Isolation and molecular identification of echovirus 13 isolated from patients of aseptic meningitis in Korea, 2002

During 2002, several epidemics of aseptic meningitis were attributed to echovirus 13 in Korea. The causative agents of these outbreaks were isolated and identified using rhabdosarcoma cells, HEp-2 and Buffalo green monkey kidney cells, and a neutralization test using monospecific antiserum. Fifty-four echovirus 13 isolates were isolated from patients with aseptic meningitis in the provinces, Seoul, Kyonggi, Gwangju, Jeonju, Busan, and Ulsan. Symptoms associated with aseptic meningitis infection in patients included the occurrence of headaches and mild fever. Molecular characterization of echovirus 13 samples was achieved by sequence and phylogenetic analyses on partial VP1 sequences from 20 Korean isolates and 10 foreign isolates listed in Genbank. Minor variation was observed among the Korean isolates, which formed a unique cluster with isolates of German and Japanese origin. The marked similarities between isolates could be attributed to a relatively recent arrival of the virus in Korea. This is the first such investigation of aseptic meningitis caused by echovirus 13 on the Korean peninsula.     

An aseptic meningitis outbreak caused by echovirus 6 in Anhui province,China

An outbreak of aseptic meningitis (AM) occurred in Jinzhai County in Anhui province from April to July in 2005. Totally, 97 children aged 3–15 years were hospitalized. To identify the etiologic agent, 77 cerebrospinal fluid specimens (CSF) and 5 fecal specimens were collected from the patients and cultured by human rhabdomyosarcoma (RD) cell line. Thirty isolates of human echovirus 6 (E6) from 27 CSF and 3 fecal specimens were confirmed by neutralization assay and sequencing analysis of the VP1 gene. The homology of VP1 gene among Anhui isolates was 99.7–100.0% and it indicated that this AM outbreak probable caused by a single transmission link of E6. Phylogenetic analysis based on all the available complete VP1 sequences indicated that E6 could be divided into clusters A, B, and C with at least 15% diversity between clusters and the C cluster could be further divided into C1, C2, C3, and C4. The Anhui isolates most resembled a 2005 strain from Russia (25465 Tambov) and belong to C4. This is the first report that E6 was responsible for an outbreak of AM in China. J. Med. Virol. 82:441–445, 2010. © 2010 Wiley‐Liss, Inc.     

Enterovirus meningitis in Brazil, 1998-2003

Acute viral infections of the central nervous system (CNS) such as acute flaccid paralysis, meningitis, and encephalitis, are responsible for a high morbidity, particularly in children. Non-Polio enteroviruses (NPEV) are known to be responsible for over 80% of viral meningitis in which the etiologic agent is identified. In the present study, we show the frequency of enterovirus meningitis in Brazil from December 1998 to December 2003. Enterovirus were isolated from 162 (15.8%), of a total of 1,022 cerebrospinal fluid (CSF) specimens analyzed. Echovirus 30 was identified in 139 of these isolates (139/162-85.2%). Other identified enteroviruses were: Coxsackievirus B5 (3.7%), Echovirus 13 (3.7%), Echovirus 18 (3%), Echovirus 6 (1.2%), Echovirus 25 (1.2%), Echovirus 1 (0.6%), and Echovirus 4 (0.6%). Patients's age ranged from 28 days to 68 years old. The most frequent symptoms were fever (77%), headache (69.5%), vomiting (71.3%), neck stiffness (41.3%), convulsion (7.1%), and diarrhea (3.7%). Although, the majority of the patients recovered without any complication or permanent squeal, five deaths occurred. Throughout the surveillance period, five viral meningitis outbreaks were confirmed: four in the Southern Brazil and one in the Northeast Brazil. Echovirus 30 was responsible for four out of the five outbreaks while Echovirus 13 caused the fifth one. Besides the outbreaks, 734 sporadic cases were also identified during the study period and 59 of these were positive for virus isolation (8%). Echovirus 30 accounted for 70% of the isolates. Our results showed that Echovirus 30 was the most prevalent etiological agent of viral meningitis in Brazil, causing both outbreaks and sporadic cases.     

