Clinical and virological features of an aseptic meningitis outbreak in North-Eastern France, 2005.

BACKGROUND: Enteroviruses (EVs) are considered as a major viral etiological cause of aseptic meningitis in children. OBJECTIVES: We assessed the clinical and virological features of an aseptic meningitis outbreak in North-East of France, 2005. STUDY DESIGN: Classical bacteriological analysis, Herpesviridae and EV PCR assays had been prospectively performed on cerebrospinal fluid (CSF) samples taken from 80 children hospitalized for aseptic meningitis. For each EV strain identified as etiological agent, a phylogenetic comparison of partial EV VP1 capsid protein coding gene was performed. RESULTS: The children older than 12 months (n=75) presented a typical aseptic meningitis syndrome, whereas the children aged less than 1 year (n=5) demonstrated only fever and hypotonia. Among the 80 studied children, EV was identified as the etiological cause of aseptic meningitis in 73 (91%) cases. Echovirus 30 (E30) was the most common isolated serotype (84% of 51 EV strains). VP1 phylogenetic analysis revealed that E30 strains were genetically closer to those isolated during 2000 aseptic meningitis outbreak comparatively to those identified during 2003 and 2006 non-epidemic years. Moreover, the genetic study demonstrated the co-circulation of four distinct lineages without any difference in temporal distribution or clinical features during the 2005 outbreak. CONCLUSIONS: The present report demonstrates the co-circulation of distinct E30 lineages during the same aseptic meningitis outbreak season. This E30 genetic diversity may be a prerequisite for the emergence of new strains potentially responsible for further aseptic meningitis outbreaks.     

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.     

无菌性脑膜炎和脑炎病人脑脊液肠道病毒RNA的检测及其临床意义

目的 检测肠道病毒（EV）在中枢神经系统感染中的致病情况,探讨检测EV感染的方法。方法 就用逆转录－聚合酶链反应（RT－PCR0和病毒培技术检测46例无菌性脑膜炎及脑炎病人脑脊液（CSF）标本。结果 RT－PCR方法敏感特异;46例无菌性脑膜炎和脑炎急性期CSF标本中,31例EV阳性（67．4％）,14例病毒培养阳性（26．1％）。统计结果显示,RT－PCR敏感性明显高于病毒培养。结论 EV是引起无菌性脑膜炎和脑炎的重要病原;RT－PCR快速敏感特异,简单易行,易于推广,是诊断EV感染的有效方法。     

Molecular epidemiological study of HEV-B enteroviruses involved in the increase in meningitis cases occurred in Spain during 2006

Human enteroviruses are one of the main etiological agents of aseptic meningitis and other central nervous system infections, particularly the serotypes included in the enterovirus B species. Molecular methods have proved useful to identify serotypes in clinical samples, facilitating the epidemiological study of these viruses. In the spring of 2006, there was a significant increase in meningitis cases caused by enteroviruses in Spain. In the present study, 138 enteroviruses directly detected in clinical samples of patients with aseptic meningitis (n = 116) and other neurological pathologies (n = 22) received by the National Center for Microbiology during the year, were genotyped by amplification and sequencing part of the VP1 region and phylogenetic analysis. Echovirus 30 was the most frequent serotype, followed in decreasing order by echovirus 6, 9, 13, 18, enterovirus 75, coxsackievirus A9, echovirus 11, 14, 29, 4, and coxsackievirus B4 and B5. Phylogenetic analysis with all Spanish echovirus 30 strains detected in 2006 and other reported echovirus 30 sequences, demonstrated that Spanish strains formed a new lineage, different from others previously described. In conclusion, echovirus 30 is the most commonly reported enterovirus serotype associated with aseptic meningitis in Spain. Direct molecular typing of clinical samples also allows rapid identification of the serotypes involved in an epidemic alert and phylogenetic analysis in the 3'-VP1 region is useful to study viral epidemiology.     

