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.     

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.     

Prevalence of infection with nonpolio enteroviruses among healthy children from a children's home and in a summer camp

The sera obtained at 6 samplings between June 1981 and May 1982 from 30 children in a children's home were tested for neutralizing antibodies to coxsackieviruses B1 through B5, echovirus serotypes 1, 2, 4, 5, 6, 7, 11, 19, 24, 30 and to enterovirus type 71. Another group of examined children comprised 19 individuals from the children's home and 63 children from families who enrolled as participants of a summer camp recreation. Samples of their sera were obtained at the beginning and the end of camping in July and August of 1981. Virus isolations were attempted in 150 stool specimens collected at various times during the observation period, and in specimens of summer camp swimming pool water collected 2-3 times a week. Overall, 10 strains of enterovirus (CB2, E2, E19, E24) were isolated from stool, but none from swimming pool water. Serologically, fourfold or greater increases in titre of antibody to at least one of the enteroviruses tested were observed 17 children, of whom 4 children showed antibody titre increases simultaneously to two enterovirus serotypes. None of the present viruses showed a tendency to mass spread. Some of the examined children showed the presence of serum antibody to enterovirus before its isolation from stool. Eight of the ten children positive for enterovirus in stool did not react by significant increases in titre of antibodies. Enteroviruses were more frequently isolated in children's home in autumn than in summer camp. Clinically, all virologically or serologically demonstrated enteroviral infections were asymptomatic. Children's home children had a significantly higher prevalence of antibodies to enteroviruses than children from families. Presumably, asymptomatic infections with nonpolio enteroviruses among children appear to be far more frequent than the results of neutralization tests in the study indicated. Moreover, enteroviruses may apparently circulate among humans who are seropositive for the respective enterovirus serotype.     

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.     

Molecular epidemiology of enteroviruses with special reference to their potential role in the etiology of insulin-dependent diabetes mellitus (IDDM). A review

Background: Several lines of evidence suggest that enterovirus infections may be involved in the etiology of the insulin-dependent diabetes mellitus (IDDM). Often in the literature, a reference is given to specifically diabetogenic strains of enterovirus but there is no systematic assessment about the generation of such strains in the course of evolution or about their abundance among the 64 enterovirus serotypes pathogenic to man. If enteroviruses truly are involved in the etiology of IDDM, a possibility to prevent the disease with enterovirus vaccines might become feasible. In such a situation it would be important to know which serotypes and strains are the most important ones, and whether there would be differences between the strains as regards the pathogenetic mechanisms involved.Objective: To present a brief summary of the basic biology of enteroviruses, on existing data of genetic variation of enteroviruses, and on molecular epidemiology of human enteroviruses with special reference to the different epidemiological modes of their putative involvement in the pathogenesis of IDDM.Conclusions: Like RNA viruses in general, enteroviruses exist as a quasispecies, a mixture of genetic microvariants with a vast potential to adapt to new environments. This means that specifically beta cell-tropic and potentially diabetogenic variants could, in theory, emerge sporadically during systemic infection of any individual. The patterns of genetic diversification of enteroviruses, cocirculation of separate genetic lineages in the human populations, and the assumed geographical restrictions of endemic transmission of the lineages, allow one to hypothesize that populations with a high persisting IDDM incidence might be endemically infected by some specific strains of enteroviruses. However, so far, there is no systematically collected data supporting this hypothesis.     

Hemagglutinating Activity of Enteroviruses Recovered in Two Primary Cell Lines and a Stable Cell Line

A microhemagglutination technique was used to detect hemagglutinating properties of enteroviruses recovered in two primary cell lines, monkey kidney (MK) and human amnion (HAm), and in a continuous cell line, human embryonic lung (HEL). During a 3-year period, 1,528 isolations of enteroviruses were tested for hemagglutinating activity and hemagglutination inhibition response; 96.3% of the viruses were also identified by virus neutralization tests. Enteroviruses recovered in HEL were far less likely to develop hemagglutinins than viruses isolated in MK or HAm. Of the enteroviruses known to agglutinate human type O cells, 77.8% of the primary viral isolates from MK, 62.1% of the isolates from HAm, and 20.3% of the isolates from HEL exhibited this property. An additional 8.1% of the isolations obtained in HEL hemagglutinated human cells after a single passage in MK. The microhemagglutination technique using microtiter equipment was simple to perform, saved time and valuable typing sera, and helped to obtain identifications rapidly.     

