Sequence comparison of echovirus type 30 isolates to other enteroviruses in the 5′noncoding region

The genetic relationship between echovirus type 30 (E30) isolates were characterised by means of amplification of a part of the 5′noncoding region by polymerase chain reaction and direct sequencing. Later, the E30 sequences were compared with other enteroviruses. Homology between recent clinical E30 isolates from different years exceeds 98% at the nucleotide level. Comparing the E30 sequence with other enteroviruses, homology varied between 68% (coxsackievirus A24) and 93% (coxsackievirus B3). E30 appeared to have coxsackie B-like characteristics in this genomic part. It is considered that E30 and coxsackie B viruses belong to the same enterovirus subgroup. © 1995 Wiley-Liss, Inc.     

Molecular characterization of human enteroviruses in clinical samples: comparison between VP2, VP1, and RNA polymerase regions using RT nested PCR assays and direct sequencing of products

Three nested RT-PCR assays were developed to permit sensitive typing of enteroviruses directly from clinical samples. These assays amplified short fragments from different genomic regions codifying for three proteins: VP2, VP1, and RNA polymerase. Given that enteroviruses have a high rate of degeneration within target codons among serotypes, the primers used consisted of mixed base and deoxyinosine residues. These techniques detected at 0.03-0.003 TCID50 of prototype Poliovirus 1 and Echovirus 30. They were used to characterize the enteroviral RNA detected in 18 CSF, stool, and throw swab samples and in 8 enterovirus isolates from patients with several syndromes. Phylogenetic analysis in each independent sequenced region grouped the enterovirus into four clusters, enabling genetic classification. A comparative study was performed among the 26 sequences obtained after direct sequencing of products with those available in the nucleotide databases. The efficiency of each assay for enterovirus identification was evaluated by both distance (Clustal) and similarity (M-NW) indices. Comparative results obtained independently in the three regions showed the highest yield of correlation between nucleotide sequences of all prototype serotypes and the analyzed genotypes in the VP1 region (26/26, 100% Clustal; 22/26, 85% M-NW). Conversely, the VP2 region failed to identify some of the circulating enteroviruses (17/26, 65% Clustal; 16/26, 62% M-NW). Using the RNA polymerase region, sequences from samples and isolates were associated with prototype strains whenever these were available (20/21, 95% Clustal; 12/21, 57% M-NW). These assays were useful for molecular identification of enterovirus directly from samples even when isolation was not possible.     

Human enterovirus isolates from an outbreak typed using heteroduplex mobility analysis

Genotyping and serotyping of enteroviruses is important for epidemiological, prognostic, and therapeutic reasons. In this study clinical isolates of enterovirus 71 during an outbreak of childhood meningoencephalitis in Sydney, Australia were identified using heteroduplex mobility analysis (HMA) of products from RT-PCR amplification of the 5' untranslated region. Five enterovirus 71 isolates shared identical heteroduplex patterns and nucleotide sequences in the 5' untranslated region. A sixth isolate exhibited minor differences in heteroduplex pattern and sequencing confirmed the isolate varied by 1% at the nucleotide level. The use of multiple reference strains and the analysis of heteroduplex patterns increased the confidence of isolate identification, and allowed identification of strain variation which could be subsequently further analyzed using sequencing. HMA can be used to accurately distinguish identical and variant isolates derived from sporadic cases and clustered infections with enteroviruses, including those causing serious infections.     

Molecular typing and epidemiology of enteroviruses identified from an outbreak of aseptic meningitis in Belgium during the summer of 2000

