV-C viruses allow for the rapid acquisition of traits that increase pathogenic and circulation potential.The enterovirus pathogenicity spectrum is related to tissue tropism and is largely determined by cellular receptor usage. Most picornaviruses use receptors from the immunoglobulin superfamily of proteins, such as intracellular adhesion molecule-1 (ICAM-1) or coxsackievirus-adenovirus receptor (CAR) (36). A distinct subgroup of HEV-C viruses, which includes coxsackievirus (CAV) A1, A19, and A22 and enterovirus 104, has not yet been grown successfully in cell culture, and the receptor molecule for this subgroup is unknown (6). HEV-C viruses are believed to be the ancestral source of poliovirus, which resulted from a capsid mutation that caused a cellular receptor switch from ICAM-1 to CD155 (poliovirus receptor [PVR]) (17).In this study, we report the discovery and characterization of a novel human enterovirus type within species HEV-C, for which we propose the designation human enterovirus 109 (EV109). Sequence analysis reveals considerable nucleotide divergence in the 5′ UTR between EV109 and other HEV-C types, and scanning bootstrap analysis supports the hypothesis that EV109 is the product of an interspecies recombination event with an ancestral member of the HEV-A group. Viral capsid amino acid alignments and homology modeling reveal the predicted three-dimensional arrangement of divergent and conserved residues of EV109 compared with other related enteroviruses. We also report highly similar EV109 isolates within multiple cases of acute pediatric respiratory illness in Managua, Nicaragua.     

Dynamics of mutation and recombination in a replicating population of complementing, defective viral genomes

In a previous study, we documented that serial passage of a biological clone of foot-and-mouth disease virus (FMDV) at high multiplicity of infection (moi) in cell culture resulted in viral populations dominated by defective genomes that included internal in-frame deletions, affecting the L and capsid-coding regions, and were infectious by complementation. In the present study, analyses of the defective genomes present in individual viral plaques, and of consensus nucleotide sequences determined for the entire genomes of sequential samples, have revealed a continuous dynamics of mutation and recombination. At some points of high genetic instability, multiple minority genomes with different internal deletions co-existed in the population. At later passages, a new defective RNA arose and displaced a related, previously dominant RNA. Nucleotide sequences of the different genomic forms found in sequential isolates have revealed an accumulation of mutations at an average rate of 0.12 substitutions per genome per passage. At the regions around the deletion sites, substantial, minor or no nucleotide sequence identity is found, suggesting relaxed sequence requirements for the occurrence of internal deletions. Competition experiments indicate a selective advantage of late phase defective genomes over their precursor forms. The defective genome-based FMDV retained an expansion of host cell tropism, undergone by the standard virus at a previous stage of the same evolutionary lineage. Thus, despite a complex dynamics of mutation and recombination, and phases of high genetic instability, a biologically relevant phenotypic trait was stably maintained after the evolutionary transition towards a primitive genome segmentation. The results extend the concept of a complex spectrum of mutant genomes to a complex spectrum of defective genomes in some evolutionary transitions of RNA viruses.     

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.     

Recombination in Enteroviruses Is a Biphasic Replicative Process Involving the Generation of Greater-than Genome Length ‘Imprecise’ Intermediates

Recombination in enteroviruses provides an evolutionary mechanism for acquiring extensive regions of novel sequence, is suggested to have a role in genotype diversity and is known to have been key to the emergence of novel neuropathogenic variants of poliovirus. Despite the importance of this evolutionary mechanism, the recombination process remains relatively poorly understood. We investigated heterologous recombination using a novel reverse genetic approach that resulted in the isolation of intermediate chimeric intertypic polioviruses bearing genomes with extensive duplicated sequences at the recombination junction. Serial passage of viruses exhibiting such imprecise junctions yielded progeny with increased fitness which had lost the duplicated sequences. Mutations or inhibitors that changed polymerase fidelity or the coalescence of replication complexes markedly altered the yield of recombinants (but did not influence non-replicative recombination) indicating both that the process is replicative and that it may be possible to enhance or reduce recombination-mediated viral evolution if required. We propose that extant recombinants result from a biphasic process in which an initial recombination event is followed by a process of resolution, deleting extraneous sequences and optimizing viral fitness. This process has implications for our wider understanding of ‘evolution by duplication’ in the positive-strand RNA viruses.     