Coxsackievirus B3-associated aseptic meningitis: an emerging infection in Hong Kong

Enterovirus (EV) infection is a common disease of childhood and associated not uncommonly with aseptic meningitis. In the summer of 2008, laboratory surveillance has detected increased number of coxsackievirus B3 (CVB3) associated aseptic meningitis in Hong Kong, constituting 11.6% of those infected. This study analyzed the epidemiology, circulating pattern, and clinical presentations of CVB3 in Hong Kong over the last 10 years with reference to the circulation of EV in the locality. Enteroviruses (EV) were isolated from respiratory, cerebrospinal fluid (CSF), stool, and vesicular samples using human rhabdomyosarcoma, human laryngeal carcinoma (HEp2-C), human lung fibroblast (MRC-5), and African green monkey kidney (Vero) cell lines. Virus isolates were identified and characterized by indirect immunofluorescence (IF) using monoclonal antibodies (mAB), neutralization test as well as partial VP1 sequencing. Different from previous years, IF test result showed that majority of the isolates from 2008 were untypeable by the mAB suggesting antigenic change. Sequence analysis revealed that these isolates were clustered with recent isolate from Fuyang, China. Review of data from 1999 to 2008 showed increased activity of CVB3 in the years 2005 and 2008, and isolates in these 2 years displayed an amino acid change from threonine to alanine at codon 277 of the VP1 gene, which may be associated with central nervous system (CNS) disease.     

Detection of varicella-zoster virus-specific DNA sequences in cerebrospinal fluid from patients with acute aseptic meningitis and no cutaneous lesions

Acute aseptic meningitis (AAM) is considered as an uncommon manifestation of varicella-zoster virus (VZV) recrudescence and is usually regarded as a complication of the cutaneous infection in patients with impaired cellular immunity. Indirect evidence suggests, however, that VZV-associated AAM may also respond to direct spread of the virus to the leptomeninges from the cells supporting the latency. The polymerase chain reaction (PCR) was used to amplify VZV-specific DNA sequences in serial cerebrospinal fluid (CSF) samples from 21 patients with AAM, who presented laboratory evidence of intrathe-cal production of VZV-specific antibody on follow-up. Eleven of these patients never showed cutaneous zosteriform lesions. VZV-DNA sequences were detected in the CSF from all patients with cutaneous zoster, as well as from six patients (55%) lacking skin lesions. Viral DNA sequences were present in six cases before the rise in specific antibody was seen in CSF, disappearing during follow-up in the seven positive cases. These results support the proposed involvement of VZV in the etiology of AAM seen among normal young adults and strongly suggest that the virus can reach directly and infect the CNS from the latently infected spinal ganglia. © 1994 Wiley-Liss, Inc.     

Echovirus 30 detection in an outbreak of acute myalgia and rhabdomyolysis,Brazil 2016–2017

Objectives

To describe an outbreak of acute myalgia accompanied by elevated levels of muscle enzymes that occurred in the northeast region of Brazil from December 2016 through to May 2017.

Molecular epidemiology of Echovirus 30 in Fujian,China between 2001 and 2011

Echovirus 30 (E‐30) was responsible for an outbreak of aseptic meningitis between April 1 and June 2, 2011 in Fujian Province, China. A molecular epidemiology study of 115 E‐30 strains was performed to characterize the genetic features of the etiologic agent of the 2011 aseptic meningitis outbreak. The phylogenetic trees of the complete VP1 gene (876 bp) from 74 of 115 isolates and 50 reference sequences were analyzed. Three lineages (E‐30_h, i, and j) were detected that had co‐circulated in Fujian in the last decade, of which E‐30_j was new. The other 72 Fujian strains and 16 representative strains from other provinces of China all belong to E‐30_h and E‐30_i. Two distinct E‐30 clusters including virus isolates obtained during adult surveillance were associated with the 2011 outbreak and differed from Fujian isolates prior to 2011, suggesting that the viruses may vary and adult infections play an important role in viral transmission. Thus, the multiple lineages of E‐30 in Fujian and variant viruses enhanced transmissibility, which may be related to the epidemic activity of E‐30. J. Med. Virol. 85:696–702, 2013. © 2013 Wiley Periodicals, Inc.     