Non-polio enteroviruses are the most common cause of aseptic meningitis worldwide. From May to September 2000, a major outbreak of aseptic meningitis occurred in Belgium. Cerebrospinal fluid samples (CSF) of 122 patients were found to contain enterovirus RNA using diagnostic RT-PCR that targeted a 231-bp gene fragment in the 5' noncoding region. In addition, a molecular typing method was developed based on RT-nested PCR and sequencing directly from CSF(a) 358-bp fragment in the aminoterminal part of the VP1 capsid protein. To identify the enterovirus type, nucleotide sequences of the VP1 amplicons were compared to all the enterovirus VP1 sequences available in GenBank. Echovirus 30 (31.2%), echovirus 13 (23.8%), and echovirus 6 (20.5%) were identified most frequently during the epidemic. Coxsackievirus B5 was present in 15.6% of the samples, and could be subdivided in two distinct epidemic clusters, coxsackievirus B5a (10.7%) and B5b (4.9%). Other enteroviruses encountered were echovirus 16 (5.7%), echovirus 18 (1.6%), coxsackievirus B4 (0.8%) and echovirus 7 (0.8%). The high prevalence of echovirus 13, considered previously a rare serotype, indicates it is an emerging epidemic type. To verify the typing results and to explore further the intratypical genetic variation, phylogenetic analysis was carried out. Geographical clustering of most of the strains within each type and subtype could be observed. The RT-nested PCR strategy, carried out directly on clinical samples, is a simple and rapid method for adequate molecular typing of the Group B enteroviruses causing aseptic meningitis.     

2011-2013年安徽省手足口病病原构成和HEV71基因特征分析

目的 了解安徽地区2011-2013年引起手足口病(hand-foot-mouth disease,HFMD)发病的病原构成情况和人肠道病毒71型(human enterovirus 71,HEV71)分离株的基因特征.方法 利用“中国疾病预防控制信息系统”,对2011-2013年报告的安徽省HFMD病例病原学特征进行统计分析.对从HFMD病例咽拭子标本中分离的26株HEV71进行VP1基因全长扩增和序列测定,使用生物软件DNASTAR 7.1比对分析同源性和应用Mega 4.1构建系统发生树.结果 安徽省2011-2013年HFMD病原构成主要为HEV71,柯萨奇病毒A组16型(coxsackie virus A16,CVA16)和其他肠道病毒,阳性检出率分别为28.74％、10.77％和15.18％.HEV71 VP1区基因序列同源性比对分析和系统发生树构建表明,26株HEV71属于C4a基因亚型,与2008年安徽阜阳流行株(EU703812)相比亲缘关系较近,VP1区核苷酸和氨基酸同源性分别为92.9％～98.8％和90.3％～99.7％.结论 安徽省2011-2013年HFMD的主要病原体为HEV71,其次为其他肠道病毒和CVA16.26株HEV71的基因型属C4a亚型,与中国大陆同期主要流行毒株基因型相一致.     

Isolation and identification of an enterovirus 77 recovered from a refugee child from Kosovo, and characterization of the complete virus genome

The complete nucleotide sequence of an enterovirus 77 isolate is reported. The virus designated FR/CF496-99 (France/Clermont-Ferrand 496-1999) was recovered from the feces of a 4-year-old child hospitalized for Salmonella gastroenteritis. The virus was identified by a molecular typing assay based on the genomic sequence encoding the VP1 capsid protein. The phylogenetic analysis based on the VP1 sequence demonstrated that the enterovirus isolated in the child clustered with viruses included in the human enterovirus B species (HEV-B) and was most closely related to enterovirus 77. A sliding window analysis of the complete genome showed an overall nucleotide similarity >80% between the P3 genomic region of the FR/CF496-99 isolate and that of the echovirus 30 prototype strain. A comparative analysis based on partial 3D(pol) sequences showed that the FR/CF496-99 virus was more closely related to recent enteroviruses from different serotypes and different geographical areas than to the prototype strains collected in the 1950s. This suggests that, in this enterovirus, the 3D(pol) encoding sequence is of recent origin.     

Detection of human enteroviruses and parechoviruses as part of the national enterovirus surveillance in the Netherlands, 1996–2011

