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.     

Isolation and characterization of a Chinese strain of human enterovirus 74 from a healthy child in the Tibet Autonomous Region of China

Human enterovirus 74 (HEV74) is a recently described serotype within the species Human enterovirus B (HEV-B). Few nucleotide sequences of HEV74 are available, and only one complete genome sequence (the prototype strain) has been published. In this study, we report the complete genome sequence of an HEV74 strain isolated from a healthy child during a stool survey in the Tibet Autonomous Region of China. The results indicated that HEV74 may be a prevalent and common enterovirus type, and that HEV74 is globally distributed, especially in Asia. Sequence analysis revealed high variability among HEV74 strains and indicated frequent recombination within HEV-B.     

Genomic characterization of an enterovirus 97 strain isolated in Shandong,China

The genomic characterization of human enterovirus 97 (EV97) strain isolated from an acute flaccid paralysis case in Shandong province, China in 1999, is described. The strain, designated as 99188/SD/CHN/1999/EV97 (abbreviated as 99188), had a genome of 7394 nucleotides. Compared with other EV97 strains, it had 81.3–83.3% nucleotide similarity and 94.0–95.4% amino acid similarity in VP1 coding region, and it had 81.4% complete genomic similarity with prototype strain BAN99-10355. The most striking feature was the deletion of 18 nucleotides in the 3′ end of VP1 coding region, combined with two deletions and one insertion in 5′ and 3′ untranslated regions. All these findings demonstrated the strain 99188 had a distant genetic relationship with other EV97 strains. In the phylogenetic trees generated from VP1 and 3D sequences of human enterovirus species B (HEV-B), the lineages of strain 99188 were not congruent, suggesting the event of recombination. Similarity plot analysis further provided the evidence of recombination with other strains of HEV-B in P2 and P3 coding region. This is the first finding of EV97 in China and the third genomic sequence of EV97 reported.     

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.     

柯萨奇B组3型病毒中国分离株VP4、3D基因序列及系统发生树分析

目的研究柯萨奇B组3型病毒（CVB3）中国分离株结构蛋白VP4、非结构蛋白3D基因序列及变异性。方法在HeLa细胞中增殖病毒，用RT-PCR扩增目的基因片段，与pMDl8-T载体连接，PCR初步鉴定后测序，进行序列同源性及系统发生分析。结果CVB3中国分离株VP4基因含207个碱基，编码69个氨基酸，与Nancy株氨基酸同源性为97．10％；3D基因含1386个碱基，编码462个氨基酸，与Nancy株氨基酸同源性为97．62％。在系统发生中，CVB3中国株VP4基因、3D基因均与Nancy株聚簇。结论CVB3中国分离株VP4和3D基因长度与Nancy株一致，进化上属同一分支。     

Immunofluorescent IgG and IgM antibodies in human sera by enterovirus infections

IgG and IgM antibodies to selected enterovirus serotypes (P2, CB 1-5, E2, 4, 20), to prototype collection strains and to fresh isolates were examined by immunofluorescence in paired sera of 11 children and 4 adults in whom enterovirus had been isolated, or a rise of specific antibodies had been proved by neutralization test. One six-month- and one seven-month-old child had antibodies of both classes to the isolated virus strain and to poliovirus only. Children from eight month onwards and adults had antibodies to the majority of enterovirus serotypes tested. Among all enteroviruses tested, heterologous reactions were observed not only with IgG but also with IgM class antibodies.     

肠道病毒71型山东临沂分离株全基因组序列分析

目的 自1例死亡患儿的标本中分离EV71,并分析其全基因组序列特点,研究其基因序列的改变是否与其神经毒力有关.方法 咽拭子标本采自山东临沂市人民医院1例死亡患儿,在人横纹肌瘤细胞(RD)上分离EV71,分段扩增获得EV71的全序列,用BLAST、Bioedit和MEGA 4进行序列分析.结果 获得1株EV71分离株SDLY107,基因组全长7405 bp,全基因组核苷酸序列与2008年的阜阳株Fuyang.Anhui.P.R.C/17.08/2同源性最高,为98.6%,与原型株BrCr/70的同源性为80.0%,与神经毒型株MS/87的同源性为86.5%.系统进化分析表明,SDLY107与中国大陆的北京株、河南株、广西株、深圳株、兰州株、阜阳株、重庆株、浙江株的亲缘关系较近,按照传统的VP1基因分型方法,可归为C4亚型,是近年来我国大陆流行的主要基因亚型.氨基酸序列分析发现,SDLY107与其他毒株相比,有2个特有的突变(E947D,K1873R).结论 SDLY107分离株属于C4亚型,氨基酸突变E947D和K1873R可能与EV71的致病性有关.