Predominance of enterovirus B and echovirus 30 as cause of viral meningitis in a UK population

Background/ObjectivesEnteroviruses are the most common cause of aseptic or lymphocytic meningitis, particularly in children. With reports of unusually severe neurological disease in some patients infected with enterovirus D68 in North America, and a recent increase in the number of paediatric enterovirus meningitis cases presenting in this UK Midlands population, a retrospective regional surveillance study was performed.Study designCerebrospinal fluid (CSF) samples received were tested using the polymerase chain reaction (PCR) for HSV-1/2, VZV, enteroviruses and parechoviruses. Enterovirus PCR positive CSF samples were sent for further serotyping. A phylogenetic tree was constructed of the echovirus 30 VP1 sequences, where sufficient sample remained for sequencing.ResultsThe number of enterovirus positive CSFs from each year were: 21 (2008), 7 (2011), 53 (2012), 58 (2013) and 31 (2014). Overall, 163 of the 170 serotyped enteroviruses belonged to the species B (echovirus 5, 6, 7, 9, 11, 13, 16, 17, 18, 21, 25, 30; coxsackie B1, B2, B3, B4, B5, A9), with only 7 belonging to species A (coxsackie A2, A6, A16 and enterovirus 71). Echovirus 30 was the predominant serotype overall, identified in 43 (25.3%) of samples, with a significantly higher proportion in the adult age group (37.3%) compared to the infant age group (12.3%). Phylogenetic analysis showed that these UK Midlands echovirus 30 VP1 sequences clustered most closely with those from Europe and China.ConclusionThis study showed a continued predominance of echovirus 30 as a cause of viral meningitis, particularly in adults, though more surveillance is needed.     

The production of IL-8 in cerebrospinal fluid in aseptic meningitis of children

Neutrophils accumulate initially in the cerebrospinal fluid (CSF) of aseptic meningitis, perhaps because of increased levels of granulocyte colony-stimulating factor (G-CSF), macrophage inflammatory protein-1α (MIP-1α), and IL-8 in the subarachnoid space. We studied levels of these cytokines in children with aseptic meningitis using ELISA. When meningeal symptoms existed, IL-8 levels (1399 ± 1600 ng/l, n= 32) in the CSF were significantly higher than those either after meningeal symptoms disappeared (61 ± 56 ng/l, n= 18) or in controls (44 ± 63 ng/l, n= 27) ( P < 0.0001). High levels of IL-8 on admission dropped sequentially. Significant correlations were found between IL-8 levels and either neutrophil counts (r= 0.612), G-CSF levels (r= 0.873) or MIP-1α levels (r= 0.623) in the CSF of the affected patients (P< 0.0001). IL-8 values in serum were lower than in the corresponding CSF samples from all individuals with meningeal symptoms. The IL-8 mRNA was detectable by reverse-transcribed polymerase chain reaction (PCR)-assisted amplification in fresh leucocytes from the CSF, but not from the peripheral blood of a healthy volunteer. The culture of CSF mononuclear cells produced high levels of IL-8 (~ 2750 ng/l). These data indicate that IL-8 levels rise transiently at the initial stage of aseptic meningitis, and that mononuclear cells that migrate into the CSF are a cellular source of this chemokine. We suppose that IL-8, in addition to G-CSF and MIP-1α, contribute to the localized neutrophil accumulation during the disease.     