Molecular characterization of enteroviruses isolated from patients with aseptic meningitis in Korea, 2005

Summary. Enteroviruses are the most common causative agents of aseptic meningitis. In 2005, an outbreak of aseptic meningitis occurred in Seoul, Korea, which appeared to be caused by certain enterovirus strains. This study analyzed the clinical isolates from 24 different aseptic meningitis patients at our center from January to September 2005. Serotyping using type-specific antisera, RT-PCR, and direct sequencing of the 5′ noncoding and VP1 regions were carried out. The serotyping study identified 15 coxsackievirus B5 (CBV5), 4 coxsackievirus B1 (CBV1), 1 coxsackievirus B3 (CBV3) and 4 echovirus 9 (ECV9). Phylogenic analysis of the VP1 region revealed CBV5 to be the most predominant strain (15 strains). Twelve of these strains were grouped together with recent strains isolated in China and France. The 12 CBV5 strains contained a novel amino acid change in the BC-loop (the 90th and 91st, AY → GN) compared to the reference strain (1954/UK/85). The amino acid changes in the BC-loop in some recent CBV5 strains tend to be located in the C-terminal part, and these changes suggest some antigenic significance. First two authors contributed equally to this work.     

Changing epidemiology of human enteroviruses (HEV) in a hand,foot and mouth disease outbreak in Vellore,south India

PurposeHand Foot and mouth disease (HFMD) is a major childhood exanthematous disease causing outbreaks that have become a major public health threat in recent years. In Vellore district of Tamil Nadu, south India, occasional outbreaks are common among the paediatric age group, most commonly in those under 5years of age (U5s). CoxsackieA6, A4, A5, A9, A10, B2 and B5 are the common serotypes causing outbreaks. This study aimed to identify the molecular serotype of the causative agent, co-circulating in this region.MethodsAdapting the WHO case definition, cases during an HFMD outbreak between October and December 2017, were identified by a clinical criterion of fever, mouth ulcers and rash in the extremities. Vesicle fluid from these lesions were collected in viral transport medium and transported cold to the Clinical Virology laboratory of a tertiary care hospital in Vellore. Identification of the causative agent was undertaken by two real time PCRs (EV1 and EV2) followed by sequencing the VP1–2C region and constructing a phylogenetic tree.ResultsAmong the 31 HFMD patients included in this study, 23 (74.2%) were U5s, 3 (9.7%) were between 6 and 15 years and the remaining 5 (16.1%) were adolescents (>15 ?yrs). The outbreak ran a mild clinical course, with 22(71%) patients having fever as a prodromal symptom. Papulovesicular lesions characteristic of HFMD were present on all 31 (100%) patients’ palms and soles, buttocks of 19 (61.3%), oral mucosa of 12 (38.7%), and all over the body in 4 (12.9%) patients. Coxsackie A6(75%) and Coxsackie A16(25%) were the pathogens associated with this outbreak.ConclusionsChanging epidemiology of HFMD was seen in this outbreak since; other serotypes apart from the classical Coxsackievirus serotypes causing HFMD outbreak were also found co-circulating. EV1 PCR was a better screening assay than EV2 PCR in this region. Continued surveillance and molecular serotyping are necessary for HFMD outbreaks in any region.     

Molecular characterization of enteroviruses detected in Gyeong-Ju and Po-Hang provinces of Korea in 2003

We identified and characterized enteroviruses by amplifying the partial VP1 gene from pediatric patients with aseptic meningitis and other enterovirus-related diseases from the Gyeong-Ju and Po-Hang provinces of Korea in 2003. We identified two strains each of coxsackievirus A (CA) 6, CA9, and CA10; three enterovirus 71 (EV71) strains; and six echovirus 30 (E30) strains. The three EV71 strains were characterized as genogroup C4. These results are the first documentation reporting the occurrence of CA10 and EV71 genogroup C4.     

Echovirus 15 and autumn meningitis outbreak among children, Patras, Greece, 2005.