Laboratories of the Dutch Working Group on Clinical Virology have routinely performed enterovirus diagnostics in the Netherlands since the early 1960s, with country-wide coverage. Enterovirus-positive samples are typed for clinical and epidemiological purposes, as well as to document the absence of poliovirus circulation. Human parechoviruses 1 and 2, initially recognized as enteroviruses, and since 2006 also the higher numbered human parechovirus types, have been detected as part of this surveillance. The purpose of this report is to describe the national enterovirus surveillance data from stool specimens collected in the Netherlands between 1996 and 2011 by all the participating laboratories. Since 2007, the average annual percentage of human enterovirus- and parechovirus-positive specimens increased from 6.5 to 10.8 % and from 0.3 to 2.5 % of the total numbers of specimens tested, respectively, following a gradual implementation of molecular diagnostics directly on clinical samples. Increased detection rates were observed for human enterovirus species A coxsackieviruses (from 0.1 to 0.5 %). Human enteroviruses of species B, C, and D were detected at average rates of 4.7, 0.04, and 0.005 %, respectively. The introduction of molecular diagnostics also resulted in an increase in the number of untyped enterovirus-positive specimens for which the presence of poliovirus was not excluded (from 1.3 to 3.1 % since 2007). To increase knowledge on human entero- and parechovirus epidemiology and type-specific pathogenesis, as well as to warrant the quality of the poliovirus surveillance in the Netherlands, it is of importance to continue the typing of enterovirus- and parechovirus-positive samples.     

Molecular identification of enteroviruses associated with aseptic meningitis in children from India

We identified and characterized enteroviruses associated with aseptic meningitis in children between April 2009 and March 2010. Enterovirus RNA was detected in 51 (45.5 %) of 112 CSF samples. Molecular typing by RT-PCR and sequencing of a partial VP1 region revealed the predominance of echovirus (ECV) 32 (n = 20), followed by ECV 11 (n = 10), ECV 13 and ECV 14 (n = 5 each), coxsackievirus (CV) B3 and CV B6 (n = 3 each), CV A2, CV A10 and ECV 30 (n = 1 each). Phylogenetic analysis of ECV 32 showed 0 to 4 % sequence divergence among strains of the present study and 20-23 % from the prototype Puerto Rico strain at the nucleotide level. This is the first report of ECV 32 associated with an aseptic meningitis epidemic and identification of seven different enterovirus serotypes (CV A2, CV A10, CV B3, CV B6, ECV 13, ECV 14 and ECV 32) in meningitis cases from India.     

Molecular Identification of Enterovirus by Analyzing a Partial VP1 Genomic Region with Different Methods

VP1 is the most suitable region for use in the identification of enterovirus. Although VP1 sequencing methods may vary, it is necessary to agree on a common strategy of sequence analysis. Identification of a strain type may be achieved by three different approaches: pairwise sequence alignment, multiple-sequence alignment, and phylogenetic inference. Other methods are also available, but they are not simple enough to be performed at a virology laboratory. The performances of these methods were evaluated with nucleotide and protein sequences obtained from 32 original samples, 8 enterovirus isolates, and 64 GenBank sequences. Pairwise sequence alignment methods had very different results. The DNASTAR package identified only 28.8% of enterovirus strains, while the Genetics Computer Group package identified 50.0 or 72.1% of enterovirus strains when nucleotide or amino acid sequences were analyzed, respectively. Multiple-sequence alignment methods identified 94.2% (Clustal W program) or 92.3% (Pileup program) of the enterovirus strains, while the phylogenetic method increased this rate to 99.0%. Comparative evaluation of these analysis methods showed that the Clustal W program (version 1.81), a freely available multiple-sequence alignment program, presented one of the best performances when used with the correct criteria. Other commercial and expensive programs did not achieve the same performances, making them less suitable for molecular typing of enteroviruses. Finally, although phylogenetic inference is the most demanding method in terms of knowledge of the user, it remained the best option analyzed.     

Genetic diversity of enterovirus subgroups

Summary Enterovirus serotypes were studied using nucleic acid hybridization and nucleotide sequence analysis. A great majority of enteroviruses could be roughly divided into two larger subgroups the first consisting of poliovirus and certain coxsackievirus A serotypes. The second subgroup included coxsackie B viruses, most ECHO viruses, enterovirus 71 and representatives of coxsackie A viruses. Enterovirus 70 showed low homology to the viruses in both groups. Interestingly, ECHO virus 22 failed to react with any of the hybridization probes indicating a relatively distant relationship. The close relationship between coxsackie B and ECHO viruses as well as between polio and certain coxsackie A viruses was also evident when nucleotide sequences of the 3 end noncoding parts were compared.     