The effect of SNP discovery method and sample size on estimation of population genetic data for Chinese and Indian rhesus macaques (<Emphasis Type="Italic">Macaca mulatta</Emphasis>)

This study was designed to address issues regarding sample size and marker location that have arisen from the discovery of SNPs in the genomes of poorly characterized primate species and the application of these markers to the study of primate population genetics. We predict the effect of discovery sample size on the probability of discovering both rare and common SNPs and then compare this prediction with the proportion of common and rare SNPs discovered when different numbers of individuals are sequenced. Second, we examine the effect of genomic region on estimates of common population genetic data, comparing markers from both coding and non-coding regions of the rhesus macaque genome and the population genetic data calculated from these markers, to measure the degree and direction of bias introduced by SNPs located in coding versus non-coding regions of the genome. We found that both discovery sample size and genomic region surveyed affect SNP marker attributes and population genetic estimates, even when these are calculated from an expanded data set containing more individuals than the original discovery data set. Although none of the SNP detection methods or genomic regions tested in this study was completely uninformative, these results show that each has a different kind of genetic variation that is suitable for different purposes, and each introduces specific types of bias. Given that each SNP marker has an individual evolutionary history, we calculated that the most complete and unbiased representation of the genetic diversity present in the individual can be obtained by incorporating at least 10 individuals into the discovery sample set, to ensure the discovery of both common and rare polymorphisms.     

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.     

A Sabin 3-derived poliovirus recombinant contained a sequence homologous with indigenous human enterovirus species C in the viral polymerase coding region

Outbreaks of poliomyelitis caused by circulating vaccine-derived polioviruses (cVDPVs) have been reported in areas where indigenous wild polioviruses (PVs) were eliminated by vaccination. Most of these cVDPVs contained unidentified sequences in the nonstructural protein coding region which were considered to be derived from human enterovirus species C (HEV-C) by recombination. In this study, we report isolation of a Sabin 3-derived PV recombinant (Cambodia-02) from an acute flaccid paralysis (AFP) case in Cambodia in 2002. We attempted to identify the putative recombination counterpart of Cambodia-02 by sequence analysis of nonpolio enterovirus isolates from AFP cases in Cambodia from 1999 to 2003. Based on the previously estimated evolution rates of PVs, the recombination event resulting in Cambodia-02 was estimated to have occurred within 6 months after the administration of oral PV vaccine (99.3% nucleotide identity in VP1 region). The 2BC and the 3D(pol) coding regions of Cambodia-02 were grouped into the genetic cluster of indigenous coxsackie A virus type 17 (CAV17) (the highest [87.1%] nucleotide identity) and the cluster of indigenous CAV13-CAV18 (the highest [94.9%] nucleotide identity) by the phylogenic analysis of the HEV-C isolates in 2002, respectively. CAV13-CAV18 and CAV17 were the dominant HEV-C serotypes in 2002 but not in 2001 and in 2003. We found a putative recombination between CAV13-CAV18 and CAV17 in the 3CD(pro) coding region of a CAV17 isolate. These results suggested that a part of the 3D(pol) coding region of PV3(Cambodia-02) was derived from a HEV-C strain genetically related to indigenous CAV13-CAV18 strains in 2002 in Cambodia.     

Genome Characterisation of Enteroviruses 117 and 118: A New Group within Human Enterovirus Species C

The more than 120 genotypes of human enteroviruses (HEVs) reflect a wide range of evolutionary divergence, and there are 23 currently classified as human enterovirus C species (HEV-C). Two new HEV-C (EV-C117 and EV-C118) were identified in the Community-Acquired Pneumonia Pediatric Research Initiative (CAP-PRI) study, and the present paper describes the characterisation of the complete genome of one EV-C117 strain (LIT22) and two EV-C118 (ISR38 and ISR10) strains. The EV-C117 and EV-C118 5′UTR sequences were related to those of EV-C104, EV-C105 and EV-C109, and were slightly shorter than those of other HEV A-D species. Similarity plot analyses showed that EV-C117 and EV-C118 have a P1 region that is highly divergent from that of the other HEV-C, and phylogenetic analyses highly supported a monophyletic group consisting of EV-C117, EV-C118, EV-C104, EV-C105 and EV-C109 strains. Phylogenetic, Simplot and Bootscan analyses indicated that recombination was not the main mechanism of EV-C117 and EV-C118 evolution, thus strengthening the hypothesis of the monophyletic origin of the coding regions, as in the case of other HEV-C. Phylogenetic analysis also revealed the emergence of a new group within HEV-C that is divided into two subgroups. Nucleotide and amino acid identity in VP1 sequences have been established as useful criteria for assigning new HEV types, but analysis of the complete P1 region improves resolution.