Social and economic significance of enterovirus infection and its role in etiologic structure of infectious diseases in the world

Human enteroviruses comprised by more than 100 serotypes, they spread everywhere and can cause wide spectrum of diseases as well as significant social and economic loss. Influenza-like illness and mild forms of enterovirus infection (herpangina, exanthema) are widespread and causes of significant number of visits in clinics. Economic cost of mild form of enterovirus infection is not high although great number of cases (10 - 15 mln cases yearly in USA) determines its important economic significance. Single cases and outbreaks of enterovirus aseptic meningitis occur less frequently but lead to significant economic burden due to hospitalization costs. Enteroviruses are also cause up to 30% of sepsis-like disease in newborns and play important role in infant morbidity and mortality. Potential of enteroviruses as a source of new diseases in humans has a special significance for practical healthcare. In XX century enteroviruses became a cause of pandemics of paralytic poliomyelitis, hemorrhagic conjunctivitis, and foot-and-mouth-like disease, which caused vast social and economic loss, and emergence of new forms of enterovirus infection is quite possible in XXI century.     

Molecular phylogeny and proposed classification of the simian picornaviruses.

The simian picornaviruses were isolated from various primate tissues during the development of general tissue culture methods in the 1950s to 1970s or from specimens derived from primates used in biomedical research. Twenty simian picornavirus serotypes are recognized, and all are presently classified within the Enterovirus genus. To determine the phylogenetic relationships among all of the simian picornaviruses and to evaluate their classification, we have determined complete VP1 sequences for 19 of the 20 serotypes. Phylogenetic analysis showed that A13, SV19, SV26, SV35, SV43, and SV46 are members of human enterovirus species A, a group that contains enterovirus 71 and 11 of the coxsackie A viruses. SA5 is a member of human enterovirus species B, which contains the echoviruses, coxsackie B viruses, coxsackievirus A9, and enterovirus 69. SV6, N125, and N203 are related to one another and, more distantly, to species A human enteroviruses, but could not be definitely assigned to a species. SV4 and SV28 are closely related to one another and to A-2 plaque virus, but distinct from other enteroviruses, suggesting that these simian viruses are members of a new enterovirus species. SV2, SV16, SV18, SV42, SV44, SV45, and SV49 are related to one another but distinct from viruses in all other picornavirus genera, suggesting that they may comprise a previously unknown genus in Picornaviridae. Several simian virus VP1 sequences (N125 and N203; SV4 and SV28; SV19, SV26, and SV35; SV18 and SV44; SV16, SV42, and SV45) are greater than 75% identical to one another (and/or greater than 85% amino acid identity), suggesting that the true number of distinct serotypes among the viruses surveyed is less than 20.     

Enteroviruses: new findings on the role of enteroviruses in type 1 diabetes

Common enterovirus infections appear to initiate or facilitate the pathogenetic processes leading to type 1 diabetes, and sometimes also precipitate the clinical disease. It is not known in detail how enterovirus infections bring about the loss of insulin-producing beta-cells, a phenomenon characteristic of the disease. Recent results from studies on pancreases from human autopsies and cultured human islets support the idea that during systemic enterovirus infections, the virus may reach pancreatic islets and cause direct beta-cell damage. Although individual enteroviruses (EV) exhibited differences in their beta-cell tropism in the cultured human islets, all serotypes studied contained highly destructive strains. The final confirmation on the role of enteroviruses in type 1 diabetes can only be obtained from intervention studies. If the association holds true then it would be possible to reduce the risk of developing type 1 diabetes by preventing enterovirus infections with a multivalent enterovirus vaccine that could be given to children soon after birth.     

National Epidemiology and Evolutionary History of Four Hand,Foot and Mouth Disease-Related Enteroviruses in China from 2008 to 2016

Hand, foot and mouth disease(HFMD) is a major public health concern in China. The most predominant enteroviruses that cause HFMD have traditionally been attributed to enterovirus A71(EVA71) and coxsackievirus A16(CVA16). Since its first large outbreak in 2008, the dominant HFMD pathogens are constantly changing. In 2013 and 2015, CVA6 exceeded both EVA71 and CVA16 to become the leading cause of HFMD in some provinces. However, there still lacks a comprehensive overview on the molecular epidemiology and evolution of HFMD-related enteroviruses at the national level. In this study, we performed systematic epidemiological analyses of HFMD-related enteroviruses using the data of 64 published papers that met the inclusion criteria, and conducted phylogenetic analyses based on 12,080 partial VP1 sequences identified in China before 31 st June 2018. We found that EVA71 prevalence has decreased sharply but other enteroviruses have increased rapidly from 2008 to 2016 and that one subtype of each enterovirus is represented during the epidemic. In addition, four genotypes EVA71_C4, CVA16_B1, CVA6_D and CVA10_C are the most predominant enterovirus strains and collectively they cause over 90% of all HFMD cases in China according to the phylogenetic trees using representative partial VP1 sequences. These four major enterovirus genotypes have different geographical distributions, and they may cocirculate with other genotypes and serotypes. These results suggest that more molecular epidemiological studies should be performed on several enteroviruses simultaneously, and such information should have implications for virological surveillance, disease management, vaccine development and policy-making on the prevention and control of HFMD.     