Molecular typing and epidemiology of non-polio enteroviruses isolated from Yunnan Province, the People's Republic of China

This report presents an overview of human enteroviruses in Yunnan Province, the People's Republic of China. A total of 210 non-polioviruses isolated under acute flaccid paralysis (AFP) surveillance during a total study period of 5 years--1997 to 2000 and 2004--were examined. Of the 210 non-poliovirus isolates, 12 adenoviruses were serologically identified, and the remaining 198 isolates were used for molecular typing. The viral genomes of 195 non-polio enteroviruses (NPEVs) on VP1 partial region of virus capsid were translated to the corresponding amino acid sequences; these were compared with those of prototype strains. Based on molecular typing, 5 isolates were classified into 5 serotypes of the human enterovirus A species, 158 isolates, into 35 serotypes of the human enterovirus B species; and 32 isolates, into 6 serotypes of the human enterovirus C species. Viruses belonging to the human enterovirus D species were not isolated. Thus, under AFP surveillance, the human enterovirus B species accounted for 75.2% of the 210 isolates, and it was considered the predominant species. This was followed by human enterovirus C (12.2%), adenovirus (5.7%), and human enterovirus A (2.4%). Further, molecular analysis suggested that several serotypes of human enteroviruses B and C that exhibited genetic polymorphism were indigenous. Molecular typing methods may aid in understanding the epidemiology of NPEVs in Yunnan Province.     

Molecular identification and full genome analysis of an echovirus 7 strain isolated from the environment in Greece

Two enteroviruses from river water and four from sewage treatment plant were isolated in Larissa, Greece, that all shared the same sequence. A full genome analysis was conducted in an attempt to reveal the evolutionary pathways of one of the isolated strains (LR11F7). VP1 nucleotide and phylogenetic analysis revealed that the isolated strain had 78% homology with the echovirus 7 prototype strain Wallace. Full genome analysis revealed that LR11F7 P1 region is related to echoviruses 7 and that P2 and P3 regions are originating from contemporary enteroviruses isolated in South Asia. Two recombination events were shown to be involved into the evolutionary history of LR11F7, the one event concerning 3A, 3B, and 2C, and the other concerning 3D genomic region, both with new types of HEV-B. The contribution of recombination to enterovirus evolution is substantial, giving rise to new genetic lineages with unknown properties.     

Isolation and genomic characterization of three enterovirus 90 strains in Shandong, China

Enterovirus 90 (EV90) is a newly identified serotype of the species Human enterovirus A, and few nucleotide sequences of EV90 are available. In this study, three EV90 strains were isolated from acute flaccid paralysis (AFP) cases in Shandong Province, China, in 2001 and 2003. Sequence analysis revealed 96.7–98.0 % VP1 nucleotide identity among themselves and 77.7–92.3 % to other EV90 strains. Complete genome analysis provided evidence of recombination in the non-capsid coding region of strain 01421. This is the first report of EV90 in China, and the low isolation rate suggests that it has not been a prevalent serotype in China.     

Revealing Molecular Targets for Enterovirus Type 71 Detection by Profile Hidden Markov Models

The enterovirus infection in 1998 claimed 78 deaths in Taiwan, with an average of 40 fatalities each year after. Traditional serum-based diagnostic methods often fail to detect enteroviruses due to antigenic changes. As a result, many isolates remain untyped and are absent from the enterovirus surveillance and epidemiological investigations. We present a profile hidden Markov model (HMM) method for molecular typing of enterovirus 71 (EV71). Based on the enteroviral sequences retrieved from GenBank, we build a nucleotide-based and an amino acid-based profile HMM for each EV71 gene using the package HMMER. HMMER bit score-based Z-scores for EV71 and non-EV71 sequences are calculated for each of these profile HMMs. In a genome-wide analysis, we find that the distribution of the EV71 Z-scores and that of the non-EV71 Z-scores have disjoint support for nucleotide-based VP1 profile HMM if the sequence is longer than 150 bases; a VP1-based molecular typing method for EV71 is thus proposed. We also report VP4 an alternative molecular target for detecting EV71, while the two UTRs and all the genes coding the internal proteins cannot be used for such purpose. To demonstrate the performance of the nucleotide-based EV71 VP1 profile HMM, 330 enterovirus VP1 nucleotide sequences newly reported to GenBank are typed with this method. All the EV71 sequences are detected with no error.     