Laboratory investigation and phylogenetic analysis of enteroviruses involved in an aseptic meningitis outbreak in Greece during the summer of 2007

BackgroundAseptic meningitis is the most commonly observed CNS infection and is mainly attributed to Non-Polio Enteroviruses (EV).ObjectiveIdentification and genetic analysis of the EV involved in the recent aseptic meningitis outbreak which occurred in Greece, during the summer of 2007.Study designIn total, 213 CSF and faecal samples were examined for EV presence by culture, while enteroviral RNA detection was performed by nucleic acid sequence-based amplification assay (NASBA). EV strains were typed by seroneutralization, as well as nested RT-PCR followed by VP1-2A gene partial sequencing. Phylogenetic analysis was carried out for the identification of the genetic relatedness among the isolated EV strains.ResultsEV detection rate in CSF and faecal samples was 43.9% and 70.8%, respectively. EV serotyping and VP1 region analysis revealed the predominance of echovirus 4 (ECV4) serotype and the circulation of ECV6, 9, 14, 25, Coxsackie A6, A15, A24 and Coxsackie B1 serotypes. All ECV4 isolates presented a 98.7% similarity in nucleotide sequence, with a Spanish ECV4 strain, isolated during a meningitis outbreak in 2006.ConclusionsIt is the first time that ECV4 is associated with an aseptic meningitis outbreak in Greece, during which 9 different EV serotypes were co-circulating. All Greek ECV4 isolates were closely related to the Spanish ECV4 strain. Genetic analysis of the VP1 gene can significantly contribute to the revelation of the endemic EV strains circulation pattern and their phylogenetic relationship with enteroviruses involved in epidemics of distant geographical areas at different time periods.     

Naive and memory CD4+ T-cells in the cerebrospinal fluid of children with aseptic meningitis following measles-mumps-rubella vaccination and enteroviral meningitis

We investigated the distribution of memory (CD45RO⁺) and naive (CD45RA⁺CD62L⁺) CD4⁺ T-cells as well as CD8⁺ T-cells and total T-cells in the CSF of children with aseptic meningitis following measles-mumps-rubella (MMW) vaccination and those with enteroviral meningitis. Flow cytometric analysis of CSF cells was performed in 12 children with MMR vaccine-associated meningitis and 11 children with enteroviral meningitis. Percentages of total T-cells, CD4⁺ and CD8⁺ T-cells and monocytes in CSF of patients from the two groups were not significantly different. The majority of CD4⁺ T-cells in the CSF of both patient groups were of memory phenotype. Percentages of CSF naive CD4⁺ T-cells were increased in children with aseptic meningitis following MMR vaccination. Further studies focused on the more detailed immunophenotyping of CSF cells are needed to fully establish the usefulness of flow cytometry in the diagnostic workup of inflammatory CNS diseases in children.     

Chemokine profiles in the cerebrospinal fluid (CSF) during the course of pyogenic and tuberculous meningitis

The concentrations of the chemokines IL-8, monocyte chemotactic protein-1 (MCP-1) and macrophage inflammatory protein-1α (MIP-1α) were measured in 120 CSF samples from 23 patients with pyogenic meningitis and from 11 patients with tuberculous meningitis (TBM) and in 10 CSF from subjects with non-infectious neurological diseases. The chemokine concentrations in patients with meningitis were significantly higher than in control subjects (P < 0.0001). The highest CSF levels were found for IL-8 (median 2917 pg/ml) and MCP-1 (median 2557 pg/ml), whereas those of MIP-1α were less significantly elevated (median 24 pg/ml) (P < 0.0001). Patients with pyogenic meningitis had higher levels of IL-8 and MCP-1 than those with TBM (P < 0.0001). In serial samples from patients with pyogenic meningitis IL-8 levels declined before MCP-1 and MIP-α. In the case of TBM, IL-8, MCP-1 and MIP-1α decreased more gradually during treatment and were detectable in the CSF for several weeks, without any characteristic time course of elimination. These data indicate that patients with pyogenic meningitis and TBM show different chemokine profiles in CSF. The distinct chemokine pattern could be responsible for a differential attraction and activation of leucocytes in the CSF which is reflected in differences in the inflammatory response and clinical course of pyogenic meningitis and TBM.     