Abstract：

Objective To isolate enterovirus 71 from a death children,and analyze whether the neurovirulence was related to the variation of nucleotide and amino acid. Methods Enterovirus 71 was isolated from throat swabs which were colleted from Shandong Linyi People's Hospital. The full length genome was sequenced by amplification with RT-PCR and sequencing of 9 overlapped gene fragments covering full length of the genomes. The nucleotide and amino acid sequenced was aligned by BLAST, Bioedit and MEGA 4. Results A strain of enterovirus 71 was isolated and named as SDLY107. The full length was 7405 bp. The results of homology analysis of overall nucleotide sequence showed that strain Fuyang. Anhui. P. R. C/17.08/2 had highest homology (98.6%)with strain SDLY107, and the homology was 80.0% between strain SDLY107 with prototype strain BrCr/70,and 86. 5% between strain SDLY107 with nerve strain MS/87. Phylogenetic analysis showed that the phylogeny was close between SDLY107 with some isolated strains from Chinese Mainland, such as Beijing, Henan, Guangxi, Sbenzhen, Lanzhou, Fuyang, Chongqing and Zhejiang strains, which was clustered for C4 subtype. The results of amino acid sequence analysis showed that there were 2 mutations, E947D and K1873R, for strain SDLY107. Conclusion SDLY107 belonged to C4 subtype, amino acid mutations E947D and K1873R of which may be relevant to the pathogenicity of EV71.     

Molecular epidemiology and type-specific detection of echovirus 11 isolates from the Americas,Europe, Africa,Australia, southern Asia and the Middle East

Echovirus 11 (E11) is among the most commonly isolated human enteroviruses. To examine the range of genetic variation within the E11 serotype, we determined the complete VP1 sequences for 53 geographically dispersed E11 strains isolated in 16 countries from 1953 to 2001. E11 sequences were monophyletic with respect to all other enterovirus serotypes. The sequences clustered into four monophyletic genogroups, A-D; members of each genogroup differed from one another by <20%. Isolates in different genogroups differed from one another by 19-28%. The E11 prototype strain, USA/CA53-Gregory, was the sole member of genogroup B. All recent US isolates were members of one of two discrete lineages within genogroup D. The well-characterized E11 antigenic variant, USA/CA63-Silva, was also a member of genogroup D. Members of genogroups A and C were antigenically similar to USA/CA53-Gregory, as measured by neutralization with anti-Gregory and anti-Silva antisera. Only USA/CA63-Silva was neutralized more efficiently by the anti-Silva antiserum; other genogroup D viruses were Gregory-like or intermediate in their neutralization phenotype. Recent non-US isolates were distributed in genogroups A, C and D. Sequence similarities among genogroup D isolates from North America, Europe, Asia, Australia and North Africa demonstrate that an E11 strain can spread rapidly over a wide geographic area. The aligned sequences were used to develop an E11-specific RT-PCR assay, using degenerate, inosine-containing primers, to amplify all members of all genogroups.     

Molecular and biological analysis of echovirus 9 strain isolated from a diabetic child

The full-length infectious cDNA clone was constructed and sequenced from the strain DM of echovirus 9, which was recently isolated from a 6-week-old child at the clinical onset of type 1 diabetes. Parallel with the isolate DM, the full-length infectious cDNA clone of the prototype strain echovirus 9 Barty (Barty-INF), was constructed and sequenced. Genetic relationships of the sequenced echo 9 viruses to the other members of the human enterovirus type B species were studied by phylogenetic analyses. Comparison of capsid protein sequences showed that the isolate DM was closely related to both prototype strains: Hill and Barty-INF. The only exception was the inner capsid protein VP4 where serotype specificity was not evident and the isolate DM clustered with the strain Hill and the strain Barty-INF with echovirus 30 Bastianni. Likewise, the nonstructural protein coding region, P2P3, of isolate DM was more similar to strain Hill than to strain Barty-INF. However, like echovirus 9 Barty, the isolate DM contained the RGD-motif in the carboxy terminus of capsid protein VP1. By blocking experiments using an RGD-containing peptide and a polyclonal rabbit antiserum to the alpha(v)beta(3)-integrin, it was shown that this molecule works as a cellular receptor for isolate DM. By using primary human islets, it was shown that the isolate DM is capable of infecting insulin-producing beta-cells like the corresponding prototype strains did. However, only isolate DM was clearly cytolytic for beta-cells. The infectious clones that were made allow further investigations of the molecular features responsible for the diabetogenicity of the isolate DM.     