Detection of enteroviruses using cDNA and synthetic oligonucleotide probes

This study compares the detection of enterovirus RNA by cDNA probes prepared from both the 5' and 3' end of the genome of coxsackie A21 and B4 with the use of synthetic oligonucleotides prepared from short but highly conserved sequences in the 5' end non-coding region of the picornavirus genome. The cDNA probes detected enteroviruses with a variable level of sensitivity which presumably depended on the degree of genomic homology with the detecting probes. Generally probes from coxsackievirus A21 detected more enteroviruses than did similar probes from coxsackievirus B4. Probes from the 5' end of the genome of both viruses were more sensitive than 3' end probes. In contrast, synthetic oligonucleotides detected all enteroviruses efficiently suggesting that these probes could be useful as 'universal' probes to detect any enterovirus. This paper discusses the application of these probes in the diagnosis and differentiation of enteroviruses.     

Complete nucleotide sequence of the genome of coxsackievirus B1

The complete nucleotide sequence of the genome of the coxsackievirus B1, a human enterovirus that belongs to the Picornaviridae, was determined by using molecular cloning and rapid sequence analysis techniques. Sequence analysis of the cloned cDNAs revealed that the virion RNA was 7389 nucleotides long and polyadenylylated at the 3' terminus. Similar to other picornavirus genomes, a single large open reading frame was identified. The translated sequence starts at nucleotide position 742 and ends at 7287 of the genome. Thus, the viral polyprotein should consist of 2182 amino acids. When the predicted amino acid sequence of the viral polyprotein was compared with those of other human enteroviruses such as polioviruses, a striking sequence homology was observed, especially in viral proteins 1B, 2C, and 3D. This allowed us to predict precise map locations of the viral structural and nonstructural proteins on the genome, although two proteolytic processing sites, between 1D and 2A and between 2B and 2C, were obscure. The result presented here implied important information with respect to the genetical variation of human enteroviruses.     

Identification of enteroviral infection among infants and children admitted to hospital with acute gastroentritis in Ho Chi Minh City, Vietnam

A total of 276 fecal specimens collected from infants and children admitted to hospital with acute gastroenteritis in Ho Chi Minh City, Vietnam from October 2002 to September 2003, were tested for the presence of enteroviruses by RT-PCR and virus isolation. Enteroviruses were detected in 27 patients by RT-PCR corresponding to 9.8%. However, only four enterovirus strains could be isolated by cell culture with two different cell lines CaCo2 and Vero, showing specific cytopathic effect (CPE). The results clearly indicate that RT-PCR is a sensitive, specific assay to investigate the true burden of acute gastroenteritis due to enteroviruses in clinical fecal specimens. In the present study, enteroviruses were identified throughout the year except in May and the highest number was in December. Enteroviruses were subjected to molecular analysis by sequencing. It was found that enterovirus strains detected were classified further into two distinct genetic clusters (I, II) and demonstrated the great genetic diversity among them. Based on genetic analysis, 5' noncoding region (5' NCR) sequences suggested the predominant presence of Vietnamese enteroviruses with the greatest similarities to coxsakieviruses (51.9%) and echoviruses (29.6%). Interestingly, two of the sequenced specimens of enteroviruses were similar to a new strain called enterovirus 74. This report is the first detection of enteroviral infection in feces from infants and children admitted to hospital with acute gastroenteritis in Vietnam.     

应用随机PCR方法鉴定一株肠道病毒

目的 从基因水平对一肠道病毒分离株进行分析鉴定,方法 提取病毒RNA,以锚定随机引物反转录合成双链cDNA,用锚定特异引物进行随机扩增,扩增产物进行克隆,测序和序列分析。结果 随机扩增的病毒基因在电泳上呈典型的smear带型;11个随机克隆经GenBank检索,与肠道病毒成员具有最高的同源性且同源率超无穷大0%;11个克隆随机分布于病毒的基因组中,排定后的序列形成4个毗连序列群,共计2261个碱基,涵盖病毒基因组的30%;部分序列与柯萨奇病毒B6型氨基酸同源率达97.3%,参照测定序列合成的引物能够特异性地扩增病毒基因。结论 经序列分析证实小RNA病毒XJ90株为肠道病毒科成员。