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.     

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.     

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.     

Genetic variation of coxsackie virus B5 strains associated with aseptic meningitis in Greece

In order to explore the genetic relationships among coxsackie virus B5 strains in Greece, the nucleotide sequences of the partial VP1 gene in strains isolated from aseptic cases of meningitis were determined and compared with those of strains isolated from other countries. Phylogenetic analysis showed a high degree of divergence (25%) among Greek strains isolated in different years, which clustered with high bootstrap values in a different subgroup of viruses, suggesting that enterovirus types vary with time rather than geographical distribution. A non-synonymous mutation present in the strains of this study was not observed in other coxsackie virus B5 strains.     

Molecular characteristics of human coxsackievirus B1 infection in Korea, 2008–2009

This study was performed to analyze epidemiological and molecular characteristics of coxsakievirus (CV) B1 infection associated with severe neonatal illness cases and death in Korea during 2008–2009. Through a nationwide surveillance program, specimens were collected from 104 patients infected with CVB1. The detection of enteroviruses (EVs) from specimens was subjected to a diagnostic real‐time polymerase chain reaction (RT‐PCR) in the 5′‐non‐coding region (NCR). A semi‐nested PCR was conducted to amplify sequences from the VP1 region and sequence comparison was performed with reference strains registered in Genbank. Male‐to‐female ratio confirmed approximately 5:4. The major clinical manifestation of patients infected with CVB1 was aseptic meningitis (55.8%). The other clinical symptoms were herpangina or hand–foot–mouth disease (22.1%) and neonatal sepsis (7.7%). The sequences of CVB1 isolates were divided into four genetic clusters (A–D) with at least 15% diversity between the clusters. Almost all the CVB1 isolates in Korea from 2008 to 2009 were in cluster D (except for 2 cases). The homology relationship was also similar between the Korean CVB1 strains and US strain (above 93%). It is possible that Korean CVB1 isolates found during 2008–2009 originated from the US strains found during 2006–2008. The identification of CVB1 in South Korea shows the potential of EVs to cause serious disease in an unpredictable fashion. J. Med. Virol. 85:110–115, 2012. © 2012 Wiley Periodicals, Inc.     

5' terminal deletions in the genome of a coxsackievirus B2 strain occurred naturally in human heart

Enteroviruses can induce human myocarditis, which can be modeled in mice inoculated with group B coxsackieviruses (CVB) and in which CVB evolve to produce defective, terminally deleted genomes. The 5' non-translated region (NTR) was enzymatically amplified from heart tissue of a fatal case of enterovirus-associated myocarditis in Japan in 2002. While no intact 5' viral genomic termini were detected, 5' terminal deletions ranged in size from 22 to 36 nucleotides. Sequence of the 5' third of this viral genome is of a modern strain, closely related to CVB2 strains isolated in Japan in 2002. A CVB3 chimera containing the 5' NTR with a 22 nt deletion produced progeny virus upon transfection of HeLa cells. When the 5' 22 nucleotide deletion was repaired, the virus induced myocarditis in mice and replicated like wild type virus in murine heart cells. This is the first report of these naturally-occurring defective enteroviral genomes in human myocarditis.     

Clinical characteristics of echovirus 11 and coxsackievirus B5 infections in Taiwanese children requiring hospitalization

BackgroundSevere illness can occur in young children infected with certain types of enteroviruses including echovirus 11 (Echo11) and coxsackievirus B5 (CoxB5). The manifestations and outcomes of Echo11 and CoxB5 diseases across all ages of children remained not comprehensively characterized in Taiwan.MethodsCulture-confirmed Echo11 (60 patients) or CoxB5 (65 patients) infections were identified in a hospital from 2010 to 2018. The demographics, clinical presentations, laboratory data and outcomes were abstracted and compared between the two viruses infections.ResultsEcho11 and CoxB5 was respectively identified in 7 (77.8%) and 2 (22.2%) of 9 calendar years. The median age of all patients was 15 months (range, 1 day–14.5 years). For infants ≤3 months old, Echo11 (23 cases) was associated with higher incidence of aseptic meningitis (35% versus 0%, P = 0.003), and a lower rate of upper respiratory tract infections (URI) (22% versus 65%, P = 0.004) compared to CoxB5 (20 cases) infections. For patients >3 months old, URI was the cardinal diagnosis (60%) for both viruses. Aseptic meningitis was also more commonly identified in elder children with Echo11 infections (27% versus 11%), though with marginal significance (P = 0.07). Acute liver failure was identified in four young infants with Echo11 infections including one neonate dying of severe sepsis and myocarditis. All patients with CoxB5 infections recovered uneventfully.ConclusionAseptic meningitis, sepsis-like illness and acute liver failure were more commonly identified in children with Echo11 than those with CoxB5 infections, suggesting greater neurological tropism and virulence toward Echo11.     