Complete genome analysis of human enterovirus B73 isolated from an acute flaccid paralysis patient in Shandong,China

Human enterovirus B73 (EV-B73) is a member of species Enterovirus B. To date, only one complete genome sequence of prototype strain CA55-1988 from California has been available. In this study, the complete genome analysis of an EV-B73 strain 088/SD/CHN/04 isolated from an acute flaccid paralysis case in Shandong Province, China in 2004 is conducted. It had 75.6 and 79.3 % nucleotide similarity with prototype strain CA55-1988 in the VP1 coding region and the complete genome, respectively. It had great VP1 nucleotide divergence (16.7–24.4 %) with EV-B73 strains from other parts of the world. Similarity plot and bootscanning analyses provided evidence of recombination with other EV-B viruses.     

Molecular comparison of echovirus 11 strains circulating in Europe during an epidemic of multisystem hemorrhagic disease of infants indicates that evolution generally occurs by recombination

We compared echovirus 11 (E11) strains implicated in a severe epidemic in Hungary in 1989 with the prototype E11 strain Gregory and with other E11 strains, most of which were isolated over the same period in Europe (Finland, The Netherlands, Romania, Russia) from sporadic cases or from environmental water. Partial sequencing indicated that the Hungarian strains were closely related to each other and to most European strains. They were particularly closely related to one Romanian strain associated with a sporadic case of hemiparesis and several Finnish strains isolated from environmental water. Sequencing of the complete genomes of one Hungarian strain, the Romanian strain, and one Finnish strain revealed differences of only a few nucleotides in the 5' half of the genome, including the 5' nontranslated region (5'-NTR) and the capsid coding region. However, significant differences were observed in the nucleotide sequences of the 3' half of the genome (nonstructural viral protein region and 3'-NTR), indicating that these strains evolved recently and independently by genetic recombination with other unknown E11 or enterovirus strains.     

Simultaneous type 1 diabetes onset in mother and son coincident with an enteroviral infection.

Enterovirus (EV) infections have been implicated in the development of type 1 diabetes. (T1D). They may cause beta-cell destruction either by cytolytic infection of the cells or indirectly by triggering the autoimmune response. Virus was isolated from a woman at diagnosis of T1D (Tuvemo 1) and in addition, virus was isolated from her son at diagnosis of T1D at the same day (Tuvemo 2). None of the isolates could initially be serotyped by conventional methods. The Tuvemo 1 virus was genotyped and after sub-cultivation it was also serotyped as Coxsackievirus B5. The mother revealed antibodies against GAD65. The boy and the father both revealed a significant increase in neutralization antibody titre against two strains of CBV-4, clearly indicating a recent or ongoing EV infection. In addition, the brother showed such a titre rise against another CBV-4 strain (E2) and against a CBV-5 strain (4429). These results show that the whole family had a proven EV infection at the time of T1D diagnosis of the mother and the 10-years-old boy, indicating that the infection might cause or accelerate the T1D.     

Molecular epidemiology of ECHO 6 virus, the causative agent of the 2006 outbreak of serous meningitis in Khabarovsk

A total of 3194 cases of enterovirus meningitis were notified in the Russian Federation in 2005, of them there were 1434 cases in the Khabarovsk Territory. Enteroviruses were isolated from 1020 out of the virologically studied 1362 patients from the Khabarovsk Territory. Viruses E6 and E30 were isolated in 80 and 14.7% of cases, respectively. E1, E3, E7, E33, Coxsackie virus B1, B4, B5, and A10 were sporadically detected. The E6 strains isolated in Komsomolsk-on-Amur were identical while E6 strains isolated in Khabarovsk belonged to two different genotypes and greatly differed from those isolated in Konsomolsk-on-Amur. The virus E30 strains isolated in Khabarovsk and Komsomolsk-on-Amur had a 1% difference in VP1 genome nucleotide sequence and belonged to E30 subtype that circulated in Russia and Kazakhstan in 2004-2005.     