BACKGROUND: Enteroviruses are the most common cause of aseptic meningitis, presenting in epidemic or endemic form. OBJECTIVES: To determine the causative agent of an aseptic meningitis outbreak in autumn, 2005 in Patras, Greece. STUDY DESIGN: Cerebrospinal fluid (CSF) samples taken during May 2005-February 2006 from children admitted to the Children Hospital of Patras with signs of aseptic meningitis were tested for the presence of enteroviral RNA. Typing was performed by nucleotide analysis. RESULTS: Enteroviruses were detected in 11 (57.9%) of 19 tested CSF samples. In a 12-day period (27 October-7 November 2005) five aseptic meningitis cases were observed. Echovirus 15 was detected in all five cases, and differed from the prototype strain by 27.6%. Enteroviruses before and after this cluster of cases were of different serotypes (Echovirus 9, Echovirus 6). All patients with Echovirus 15 infection were male with a mean age of 7.7 years (2 months-13 years), all recovered successfully. CONCLUSIONS: This is the first report of a cluster of aseptic meningitis cases caused by Echovirus 15. The causative agent was a new variant of Echovirus 15.     

Prospective identification of enteroviruses involved in meningitis in 2006 through direct genotyping in cerebrospinal fluid

Enterovirus infections were investigated with special emphasis on performing rapid molecular identification of enterovirus serotypes responsible for aseptic meningitis directly in cerebrospinal fluid (CSF). Enterovirus genotyping was carried out directly with specimens tested for the diagnostic procedure, using two seminested PCR assays designed to amplify the complete and partial gene sequences encoding the VP1 and VP4/VP2 capsid proteins, respectively. The method was used for identifying the enterovirus serotypes involved in meningitis in 45 patients admitted in 2005. Enterovirus genotyping was achieved in 98% of the patients studied, and we obtained evidence of 10 of the most frequent serotypes identified earlier by genotyping of virus isolates. The method was applied for the prospective investigation of 54 patients with meningitis admitted consecutively in 2006. The enterovirus serotypes involved were identified with the cerebrospinal fluid (CSF) of 52 patients (96%) and comprised 13 serotypes within the human enterovirus B species and 1 within the human enterovirus A species. The three most common serotypes were echovirus 13 (E13; 24%), E6 (23%), and coxsackievirus B5 (11.5%), a pattern different from that observed in 2005. Genotyping of virus isolates was also performed in 35 patients in 2006 (meningitis, n = 31; other diseases, n = 4). By comparison, direct genotyping in CSF yielded a more complete pattern of enterovirus serotypes, thereby allowing the detection of rare serotypes: three less common serotypes (CB2, E21, and E27) were not detected by indirect genotyping alone. The study shows the feasibility of prospective enterovirus genotyping within 1 week in a laboratory setting.     

2001年徐州地区暴发性病毒性脑膜炎病原的研究

目的 确定引起2001年江苏省徐州地区无菌性脑膜炎流行的病原体。方法 组织培养法从患者脑脊液分离病毒，标准血清中和试验鉴定分离毒株；中和试验检测双份血清中和抗体效价；逆转录聚合酶链反应（RT-PCR）检测肠道病毒特异性基因片段。结果 22份脑脊液中分离出4株柯萨奇B5型、2株柯萨奇B3型、1株艾可7型肠道病毒，分离阳性率31．8％。RT-PCR检测脑脊液21份，肠道病毒阳性11份。阳性率52．4％。19例双份血清中11例中和效价呈4倍以上增长或转阳，阳性率57．9％。结论 此次江苏省徐州地区无菌性脑膜炎流行的病原体是以柯萨奇B5为主要血清型的肠道病毒。     

Genome analysis of two type 6 echovirus (E6) strains recovered from sewage specimens in Greece in 2006

Echovirus 6 (E6) is one of the main enteroviral serotypes that was isolated from cases of aseptic meningitis and encephalitis during the last years in Greece. Two E6 (LR51A5 and LR61G3) were isolated from the sewage treatment plant unit in Larissa, Greece, in May 2006, 1 year before their characterization from aseptic meningitis cases. The two isolates were initially found to be intra-serotypic recombinants in the genomic region VP1, a finding that initiated a full genome sequence analysis. In the present study, nucleotide, amino acid, and phylogenetic analyses for all genomic regions were conducted. For the detection of recombination events, Simplot and bootscan analyses were carried out. The continuous phylogenetic relationship in 2C–3D genomic region of strains LR51A5 and LR61G3 with E30 isolated in France in 2002–2005 indicated that the two strains were recombinants. SimPlot and Bootscan analyses confirmed that LR51A5 and LR61G3 carry an inter-serotypic recombination in the 2C genomic region. The present study provide evidence that recombination events occurred in the regions VP1 (intraserotypic) and non-capsid (interserotypic) during the evolution of LR51A5 and LR61G3, supporting the statement that the genomes of circulating enteroviruses are a mosaic of genomic regions of viral strains of the same or different serotypes. In conclusion, full genome sequence analysis of circulating enteroviral strains is a prerequisite to understand the complexity of enterovirus evolution.     