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.     

Synthesis and antienteroviral activity of a series of novel, oxime ether-containing pyridyl imidazolidinones

A series of novel, oxime ether-containing pyridyl imidazolidinones were synthesized and their antiviral activity was evaluated in a plaque reduction assay. It is very interesting that this class of compounds is specific for human enteroviruses, in particular, enterovirus 71 (EV71). Some derivatives strongly inhibited enterovirus replication with activities higher or comparable to those of the reference compounds such as A1 and A2. Preliminary SAR studies revealed that the chain length of the alkyl linker and the alkyl substituent at the oxime ether group largely influenced the in vitro anti-EV71 activity of this new class of potent antiviral agents. Among this series of compounds synthesized, the pyridyl imidazolidinone with an ethyl oxime ether group located at the para position of the phenoxyl ring (8b) was identified as the most potent enterovirus 71 inhibitor (IC50=0.001 microM) with no apparent cytotoxic effect toward RD (rhabdomyosarcoma) cell lines (CC50>25 microM). Furthermore, this compound has been shown broad-spectrum activity against most of the serotypes of enteroviruses tested in the nanomolar range.     

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.     

Studies on Swine Enteroviruses

Of 38 new isolates of enterovirus recovered from fecal specimens of Japanese pigs, 14 isolates were found to be strains of a new serotype (represented by strain IPI) distinct from the five serotypes of swine enterovirus hitherto recognized in Japan. The remaining isolates included serotypes Teschen, T80, SF1 and V13. The two different types of cytopathic effect, which have been recently recognized in swine kidney cell cultures, were also observed with our isolates; type I is characterized by a rounding of cells followed by aggregation and destruction of affected cells, and type II by granulation and degradation of cells with poor clumping of affected cells. Of interest is the finding that the type I strains possess a higher heat stability than the type II strains when heated at 50 C for one hr.     

Detection of adenoviruses and enteroviruses in polluted waters by nested PCR amplification.

A procedure has been developed for the rapid detection of enteroviruses and adenoviruses in environmental samples. Several systems for virus concentration and extraction of nucleic acid were tested by adding adenovirus type 2 and poliovirus type 1 to different sewage samples. The most promising method for virus recovery involved the concentration of viruses by centrifugation and elution of the virus pellets by treatment with 0.25 N glycine buffer, pH 9.5. Nucleic acid extraction by adsorption of RNA and DNA to silica particles was the most efficient. One aliquot of the extracted nucleic acids was used for a nested two-step PCR, with specific primers for all adenoviruses; and another aliquot was used to synthesize cDNA for a nested two-step PCR with specific primers for further detection of seeded polioviruses or all enteroviruses in the river water and sewage samples. The specificity and sensitivity were evaluated, and 24 different enterovirus strains and the 47 human adenovirus serotypes were recognized by the primers used. The sensitivity was estimated to be between 1 and 10 virus particles for each of the species tested. Twenty-five samples of sewage and polluted river water were analyzed and showed a much higher number of positive isolates by nested PCR than by tissue culture analysis. The PCR-based detection of enteroviruses and adenoviruses shows good results as an indicator of possible viral contamination in environmental wastewater.     

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.     

Enteroviruses in sludge: multiyear experience with four wastewater treatment plants.

We describe our experience with the isolation of viruses from four treatment plants located in different geographic areas. Over a period of 3 years, 297 enteroviruses were isolated from 307 sludge samples. The highest frequency of viral isolation (92%), including multiple isolates from single samples, was obtained from a treatment plant serving the smallest population. Excluding the polioviruses, 22 different enterovirus serotypes were isolated. The methods used to isolate the viruses were relatively simple and included an elution procedure in which beef extract was used and a disinfection step. No concentration procedure was used. Of three cell culture systems used, the RD line of human rhabdomyosarcoma cells was by far the most useful for the isolation of echoviruses; BGM and HeLa cells were particularly useful for the isolation of group B coxsackieviruses. A seasonal effect on viral isolation rates from sludge was observed.     

Enteroviruses in sludge: multiyear experience with four wastewater treatment plants

We describe our experience with the isolation of viruses from four treatment plants located in different geographic areas. Over a period of 3 years, 297 enteroviruses were isolated from 307 sludge samples. The highest frequency of viral isolation (92%), including multiple isolates from single samples, was obtained from a treatment plant serving the smallest population. Excluding the polioviruses, 22 different enterovirus serotypes were isolated. The methods used to isolate the viruses were relatively simple and included an elution procedure in which beef extract was used and a disinfection step. No concentration procedure was used. Of three cell culture systems used, the RD line of human rhabdomyosarcoma cells was by far the most useful for the isolation of echoviruses; BGM and HeLa cells were particularly useful for the isolation of group B coxsackieviruses. A seasonal effect on viral isolation rates from sludge was observed.