柯萨奇病毒B3通过caspase依赖途径诱导HeLa细胞凋亡

目的 评价柯萨奇病毒B3（CVB3）致HeLa细胞死亡的方式及分子机制。方法 用CVB3作用于HeLa细胞,在不同时间收集细胞,通过相差显微镜、电子显微镜、流式细胞仪以及分子生物学手段,HeLa细胞的病变及caspase-3基因mRNA和蛋白质的表达。结果 CVB3作用于HeLa细胞后,细胞很快发生变性和坏死,24h后较多细胞凋亡;caspase基因在病毒作用早期即被活化,表现在caspase-3 mRNA在病毒作用后6h内,迅速增高达峰值。在24h内,又降至接近病毒作用前的水平。caspase-3蛋白表达在42h内逐渐增高。结论CVB3可诱导HeLa细胞发生坏死和凋亡两种反应,坏死早于凋亡,细胞凋亡与caspase-3的表达密切相关。     

苦参总碱体外抗柯萨奇B病毒3型作用测定及其机理的初步研究

先用酸水浸泡、氯仿萃取等处理提取苦参总碱，然后通过微量细胞培养法，病毒蛋白质Ｗｅｓｔｅｒｎｂｌｏｔ，分析了不同浓度苦参总碱对病毒增殖和衣壳蛋白含量的影响，表明苦参总碱在体外试验中能抑制柯萨奇Ｂ病毒３型（ＣＶＢ３）的增殖，并呈一定的剂量依赖性。通过病毒吸附和穿入细胞前后苦参总碱作用的比较，显示苦参总碱能进入细胞内发挥抗病毒作用，它可能不影响ＣＶＢ３吸附、穿入等环节，而是影响ＣＶＢ３侵入细胞后某环节，特别是病毒的生物合成。     

Cardioprotective effect of NO-metoprolol in murine coxsackievirus B3-induced myocarditis

The effect of NO-metoprolol, that is, 3-nitrooxypivaloyl metoprolol-amide, a novel NO-releasing derivative of the β1-blocking drug metoprolol was investigated in A.CA/SnJ mice infected with coxsackievirus B3 (CVB3) and compared to metoprolol and placebo. Daily treatment of mice with the respective drug started immediately (experiment A) or 3 days after virus infection (experiment B) and was continued until day 13 post-infection (p.i.). Two doses of NO-metoprolol were administered. Body mass differences, viral load, and histopathological signs of myocarditis were compared between the several groups. As a result, NO-metoprolol diminished significantly the body weight loss, the viral load and the histopathology, whereas metoprolol treatment led solely to a significant attenuation of myocardial damage. In experiment A, low dose NO-metoprolol decreased significantly enteroviral copy numbers. Both doses of NO-metoprolol had a significant effect on reduction of myocardial infiltrates and fibrosis. The data suggest that delayed drug administration might more advantageous. Both doses of NO-metoprolol reduced significantly the scores of four tested parameters compared to placebo. Body weight loss, virus titers, plus-strand as well as minus-strand enteroviral RNA levels, infiltration and fibrosis scores were diminished significantly when NO-metoprolol was given 3 days p.i. In addition, a significant difference regarding the enteroviral copy numbers was observed between low dose NO-metoprolol- and metoprolol-treated mice. Treatment with metoprolol reduced insignificantly the viral load and body weight loss (experiment A and B) but led to a significant reduction of myocardial histopathology in experiment A. The results indicate that NO-metoprolol treatment has a greater therapeutic benefit than metoprolol.     