山西省临汾市ECHO30病毒所致病毒性脑炎疫情的病原学分析

目的 对山西省临汾市一起病毒性脑炎疫情进行病原学分析.方法 用Real-timePCR方法对9份咽拭子标本进行肠道病毒核酸检测.对肠道病毒核酸阳性的标本,分别扩增肠道病毒5'UTR区和VP2基因区片段,PCR扩增产物测序后,经BLAST比对初步确定病毒型别为ECHO30;使用ECHO30的特异性引物扩增VP1基因部分片段,测序后使用MEGA 4.0软件与NCBI数据库的其他ECHO30毒株进行进化分析.结果 9份病毒性脑炎标本中有4份为肠道病毒阳性;经肠道病毒5'UTR片段、VP2基因和VP1基因测序,确定4条临汾株ECHO30病毒核苷酸序列与2004年浙江ECHO30株和ECHO30标准株Bastianni同源性较高,且4条ECHO30病毒VP1基因的差异性为0.5％.进化分析显示,本地区的4株ECHO30病毒自成一簇,且与中国大陆地区的其他参考株亲缘关系较近.结论 肠道病毒属ECHO30病毒引发山西省临汾市病毒性脑炎疫情,有必要监测本地区的病毒性脑炎病原谱.     

无菌性脑膜炎暴发中柯萨奇B3病毒的基因特征分析

目的 明确2008年山东省临沂市郯城县无菌性脑膜炎暴发的病原,对分离到的柯萨奇B3病毒的基因特征、遗传变异规律及进化来源进行分析.方法 采集暴发病例的粪便、脑脊液标本,应用RD、Hep-2细胞进行病毒分离;阳性分离物分别采用中和试验、逆转录-聚合酶链式反应(RT-PCR)和核苷酸序列测定方法进行定型,最后与GenBank中检索到的其他CVB3毒株进行同源性分析,并构建VP1完整编码区亲缘进化树研究其遗传进化规律.结果 共采集22份粪便和120份脑脊液标本,分离到病毒35株,经鉴定34株为CVB3,1株为ECH030.34株CVB3分离株VP1区部分序列(381 bp)核苷酸同源性为90.5%～100%,氨基酸同源性为98.4%～100%;与Nancy原型株的核苷酸同源性为79.5%～81.6%,氨基酸同源性为98.4%～99.2%.012/2008TC/SD/CHN和177/2008TC/SD/CHN与安徽2008年分离的Fuyang19株同源性最高,核苷酸同源性分别为98.2%和91.0%,氨基酸同源性分别为99.2%和98.9%;进化树分析显示,中国大陆分离株独处一支,且呈单源性发生关系.结论 此次脑膜炎暴发的病原是CVB3.它与其他的中国分离株同源性高,与国外分离到的CVB3具有不同的基因特征.     

Complete nucleotide sequence analysis of the VP1 genomic region of Echoviruses 6 isolated from sewage in Greece revealed 98% similarity with Echoviruses 6 that were characterized from an aseptic meningitis outbreak 1 year later

The molecular characterization of two enterovirus strains (LR51A5 and LR61G3) isolated from the sewage treatment plant unit in Larissa, Greece, in May and June 2006 and the investigation of their relationship with enteroviruses of the same serotype isolated in Greece in 2001 and 2007 were performed by complete VP1 sequence analysis of the isolates. The close phylogenetic relationship and the high nucleotide similarity (98%) led to the conclusion that the virus isolated from sewage in 2006 was associated with that isolated from an aseptic meningitis outbreak 1 year later. Bootscan analysis of the VP1 genomic region revealed that intraserotypic multi-recombination events might have been involved in the evolutionary past history of the LR51A5 and LR61G3 isolates.     