Evaluation of combining upper respiratory tract swab samples with cerebrospinal fluid examination for the diagnosis of enteroviral meningitis in children

In a prospective comparative study, the use of combined analysis of upper respiratory tract swab samples and cerebrospinal fluid (CSF) samples was assessed to improve the detection rate of enteroviral meningitis in children. An enterovirus was detected in 32% of patients with aseptic meningitis when testing CSF samples alone compared with 71.5% when combining CSF and respiratory tract findings. An enterovirus was detected in 17% of respiratory tract samples in an age- and sex-matched control group without meningitis. Thus, combining the examination of upper respiratory tract with CSF findings may improve the detection rate of enteroviral meningitis. Upper respiratory tract samples should be included in the diagnosis scheme to differentiate benign enteroviral meningitis from other life-threatening infections of the central nervous system. J. Med. Virol. 57:193–197, 1999. © 1999 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.     

Intrathecal synthesis of IgG antibodies to varicella-zoster virus in two cases of acute aseptic meningitis syndrome with no cutaneous lesions

IgG antibodies to varicella-zoster virus (VZV) were detected by indirect enzyme-immunoassay (EIA) in CSF of two patients with acute aseptic meningitis syndrome (AAMS) not associated with evident cutaneous lesion or recent history of zoster infection. Their characteristic features and serological data are compared with those observed in two patients with AAMS and zoster cutaneous lesions, and in 13 patients with AAMS of unknown or other etiology. According to several indexes applied to assess the origin of the detected antibodies, it is concluded that VZV IgG antibodies are of intrathecal production. The possible etiologic relationship between the neurological syndrome and the detection of VZV antibodies in CSF is discussed. Routine serological procedures are proposed for the diagnosis of CNS acute infections of probable viral etiology that should provide additional data on these rarely described cases.     

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.     

Local production of mumps IgG and IgM antibodies in the cerebrospinal fluid of meningitis patients

Immunoglobulin G (IgG) and M (IgM) antibodies against mumps virus were measured by an enzyme-linked immunosorbent assay (ELISA) in the serum and cerebrospinal fluid (CSF) specimens of patients with mumps meningitis. The CSF IgG antibodies correlated well with the respective antibody titers in serum. On the contrary, in only about half of the patients a moderate correlation was found between the CSF and serum IgM antibody titers, while the other patients did not have detectable mumps IgM antibodies in CSF irrespective of intermediate to high titers in serum. Two different immunologic mechanisms may be involved in these two groups which, however, did not show any clinical differences. The lack of IgM antibodies in the CSF of many patients diminished the value of CSF in the laboratory diagnosis of mumps meningitis compared to use of serum specimens. Intrathecal synthesis of mumps IgG antibodies was demonstrated in 83% of the patients, and of IgM antibodies in at least 67% of those patients with detectable IgM antibodies in CSF. The ratio between mumps IgG and IgM antibodies was higher in CSF than in serum, suggesting that the synthesis of IgG antibodies in central nervous system was more efficient than that of IgM antibodies.     

深圳市2004—2012年流行性脑脊髓膜炎疫情特征分析

目的根据深圳市2004—2012年流行性脑脊髓膜炎（简称“流脑”）的发病情况,分析实施传染病网络直报后流脑的流行特点,为防控提供科学依据。方法收集2004—2012年疫情报告数据与资料,统计汇总后进行流行病学特征分析。结果2004—2012年深圳市共报告40例流脑病例,各年发病率分别为0．043／10万、0．187／10万、O．080／10万、0．066／10万、0．026／10万、0．026／10万、0．039／10万、0．029／10万、0．019／10万,发病特点呈常态散发,发病时间集中在冬春季节,病例主要以工人、散居儿童为主。结论加强对流脑的预警监测和对专业人员的技术培训,及对重点人群进行疾病干预,可有效防控流脑疫情。     

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.