Detection of non-polio enteroviruses from 17 years of virological surveillance of acute flaccid paralysis in the Philippines

Acute flaccid paralysis (AFP) surveillance has been conducted as part of the World Health Organization (WHO) strategy on poliomyelitis eradication. Aside from poliovirus, which is the target pathogen, isolation, and identification of non-polio enteroviruses (NPEVs) is also done by neutralization test using pools of antisera which can only identify limited number of NPEVs. In the Philippines, despite the significant number of isolated NPEVs, no information is available with regard to its occurrence, diversity, and pattern of circulation. In this study, a total of 790 NPEVs isolated from stool samples submitted to the National Reference Laboratory from 1992 to 2008 were analyzed; neutralization test was able to type 55% (442) of the isolates. Of the remaining 356 isolates, which were untyped by using neutralization test, 348 isolates were analyzed further by RT-PCR targeting the VP1 gene. A total of 47 serotypes of NPEV strains were identified using neutralization test and molecular typing, including 28 serotypes of human enterovirus B (HEV-B), 12 serotypes of HEV-A, and 7 of HEV-C. The HEV-B group (625/790; 79%) constituted the largest proportion of isolates, followed by HEV-C (108/790; 13.7%), HEV-A (57/790; 7.2%), and no HEV-D. Coxsackievirus (CV) B, echovirus (E)6, E11, and E13 were the most frequent isolates. E6, E11, E13, E14, E25, E30, E33, CVA20, and CVA24 were considered as endemic strains, some NPEVs recurred and few serotypes existed only for 1-3 years during the study period. Despite some limitations in this study, plural NPEVs with multiple patterns of circulation in the Philippines for 17 years were identified.     

A role of recombination in the evolution of enteroviruses

Enteroviruses, members of the family Picornaviridae (more than 70 serotypes), are distributed throughout the world and cause a broad spectrum of clinical manifestations in man and animals. Human enteroviruses are classified into 5 species: human enteroviruses A to D, and Poliovirus. Recombination has long been known to be an important property of poliovirus genetics; however, recombination has been recently shown to be also ubiquitous in non-polio enteroviruses. Prototype enterovirus strains have complex phylogenetic relations and all currently available enterovirus strains are recombinant to prototype strains. The extremely high rate of recombination allows the fragments of the enterovirus genome to evolve independently at a microevolutionary scale. Recombination strictly takes place between the members of the same species, usually outside the capsid-encoding genome region. Therefore, it can be concluded that the species enterovirus exists as a worldwide reservoir of the genetic information that shuffles frequently to produce new virus variants. This new model of the genetics of enterovirus accounts for failures to associate the serotype of enteroviruses and the form of the disease caused. The contents of this review have been published in the Reviews in Medical Virology, 2005.     

风疹病毒JR23株E1包膜糖蛋白的基因克隆与序列分析

目的：建立风疹病毒包膜糖蛋白E1的克隆载体，研究E1基因变异情况，并对其序列进行系统发生树分析。方法：利用RT－PCR方法扩增并回收风疹病毒JR23株的包膜糖蛋白E1的基因片段，将其与PMD－18T载体连接，经氨苄青霉素筛选，酶切鉴定，以获得风疹病毒E1蛋白基因的克隆，将此基因测序后，利用DNASTAR和WINSTAR软件包绘制系统发生树进行序列之间的比较分析。结果：筛选出含有风疹病毒E1蛋白基因的克隆，序列分析及发生树的绘制表明：JR23株与日本TCRB株及英国THOMAS株差别最小，分别为0．9％和1．2％，与北京BRD2株及香港XG379株差别最大，分别为7．6％和7．3％，与其它各株的差别均小于3％（除NC株为3．7％外），系统发生也与THO－MAS株、TCRB株最近，与BRD2株最远。结论：克隆载体的建立为进一步研究E1基因与糖蛋白功能的关系提供基础。系统发生表明中国不同地区风疹流行株基因序列存在明显差异，这对风疹病毒遗传与变异，分子流行病学研究，以及制备有效的亚单位疫苗提供了资料。     

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.     

Partial 3D gene sequences of Coxsackie viruses reveal interspecies exchanges

The 3D region of 46 clinical Coxsackievirus strains, primarily belonging to the human enterovirus B species (HEV-B), were analyzed using nucleotide distance matrices and phylogeny software. The conclusions from previously analyzed genomic regions (VP1—2A-2B-2C) of the aforementioned strains revealed that enteroviruses’ inheritance is being guided by gene adaptation among viruses of different serotypes. In this report the comparison of partial VP1 and 3D gene phylogenies presented an obvious incongruence. Moreover, the phylogeny of 3D sequences of the strains revealed an unexpected (and for the first time reported) homology among strains of different species. The observations of our study indicate that conversion events such as multiple mutations or recombination among strains and unknown donors may occur during the evolution of circulating strains, leading, probably, to viruses with altered genome and virulence.