In‐utero coxsackievirus B4 infection of the mouse thymus

Type B coxsackievirus (CV‐B) infections are involved frequently in the triggering of several autoimmune diseases such as myocarditis, dilated cardiomyopathy, pericarditis, pancreatitis, type 1 diabetes, encephalitis, thyroiditis or Sjögren's syndrome. Serological and virological evidence suggests that maternal infections during pregnancy can play a role in the appearance of these diseases in offspring. The current study aims to explore the effect of an in‐utero CV‐B infection on the fetal thymus, the central site for programming immunological self‐tolerance. In this perspective, female Swiss albino mice were inoculated intraperitoneally or orally with the diabetogenic CV‐B4 E2 strain at gestational days 10 or 17. Offspring were killed at different post‐inoculation times, and their thymuses were analysed for evidence of infection and alterations in thymic T cell subsets. In‐utero CV‐B infection of the thymus was demonstrated during the course of vertical transmission, as attested by viral RNA and infectious virus detection in most analysed samples. No histopathological changes were evident. Thymic T cells were not depleted, despite being positive for viral RNA. As evidenced by flow cytometry analysis, CV‐B infection of the fetal thymus induced significant changes of thymic T cell populations, particularly with maternal inoculation at gestational day 10. Altogether, these findings suggest that CV‐B infection of the fetal thymus may play an important role in the genesis of autoimmune diseases.     

S100B in the cerebrospinal fluid—A marker for glial damage in the rabbit model of pneumococcal meningitis

The rabbit model provides an important experimental setting for the evaluation of antibiotic agents against pneumococcal meningitis. One of the primary targets of this model is the study of neuronal and glial cell damage in bacterial meningitis. The aim of this investigation was to evaluate whether a significant increase of S100B in the cerebrospinal fluid (CSF) as an indicator of white matter damage could be observed in this meningitis model. Seven rabbits were infected intracisternally with S. pneumoniae, and CSF S100B concentrations were examined serially before infection, at 12 h, 14 h, 17 h, 20 h, and at 24 h after infection. The course of CSF S100B increase and its relation to other parameters of brain tissue destruction and CSF inflammation were measured. Axonal damage was visualized by amyloid precursor protein (APP) immunostaining and demyelination by Luxol Fast Blue/Periodic Acid Schiff (LFB-PAS) stain. In each animal, we observed a distinct rise in S100B concentration in the CSF due to pneumococcal meningitis. We conclude that the CSF concentration of the glial S100B protein can be used as an additional parameter for future interventional studies focusing on glial cell damage in the rabbit model of bacterial meningitis.     

A 5-year molecular epidemiology survey of human enterovirus 71 before vaccine application in Yunnan Province,China

Enterovirus A71 (EV-A71) infection is known to cause hand, foot, and mouth disease (HFMD). Last year, an inactivated EV-A71 whole virus vaccine was used to prevent this disease in Yunnan, China. To obtain a viral genetic background for evaluating vaccine protection and monitor the adaptive evolution of the virus after the vaccination, a 5-year molecular epidemiology survey was performed before the vaccination. Twenty-six EV-A71 strains were separated from 561 stool specimens of patients with serious HFMD. The whole-genomic sequences of these strains were sequenced. Phylogenetic trees were constructed, and the mutation spectra were analyzed based on these viral sequences. There was no obvious mutation for the circular EV-A71 strains of the same year. Pathogenic EV-A71 strains may arise from a “subgroup” randomly each year. Whole-genomic analyses showed that a hotspot nonsynonymous substitution potentially affecting the immunogenicity of vaccines was found in the 2A gene, but not in genes of the viral capsid proteins, and the genetic diversity of whole viral genomes associated with the incidence of HFMD. Therefore, it will be valuable to monitor the genome-wide changes of EV-A71 to detect the adaptive mutations affecting immunogenicity or perform investigations using genetic diversity as a parameter.     

Molecular characterization of Coxsackievirus A16 strains isolated from children with severe hand,foot, and mouth disease in Yunnan,Southwest China,during 2009-2015

Coxsackievirus A16 (CV-A16) commonly causes mild symptoms, but severe diseases, such as aseptic meningitis, encephalitis, and even fatal cases, have been reported. Thirteen CV-A16 strains were isolated from patients with severe hand, foot, and mouth disease in Yunnan, Southwest China, from 2009 to 2015. Subgenotype B1a and B1b of CV-A16 were predominantly circulating the region with B1b the predominant strain in recent years. The mean rate of nucleotide substitution based on the VP1 gene sequence was 4.545 × 10 ⁻³ substitution per site per year from 2009 to 2015. These results may help in understanding the genetic diversity of CV-A16 and develop a CV-A16 vaccine.