Epidemiology of enterovirus types causing neurological disease in Austria 1999–2007: Detection of clusters of echovirus 30 and enterovirus 71 and analysis of prevalent genotypes

Between 1999 and 2007 1,388 stool specimens from patients with acute flaccid paralysis or aseptic meningitis were submitted to the Austrian reference laboratory for poliomyelitis. Samples (201) yielded non‐poliovirus enterovirus in culture. One hundred eighty‐one viruses were available for typing and 78 isolates which remained serologically untyped were further analyzed by CODEHOP‐PCR and sequencing of the VP1 gene and the 5′‐untranslated region (5′‐UTR). Typing revealed an Echovirus 30 outbreak in northwestern Austria in 2000, which was in accordance with the situation in Europe, and no dramatic seasonal changes of Coxsackie viruses were observed. In 2002/2003 a small outbreak of enterovirus 71 (EV71), affected 12 patients in the province of Styria. This virus was identified as genotype C1 and appeared to be genetically distinct from the isolates observed in 2001/2002 in Vienna. In 2004 two unrelated cases occurred in Lower Austria, which were identified as genotype C4, which has been described associated with high mortality most recently in China. In contrast to the situation in Asia the detected EV71 cases were not associated with hand–foot–mouth disease, but with serous meningitis only. This was surprising as a recent publication suggested a reduced neurovirulence of C1 genotype in children in Norway, presumably due to alterations in 5′‐UTR and polymerase gene. However, comparing the 5′‐UTR of the Austrian isolates and established virulent reference strains to the Norwegian isolate and an attenuated EV71 laboratory strain we did not find an indication that the genotype C1 possesses a RNA structure in its 5′‐UTR leading to reduced neurovirulence. J. Med. Virol. 81:317–324, 2009. © 2008 Wiley‐Liss, Inc.     

Update on molecular characterization of coxsackievirus B5 strains

Among Coxsackie B viruses, Coxsckievirus B5 is one of the most predominant serotypes in human, it is frequently associated with cases of neurological diseases, epidemics of meningitis and is a common cause of cardiomyopathy and diabetes. In the present study 27 isolates of Coxsackievirus B5 from North Africa, obtained from cerebrospinal fluid and stool samples of healthy individuals, patients with acute flaccid paralysis or aseptic meningitis were investigated by partial sequencing in the 5' half of the VP1 region and compared to the up‐to‐date published Coxsackievirus B5 sequences in the same genomic region. Four distinct genomic groups and ten different clusters were individualized. Most of the isolates from Algeria and Tunisia belonged to two clusters. For both, the sequences from North Africa clustered mainly with sequences from European countries, the majority isolated recently during the 2000s. The analysis of the alignment of amino‐acids sequences in the VP1 gene revealed four major substitutions in strains from different clusters, we also noticed changes in the BC‐loop region; this region is associated with viral antigenicity. This study permit to better identify circulating Coxsackievirus B5 strains throughout the world and their genetic relationship. The protein analysis showed changes that could imply some antigenic significance. J. Med. Virol. 83:1247–1254, 2011. © 2011 Wiley‐Liss, Inc.     

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.     

Echovirus 30 meningitis epidemic followed by an outbreak-specific RT-qPCR

BackgroundAn outbreak of enteroviral aseptic meningitis emerged in Southwestern Finland in August 2009. The same enterovirus reappeared with increasing incidence of meningitis in other parts of Finland in 2010.ObjectivesTo identify the incidence and molecular epidemiology of enteroviral meningitis outbreak.Study designThe causative agent was identified as echovirus 30 (E-30) by sequencing partial viral protein 1 capsid genome, and a virus type-specific RT-qPCR was set up for sensitive detection of the virus in cerebrospinal fluid specimens. Enterovirus positive CSF specimens were subjected to the E-30-specific assay to investigate this unusual occurrence of aseptic meningitis and facilitate case confirmation during the outbreaks between August 2009 and September 2010.ResultsE-30 was detected in 106 (72%) enterovirus positive cerebrospinal fluid specimens. All the meningitis cases in 2009 and most of them in 2010 were among adolescents and several were members of sport teams.ConclusionsBetween August 2009 and September 2010, E-30 caused an extensive outbreak with two peaks in Finland. Type-specific RT-PCR allowed rapid diagnostic follow-up of the epidemic.